CA2083393A1 - Cell assay device - Google Patents

Abstract

This invention encompasses porous microchambers which contain cells and permit liquid to flow in and out of the chamber while retaining cells within the chamber. These porous microchambers serve as disposable devices for placing cells in a micro flow chamber so that properties of the cells within the porous microchamber can be measured.

Description

PCI / ~S91 t03729 20~3393 CELL ASSAY DEVICE
This is a continuation-in-part of pending application Serial No.
07/532,571, filed June 4, 1990.

BACKGROUND OF THE INVENTION
S Ei~ of the Invention This invention is in the field of microphysiometers and, in particular, it relates to single-use disposable devices and reagents, and reusab]e peripheral pa;rts, used in conjunction with a microphysiometer.

escription of the Prior Art The prior art describes cups which have a filter membrane on the bottom and such cups are usable for filtering and grouping cells on the inner surface of the membrane ("Selected Methods in Cellular ~mmunology", Edited by Bar'oara B. Mishell and Stanley M. Shiigi, University of California, Beskeley, Editosial Consultants: Claudia Henry and Robert I. Mishell, University of California, Berlceley, Published by W. H. Freeman and Company, San Francisco, Copyright 1980, pp. 37, 40, 43, 61, 62, 63 and 64). Also of interest are siliconelectrodes for use in microflow cells described in U.S. Patents Nos 4,591,550;
4,737,464; 4,741,619; 4,704,353 and 4,519,890. These patents are incorporated herein by reference.
Also, see U.S. Patent No. 4,172,770 (Semersky) which describes a flow system and lapanese Patent 1002-06~-A which also describes a flow system.
Further prior art ~alces the forrn of commercially-available single use vessels for the culture of living cells. In general, because of the extreme sensitivity of living cells ~o the chemical and physical nature of their environment, including potential problems of contamination by endotoxin microorganism or by cleaning solutions, it is preferable to use culture vessels that have been manufactured undes carefully controlled conditions and then thrown away rather than cle~ned and recycled. Although commercially-available single-use vessels take many forms, including bottles, tubes, and single or multiwell covered dishes, the closest commercially-available prior art items are all essentially manufactured versions Gf the design described jn the first reference given in the previnus paraglaph (specifically pages 61, 62, 63, 64 of that reference). Manufacturers of such items include Costar Corp., Cambridge, MA, (product name: Transwell) and Millipore Corp., Pedford, MA, (product name: Millicell). This device is further described in EPA 239,697.

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2 0 8 3 3 9 3 PCT/~!Sg l /0372_ ~ef. Descn~tion of the Drawines Figure 1 is a cross-sectional schematic of components of the device of e~ample l;
Figure la is a cross-sectional schematic of the microflowchamber;
Figure 2 is a cross-sectional view of the device showing the spring loading mechanism;
Figure 2a is a cut-away top plan view of 2;
Figure 2b is a bottom plan view 2;
Figure 3 is a cross-sectional view showing integral spacer means;
Figure 3a is a cross-sectional view showing integral spacer means;
Figure 4 is a schematic of a bactenal indicator device;
Figure 4a is a capsule of porous membrane material containing cells;
Figure S is the effect of carbonyl cyanide m-nitrophenylhydrazine (CCCP) on 3T3 cells;
Figure 6 shows the effects of ethanol on P388D-1 cells; and Figure 7 shows continuous monitoring of adherent and non-adherent cells;
Figure 8 is a plot of acidification rates verses time for P388D-1 cells in a discontinuous collagen matrix;
Figure 9 is a semilog plot of acidification rates verses time;
Figure 10 shows a three pronged cup which serves as an outer ~leeve;
Figure ll shows a cell focusing insert which fits into the cup of Figure 10 or 1;
Figure l2 shows graphs of focusing experiment in fibrinogen; and Figure 13 shows graphs of focusing experiment in chopped collagen.

Summary of the Invention This invention encompasses porous microchambers of which at least part of the chamber wall is constructed from a porous material such that fluids and agents contauned therein can entu the porous microchamber when placed in a flowing stream, but cells cannot escape.
One embodiment of the invention provides for convenient assembly (or closure) of the microchamber, c~>ntaining cells of choice, prior to use in the microphysiometer. In this embodiment, rigid inner and outer sleeves covered at one end with a porous membrane, together with spacer means, are fitted together , " . ' . : .
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~' ~l/t8~53 pCI /1 1~;9t/03729 2~3!~3 such that when the inner sleeve is fully inserted in the outer sleeve, the membranes are separated by a spacer means. Thus, the spacer means and the inner and outer membrane form a microchamber having living cells trapped within. The sleeves are adapted for holding the microchambel containing the 5 cells adjacent to a silicon electrode that forms one wall of a flat microflowchamber. Cells ue retained within the internal cavity of the porous microchamber while liquid is permitted to flow through, above, between, and below the membranes and around the cells. (See Figure 1). The principal direction of flow of the liquid is parallel to the plane of the membranes and the 10 silicon surface. Changes in the media surrounding the cells (such as pH changes) can be measu~ed by the silicon electrode. Design objectives include (a) maximizing cell volumelmedium volume ratio to increase measurement sensitivity, (b) optimizing exchange rate of flowing liquid to flush out spent medium and/or introduce reagents, (c) minimizing the distance that measurable species such as 15 protons must diffuse to reach the silicon electrode. The device of this invention provides a convenient disposable reagent for use in a microphysiometer of the type described in United States Serial No. 07/408.896, assigned to the same assignee as this application and incorporated herein by reference.
In another embodiment of the invention, certain cells, such as bacterial 20 endospores, would be trapped between two membrane discs that are welded or stuck together at their edges to form a preassembled package that could be dropped into a microphysiometer flow chamber. Use of such a device would include, but not be limited to, validation of sterilization procedures; the preassembled endospore paclcage would be used as a biological indicator (BI) 25 included in a load of items to be sterilized. ln a further irnprovement of the BI
for use in the microphysi~meter, feed and waste lines would be firmly anached to the endospore package, as well as filters to prevent entry of contaminating micro-organisms from the feed line and the waste line exit. Thus, cells encapsulated in porous material to form a porous microcha nber can be placed in 30 the microflowchamber and advantageously studied in accord with this invention.

Detailed Description of the Inv ntion Several embodiments of this invention are described with reference to Figure 1. ln its simplest embodiment outer sleeve 1 has an upper opening 2 and a lower opening 3 which is covered with porous membrane 4. An inner sleeve 10 has an upper openin~ 11 and a l~wer opening 12 which is covered with a - . . . - ~ .. : .
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porous membrane 13. Inner sleeve 10 fits within outer sleeve 1- A spacer means ~L in this instance a piece of plastic sheet material which defines an opening ~L fits into the outer sleeve. The spacer means has a thickness of about25 to 200 micrometers, preferably about 50 micrometers, and holds the cells S within a~oout 1~300 micrometers from the silicon electrode. The height of the microflowchamber is about 5~300 micrometers. These sizes permit detection in pH change of about 0.1 to 0.5 pH units over about 1 to 5 minutes. Typical flow rates are about 10 to 200 microliters per minute. Thus, when the inner sleeve is inserted into the outer sleeve membranes 13 and 4 and the spacer means definea porous microchamber ~ in Figure la. In operation a plunger 30 is inserted into the inner sleeve. Plunger ~ has an inlet ~1 and an outlet ~ and when the plunger is tightly pressed against the membrane 13 a seal is formed by ridge or, alternatively, an ~nng sealed in a gland at the same position on the plunger.
Liquid can flow from the inlet in 31 above, through, between, and below membranes 13 and _ and the through outlet 32. A silicon sensor of the type described in U.S. Patents Nos. 4,519,550, 4,737,464, 4,741,619, 4,704,353 and 4,519,890 is adjacent and parallel to the outer surface of rnembrane 4 so that changes in the solution caused by the cells, such as pH, can be detected by the sensor 40. After measurement the inner and outer sleeve containing the cells canbe discarded.
In one embodiment spontaneously adhering cells are placed on the - inner surface of membrane 4. In another embodiment the cells are deposited in a polymes matrix, such as a collagen sponge, in the opening 2] of the spacer means. The cells in the polymer matrix are then incorporated into the porous microchamber ~ This embodiment is primarily used with cells that do not spontaneously adhere to porous membranes~ While in the embodiment illustrated in Figure 1 the spacer means is a separately inserted disk, the spacer means canalso be integlal with the inner and outer sleeve~ ln z third embodiment, a mixture of non-adherent cells and a preparation of discontinuous matrix such as particles of collagen sponge with diameters t~pically 10-10000 times that of thecells they are to en'~ap, are c~untrifuged into the outer sleeve before insertion of ~he inner sleeve ~o form the chamber.
The porous membranes are made of biocompatible porous polymer material~ A preferred material is a porous polycarbonate membrane. The pore size of this membrane can be selected for use with different cells. For example,a small pore size (0~4~ micron or less) is suitable for use with bacteria~ while a , ~ .....

W~ /18653 Pcr/~s91/03729 5 2~83393 larger pore size (generally in the range 3-12 micron) is chosen for eukaryotic cells.
The inner and outer sleeve may have a variety of shapes, for example, they may be circular, oval, square or rectangular. A preferred shape because ~f flow pattern considerations is oval. The inner sleeve may fit quite loosely intothe outer sleeve or, preferably the inner sleeve and the outer sle~ve should have spacing element such as 14 in the form of bumps or ridges either on the outer diameter of the inner or on the inner diameter of the outer sleeve to accuratelyposition one sleeve with respect to the other but allowing fluid to press ~etween the sleeves~ The sleeves are generally made of Agid polymer maserial such as polystyrene. It is important that the outer and inner sleeves in combination arespecifically structured to bring the microchamber and, in particular, the outer surface of the membrane of the outer sleeve in intimate contact with the detecting elect~ode. It is also important that the plunger, together with the plunger ridge 33 or O-ring and the spacer means form a seal to provide a leak tight compartment defining the microflowchamber 26 so that solution can be flowed in and out of the microflowchamber 26 by way of inlet 31 and outlet 32. Thus, the silicon electrode surface 40 forms one wall of the microflowchamber 26 and the bottom surface of the plunger 34 forms the t~ surface of the microflowchamber ~ within which a porous microchamber containing cells is removably plaoed.
The microflowchamber 26 has dimensions of between 10 nanoliters and 10 microliters. Although for certain applications in which analysis of eMuent from the chamber is desired and cells are trapped by a polymer matrix, larger volumesmay be preferable as long as ~he cell larger volume/flow chamber volume can be kept high enough to attain sufficient pH change rate for measurement. The plunger is made of rigid, polymer materials and is spring loaded to ensure a seal on the outer edges of the membranes~ The inner and outer sloeves and the spacer with or without the cell trapping polymer matrix are intended to be disposable items.
Figure 2 illustrates a spring loading mechanism for maintaining the plunger in a position to form ~nd seal the mic~oflowchamber. The flow chamber casing 50 re~eives the outer sleeve 1. The inner sle~ve L fits within the outersleeve 1 and the plunger ~0 fits within the inner sleeve L0 sufficiently loo~ely that air readily exits from between the pieces when they are being positioned. Tubing31 and 32 de~iver solution thr~ugh the plunger to and frorn the microflowchamber26. Spring 51 is attached to the top 52 of the plun~er and the plunger retainer .
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2083~3 . The plunger retainer means ~ has an opening ~ for retaining the plunger in the down position by engaging post ~. A top view is shown in Figure 2a.
Figure 2b shows a bottom pla~e 6Q and silicon electrode 40. Hole 61 provides for locking plate ~ to the flow chamber casing ~Q.
S Figure 10 shows an advantageous outer sleeve 2_ with prongs 21 to support the sleeve portion ~. Membrane 4 is shown at the bottom of the sleeve . Figure 10a is a top plan view of the device and Figure lOb is an AA
sectional view. Figure lOc is a bonom plan view showing the ~uter surface of membrane 4 which rest against the electrode. Turning plunger retainer ~ counter clockvise releases the plunger retainer and the plunger can be removed.
lt is also advantageous to focus the cells in the center of the outer membrane. This can be done by placing the focusing insert 80 of Figure I I into the outer sleeve l so that the bottom surface 81 rests on membrane 4 and cells 83 are located in the opening 82 so that only part of membrane 4 has cells located on it. The cells are centrifuged on to the membrane and the insert 80 isremoved from the outer sleeve. This serves to focus cells on a smaller area of the membrane. Figure 12 and 13 illustrate how 1lS to l/10 the number of cells can give comparable results when the cells are focused to 1/10 the area of membrane 4. Typically cells are focused in an area 5 to 15 mm~, preferably about 10 mm2, centered over the interrogated circular area of the silicon sensing device, an area of about 5 mm. The focusing insert 80 can be placed in the sleeve 1 of Figure I or sleeve ~ of Figure 10.
Figure 3, and 3a illustrate other types of integral spacer means. In Figure 3 an integr~lly molded spacer 16 is on the bottom opening inner sleeve 10before membrane 13. ln Figure 3a the spacer L7 is integrally molded on the bottom opening of sleeve 1 with membrane 4 on the outside of 17 and membrane 13 on sleeve 10 on the inside of the spacer. Cells are placed on the inner surface of the membrane 4 of the outer sleeve 1 and the spacer 20 is inserted inthe outer sleeve. The inner sleeve 16 is inserted to press against the spacer 20.
The plunger 30 is pressed into the inner sleeve to foTTn a seal between the spacer and the membranes and define microflowchamber 26. The membrane 4 rests on the silicon electrode 40 arld fluid is pumped over the cells. ~arious cell affecting agents are contained in the fluid and the effects of those agents on the cells is measured by silicon electrode 40. The small volume of the microtlowchamber provides for extremely sensitive or responsive measurements.
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, ' W~ ~1/18653 PCr/USs1/037~9 7 2~J83~93 Figure 4 illustrates details of a porous microchamber intended for use as a Biological Indicator. ln Figure 4 spores 61 are trapped between upper 60 and lower 70 porous membranes and a feed line or inlet 62 with in-line filter and a waste line or outlet 64 with in-line filter 65. The filter prevents bacteria 5 from entering the microchamber 66 and is made from a material such as nitrocellulose or polycarbonate mesh material having pore size of 0.2 - 0.45 microns, typically. In this embodiment liquid can be flowed through feed line 62through the microchamber 66 and out the waste line.
~ndospores are typically of the genus Bacilllls, such as Bacillus 10 ~ubtillis, ATCC #9372, subspecies niger. Figure 4a illustrates a capsule of porous material 75 cortaining cells 76. This porous capsule fits in the microflowchamber. The invention is further illustrated by the following examples.

Examgle 1: Effects of Exo~enous Cell-Affectin~ Agent on the Metabolism of Adherent ~ells in a Single Use Cell Assav Device as Measured with the Micro~hvsiQmç~ç~
Adherent cells, sometimes referred to as anchorage-dependent cells, generally must be attached to a biocompatible surface in order to m~intain stable metabolic rates and inc$ease in numbers. Adherent cell lines have been used f~r a wide variety of studies, including toxicological, pharmacological and 20 environmental applications. In order to study whether adherent cells could beused in a single use cell assay device designed for the microphysiometer, the following procedure was performed. Outer sleeves I were placed in a 12-well tissue culture ~late and Dulbecco's modification of Eagles medium (DME) containing 5% fetal bovine serum was added to the wells to 50% of capacity.
25 The sleeves were seeded with mouse fibroblast 3T3 cells (an adherent cell line) and the entire plate was incubated at 3TC in 5% CO. for 2 or more days During this time the cells settled to the bottom of the sleeves and attached to the porous membranes, whereupon they were allowed to grow to approximately 70 confluency. At that time each outer sleeve was removed from the plate and 30 spacer means 20 and inner sleeve l0 were placed on top of the layer of livingcells. This entire assembly was then placed inside the flow chamber casing 50.
lt was importanl during assembly to prevent air bubbles from being introduc~d into the flow chamber. This is accomplished by the following means:
(I) a small amount of mcdium must be present on top of the silicon electrode 35 40 so that when sleeve 1 is placed on top no air is trapped between the two .. . . ~ .
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2083~93 surfaces; (2) a small amount of medium must be present on top of the layer of cells and the spacer means on top of membrane 4, and membrane 1~ must be dry before inserting the inner sleeve 10 into outer sleeve I --care must be taken tovisually ensure that there are no bubbles trapped between the membranes or belowS the bottom memb~ane ~; (3~ the inlet line ~ must be filled with medium and a small drop of medium should suspend under the plunger surface ;~ before introduction of the plunger assembly into the inner sleeve. Additional components were assembled as described in the text and shown in Figures 1 and 2, and the whole placed within the microphysiometer and maint~uned at 3TC.
DME without bicarb~nate ~having a buffer capacity of approximately 2mM) was then perfused into the chamber and acidificati~n rates were measured periodically as described by Parce et al. (Science 246, 243 (1989)). Dunng the indicated period the DME was supplemented with the metabolic uncoupler car~onyl cyanide m-nitrophenylhydrazine (CCCP; S micromolar ~M). The rate of medium acidification increased while the eells were exposed and then returned to the original acidification rate after replacement of the CCCP-containing perfusion medium with the original medium (See Figure 5). :
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Example 2: Immobiliution and Monitorine of the Metabolism of Non-adherent Cells in a Continuous Polvmer Matrix Non-adherent cells (also known as anchorage-independent cells) generally do not become anchored to adjacent surfaces. Nevertheless, it may be desirable to use a microphysiometer to monitor the metabolism of some of these ~ypes of cells for toxicological, pharmacological, environmental. etc., purposes.
In initial experiments in which non-adherent cells were trapped between two membranes as described in Example 1, reproducible acidification was not observedand subsequent studies revealed that the cells were swept to the sides of the porous microchamber away from the central detector ~y the movement of fluid through this volume. In order to prevent this loss of non-adherent cells due to movement of fluid, some restriction of cell movement was deemed necessary.
A number of biocompatible sponge-like matenals have been made commercially available for use as hemostatic agents. These materials, include, but are not limited to, collagen, polyvinyl alcohol and polyurethane. When applied to a surgical wDund, these materials promote the deposition of new tissue. The tortuous network and proven biocompatible nature of these maserials might allow them to serve as cell immobilization rnatrices, and thus some of these materials " ' ''' ~ . ~ .

wr ~l/l8653 Pcr/~lss1/o3729 -9- 2~833~3 were tested for their ability to immobilize non-adherent cells within the microphysiometer. To accomplish this, a spacer means 20 was placed within an outer sleeve 1 containing a 5 ~ pore polycarbonate membrane. Into the center hole of the spacer means a 6 mm diameter disk of polymer matnx, approximately 150 ~m thick, was placed. The s)uter sleeve 1 and polymer matrix were placed within a well of a 12-well microtiter plate. For experiments with mammalian cells, a spacer means 20 was inserted in the outer sleeve prior to addition of the polymer matrix. One ml of a suspension containing either P388D-l cells (non-adherent mammalian cells; approximately 107 cellslml) or Sacch~rornyces cerewseae (non-adherent yeast cells; approximately 107 cells/ml) was pipetted into the outer sleeve. The microplate was then centrifuged at 400 x g for 5 min., after which an inner sleeve lQ was placed within the outer sleeve and thus a s~cond membrane was located above the cells trapped within the polymer matnix.
The inner and outer sleeves, spacer means (when present), polymer matrix and cells were inserted into the flow chamber casing 5~ and acidification rates of the cells were determined as described in Example 1. Wilh the mammalian cell line stable metabolic rates were achieved for the duration of the experiment. ln the yeast experiment the rates of acidification determined with the microphysiometerincreased with time, indicating an increase in cell number within the microflowchasnber for the duration of the experiment. Both of these experiments suggest that the oells were properly immobilized and their metabolism not adversely affected by the immo~ilization procedure or the microphysiometer environment.

Example 3: Effects of Ethano] on the Metabolism of a Po~ulation of Non-adherent Cells P388D-1 cells were centrifuged into a polymer matrix and their metabolism monitored with a microphysiometer as described in Example 2. After a period of adjustment to the microphysiometer environment during which time the rates of acidification of the cells' environments stabilized, the DME with 5%
fetal bovine serum ~/as replaced with DME containin~ ~% fetal bovine serum and 10% ethanol for a period of 5 min. Following the exposure to this potentially harmful A~ormulation, the medium lacking ethanol was reintroduced into the microphysiometer and us~d to flush out the ethasol-con~inin~ medium. The metabolism of the P388D-I cells was continuously monitored during the medium exchange. As can be seen in Figure 6, this concentration of ethanol suppressed WO 91/186S3 Pcr/uss1/03~
20~393 the metabolism of the cells, and this effect was not reversed by ~he removal of ethanol from the perfusion medium, suggesting that irreversible damage may have been done to the cells immobilized in the microphysiometer.

Example 4: Lon~:~rm_Continuous Monitorin~ of Both Adherent and Non-Adherent Cell ~etabolism Non-adherent (P388D-1) and adherent (NRK) cells were placed in each of two disposable cell assay devices in the manners described in Examples l and 2 and placed within each of two cell chambers in a microphysiometer. The respective acidification rates of the two populations were measured for a periodof over l2 hours while DME containing 5% fetal bovine serum was perfused through the cell chambers. Figure 7 represents a plot of the acidification ratesof these cell populations, both of which demonstrated stable rates for the duration of the experiment. The non-adherent cell population required a period of adjustment to the microphysiometer environment (approximately 2.5 h), during lS which the acidification rate for this population increased rapidly. This was followed by a long period of less drasnatic increase in the: rate of acidification, said state occurring for the remainder of the experiment.

E~amDle 5: Immobilization of Non-adherent Cel!s and Continuous l~onitorin~
of Metabolism in a Discontinuous Matrix A polymer ma~rix which would form around non-adherent cells and thus immobilize thesn would eliminate the need to manufacture and position disksof precast polymer matrix. in order to test this concept, the procedure described in E~xample 2 was performed with the following modification. No precast polymer matrix was inserted into the spacer means 20. An collagen hemostatic sponge (Collastat, Vitaphore Corp., Chicago, Ill.) was suspended in phosphate-buffered saline solution pH 7.2) and finely chopped in a blender for six 3 sec.
cycles. A volume (0.5 ml) of this suspension, which consisted o~ particles of collagen matrix of various sizes, mostly in the range of 0.2 to l.0 mm diameter and representing about 0.5% (vlv) of the suspension, was mixed with 0.5 ml of a P388D-1 cell suspension (approximately 107 cells/ml). The cell/polymer suspension was centrifuged at 400 x g for 5 min. into the outer sleeve ] with a spacer means 2~; an inner sleeve 10 was added and this assembly was inserted into the flow chamber casing 50. The metabolism of the cel]s as indicated by therate at which they acidified their environment was monitored as described in - . .
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Example 1~ After a period of acclimation to the microphysiometer environment, stable rates of acidification were measured for the duration of the experiment (~:igure 8). These results demonstrate that the discontinuous matrix used in this e1~periment did immobilize the non-adherent cells and that cellular metabolism was S apparently not adversely affected by this procedure. Such a discontinuous matrix preparation may thus be appropriate for deterrnining the effects of cell-affecting agents as described in Example 3.

Example 6: Monitorin~ of Bacterial Growth and Metabolism in a Microphysiometer ln order to measure the rate of acidification of the microflowchamber environment by a bacterial population, a number of modific~tions to the e~perimental and single-use element design described in Example I were made.
A suspension of Bacillus su~ti1is endospores which had been enumerated with a Petroff-Hauser direct counting chamber was centrifuged into an outer sleeve 1 with a spacer means 20. The suspension had been diluted to a density so that aprpoximately 250 endospores would be present in the microtlowchamber when the microflowchamber was completely assembled. No polymer matrix was present, the lower membrane 4 had a pore size of 0.4 micron and the membrane above the endosp~res 13 had a pore size of 0.22 micron. Instead of DM~ with 5% fetal bovine serum, a complex bacteriological nutrient mediurn was substituted and the cells were incubated at 370C in the microphysiometer. This example provides evidence that the invention has utility in the field of validati~n of desterili~aticn procedures when used as a biological indicator. No discernible metabolic rate could be observed until approximately four hours after the experiment began, after which a rapid increase in acidification was seen. From a semi-log plot of these rates a determina~ion of the doubling time of B. subti~is in the microphysiometer could be determined (Figure 9).

Example 7: Concentratin~ Cells into the Readin~ Area of the Microphysiometer Ce]] Capsule Usin~ a "Cell Focusin~" Device and Immobilization of Cells usin~ a Fibrin Gel When the standard cell loading procedure is used for loading cells into the cell capsule, --1x 106 cells (TF-1, bone marrow cells) must be used to have acidification rates of ~ LVIsec (medium buffer I mM). The large number of cells are required because they are deposited over the entire membrane area , . ~ , ~ . . -wo 91/18653 PCr/US91103'U9 20833~3 -12-(113 mm2) during the cell loading. The actual reading area is much less, --5 mm2 In order to decrease the cell number required to obtain equivalent rates which are necessary to obtain optimum precision of acidification ra~e measYrement, S we have designed a "cell focuser" which is a plastic insert that directs cells during the loading procedure into a small area in the mjddle of the cell capsule membrane. It is shaped like a cylindes so that cells are evenly distributed in the circular area at the base of the focuser, and the top is shaped like a funnel toallow a larger volume of cells to be used if necessary to reach the required cell 10 loading numbers (Figure 11). There is a ridge on the outer edge of the top portion of the focuser which rests on the upper edge of the cell capsule to support the device during cell loading, and the barrel of the focuser extending down into the cell capsule is of the appropriate length to contact and press against the bottom membrane. The ce~ls are depositod in a circular area of 11.4 mm.
Cell capsules with spacer in a l2 well titre plate are filled with 0.7 ml of loading medium, and the cell focuser is placed in the capsule well, aligning it in the center. The O.l to 0.2 ml of cell suspension containing lO0,000 -200,000 cells is placed in the cell focuser. The assemb]y is then placed into a well containing 2 ml of loading medium. The plate is then cenlrifuged at 500 20 x g for 5 min. at room temperature. The cell focuser is casefully removed from the capsule using a forceps, and the cells are overlaid with 50 ~L of fibrinogen/thrombin mixed reagent. The fibrinogen/thrombin reagent is psepared immediately before use by mixing equal volumes of purified fibrinogen (microliter mg/ml, dissolved in RPMI 1640 culture medium containing lO mg/ml human 25 serum albumin and lq~ D-glucose) solution with purified thrombin solution (--0.8 NlH units/ml, dissolved in RPMI l640). All reagents are endotoxin free. After the fibrin gel is hardened, ~5 min., the membrane insert is sunk down over the entrapped cells. The capsule is then assembled into the microphysiometer.
When cells are loaded using the cell focuser, they adhere suMciently 30 to the membrane so that they are not remixed when the ~ocuser is removed. Thefibrin gel overlaying the cells in the circular pattem holds the cells tightly into place, and there is no cell movement when observed under the microscope using a flow rate of 100 ~L per min. This procedure was also successfully done using chopped collagen, by precoating the bottom membrane with a carpet of chopped 35 collagen according to standard procedures. Figure 12 shows the stimulation ofTF-] cells by GM-csf using the standard fibrin gel entrapment procedure (1 x 106 , W~ ~1/18653 pcr/us91/o3729 2~833~
-l3-cells) and the cell focuser wi~h ll~th ~he cell number (2 x ld cells). Equiva}ent results were obtained, and the cell focuser gave 34% higher rates. Figure 13 shows the same experiment done with chopped collagen. Again, equivalent results were obtained, and the cell focuser rates were the same as the standard method.
Using the cell focuser, it is possible to get equivalent resu1ts to the standard method with 1/5 to 1/10 the number of cells (IOQ,000 to 200,000) cells.This is very important when cells are difficult to grow as in primary cell cultures.
A calcium alginate gel is suitable to fix the cells.

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Claims (19)

What is claimed is:

1. A device for removably placing cells in a microflowchamber having a volume of about 10 nanoliters to about 10 microliters of a microphysiometer comprising a porous microchamber which contains cells and retains cells within the microchamber when liquids flow in and out of the microchamber wherein the porous microchamber comprises inner and outer porous membranes separated by a spacer means which is about 25 to 200 micrometers thick.

2. A device for removably placing cells in a microflowchamber having a volume of about 10 nanoliters to about 10 microliters of a microphysiometer comprising a porous microchamber which contains cells and retains cells within the microchamber when liquids flow in and out of the microchamber wherein the porous microchamber comprises a capsule of porous membrane material.

3. A device according to claim 2 with inlet and outlet flow lines directly attached to the porous microchamber whereby liquid is flowed through the inlet flow line into the porous microchamber to contact the cells in the porous microchamber and out of the porous microchamber through the outlet flow line.

4. In combination the porous microchamber of claims 1, 2, or 3 within a microflowchamber having:
(a) a means for flowing liquid in and out of the microflowchamber and (b) a means for detecting changes in properties of the liquid within the microflowchamber.

5. The combination of claim 4 wherein the height of the microflowchamber is about 50 to 300 micrometers.

6. The combination of claim 4 wherein the cells are held about 10 micrometers to about 300 micrometers from a silicon electrode by the porous microchamber.

7. A device for removably placing cells in a microflowchamber of a microphysiometer having a volume of about 10 nanoliters to 10 microliters comprising:

(a) an outer sleeve having a top and bottom opening wherein the bottom opening is covered with a porous member;

(b) an inner sleeve fitting within the outer sleeve and having a top and bottom opening wherein the bottom opening is covered with a porous membrane; and (c) a spacer means having a thickness of about 25 to 2 micrometers between the porous membranes of the inner and outer sleeves which defines an opening and which together with the porous membranes defines a porous microchamber when the inner sleeve is placed within the outer sleeve and both porous membranes are in contact with the spacer means and wherein cells are maintained in the porous microchamber when liquid flows through the porous membranes.

8. A device according to claim 7 wherein the spacer means is a continuous strip of thin plastic material fitting the inner wall of the outer sleeve, defining an opening and having a thickness of about 25 to 200 micrometers.

9. A device according to claim 7 wherein the spacer means is integral with the outer or inner sleeve.

10. A device according to claims 8 or 9 wherein the height of the microflowchamber is about 50 micrometers to about 300 micrometers.

11. A device according to claim 7 wherein the cells are held about 10 to about 300 micrometers from a silicon electrode by the porous microchamber.

WO 91/18653 PCT/US91/0372?

12, A device according to claim 7 wherein cells are focused on a portion of the membrane in the outer sleeve.

13. A device according to claim 7 wherein the opening defined by the spacer means is covered with a polymer mesh which entraps cells.

14. A device according to claim 13 wherein the polymer mesh is a collagen sponge.

15. A device according to claim 7 wherein non-adherent cells are fixed in a fibrin gel.

16. A device according to claim 14 wherein the cells to be tested are intermixed with polymer matrix particles and cocentrifuged on the porous membrane of the outer sleeve before inserting the inner sleeve.

17. A device according to claim 16 wherein the polymer matrix particles are particles of collagen sponge.

18. In combination with the device of claim 7, a plunger with a top and bottom surface which has openings for directing a stream of liquid through the plunger and which fits into the inner sleeve and the bottom surface of the plunger contacts the porous membrane of the inner sleeve; a silicon electrode which contacts the outer surface of the porous membrane of the outer sleeve wherein when the plunger is pressed a seal is formed between the porous membranes, the spacer means, the plunger and the silicon electrode to define a microflowchamber where liquids are flowed into and out of the microflowchamber having a volume of about 10 nanoliters to about 10 microliters thorugh the openings in the plunger.

19. A device according to claim 7 wherein the inner and outer sleeve is separated by a spacing element.