WO1990013625A1 - Procede de preparation de microporteurs a base de gelatine - Google Patents

Procede de preparation de microporteurs a base de gelatine Download PDF

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Publication number
WO1990013625A1
WO1990013625A1 PCT/DK1990/000112 DK9000112W WO9013625A1 WO 1990013625 A1 WO1990013625 A1 WO 1990013625A1 DK 9000112 W DK9000112 W DK 9000112W WO 9013625 A1 WO9013625 A1 WO 9013625A1
Authority
WO
WIPO (PCT)
Prior art keywords
gelatin
particles
coated
particulate material
microcarrier
Prior art date
Application number
PCT/DK1990/000112
Other languages
English (en)
Inventor
Kim Jacobsen
Original Assignee
Danochemo A/S
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
Application filed by Danochemo A/S filed Critical Danochemo A/S
Publication of WO1990013625A1 publication Critical patent/WO1990013625A1/fr

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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using microcarriers
    • 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
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • This invention relates to a method of preparing gelatin microcar ⁇ riers for the cultivation of cells.
  • a microcarrier is a solid particle capable of supporting the growth of anchorage-dependent cells contained in a liquid culture medium.
  • Microcarriers are typically spherical particles having a diameter of 50-500 ⁇ m and preferably 100-400 ⁇ m. Microcarriers may have a porous structure or a dense surface with dents.
  • Cultivation methods based on the use of microcarriers are considered to be some of the most suitable methods for large scale production of cell products.
  • the large surface area per weight unit the possibility of optimizing the microcarrier surface to adapt it to different types of cells, the relative simple up-scaling procedures which are required in microcarrier-based cultivations, the fact that the microcarrier based production technique is similar to ordinary fermentation techniques and can be effected in only slightly odi- fied fermentors are factors which strongly favour microcarrier based production methods.
  • the most critical problem involved in microcarrier based large scale cell production in conventional fermentors is concerned with the stirring of the culture medium in order to continuously expose the cells to fresh culture medium, since the cells are sensitive to the mechanical treatment of the medium.
  • European patent publication No. 066.726 discloses the use of a cross-linked dextran microcarrier, which has been modified by reaction with a tertiary ammonium compound to provide the micro- carrier particles with an outer layer of quaternary amino groups so as to obtain a desired surface charge.
  • an aqueous solution of a colloid e.g. gelatin, pectin, dextran, agarose or gum arabic, and optionally a sugar is emulsified in a liquid which is immiscible with water such as peanut oil, castor oil, and mineral oil or an organic solvent.
  • a colloid e.g. gelatin, pectin, dextran, agarose or gum arabic
  • a sugar is emulsified in a liquid which is immiscible with water such as peanut oil, castor oil, and mineral oil or an organic solvent.
  • the emulsion thus formed is then cooled so as to cause the colloid to form gel par ⁇ ticles.
  • the particles formed are then separated and dried by well known techniques.
  • the above mentioned method suffers form the drawback that the microcarrier particles are not free-flowing but tend to form agglomerates.
  • gelatin particles thus formed are free-flowing but they have been found unsuitable for use as microcarriers primarily because the coating on the gelatin particles prevent the cells to be cultivated from contacting the gelatin surface.
  • the object of the invention is to prepare a free-flowing gelatin microcarrier.
  • a further object of the invention is to provide non-coated gelatin microcarrier particles.
  • a still further object of the invention is to provide gelatin microcarrier particles having a surface which is suitable as a support for anchorage-dependent cells.
  • the method of the invention comprises the steps of atomizing an aqueous gelatin solution in a stream of air while introducing into the atomizing zone a dry particulate material to form gelatin particles coated with said dry particulate material, recovering the coated gelatin particles and treating the coated gelatin particles with a medium capable of removing the coating therefrom without substantially decomposing the gelatin particles.
  • the invention is based on the discovery that by providing gelatin particles formed by atomizing a gelatin solution with a coating of a solid particulate material, a build up of material on the walls of the atomizing chamber can be avoided and that the coating on the gelatine particles can be removed so as to form non-coated gelatin particles which are excellently suitable as a substrate for ancho ⁇ rage-dependent cells. Thus it has been found that such cells adhere well and uniformly to the surface of such non-coated gelatin par ⁇ ticles.
  • the gelatin solution is preferably atomized by means of an atomizer or an atomizing wheel mounted for rotation on a spray drying apparatus.
  • the temperature of the gelatin solution is preferably between 20 and 90°C, and the viscosity between 50 and 200 cps.
  • the temperature of the stream of air is preferably between 5 and
  • the dry particulate material such as a powder, is introduced into the atomizing zone so as to bring the solid particulate material in direct contact with the droplets shortly after their formation.
  • the dry particulate material is preferably a powder such as calcium carbonate, dicalcium phosphate, tricalcium phosphate, calcium lactate or magnesium carbonate.
  • a particularly preferred group of particulate materials are compounds which are soluble in acidic aqueous solutions, because gelatin is relatively insoluble in such solutions.
  • starch such as corn starch, rice starch, and wheat starch
  • starch derivatives which are enzymatically degradable.
  • the dry particulate material is preferably used in an amount of from 5 to 50% by weight based on the weight of the gelatin particles.
  • the coated gelatin particles are preferably introduced into a fluid-bed and are dried therein in a conventional way.
  • the tempera ⁇ ture of the fluid-bed is preferably within the range of from 10 to 60°C.
  • the removal of the coating on the gelatin particles is preferably effected by treating the coated particles with a liquid medium capable of dissolving or degrading the coating.
  • the coating material is preferably soluble in acidic solutions and in that case the coated gelatin particles are preferably treated with a solution of a mineral acid such as hydrochloric acid.
  • the particles are preferably treated with an aqueous medium containing a starch degrading enzyme, such as ⁇ -amylase.
  • a starch degrading enzyme such as ⁇ -amylase
  • the gelatin material may be cross-linked either in coated or non- coated state by chemical reaction with e.g. an aldehyde such as formaldehyde or glutaraldehyde or by heating to an elevated tempe- rature e.g. a temperature of from 150 to 200°C for a period of from 30 minutes to 3 hours.
  • an aldehyde such as formaldehyde or glutaraldehyde
  • an elevated tempe- rature e.g. a temperature of from 150 to 200°C for a period of from 30 minutes to 3 hours.
  • the cross-linking may be effected immediately following the forma ⁇ tion of the coated gelatin particles or prior to or subsequent to the removal of the coating from the particles.
  • the removal of the coating may be a step in the conventional pretreatment of a microcarrier before inoculation with cells.
  • microcarrier is ordinarily subjected to a series of washes with different buffers systems, media, etc.
  • the particles thus obtained having a coat of tricalcium phosphate (20-40% w/w) are suspended in 5 1 of water, whereafter 7 1 of IN hydrochloric acid are added in order to dissolve the tricalcium phosphate.
  • the naked gelatine particles thus formed are washed with water until all hydrochloric acid and salts have been removed. Subsequently the particles are filtered off and dried in a fluid- bed.
  • a starch degrading enzyme (Termanyl 120L, N0V0 Industri A/S) is added so as to dissolve the corn starch.
  • the naked gelatin particles thus formed are washed with water until all enzyme and starch residues have been removed and the particles are then filtered off and dried in a fluid-bed.
  • microcarrier was washed with 200 ml PBS buffer, separated by decanting, and re-slurried in 100 ml PBS buffer. Subsequently the microcarrier was sterilized and autoclaved at 121°C for 20 min.
  • a kidney cell line was introduced into 25 ml Erlenmayer flasks to obtain a cell density of 4,4 • 10 cells/ml together with the pretreated microcarrier or a well known Sephadex microcarrier (Cytodex 3 (Pharmacia)) (microcarrier density: 0,1 g/20 ml) and a DME/HAM culture medium.
  • the total volume of the contents of the flasks was 7.5 ml.
  • the flasks were shaken in a water bath at a temperature of 35°C and at 65 rpm.
  • the number of adhering cells will appear from the following table.
  • the adherence of the cells to the microcarrier according to the invention is superior to the adherence of the cells to the known microcarrier.

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

Abstract

Procédé de préparation de microporteurs à base de gélatine servant à la culture de cellules consiste à atomiser une solution de gélatine aqueuse et en même temps à ajouter une matière anti-bouchage particulaire et sèche afin de former des particules de gélatine enrobées, ainsi qu'à traiter les particules enrobées avec un milieu capable d'enlever la couche des particules.
PCT/DK1990/000112 1989-05-01 1990-05-01 Procede de preparation de microporteurs a base de gelatine WO1990013625A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK210989A DK210989D0 (da) 1989-05-01 1989-05-01 Microcarriers fremstillet ved sprayteknik
DK2109/89 1989-05-01

Publications (1)

Publication Number Publication Date
WO1990013625A1 true WO1990013625A1 (fr) 1990-11-15

Family

ID=8110129

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK1990/000112 WO1990013625A1 (fr) 1989-05-01 1990-05-01 Procede de preparation de microporteurs a base de gelatine

Country Status (3)

Country Link
AU (1) AU5654090A (fr)
DK (1) DK210989D0 (fr)
WO (1) WO1990013625A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012510A1 (fr) * 1994-10-25 1996-05-02 Boehringer Mannheim Gmbh Biomateriau contenant des cellules epitheliales et son utilisation en tant que materiau de greffe
US6224629B1 (en) * 1998-12-09 2001-05-01 Purzer Pharmaceuticals Co. Ltd. Bone substitute composition and process of preparation thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1392135A (en) * 1972-03-24 1975-04-30 Hoechst Ag Vinyl acetate/ethylene polymer powders
WO1982000660A1 (fr) * 1980-08-20 1982-03-04 Mosbach K Immobilisation de cellules animales
EP0239648A1 (fr) * 1985-10-03 1987-10-07 Nauchno-Proizvodstvennoe Obiedinenie "Biolar" Procede d'obtention de micro-porteurs pour la culture de cellules, et micro-porteurs obtenus par ce procede

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1392135A (en) * 1972-03-24 1975-04-30 Hoechst Ag Vinyl acetate/ethylene polymer powders
WO1982000660A1 (fr) * 1980-08-20 1982-03-04 Mosbach K Immobilisation de cellules animales
EP0239648A1 (fr) * 1985-10-03 1987-10-07 Nauchno-Proizvodstvennoe Obiedinenie "Biolar" Procede d'obtention de micro-porteurs pour la culture de cellules, et micro-porteurs obtenus par ce procede

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 351, World Patent Index 81-90, Dialog accession no. 89-209258/29, (LION CORP): "Improving fluidity of powdered carotene compsn. - by covering with impalpable powder of a least one of calcium and magnesium carbonate(s)"; & JP,A,01 144 953, 07-06-1989, 8929. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012510A1 (fr) * 1994-10-25 1996-05-02 Boehringer Mannheim Gmbh Biomateriau contenant des cellules epitheliales et son utilisation en tant que materiau de greffe
US6224629B1 (en) * 1998-12-09 2001-05-01 Purzer Pharmaceuticals Co. Ltd. Bone substitute composition and process of preparation thereof

Also Published As

Publication number Publication date
AU5654090A (en) 1990-11-29
DK210989D0 (da) 1989-05-01

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