EP0247077A1 - Process for cell immobilisation - Google Patents

Process for cell immobilisation

Info

Publication number
EP0247077A1
EP0247077A1 EP19860906378 EP86906378A EP0247077A1 EP 0247077 A1 EP0247077 A1 EP 0247077A1 EP 19860906378 EP19860906378 EP 19860906378 EP 86906378 A EP86906378 A EP 86906378A EP 0247077 A1 EP0247077 A1 EP 0247077A1
Authority
EP
European Patent Office
Prior art keywords
microparticles
cells
matrix
cell
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP19860906378
Other languages
German (de)
French (fr)
Inventor
Eric Robinson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB858526095A external-priority patent/GB8526095D0/en
Priority claimed from GB868616881A external-priority patent/GB8616881D0/en
Application filed by Individual filed Critical Individual
Publication of EP0247077A1 publication Critical patent/EP0247077A1/en
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier

Definitions

  • the present invention relates to entrapment of materials, for example viable cells, within a solid framework.
  • immobilised microbial, plant or animal cells for the production of pharmaceutical products and fine chemicals is gaining in commercial importance.
  • advantages of the use of immobilised cells include repeated use, continuous process operation and the elimination of the separation steps necessary if free cells are used which must be removed from product solutions.
  • the object of the present invention is to provide an improved process for cell immobilisation which overcomes this difficulty, producing a strong porous support matrix for the cells which has good mass transfer characteristics.
  • a process for immobilising cells which comprises the steps of mixing the cells with microparticles which are substantially insoluble in aqueous media, blending thoroughly to uniformly disperse the cells and microparticles then bonding the microparticles at points of contact to form a permanent porous matrix entrapping the cells within the cavities therein. It is preferred but not re ⁇ uired that the microparticles are spherical in shape and uniform in diameter. Where uniform microspheres are used their diameter should be less than six times the smallest dimension of the cell.
  • the microparticles used may be composed of inorganic oxides, hydroxides, carbonates or sulphates which are substantially insoluble in an aqueous medium such as silica, alumina, aluminium hydroxide, calcium carbonate or calcium sulphate or of an organic polymer such as polyacrylamide, polystyrene, polyvinyl chloride, polyvinyl acetate, dextran, cellulose or starch. Mass transfer through the matrix is improved if the microparticles are themselves porous.
  • microparticles and cells are mixed and blended in water or in a medium compatible with the viability of the cells.
  • microparticles are silica microspheres produced according to my copending International Application No. FCT/GB86/00319. Microspheres of the appropriate size held in aqueous slurry may be close packed and bonded at points of contact by dewatering and drying. Cells mixed homogeneously with these microspheres before dewatering are entrapped within the cavities in this matrix. Dewatering is achieved by aspiration or by the application of pressure after which the mass is dried in a current of air at 20° to 30oC.
  • microparticles do not bond naturally they may be precoated with an adhesive which softens in the medium employed for mixing and dispersing the cells and microparticles.
  • the microparticie should not be more than six times larger than the c e lls and ideal ly not more than three times the smallest dimension of the cells otherwise cells may escape from the matrix by moving through the interstitial pores.
  • the plastic mass may be formed into any required shape and size by for example extrusion, by pressing into pellets or molding into beads or tablets.
  • Figure 1 illustrates a small element of an entrapment matrix.
  • a cell 10 is entrapped in the cavity formed by six close packed spherical microparticles 11.
  • the ratio of the diameter of the microparticle to that of the cell is greater than 2.3:1.0 but no more than 6.0:1.0 otherwise cells will escape through the interstitial pores 12 between cavities.
  • Figure 2 illustrates in cross-section a small element of an entrapment matrix in which a larger cell 13 creates a cavity by replacing a microparticle 14 in the matrix.
  • microparticles similar in size to the cells are used.
  • access to the entrapped cell is by way of six interstitial pores 12 through which nutrients, expressed proteins or gases may pass.
  • access to the cell is gained through at least 24 such interstitial pores.
  • the strength of the porous matrix may be improved by treatment with a solution of a polymer, or a solution of a monomer which can be polymerised, to form a porous skin or coating around the package of cells and microparticles.
  • the treatment is carried out after shaping into beads, pellets or lumps which are immersed in a solution of a polymer in an appropriate solvent such that at least part of the solution is taken up by the entrapment matrix after which the excess solution is removed.
  • the polymer used may be for example cellulose acetate, polystyrene, polyacrylonitriie, polyacrylamide, polyamide or polyvinylchloride. Vhere the polymer is applied in a solvent, appropriate solvents include acetone, chloroform or dimethylformamide. The solvent Chosen should not affect the immobilising matrix.
  • the strength of the polymer solution used may be from 0.1% to 20% and is preferably from 1% to 5%.
  • the package of cells and microparticles may be treated with a solution of a monomer which is subsequently polymerised.
  • beads or pellets may be treated with a solution of sebacoyl chloride in chloroform. These are then transferred to an aqueous solution of hexamethylene diamine to form a skin of polyamide around the beads or pellets.
  • the benefits of forming a porous skin or coating within the outer layers of the matrix containing immobilised cells are tvofold. First it acts by tying in cells exposed on the surface which otherwise break free from the immobilising matrix. Second it adds strength to the package, in particular it improves the retention of strength over long periods of use.
  • the skin or coating may be applied before or after drying the matrix holding entrapped cells. In addition it may be semi-permeable.
  • the beads were packed into a fixed bed reactor of approximately 1 1 capacity through which was pumped a solution containing 200 g/l sucrose at 30°C, The conversion of sucrose into ⁇ thanol was found to be 8g ethanol/l/hour.
  • the mass was broken and sieved through a 4 mesh sieve.
  • the cell density within the matrix was approximately 5 x 10 9 cells/cm .
  • the material was packed into a fixed bed reactor of approximately 1 1 capacity through which was pumped a solution containing 200 g/l sucrose.
  • the conversion of sucrose to ⁇ thanol was found to be 10g ethanol/l/hour.
  • Vhem set and dried the tablets were used to ferment a sucrose malt solution to a beer containing approximately 4% ethanol.

Abstract

Procédé pour immobiliser des cellules dans une matrice structurée conçue pour convenir aux cellules, en produisant un puissant support poreux pour lesdites cellules, lequel présente de bonnes caractéristiques de tansfert en masse. Les cellules sont emprisonnées dans des cavités entre des micro-particules qui sont liées aux points de contact pour construire la matrice rigide.A method of immobilizing cells in a structured matrix designed to be suitable for cells, producing a powerful porous support for said cells, which has good bulk transfer characteristics. The cells are trapped in cavities between micro-particles which are linked to the contact points to build the rigid matrix.

Description

PROCESS FOR GELL IMMOBILISATION
The present invention relates to entrapment of materials, for example viable cells, within a solid framework.
The use of immobilised microbial, plant or animal cells for the production of pharmaceutical products and fine chemicals is gaining in commercial importance. The advantages of the use of immobilised cells include repeated use, continuous process operation and the elimination of the separation steps necessary if free cells are used which must be removed from product solutions.
Amongst the various methods devised for immobilising cells, entrapment within polymer gels, for example polyacrylamide gels, has been most extensively applied. A disadvantage of existing systems is that to obtain adequate strength the gel structure must be dense and this reduces the porosity of the system. Mass transfer within the immobilising ma trix is thus limited.
The object of the present invention is to provide an improved process for cell immobilisation which overcomes this difficulty, producing a strong porous support matrix for the cells which has good mass transfer characteristics.
According to the present invention there is provided a process for immobilising cells which comprises the steps of mixing the cells with microparticles which are substantially insoluble in aqueous media, blending thoroughly to uniformly disperse the cells and microparticles then bonding the microparticles at points of contact to form a permanent porous matrix entrapping the cells within the cavities therein. It is preferred but not reαuired that the microparticles are spherical in shape and uniform in diameter. Where uniform microspheres are used their diameter should be less than six times the smallest dimension of the cell.
The microparticles used may be composed of inorganic oxides, hydroxides, carbonates or sulphates which are substantially insoluble in an aqueous medium such as silica, alumina, aluminium hydroxide, calcium carbonate or calcium sulphate or of an organic polymer such as polyacrylamide, polystyrene, polyvinyl chloride, polyvinyl acetate, dextran, cellulose or starch. Mass transfer through the matrix is improved if the microparticles are themselves porous.
Normally the microparticles and cells are mixed and blended in water or in a medium compatible with the viability of the cells.
Particularly suitable microparticles are silica microspheres produced according to my copending International Application No. FCT/GB86/00319. Microspheres of the appropriate size held in aqueous slurry may be close packed and bonded at points of contact by dewatering and drying. Cells mixed homogeneously with these microspheres before dewatering are entrapped within the cavities in this matrix. Dewatering is achieved by aspiration or by the application of pressure after which the mass is dried in a current of air at 20° to 30ºC.
Where the microparticles do not bond naturally they may be precoated with an adhesive which softens in the medium employed for mixing and dispersing the cells and microparticles.
Preferably the microparticie should not be more than six times larger than the c e lls and ideal ly not more than three times the smallest dimension of the cells otherwise cells may escape from the matrix by moving through the interstitial pores.
After the cells and microparticles are mixed and before the microparticles are finally bonded together the plastic mass may be formed into any required shape and size by for example extrusion, by pressing into pellets or molding into beads or tablets.
The means whereby cells are entrapped within the matrix of microparticles will be further apparent from the accompanying drawings of preferred embodiments of the invention. Figure 1 illustrates a small element of an entrapment matrix. A cell 10 is entrapped in the cavity formed by six close packed spherical microparticles 11. The ratio of the diameter of the microparticle to that of the cell is greater than 2.3:1.0 but no more than 6.0:1.0 otherwise cells will escape through the interstitial pores 12 between cavities. Figure 2 illustrates in cross-section a small element of an entrapment matrix in which a larger cell 13 creates a cavity by replacing a microparticle 14 in the matrix. Here microparticles similar in size to the cells are used. In the first case access to the entrapped cell is by way of six interstitial pores 12 through which nutrients, expressed proteins or gases may pass. In the second case access to the cell is gained through at least 24 such interstitial pores.
The strength of the porous matrix may be improved by treatment with a solution of a polymer, or a solution of a monomer which can be polymerised, to form a porous skin or coating around the package of cells and microparticles. The treatment is carried out after shaping into beads, pellets or lumps which are immersed in a solution of a polymer in an appropriate solvent such that at least part of the solution is taken up by the entrapment matrix after which the excess solution is removed. The polymer used may be for example cellulose acetate, polystyrene, polyacrylonitriie, polyacrylamide, polyamide or polyvinylchloride. Vhere the polymer is applied in a solvent, appropriate solvents include acetone, chloroform or dimethylformamide. The solvent Chosen should not affect the immobilising matrix. The strength of the polymer solution used may be from 0.1% to 20% and is preferably from 1% to 5%.
Alternatively the package of cells and microparticles may be treated with a solution of a monomer which is subsequently polymerised. Thus for example beads or pellets may be treated with a solution of sebacoyl chloride in chloroform. These are then transferred to an aqueous solution of hexamethylene diamine to form a skin of polyamide around the beads or pellets.
The benefits of forming a porous skin or coating within the outer layers of the matrix containing immobilised cells are tvofold. First it acts by tying in cells exposed on the surface which otherwise break free from the immobilising matrix. Second it adds strength to the package, in particular it improves the retention of strength over long periods of use.
The skin or coating may be applied before or after drying the matrix holding entrapped cells. In addition it may be semi-permeable.
The invention will be further apparent from the following examples.
EXAMPLE 1
Cells of Saccharomyces cerevisiae were mixed with silica microspheres 7 pn in diameter and the mixture was carefully blended for 15 minutes. The serai fluid mass was transferred to a vessel fitted with an oriface 3mm in diameter. The vessel was vibrated to fluidise the mixture which formed droplets as it passed through the oriface and fell onto an absorbant surface which aspirated interstitial water leaving moist beads which were dried in a current of air at 20ºC. After drying the 4mm beads are hard and strong with a crushing strength of 0.75 Kg. The porosity was 0.5 ml/ml and the cell density of S. cerevisiae was approximately 3 x 109cells/cm3.
The beads were packed into a fixed bed reactor of approximately 1 1 capacity through which was pumped a solution containing 200 g/l sucrose at 30°C, The conversion of sucrose into βthanol was found to be 8g ethanol/l/hour.
EXAMPLE 2
Cells of S. cerevisiae were mixed and blended carefully with silica microspheres 7 μm in diameter for 15 minutes in aqueous slurry. The fluid mass was spread on filter paper to remove interstitial water after which the mass was dried at 25°C.
The mass was broken and sieved through a 4 mesh sieve. The cell density within the matrix was approximately 5 x 109 cells/cm .
The material was packed into a fixed bed reactor of approximately 1 1 capacity through which was pumped a solution containing 200 g/l sucrose. The conversion of sucrose to βthanol was found to be 10g ethanol/l/hour.
EXAMPLE 3
20g of 4 mm beads made from silica microspheres and containing 2g of yeast cells held in the interstitial cavities was treated with 50ml 4% polystyrene in chloroform for 2 minutes. The beads were removed and air dried for 48 hours at 20ºC. These beads were used in a continuous fermentation system behaving as an active biocatalyst for 4 months. The production of ethanol from sucrose was measured at 5g/1/hour.
EXAMPLE 4
Cells of aspergillus niger were mixed carefully with a neutral slurry of 3 μm silica microspheres and the mass was spread on filter paper to remove excess water. The mass was allowed to dry for 2 days in a current of air at 20ºC, before being broken and sieved through a 4 mesh sieve.
When packed in a column through which a nutrient liquor was circulated the cells were observed to express a mixture of enzymes including pectinase, cellulase, and hemicellulase.
EXAMPLE 5
Spores of Phytophthora infestans were mixed thoroughly with 8 um silica microspheres and the mass was formed into beads. These were dried at 25°C for 2 days. The beads were then moistened with a nutrient solution and held at 25°C for a further 7 days after which each bead was found to contain a mycelium.
Vhen the beads were packed in a column and had a nutrient liquor circulated over them the immobilised microorganism produced cellulase.
EXAMPLE 6
20 g of 2mm diameter beads made from 7 μm silica microspheres and holding 3 g yeast cells in interstitial cavities were treated for 2 minutes with 50 ml of a 2% solution of cellulose acetate in acetone before the beads were completely dried. The The beads were separated from the solution and the solvent removed before they were used in a batch fermentation to produce an aqueous ethanol solution.
EXAMPLE 7
1.7g S. cerevisiae was mixed with 30g 7μm particles of calciumsulphatβ hernihydrate in 20 ml water. Cells and microparticles were dispersed carefully and the mixture pressed into 4mm diameter tablet molds.
Vhem set and dried the tablets were used to ferment a sucrose malt solution to a beer containing approximately 4% ethanol.
EXAMPLE 8
10g of alumina with a spheroidal particle shape approximately 7 μm across were added to 25ml of 8% polystyrene in chloroform. All the polystyrene solution was taken up by the raicroparticles which were kept in motion while the chloroform was removed. 1g yeast was suspended in 2.5ml water and this was added to 25ml acetone. The polystyrene coated alumina was added and the cells and alumina worked together for 5 mins. The paste was then pressed at 12 x 104 Pa to expel excess water/acetone The softened polystyrene coating behaved as an adhesive bonding the microparticles to entrap the yeast. The entrapped cells were used to ferment a glucose solution.
The method demonstrated by this example has been used successfully to form entrapment matrices using other inorganic microparticles and microspheres of organic polymers.

Claims

1. A process for immobilising cells which comprises the steps of mixing the cells with microparticles which are substantially insoluble in aαueous media, blending thoroughly to uniformly disperse the cells and microparticles then bonding the microparticles at points of contact to form a permanent porous matrix entrapping the cells within the cavities therein,
2. A process as claimed in claim 1 wherein the microparticles are spherical in shape.
3. A process as claimed in claim 1 wherein the microparticles are uniform in diameter.
4 A process as claimed in claim 1 wherein the diameter of the microparticles used is less than six times the smallest dimension of the cell.
5. A process as claimed in claim 1 wherein the microparticles are composed of inorganic oxides, hydroxides, carbonates or sulphates which are substantially insoluble in aqueous media.
6. A process as claimed in claim 1 wherein the microparticles are composed of silica, alumina, aluminium hydroxide, calcium carbonate or calcium sulphate.
7. A process as claimed in claim 1 wherein the microparticles are composed of a polymer.
8. A process as claimed in claim 1 wherein the microparticles are composed of polyacrylamide, polystyrene, polyvinyl chloride, polyvinyl acetate, dextran, cellulose or starch.
9. A process as claimed in claim 1 wherein the microparticles and cells are mixed and blended in a liquid medium compatible with the viability of the cells.
10. A process as claimed in claim 1 wherein the microparticles are pretreated with an adhesive.
11. A process as claimed in claim 1 wherein the microparticles and cells are mixed and blended in a medium which softens an adhesive applied to the microparticles.
12. A matrix containing immobilised cells prepared by the process as claimed in any preceding claim.
13. A matrix as claimed in claim 12 which has been treated with a polymer solution.
14. A matrix as claimed in claim 12 which has been treated with a solution of cellulose acetate, polystyrene, polyamide, or polyvinyl chloride.
15. A matrix as claimed in claim 12 which has a polymer coating deposited thereon.
16. A matrix as claimed in claim 15 which has a semi-permeable coating deposited thereon. PROCESS FOR CELL IMMOBILISATION
A process for immobilising cells in a structured matrix engineered to suit the cell, producing a strong porous support for the cells which has good mass transfer characteristics. Cells are entrapped in cavities between microparticles which are bonded at points of contact to construct the rigid matrix.
EP19860906378 1985-10-22 1986-10-21 Process for cell immobilisation Ceased EP0247077A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB858526095A GB8526095D0 (en) 1985-10-22 1985-10-22 Cell immobilisation
GB8526095 1985-10-22
GB868616881A GB8616881D0 (en) 1986-07-10 1986-07-10 Cell immobilisation
GB8616881 1986-07-10

Publications (1)

Publication Number Publication Date
EP0247077A1 true EP0247077A1 (en) 1987-12-02

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP19860906378 Ceased EP0247077A1 (en) 1985-10-22 1986-10-21 Process for cell immobilisation

Country Status (2)

Country Link
EP (1) EP0247077A1 (en)
WO (1) WO1987002704A1 (en)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8729889D0 (en) * 1987-12-22 1988-02-03 Unilever Plc Bio-catalysts support systems
GB9718415D0 (en) * 1997-08-29 1997-11-05 Smithkline Beecham Plc Formulation
GB9910499D0 (en) 1999-05-06 1999-07-07 Azur Env Ltd Assay reagent
CA2518148A1 (en) * 2003-03-04 2004-09-16 The Technology Development Company Ltd. Long acting injectable insulin composition and methods of making and using thereof
SE0600091L (en) 2006-01-18 2007-04-17 Bows Pharmaceuticals Ag Process for the preparation of a dextran matrix for controlled release of insulin
US8283153B2 (en) 2010-06-09 2012-10-09 Ford Global Technologies, Llc Mycelium structures containing nanocomposite materials and method
US8227224B2 (en) 2010-06-09 2012-07-24 Ford Global Technologies, Llc Method of making molded part comprising mycelium coupled to mechanical device
US8313939B2 (en) 2010-06-09 2012-11-20 Ford Global Technologies, Inc. Injection molded mycelium and method
US8298810B2 (en) 2010-06-09 2012-10-30 Ford Global Technologies, Llc Mycelium structure with self-attaching coverstock and method
US8227225B2 (en) 2010-06-09 2012-07-24 Ford Global Technologies, Llc Plasticized mycelium composite and method
US8227233B2 (en) 2010-06-09 2012-07-24 Ford Global Technologies, Llc Method of making foamed mycelium structure
US8298809B2 (en) 2010-06-09 2012-10-30 Ford Global Technologies, Llc Method of making a hardened elongate structure from mycelium
US20170106129A1 (en) * 2014-05-14 2017-04-20 1866402 Ontario Limited Bonded microsphere filter
CN115448348B (en) * 2022-08-29 2023-07-14 东华大学 Solid morphology control agent and preparation method and application thereof

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Publication number Priority date Publication date Assignee Title
GB1415301A (en) * 1971-11-18 1975-11-26 Unilever Ltd Enzyme-containing granule
DK146481C (en) * 1978-08-14 1984-03-26 Novo Industri As PROCEDURE FOR THE PREPARATION OF AN IMMOBILIZED ENZYME PRODUCT
DE2912827A1 (en) * 1979-03-30 1980-10-09 Basf Ag METHOD FOR IMMOBILIZING ENZYMATICALLY ACTIVE PREPARATIONS

Non-Patent Citations (1)

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Title
See references of WO8702704A1 *

Also Published As

Publication number Publication date
WO1987002704A1 (en) 1987-05-07

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