WO2010105845A2

WO2010105845A2 - A cell cultivation receptacle and related apparatuses

Info

Publication number: WO2010105845A2
Application number: PCT/EP2010/001746
Authority: WO
Inventors: Jan De Sonneville; Maxim Emile Kuil
Original assignee: Universiteit Leiden
Priority date: 2009-03-19
Filing date: 2010-03-19
Publication date: 2010-09-23
Also published as: GB0904720D0; WO2010105845A3; GB2468701A

Abstract

A cell cultivation receptacle (10) comprises a recess layer (11) having formed therein a downwardly extending recess (12) defining an opening (13) on an upper side of the recess layer (11). The recess (12) further defines one or more lateral boundaries (14) that taper inwardly in the downward direction, from the opening (13), to intersect the bottom (16) of the recess. The bottom (16) is defined by a bottom layer (17); and the receptacle (10) a removable closure (18), for the opening, including a closure member (19) that is moveable from an open position spaced from the aperture (13) to a closed position in which it closes off the aperture (13). The closure member (19) includes extending downwardly from an underside a displacement member (21) that when the closure member (19) occupies its closed position occupies part of the interior of the recess (12), the displacement member terminating in a lower surface (22) that is spaced from the recess bottom (16) to define a cell cultivation space in the recess, and the displacement member (21) including at least a portion that tapers in the said downward direction at a more acute taper angle than the taper angle of the or each said lateral boundary (14).

Description

A CELL CULTIVATION RECEPTACLE AND RELATED APPARATUSES

This invention relates to a cell cultivation receptacle. The invention also relates to an apparatus including such a receptacle.

In numerous branches of scientific research it is commonplace to employ cell cultivation receptacles in which researchers cultivate cell cultures intended for study. One very common form of cultivation receptacle is a so-called titre well that typically is a straight- sided cylindrical well formed in a plate, known as a titre plate, that is specially shaped for locking retention in an analytical apparatus.

In practice, a titre plate typically includes an array of titre wells set in a grid-like pattern. One well known arrangement includes 96 titre wells defining an array of 8 rows containing 12 titre wells each.

A titre plate is typically manufactured from a transparent polymer such as polystyrene, polycarbonate, a range of cyclo-olefins or acrylonitrile-butadiene-styrene ('ABS'). The transparency permits researchers to perform various optical tests on cells cultivated in the titre wells. In addition titre wells are suitable for carrying out numerous tests and investigations that do not involve cell material. The invention relates to receptacles that are suitable for cell cultivation purposes, regardless-of whether in practice they are used in conjunction with cell material.

The titre wells are open-ended at their in-use upper ends. Electronically-controlled dosing apparatuses may be employed to inject each of the wells of a titre plate with a culture solution and eg. reagents, enzymes or other additives the effect of which on cells in the titre wells it is desired to study.

As used herein terms such as "upper", "lower", "verticar, "horizontar, "upwardly and "downwardly for convenience are construed with reference to a titre well or cell cultivation receptacle in its upright orientation, as would arise when a titre plate is placed flat on a horizontal surface such as a laboratory bench. It is however recognised that in use of a titre plate or other cell cultivation receptacle its orientation may change eg. as a result of being centrifuged or otherwise agitated, or by reason of being tilted or inverted as part of an experimental or observational procedure. The terms mentioned, and related terms, are not to be construed as limiting the scope of the invention to any particular orientation of the cultivation receptacles, or to any particular mode of use. Since the known titre plate is shaped for locking retention in laboratory apparatuses, following dosing of the titre wells with the desired mixture of substances the titre plate may be treated in accordance with the aims of the experiment in question. Such treatment may include centrifuging the titre plate, warming/heating it and/or subjecting it to controlled exposure to radiation, magnetic fields, electrical fields or other forms of energy.

It is in addition usually particularly important that the titre plate material is transparent, since numerous optical tests have become commonplace in order to assess the behaviour of cell cultures under varying conditions. One particular class of optical tests is spectrophotometric observation. Another, similarly important, technique is flurometric observation. In each case it is necessary to illuminate the insides of the wells using light of a chosen wavelength. For this purpose the material of the titre plate must be transparent to the wavelength in question. Furthermore such techniques often involve reflectance and/or scattering of incident light, sometimes in wavelengths that differ from the incident light wavelengths. Clearly the material of the titre plate must also be transparent to such shifted wavelengths so that observable phenomena may be detected by apparatuses located outside the receptacles.

Conventional titre plates have been highly successful for many years, since they are cheap to manufacture, they are familiar to researchers in many branches of science; and they provide for good repeatability of experiments since the wells of a titre plate are all of the same dimensions and characteristics.

However the performance of conventional titre plates may be sub-optimal in certain situations.

One reason for this is that the capacity of each well of a conventional 96-well titre plate is relatively large at approximately 400 microlitres. Such a volume if even only partially filled with a culture under investigation gives rise to numerous disadvantages.

Several of the disadvantages are discussed in US 6,027,695, which identifies that there is a general desire, in scientific research, to assay ever larger numbers of samples. Partly because of the cost of cell cultures and reagents, a need to assay more samples suggests that it is desirable to assay smaller amounts in each titre well. Moreover when assessing cultures fluorometrically, the material of the titre plate contributes a background fluorescence that becomes statistically significant when the volume in each titre well is reduced compared with conventional quantities.

A further problem associated with simply reducing the quantity of material in a titre well is that the concentration of organic molecules depending on the circumstances may decrease below or may exceed a statistically acceptable threshold level.

When coating a surface with a given surface density of cells the dispensed number of cells is proportional to the surface area. When a small surface is covered it is advantageous to dispense a small volume containing the cells, allowing the use of smaller titre wells and thus smaller quantities of reagents. Scaling down in this way results in an increased ratio of surface area to volume, which means that a higher volume concentration of cells is needed to cover the smaller surface with the same surface density of cells.

A smaller volume however results in a higher concentration of cells, that is the average distance between cells in suspension is less, and therefore the probability of (undesirable) coagulation is higher in such a suspension.

A high and evenly dispersed cell concentration is difficult to achieve, and coagulation of cells results in an unpredictable distribution of these cells.

For the foregoing reasons (ie. a desire for increased testing throughput, without suffering other disadvantages associated with simply reducing the quantities of materials in the titre wells), US 6,027,695 proposes the use of so-called "microwells" the capacities of which are several multiples smaller than the approximately 400 microlitre capacities of conventional titre wells.

In such wells the culture concentrations may be controlled to acceptable levels in order to overcome at least one of the problems identified in US 6,027,695. However, under such circumstances further, practical problems then arise.

When employing microwells it is possible to devise titre plates having considerably more than the conventional 96 wells per plate. In such a design the opening of each well on the upper face of the titre plate is much smaller than in the case of the conventional titre well. This creates a requirement for significantly greater precision in the dosing of the titre wells than is required when considering conventional titre plates. Although it is possible to achieve accurate microdosing, the equipment required to do so is expensive and complicated. Furthermore, as proposed in US 6,027,695, it is often necessary to create special designs of boundaries between adjacent wells for the purpose of avoiding the collection of cell culture on parts of the titre plates outside the wells.

US 6,027,695 identifies further difficulties associated with the use of microwells.

In particular, it is necessary to dose all the wells of potentially a large number virtually simultaneously, in order to minimise the risk of variability of conditions from one well to the next. The apparatuses required for microdosing purposes, however, precisely because they dose only small quantities, typically can fill the wells only quite slowly.

Unless it is possible to arrange dosing of all the wells simultaneously through employing a very large number of injection nozzles (which in itself brings further problems), simultaneous dosing of a large number of microwells may not be possible.

Since when using microwells only tiny quantities of the target cultures are employed, evaporation can become highly significant to the results of an experiment. Especially if it takes a long time to fill all the wells of a microwell titre plate, those dosed initially with cultures and/or reagents may end up with different compound concentrations than those intended by the experimenters.

The paper "Understanding MicroChannel Culture: Parameters Involved in Soluble Factor Signalling" Yu et al, Lab Chip, 2007, 7, 726-730, further identifies that in microchannels (that are similar to microwells) a culture - air interface is undesirable because it creates convective flows in the culture. These tend to distribute both large and small particles throughout the culture, whereas typically it is desired that the large particles remain at the walls of the wells and/or channels so that only the smallest particles are suspended in the culture for optical evaluation.

"Understanding MicroChannel Culture ..." suggests therefore that when considering microwells and similar small-volume assay spaces it is necessary to avoid liquid-air interfaces, for this reason.

US 2008/0145922 A1 proposes a culture receptacle akin to a single well of a conventional 96-well titre plate, that is modified in order to include a cover that closes off the open upper end of the well. The cover includes protruding downwardly into the well a bobbin-like piston. The length of the piston is slightly less than the depth of each well, such that when the cover is fitted in place a small space exists between the underside of the piston and the adjacent bottom of the well.

The piston includes extending longitudinally through it a dosing channel via which culture and/or other substances may be introduced into the well.

The small space that results between the lowermost end of the piston and the well bottom is suitable as a microchannel cell cultivation space. The arrangement of US 2008/0145922, however, does not address the requirement, that is prevalent in the art, to increase the number of assays per experiment through the use of smaller quantities, overall, of liquid substances. On the contrary, it appears that in the case of US 2008/0145922 the quantity of liquid placed into each titre well is the same as in the prior art; and like the prior art devices the US 2008/0145922 plate contains only 96 wells in an 8 x 12 array.

The bobbin-like piston simply serves to create microchannel conditions in a region of the interior of each well. It appears therefore firstly that there is considerable wastage of liquid since a conventional titre well dose is required in order to create microwell or microchannel conditions on insertion of the piston into the interior of the well. It appears that unused liquid simply flows to the "reverse" side of the piston, where it is of limited or no benefit in the experiment under consideration, and may also give rise to varying substance concentrations within the interior of the well.

Furthermore the piston of US 2008/0145922 is complicated in structure, being equipped with a dosing channel and an interface for connection to a dosing machine. Apart from the added cost and complexity of such an arrangement, compared with a planar titre plate cover, the dosing channel may be less reliable in use than other means for introducing substances into the interiors of the wells.

When transmission microscopy is required for the purpose of imaging the interiors of the wells the dosing channel and interface connection block the light path through the wells.

It follows that, overall, there is a need for a simple arrangement that is capable of reliably creating microwell or microchannel cell culture environments, without wasting fluid. According to the invention in a first aspect there is a provided a cell cultivation receptacle comprising a recess layer having formed therein a downwardly extending recess defining an opening on an upper side of the recess layer and further defining one or more lateral boundaries that taper inwardly in the downward direction, from the opening, to intersect the bottom of the recess; a bottom layer defining the said bottom; and a removable closure for the opening including a closure member that is moveable from an open position spaced from the aperture to a closed position in which it closes off the aperture, the closure member including extending downwardly from an underside a liquid displacement member that when the closure member occupies its closed position occupies part of the interior of the recess, the liquid displacement member terminating in a lower surface that is spaced from the recess bottom to define a cell cultivation space in the recess, and the liquid displacement member including at least a portion that tapers in the said downward direction at a more acute taper angle than the taper angle of the or each said lateral boundary.

Such an arrangement is advantageous firstly because the liquid displacement member on insertion into the interior of the recess occupies a substantial percentage of its volume. This means that a relatively small quantity of culture liquid placed in the bottom of the recess may become displaced until only a thin layer of liquid lies in a narrow space between the lowermost extremity of the displacement member and the bottom of the receptacle, thereby efficiently defining a microwell region that is highly suitable for cell cultivation and subsequent experiments / observations.

The fact that the liquid displacement member tapers at a more acute angle than the taper angle of the lateral boundary means that the displacement member cannot become stuck in the receptacle. Also the difference in taper angles means that above the aforementioned microwell region the receptacle having the liquid displacement member received therein defines a well of liquid. The displacement member may, despite its difference of taper angle from the lateral boundary, be arranged to be spaced only a short distance from the lateral boundary along its length. In consequence the concentration of liquid in the well is essentially the same as in the microwell defined between the lowermost extremity of the displacement member and the bottom of the receptacle. The provision of a tapered lateral boundary of the receptacle means that the transport of molecules and organic material in the culture liquid adopts desirable characteristics, as explained in more detail below.

Preferably the horizontal cross-section of the liquid displacement member is essentially complementary to the horizontal cross-section of the recess at the same depth in the receptacle. As indicated, however, advantageously the cross-sections are not mutually congruent so as to allow for a space between the liquid displacement member and the lateral boundary that defines the aforesaid well.

Conveniently the recess is circular in horizontal cross-section and is frustoconical in shape. In view of the foregoing it is also therefore preferable that the liquid displacement member is circular in horizontal cross-section and is frustoconical in shape.

In an alternative arrangement, however, the recess may be polygonal in horizontal cross- section and therefore frusto-pyramidal in shape. Under such circumstances preferably the liquid displacement member is polygonal in horizontal cross-section and frusto- pyramidal in shape.

Almost any polygonal shape is suitable for the cross-section of the receptacle. Preferred polygons include triangles, squares and hexagons. It is believed that when more than six lateral boundaries are defined, by reason of using a higher-ordered polygon than a hexagon, manufacturing of the receptacle may become problematic.

Yet a further possibility is for the cross-sections of the recess and/or the liquid displacement member to be ovaloid.

Regardless of the precise cross-sectional shape adopted, the or at least one said lateral ^■boundary preferably subtends an angle of between 170 degrees and 100 degrees, and especially 150 degrees to 110 degrees, to the bottom of the recess.

The precise angle may be varied for example in order to suit the liquid to be dosed into the receptacle. Many experiments rely on gravitational attraction, when the well is in an initial, upright position, in order to convey particles to the bottom so that they are correctly positioned for growth when the microwell is defined following insertion of the displacement member as described above. The taper angle of the lateral boundary may be chosen to suit the transport characteristics of particular types of particle. In another arrangement, that is of particular benefit when the material of the recess is transparent, the bottom of the recess is concavely curved. In such an arrangement the recess bottom may act as a lens the precise optical characteristics of which may be chosen to suit the type of optical observation required.

When the recess bottom is concavely curved, the or a said lateral boundary preferably subtends an angle of between 170 degrees and 100 degrees, and especially 150 degrees to 110 degrees, to a tangent to the bottom of the recess.

An advantage of a concavely curved recess bottom is that particles can be more evenly dispersed on the said bottom when using gravitational or centrifugal forces to position them in the recess.

In one embodiment of the invention the bottom surface of the displacement member is also convexly curved. Such an arrangement further is advantageous when the displacement member is transparent since it may then also act as a lens the optical characteristics of which may be chosen to suit the precise experiment under consideration.

In view of the foregoing therefore it is advantageous that the recess layer optionally is transparent, and is supported on a base layer. The base layer also may be transparent in order to provide for appropriate optical transmission characteristics of the receptacle.

In accordance with one embodiment of the invention the recess layer is integral with the base layer. In an alternative arrangement the recess layer is formed separately from the base layer. In the latter case optionally the recess layer and the base layer may be releasably securable to one another.

It is further preferable, especially when considering optical observations, that the closure member and the displacement member are formed from one or more transparent materials.

The nature of the transparency (for example in terms of the transmission and/or blocking of particular light wavelengths) may be chosen to suit the experiment and the culture under consideration. In particular it is desirable that the materials of the receptacle are transparent both to incident light (as may be applied eg. during transmission microscopy) and reflected or scattered light (as may result eg. from fluorescing of material in the recess).

Conveniently the closure member and the liquid displacement member are integral with one another. This permits a simple structure that may be straightforwardly moulded using per se known manufacturing techniques.

In a practical embodiment of the invention there is provided a plurality of the recesses formed in the recess layer.

In such an arrangement preferably there is provided a respective said liquid displacement member for each recess.

Such a configuration may conveniently be provided in a receptacle in which a common closure member has extending downwardly from an underside a plurality of liquid displacement members each located so as to penetrate a said recess on moving of the closure member to its closed position.

This arrangement is convenient since it permits the simultaneous closing off of all the receptacles of a test plate in such a way that microwell conditions are simultaneously created in each of them.

In another arrangement, within the scope of the invention, however it may be desirable to provide a plurality of closure members each having one or more liquid displacement members extending downwardly from an underside.

Preferably the receptacle of the invention includes one or more microchannels interconnecting two or more of the recesses.

When as specified herein the recess layer and the base layer are formed separately from one another, the or each microchannel desirably may be defined at the boundary of the recess layer and the base layer.

Numerous options exist for creating the microchannels. For example a half-round channel may be formed in each of the recess layer and the base layer so that on assembly of the layers together the respective channels may align in register with one another to define a circular cross-section microchannel. In another arrangement a channel may be formed (eg. by milling) in the surface of one of the base and recess layers, the other of which is planar to provide a flat boundary to the microchannel.

Conveniently the receptacle includes defined in the closure member an observation region at which optical signals generated in the microchannel are detectable.

Multiple microchannels can form a microfluidic circuit, and can be used as "micro-total analysis" (μTAS) systems such as described in the journal "Lab on a Chip" (Royal Society of Chemistry, 2007 (www.rsc.org/loc)). In such applications the recesses are used to load one or more microfluidic circuits in an automated fashion.

Optionally the receptacle may include one or more elements selected from the list comprising optical sensors, chemical sensors and flow control elements located so as to act on or be influenced by fluid in a said fluidic microchannel. Such features are particularly beneficial when considering μTAS system applications of the receptacle of the invention.

Regardless of whether the microchannels are present or not, and regardless of the microchannel construction, when a plurality of the recesses is provided they preferably are arranged in a regular pattern in the recess layer.

More particularly, the plurality of recesses may advantageously be arranged in mutually orthogonal rows and columns so as to define a grid-like array. Such an arrangement has been shown to be highly suitable for experimental purposes since each recess may be identified by coordinates referring to its column and row whereby assay and screening results may readily be compared, stored and transmitted.

In another arrangement according to the invention, a well may be provided underlying the or a said recess and in register therewith. Such a well may optionally be defined by a well layer that underlies the recess layer and has formed therein the well.

The provision of a well as aforesaid may be advantageous when it is required to provide an enlarged cell culture space. Conveniently the well layer is transparent. The well layer may additionally include formed or defined therein one or more optional optical prisms, mirrors and/or other optical elements.

Such features allow for side viewing of the well, or so-called "dark field" microscopy.

When plural recesses are provided, preferably the receptacle includes a respective said well underlying each recess.

In an embodiment of the invention the displacement member includes formed therein one or more fluid flow passages that permit the conveyance of fluid to and/or from the interior of the recess.

Preferably the or each said passage terminates in a further opening in the said lower surface.

Conveniently the liquid displacement member includes a plurality of the passages and a plurality of the further openings that are arranged in a regular or irregular pattern in the lower surface.

Preferably the or each passage is connectable to a source and/or a drain of fluid.

In one preferred arrangement the receptacle includes a plurality of liquid displacement members two or more of which each include a said passage and are fluidically connected to one another.

Optionally the displacement member may substantially occupy the recess when the closure closes off the opening.

In an embodiment of the invention the receptacle includes received within the recess one or more spacer members that space the liquid displacement member from the bottom of the recess.

Preferably the receptacle includes in the recess a fluid of lower density than the material of the spacer. This ensures that the spacer remains in place at the bottom of the recess after being positioned therein. The spacer member may optionally be formed from a transparent material. The spacer additionally or alternatively may include formed therein one or more through-going holes permitting the passage of flowable matter from one side of the spacer to another. One or more of the holes may be frustoconical in shape. Such holes permit the dosing of specific areas of the recess bottom with cells, cultures, enzymes, reagents or other fluids.

Conveniently the spacer member includes depending downwardly therefrom one or more support feet defining a gap between the spacer member and the bottom of the recess. It is also preferable that the spacer member and the lateral boundary of the recess each include one or more mutually engageable positioning members for locating the spacer member in a predetermined location and/or orientation in the recess.

Preferably the spacer member is removably positionable in the recess. It is also preferable that the spacer member and the recess each include one or more mutually engageable positioning members for locating the spacer member in a predetermined location and/or orientation in the recess.

Such features facilitate insertion of the spacer into the recess and assure that it remains in position after insertion.

In accordance with one embodiment of the invention the recess is frustopyramidal in horizontal cross-section and the displacement member is also frustopyramidal in horizontal cross-section, the liquid displacement member when the closure adopts a closed configuration on two opposite sides engaging and sealing against respective, mutually opposed lateral boundaries of the recess so as to define on two further, mutually opposed lateral boundaries a pair of fluid flow regions that are interconnected by the cell cultivation space whereby to define a fluid flow path extending from one side of the recess to another, the fluid flow path being connectable to one or more devices for effecting flow of fluid in the path.

Such an embodiment is of particular use for example when performing an experiment aimed at capturing particles by a technique involving coating the recess bottom with antibodies and causing fluid in the fluid flow path to flow over the recess bottom. Conveniently, to this and related ends, the receptacle optionally includes one or more fluid flow ports that are fluidically connectable for supplying fluid to and/or from the fluid flow path.

Preferably the area of the bottom of the recess is such as to permit at least 50% of it to be covered by between 3 and 300 cells, especially 50 to 150 cells.

The invention also resides in a closure member having extending downwardly from an in- use underside a liquid displacement member that when the closure adopts a closed position closing off a cell cultivation receptacle occupies part of the interior of the recess, the liquid displacement member terminating in a lower surface and including at least a portion that tapers in the said downward direction. Various features of such closures as are described herein in conjunction with other features of the receptacle are within the scope of the invention when forming part of such a closure when considered independently of the other closure features.

The invention is also considered to reside in a cell culture apparatus including one or more receptacles as defined herein.

Conveniently such an apparatus includes one or more clamps for securing the or each receptacle relative to the apparatus. A preferable form of clamp is or includes a perforated suction plate that is operatively connected to a suction pump. Other forms of clamping device, including mechanical fastenings and adhesive compounds, may in other embodiments be employed.

Preferably the apparatus includes an injector having one or more cannulae for perforating the closure member so as to permit injection of matter into the interior of the or a said recess through the closure member.

An advantage of such an arrangement is that the recesses may be provided in a pre-closed (ie. sealed or sterile) condition. The apparatus may include eg. a manipulator or other support for the injector that pierces the cover member for the purpose of dosing the receptacles. In such an arrangement the disadvantages of evaporation may be reduced or eliminated, especially if the cover member is manufactured from a material the resilient deformability of which permits it to seal any perforation created by the injector. Preferably the injector includes two cannulae, that respectively inject material into the interior of the recess and extract material therefrom.

As indicated it is preferable that the cannulae are supported on a common support that is moveable relative to at least one said recess so as to permit insertion of the cannulae into and withdrawal of the cannulae from the said recess. Even more preferably the support supports a plurality of injectors arranged in a pattern corresponding to the pattern of a plurality of the recesses.

When the receptacle includes a fluid flow path as defined hereinabove optionally one or more said cannulae in use is fluidically connectable to the fluid flow path. Such an arrangement is beneficial when performing a particle capture experiment as outlined hereinabove.

According to a further aspect of the invention a method of treating particles comprises the steps of:

(i) coating the recess bottom of a receptacle or of a receptacle of an apparatus as defined herein, having its closure member in an open position, with antibodies; (ii) causing the closure member to adopt its closed position thereby defining the fluid flow path;

(iii) causing a first fluid, containing one or more particles to which the said antibodies are antigenic, to flow in the fluid flow path;

(iv) causing a second, rinsing fluid to flow in the fluid flow path; and (v) detecting the particles in the recess.

Another method in accordance with the invention includes the steps of:

(i) filling a receptacle as defined herein to a chosen depth with a hydrogel having cells suspended therein; (ii) applying the closure member to the receptacle so that the liquid displacement member contacts the hydrogel;

(iii) causing the displacement member to oscillate towards and away from the bottom of the recess so as to impart mechanical stress to the cells in the hydrogel.

Yet a further method in accordance with the invention includes the steps of:

(i) filling a receptacle as defined herein to a chosen depth with a hydrogel; (ii) causing a plurality of cells to become adhered to the bottom of the recess; (iii) applying the closure member to the receptacle so that the liquid displacement member contacts the hydrogel;

(iv) causing the displacement member to oscillate towards and away from the bottom of the recess so as to impart shear forces on the cells.

A variant of this method involves omitting the hydrogel and therefore includes the steps of:

(i) causing a plurality of cells to become adhered to the bottom of the recess of a receptacle as defined herein; (ii) applying the closure member of the receptacle thereto so that the liquid displacement member is proximate the adhered cells so as to cause displacement of fluid surrounding the cells; and

(iii) causing the displacement member to oscillate towards and away from the bottom of the recess so as to impart shear forces to the cells.

To this end the cells are surrounded by a fluid that is capable of transferring forces generated by the displacement member.

Conveniently, in any of the foregoing cases, the liquid displacement member is shaped so as to contact the hydrogel to varying depths. This feature conveniently permits differing amounts of stress or shear to be applied to cells in different parts of the receptacle.

Optionally the method may include the further steps of: (vi) causing a third fluid, containing optically detectable, further antibodies, that are antigenic to the particles, to flow in the fluid flow path; and

(vii) detecting the particles using one or more optical detection techniques.

In accordance with a further aspect of the invention there is provided a method of forming a tapered recess in a recess layer comprising the steps of:

(i) forming or providing a parallel-sided, circular-section recess extending from the recess layer so as to define an opening in the recess layer and a flat bottom to the recess;

(ii) placing a settable material into the recess so as to lie in a formable material layer on the flat bottom; (iii) inserting a circular frustoconical mould member, having a largest diameter that is less than the diameter of the parallel-sided recess, into the recess so as to displace the settable material and define a tapered, frustoconical shape therein;

(iv) causing setting of the settable material; and (v) removing the mould member from the recess.

Preferably at least the recess layer, the recess and the settable material are inert to one or more of: cells; cellular nuclei; mitochondrial material; nucleic acids; amino acids; proteins; enzymes; embryos.

Conveniently the settable material is or includes a liquid containing a buffer or cell culture medium mixed with one or more gelling agents.

In preferred embodiments the gelling agent is agarose, collagen or matrigel (RTM), or mixtures of two or more of these materials.

Preferably the gelling agent constitutes agarose, collagen or matrigel (RTM).

In preferred embodiments of the invention the surface finish of the mould member is sufficiently smooth as to create tapered walls of the recess that do not inhibit, or inhibit only minimally, the movement of cells towards the bottom layer. The worker of skill is able, based on his own knowledge, to determine the appropriate surface finish specification based on:

• the material of the mould member; • the material of the recess layer; and

• the choice of the particles undergoing study and/or experimentation.

The terms "microwelf and "microchanner as used herein will be understood by the worker of skill in the art; and furthermore may be interpreted by reference to the prior art documents mentioned.

The phrase "liquid displacement member" as used herein refers to any member that operates to displace liquid in the recess forming part of the invention. In particular the phrase refers to a member that directly contacts the liquid in order to displace it although other arrangements, in which the member acts indirectly on the liquid, are possible within the scope of the invention. The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

There now follows a description of preferred embodiments of the invention, by way of non-limiting example, with reference being made to the accompanying drawings in which:

Figure 1 is a schematic, vertically sectioned view of a first embodiment of receptacle according to the invention; Figure 2 is a view similar to Figure 1 of a second embodiment of receptacle according to the invention and including various optional features that may be employed singly or in combination in eg. the Figure 1 arrangement;

Figure 3 is a further, vertically sectioned view of a receptacle including additional, optional features; Figure 4 is a schematic, vertically sectioned view of a cell culture apparatus according to the invention and including a plurality of receptacles such as those shown in Figures 1 to 3;

Figure 5 shows further features of an apparatus according to the invention, including an injection device and a clamp for holding a receptacle according to the invention in the apparatus;

Figure 6A shows in schematic, vertically sectioned view a modified form of closure, in accordance with the invention, including one or more fluid flow channels;

Figures 6B - 6D show possible patterns of the fluid flow channels in the Figures 6A arrangement, in plan view; Figure 7 shows in schematic, vertically sectioned view a receptacle in accordance with the invention in which the displacement member occupies substantially all of the interior of the recess so as to define one or more fluid flow channels;

Figure 8 shows a closure, in accordance with an aspect of the invention, in use in a titre well of conventional design; Figure 9 shows in vertically sectioned view a spacer that may optionally form part of the receptacle of the invention;

Figure 10 shows in schematic, vertically sectioned view a modified form of the spacer arrangement of Figure 9;

Figure 11 shows, in similar view to Figures 9 and 10, another arrangement for supporting a spacer member;

Figures 12 and 13 are views similar to Figures 1 to 3 and 6 to 10 showing injecting and/or pipetting of cells and an embryo respectively; Figure 14 shows in schematic, vertically sectioned view a variant, of the receptacle of the invention, in which a closure member defines a fluid flow path;

Figure 15 shows an apparatus including a receptacle that is similar to the Figure 14 arrangement; Figures 16 to 20 show steps in a preferred method of manufacturing apparatus in accordance with the invention; and

Figures 21 and 22 respectively show two modes of use of the apparatus of the invention.

Referring to the drawings, a receptacle 10 in accordance with the invention comprises a recess layer 11 having formed therein a recess 12 that extends downwardly from an opening 13 formed in the upper surface of recess layer 11 when the receptacle 10 is in its upright orientation as shown in Figure 1.

The recess includes in the embodiment shown a single lateral boundary 14 that terminates at its lowermost extremity by intersecting the bottom 16 of the recess 12.

The single lateral boundary (wall) 14 in the embodiment illustrated arises because the recess bottom 16 and the opening 13 are each circular or ovoid in shape. Therefore a single, continuous lateral boundary 14 defines the recess 12 as essentially frustoconical in shape in the embodiment shown.

Recess bottom 16 is planar, and extends horizontally although as described herein other arrangements of the recess bottom are possible.

The diameter of recess bottom 16 is less than that of opening 13 such that recess 12 tapers inwardly in the downward direction when the receptacle 10 is orientated as shown in Figure 1.

The recess bottom 16 is defined in the embodiment shown by a bottom layer 17 extending along the length of the receptacle 10 underneath the recess layer 11.

The receptacle 10 also includes a removable closure 18 in the form of a horizontally extending, planar closure member 19 having extending downwardly from its underside (that in the embodiment shown is flat) a displacement member 21. Displacement member 21 is in the Figure 1 arrangement a circular frustocone, the centre axis of which is aligned with the centre of the circular recess bottom 16. Displacement member 21 is arranged to displace liquid in the bottom of recess 12, preferably by direct contact therewith.

Since displacement member 21 is a frustum of a cone, it terminates at its lowermost end in a lower surface 22 that extends horizontally when the components of the receptacle 10 are orientated as shown in Figure 1.

The length of displacement member 21 is such that when the closure member 19 closes opening 13 lower surface 22 lies a short distance above recess bottom 16 so as to define a microwell cell cultivation space between the respective surfaces 16 and 22.

As signified by the symbol "α" in Figure 1 , the exterior of displacement member 21 tapers at an angle to the vertical that is less than the angle β subtended to the vertical by the lateral boundary of the recess 12.

As explained, this feature ensures firstly that even though the displacement member 21 tapers in a similar fashion to lateral boundary 14, a gap exists between displacement member 21 and lateral boundary 14 all around the interior of receptacle 12.

Various advantages of such an arrangement are explained above.

The closure member 19 is essentially planar on its underside such that when placed in position to close off the opening 13 it seals around the periphery of the latter. This minimises the risk of evaporation of liquids contained within the recess 12.

The sealing also minimises the risk of cross contamination between multiple recesses 12.

As is apparent from study of Figure 1 , the angle subtended by the lateral boundary 14 relative to the recess bottom 16 is equal to (90 + β) degrees. In the embodiment shown the subtended angle 90 + β lies in the range 170 - 100 degrees, and especially 150 - 110 degrees. Typically the depth of the recess layer 11 is in the range 100 micrometre - 1 cm, and preferably is between 500 micrometer and 5 mm. The most preferred thickness is in the range 1 - 3 mm.

The thickness of the bottom layer preferably is 1 nanometer - 3 millimeter and preferably is between 100 micrometer and 1 mm. A particularly preferred thickness for the bottom layer is 180 micrometer.

The components of the receptacle 10 may be made from eg. glass, quartz, mica or a polymer material such as polypropylene, polycarbonate and cyclo-olefin polymers, polystyrene, polyurethane, PDMS and similar materials. The light transmission characteristics of the materials may be chosen, as indicated, to suit the experiment under consideration.

In the embodiment shown the displacement member 21 is formed separately from the closure member 19. Similarly, the recess layer 11 is formed separately from bottom layer 17.

An advantage of forming the parts of the receptacle 10 in this fashion is that the optical characteristics of different parts of the receptacle may be selected as desired.

In alternative arrangements, however, it is possible within the scope of the invention for example to form the displacement member 21 integrally with the closure member 19 eg. by injection moulding. Similarly if required the recess layer 11 and the bottom layer 17 may be formed integrally with one another.

The distance between the lower surface of displacement member 21 and the recess bottom 16 is, for example, 20 micrometer.

One advantage of the choice of taper angle of the frustoconical shape of recess 12 is that when the receptacle 10 is subjected to treatment in a centrifuge a desired distribution of particles in the cell culture arises. In order to assist in this, the surface roughness of the lateral boundary 14 is chosen to be typically smaller than half of the diameter of the particles in the suspension to be analysed, such that particles cannot become trapped on the surface while gravity or centrifugal force acts to drive the particles towards the recess bottom 16. The closure 18 is advantageous firstly because it enables an optimal view when the receptacle 10 is used in a transmission microscope apparatus, as reflected light is scattered less than in the prior art by the suspension. This is in turn because the overall quantity of the suspension in the light path is less than in the prior art.

As explained herein, the space between the lower surface 22 and the recess bottom 16 is well suited to act as a cell cultivation region. This is partly because the diffusion of secreted soluble signals into the bulk suspension is reduced. Furthermore the particle concentrations may be arranged to be optimal, for example from the standpoint of avoiding coagulation.

The optical properties of the receptacle 10 can however be further improved by using one or more optional features as shown in Figure 2.

In Figure 2 a receptacle 10' is shown that in many respects is similar to receptacle 10 of Figure 1.

Thus in Figure 2 the recess layer 11 , opening 13, closure 18 and closure member 19 are essentially the same as their counterparts in Figure 1. On the other hand the shapes of the recess 12', lateral boundary 14', recess bottom 16', displacement member 20' and lower surface 22' differ from their counterparts in Figure 1 and therefore are represented in Figure 2 through the use of primed numbers.

More particularly, the lateral boundary 14 is not straight-walled when viewed in vertical cross-section as shown in Figure 2, and instead is curved.

The curvature of lateral boundary 14' in Figure 2 is fundamentally parabolic, although other types of curved surface maybe employed. The use of a parabolic curve however is desirable since its dimensions are easy to calculate.

The recess bottom 16' is curved in a continuation of the curvature of the lateral boundary shape 14'. Since in preferred embodiments the recess bottom 16' is made of a transparent material this form of recess bottom acts as a lens the optical characteristics of which can be optimised to suit the measurement apparatus in conjunction with which the receptacle 10' is employed. In a similar fashion, the displacement member 21' includes a lower surface 22' that also is curved, generally in congruity with the curvature of the modified recess bottom 16'.

The transparency of the displacement member 21' and closure member 19 means that the closure 18 also acts as a lens.

The curvature of the recess bottom 16' in addition allows for collection of the particles 23 of interest in the lowermost part of the recess 12'.

This can lead to a more controlled concentration of the particles 23 in a preferred part of the recess 12'. Figure 2 schematically shows a higher concentration of particles 23 at the lowermost part of recess 12' than in the corresponding region of recess 12 of Figure 1.

The curvature of lowermost surface 22' of Figure 2 causes refraction of incident light. It may form a condenser lens that focuses the illuminating light to form a controlled spot or line in a transmission microscope apparatus. The curvature of recess bottom 16' refracts the light transmitted onto it from the displacement member 21', such that light containing experimental information may be studied, absorbed and/or recorded using appropriate apparatus located on the lowermost side of the receptacle 10'.

Although in Figure 2 the curvature of the lateral boundary 14 is shown in conjunction with both curvature of the lower surface 22' and curvature of the recess bottom 16', if desired each of these features may be employed in isolation in an embodiment of the invention or in combination with only one of the other such features.

Figure 3 shows in vertically sectioned view a further embodiment of receptacle 10" according to the invention.

The construction of receptacle 10" is very similar to that of receptacle 10 of Figure 1, such that the recess layer 11 , recess 12, opening 13, lateral boundary 14, recess bottom 16, bottom layer 17, closure 18, closure member 19, displacement member 21 and lower surface 22 may if desired be constructed exactly as shown in Figure 1 , or in accordance with designs that differ from the Figure 1 designs only in relatively small details. The primary difference between receptacle 10" of Figure 3 and receptacle 10 of Figure 1 is that interposed between recess layer 11 and bottom layer 17 is a well layer 24 having formed therein a downwardly extending well 26.

Well 26 is formed as a straight-sided cylindrical bore passing through well layer 24. The diameter of well 26 (assuming its horizontal cross-section is circular) is the same as that of recess bottom 16 and the narrowest point of recess 12. As a result well 26 extends directly downwardly from a location slightly below lower surface 22.

Well 26 is suited eg. for cell tissue culture generation, the study of biopsy materials and the study of cells that are cultivated in gels, the dimensions of which may be chosen to match those of well 26.

Well layer 24 may be manufactured integrally with either or both of recess layer 11 and bottom layer 17. Well layer 24 may be made from e.g. glass, quartz, a polymer such as polypropylene, polycarbonate, cyclo-olefin polymers, polystyrene, polyurethane or PDMS.

Also visible schematically in Figure 3 and set into well layer 24 spaced a short distance radially outwardly from well 26 is a pair 27, 28 of optical elements. The optical elements 27, 28 may be e.g. optical prisms, mirrors or combinations of such devices. One purpose of the optical elements 27, 28 is to permit side viewing of the contents of the well 26. Such elements also are suited to dark field microscopy.

In addition the lower surface 22 and/or recess bottom 16 of the Figure 3 embodiment may if desired include curved surfaces, in like manner to their counterparts shown in Figure 2, so as to act as lenses.

As stated above, the horizontal cross-section of the recess 12 may be circular or ovoid. When so configured the receptacle 10" is best suited for incorporation of a well such as well 26 since this may readily be constructed as a cylindrical chamber.

It is however possible for the cross-section of the receptacle 12 and, if desired, of the displacement member 21, to be polygonal. In such an arrangement the well 26 may also be constructed as a polygon in horizontal cross-section (although it is believed that such an arrangement is more difficult to manufacture reliably than the cylindrical version described above). Figure. 4 shows an apparatus 30 according to the invention including a plurality of receptacles 10/10710" that may be of any of the designs disclosed herein, or may be hybrids of such designs as would occur to the worker of ordinary skill in the relevant art.

In Figure 4 the apparatus 30 is shown as having only a pair of the receptacles 10. In a practical embodiment a significant number of the receptacles would be arranged in a grid-like array, in ways as described herein.

The recesses 12 of the adjacent receptacles 10 shown in Figure 4 are fluidically interconnected adjacent the respective recess bottoms 16 by way of microfluidic channels 31 formed between the bottom layer 17 that is common to the receptacles 10 shown and the recess layers 11.

The microfluidic channels may be formed e.g. by milling, etching or otherwise forming in- register eg. semi-round or rectangular channels in the underside of each recess layer 11 and the upper surface of the bottom layer 17. In an alternative arrangement the microfluidic channels 31 may be formed in only one of these components, with the other serving to define a planer boundary of the channels 31. In either case, however, the formation of microfluidic channels 31 is best suited to embodiments of the receptacles 10 in which the recess layers 11 are formed separately from the bottom layer 17 and subsequently adhered together.

Since the closure member 19 (that in the embodiment shown is common to a plurality of the displacement members 21 , although this need not necessarily be so), and the recess layers 11 preferably are formed of transparent materials it is possible to define an optical observation zone 32 identified by arrowheads in Figure 4. In the optical observation zone, that preferably lies between adjacent recesses 12, it is possible to obtain optical data (e.g. through using transmission microscopy) on the status of material in the microfluidic channel 31 interconnecting the two adjacent recesses 12.

In the embodiments of the invention including microfluidic channels it is possible to incorporate microsensors and fluid flow control elements into the channels or other components of the receptacle such as the bottom layer 17 and/or the recess 12 or other parts of the receptacle 10 that in use are proximate fluid. Such parts include but are not limited to a well 26 described below in relation to Figure 7. Such sensors may include chemical or optical detectors; and such elements may include powered (active) or non-powered (passive) flow control devices.

When the receptacle 10 is modified as aforesaid it is useable for experiments involving eg. electrophoresis, transcriptome analysis, polymerase chain reaction amplification and liquid chromatography.

If it is possible to cause migration of liquid from one recess 12 towards another, it becomes possible using the apparatus of the invention to perform reaction studies in the region of the microfluidic channel 31 that corresponds to the optical observation zone 32.

Migration may be effected for example by creating an electroosmotic gradient in the microfluidic channel 31. One way of achieving this is to provide a voltage difference by use of two or more conducting rods 33 that penetrate through the closure member 19 and recess layer 11 to contact fluid in the microfluidic channel 31. Connection of conducting rod 33 to a controlled voltage source can create the aforesaid electroosmotic gradient that can in turn cause migration of material from one recess 12 towards another, such that reaction effects can be observed by way of the optical observation zone 32.

Numerous arrangements for connecting conducting rods 33, that preferably are provided in plural numbers, to a voltage source will occur to the worker of skill in the relevant art. Instead of penetration of the closure member 19 and recess layer 11 , the electrical wire/path 31 may end on the side of the device where other arrangements of connecting conducting rods 33 may be provided.

In Figure 5 an apparatus 40, that is similar to apparatus 30, is shown in vertically sectioned view.

In apparatus 40 a practical number of receptacles 10, that each may be of a design similar to those disclosed hereinabove, is provided in an array. The array extends as is visible in Figure 5 from one end of the apparatus 40 to the other and also into the plane of Figure 5.

Apparatus 40 may include a clamp in the form of a hold-down plate 41. Hold-down plate 41 may be embodied in any of a range of per se known ways in order to secure the array of receptacles 10. One method of operation of the hold-down plate involves the formation of a matrix of apertures extending through the hold-down plate. On its underside the apertures are connected to a suction pump that when operated draws the array of receptacles 10 downwardly into gripping engagement with the plate 41.

Other means of securing the array however are within the scope of the invention and include (but are not limited to) mechanical fastenings and adhesive compounds.

The apparatus 40 of Figure 5 includes a fluid injector 42 having protruding downwardly from its underside a pair of cannulae 43, 44.

Injector 42 may be moved in an indexing motion to overlie any of the receptacles 10. Injector 42 is also moveable in a vertical direction such that the cannulae 43, 44 may perforate the closure member 19 of each receptacle 10 for the purpose of injecting fluid into each recess 12 and/or removing fluid therefrom.

In another arrangement a plurality of the receptacles 10 may be moveable laterally so as sequentially to underlie an injector 42 that is constrained to move only in the vertical direction. This represents an alternative way of achieving multiple receptacle dosing.

Injector 42 is shown in schematic form, although it may adopt any of range of practical formats that will occur to the worker of skill in the art. Such formats include multiple-head injectors, that are capable of injecting plural numbers of the recesses 12 simultaneously.

In the arrangement shown in Figure 5 the recesses 12 may be pre-sealed by way of the closure members 19. In practice a common closure member would be provided for all the recesses 12.

Perforation of the closure member(s) 19 by the cannulae 43, 44 minimises the possibility of evaporation from the recesses 12. As explained hereinabove if the material of the closure member 19 is "self-sealing" evaporation would not occur even after withdrawal of the cannulae 43, 44 as a result of the vertical motion of the injector 42.

The injector 42 may be mechanically, electrically and fluidically connected to a control and supply arrangement the precise design of which will be within the capability of the worker of skill in the art. Figures 6 to 10 disclose various optional features of receptacles, closures and apparatuses in accordance with the invention.

In Figure 6A there is shown in vertically sectioned view a closure 18' according to the invention, that includes an in-use horizontally extending closure member 19'.

As specified herein, the invention resides in a closure as defined and/or described herein when considered on its own, ie. separately from the remainder of the components of the receptacle 10. Figure 6A is illustrative of this concept; and also shows further, optional features of the closure. Such features may be present either when the closure is considered on its own or as part of a receptacle 10.

In particular, in Figure 6A displacement member 21' includes a core 46 that is of approximately the overall shape of the downwardly extending displacement member 21'; and an outer wall 47.

Outer wall 47 is essentially congruent with the shape of core 46 but in certain regions of the displacement member 21' is spaced therefrom so as to define one or more downwardly extending fluid flow channels 48, 49.

The flow channels 48, 49 extend upwardly to merge with horizontal flow supply channels 48', 49' formed a similar manner to the channels 48, 49 on the underside of closure member 19'.

In Figure 6A since two fluid flow channels 48, 49 are visible it will be apparent that these may be allocated respectively for the purpose of feeding fluid into and extracting fluid from the interior of the recess 12 of a receptacle 10 as shown in Figures 1 to 5.

In order to achieve such an arrangement, the passages 48', 49' may be operatively connected to sources of fluid, pumps and extraction devices as appropriate. To this end the passages 48', 49' may terminate in appropriate connectors eg. on the exterior of closure member 19'.

Each of the passages 48, 49 terminates in a further opening in the lower surface 22' of the displacement member 21 '. In Figure 6A, the passages 48, 49 extend on opposite sides of the core 46. As is apparent from Figures 6B, 6C and 6D, however, in which the shaded parts represent the material of the core 46 and the dotted lines the outer wall 47, more or fewer than two passages 48, 49 may be provided. They may be established in a range of regular or irregular patterns, although it is preferable that the passages open at the outer edges of the lower surface 22' of the displacement member 21' so as to take advantage of their formation towards the exterior of the displacement member 21' by reason of the construction involving outer wall 47.

The closure 18' may include depending downwardly therefrom a plurality of displacement members 21' as exemplified by the designs shown in Figures 6A to 6D. In such a case, the passages 48', 49' may as desired be fluidically interconnected so as to define a supply and/or drain network feeding fluid to and extracting fluid from the interior of a recess 12 of a receptacle 10 as described herein. As will be apparent to the worker of skill in the art, a wide variety of arrangements of the fluid passages and their interconnections is possible within the scope of the invention.

Figure 7 shows another variant that is within the scope of the invention.

In Figure 7 receptacle 10" is substantially of the same design as that of Figure 3, in that a well layer 24 defines a well 26 in which eg. a cell (exemplified by numeral 51), embryo or other organism may be cultivated. The prisms 27, 28 that optionally are present as disclosed in relation to Figure 3 are also visible in Figure 7.

In the Figure 7 arrangement a pair of fluid flow passages 52, 53 is defined as a pair of open channels formed both in the outer surface of the displacement member 21" and on the underside of the closure member 19". The fluid flow passages 52, 53 therefore become fully defined only on insertion of the displacement member 21" into the recess 12. To this end, the displacement member 21" occupies substantially all of the interior volume of the recess 12 when inserted therein, save for the portions defined as the fluid flow passages 52, 53.

One or more connection ports 54 may be defined eg. as through-going bores extending through the closure member 19' so as to be in fluid communication with one or more of the passages 52, 53 as illustrated schematically in Figure 7. Various means are known in the art for connecting eg. a source of fluid via such a port. Such means include but are not limited to clamps, screw-connectors and adhesive connections. When the displacement member 21' is formed from a material such as PDMS or the other polymers mentioned herein, except in the regions represented by the passages 52, 53 the displacement member 21' seals against the lateral boundary 14 of the recess 12. In consequence the environment in the well 26 may be carefully controlled.

As is apparent from the schematic illustration of port 54, this feature may be connected to only a single well 26 via a single passage 53; or it may be connected to a plurality of such wells 26 that are part of an array of recesses 12. The means for achieving such connection will be known to the worker of skill.

When considering arrangements such as those shown in Figures 6 and 7, in which channels are formed in or defined by the displacement member 21, it may be desirable to secure the closure member 19 against leakage. Various means, within the knowledge of the worker of skill in the art, may be employed to achieve such sealing. As examples one may consider the use of clamping members, fasteners and adhesive compounds.

Among other things, the advantages of the arrangement shown in Figure 7 are that the well dimensions may be chosen to suit the experiment under consideration. Thus, for example, if zebrafish embryos are cultivated, the wells 26 of an array of receptacles 10 according to the invention may be between 0.8 millimetres and millimetres in diameter, and preferably between 1 and 1.5 millimetres in diameter. For such applications, the most preferred embodiment is 1.3 millimetres in diameter.

When considering a smaller embryo (such as C Elegans) the dimensions of the wells 26 may be adjusted appropriately during manufacture of the receptacles 10.

Such an arrangement may be readily manufactured using the well layer 24 of the invention, since the remaining parts of the receptacle 10 may be standardised and only the well layer 24 modified in accordance with the requirement of the precise experiment under consideration, before assembly of the receptacle takes place.

Advantages of selecting the well dimensions specifically for a particular experiment include:

less precision is required to inject embryos into the well; there is less scattering of light by the culture medium than would otherwise occur;

the fluid channels of the kind described herein may be accurately located for providing nutrients to the cell and/or removing waste products;

aside from the benefit of providing mirrors and/or prisms 27, 28, the well layer 24 additionally may provide for possible differential interference contrast (DIC) microscopy.

The foregoing advantages arise in addition to the benefits of good soluble signalling and the provision of adequate nutrients for a prolonged period of activity, that are explained hereinabove.

Figure 8 shows in schematic, vertically sectioned view a closure 18 in accordance with the invention and as defined herein in use to close a standard titre well, of the type that is conventionally encountered in a 96-well titre plate.

Among other advantages that derive from use of the closure 18 with a standard titre well are that good soluble signalling arises yet there remains enough cell medium volume around the displacement member 21 to provide enough nutrients for a growing cell for a prolonged period (eg. two days or more).

The displacement member 21 may be made eg. from a gas permeable material such as PDMS. A gas permeable material allows for gas exchange (oxygen and CO₂) between the medium displaced by the displacement member (and hence residing at a high level in the recess 12) and that located adjacent the recess bottom 16.

In addition, the entire closure 18 may be made from a gas permeable material, such as PDMS, or a combination of materials. This enables optional gas exchange when the cell cultivation device is placed in a cell incubation oven.

As best shown in Figure 9, the receptacle 10 of the invention may optionally include one or more spacer members 57 that in the preferred embodiments are removably positionable in the recess 12 so as to lie on recess bottom 16 as shown. An advantage of using the spacer member 57 is that it may correctly space the displacement member 21 from the recess bottom 16, so as to define the desired cell cultivation space and also to control the displacement of fluid in the recess 12.

Although the spacer member 57 may optionally be fixed to the recess bottom 16, it is preferred that the spacer member 57 is removably insertable. To this end the spacer member 57 may be manufactured from a material whose density is greater than that of the fluid in the recess 12, such that it sinks into place in the recess 12.

Spacer member 57 may if desired be manufactured from a transparent material, in accordance with the criteria, for transparency, described herein.

Preferred materials for the spacer member 57 include quartz and glass.

Spacer member 57 includes formed therein one or more through-going bores 58 permitting the transport of fluid from one side of the spacer member 57 to the other. This permits the re-supplying of nutrients, etc. to cells in the cultivation space.

A modified version of the spacer member 57' is shown in schematic, vertically sectioned view in Figure 10.

The Figure 10 spacer member 57' includes modified bores 58' that taper, as illustrated, in the downward direction. Such bores are suitable for targeting of introduced nutrients onto the cells or other particles 23 being cultivated within the receptacle 10. The use of the frustoconical or other tapered shapes shown assists in accurate targeting of injected fluids.

Figure 10 also illustrates somewhat schematically a further, optional feature of the spacer member 57' namely the presence of features such as feet 59 that engage with complementary features (such as recesses) formed in or secured to the interior of recess 12.

The purpose of such engagement is to assure that the spacer member 57' is always inserted into the recess 12 at the correct location and in the right orientation for targeting of fluids on to the particles 23. Other forms of inter-engagement members may of course be provided acting between the spacer member 57' and the structure of the recess 12. As a further example the sides of the spacer member 57' may be arranged to engage the lateral boundary 14 so as to prevent rotation of the spacer member.

Yet a further arrangement is shown in Figure 11 , in which the spacer member 57 omits the feet 59 and instead is supported on one or more horizontally extending shoulders 62 extending radially inwardly from the lateral boundary 14 at the lower end of recess 12. Such a means of supporting the spacer member 57 may be of particular benefit when the receptacle 10 includes a well 26 of the kind described above.

The feet 59 are also present in the Figure 9 embodiment, but in that case they serve simply to raise the spacer member 57 off the recess bottom 16 so as to define an adequate cell cultivation space.

In Figure 10 the particles 23 reside on a gel layer 61 as illustrated. In some modes of use of the receptacle shown in Figure 10 the spacer layer 57' may be removed after seeding and adhering of the cells has occurred. The spacer member 57 or another design of spacer member may subsequently be inserted into the recess 12, eg. for the purpose of seeding further cells or for other activities as will occur to the worker of skill.

One purpose of using a different design of spacer member 57 at a second stage of the experiment is to provide a shifted pattern of the bores 58' such that subsequently seeded cells / particles may be adhered to a different location of the gel layer 61 than that originally used.

Yet a further reason for re-inserting a spacer member 57 of modified design compared with that originally used during seeding of the particles 23 is to block selected regions of the gel 61 or recess bottom 16 from the culture medium, for the purpose of assessing the effects of deprivation of medium on the cells 23. Also under such circumstances the tendency of the cells 23 to migrate to regions that are rich in nutrients may be observed.

In applications in which the cells seek to migrate to nutrient spots it is possible to study protein interactions in vitro.

The use of a variety of designs of spacer member 57, 57' may furthermore provide for a 3D cell tissue comprising different cell types at different locations. The spacer member 57, 57' may in addition be formed with one or more recesses and/or projections the purpose of which is to assist when inserting the spacer member 57' into and removing it from the recess 12.

The spacer member 57, 57' may be manufactured from a gas-permeable material, or from a mixture of gas-permeable and non-permeable materials (such as but not limited to quartz, glass and the various polymeric materials mentioned herein).

As best illustrated in Figures 12 and 13, the taper formed in at least a portion of the lateral boundary 14 is of benefit when it is desired to inject cells (Figure 12) or an embryo (Figure 13) or otherwise apply a flowable substance using a hypodermic-type injector needle 63 or as an alternative, a pipette or similar device. The angle of the taper may be chosen to facilitate manipulation of the injector needle, etc, 63 in the recess 12.

Figure 14 shows a variant of the receptacle 10 lying within the scope of the invention.

In Figure 14 the recess 12 and the displacement member 21 are each frustopyramidal in shape. The two flanks of the frustopyramidal closure member 21 that are not visible in Figure 14 seal against the adjacent lateral boundaries 14 of the recess 12 in a leak-proof manner. If necessary the closure 18 may be clamped or otherwise secured shut in order to achieve such sealing.

The other two flanks 21a, 21b of displacement member 21 are spaced from the lateral boundaries 14. As a result in conjunction with the cell cultivation space defined beneath its lower surface 22 the displacement member 21 defines a fluid flow path 64.

Such a path is useful in experiments in which flow of fluid, signified schematically by arrows 66, is desired.

Figure 14 shows an exemplary connection port 67 (that may be of the kinds described above) the purpose of which is to permit the supply and/or drawing of fluid in the path 64.

Figure 14 shows the path 64 connecting, as signified by numeral 64a, to a similar fluid flow path defined in another receptacle 10. In an alternative arrangement however a further port 67 may be provided. Figure 15 shows an arrangement that is similar to Figure 13 except that the fluid flow port(s) 67 is/are replaced or augmented by an injector 68 that is similar to injector 42 of Figure 5. In the Figure 15 embodiment the cannulae 69, 71 of injector 68 penetrate respective ends of the fluid flow path 64 so as to act respectively as infeed and outfeed cannulae.

As signified by arrow 72 injector 68 may be raised and lowered (eg. under the control of programmable motor drives) to cause penetration and withdrawal of the cannulae. The injection of fluid may itself be controlled eg. electronically; and the injector 68 and/or the recess layer 11 may be moveable laterally so as to effect dosing of multiple cell cultivation spaces via multiple examples of the fluid flow path 64.

The arrangements of Figures 14 and 15 are useful for example when it is desired to capture particles such as proteins, microvesicles or cells having exposed proteins.

In such experiments in accordance with the method of the invention the recess bottom 16 may be initially coated with antibodies that are in some way antigenic, or at least mutagenic, to the particles of interest. Such coating may be achieved by pipetting the antigens onto the recess bottom 16 with the closure 18 in its open position.

Thereafter the closure 18 is closed so as to define the fluid flow path 64. The port 67 may be connected to a fluid supply containing the particles of interest, or an injector 68 employed to charge the fluid flow path with fluid. As the particles resultingly flow over the recess bottom 16 they become attached to the antibodies.

The fluid flow path 64 may then be further employed for the purpose of flushing un- adhered particles away from the cell cultivation space. The adhered particles are then available for detection, experimentation and/or study.

Using the arrangement of Figure 14 multiple cell cultivation spaces may be treated using a single source.

In a refinement of the foregoing method the fluid flow path 64 may subsequently be employed for the purpose of conveying further antigens to the target particles into the cell cultivation space(s). Such further antigens may be eg. radiolabeled or tagged with fluorescent tags. Subsequent scintigraphy or optical detection tests may be employed to identify the particles, of interest, to which the further antibodies have adhered. Among other advantages of the foregoing technique are the following:

• only a small area of the recess 12 is initially coated with antibodies, thereby saving on materials and energy;

• at the coated area the cell cultivation space may be eg. only two or three particles high, leading to good particle adherence;

• the Reynolds number of fluid outside the (narrow) fluid flow path 64 is relatively high, giving rise to low pumping pressure requirements and minimal shear forces;

• with the closure member 19 in its open position flushing with air or water / other solvents is readily possible;

• fluid flow can be effected in either direction along the fluid flow path 64, and multiple fluid passes are possible;

• as stated, a single fluid source can be used to supply fluid to multiple, interconnected recesses.

One possible method for manufacturing the recess 10 visible eg. in Figure 1 will now be described with reference to Figures 16 to 20.

In Figure 16 a conventional, parallel-sided, open-ended cylindrical well 12' extends away from a recess layer 11 so as to define a bottom wall 17 that is spaced from layer 1 1 and an opening 13 formed in recess layer 11. In a preferred embodiment well 12' is a titre well or microwell and recess layer 11 is the structural upper plane of a well plate such as but not limited to a titre plate.

As a first step in manufacture of the recess 10a liquid 81 is filled into well 12' to a depth below opening 13.

Liquid 81 is settable by reason of being a mixture of a gelling agent and a cell culture liquid or a buffer. Typically the gelling agent is agarose, collagen or matrigel (RTM) or mixtures of two or more such components. The gelling agent typically is present in a quantity of between 1% and 2% by mass of the liquid 81. The liquid is at least inert to material to be studied, cultivated or otherwise treated in the recess 10 at least in the sense of being harmless to such material. The presence of ingredients such as culture medium, buffer, collagen and/or matrigel actively promotes the survival of biological material.

As shown in Figure 17 a circular frustoconical mould member 82 is inserted into well 12' in order to contact the liquid 81 and impress a tapered, frustoconical shape. During this process the flattened, free end of the mould member 82 contacts the bottom layer 17 of well 12' so as to displace the liquid entirely away from this region. In other embodiments of the method this need not be so.

The diameter D of the free end preferably is in the range 10 to 400 micrometers and preferably about 200 micrometers.

In the condition shown in Figure 17 solidification (setting) of the liquid is initiated. This might typically involve heating, cooling, combinations of heating and cooling, or other techniques depending on the chosen composition of the liquid 81.

Following setting of the liquid 81 the mould member 82 is removed. The result is the tapered, frustoconical recess 12 described above and as shown in Figure 19. This may be dosed with a cell medium 83 containing cells, embryos or other biological material 84 requiring study or treatment.

As shown in Figure 20 a closure member 19 including a liquid displacement member 21 may be employed, in accordance with the principles described herein, in order to promote efficient survival of the biological material that by reason of tapering of recess 12 is located on the recess bottom as illustrated in Figures 19 and 20. To this end the taper angle E shown in the drawings typically is between 30 and 60 degrees, and preferably is around 45 degrees.

Figures 21a - 21 d show sequentially, in schematic view, one mode of use of apparatus in accordance with the invention in which a receptacle 10, closure member 18 and attached liquid displacement member 19 are operated in accordance with the following method steps:

In Figures 21c and 21 d the arrows show the oscillations of the closure member 18, that preferably are repeated multiple times.

The hydrogel is shown schematically by way of the shading in the figures.

Figures 22a - 22d show a similar mode of use, the primary difference between the Figures 21 and the Figures 22 being that in the Figures 22 the cells are adhered to the receptacle bottom instead of being suspended in the hydrogel as in Figures 21a - 21d. This means that in Figures 22a - 22d the cells undergo shear, in preference to stress, as indicated by horizontal arrows in Figure 22c.

The method shown in Figure 22 omits a hydrogel entirely but in yet a further variant of the invention the adhered cells could be present in the receptacle with a hydrogel also present.

In any of the aforementioned cases the depth of displacement member 19 need not be constant and it may instead be eg. stepped at its free end or otherwise shaped so as to impart different stresses or shear forces on cells in different parts of the receptacle.

Claims

1. A cell cultivation receptacle comprising a recess layer having formed therein a downwardly extending recess defining an opening on an upper side of the recess layer and further defining one or more lateral boundaries that taper inwardly in the downward direction, from the opening, to intersect the bottom of the recess; a bottom layer defining the said bottom; and a removable closure for the opening including a closure member that is moveable from an open position spaced from the aperture to a closed position in which it closes off the aperture, the closure member including extending downwardly from an underside a liquid displacement member that when the closure member adopts its closed position occupies part of the interior of the recess, the liquid displacement member terminating in a lower surface that is spaced from the recess bottom to define a cell cultivation space in the recess, and the liquid displacement member including at least a portion that tapers in the said downward direction at a more acute taper angle than the taper angle of the or each said lateral boundary.

2. A receptacle according to Claim 1 wherein the horizontal cross-section of the liquid displacement member is essentially complementary to the horizontal cross section of the recess at the same depth in the receptacle.

3. A receptacle according to Claim 1 wherein the recess is circular in horizontal cross-section and is frustoconical in shape.

4. A receptacle according to Claim 3 wherein the displacement member is circular in horizontal cross-section and is frustoconical in shape.

5. A receptacle according to Claim 1 wherein the recess is polygonal in horizontal cross-section and is frusto-pyramidal in shape.

6. A receptacle according to Claim 6 wherein the displacement member is polygonal in horizontal cross-section and is frusto-pyramidal in shape.

7. A receptacle according to any preceding claim wherein the or a said lateral boundary subtends an angle of between 170 degrees and 100 degrees, and especially 150 degrees to 110 degrees, to the bottom of the recess.

8. A receptacle according to any preceding claim wherein the bottom of the recess is concavely curved.

9. A receptacle according to any preceding claim, wherein the lateral boundary is concavely curved.

10. A receptacle according to Claim 9 wherein the shape of the lateral boundary is parabolic when viewed in vertical section.

11. A receptacle according to Claim 8 wherein the or a said lateral boundary subtends an angle of between 170 degrees and 100 degrees, and especially 150 degrees to 110 degrees, to a tangent to the bottom of the recess.

12. A receptacle according to any of Claims 8 to 11 wherein the lower surface of the displacement member is convexly or concavely curved.

13. A receptacle according to any preceding claim wherein the recess layer is transparent, and is supported on a base layer.

14. A receptacle according to Claim 13 wherein the base layer is transparent.

15. A receptacle according to Claim 13 wherein the recess layer is integral with the base layer.

16. A receptacle according to Claim 13 wherein the recess layer is formed separately from the base layer.

17. A receptacle according to Claim 16 wherein the recess layer and the base layer are releasably securable one to the other.

18. A receptacle according to any preceding claim wherein the closure member and the liquid displacement member are formed from one or more transparent materials.

19. A receptacle according to Claim 13 wherein the closure member and the liquid displacement member are integral with one another.

20. A receptacle according to any preceding claim including a plurality of the recesses formed in the recess layer.

21. A receptacle according to Claim 20 including a respective said liquid displacement member for each said recess.

22. A receptacle according to Claim 21 including a common closure member having extending downwardly from an underside a plurality of liquid displacement members each located so as to penetrate a said recess on moving of the closure member to its closed position.

23. A receptacle according to Claim 21 including a plurality of closure members each having one or more liquid displacement members extending downwardly from an underside.

24. A receptacle according to any of Claims 20 to 23 including one or more microchannels interconnecting two or more of the recesses.

25. A receptacle according to Claim 24 when dependent from Claim 16 or any preceding claim depending therefrom, wherein the or each microchannel is defined at the boundary of the recess layer and the base layer.

26. A receptacle according to Claim 24 or Claim 25 including defined in the closure member an observation region at which optical signals generated in the microchannel are detectable.

27. A receptacle according to any of Claims 24 to 26 including one or more elements selected from the list comprising optical sensors, chemical sensors and flow control elements located so as to act on or be influenced by fluid in a said fluidic microchannel.

28. A receptacle according to any of Claims 24 to 27 including an electroosmosis element extending from the exterior of a said receptacle to the interior of a said microchannel and being connected to a voltage source.

29. A receptacle according to any of Claims 20 to 28, wherein the plurality of recesses are arranged in a regular pattern in the recess layer.

30. A receptacle according to Claim 29 wherein the plurality of recesses are arranged in mutually orthogonal rows and columns so as to define a grid-like array.

31. A receptacle according to any preceding claim further including a well underlying the or a said recess and in register therewith.

32. A receptacle according to Claim 31 including a well layer underlying the recess layer and having formed therein the well.

33. A receptacle according to Claim 32 wherein the well layer is transparent.

34. A receptacle according to Claim 32 wherein the well layer includes formed or defined therein one or more optical prisms and/or mirrors.

35. A receptacle according to any of Claims 31 to 34 when dependent from Claim 20, including a respective said well underlying each said recess.

36. A receptacle according to any preceding claim, wherein the liquid displacement member includes formed therein one or more fluid flow passages that permit the conveyance of fluid to and/or from the interior of the recess.

37. A receptacle according to Claim 36 wherein the or each said passage terminates in a further opening in the said lower surface.

38. A receptacle according to Claim 37 including a plurality of the passages and a plurality of the further openings that are arranged in a regular or irregular pattern in the lower surface.

39. A receptacle according to any of Claims 36 to 38 wherein the or each passage is connectable to a source and/or a drain of fluid.

40. A receptacle according to any of Claims 36 to 39 including a plurality of liquid displacement members two or more of which each include a said passage and are fluidically connected to one another.

41. A receptacle according to Claim 31 or any preceding claim depending therefrom, wherein the liquid displacement member substantially occupies the recess when the closure closes off the opening.

42. A receptacle according to any preceding claim including received within the recess one or more spacer members that space the liquid displacement member from the bottom of the recess.

43. A receptacle according to Claim 42 including in the recess a fluid of lower density than the material of the spacer member.

44. A receptacle according to Claim 42 or Claim 43 wherein the spacer member is formed from a transparent material.

45. A receptacle according to any of Claims 42 to 44, wherein the spacer member includes formed therein one or more through-going holes permitting the passage of flowable matter from one side of the spacer to another.

46. A receptacle according to Claim 45 wherein one or more said through-going holes is frustoconical in shape.

47. A receptacle according to any of Claims 42 to 46 wherein the spacer member includes depending downwardly therefrom one or more support feet defining a gap between the upper member and the bottom of the recess.

48. A receptacle according to any of Claims 42 to 47 wherein the spacer member is removably positionable in the recess.

49. A receptacle according to Claim 48 wherein the spacer member and the recess each include one or more mutually engageable positioning members for locating the spacer member in a predetermined location and/or orientation in the recess.

50. A receptacle according to any preceding claim wherein the recess is frustopyramidal in horizontal cross-section and the liquid displacement member is also frustopyramidal in horizontal cross-section, the liquid displacement member when the closure adopts a closed configuration on two opposite sides engaging and sealing against respective, mutually opposed lateral boundaries of the recess so as to define on two further, mutually opposed lateral boundaries a pair of fluid flow regions that are interconnected by the cell cultivation space whereby to define a fluid flow path extending from one side of the recess to another, the fluid flow path being connectable to one or more devices for effecting flow of fluid in the path.

51. A receptacle according to Claim 50 including one or more fluid flow ports that are fluidically connectable for supplying fluid to and/or from the fluid flow path.

52. A receptacle according to any preceding claim wherein the area of the bottom of the recess is such as to permit at least 50% of it to be covered by between 3 and 300 cells, especially 50 to 150 cells.

53. A closure for a cell cultivation receptacle, including a closure member having extending downwardly from an in-use underside a liquid displacement member that when the closure adopts a closed position closing off a cell cultivation recess occupies part of the interior of the recess, the liquid displacement member terminating in a lower surface and including at least a portion that tapers in the said downward direction.

54. A closure according to Claim 53 that is circular in horizontal cross-section and frustoconical in shape.

55. A closure according to Claim 53 that is polygonal in horizontal cross-section and frusto- pyramidal in shape.

56. A closure according to any of Claims 53 to 55 wherein the lower surface of the liquid displacement member is convexly or concavely curved.

57. A closure according to any of Claims 53 to 56 that is formed from one or more transparent materials.

58. A closure according to any of Claims 53 to 57 wherein the closure member and the liquid displacement member are formed integrally with one another.

59. A closure according to any of Claims 53 to 58 wherein the closure member includes extending downwardly therefrom a plurality of liquid displacement members.

60. A closure according to any of Claims 53 to 59 wherein the liquid displacement member includes formed therein one or more fluid flow passages.

61. A closure according to Claim 60 wherein the or each said passage terminates in a further opening in the said lower surface.

62. A closure according to Claim 61 including a plurality of the passages and a plurality of the further openings that are arranged in a regular or irregular pattern in the lower surface.

63. A closure according to any of Claims 60 to 62 wherein the or each passage is connectable to a source and/or a drain of fluid.

64. A closure according to Claim 59 or any preceding claim depending therefrom, including a plurality of the said passages that are fluidically mutually interconnected.

65. A cell culture apparatus including one or more receptacles according to any of Claims 1 to 52.

66. An apparatus according to Claim 65 including one or more clamps for securing the or each receptacle relative to the apparatus.

67. An apparatus according to Claim 66 wherein the clamp is or includes a perforated suction plate that is operatively connected to a suction pump.

68. An apparatus according to any of Claims 65 to 67 including an injector having one or more cannulae for perforating the closure member so as to permit injection of matter into the interior of the or a said recess through the closure member.

69. An apparatus according to Claim 68 wherein the injector includes two cannulae, that respectively inject material into the interior of the recess and extract material therefrom.

70. An apparatus according to Claim 69 wherein the cannulae are supported on a common support that is moveable relative to at least one said recess so as to permit insertion of the cannulae into and withdrawal of the cannulae from the said recess.

71. An apparatus according to Claim 70 wherein the support supports a plurality of injectors arranged in a pattern corresponding to the pattern of a plurality of the recesses.

72. An apparatus according to any of Claims 68 to 71 when depending from Claim 50 or Claim 51 wherein one or more said cannulae in use is fluidically connectable to the fluid flow path.

73. A method of treating particles comprising the steps of:

(i) coating the recess bottom of a receptacle according to any of Claims 50 to 52 or of a receptacle of an apparatus according to Claim 71 , having its closure member in an open position, with antibodies;

(ii) causing the closure member to adopt its closed position thereby defining the fluid flow path;

74. A method according to Claim 73 including the further steps of: (vi) causing a third fluid, containing optically detectable, further antibodies, that are antigenic to the particles, to flow in the fluid flow path; and

(vii) detecting the particles using one or more optical detection techniques.

75. A method of treating particles comprising the steps of: (i) filling a receptacle according to any of Claims 1 to 52 to a chosen depth with a hydrogel having cells suspended therein;

(ii) applying the closure member to the receptacle so that the liquid displacement member contacts the hydrogel;

76. A method of treating particles comprising the steps of:

(i) filling a receptacle according to any of Claims 1 to 52 to a chosen depth with a hydrogel; (ii) causing a plurality of cells to become adhered to the bottom of the recess;

(iii) applying the closure member to the receptacle so that the liquid displacement member contacts the hydrogel; (iv) causing the displacement member to oscillate towards and away from the bottom of the recess so as to impart shear forces on the cells.

77. A method of treating particles comprising the steps of: (i) causing a plurality of cells to become adhered to the bottom of the recess of a receptacle as defined herein;

(ii) applying the closure member of the receptacle thereto so that the liquid displacement member is proximate the adhered cells so as to cause displacement of fluid surrounding the cells; and (iii) causing the displacement member to oscillate towards and away from the bottom of the recess so as to impart shear forces to the cells.

78. A method according to any of Claims 75 to 77 wherein the liquid displacement member is shaped so as to contact the hydrogel to varying depths.

79. A method of forming a tapered recess in a recess layer comprising the steps of: (i) forming or providing a parallel-sided, circular-section recess extending from the recess layer so as to define an opening in the recess layer and a flat bottom to the recess; (ii) placing a settable material into the recess so as to lie in a formable material layer on the flat bottom;

(iii) inserting a circular frustoconical mould member, having a largest diameter that is less than the diameter of the parallel-sided recess, into the recess so as to displace the settable material and define a tapered, frustoconical shape therein; (iv) causing setting of the settable material; and

(v) removing the mould member from the recess.

80. A method according to Claim 79 wherein at least the recess layer, the recess and the settable material are inert to one or more of: cells; cellular nuclei; mitochondrial material; nucleic acids; amino acids; proteins; enzymes; embryos.

81. A method according to Claim 79 or Claim 80 wherein the settable material is or includes a liquid containing a buffer or cell culture medium mixed with one or more gelling agents.

82. A method according to Claim 81 wherein the gelling agent is selected from the list consisting of agarose, collagen or matrigel (RTM).

83 A method according to Claim 80 or Claim 81 wherein the gelling agent constitutes 1-2% by mass of the settable material.