WO2009034186A2

WO2009034186A2 - Process for cell cultivation

Info

Publication number: WO2009034186A2
Application number: PCT/EP2008/062212
Authority: WO
Inventors: Nadia Zghoul; Kurt Dittmar; Werner Lindenmaier
Original assignee: Helmholtz-Zentrum für Infektionsforschung GmbH
Priority date: 2007-09-13
Filing date: 2008-09-12
Publication date: 2009-03-19
Also published as: WO2009034186A3

Abstract

The present invention provides a process for the production of a structurally stable porous matrix material, the inner surfaces of which are at least partially colonized by adhering cells, the process involving the cultivation of mammalian cells under cell culture conditions in cell culture medium in the bio-reactor, which is agitated by a rocking movement. Accordingly, the bio-reactor used in the process of the invention is not provided with movable stirrers or static mixing elements, but rather provides essentially flat or curved inner surfaces, i.e. without elements projecting or protruding over the inner surface of the bio-reactor.

Description

Process for cell cultivation

The present invention relates to a process for producing an implant suitable for medical purposes, e. g. for replacing or filling of tissue defects, e.g. bone, cartilage or fatty tissue defects in humans. The implant comprises and preferably essentially consists of a porous material that is colonized with tissue forming cells, e.g. derived from stem cells, which are preferably immunologically compatible with the eventual implant recipient, more prefereably autologous cells.

State of the art

In addition to classic stirred vessel bio-reactors, WO2007/001173 discloses a bioreactor, having a volume essentially confined by a container, which may be lined with a plastic bag, which container is agitated by a rocking movement provided by a supporting table tilting about an essentially horizontally arranged axis. The bio-reactor is used for cultivating cells in suspension. Further bio-reactors for use in suspended cell culture, i.e. for supporting planctonic growth of mammalian cells or microorganisms are also known from the following documents:

WO 2005/0118771 A2 relates to a disposable bioreactor essentially consisting of the double layer plastic bag equipped with inlet and outlet tubings and a magnetic stirrer in order to overcome mixing by a rocking movement.

US6544788 B2, trying to improve perfusion bio-reactors, discloses a bio-reactor that is agitated by being placed on a rocking table to generate a wave, and a filter connected to an outlet opening in the inner surface of the bioreactor vessel by an elastic tubing, allowing the filter to float along with the wave movement of the cell culture medium. The floating filter is used for withdrawing medium components, e.g. secreted cellular products while avoiding clogging.

US 2005/0186669 Al describes a bioreactor for the cultivation of mammalian cells which is agitated by a seesaw or rocking movement of the bio-reactor about a fulcrum. For maximising the surface area for cell adhesion and for maximising the interface between air and growth medium, there is porous cell growth substrate fixed to two opposite sides of the bio-reactor. During the cultivation, the seesaw movement or rocking of the bioreactor alternatingly fully or partially lifts the porous cell growth substrates on either side of the bio-reactor out of the growth medium.

For the cultivation of bone, chondrogenic tissue and tendons, WO2007/045619 describes a bio -reactor, wherein a porous matrix is supported on a carrier. The carrier and the matrix thereon essentially cover the cross-section of the bio-reactor, allowing a forced perfusion of the porous matrix with the cell culture medium and mammalian cells suspended therein, without medium flowing around the matrix.

Martin et al. (Trends in Biotechnology 22, 80-86 (2004)) reports that cellular ingrowth into a porous matrix material arranged in a bio-reactor to perfuse the matrix material with medium containing suspended cells, was found to be limited to a maximum distance of 0.2 to 0.4 mm from the outer perimeter of the matrix. Objects of the invention

In view of the fermentation processes that are designed to allow the ingrowth of mammalian cells onto inner surfaces and into the interstices of a porous matrix material, the present invention seeks to provide a simplified production process for porous matrix material provided with mammalian cells attached to the interstices, e.g. the inner surface. Preferably, cells colonize the entire inner surface of the porous matrix material, more preferably essentially filling the inner volume of the porous matrix material while adhering to the inner pore surfaces, preferably with the cells forming interconnective tissue within the inner volume of the matrix material.

A preferred object of the present invention is the provision of a production process for artificial bone tissue, adipose tissue or chondroitic tissue comprising a structurally stable rigid or elastic porous matrix material and cells, especially bone forming cells such as osteoblasts, adipose cells, and chondroblasts, respectively.

General description of the invention

The present invention achieves the above-mentioned objects by providing a process for use in the production of a structurally stable porous matrix material, e.g. for medical use as an implant, the inner surfaces of which are at least partially colonized by adhering cells, the process involving the cultivation of mammalian cells under cell culture conditions in cell culture medium in the bioreactor, which is agitated by a rocking movement. The rocking movement can also be referred to as a tilting or pivoting movement, e.g. a periodic and preferably symmetrical movement about an essentially horizontally arranged axis of the bioreactor. In the process for production, the longitudinal axis bio-reactor preferably is periodically tilted from a horizontal position to an inclined position. Preferably, tilting is performed symmetrically about a horizontal axis, in relation to which the bio-reactor (1) is positioned to have its longitudinal axis (2) arranged in an angle of 30° to 90°. The bio-reactor can additionally be moved to have it tilt sideways, e.g. to move the bio-reactor periodically with its longitudinal axis moving about a vertical axis, e.g. by 30° to 90° of its longitudinal axis in relation to a vertical axis. At present it is assumed that it is the non-turbulent, i.e. laminar flow of the medium containing the cells as well as the porous matrix material suspended therein that is the cause for the cell growth within the matrix material.

Generally, the invention relates to a process for producing a porous matrix material comprising on the surface of its interstices cells, for medical use as a bone implant, the process comprising the steps of cultivating cells in a bio-reactor having a first continuous inner surface essentially extending essentially in the direction of a longitudinal axis and second continuous inner surfaces sealing the cross-section spanned by the first inner surface at two spaced apart sections of the bio-reactor, with at least one inlet and one outlet port for gas arranged within the first and/or second inner surface, providing the porous matrix material within the bio-reactor, periodically tilting the longitudinal axis of the bio-reactor from a horizontal position to an inclined position, and providing a volume of cell culture medium within the bio -reactor to at least cover the porous matrix material in the inclined position at a temperature and in an atmosphere suitable for growth of the cells. The step of periodically tilting the longitudinal axis of the bio-reactor from a horizontal position to an inclined position can also be referred to as periodically tilting the bio-reactor for movement of its longitudinal axis from a horizontal position to an inclined position.

Accordingly, the bio-reactor used in the process of the invention is not provided with movable stirrers or static mixing elements, but rather provides essentially flat or curved inner surfaces, which are also referred to as continuous surfaces, i.e. without elements projecting or protruding over the inner surface of the bio -reactor. Further, the bio -reactor used in the practice of the invention preferably does not have sensors, e.g. a pH-sensor, or ports protruding above its inner surfaces, which sensors and ports, if necessary, are preferably formed within the plane of the inner surface, or in recesses of the inner surface of the bio- reactor. In the alternative to sensors or ports arranged in the wall of the bio-reactor, these can be arranged in pipes or tubing connected in fluid communication with the volume within the bio-reactor containing medium, e. g. in the form of a by-pass fluid line or within a fluid line connected to an exit port of the bio-reactor. In accordance with no elements or surface sections of the inner surface of the bio-reactor protruding into its volume, the bio-reactor has a first continuous inner surface essentially extending in the direction of a longitudinal axis and second continuous inner surfaces sealingly arranged to cover the cross-section spanned by the first inner surface at two spaced apart sections of the bio-reactor. The closed inner surface of the bio-reactor can be provided with inlet and outlet ports for gas and fluids, which ports can be arranged within the first and/or second inner surfaces.

Preferred geometries of the inner volume of the bio-reactor are selected from a cylindrical, rectangular or square cross-section of the first inner surface along a longitudinal axis with the spaced apart second inner surfaces forming end sections, e. g. as flat, round or cone shaped walls, preferably essentially perpendicular to the longitudinal axis. Alternatively, the bio- reactor can have a generally oval inner volume shape or cross-section. Examples of inner volume geometries are tubes with a round or oval cross-section with both ends being sealed by rounded, flat or cone shaped walls.

At present, bio-reactors with walls that are essentially non-deformable under the cultivation conditions are preferred, i.e. bio-reactors with an essentially pre-determined or fixed inner volume geometry, because it is believed that the tilting movement generates best mass transfer for sustaining cell growth within the matrix material along with suspending the matrix material in a gentle manner to avoid damaging contacts with the bio-reactor. However, a portion of the bio-reactor wall or the entire bio-reactor wall can be elastic or deformable under cell culture conditions in the tilting movement, e.g. an elastic material in the general shape of a closed bag.

Inlet and outlet ports can be arranged within any wall section of the bio-reactor, preferably within the end-sections, which most preferably are removable. As one embodiment, an inlet port and/or outlet port for gases is provided by a wall section of the bio-reactor being formed of gas-permeable and liquid-tight membrane, wherein the gas-permeability is at least for oxygen and carbon dioxide, and preferably only marginally for water vapour. Accordingly, the bio-reactor, e.g. in embodiments in which its walls are partially or completely formed of an elastic material, e. .g a foil, the elastic material can essentially consist of a gas-permeable membrane material.

Surprisingly, it has been found that the rocking movement provided to the bio-reactor results in drastically increased colonization of the inner surface of the porous matrix material when compared to cultivation under stirring or rolling. It is assumed that the rocking movement at the same time provides for a laminar flow sufficient to generate sufficient mass transport for nutrients and dissolved gaseous components, e. g. of oxygen and carbon dioxide between the cells and the liquid phase of the medium while preventing the growth of a cellular layer about the perimeter of the matrix material which layer can be observed in other cultivation procedures to shield the inner volume of the matrix. Concurrently, the rocking movement has been observed to essentially maintain the porous matrix material in suspension, which is assumed to be the reason for avoiding abrasion of the matrix that may occur by contacting the bio -reactor or mixing elements.

The production process according to the invention therefore provides growth conditions sustaining cellular growth within the interstices of the porous matrix material, i.e. suitable for cells to adhere to the inner surfaces of the porous matrix material, essentially across the entire cross-section of porous matrix material that is dimensioned to be of practical use as a bone implant, e.g. growth of cells into the inner volume of the matrix to a distance of at least 5 mm from the outer perimeter of the matrix material.

Preferably, the tilting or rocking movement acting onto the bio-reactor is supplemented by a rotating movement about an axis essentially perpendicular to the axis of tilting movement.

Preferably, the porous matrix material is selected from a poly(lactide-co-glycolide)-calcium phosphate composite, acellularised spongiosa, calcium-hydroxyl apatite, calcium-deficient hydroxyapatite, β-tri-calcium phosphate and bioresorbable ceramics, and collagen, agarose, chitosan, hyaluronic acid, poly caprolactone, poly glycolide, poly lactides, and co-polymers and mixtures thereof. Preferably, the porous matrix material has a porosity of 5 - 80% volume interstices per total volume. Preferably, the inner volume of the matrix material has interconnected pores having diameters in the range of 20 to 4000 μm, preferably 50 to 2000 μm. Preferably, the porous matrix material has a minimum thickness of 5 to 20 mm, and a maximal thickness up to 40 mm, preferably up to 30 or 25 mm.

Additionally, the porous matrix material can be coated with serum protein and/or extracellular matrix protein that is absorbable or producible by the cells cultivated with the matrix. Examples for protein for coating the matrix material are fibronectin and collagen. The coating has been found to enhance adhesion by differentiating cells, e.g. by bone forming cells.

Further, it is preferred to contact the matrix material with serum of the same species as the cells used for cultivation, e.g. human serum in the case of human cells, prior to contacting the cells with the matrix material, because differentiation of human trabecular bone cells on matrix material was enhanced by a human serum coating of the matrix as measured by increased collagen type I medium levels (CICP). Alternatively, fetal bovine serum can be used in the place of human serum for human cells, giving a similar enhancement of cell differentiation. Also preferred is that the medium contains serum of the same species as the osteogenic cells, e.g. human serum in the case of human cells, e.g. at 5 to 20 vol.-%.

Mammalian cells for use in the production process of the invention preferably are bone generating cells, e.g. osteoblasts. Bone generating cells can be generated by differentiating stem cells, e. g. by inducing differentiation in stem cells by adding differentiation inducing substances to the medium. Examples for preferred human cells are stem cells, derived from e.g. trabecular bone or bone marrow, mesenchymal and/or mesodermal stem and/or progenitor cells or stem cells, or cells comprising endodermal and ectodermal progenitor cells. Using differentiation inducing substances in the medium, mesenchymal progenitor cells or stem cells can be induced to differentiate into osteoblasts, chondrogenic cells and chondrocytes, or adipogenic and adipose cells; mesodermal progenitor cells or stem cells can be induced to differentiate into osteoblasts, hematopoietic cells or endothelial cells.

In order to generate a suitable implant, it is preferred to adapt the selected matrix material to the differentiated cell type desired, e. g. an elastic porous matrix material is preferred for cultivation with adipogenic cells to obtain an elastic implant which is for example suitable for replacing adipose tissue in reconstrutive surgery. For cultivation of chondrogenic cells, it is preferred to use an elastic or rigid matrix material to obtain an implant having sufficient structural stability to replace chondrogenic tissue. When cultivating osteogenic cells, it is preferred to use a structurally stable matrix material to obtain a rigid implant suitable for filling a bone defect. Generally, it is preferred to use a matrix material that is bioresorbable by the implant recipient.

The process of the invention can be operated batchwise with full or partial replacement of the cell culture medium during the cultivation period, or with continuous inflow and withdrawal of medium during the cultivation process using inlet and outlet ports arranged within the bioreactor walls, e.g. connected by pipes, preferably by elastic tubing. For aeration, a port in the wall of the bio-reactor, e.g. its lid section is preferred, which may be covered with an oxygen permeable membrane having a low cut-off size, e.g. of 1 - 10 nm pore size, preventing the entry of contaminants into the inner bioreactor volume, i.e. acting as a sterile filter. It has been found that for cultivation processes using up to 30 - 50 mL cell culture medium at 30 to 70% filling of the bio-reactor volume, sufficient aeration is provided by a liquid tight and gas permeable sterile filter membrane of a total surface of 0.01 to 5 cm², even when the tilting movement causes the periodic immersion of the surface of the sterile filter membrane, which is oriented towards the inner bio-reactor volume, e.g. forming a section of the inner surface of the bio-reactor.

For the cultivation process, it is preferred that the bio-reactor has a generally longitudinal shape, and its inner volume is filled with cell culture medium to at least cover the porous matrix material, and preferably filling the inner volume of the bio-reactor by at least 30 - 70%, preferably from 40 - 60%, allowing a sufficiently large interface between the liquid medium phase and the gas phase.

In contrast to known bio-reactors, which are especially adapted to keep a porous matrix material in a fixed position for perfusion by cell culture medium, the process of the present invention provides for a drastically reduced complexity of the bioreactor, as well as for greatly improved ease of handling.

Surprisingly, it has been found that the tilting or rocking movement applied to the bio-reactor, preferably in a symmetric fashion by about 50 to 20° from the horizontal into each direction, i.e. upwards and downwards, provides for efficient mass transfer to allow cell growth within the inner volume across the porous matrix material while also preventing abrasion to the matrix material, maintaining its structural integrity. In contrast, comparative tests using a rotary movement, about an essentially horizontal axis in the same tube resulted in significant damages to the porous matrix material, e.g. in breaking of tips and edges.

In a simple embodiment, the present invention uses a conventional plastic vessel, e.g. having a circular cross-section of about 2 to 4 cm and a length of about 5 to 10 cm, i.e. available under the name of Falcon tube, with at least one port for allowing gas exchange for aeration of the culture medium, in a means for tilting the vessel, preferably in combination with rotating. For tilting, the bioreactor vessel can be moved periodically around an essentially horizontal axis, preferably perpendicular to its longitudinal axis. Pivoting of the bio-reactor about its longitudinal axis can also be referred to as pivoting the bio-reactor with its longitudinal axis about an essentially horizontal axis, e.g. by an angle of 30° to 90° of the longitudinal axis of the bio-reactor to the horizontal axis of tilting. The moving can be provided by a rocking or tilting means, by pivoting the longitudinal axis of the bioreactor, e.g. about an essentially horizontal axis which may be arranged perpendicular to its longitudinal axis. Preferably, tilting the bio-reactor is performed in combination with rotating the bioreactor, preferably along its longitudinal axis. The process of the invention is generally under cell culture conditions, e.g. at 37°C in a 5 to 10 % CO₂ atmosphere.

Preferably, the tilting movement, preferably in combination with the rotating movement, is at about 5 to 20 per min, more preferably at about 10 to 15 per min, depending on the ratio of the dimension of the bioreactor along its longitudinal axis in respect of its cross-section. In detail, the larger longitudinal dimension of the bioreactor in relation to its cross-section typically requires lower tilting frequencies than a bioreactor having a shorter longitudinal dimension. Generally, it is preferred to adjust the dimensions of the bio-reactor and the tilting frequency to obtain laminar flow conditions while preferably maintaining the matrix material in suspension, i.e. avoiding it contacting the bio-reactor. In accordance with the movement of the matrix material within the bio -reactor without any mechanical restrictions by fixation to a support, the process of the invention allows the unrestricted movement of the matrix material within the agitated culture medium.

Detailed description of the invention

The present invention is now described by way of examples with reference to the figures, showing in

- Figure 1 a bio-reactor in subsequent positions during the tilting movement,

- Figure 2 a comparative bio-reactor setup,

- Figure 3 transverse sections of matrix material after cultivation with cells,

- Figure 4 a micrograph of a transverse section of matrix material after cultivation according to the invention with cells in haematoxylin and eosin staining,

- Figure 5 micrographs of sections of matrix material after cultivation according to the invention with cells in van Gieson staining, - Figure 6 micrographs of sections of matrix material after cultivation under static conditions with cells in van Gieson staining,

- Figure 7 micrographs of sections of matrix material after cultivation according to the invention with immunohistochemical staining for connexin-43, and

- Figure 8 scanning electron micrographs of sections of matrix material after cultivation.

Example 1 : Cultivation of human osteogenic cells within a porous matrix material to produce a porous matrix essentially colonized throughout its inner surface, and at least partially throughout its inner volume, suitable as a bone implant

As an example for an implant according to the present invention comprising cells obtainable from mesenchymal stem cells, osteoblasts were generated by differentiation of human trabecular bone derived cells into osteogenic cells. In detail, human trabecular bone derived cells were obtained from biopsies and could be expanded by seeding to 1 x 10⁴ cells /cm² and cultivation in cell culture basal medium (e.g. Iscove's modified Dulbecco's medium 1:1 with Ham's medium F12), supplemented with 15% human serum, 1% penicillin/streptomycin, until 80 - 90% confluence, which was reached after 5 to 7 days of culture. Subsequently, the medium was changed and osteogenic differentiation was induced by cultivation of cells for least 14 to up to 30 to 40 days in osteogenic differentiation medium, containing I x IO ⁸ M dexamethason, 10 mM beta -glycerol phosphate and 200 μM ascorbic acid in cell culture basal medium, supplemented with 10% human serum and 1% penicillin/streptomycin. All chemicals were cell culture quality.

Osteogenic differentiation was assessed by C-terminal type I collagen levels, and alkaline phosphatase as well as calcein, von Kossa staining and alizarin red staining.

Figure 1 A schematically shows a bio-reactor 1 of the invention, which has a circular or rectangular cross-section with a longitudinal axis 2. The end sections are closed sealingly by lids 3, 4, except for gas permeability. The pivoting movement reaches the positions shown left and right, with the straight short arrow indicating the flow of the medium in the tilted end positions, whereas in the central picture, the longitudinal axis 2 is essentially horizontal, as can be seen from the medium 5 having its surface parallel to the longitudinal axis of the bio- reactor. The cell culture medium 6 is shown to share the bio-reactor volume with the oxygen- containing gas phase 7. A matrix material 8 is shown to freely float within the medium 6. In Figure 1 B, a bioreactor performing the cultivation process according to the invention is shown in different positions of the tilting movement of the bioreactor. For tilting, the bioreactor, which was a 50 mL plastic reaction vessel (50 mL tube available from Falcon with a gas-permeable 0.22 μm filter arranged in the lid, obtained from TPP, Switzerland) was arranged between two parallel rotating discs, at approximately the same distance from the axis of the rotating discs, such that the longitudinal axis of the bio-reactor was positioned at an angle of approximately 30° to the axis of rotation. The rotating discs were arranged to rotate around the same horizontally oriented axis.

For aeration, the bio-reactor vessel was equipped with a lid containing a sterile filter (0.22 μm) to allow sterile gas exchange (available from Millipore Corp., USA). During cultivation, the tilting actuator shown in Figure 1 (available as MACSmix from Miltenyi Biotech, Germany) was positioned in a 37°C incubator for cell culture. Initially, 25 to 35 mL cell culture medium, containing about 5 x 10⁶ cells and a 10 x 10 mm porous matrix cube of poly(lactide-co-glycolid)-calcium phosphate composite having pore diameters in the range of below 100 μm to 2000 μm were positioned into the bio-reactor and the tilting movement was started at 10 to 12 rpm of the rotating discs.

For comparison, static cultivation was used, or perfusion cultivation in a bio-reactor suitable forced perfusion of the matrix arranged in a chamber located within a conventionally controlled stirred vessel bio-reactor. Figure 2 schematically shows a gas mixing unit equipped with a filter, a gas exhaust, a cell culture medium flask with a pump, and a waste receiving flask with a pump each provided with a connective pipe to the inner volume of the bioreactor, which is provided with a temperature control thermostat, as well as with sensors for pH, dissolved oxygen concentration, and for filling level. These elements can also be used for the bio-reactor of the invention. In the comparative cultivation process, the matrix is enclosed within the scaffold chamber where it is exposed to the forced circulation of culture medium that it pumped through it.

Preferably, cells were allowed to the settle onto the matrix material prior to introduction into the bio-reactor by dropwise adding the cells within a medium volume corresponding to the total pore volume of the matrix. For the 10 x 10 mm matrix cube, about 0,4 mL cell suspension containing about 5 x 10⁶ cells were used, followed by incubation for about 90 min at 37°C and 12% CO₂ under sterile conditions, and then transferring the cell containing matrix to 15 mL expansion medium of cell culture basal medium with 15% human serum, 1% penicillin-streptomycin.

Preferably, the medium was first restricted to a small volume, e.g. 15 mL, with an additional identical volume following twenty-four hours initial cultivation before the tilting movement of the bio-reactor was started.

Under the cultivation conditions, the matrix was periodically gently moved by the tilting movement of the bio-reactor, while generally avoiding turbulent flow conditions.

With a total medium change approximately every 2 to 4 days, preferably every 3 days, cultivation in the tilting bio -reactor was continued for approximately 35 days under tilting. Tilting was adjusted by setting the rotation of the parallel discs to 12 rpm, resulting in a rate of 24 /min of each of the two end sections of the bio-reactor from the horizontal position. Analysis of the matrix material colonized with bone forming cells according to the process of the invention was done by histological analysis using cross-sections of the matrix, fixing in 4% buffered paraformaldehyde solution (PFA) at 4 ⁰C overnight and then processed for paraffin embedding. In detail, matrix samples were washed in water for 2 hours, dehydrated in successive ethanol washes at 70%, 80%, two changes of 96%, for one hour each, then transferred to 100% ethanol (two washes), and finally immersed by two washes into xylene substitute (Histoclear, available from Thermo Electron Corporation, Dreieich, Germany). Immersion into liquid paraffin at 56 ⁰C was twice for 2 hours each, followed by cooling and sectioning to 4, 10 and 20 μm on a rotary microtome (Shandon 0325). Sections were mounted onto poly-L-lysine coated slides to improve adhesion, incubated a hot plate at 50 ⁰C and oven dried at 60 ⁰C for 30 to 40 min.

For immunohistochemical staining for analysis in confocal laser scanning microscopy, paraffin sections were subjected to a heat induced epitope retrieval step using an antigen retrieval solution (available from DakoCytomation, S 1700), according to the instructions of the manufacturer. Briefly, sections were de-paraffmized by dipping sections two times, 10 min each, into xylene substitute (Histoclear), followed by four consecutive washes in 100%, 90%, 80%, 70% ethanol, for 5 min each, then rinsed in distilled water for rehydration before haematoxylin and eosin staining. Confocal imaging was done on sections with an LSM META 510 confocal scanning laser system (Zeiss, Jena) on an Axiovert 200M microscope (Zeiss, Jena) with viewing and editing by LSM 5 image browser (Zeiss, Jena).

For haematoxylin and eosin staining, standard protocols were used.

Micrographs of paraffin transverse cross-sections of human trabecular bone derived cells colonizing the matrix after cultivation over 35 days are shown in Figure 3 obtained by the cultivation process in the tilting bioreactor and, as a comparison, by static culture. In detail, Figure 3 A shows the result of the tilting bioreactor cultivation process in haematoxylin and eosin staining (thickness 10 μm) with cells (grey) only along the inner surface of the matrix (black). Essentially, cells can be seen to grow on inner surfaces of the matrix across the entire cross-section of the matrix, with different thicknesses of the cellular layer. Extended cultivation could be used to increase the thickness of the cell layer within the inner volume of the matrix, finally filling the inner volume, i.e. the pores of the matrix.

In contrast, Figure 3 B shows that static cultivation only resulted in cellular colonization of the outer perimeter of the matrix, whereas essentially no cell layer is detected in the inner volume of the matrix. Forced perfusion cultivation lead to patch- wise colonization of the inner volume of the matrix but no uniform colonization as was obtained by the tilting cultivation process of the invention.

Figure 4 shows an enlargement (scale bar 5 mm) of the central part of a transverse section of the matrix colonized by cells in the tilting bioreactor process, demonstrating ubiquitous cell and tissue distribution (grey) in the interstices formed by the matrix material (dark grey).

Further, van Gieson staining of the matrix with cells cultivated according to the invention is shown in Figure 5. Here, pores of the matrix completely filled with positive van Gieson staining of the intercellular spaces are shown across the inner section of the matrix, indicating de novo laid down tissue matrix. Dark (originally violet) staining cell nuclei and the matrix material (originally pink) are indicated by an asterisk. Scale bar in picture A is 200 μm. The higher magnification of picture B (scale bar 100 μm) of a section of picture A shows that cells are organised in a collagen rich matrix, squeezing through a pore, probably alongside medium flow. Picture C (scale bar 100 μm) shows that the cells form a network, contacting each other by short processes, and attaching strongly to the matrix surface, as e.g. indicated by the arrow. Picture D (scale bar 50 μm) shows that osteocyte-like cells (OC) having branching fϊlapodia could be identified at several locations. Collagen-like fibres are indicated by CF.

Analysis of the matrix colonized with cells in cultivation under static conditions identified several layers of cells around the outer perimeter of the matrix material, which layers of cells (approximately 200 μm thick) are assumed to shield the inner volume of the matrix from inflow of nutrients and oxygen. Connective tissue staining confirmed that cell ingrowth was restricted to the periphery of the matrix, with only pores directly connected to the outer matrix perimeter being contacted by cells, but no infiltration of cells into the inner volume of the matrix. Further, the cells of the comparative cultivation process had an increasingly weak and brittle occurrence with increasing distance from the outermost cell layer around the outer perimeter of the matrix, i.e. cell morphology was drastically impaired close to the matrix. Further, extracellular collagen matrix was seen to a far lesser extent than that generated in the process using the tilting bio-reactor processing. Analysis of the comparative colonized matrix is shown in Figure 6, with picture A (scale bar 2 mm) showing the essentially empty interior volume of the matrix, picture B (scale bar 500 μm) of a section at the perimeter of the scaffold with P indicating a pore, CL a cell layer and the asterisk indicating the matrix. Picture C (scale bar 200 μm) shows that cells were loosely unorganized (indicated by arrow), when further away from the perimeter of the matrix.

Further, the osteogenic character of the tissue within the matrix material generated by the tilting cultivation process of the invention could be confirmed by immunohistochemical staining of connexin 43, showing gap junctions between the cells.

In detail, paraffin embedded sections were stained with antibody specific for human connexin 43, followed by identification of the antibody by specific dyeing (secondary antibody- Alexa 488) in confocal microscopy. Figure 7 shows a microscopic analysis, additionally containing staining for nuclei with DRAQ5 (obtainable from Biostatus Ltd, GB) and cytosceleton staining with phalloidine 546. Matrix material is indicated by the asterisk; some of the points of contact between cells are indicated by arrows. Picture B (scale bar 50 μm) is an enlargement of a section in the upper left corner of picture A (scale bar 20 μm). Further, it was only in the tilting cultivation process that cells produced a thick fibrous extracellular matrix layer forming clusters of crystal- like structures bulging out and aligning collagen fibrils, as shown in Figure 8, picture A (scale bar 10 μm) for the static cultivation process, and picture B for the tilting cultivation process (scale bar 5 μm).

Example 2: Cultivation of human adipogenic cells within a porous matrix material to produce a porous matrix essentially colonized throughout its inner surface, and at least partially throughout its inner volume, suitable as an adipose tissue implant

As a further example for a matrix containing cells derived from mesenchymal stem cells obtainable by the process of the present invention, of human trabecular derived cells were subjected to adipogenic differentiation. For generation of an adipose tissue implant, Example 1 was repeated with replacing osteogenic differentiation by adipogenic differentiation, using cell culture basal medium supplemented with 10 % fetal bovine serum and 1% penicillin- streptomycin, 5 μg/mL insulin, 0.5 mM isobutylbethylxanthine, 200 μM indomethacin, and 0.5 mM hydrocortisone for replacing the initial expansion medium. After 21 days of induction, adipogenic differentiation was found by histologically visualising lipid vacuoles in Nile Red staining. In short, cells were rinsed in PBS and fixed with 4 % PFA for 30 min, then rinsed with distilled water, followed by incubation with the Nile Red derivative AdipoRed solution (obtainable from Cambrex), 30 min at room temperature. The staining solution was aspirated and the cell layer was kept moist with PBS for fluorescent microscopy analysis.

As matrix material, the poly(lactide-co-glycolid)-calcium phosphate composite of Example 1 can be used. Preferably, the poly(lactide-co-glycolid)-calcium phosphate composite matrix was replaced by a more flexible matrix material, namely porous collagen matrix. It was found that the behaviour of fat cells in the cultivation process of the invention is similar to that of bone and cartilage cells.

In microscopic analysis, cultivation with tilting according to the invention was found to produce adipose cell layers and adipose tissue within the interstices of the matrix material, whereas comparative cultivation processes in horizontally rotating cultivation flasks or stirred vessel bio-reactors did not result in a significant colonization of the inner surfaces of the matrix material. Example 3: Cultivation of human chondrogenic cells within a porous matrix material to produce a porous matrix essentially colonized throughout its inner surface, and at least partially throughout its inner volume, suitable as chondroid tissue implant

As a further example for a matrix material containing cells obtained by the process of the invention, chondrocytes were generated from mesenchymal stem cells. In detail, for chondrogenic differentiation, the human trabecular derived cells were expanded according to Example 1 and then were changed from the initially used medium to chondrogenic induction medium, as applicable, consisting of cell culture basal medium supplemented with 1% penicillin/streptomycin, 50 μL /mL ascorbic acid, 1.25 μg/mL bovine serum albumin, 1 x 10^~7 M dexamethason, 6.25 μg/mL insulin, 40 μg/mL prolin, 100 μg/mL sodium pyruvate, 6.25 μg/mL transferrin, 10 ng/niL TGF-β, with medium exchange for the culture every 72 hours.

Chondrogenic potential could be demonstrated histologically by Alcian blue staining after twenty-one days cultivation. In short, Alcian blue stains sulphated glycoaminoglycans and proteoglycans typical for chondrocytes. Alcian blue was dissolved in 3 % acetic acid and filtrated through a 0.22 μm membrane. Cells were fixed in ice-cold methanol at -20⁰C for 30 min at room temperature, rinsed with distilled water and stained for 10 min with the Alcian blue solution, followed by rinsing with deionized water.

As a matrix material, the poly(lactide-co-glycolid)-calcium phosphate composite of Example 1 was used.

In microscopic analysis, cultivation with tilting according to the invention was found to produce chondrocyte cell layers within the interstices of the matrix material, whereas comparative cultivation processes in horizontally rotating cultivation flasks or stirred vessel bio-reactors did not result in a significant colonization of the inner surfaces of the matrix material.

Claims

1. Process for producing a porous matrix material (8) comprising on the surface of its interstices cells, for medical use as a bone implant, the process comprising the steps of cultivating cells in a bio -reactor (1) having a first continuous inner surface essentially extending essentially in the direction of a longitudinal axis (2) and second continuous inner surfaces (3, 4) sealing the cross-section spanned by the first inner surface at two spaced apart sections of the bio-reactor (1), with at least one inlet and one outlet port (3, 4) for gas, which ports are arranged within the first and/or second inner surface, providing the porous matrix material (8) within the bio-reactor (1), providing a volume of cell culture medium (5) within the bio-reactor (1) to at least cover the porous matrix material (8) in a position of the bio-reactor with its longitudinal axis inclined, providing a temperature and an atmosphere suitable for growth of the cells, and periodically tilting the longitudinal axis (2) of the bio-reactor (1) from a horizontal position to an inclined position.

2. Process according to claim 1, characterized in that the matrix material (8) is structurally stable.

3. Process according to claim 2, characterized in that the matrix material (8) is selected from poly(lactide-co-glycolide)-calcium phosphate composite, acellularised spongiosa, calcium-hydroxyl apatite, calcium-deficient hydroxyl apatite, β-tri-calcium phosphate and bioresorbable ceramics.

4. Process according to claim 1, characterized in that the matrix material (8) is elastic.

5. Process according to claim 4, characterized in that the matrix material (8) is selected from collagen.

6. Process according to one of the preceding claims, characterized in that cross-section spanned by the first inner surface is circular.

7. Process according to one of the preceding claims, characterized in that the matrix material (8) floats in the medium (5) without mechanical restriction.

8. Process according to one of the preceding claims, characterized in that periodically tilting is at an average frequency of 5 to 40 tilting movements per minute of the longitudinal axis (2) of the bio-reactor (1) to one inclined position.

9. Process according to one of the preceding claims, characterized in that tilting is performed symmetrically about a horizontal axis, in relation to which the bio-reactor (1) is positioned to have its longitudinal axis (2) arranged in an angle of 30° to 90°.

10. Process according to one of the preceding claims, characterized in that the bio-reactor (1) is additionally rotated along its longitudinal axis (2).

11. Process according to one of the preceding claims, characterized in that the bio-reactor (1) has at least one sensor, the surface of which is essentially arranged within the plane of the first and/or second inner surface.

12. Process according to one of the preceding claims, characterized in that the cells are stem or progenitor cells.

13. Process according to claim 12, characterized in that the stem or progenitor cells are selected from mesodermal progenitor cells and mesenchymal progenitor cells.

14. Process according to one of the preceding claims, characterized in that prior to providing the matrix material (8) within the bio-reactor (1), the cells are contacted with the matrix material (8) in a volume of cell culture medium essentially corresponding to the pore volume of the matrix material (8), followed by cultivation of the matrix material (8) in medium without forced movement of the medium.