CA1269628A - Cell culture microcarrier, method for preparing same and use thereof for cultivating anchorage-dependent cells - Google Patents
Cell culture microcarrier, method for preparing same and use thereof for cultivating anchorage-dependent cellsInfo
- Publication number
- CA1269628A CA1269628A CA000472941A CA472941A CA1269628A CA 1269628 A CA1269628 A CA 1269628A CA 000472941 A CA000472941 A CA 000472941A CA 472941 A CA472941 A CA 472941A CA 1269628 A CA1269628 A CA 1269628A
- Authority
- CA
- Canada
- Prior art keywords
- microcarrier
- cell culture
- positively charged
- basic groups
- dependent cells
- 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.)
- Expired - Lifetime
Links
- 238000004113 cell culture Methods 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 18
- 230000001419 dependent effect Effects 0.000 title claims description 11
- 150000004676 glycans Chemical class 0.000 claims abstract description 15
- 239000005017 polysaccharide Substances 0.000 claims abstract description 15
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 13
- 125000004432 carbon atom Chemical group C* 0.000 claims abstract description 9
- 125000001183 hydrocarbyl group Chemical group 0.000 claims abstract 3
- 150000001450 anions Chemical class 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 3
- 150000004804 polysaccharides Polymers 0.000 claims description 2
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 claims 2
- 125000000816 ethylene group Chemical group [H]C([H])([*:1])C([H])([H])[*:2] 0.000 claims 1
- 230000003472 neutralizing effect Effects 0.000 claims 1
- 125000000217 alkyl group Chemical group 0.000 abstract description 5
- 125000003710 aryl alkyl group Chemical group 0.000 abstract description 4
- 125000003118 aryl group Chemical group 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 28
- 229920002307 Dextran Polymers 0.000 description 14
- 239000011324 bead Substances 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- YMDNODNLFSHHCV-UHFFFAOYSA-N 2-chloro-n,n-diethylethanamine Chemical compound CCN(CC)CCCl YMDNODNLFSHHCV-UHFFFAOYSA-N 0.000 description 5
- 229920005654 Sephadex Polymers 0.000 description 5
- 239000012507 Sephadex™ Substances 0.000 description 5
- 239000012091 fetal bovine serum Substances 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 231100000331 toxic Toxicity 0.000 description 5
- 230000002588 toxic effect Effects 0.000 description 5
- 231100000433 cytotoxic Toxicity 0.000 description 4
- 230000001472 cytotoxic effect Effects 0.000 description 4
- 239000007758 minimum essential medium Substances 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 230000003416 augmentation Effects 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- -1 diethylaminoethyl groups Chemical group 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 150000002430 hydrocarbons Chemical group 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 229960005419 nitrogen Drugs 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 241000867607 Chlorocebus sabaeus Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 229940095679 poly-dex Drugs 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000000954 titration curve Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- QLOKJRIVRGCVIM-UHFFFAOYSA-N 1-[(4-methylsulfanylphenyl)methyl]piperazine Chemical compound C1=CC(SC)=CC=C1CN1CCNCC1 QLOKJRIVRGCVIM-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 241000501458 Cultus Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000010934 O-alkylation reaction Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002152 alkylating effect Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 150000001449 anionic compounds Chemical class 0.000 description 1
- 230000003190 augmentative effect Effects 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 210000001840 diploid cell Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- HKIOYBQGHSTUDB-UHFFFAOYSA-N folpet Chemical group C1=CC=C2C(=O)N(SC(Cl)(Cl)Cl)C(=O)C2=C1 HKIOYBQGHSTUDB-UHFFFAOYSA-N 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 150000002891 organic anions Chemical class 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 125000000542 sulfonic acid group Chemical group 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 125000001302 tertiary amino group Chemical group 0.000 description 1
- 125000005425 toluyl group Chemical group 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
- C08B37/0021—Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
-
- 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
- C12N2533/70—Polysaccharides
Abstract
Cell Culture Microcarrier, Method for Preparing Same and Use Thereof for Cultivating Anchorage-Dependent Cells Abstract of the Disclosure A cell culture microcarrier having positively charged cross-linked polysaccharides and basic groups linked therewith wherein the basic groups have the formula (I)
Description
Cell Culture Microcarrier, Method for_Preparing Same an~ Use Thexeof for Cultivating Anchorage-Dependent Cells The present invention relates to a cell culture microcarrier having positively charged chemical portions comprising cross-linked polysaccharides and basic groups linked therewith, a method for preparing same, and -the use thereof for cultivating anchora~e-dependent cells in a microcarrier cul-ture.
BACKGROUND OF THE INVENTION
The fundamentals of maintaining and augmenting human and animal cells in cell cultures have been systematically developed during the last twenty years. Methods for cultivating cells have now been firmly established for the production of vaccine sub-stances, antibodies, interferon, enzymes, and hormones in many laboratories.
Primary cells and diploid cells require a solid substrate having a distinct surface charge for their growth. These cells are designated as "anchorage-dependent" cells since they are only able to grow if they can adhere to a carrier.
~0 Initially, glass in the form of Petri dishes, culture bot-tles, or roll-culture flasks was used as the carrier. However, there can be formed only a monolayer on the surfaces of the equipment so that the surface available per unit will be limited.
Thus, for such cell augmentation cultures on a commercial scale, thousands of roll-culture flasks have to be cleaned, sterilized, filled with nutrient medium, inoculated with cells, and harvested once the cell augmentation is finished. Since all of these steps have to be carried out under sterile conditions, they are very labor- and cost-intensive.
DISCUSSION OF THE PRIOR ART
By the introduction of the cell culture augmentation on microcarriers by A.L. van Wezel, Nature 216 (1967), p. 64, an improved technology has been developed that coul~ be suitable to eliminate the described problems. In this technique, microcarri-ers having an average particle size in the range from 100 to fn 300 ~ are suspended in a nutrient medium. In as far as the microcarriers have a suitable surface charge, the anchorage-dependent cells will adhere to the microcarriers and grow thereon. Two to 5 g/l (dry weight) of microcarrier provide a sur~ace in the nutrient medium of from 0.76 to 1.9 m2/l of cul-ture volume. In contrast, the largest roller flasks will only provide a surface of about 0.16 m2. Thus, 1 l of a culture containing microcarriers will be able to replace from 5 to 12 large roller flasks. However, on the commercial scale, microcar-riers so far have not yet gained the importance having been expected, since the microcarriers as so far developed still have a number of inherent drawbacks.
A.L. van Wezel originally used insoluble dextran beads substituted with diethylaminoethyl groups and commercially avail-able as DEAE Sephadex~A-50 ~Pharmacia AB, Sweden). However, in _i the course of the use thereof, cytotoxic and nutrient adsorption effects appeared, manifested by an initial death of cells and an unsatisfactory cell growth; cf. C.-B. Horng, W. Melimaus (Biotechnology and Bioengineering~ Vol. 17 (1975), pp. 715-732).
In the meantime, various improvements for microcarriers have been developed, which are capable of eliminating some of the original disadvantages. Thus, in the German Offenlegungsschrift 29 09 340 there has been described a process for pre-treating microcarriers for cell cultures wherein the beads are impregnated with fetal calf serum and heated in the serum at 75C to 90C for about 10 minutes.
Levine, Wong, Wang and Thilly experimented with microcarri-ers of various charge densities and concluded that DEAE dextran beads having a diameter of 150 ~m and a charge density of 2 meq/g ~ fr~O~e ~lar~ -2-~ C~W-19148 of the dry dextran matrix constitute the optimum characteristics for the adhesion of the cells and the growth thereof; cf~ D.F-Levine et al., Somatic Cell Genetics, Vol. 3 (1977), pp. 1~9-155;
U.S. Patent 4,189,534 and German Offenlegungsschri.ft ~7 49 989-~ licrocarriers that contain a charge density of only 1.5 to2.0 meq/g instead of 5.~ meq/g, in the meantime, have been mar-keted by the firm Pharmacia AB under the designation "Cytodex 1"
and by the firm Flow Labora~ories under the designation "Super~
,~ts beads". A survey of the properties and possible applications of these products and an introduction into th~ development of the microcarrier technology is presented by the company brochure "~licrocarrier Cell Culture, Principles & Methods", edition 1981, available from the firm Pharmacia AB, Uppsala, Sweden.
Unfortunately, there has been shown that even these improved microcarriers having a significantly reduced charge density, in practice, still display cytotoxic effects, particularly with sensitive cells.
The European Offenlegungsschrift (Published Unexamined Patent Application~ 0 066 726 premises that these cytotoxic ~o properties of the microcarriers based on DEAE dextran reside in the chemical structures of the microcarriers themselves. Thus, it has been known that DEAE microcarriers, in addition to the DEAE substituent comprising a tertiarily bonded nitrogen, also contain groups comprising a quaternarily bonded nitrogen, which latter groups are formed in the synthesis of the microcarriers by a further reaction with more chloroethyl-diethylamine, a reaction which never can be completely avoided. These so called "tandem groups" are supposed to have an alkylating action and to be toxic; cf. L. Ahlgren et al., "Polymeric Amines and Ammonium Salts", E.J. Goethals, Pergamon Press, pp. 293-294. In the com-pany brochure "Microcarrier Cell Culture, Principles & Methods"
by Pharmacia AB, at page 27, there has further been mentioned ~ J~ k -3-9 ~ ~ ~ CAW-]91~8 that, in the known preparation procedures for DE~ dextr~n micro-carriers, up to 35~ of -tandem groups are formed. In the product Cytodex 1, the tandem group content is reduced to about 15~.
In order to completely exclude the aforementioned cytotoxic effect, there have been described in European offenlegungsschrift 0 066 726 microcarriers that only contain quaternary amino groups. Such microcarriers have been marketed under the designa-*
tion "Cy~odex 2" by Pharmacia AB, Sweden. They have a chargecapacity of from 0.5 to 0.8 meq/g.
However, in spite of this further reduced charge capacity, these microcarriers in practice still have displayed disadvanta-geous ef~ects that are attributed to an adsorp~ion of components of the culture medium to the carrier~ Thus, in German Offen-legungsschrift 30 33 885, charge-free microcarriers have been described that have been coated with polypeptides such as collagen or gelatin. However, to these microcarriers there can only adhere cells that on the surface thereof contain structural elements that have a sufficient bonding affinity to the polypeptide layer on the microcarrier.
~o GENERAL DISCUSSION OF THE INVENTIOM
The present invention seeks to provide a cell culture microcarrier comprising positively charged portions oE
cross-linked polysaccharides and basic groups linked therewith, which do not have any of the aforementioned drawbacks. More spe-cifically, the present invention seeks to develop a microcarrier that may be employed even on a commercial scale for the cultivation of sensitive cells and, thus, substantially have no toxicity.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. l is a number of plots of percent of adhered cells over time for five microcarriers of the present invention and two ~ -~ * Trade Mark CAW~191~8 prior art microcarr.iers. The ma-tter i8 discussed in Example 3, infra.
Fig. 2 is a plot oE cell concentra~ion over time for t~o microcarriers of the present invention and one prior ark micro~
carrier; see Example 4r infra.
DETAIL~D DESCRIPTION OF THE I~lVENTION
In accordance with the present invention, there i5 provided `a polysaccharide with positively chargedlbasic groups of the formula (I) o (Z I )n wherein Z is an optionally substituted hydrocarbon chain having at least 2 carbon atoms, preferably a hydrocarbon chain of 2 to 5 carbon atoms, Rl and R2 are the same or diffe.rent and represent alkyl, ryl, or aralkyl group~, preferably Cl 4 alkyl, and n is at least 1 , pre~erably 1 to 4.
Cell culture microcarriers comprising such positively charged basic groups surprisingly have proven to be not toxic, although the art presumed that such groups would be particularly toxic and, therefore, if possible, should not be present at all or only in a small number on the cell culture microcarriers.
Unexpectedly, these positively charged basic groups may even be present in a large number, so that it is not necessary to li.mit the value of the meq/g of dry cross-linked polysaccharides. One reason for the surprisingly good properties of the cell cultu.re microc~rrier of the present invention appears to reside in that it is not the charge density which i5 of crucial i~portance, but the pKs value of the positively charged basic groups. While the pKs value of the basic yroups of DEAE Sephadex is about 9.2, the pKs values of the ce].l culture microcarrier according to the .' ~ CA~7-19148 invention are in the range of from 5 to 8. A preferred, and readily preparable, cell culture microcarrier of the invention is a polysaccharide containing positively charyed basic groups of formula (I) wherein Z is an ethylene yroup, R1 and R2 represent ethyl, and n is equal to 1; the pKs value is 6.2. Such a cell culture microcarrier is substituted virtually with tandem groups only.
In addition to the mentioned simplest case of tandem groups, ~ccording to the invention there may be also used such groups wherein Z contains 3, 4 or more carbon atoms, and wherein R1 and R~, in addition to the ethyl group, also represent methyl groups, propyl groups, isopropyl groups and butyl groups. Since the pKs values will still be in the range of from 5 to 8, R1 and R2 may also be aryl groups such as phenyl and toluyl groups and aralkyl groups such as, more specifically, benzyl groups. Since the pKs values are in the range of from 5 to 8, n may also be larger than 1, so that basic groups comprising 3 or 4 amino ~roups may also be employed.
As the negative counterions to the positively charged nitro-gen atoms, basically all physiologically compatible inorganic and organic anions may be used. Suitable are, more particularly, chloride, phosphate, sulfate, and acetate ions.
The pKs values of the basic substituents may be readily determined by titration. The titration curves show the ~nown point of inflexion at the pKs value.
The present invention further relates to a method of prepar-ing the new cell culture microcarrier. The preparation is accom-plished by the E~ se known reaction of a cross-linked polysaccharide with a compound of the formula (II) 1l Y _ Z - N (II) C~W-191~8 wherein Z is an op-tionally substitutecl hyclrocarbon chain having at least 2 carbon atoms, preferably a hydrocarbon chain of 2 to 5 carbon atoms, Rl and R2 are -the same or different and represent alkyl, aryl or aralkyl groups, preferably Cl 4 alkyl, and Y is a reactive group.
The reactive group, more speci~ically, may be chloxide, bromide, but also a sulfonic acid group etc., which i5 capa~le of ef~ect-ing an O-alkylation of the cross-linked polysaccharide.
According to the invention the reaction is carried out in the presence of a strong base in at leagt two steps ànd using at least a two times molar excess of compound (II). It is preferred that the aqueous alkaline suspension o~ the cross-linked poly-saccharide is poured into a suspension medium such as toluene, paraffins, water, and reacted with compound (II) while stirring.
Subsequently it is preferred that the positively charged basic groups are neutralized by physiologically compatible anions.
As the cross-linked polysaccharides, there may preferably be used the known cross-linked polydextrans. However, basically any ~o other swellable and cross-linked polysaccharide may also be used as the carrier of the positive basic groups.
The new cell culture microcarrier, a method for preparing same and~he use thereof are further illustrated by way of the following examples, although the present invention is not limited thereto.
Preparation of the microcarrier:
Dry cross-linked dextran beads (Polydex PL-50 or Sephadex G 50) having a water absorption of about 5 g/g and a particle size of from 80 to 100 ~m (13 g) are allowed to swell in distilled water overnight. After removal by suction oE the excess water the swellecl beads are admixed with 5.3 g oE a 43% by ~~ * Trade Mark -7-~, , ~ CAW~1914 weight sodium hydroxide solution~ The thus-prepared alkaline suspension of dextran beads is poured into 120 g of toluene pre-warmed to 50C. Then, 8 ml of chloroethyl-diethylamine are added to the reaction mixture with vigorous stirriny. After a reaction period of 3 hours at 50C, a further 10 ml of chloroethyl-diethylamine are added.
The reaction is completed after another 3 hours of stirring at 50C. The reacted dextran beads are separated from the re-action mixture by filtration. The microcarriers are purified by lo washing several times with alcohol and distilled water and ad-justing the pH to 5.0 using diluted hydrochloric acid.
De-swelling is eventually effected by repeated treatment of the beads with washing solutions having increasing alcohol concen-trations. The de-swelled beads are dried at 80C overnight.
The microcarriers prepared in accordance with the described method contain about 4.0% of nitrogen and have a charge capacity of 2.86 meq/g of the microcarrier and 4.66 meq/g of the dry untreated dextran, respectively.
The exchange capacity of the thus prepared microcarriers is ~o 2.4 meq/g of the microcarrier and 3.91 meq/g of the dry untreated cross-linked dextran, respectively.
For the determination of the exchange capacity, 1.0 g of the dried microcarriers is allowed to swell in distilled water, transferred to a small column, and washed with 0.1 N diluted hydrochloric acid several times. The removal of the non-bonded chloride ions is accomplished by rinsing the beads with 10 4 N
hydrochloric acid.
The bonded chloride ions are displaced by the addition of a sufficient amount of 10~ sodium sulfate solution and then de-termined by titrating the eluate with 0.1 N silver nitrate so-lution with potassium chromate as indicator.
The obtained result indicates the number of milliequivalents of C1 per gram of the microcarrier. The calculation of the ~2~ 2~
millie~uivalents of Cl per gram of the untreated dextran beads is made using the following formula:
Exchange capacity/g - dry untreate~ cross-linked polydextran =
meq Cl / g microcarrier 1, 000 x - -- - ------------- ---~
% N x 1,000 1,000 - 135 x 100 x 1~
The determination of the structure of the thus-bound nitro-gen compounds is effected by titration of 1 g of the dry micro-carrier in 20 ml of a 1 M potassium chloride solution after adjustment of the initial pH to 12 using 1 N hydrochloric acid.
The titration curve reveals, by the absence of a step in the pH
range of from 10 to 8.5, that tertiary amino groups are no longer present. The pKs value of the amino compound is be-tween 5 and 7.
Example 2 By way of variation of the used amounts of sodium hydroxide solu~ion and chloroethyldiethylamine, the following microcarriers No. 434, 416, 432, and 435 are prepared.
Microcarrier Polydex PL 50 NaOH Chloroethyldiethylamine No. g g (1) ml (2) ml 434 9.5 0.2751.0 1.25 416 4.0 0.22 0.8 1.0 432 13.0 2.1 7.8 9.5 421 13.0 2.2 8.0 10.0 435 9.5 3.7613.0 16.5 ~ %~ CAW-191~8 Substance ~ N A B C D
434 1.0 0.66 0.71 0.73 0-79 416 1.8 1.14 1.29 1.38 1.56 432 3.8 2.33 2.71 3.68 ~.27 421 4.0 2.4 2.86 3.91 4.66 435 408 3.0 3.43 5.sg 6.39 Sephadex A-50 4.2 2.7 3.0 4.54 5.04 Cytodex 1 1.8 1.2 1.29 1.45 1.56 10 Notes:
A: Exchange capacity per gram of microcarrier;
B: Charge capacity per gram of microcarrier;
C: Exchange capacity per gram of untreated cross-linked dextran;
D: Charge capacity per gram of untreated cross-linked dextran.
Example 3 Kinetics of adhesion of GMK (Green Monkey Kidney~ cells:
Microcarriers, prepared as in Examples 1 or 2, respectively, ~n each in a quantity of 5 mg/ml were incubated in Petri dishes for bacteriological use having a diameter of 6 cm with 5 ml of medium MEM (Minimum Essential Medium) and 8~ of FBS (Fetal Bovine Serum) at 37C for 1 hour.
After the addition of fresh GMK cells stripped in trypsin/versene so that a concentration of 2.6 x 105 cells/ml of medium was obtained, samples were taken every 15 minutes. The degree of adhesion can be determined by counting the non-bonded cells.
In comparison to the cell carrier materials according to the invention, the adhesion kinetics were also determined using the commercially available microcarriers based on dextran (Cytodex~) ~ CAW-191 having a charge density of 2.0 meq/g of neutral dextran matrix and using the anion exchanger Sephadex A-5 ~
The results as presented in Fig. 1 show that the new micro-carriers are not toxic even with a high charge capaci-ty, whereas ~ephadex A-50 displays the ~nown toxic effects after about 50 minutes.
Example 4 Growth of anchorage-dependent cells, exemplified by G~IX
cells on the microcarriers No. 416 and 421 having different charge densities.
Each of the dried microcarriers in a quantity of 0.3 g was allowed to swell in 20 ml of PBS and then sterilized at 115C
under 15 psi for 15 minutes.
The PBS is decanted and replaced by warm culture medium.
For example, MEM (Minimum Essential Medium) with an addition of 8~ of Fetal Bovine Serum (FBS) serves as the culture medium. The bead suspensions transferred into culture vessels, and preferably into special spinner flasks. The volume was replenished with the culture medium + 8% of FBS to 100 ml, and then gas was passed into the batch, and the temperature was adjusted. Inoculation was effected with a cell inoculum of 1.3 x 10 GMK (Green Monkey Kidney) cells originating from passage No. 127 of the Institut f~r Virologie of the University of Cologne, West Germany, and had been pre-cultivated in plastic flasks containing culture medium for 5 days.
After an initial static phase of adhesion, a stirring speed of 30 rpm was adjusted. The medium was changed every 48 hours.
The cell growth was monitored through a period of 170 hours by removing the cells from the carriers by trypsinating and counting them in accordance with a modified Sanfort et al. method (J.
Natl. Cancer Inst. II, 737 (1951)). For comparison, the growth on the known Cytodex 1 microcarriers was determined at the same 1269~ CAW-19148 time. ~he result is seen from Fig. 2. It is apparent fro~ Fig-
BACKGROUND OF THE INVENTION
The fundamentals of maintaining and augmenting human and animal cells in cell cultures have been systematically developed during the last twenty years. Methods for cultivating cells have now been firmly established for the production of vaccine sub-stances, antibodies, interferon, enzymes, and hormones in many laboratories.
Primary cells and diploid cells require a solid substrate having a distinct surface charge for their growth. These cells are designated as "anchorage-dependent" cells since they are only able to grow if they can adhere to a carrier.
~0 Initially, glass in the form of Petri dishes, culture bot-tles, or roll-culture flasks was used as the carrier. However, there can be formed only a monolayer on the surfaces of the equipment so that the surface available per unit will be limited.
Thus, for such cell augmentation cultures on a commercial scale, thousands of roll-culture flasks have to be cleaned, sterilized, filled with nutrient medium, inoculated with cells, and harvested once the cell augmentation is finished. Since all of these steps have to be carried out under sterile conditions, they are very labor- and cost-intensive.
DISCUSSION OF THE PRIOR ART
By the introduction of the cell culture augmentation on microcarriers by A.L. van Wezel, Nature 216 (1967), p. 64, an improved technology has been developed that coul~ be suitable to eliminate the described problems. In this technique, microcarri-ers having an average particle size in the range from 100 to fn 300 ~ are suspended in a nutrient medium. In as far as the microcarriers have a suitable surface charge, the anchorage-dependent cells will adhere to the microcarriers and grow thereon. Two to 5 g/l (dry weight) of microcarrier provide a sur~ace in the nutrient medium of from 0.76 to 1.9 m2/l of cul-ture volume. In contrast, the largest roller flasks will only provide a surface of about 0.16 m2. Thus, 1 l of a culture containing microcarriers will be able to replace from 5 to 12 large roller flasks. However, on the commercial scale, microcar-riers so far have not yet gained the importance having been expected, since the microcarriers as so far developed still have a number of inherent drawbacks.
A.L. van Wezel originally used insoluble dextran beads substituted with diethylaminoethyl groups and commercially avail-able as DEAE Sephadex~A-50 ~Pharmacia AB, Sweden). However, in _i the course of the use thereof, cytotoxic and nutrient adsorption effects appeared, manifested by an initial death of cells and an unsatisfactory cell growth; cf. C.-B. Horng, W. Melimaus (Biotechnology and Bioengineering~ Vol. 17 (1975), pp. 715-732).
In the meantime, various improvements for microcarriers have been developed, which are capable of eliminating some of the original disadvantages. Thus, in the German Offenlegungsschrift 29 09 340 there has been described a process for pre-treating microcarriers for cell cultures wherein the beads are impregnated with fetal calf serum and heated in the serum at 75C to 90C for about 10 minutes.
Levine, Wong, Wang and Thilly experimented with microcarri-ers of various charge densities and concluded that DEAE dextran beads having a diameter of 150 ~m and a charge density of 2 meq/g ~ fr~O~e ~lar~ -2-~ C~W-19148 of the dry dextran matrix constitute the optimum characteristics for the adhesion of the cells and the growth thereof; cf~ D.F-Levine et al., Somatic Cell Genetics, Vol. 3 (1977), pp. 1~9-155;
U.S. Patent 4,189,534 and German Offenlegungsschri.ft ~7 49 989-~ licrocarriers that contain a charge density of only 1.5 to2.0 meq/g instead of 5.~ meq/g, in the meantime, have been mar-keted by the firm Pharmacia AB under the designation "Cytodex 1"
and by the firm Flow Labora~ories under the designation "Super~
,~ts beads". A survey of the properties and possible applications of these products and an introduction into th~ development of the microcarrier technology is presented by the company brochure "~licrocarrier Cell Culture, Principles & Methods", edition 1981, available from the firm Pharmacia AB, Uppsala, Sweden.
Unfortunately, there has been shown that even these improved microcarriers having a significantly reduced charge density, in practice, still display cytotoxic effects, particularly with sensitive cells.
The European Offenlegungsschrift (Published Unexamined Patent Application~ 0 066 726 premises that these cytotoxic ~o properties of the microcarriers based on DEAE dextran reside in the chemical structures of the microcarriers themselves. Thus, it has been known that DEAE microcarriers, in addition to the DEAE substituent comprising a tertiarily bonded nitrogen, also contain groups comprising a quaternarily bonded nitrogen, which latter groups are formed in the synthesis of the microcarriers by a further reaction with more chloroethyl-diethylamine, a reaction which never can be completely avoided. These so called "tandem groups" are supposed to have an alkylating action and to be toxic; cf. L. Ahlgren et al., "Polymeric Amines and Ammonium Salts", E.J. Goethals, Pergamon Press, pp. 293-294. In the com-pany brochure "Microcarrier Cell Culture, Principles & Methods"
by Pharmacia AB, at page 27, there has further been mentioned ~ J~ k -3-9 ~ ~ ~ CAW-]91~8 that, in the known preparation procedures for DE~ dextr~n micro-carriers, up to 35~ of -tandem groups are formed. In the product Cytodex 1, the tandem group content is reduced to about 15~.
In order to completely exclude the aforementioned cytotoxic effect, there have been described in European offenlegungsschrift 0 066 726 microcarriers that only contain quaternary amino groups. Such microcarriers have been marketed under the designa-*
tion "Cy~odex 2" by Pharmacia AB, Sweden. They have a chargecapacity of from 0.5 to 0.8 meq/g.
However, in spite of this further reduced charge capacity, these microcarriers in practice still have displayed disadvanta-geous ef~ects that are attributed to an adsorp~ion of components of the culture medium to the carrier~ Thus, in German Offen-legungsschrift 30 33 885, charge-free microcarriers have been described that have been coated with polypeptides such as collagen or gelatin. However, to these microcarriers there can only adhere cells that on the surface thereof contain structural elements that have a sufficient bonding affinity to the polypeptide layer on the microcarrier.
~o GENERAL DISCUSSION OF THE INVENTIOM
The present invention seeks to provide a cell culture microcarrier comprising positively charged portions oE
cross-linked polysaccharides and basic groups linked therewith, which do not have any of the aforementioned drawbacks. More spe-cifically, the present invention seeks to develop a microcarrier that may be employed even on a commercial scale for the cultivation of sensitive cells and, thus, substantially have no toxicity.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. l is a number of plots of percent of adhered cells over time for five microcarriers of the present invention and two ~ -~ * Trade Mark CAW~191~8 prior art microcarr.iers. The ma-tter i8 discussed in Example 3, infra.
Fig. 2 is a plot oE cell concentra~ion over time for t~o microcarriers of the present invention and one prior ark micro~
carrier; see Example 4r infra.
DETAIL~D DESCRIPTION OF THE I~lVENTION
In accordance with the present invention, there i5 provided `a polysaccharide with positively chargedlbasic groups of the formula (I) o (Z I )n wherein Z is an optionally substituted hydrocarbon chain having at least 2 carbon atoms, preferably a hydrocarbon chain of 2 to 5 carbon atoms, Rl and R2 are the same or diffe.rent and represent alkyl, ryl, or aralkyl group~, preferably Cl 4 alkyl, and n is at least 1 , pre~erably 1 to 4.
Cell culture microcarriers comprising such positively charged basic groups surprisingly have proven to be not toxic, although the art presumed that such groups would be particularly toxic and, therefore, if possible, should not be present at all or only in a small number on the cell culture microcarriers.
Unexpectedly, these positively charged basic groups may even be present in a large number, so that it is not necessary to li.mit the value of the meq/g of dry cross-linked polysaccharides. One reason for the surprisingly good properties of the cell cultu.re microc~rrier of the present invention appears to reside in that it is not the charge density which i5 of crucial i~portance, but the pKs value of the positively charged basic groups. While the pKs value of the basic yroups of DEAE Sephadex is about 9.2, the pKs values of the ce].l culture microcarrier according to the .' ~ CA~7-19148 invention are in the range of from 5 to 8. A preferred, and readily preparable, cell culture microcarrier of the invention is a polysaccharide containing positively charyed basic groups of formula (I) wherein Z is an ethylene yroup, R1 and R2 represent ethyl, and n is equal to 1; the pKs value is 6.2. Such a cell culture microcarrier is substituted virtually with tandem groups only.
In addition to the mentioned simplest case of tandem groups, ~ccording to the invention there may be also used such groups wherein Z contains 3, 4 or more carbon atoms, and wherein R1 and R~, in addition to the ethyl group, also represent methyl groups, propyl groups, isopropyl groups and butyl groups. Since the pKs values will still be in the range of from 5 to 8, R1 and R2 may also be aryl groups such as phenyl and toluyl groups and aralkyl groups such as, more specifically, benzyl groups. Since the pKs values are in the range of from 5 to 8, n may also be larger than 1, so that basic groups comprising 3 or 4 amino ~roups may also be employed.
As the negative counterions to the positively charged nitro-gen atoms, basically all physiologically compatible inorganic and organic anions may be used. Suitable are, more particularly, chloride, phosphate, sulfate, and acetate ions.
The pKs values of the basic substituents may be readily determined by titration. The titration curves show the ~nown point of inflexion at the pKs value.
The present invention further relates to a method of prepar-ing the new cell culture microcarrier. The preparation is accom-plished by the E~ se known reaction of a cross-linked polysaccharide with a compound of the formula (II) 1l Y _ Z - N (II) C~W-191~8 wherein Z is an op-tionally substitutecl hyclrocarbon chain having at least 2 carbon atoms, preferably a hydrocarbon chain of 2 to 5 carbon atoms, Rl and R2 are -the same or different and represent alkyl, aryl or aralkyl groups, preferably Cl 4 alkyl, and Y is a reactive group.
The reactive group, more speci~ically, may be chloxide, bromide, but also a sulfonic acid group etc., which i5 capa~le of ef~ect-ing an O-alkylation of the cross-linked polysaccharide.
According to the invention the reaction is carried out in the presence of a strong base in at leagt two steps ànd using at least a two times molar excess of compound (II). It is preferred that the aqueous alkaline suspension o~ the cross-linked poly-saccharide is poured into a suspension medium such as toluene, paraffins, water, and reacted with compound (II) while stirring.
Subsequently it is preferred that the positively charged basic groups are neutralized by physiologically compatible anions.
As the cross-linked polysaccharides, there may preferably be used the known cross-linked polydextrans. However, basically any ~o other swellable and cross-linked polysaccharide may also be used as the carrier of the positive basic groups.
The new cell culture microcarrier, a method for preparing same and~he use thereof are further illustrated by way of the following examples, although the present invention is not limited thereto.
Preparation of the microcarrier:
Dry cross-linked dextran beads (Polydex PL-50 or Sephadex G 50) having a water absorption of about 5 g/g and a particle size of from 80 to 100 ~m (13 g) are allowed to swell in distilled water overnight. After removal by suction oE the excess water the swellecl beads are admixed with 5.3 g oE a 43% by ~~ * Trade Mark -7-~, , ~ CAW~1914 weight sodium hydroxide solution~ The thus-prepared alkaline suspension of dextran beads is poured into 120 g of toluene pre-warmed to 50C. Then, 8 ml of chloroethyl-diethylamine are added to the reaction mixture with vigorous stirriny. After a reaction period of 3 hours at 50C, a further 10 ml of chloroethyl-diethylamine are added.
The reaction is completed after another 3 hours of stirring at 50C. The reacted dextran beads are separated from the re-action mixture by filtration. The microcarriers are purified by lo washing several times with alcohol and distilled water and ad-justing the pH to 5.0 using diluted hydrochloric acid.
De-swelling is eventually effected by repeated treatment of the beads with washing solutions having increasing alcohol concen-trations. The de-swelled beads are dried at 80C overnight.
The microcarriers prepared in accordance with the described method contain about 4.0% of nitrogen and have a charge capacity of 2.86 meq/g of the microcarrier and 4.66 meq/g of the dry untreated dextran, respectively.
The exchange capacity of the thus prepared microcarriers is ~o 2.4 meq/g of the microcarrier and 3.91 meq/g of the dry untreated cross-linked dextran, respectively.
For the determination of the exchange capacity, 1.0 g of the dried microcarriers is allowed to swell in distilled water, transferred to a small column, and washed with 0.1 N diluted hydrochloric acid several times. The removal of the non-bonded chloride ions is accomplished by rinsing the beads with 10 4 N
hydrochloric acid.
The bonded chloride ions are displaced by the addition of a sufficient amount of 10~ sodium sulfate solution and then de-termined by titrating the eluate with 0.1 N silver nitrate so-lution with potassium chromate as indicator.
The obtained result indicates the number of milliequivalents of C1 per gram of the microcarrier. The calculation of the ~2~ 2~
millie~uivalents of Cl per gram of the untreated dextran beads is made using the following formula:
Exchange capacity/g - dry untreate~ cross-linked polydextran =
meq Cl / g microcarrier 1, 000 x - -- - ------------- ---~
% N x 1,000 1,000 - 135 x 100 x 1~
The determination of the structure of the thus-bound nitro-gen compounds is effected by titration of 1 g of the dry micro-carrier in 20 ml of a 1 M potassium chloride solution after adjustment of the initial pH to 12 using 1 N hydrochloric acid.
The titration curve reveals, by the absence of a step in the pH
range of from 10 to 8.5, that tertiary amino groups are no longer present. The pKs value of the amino compound is be-tween 5 and 7.
Example 2 By way of variation of the used amounts of sodium hydroxide solu~ion and chloroethyldiethylamine, the following microcarriers No. 434, 416, 432, and 435 are prepared.
Microcarrier Polydex PL 50 NaOH Chloroethyldiethylamine No. g g (1) ml (2) ml 434 9.5 0.2751.0 1.25 416 4.0 0.22 0.8 1.0 432 13.0 2.1 7.8 9.5 421 13.0 2.2 8.0 10.0 435 9.5 3.7613.0 16.5 ~ %~ CAW-191~8 Substance ~ N A B C D
434 1.0 0.66 0.71 0.73 0-79 416 1.8 1.14 1.29 1.38 1.56 432 3.8 2.33 2.71 3.68 ~.27 421 4.0 2.4 2.86 3.91 4.66 435 408 3.0 3.43 5.sg 6.39 Sephadex A-50 4.2 2.7 3.0 4.54 5.04 Cytodex 1 1.8 1.2 1.29 1.45 1.56 10 Notes:
A: Exchange capacity per gram of microcarrier;
B: Charge capacity per gram of microcarrier;
C: Exchange capacity per gram of untreated cross-linked dextran;
D: Charge capacity per gram of untreated cross-linked dextran.
Example 3 Kinetics of adhesion of GMK (Green Monkey Kidney~ cells:
Microcarriers, prepared as in Examples 1 or 2, respectively, ~n each in a quantity of 5 mg/ml were incubated in Petri dishes for bacteriological use having a diameter of 6 cm with 5 ml of medium MEM (Minimum Essential Medium) and 8~ of FBS (Fetal Bovine Serum) at 37C for 1 hour.
After the addition of fresh GMK cells stripped in trypsin/versene so that a concentration of 2.6 x 105 cells/ml of medium was obtained, samples were taken every 15 minutes. The degree of adhesion can be determined by counting the non-bonded cells.
In comparison to the cell carrier materials according to the invention, the adhesion kinetics were also determined using the commercially available microcarriers based on dextran (Cytodex~) ~ CAW-191 having a charge density of 2.0 meq/g of neutral dextran matrix and using the anion exchanger Sephadex A-5 ~
The results as presented in Fig. 1 show that the new micro-carriers are not toxic even with a high charge capaci-ty, whereas ~ephadex A-50 displays the ~nown toxic effects after about 50 minutes.
Example 4 Growth of anchorage-dependent cells, exemplified by G~IX
cells on the microcarriers No. 416 and 421 having different charge densities.
Each of the dried microcarriers in a quantity of 0.3 g was allowed to swell in 20 ml of PBS and then sterilized at 115C
under 15 psi for 15 minutes.
The PBS is decanted and replaced by warm culture medium.
For example, MEM (Minimum Essential Medium) with an addition of 8~ of Fetal Bovine Serum (FBS) serves as the culture medium. The bead suspensions transferred into culture vessels, and preferably into special spinner flasks. The volume was replenished with the culture medium + 8% of FBS to 100 ml, and then gas was passed into the batch, and the temperature was adjusted. Inoculation was effected with a cell inoculum of 1.3 x 10 GMK (Green Monkey Kidney) cells originating from passage No. 127 of the Institut f~r Virologie of the University of Cologne, West Germany, and had been pre-cultivated in plastic flasks containing culture medium for 5 days.
After an initial static phase of adhesion, a stirring speed of 30 rpm was adjusted. The medium was changed every 48 hours.
The cell growth was monitored through a period of 170 hours by removing the cells from the carriers by trypsinating and counting them in accordance with a modified Sanfort et al. method (J.
Natl. Cancer Inst. II, 737 (1951)). For comparison, the growth on the known Cytodex 1 microcarriers was determined at the same 1269~ CAW-19148 time. ~he result is seen from Fig. 2. It is apparent fro~ Fig-
2 that the growth on the new microcarriers is good even at a high charge capacity.
Claims (11)
1. A cell culture microcarrier having a pK of 5-8 and having positively charged chemical portions comprising cross-linked polysaccharides and positively charged basic groups linked therewith, said positively charged basic groups having the formula (I):
(I) wherein z is a hydrocarbon chain having 2 to 5 carbon atoms, R1 and R2 are the same or different and represent C1-4 alkyl, and n is 1 to 4.
(I) wherein z is a hydrocarbon chain having 2 to 5 carbon atoms, R1 and R2 are the same or different and represent C1-4 alkyl, and n is 1 to 4.
2. The cell culture microcarrier of claim 1, wherein Z is an ethylene group, R1 and R2 each represent an ethyl group, and n is 1.
3. The cell culture microcarrier of claim 1, wherein said positively charged basic groups have been neutralized by physiologically compatible anions.
4. The cell culture microcarrier of claim 2, wherein said positively charged basic groups have been neutralized by physiologically compatible anions.
5. The cell culture microcarrier of claim 1, wherein Z
contains 2, 3 or 4 carbon atoms.
contains 2, 3 or 4 carbon atoms.
6. The cell culture microcarrier of claim 5, wherein n is 1, 2 or 3.
7. The cell culture microcarrier of claim 5 or 6, wherein said positively charged basic groups have been neutralized by physiologically compatible anions.
8. A method for preparing a cell culture microcarrier having a pK of 5-8 and having positively charged chemical portions comprising cross-linked polysaccharides and basic groups linked therewith comprising pouring an aqueous alka-line suspension of a cross-linked polysaccharide into a suspension medium and reacting said cross-linked polysac-charide with an at least two times molar excess of a compound of the formula (II):
(II) wherein Z is a hydrocarbon chain having 2 to 5 carbon atoms, R1 and R2 are the same or different and represent C1-4 alkyl, and Y is a reactive group, while stirring, and subsequently neutralizing positively charged basic groups with physio-logically compatible anions.
(II) wherein Z is a hydrocarbon chain having 2 to 5 carbon atoms, R1 and R2 are the same or different and represent C1-4 alkyl, and Y is a reactive group, while stirring, and subsequently neutralizing positively charged basic groups with physio-logically compatible anions.
9. A process for cultivating anchorage-dependent cells in a microcarrier culture comprising cultivating anchorage-dependent cells with the cell culture microcarrier of claim 1, 2 or 3.
10. A process for cultivating anchorage-dependent cells in a microcarrier culture comprising cultivating anchorage-dependent cells with the cell culture microcarrier of claim 4.
11. A process for cultivating anchorage-dependent cells in a microcarrier culture comprising cultivating anchorage-dependent cells with the cell culture microcarrier of claim 5 or 6.
Applications Claiming Priority (2)
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DE19843402927 DE3402927A1 (en) | 1984-01-28 | 1984-01-28 | CELL CULTURE MICROCARRIERS, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE FOR GROWING ANCHOR-DEPENDENT CELLS |
DEP3402927.3 | 1984-01-28 |
Publications (1)
Publication Number | Publication Date |
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CA1269628A true CA1269628A (en) | 1990-05-29 |
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CA000472941A Expired - Lifetime CA1269628A (en) | 1984-01-28 | 1985-01-25 | Cell culture microcarrier, method for preparing same and use thereof for cultivating anchorage-dependent cells |
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US (2) | US4824946A (en) |
EP (1) | EP0150796B1 (en) |
JP (1) | JPS60237991A (en) |
AT (1) | ATE46717T1 (en) |
CA (1) | CA1269628A (en) |
DE (2) | DE3402927A1 (en) |
DK (1) | DK37985A (en) |
ES (1) | ES8602039A1 (en) |
IE (1) | IE850175L (en) |
PT (1) | PT79884B (en) |
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JPS6410979A (en) * | 1987-07-03 | 1989-01-13 | Mitsubishi Chem Ind | Microcarrier for cultivating cell |
WO1989012070A1 (en) * | 1988-06-03 | 1989-12-14 | Italfarmaco S.P.A. | Glycosaminoglycan salts, processes for the preparation thereof and pharmaceutical compositions containing them |
US5227481A (en) * | 1989-07-07 | 1993-07-13 | National Starch And Chemical Investment Holding Corporation | Cationic polysaccharides and reagents for their preparation |
US5349089A (en) * | 1989-07-07 | 1994-09-20 | National Starch And Chemical Investment Holding Corporation | Reagent for preparing polycationic polysaccharides |
ATE175242T1 (en) * | 1991-02-28 | 1999-01-15 | Anticancer Inc | A TISSUE CULTURE PROCEDURE FOR SKIN IN A NATURAL STATE |
DE19954357C2 (en) * | 1999-11-11 | 2002-09-05 | Cosmedica Consulting Gmbh | Composition containing heteropolysaccharide and pearl-shaped cross-linked polysaccharide, and their use for medical / cosmetic purposes |
DE10307925A1 (en) * | 2002-02-25 | 2003-09-04 | Pentax Corp | Cell culture carrier and cell culture method |
US20080020049A1 (en) * | 2005-02-25 | 2008-01-24 | Andrew Darling | Super-sparger microcarrier beads and precision extrusion deposited poly-epsilon-caprolactone structures for biological applications |
US20090047260A1 (en) * | 2007-08-17 | 2009-02-19 | Wake Forest University Health Sciences | Keratin biomaterials for cell culture and methods of use |
DK3046628T3 (en) | 2013-09-19 | 2020-02-10 | Univ Leland Stanford Junior | Methods and compositions for preparing hepatocyte-like cells |
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US3910819A (en) * | 1974-02-19 | 1975-10-07 | California Inst Of Techn | Treatment of surfaces to stimulate biological cell adhesion and growth |
JPS6026121B2 (en) * | 1977-02-28 | 1985-06-21 | 名糖産業株式会社 | New dextran derivatives and their production method |
FR2400063A1 (en) * | 1977-08-08 | 1979-03-09 | Pasteur Institut | PROCESS FOR OBTAINING SUPPORTS FOR CELLULAR CULTURES AND SUPPORTS OBTAINED |
US4293654A (en) * | 1977-10-17 | 1981-10-06 | Massachusetts Institute Of Technology | Cell culture microcarriers |
SE445116B (en) * | 1979-09-12 | 1986-06-02 | Pharmacia Fine Chemicals Ab | MAKE CULTURE CELLS ON MICROBATORS WITH FIBRONECTINE LAYERS |
US4335215A (en) * | 1980-08-27 | 1982-06-15 | Monsanto Company | Method of growing anchorage-dependent cells |
US4415668A (en) * | 1981-04-20 | 1983-11-15 | President And Fellows Of Harvard College | Cell culture |
SE8103137L (en) * | 1981-05-19 | 1982-11-20 | Pharmacia Ab | POLYMER WITH QUARTER AMINOGRUPS |
SE8103138L (en) * | 1981-05-19 | 1982-11-20 | Pharmacia Fine Chemicals Ab | MICROBATCHERS FOR CULTURE OF ANCHORING-DEPENDENT CELLS |
IL74259A0 (en) * | 1984-02-06 | 1985-05-31 | Surface Concepts Pty Ltd | Improved method for cell culture |
SE454518B (en) * | 1984-05-21 | 1988-05-09 | Statens Bakteriologiska Lab | PROCEDURE FOR CULTURING DIPLOID CELLS IN THE PRESENTATION OF CELLULOSA FIBERS |
US4661407A (en) * | 1985-01-07 | 1987-04-28 | Kms Fusion, Inc. | Glass-surface microcarrier for anchorage-dependent cell cultivation |
JPS63209582A (en) * | 1987-02-27 | 1988-08-31 | Teijin Ltd | Cultivation of attaching animal cell |
JPS63237782A (en) * | 1987-03-27 | 1988-10-04 | Teijin Ltd | Culture of anchorage dependent animal cell |
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1984
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1985
- 1985-01-14 US US06/691,006 patent/US4824946A/en not_active Expired - Fee Related
- 1985-01-22 EP EP85100611A patent/EP0150796B1/en not_active Expired
- 1985-01-22 AT AT85100611T patent/ATE46717T1/en not_active IP Right Cessation
- 1985-01-22 DE DE8585100611T patent/DE3573273D1/en not_active Expired
- 1985-01-25 JP JP60013387A patent/JPS60237991A/en active Pending
- 1985-01-25 CA CA000472941A patent/CA1269628A/en not_active Expired - Lifetime
- 1985-01-25 IE IE850175A patent/IE850175L/en unknown
- 1985-01-25 ES ES539867A patent/ES8602039A1/en not_active Expired
- 1985-01-25 PT PT79884A patent/PT79884B/en unknown
- 1985-01-28 DK DK37985A patent/DK37985A/en not_active Application Discontinuation
-
1989
- 1989-01-04 US US07/293,197 patent/US4910142A/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
DK37985A (en) | 1985-07-29 |
PT79884A (en) | 1985-02-01 |
ES539867A0 (en) | 1985-11-16 |
DE3402927C2 (en) | 1989-03-09 |
US4910142A (en) | 1990-03-20 |
JPS60237991A (en) | 1985-11-26 |
EP0150796A2 (en) | 1985-08-07 |
EP0150796B1 (en) | 1989-09-27 |
IE850175L (en) | 1985-07-28 |
ES8602039A1 (en) | 1985-11-16 |
ATE46717T1 (en) | 1989-10-15 |
PT79884B (en) | 1986-10-23 |
US4824946A (en) | 1989-04-25 |
DE3573273D1 (en) | 1989-11-02 |
DK37985D0 (en) | 1985-01-28 |
DE3402927A1 (en) | 1985-08-08 |
EP0150796A3 (en) | 1988-01-07 |
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