EP0247077A1 - Procede pour immobiliser des cellules - Google Patents

Procede pour immobiliser des cellules

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)
English (en)
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/fr
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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Dispersion Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

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.
EP19860906378 1985-10-22 1986-10-21 Procede pour immobiliser des cellules Ceased EP0247077A1 (fr)

Applications Claiming Priority (4)

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

Publications (1)

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

Family

ID=26289918

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19860906378 Ceased EP0247077A1 (fr) 1985-10-22 1986-10-21 Procede pour immobiliser des cellules

Country Status (2)

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

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
EA200501422A1 (ru) 2003-03-04 2006-04-28 Дзе Текнолоджи Девелопмент Компани Лтд. Длительнодействующая инъецируемая композиция инсулина и способы её изготовления и применения
SE0600091L (sv) 2006-01-18 2007-04-17 Bows Pharmaceuticals Ag Förfarande för framställning av en dextranmatris för kontrollerad frisättning av insulin
US8298809B2 (en) 2010-06-09 2012-10-30 Ford Global Technologies, Llc Method of making a hardened elongate structure from mycelium
US8227225B2 (en) 2010-06-09 2012-07-24 Ford Global Technologies, Llc Plasticized mycelium composite 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
US8283153B2 (en) 2010-06-09 2012-10-09 Ford Global Technologies, Llc Mycelium structures containing nanocomposite materials and method
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
US8227233B2 (en) 2010-06-09 2012-07-24 Ford Global Technologies, Llc Method of making foamed mycelium structure
WO2015172232A1 (fr) * 2014-05-14 2015-11-19 1866402 Ontario Limited Filtre en microsphères liés
CN115448348B (zh) * 2022-08-29 2023-07-14 东华大学 一种固体型形貌控制剂及其制备方法和应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1415301A (en) * 1971-11-18 1975-11-26 Unilever Ltd Enzyme-containing granule
DK146481C (da) * 1978-08-14 1984-03-26 Novo Industri As Fremgangsmaade til fremstilling af et immobiliseret enzymprodukt
DE2912827A1 (de) * 1979-03-30 1980-10-09 Basf Ag Verfahren zur immobilisierung von enzymatisch aktiven praeparaten

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8702704A1 *

Also Published As

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

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