CA2025798A1 - Method for cultivating cells in microporous beads - Google Patents
Method for cultivating cells in microporous beadsInfo
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- CA2025798A1 CA2025798A1 CA002025798A CA2025798A CA2025798A1 CA 2025798 A1 CA2025798 A1 CA 2025798A1 CA 002025798 A CA002025798 A CA 002025798A CA 2025798 A CA2025798 A CA 2025798A CA 2025798 A1 CA2025798 A1 CA 2025798A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0012—Cell encapsulation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
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Abstract
Abstract of the disclosure A method for cultivating cells in microporous beads The invention relates to a method for cultivating cells entrapped in microporous beads, which are located closely packed in a fixed bed and are supplied with nutrients discontinuously or continuously by the flow of medium in a vessel. Suitable microporous beads have a membrane composed of a gel or of a polyelectrolyte.
Description
HOECHST AKTIENGESELLSCHAFT HOE 89/F 311 Dr.TH/PP
Description A method for cultivating cells in microporous beads The invention relates to a method for cultivating cells in microporo-ls beads which are located in a fixed bed and are supplied discontinuously or continuously with nut-rients.
A number of different methods with which whole cells can be immobilized are known. Thus, cells can be entrapped by suitable measures in a crosslinked gel and continue to be viable and active therein (Klein, Wagner ~Methods for the Immobilisation of Microbial Cells" in Appl. Biochem.
Bioeng. 4~ 51 (1983)). However, it has emerged in practice that live cells disrupt the gel matrix by growing and may then enter the surrounding nutrient solution. There they are able to grow further and thus greatly impede cell retention in continuous processes.
US Patent 2,958,517 discloses a device for the cultiva-tion of free mammalian cells, in which the nutrient solution is mixed with a magnetically driven stirrer bar.
However, with this procedure there are always mutual collisions of the cells and, consequently, impairment of vital functions, which may lead to death of the cells.
US 706,872 describes a continuous cultivation of mEm-malian cells on porous sponge-like particles. About 1 to 5~ of the cell population in the reactor appear free in the nutrient solution, which may easily lead to the free cells settling on and blocking the retaining devices for the immobilizates, such as membranes or sintered glass disks.
It is ve~y difficult to culture plant or animal cells in particular. These cells have very sensitive and fragile membranes which can easily be weakened or damaged by only slight mechanical effects. This greatly impairs the viability and the productivity of these cells.
Another method of Lmmobilization is represented by the technique of entrapment of cells in semipermeable mem-branes which are called microporous beads hereinafter.
Examples of this technique are specified in European Patent Application 0,173,915 or 0,280,155 and in German Offenlegungsschrift 3,529,203. It is also shown in the German Offenlegungsschrift how immobilized cells can be cultured in a stirred reactor. However, even in this reactor the mechanical stress on the cells from the stirrer is still too great. There may be damage to the capsules and release of the entrapped cells and, finally, damage to the cells.
It has now been found, surprisingly, that the above-mentioned difficul~ies can be overcome by the close-packed arrangement of microporous beads in a fixed bed.
Despite the close packing, the microporous beads retain their stability and thus permit optimal growth and production conditions for the cells contained therein.
This close arrangement of the microporous beads in the reactor surprisingly also preventR the formation of channels between the microporous beads. This ensures a uniform and readily controllable supply of all the required nutrient~ to the cells. Hence this also makes it possible to cultivate the cells without interruption over a long period, covering many generations.
The invention thus relates to a method for cultivating cells in microporous bead~ whose membrane i9 composed of an anionic polysaccharide gel or of a polyelectrolyte membrane, wherein the microporous beads are located in a fixed bed.
The microporous beads lie in the fixed bed as close-packed beads in the stream of nutrient solution. They ;~J " ~
experience only slight spatial changes in position and thus only slight mechanical friction, which might lead to damage, occurs on the membranes of the microporous beads.
The invention is described in detail hereinafter, espe-5 cially in its preferred embodiments. The in~ention is furthermore defined in the claims.
The microporous beads suitable for the invention are composed of a biocompatible, non-toxic, semipermeable, water-insoluble membrane. The preparation of such mem-branes is described, for example, in European Patent Application 0,173,915. For this purpose, the cells are suspended in, in particular, an aqueous solution of the core polymer. The core polymer increases the viscosity of the cell suspension so that, during the subsequent dropwise additivn to the anionic polysaccharide solution, mixing of the solutions is prevented. Suitable core polymers are all neutral, water-soluble, biocompatible polymers which increase the viscosity. Examples of these are hydroxypropylmethylcellulose or hydroxymethyl-cellulose. These core polymers are mixed with one or more divalent cations such as, for example, CaCl2. The mixture is then converted into the form of drops and introduced into an anionic polysaccharide solution composed of, for example, alginate, carrageenan, chitosan, pectinate or carboxymethylcellulose. Alginate is preferably used. A
semipermeable membrane is formed at the phase boundary between core polymer and polysaccharide solution owing to the presence of one or more divalent cations, and then entraps the cells. The membrane is compo~ed of the anionic polysaccharide which is converted by the divalent cations into the form of a gel. On the other hand, the core polymer in which the cells are suspended remains fluid.
It is also possible in an analogous manner, as described in European Patent Application 0,280,155, for cells to be suspended in an aqueous solution of an anionic polymer ~ 6~ ~ r (polyacid) such as, for example, alginate, carrageenan, hyaluronic acid, carbo~ymethylcellulose, xanthan or furcellaran, converted into the form of drops and sub-sequently introduced into an aqueous solution of a cationic polymer (polybase) such as, for example, of a copolymer of l-vinyl-3-methylimidazolium chloride and 1 vinyl-2-pyrrolidone or of a polyallylamine/2-hydroxy-propylene copolymer. A copolymer of l-vinyl-3-methyl-imidazolium chloride and l-vinyl-2-pyrrolidone i8 prP~
ferably used. A semipermeable polyelectrolyte membrane is formed at the phase boundary between polyacid and polybase and entraps the cells. In this case too the core polymer in which the cells are suspended remains fluid.
The membrane prevents the passage of cells but is freely permeable to gases and constituents of the medium.
It is possible for all viable cells to be entrapped and cultivated in these microporous beads. These are bac-teria, fungi or yeasts and, in particular, all cell lines of animal or plant origin, as well as, particular pre-ferably, hybridoma cells. Under suitable conditions, thecells grow by cell division in the microporous beads.
Supply with nutrients and transport away of formed products takes place by diffusion through the semiper-meable membrane.
Cultivation is carried out in a vessel in which the microporous beads are closely packed. The vessel can be made of inert material such as, for example, metal, ceramic, glass or a plastic material which can be steri-lized by chemical or physical methods. The vessel can be, in particular, in a form resembling a cylinder. The ratio of hei~ht to diameter can vary within a wide range. The ratio of height to width can vary within the range from 100:1 to 1:100, in particular from 2:1 to 20:1.
The cultivation vessel has at opposite ends inlet and outlet openings; between them are located the microporous beads. The openings are arranged so that the nutrient r~ i rf ' ~ I !
-- S --solution can flow through all the microporous beads located between them. Between the openings and the microporous beads are located retaining devices for the microporous beads, such as, for example, membranes or sintered glass disks, which are, however, easily per-meated by the nutrient solution. It is possible with an arrangement of this type to allow nutrient solution to flow continuously or discontinuously at any desired rate through the vessel. The volume of liquid remains constant during this time. The nutrient solution can also be circulated in a loop by sn appropriate device, so that better utilization of nutrient constituents, which are often costly, is possible.
A11 control, measurement and adjustment procedures can, if necessary, take place outside the cultivation vessel.
These are, in particular, the measurement of temperature, of pH and of oxygen partial pressure, but also of gas exchange f 2 and CO2, the control of the pH and the renewal of constituents of the nutrient solution.
It is possible, without impairing the encapsulated cells, to branch off a portion of the nutrient solution in order to remove and process the products which have been produced. It is also possible to replace the branched-off nutrient solution by fresh. The nutrient replacemen~ and the product isolation can take place discontinuously or continuously from the device for recycling the nutrient stream.
The choice and adjustment of the chemical and physical parameters for the cultivation takes place in accordance with the requirements for gro~Yth and prvd~ction of the cells entrapped in the microporous beads. The limits of these cultivation para~eters can vary within a wide range. However, it will not be difficult for a person skilled in the art to discover the parameters which are optimal for the cells used in each case.
For hybridoma cells for example, the following physical and chemical cultivation parameters have proven suitable.
The cultivation temperature is between 35 and 39C, preferably at 37C. Adequate growth is attained at a pH
between 6.8 and 7.2, preferably at 7Ø The flow rate through the reactor can vary within a wide range depen-ding on the stage of growth. Good results are obtained at between 0.1 and 50 liters of medium per hour and liter of reactor vol~me, preferably between 5 and 20 l/h.l.
It has been found, surprisingly, that the microporous beads retained their stability despite the close packing in the cultivation vessels. There was no channel forma-tion and no clumping of the capsules. It was thus pO8-sible to carry out the cultivation over a long period.
The close packing of the microporous beads and the continuous recycling of nutrients make it possible to cultivate ~ells and produce products in a very small space with a simple apparatus under sterile conditions.
In particular, the nutrient recycling results in effi-cient and economic utilization of the required nutrient~, which are often very costly. Regeneration of the nutrient solution is also possible owing to the selective removal of the final products of metabolism, such as, for exam-ple, lactic acid, CO2 or ammonium ions, by ionic exchan-gers or other separation methods in the cell-free nut-rient stream.
The examples which are detailed below serve to illustrate the invention further.
~xample 1 A suspension of hybridoma cells is diluted 1:2 ~ratio by mass) with a 3% strenqth carrageenan solution (Sigma Chemie GmbH, Munich) in Dulbecco'æ medium ~Dulbecco &
Freeman, ~1959), Virology 8, 396). The solution is converted into drops through a nozzle. The nozzle com-prises a needle of internal diameter 0.2 mm and external 2;~ ?3 diameter 0.4 mm. It is concentrically inserted into a hollow cylinder so that a tangential stream of air can be generated through the resulting annular space, which stream forces off the drops emerging from the needle. The diameter of the drops was 0.1 mm - 3 mm depending on the speed of the air. The drops fell into a solution of the polybase.
The polybase is prepared as follows:
1443 g (lO mole) of l-vinyl-3-methylimidazolium chloride and 56 g (0.5 mole) of l-vinyl-2-pyrrolidone are dis-solved in 3.8 1 of water which contains 38 g of potassium peroxodisulfate as initiator in a 4 liter glass flask.
The mixture is polymerized under nitrogen at 60C for 5 h. A clear yellow-brown 40% strength solution of neutral pH is obtained. 100 ml of the 40% strength solution are made up to 2 1 with a 0.9% strength aqueous NaCl solution. About 100-120 ml of carrageenan/cell suspension are added dropwise to 1 liter of the diluted polymer solution.
After the dilution, the capsules are allowed to settle out, the solution is decanted off, suspension is carried out 3 times with 0.9~ NaCl solution. It is subsequently possible to cultivate cells in the microporous beads.
~ample 2 28.2 g of hydroxypropylmethylcellulose are dissolved in 675 ml of 0.9% strength NaCl solution. Then 75 ml of a 13.3% strength CaCl2 x 2H2O solution are added. ~his solution is mixed w;th the hybridoma cell suspension in the ratio of 3:1 (ratio by mass). This suspe~sion is converted into drops as described in ~xample 1. The drops are collected in an alginate solution. The alginate solution is prepared as follows. 15 g of alginate are dissolved in S00 ml of 0.9~ strength NaCl solution and then diluted to 2 l with O.g% strength NaCl solution.
80 ml of the above cell suspension are added dropwise to ~ J ;
1 1 of alginate solution. An alginate gel polymerizes out at the phase boundary between drops and alginate solution and holds the entrapped cells. The microporous beads are washed 3 times with 0.9% strength NaCl solution and then transferred into a 2% strength ~aCl2 x 2H2O solution and stirred for 2 min. The solution is then decanted off, and the microporous beads are washed several times with 0.9%
strength NaCl solution.
Fxample 3 The cells encapsulated as in Example 2 are transferred under sterile conditions into a sterile reactor column.
The column is completely filled with capsules and then medium is continuously passed through in a closed system.
The adjustment and control of the parameters takes place in a 5 l reactor which is connected to the column via lines. The gas exchange is brought about in the reactor by tubular membranes. Cultivation is carried out under the following conditions:
Medium Dulbecco's nutrient solution (Dulbecco (1959), Virology, 8, 296) 10% fetal calf serum Volume of the column 0.4 l Medium flow rate 7 l/h pH 7.0 PO2 60% air satur~tion The PO2 content is measured at the end of the column with an oxygen electrode. ~ peristaltic pump ensures the flowing over. Ihe experiment lasts 25 days. The medium i8 changed 4 tLmes. For this, 50% of the old medium was replaced by new each time. The timing of the medium change is determined by measuring metabolic products. A
medium change is carried out at ammonium concentrations above 3 mmol/l and glutamine amounts of less than 1.5 mmol/l. The cell count at the start is 20,500 cells r J 1~) _ g _ per capsule and rises at the end of the cultivation to about 200,000 cells per capsule. The mean antibody production is 0.08 mg/ml of reactor volume and day.
The amount of ammonium and glutamine was determined with an enzymatic standard determination supplied by Boehringer, Mannheim.
~xample 4 The cells entrapped in microporous capsules are culti-vated as indicated in Example 3. A cylindrical glass column with an effective volume of 400 ml is used as reactor. The height to diameter ratio is 3 to 1. Sintered glass disks with a pore diameter of 0.1 mm are installed to retain the microporous capsules of the entry and exit of the glass column. The area of the sintered glass disk at the exit is twice that at the entry.
Example 5 The cells encapsulated as in Example 1 are transferred under sterile conditions into a sterile reactor column as described in Example 3. The cultivation is carried out as indicated in Example 3 although in Iscove's nutrient solution (Iscove & Melchers (1978), J. Exp. Med., 147:
923). The mean antibody production is Q.06 mg/ml of reactor volume and day. The cultivation lasted 4 weeks.
Description A method for cultivating cells in microporous beads The invention relates to a method for cultivating cells in microporo-ls beads which are located in a fixed bed and are supplied discontinuously or continuously with nut-rients.
A number of different methods with which whole cells can be immobilized are known. Thus, cells can be entrapped by suitable measures in a crosslinked gel and continue to be viable and active therein (Klein, Wagner ~Methods for the Immobilisation of Microbial Cells" in Appl. Biochem.
Bioeng. 4~ 51 (1983)). However, it has emerged in practice that live cells disrupt the gel matrix by growing and may then enter the surrounding nutrient solution. There they are able to grow further and thus greatly impede cell retention in continuous processes.
US Patent 2,958,517 discloses a device for the cultiva-tion of free mammalian cells, in which the nutrient solution is mixed with a magnetically driven stirrer bar.
However, with this procedure there are always mutual collisions of the cells and, consequently, impairment of vital functions, which may lead to death of the cells.
US 706,872 describes a continuous cultivation of mEm-malian cells on porous sponge-like particles. About 1 to 5~ of the cell population in the reactor appear free in the nutrient solution, which may easily lead to the free cells settling on and blocking the retaining devices for the immobilizates, such as membranes or sintered glass disks.
It is ve~y difficult to culture plant or animal cells in particular. These cells have very sensitive and fragile membranes which can easily be weakened or damaged by only slight mechanical effects. This greatly impairs the viability and the productivity of these cells.
Another method of Lmmobilization is represented by the technique of entrapment of cells in semipermeable mem-branes which are called microporous beads hereinafter.
Examples of this technique are specified in European Patent Application 0,173,915 or 0,280,155 and in German Offenlegungsschrift 3,529,203. It is also shown in the German Offenlegungsschrift how immobilized cells can be cultured in a stirred reactor. However, even in this reactor the mechanical stress on the cells from the stirrer is still too great. There may be damage to the capsules and release of the entrapped cells and, finally, damage to the cells.
It has now been found, surprisingly, that the above-mentioned difficul~ies can be overcome by the close-packed arrangement of microporous beads in a fixed bed.
Despite the close packing, the microporous beads retain their stability and thus permit optimal growth and production conditions for the cells contained therein.
This close arrangement of the microporous beads in the reactor surprisingly also preventR the formation of channels between the microporous beads. This ensures a uniform and readily controllable supply of all the required nutrient~ to the cells. Hence this also makes it possible to cultivate the cells without interruption over a long period, covering many generations.
The invention thus relates to a method for cultivating cells in microporous bead~ whose membrane i9 composed of an anionic polysaccharide gel or of a polyelectrolyte membrane, wherein the microporous beads are located in a fixed bed.
The microporous beads lie in the fixed bed as close-packed beads in the stream of nutrient solution. They ;~J " ~
experience only slight spatial changes in position and thus only slight mechanical friction, which might lead to damage, occurs on the membranes of the microporous beads.
The invention is described in detail hereinafter, espe-5 cially in its preferred embodiments. The in~ention is furthermore defined in the claims.
The microporous beads suitable for the invention are composed of a biocompatible, non-toxic, semipermeable, water-insoluble membrane. The preparation of such mem-branes is described, for example, in European Patent Application 0,173,915. For this purpose, the cells are suspended in, in particular, an aqueous solution of the core polymer. The core polymer increases the viscosity of the cell suspension so that, during the subsequent dropwise additivn to the anionic polysaccharide solution, mixing of the solutions is prevented. Suitable core polymers are all neutral, water-soluble, biocompatible polymers which increase the viscosity. Examples of these are hydroxypropylmethylcellulose or hydroxymethyl-cellulose. These core polymers are mixed with one or more divalent cations such as, for example, CaCl2. The mixture is then converted into the form of drops and introduced into an anionic polysaccharide solution composed of, for example, alginate, carrageenan, chitosan, pectinate or carboxymethylcellulose. Alginate is preferably used. A
semipermeable membrane is formed at the phase boundary between core polymer and polysaccharide solution owing to the presence of one or more divalent cations, and then entraps the cells. The membrane is compo~ed of the anionic polysaccharide which is converted by the divalent cations into the form of a gel. On the other hand, the core polymer in which the cells are suspended remains fluid.
It is also possible in an analogous manner, as described in European Patent Application 0,280,155, for cells to be suspended in an aqueous solution of an anionic polymer ~ 6~ ~ r (polyacid) such as, for example, alginate, carrageenan, hyaluronic acid, carbo~ymethylcellulose, xanthan or furcellaran, converted into the form of drops and sub-sequently introduced into an aqueous solution of a cationic polymer (polybase) such as, for example, of a copolymer of l-vinyl-3-methylimidazolium chloride and 1 vinyl-2-pyrrolidone or of a polyallylamine/2-hydroxy-propylene copolymer. A copolymer of l-vinyl-3-methyl-imidazolium chloride and l-vinyl-2-pyrrolidone i8 prP~
ferably used. A semipermeable polyelectrolyte membrane is formed at the phase boundary between polyacid and polybase and entraps the cells. In this case too the core polymer in which the cells are suspended remains fluid.
The membrane prevents the passage of cells but is freely permeable to gases and constituents of the medium.
It is possible for all viable cells to be entrapped and cultivated in these microporous beads. These are bac-teria, fungi or yeasts and, in particular, all cell lines of animal or plant origin, as well as, particular pre-ferably, hybridoma cells. Under suitable conditions, thecells grow by cell division in the microporous beads.
Supply with nutrients and transport away of formed products takes place by diffusion through the semiper-meable membrane.
Cultivation is carried out in a vessel in which the microporous beads are closely packed. The vessel can be made of inert material such as, for example, metal, ceramic, glass or a plastic material which can be steri-lized by chemical or physical methods. The vessel can be, in particular, in a form resembling a cylinder. The ratio of hei~ht to diameter can vary within a wide range. The ratio of height to width can vary within the range from 100:1 to 1:100, in particular from 2:1 to 20:1.
The cultivation vessel has at opposite ends inlet and outlet openings; between them are located the microporous beads. The openings are arranged so that the nutrient r~ i rf ' ~ I !
-- S --solution can flow through all the microporous beads located between them. Between the openings and the microporous beads are located retaining devices for the microporous beads, such as, for example, membranes or sintered glass disks, which are, however, easily per-meated by the nutrient solution. It is possible with an arrangement of this type to allow nutrient solution to flow continuously or discontinuously at any desired rate through the vessel. The volume of liquid remains constant during this time. The nutrient solution can also be circulated in a loop by sn appropriate device, so that better utilization of nutrient constituents, which are often costly, is possible.
A11 control, measurement and adjustment procedures can, if necessary, take place outside the cultivation vessel.
These are, in particular, the measurement of temperature, of pH and of oxygen partial pressure, but also of gas exchange f 2 and CO2, the control of the pH and the renewal of constituents of the nutrient solution.
It is possible, without impairing the encapsulated cells, to branch off a portion of the nutrient solution in order to remove and process the products which have been produced. It is also possible to replace the branched-off nutrient solution by fresh. The nutrient replacemen~ and the product isolation can take place discontinuously or continuously from the device for recycling the nutrient stream.
The choice and adjustment of the chemical and physical parameters for the cultivation takes place in accordance with the requirements for gro~Yth and prvd~ction of the cells entrapped in the microporous beads. The limits of these cultivation para~eters can vary within a wide range. However, it will not be difficult for a person skilled in the art to discover the parameters which are optimal for the cells used in each case.
For hybridoma cells for example, the following physical and chemical cultivation parameters have proven suitable.
The cultivation temperature is between 35 and 39C, preferably at 37C. Adequate growth is attained at a pH
between 6.8 and 7.2, preferably at 7Ø The flow rate through the reactor can vary within a wide range depen-ding on the stage of growth. Good results are obtained at between 0.1 and 50 liters of medium per hour and liter of reactor vol~me, preferably between 5 and 20 l/h.l.
It has been found, surprisingly, that the microporous beads retained their stability despite the close packing in the cultivation vessels. There was no channel forma-tion and no clumping of the capsules. It was thus pO8-sible to carry out the cultivation over a long period.
The close packing of the microporous beads and the continuous recycling of nutrients make it possible to cultivate ~ells and produce products in a very small space with a simple apparatus under sterile conditions.
In particular, the nutrient recycling results in effi-cient and economic utilization of the required nutrient~, which are often very costly. Regeneration of the nutrient solution is also possible owing to the selective removal of the final products of metabolism, such as, for exam-ple, lactic acid, CO2 or ammonium ions, by ionic exchan-gers or other separation methods in the cell-free nut-rient stream.
The examples which are detailed below serve to illustrate the invention further.
~xample 1 A suspension of hybridoma cells is diluted 1:2 ~ratio by mass) with a 3% strenqth carrageenan solution (Sigma Chemie GmbH, Munich) in Dulbecco'æ medium ~Dulbecco &
Freeman, ~1959), Virology 8, 396). The solution is converted into drops through a nozzle. The nozzle com-prises a needle of internal diameter 0.2 mm and external 2;~ ?3 diameter 0.4 mm. It is concentrically inserted into a hollow cylinder so that a tangential stream of air can be generated through the resulting annular space, which stream forces off the drops emerging from the needle. The diameter of the drops was 0.1 mm - 3 mm depending on the speed of the air. The drops fell into a solution of the polybase.
The polybase is prepared as follows:
1443 g (lO mole) of l-vinyl-3-methylimidazolium chloride and 56 g (0.5 mole) of l-vinyl-2-pyrrolidone are dis-solved in 3.8 1 of water which contains 38 g of potassium peroxodisulfate as initiator in a 4 liter glass flask.
The mixture is polymerized under nitrogen at 60C for 5 h. A clear yellow-brown 40% strength solution of neutral pH is obtained. 100 ml of the 40% strength solution are made up to 2 1 with a 0.9% strength aqueous NaCl solution. About 100-120 ml of carrageenan/cell suspension are added dropwise to 1 liter of the diluted polymer solution.
After the dilution, the capsules are allowed to settle out, the solution is decanted off, suspension is carried out 3 times with 0.9~ NaCl solution. It is subsequently possible to cultivate cells in the microporous beads.
~ample 2 28.2 g of hydroxypropylmethylcellulose are dissolved in 675 ml of 0.9% strength NaCl solution. Then 75 ml of a 13.3% strength CaCl2 x 2H2O solution are added. ~his solution is mixed w;th the hybridoma cell suspension in the ratio of 3:1 (ratio by mass). This suspe~sion is converted into drops as described in ~xample 1. The drops are collected in an alginate solution. The alginate solution is prepared as follows. 15 g of alginate are dissolved in S00 ml of 0.9~ strength NaCl solution and then diluted to 2 l with O.g% strength NaCl solution.
80 ml of the above cell suspension are added dropwise to ~ J ;
1 1 of alginate solution. An alginate gel polymerizes out at the phase boundary between drops and alginate solution and holds the entrapped cells. The microporous beads are washed 3 times with 0.9% strength NaCl solution and then transferred into a 2% strength ~aCl2 x 2H2O solution and stirred for 2 min. The solution is then decanted off, and the microporous beads are washed several times with 0.9%
strength NaCl solution.
Fxample 3 The cells encapsulated as in Example 2 are transferred under sterile conditions into a sterile reactor column.
The column is completely filled with capsules and then medium is continuously passed through in a closed system.
The adjustment and control of the parameters takes place in a 5 l reactor which is connected to the column via lines. The gas exchange is brought about in the reactor by tubular membranes. Cultivation is carried out under the following conditions:
Medium Dulbecco's nutrient solution (Dulbecco (1959), Virology, 8, 296) 10% fetal calf serum Volume of the column 0.4 l Medium flow rate 7 l/h pH 7.0 PO2 60% air satur~tion The PO2 content is measured at the end of the column with an oxygen electrode. ~ peristaltic pump ensures the flowing over. Ihe experiment lasts 25 days. The medium i8 changed 4 tLmes. For this, 50% of the old medium was replaced by new each time. The timing of the medium change is determined by measuring metabolic products. A
medium change is carried out at ammonium concentrations above 3 mmol/l and glutamine amounts of less than 1.5 mmol/l. The cell count at the start is 20,500 cells r J 1~) _ g _ per capsule and rises at the end of the cultivation to about 200,000 cells per capsule. The mean antibody production is 0.08 mg/ml of reactor volume and day.
The amount of ammonium and glutamine was determined with an enzymatic standard determination supplied by Boehringer, Mannheim.
~xample 4 The cells entrapped in microporous capsules are culti-vated as indicated in Example 3. A cylindrical glass column with an effective volume of 400 ml is used as reactor. The height to diameter ratio is 3 to 1. Sintered glass disks with a pore diameter of 0.1 mm are installed to retain the microporous capsules of the entry and exit of the glass column. The area of the sintered glass disk at the exit is twice that at the entry.
Example 5 The cells encapsulated as in Example 1 are transferred under sterile conditions into a sterile reactor column as described in Example 3. The cultivation is carried out as indicated in Example 3 although in Iscove's nutrient solution (Iscove & Melchers (1978), J. Exp. Med., 147:
923). The mean antibody production is Q.06 mg/ml of reactor volume and day. The cultivation lasted 4 weeks.
Claims (10)
1. A method for cultivatinq cells in microporous beads whose membrane is composed of an anionic polysaccharide gel or of a polyelectrolyte membrane, wherein the microporous beads are located in a fixed bed.
2. The method as claimed in claim 1, wherein cells of plant or animal origin are cultivated.
3. The method as claimed in claim 2, wherein hybridoma cells are cultivated.
4. The method as claimed in one or more of claims 1 to 3, wherein the membrane of the microporous beads is composed of an alginate gel.
5. The method as claimed in claim 4, wherein the alginate membrane is formed by complexation with a crosslinking agent from the drop phase.
6. The method as claimed in one or more of claims 1 to 3, wherein the membrane of the microporous beads is composed of a copolymer composed of l-vinyl-3-methylimidazolium chloride and l-vinyl-2-pyrrolidone.
7. The method as claimed in one or more of claims 1 to 6, wherein the nutrient supply is effected by a closed flow of nutrient solution.
8. The method as claimed in claim 7, wherein the nutrient supply takes place discontinuously or continuously.
9. The method as claimed in one or more of claims 1 to 7, wherein the nutrient replacement takes place discon-tinuously or continuously.
10. The method as claimed in claim 1 and substantially as described herein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3931433.2 | 1989-09-21 | ||
| DE3931433A DE3931433A1 (en) | 1989-09-21 | 1989-09-21 | METHOD FOR CULTIVATING CELLS IN MICROHOLE BALLS |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2025798A1 true CA2025798A1 (en) | 1991-03-22 |
Family
ID=6389834
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002025798A Abandoned CA2025798A1 (en) | 1989-09-21 | 1990-09-20 | Method for cultivating cells in microporous beads |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0418796B1 (en) |
| JP (1) | JP2854951B2 (en) |
| KR (1) | KR910006475A (en) |
| AT (1) | ATE100856T1 (en) |
| CA (1) | CA2025798A1 (en) |
| DE (2) | DE3931433A1 (en) |
| DK (1) | DK0418796T3 (en) |
| IE (1) | IE64854B1 (en) |
| PT (1) | PT95376B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7851189B2 (en) * | 2005-03-07 | 2010-12-14 | Boston Scientific Scimed, Inc. | Microencapsulated compositions for endoluminal tissue engineering |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987000197A1 (en) * | 1985-06-24 | 1987-01-15 | Karyon Technology, Incorporated | Entrapment of anchorage-dependent cells |
| DE3735397A1 (en) * | 1987-10-20 | 1989-05-03 | Hoechst Ag | MAGNETIC MEMBRANE CAPSULES AND THEIR USE |
-
1989
- 1989-09-21 DE DE3931433A patent/DE3931433A1/en active Granted
-
1990
- 1990-09-18 AT AT90117893T patent/ATE100856T1/en not_active IP Right Cessation
- 1990-09-18 DE DE90117893T patent/DE59004397D1/en not_active Expired - Fee Related
- 1990-09-18 DK DK90117893.9T patent/DK0418796T3/en active
- 1990-09-18 EP EP90117893A patent/EP0418796B1/en not_active Expired - Lifetime
- 1990-09-19 KR KR1019900014794A patent/KR910006475A/en not_active Withdrawn
- 1990-09-20 JP JP2249019A patent/JP2854951B2/en not_active Expired - Lifetime
- 1990-09-20 CA CA002025798A patent/CA2025798A1/en not_active Abandoned
- 1990-09-20 IE IE340490A patent/IE64854B1/en not_active IP Right Cessation
- 1990-09-20 PT PT95376A patent/PT95376B/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| IE64854B1 (en) | 1995-09-06 |
| EP0418796B1 (en) | 1994-01-26 |
| PT95376A (en) | 1991-05-22 |
| EP0418796A1 (en) | 1991-03-27 |
| KR910006475A (en) | 1991-04-29 |
| DE3931433C2 (en) | 1991-12-12 |
| JPH03119990A (en) | 1991-05-22 |
| IE903404A1 (en) | 1991-04-10 |
| ATE100856T1 (en) | 1994-02-15 |
| DE59004397D1 (en) | 1994-03-10 |
| PT95376B (en) | 1997-07-31 |
| JP2854951B2 (en) | 1999-02-10 |
| DK0418796T3 (en) | 1994-04-11 |
| DE3931433A1 (en) | 1991-04-04 |
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| Date | Code | Title | Description |
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| FZDE | Discontinued |