CA2098255A1 - Serum-free cell culture medium - Google Patents
Serum-free cell culture mediumInfo
- Publication number
- CA2098255A1 CA2098255A1 CA 2098255 CA2098255A CA2098255A1 CA 2098255 A1 CA2098255 A1 CA 2098255A1 CA 2098255 CA2098255 CA 2098255 CA 2098255 A CA2098255 A CA 2098255A CA 2098255 A1 CA2098255 A1 CA 2098255A1
- Authority
- CA
- Canada
- Prior art keywords
- medium
- added
- reached
- nutrient medium
- amount
- 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.)
- Abandoned
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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
-
- 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
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0056—Xeno-free medium
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
BEHRINGWERKE AKTIENGESELLSCHAFT 92/B 015 - Ma 930 Auslandstext Abstract of the disclosure Serum-free cell culture medium The invention relates to a nutrient medium for growing cells, which contains amino acids, vitamins, cofactors and inorganic salts but is free of animal proteins.
Description
2~1 9 ~
BEHRI~GWERRE ARTIENGE~ LLSCl~AFr 92/B 015 - Ma 930 Serum-freQ cell culture medium The invention relates to a nutrient medium for growing cells, which contains amino acids, vitamins, cofactors and inorganic salts but is free of animal proteins.
The production of modern drugs on a biotechnological basis is dependent in many cases on complicated eukary-otic cell culture production systems because many glyco-proteins cannot be produced in functionally active form in simple prokaryotes. These complex eukaryotic cell culture systems require nutrient media which, besides defined salts, vitamins, cofactors and amino acids (basal medium), contain a mixture of high molecular weight proteins or lipids, especially the costly fetal bovine serum (FBS). These high molecular weight constituents, which are required in large number and high concentration in the nutrient medium but are poorly defined and originate, for example, from fetal bovine serum, must after the fermenter production phase be separated by elaborate puri.fication processes from the drug which is to be produced. It is furthermore neces~ary to ensure that the FBS used contains no pathogenic agents.
It would be desirable to have a low-cost nutrient medium which makes it possible to grow a wide range of eukary-otic cell lines just as well as c09tly FB5-containing nutrient medium and which contains no animal foreign proteins.
It has been found, surpri~ingly, that it is possible to produce a defined, rea onably priced nutrient medium which permits the permanent growth of a large number of eukaryotic cell lines, comparably as wPll as FBS-contain-ing nutrient medium. It contains no animal proteins but, apart from amino acids, vitamins, cofactors and usual 20~'32~
inorganic salts, contains human transferrin, human insulin, ethanolamine and sodium selenite.
The invention relates to a nutrient medium for cell cultures containing amino acids, vitamins, cofactors and usual inorganic salts, which contains human transferrin, human insulin, ethanolamine and sodium selenite.
Clinically approved transferrin and insulin are preferably used.
It is possible to employ as medium which contains amino acids, vitamins, cofactors and inorganic salts ~basal medium) one of the media known in the prior art.
Basal media of this type are known, for example MEM alpha medium, DMEM/F 12 (1:1) medium, DME~ (Dulbecco's modified Eagle medium), William's medium E, Ham's F12 medium~
Other basal media can be used if sodium pyruvate is added in a concentration of 25-500, preferably 110 ~ g/l.
Examples of 1:hese basal media are: RPMI 1640 medium, CMRL 1066 medium, BME basal medium, Waymouth's medium, NCTC 135 medium, MEM (minimum essential medium).
Insulin and transferrin are added to one of these basa]
media so that the concentration reaches 6-20, preferably 8-12 mg/l insulin and 20-50, preferahly 30-40 mg/l transferrin in the medium.
Ethanolamine and sodium selenite are added to the basal medium so that the concentration reaches 1.3-20, prefer-ably 8-14 mg/l ethanolamine and 2-20, preferably 6-10 ~g/l sodium selenite in the medium.
L-Glutamine is added to the medium before use so that the concentration of this ~mino acid reaches 140-1200 mg/l, preferably 600 mg/l.
2~9~25~
BEHRI~GWERRE ARTIENGE~ LLSCl~AFr 92/B 015 - Ma 930 Serum-freQ cell culture medium The invention relates to a nutrient medium for growing cells, which contains amino acids, vitamins, cofactors and inorganic salts but is free of animal proteins.
The production of modern drugs on a biotechnological basis is dependent in many cases on complicated eukary-otic cell culture production systems because many glyco-proteins cannot be produced in functionally active form in simple prokaryotes. These complex eukaryotic cell culture systems require nutrient media which, besides defined salts, vitamins, cofactors and amino acids (basal medium), contain a mixture of high molecular weight proteins or lipids, especially the costly fetal bovine serum (FBS). These high molecular weight constituents, which are required in large number and high concentration in the nutrient medium but are poorly defined and originate, for example, from fetal bovine serum, must after the fermenter production phase be separated by elaborate puri.fication processes from the drug which is to be produced. It is furthermore neces~ary to ensure that the FBS used contains no pathogenic agents.
It would be desirable to have a low-cost nutrient medium which makes it possible to grow a wide range of eukary-otic cell lines just as well as c09tly FB5-containing nutrient medium and which contains no animal foreign proteins.
It has been found, surpri~ingly, that it is possible to produce a defined, rea onably priced nutrient medium which permits the permanent growth of a large number of eukaryotic cell lines, comparably as wPll as FBS-contain-ing nutrient medium. It contains no animal proteins but, apart from amino acids, vitamins, cofactors and usual 20~'32~
inorganic salts, contains human transferrin, human insulin, ethanolamine and sodium selenite.
The invention relates to a nutrient medium for cell cultures containing amino acids, vitamins, cofactors and usual inorganic salts, which contains human transferrin, human insulin, ethanolamine and sodium selenite.
Clinically approved transferrin and insulin are preferably used.
It is possible to employ as medium which contains amino acids, vitamins, cofactors and inorganic salts ~basal medium) one of the media known in the prior art.
Basal media of this type are known, for example MEM alpha medium, DMEM/F 12 (1:1) medium, DME~ (Dulbecco's modified Eagle medium), William's medium E, Ham's F12 medium~
Other basal media can be used if sodium pyruvate is added in a concentration of 25-500, preferably 110 ~ g/l.
Examples of 1:hese basal media are: RPMI 1640 medium, CMRL 1066 medium, BME basal medium, Waymouth's medium, NCTC 135 medium, MEM (minimum essential medium).
Insulin and transferrin are added to one of these basa]
media so that the concentration reaches 6-20, preferably 8-12 mg/l insulin and 20-50, preferahly 30-40 mg/l transferrin in the medium.
Ethanolamine and sodium selenite are added to the basal medium so that the concentration reaches 1.3-20, prefer-ably 8-14 mg/l ethanolamine and 2-20, preferably 6-10 ~g/l sodium selenite in the medium.
L-Glutamine is added to the medium before use so that the concentration of this ~mino acid reaches 140-1200 mg/l, preferably 600 mg/l.
2~9~25~
~xample:
A medium with the following composition was prepared. The amounts (in gram) relate to 200 liters of medium.
Basal medium components:
NaCl 1 280.00 KCl 80.00 MgSO4 7 H2O
NaH2PO4 H2O 25.00 CaCl2 2 H2O 52.80 Fe~NO3)3 9 H2O 0.02 Phenol red 3.70 Glucose H20 Sodium pyruvate 22.10 L-Alanine 7.14 L-Arginine HCl 16.80 L-Asparagine H2O 13.66 L-Aspartic acid 10.63 L-Cysteine 9.60 L-Glutamic acid 11.83 Glycine 6.02 L-Hi~tidine HCl H2O 8.40 L-Isoleucine 21.00 L-Leuclne 21.00 L-Ly~ine HCl 29.20 L-Methionine 6.00 L-Phenylalanine 13.20 L-Proline 9.22 L-Serine 8.42 L-Threonine 19.00 L-Tryptophan 3.20 L-Tyrosine 14.40 L-Valine 18.80 Calcium pantothenate 0.80 Choline chloride 0.80 Folic acid 0.80 .
.. ~
., .
2~9~
A medium with the following composition was prepared. The amounts (in gram) relate to 200 liters of medium.
Basal medium components:
NaCl 1 280.00 KCl 80.00 MgSO4 7 H2O
NaH2PO4 H2O 25.00 CaCl2 2 H2O 52.80 Fe~NO3)3 9 H2O 0.02 Phenol red 3.70 Glucose H20 Sodium pyruvate 22.10 L-Alanine 7.14 L-Arginine HCl 16.80 L-Asparagine H2O 13.66 L-Aspartic acid 10.63 L-Cysteine 9.60 L-Glutamic acid 11.83 Glycine 6.02 L-Hi~tidine HCl H2O 8.40 L-Isoleucine 21.00 L-Leuclne 21.00 L-Ly~ine HCl 29.20 L-Methionine 6.00 L-Phenylalanine 13.20 L-Proline 9.22 L-Serine 8.42 L-Threonine 19.00 L-Tryptophan 3.20 L-Tyrosine 14.40 L-Valine 18.80 Calcium pantothenate 0.80 Choline chloride 0.80 Folic acid 0.80 .
.. ~
., .
2~9~
I-Inositol 1.44 Nicotinamide 0.80 Pyridoxal HCl 0.80 Riboflavin 0.08 ~hiamine HCl 0.80 NaHCO3 750 00 Additional components:
Human insulin (Hoechst:PHFM) 2.00 Human transferrin (Behringwerke AG, p~oduct identifier: 0 TRE)7.00 Ethanolamine 1.68 ml Sodium selenite (1 mM) 9.4 ml Distilled water ad 200 l After addition of 600 mg/l glutamine, hybridomas and transfectomas were grown in this medium and, for compar-ison therewith, in FBS-containing basal medium (Dulbecco's medium + FBS). The result is ~hown in Table I. The cells were cultivated under standard cell culture condit;ion~ at 37C and 5 % CO2 in vapor-saturated atmosphere for the periods noted in Table I, and the live cell count and the product concentration were determined on aliquots.
The data demonstrate the equivalence of the DKHI medium in respect of cell growth and production efficiency to the D-FBS complete medium. The growth rates are the same for both media.
It was furthermore possible to cultivate human and rat tumor cell lines in the medium (Table II). The comparison media were DMEM + 10 % FBS and an ITES medium published by Murakami et al. (Proc. natl. Acad. Sci. USA 79, 2~2~
1158-116~, 1982).
The DK~I medium dif fer6 from the ITES medi~m in that it contains 13 mg/l insulin, 200 ~mol/l ethanolamine, 1 mmol/l pyru~ic acid and 3.75 g of sodium hydrogen carbonate/l, and no HEPES buffer (see Table III for comparison). These differences lead to an improved cell growth, as Table II documents.
209~2~
X X ~
".: 8 8 ~a ~ ¢ O ~ 0 E
.g ooo I
u~ E
C ~t ~ X X, o ;~ ~ ~ EE -~ ~-oo oo ~ .s:
~ E ~ o # . ~~ ~ ~ ~ ' ~ D
t.~ ~ N ~ t~ t,~
t~
. '~
E .
i V ~
~ ~i ~ 'C ~ ~
~ ~ o ~ o ~ ~ X o ~ ~ ..
t'~ .. X
~ol ~ c 0 m m m, 5 ~ z ~ ~ x~ ~ Xx ~ m 01 =
~ ~1 I , , , o +
~ -l l l l l l l l l ~
G ~ '3 ~ ~ o ~ ~ ~
_1 O ~ o o o ~ ~0 a~ ~ ) g ~ ~ ~
E-2~
Table III
Compari~on of the added componentæ in the IT~S medium ~nd in the DR~I mediu~
ITES DKHI
Basal medium DMEM/F12 (1:1) DMEM
HEPES 15 mM - *
NaHCO3 1.2 g/l 3.75 g/l *
Insulin 5 mg/l 10 mg/l *
10 Transferrin 35 mg/l 35 mg/l Na selenite 2.5 nM - 2.5 mM 50 nM
Ethanolamine 20 ~M 200 ~M *
Na pyruvate - 1 mM *
L-Glutamine - 2 mM *
* significant dif ferences between ITES medium and DKHI
medium
Human insulin (Hoechst:PHFM) 2.00 Human transferrin (Behringwerke AG, p~oduct identifier: 0 TRE)7.00 Ethanolamine 1.68 ml Sodium selenite (1 mM) 9.4 ml Distilled water ad 200 l After addition of 600 mg/l glutamine, hybridomas and transfectomas were grown in this medium and, for compar-ison therewith, in FBS-containing basal medium (Dulbecco's medium + FBS). The result is ~hown in Table I. The cells were cultivated under standard cell culture condit;ion~ at 37C and 5 % CO2 in vapor-saturated atmosphere for the periods noted in Table I, and the live cell count and the product concentration were determined on aliquots.
The data demonstrate the equivalence of the DKHI medium in respect of cell growth and production efficiency to the D-FBS complete medium. The growth rates are the same for both media.
It was furthermore possible to cultivate human and rat tumor cell lines in the medium (Table II). The comparison media were DMEM + 10 % FBS and an ITES medium published by Murakami et al. (Proc. natl. Acad. Sci. USA 79, 2~2~
1158-116~, 1982).
The DK~I medium dif fer6 from the ITES medi~m in that it contains 13 mg/l insulin, 200 ~mol/l ethanolamine, 1 mmol/l pyru~ic acid and 3.75 g of sodium hydrogen carbonate/l, and no HEPES buffer (see Table III for comparison). These differences lead to an improved cell growth, as Table II documents.
209~2~
X X ~
".: 8 8 ~a ~ ¢ O ~ 0 E
.g ooo I
u~ E
C ~t ~ X X, o ;~ ~ ~ EE -~ ~-oo oo ~ .s:
~ E ~ o # . ~~ ~ ~ ~ ' ~ D
t.~ ~ N ~ t~ t,~
t~
. '~
E .
i V ~
~ ~i ~ 'C ~ ~
~ ~ o ~ o ~ ~ X o ~ ~ ..
t'~ .. X
~ol ~ c 0 m m m, 5 ~ z ~ ~ x~ ~ Xx ~ m 01 =
~ ~1 I , , , o +
~ -l l l l l l l l l ~
G ~ '3 ~ ~ o ~ ~ ~
_1 O ~ o o o ~ ~0 a~ ~ ) g ~ ~ ~
E-2~
Table III
Compari~on of the added componentæ in the IT~S medium ~nd in the DR~I mediu~
ITES DKHI
Basal medium DMEM/F12 (1:1) DMEM
HEPES 15 mM - *
NaHCO3 1.2 g/l 3.75 g/l *
Insulin 5 mg/l 10 mg/l *
10 Transferrin 35 mg/l 35 mg/l Na selenite 2.5 nM - 2.5 mM 50 nM
Ethanolamine 20 ~M 200 ~M *
Na pyruvate - 1 mM *
L-Glutamine - 2 mM *
* significant dif ferences between ITES medium and DKHI
medium
Claims (11)
1. A nutrient medium for cell cultures containing a basal medium containing amino acids, vitamins, cofactors and usual inorganic salts, which additionally contains human transferrin, human insulin, ethanolamine and sodium selenite.
2. A nutrient medium as claimed in claim 1, which con-tains clinically approved transferrin and insulin.
3. A nutrient medium as claimed in claim 1, wherein the basal medium used is DMEM/F 12 (1:1) medium, DMEM
(Dulbecco's modified Eagle medium), William's medium E or Ham's F12 medium.
(Dulbecco's modified Eagle medium), William's medium E or Ham's F12 medium.
4. A nutrient medium as claimed in claim 1, wherein the basal medium used is RPMI 1640 medium, CMRL 1066 medium, BME basal medium, Waymouth's medium, NCTC 135 medium or MEM (minimum essential medium), and sodium pyruvate is added in an amount such that a concentration of 25-500, preferably 110 mg/l sodium pyruvate is reached in the medium.
5. A nutrient medium as claimed in claim 1, wherein insulin is added in an amount such that a concentration of 6-20, preferably 8-12 mg/l insulin is reached in the medium.
6. A nutrient medium as claimed in claim 1, wherein transferrin is added in an amount such that a concentra-tion of 20-50, preferably 30-40 mg/l transferrin is reached in the medium.
7. A nutrient medium as claimed in claim 1, wherein ethanolamine is added in an amount such that a concentra-tion of 1.3-20, preferably 10-14 mg/l ethanolamine is reached in the medium.
8. A nutrient medium as claimed in claim 1, wherein the sodium selenite is added in an amount such that a concen-tration of 2-20, preferably 6-10 µg/l sodium selenite is reached in the medium.
9. A nutrient medium as claimed in claim 1, wherein L-glutamine is added in an amount such that a concentration of 140-1200, preferably 600 mg/l L-glutamine is reached in the medium.
10. A nutrient medium as claimed in claim 1, which contains the following constituents (in gram) in 200 liters of liquid:
Basal medium components:
NaCl 1 280.00 KCl 80.00 MgSO4 ? 7 H2O 40.00 NaH2PO4 ? H2O 25.00 CaCl2 ? 2 H2O 52.80 Fe(NO3)3 ? 9 H2O 0.02 Phenol red 3.70 Glucose ? H2O 990.00 Sodium pyruvate 22.10 L-Alanine 7.14 L-Arginine HCl 16.80 L-Asparagine ? H2O 13.66 L-Aspartic acid 10.63 L-Cysteine 9.60 L-Glutamic acid 11.83 Glycine 6.02 L-Histidine HCl ? H2O 8.40 L-Isoleucine 21.00 L-Leucine 21.00 L-Lysine HCl 29.20 L-Methionine 6.00 L Phenylalanine 13.20 L-Proline 9.22 L-Serine 8.42 L-Threonine 19.00 L-Tryptophan 3.20 L-Tyrosine 14.40 L-Valine 18.80 Calcium pantothenate 0.80 Choline chloride 0.80 Folic acid 0.80 I-Inositol 1.44 Nicotinamide 0.80 Pyridoxal HCl 0.80 Riboflavin 0.08 Thiamine HCl 0.80 NaHCO3 750.00 Additional components:
Human insulin (Hoechst:PHFM) 2.00 Human transferrin (Behringwerke AG, product identifier: ? TRE) 7.00 Ethanolamine 1.68 ml Sodium selenite (1 mM) 9.4 ml Distilled water ad 200 l and, before use, L-glutamine is added in an amount such that a concentration of 140-1200 mg/l, preferably 600 mg/l is reached.
Basal medium components:
NaCl 1 280.00 KCl 80.00 MgSO4 ? 7 H2O 40.00 NaH2PO4 ? H2O 25.00 CaCl2 ? 2 H2O 52.80 Fe(NO3)3 ? 9 H2O 0.02 Phenol red 3.70 Glucose ? H2O 990.00 Sodium pyruvate 22.10 L-Alanine 7.14 L-Arginine HCl 16.80 L-Asparagine ? H2O 13.66 L-Aspartic acid 10.63 L-Cysteine 9.60 L-Glutamic acid 11.83 Glycine 6.02 L-Histidine HCl ? H2O 8.40 L-Isoleucine 21.00 L-Leucine 21.00 L-Lysine HCl 29.20 L-Methionine 6.00 L Phenylalanine 13.20 L-Proline 9.22 L-Serine 8.42 L-Threonine 19.00 L-Tryptophan 3.20 L-Tyrosine 14.40 L-Valine 18.80 Calcium pantothenate 0.80 Choline chloride 0.80 Folic acid 0.80 I-Inositol 1.44 Nicotinamide 0.80 Pyridoxal HCl 0.80 Riboflavin 0.08 Thiamine HCl 0.80 NaHCO3 750.00 Additional components:
Human insulin (Hoechst:PHFM) 2.00 Human transferrin (Behringwerke AG, product identifier: ? TRE) 7.00 Ethanolamine 1.68 ml Sodium selenite (1 mM) 9.4 ml Distilled water ad 200 l and, before use, L-glutamine is added in an amount such that a concentration of 140-1200 mg/l, preferably 600 mg/l is reached.
11. The use of a nutrient medium as claimed in claim 1 for growing hybridomas, transfectomas or tumor cell lines.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4219250A DE4219250A1 (en) | 1992-06-12 | 1992-06-12 | Serum-free cell culture medium |
| DEP4219250.1 | 1992-06-12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2098255A1 true CA2098255A1 (en) | 1993-12-13 |
Family
ID=6460869
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2098255 Abandoned CA2098255A1 (en) | 1992-06-12 | 1993-06-11 | Serum-free cell culture medium |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0573812A2 (en) |
| JP (1) | JPH0678759A (en) |
| AU (1) | AU4018393A (en) |
| CA (1) | CA2098255A1 (en) |
| DE (1) | DE4219250A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5292655A (en) * | 1990-01-29 | 1994-03-08 | Wille Jr John J | Method for the formation of a histologically-complete skin substitute |
| US5512477A (en) * | 1994-04-21 | 1996-04-30 | Genzyme Corporation | Serum-free medium supplement |
| JP4849408B2 (en) | 2004-03-01 | 2012-01-11 | 財団法人北九州産業学術推進機構 | Protein production novel human cell line, selection method of novel human cell line, use of novel human cell line, protein production method and purification method therefrom, and pharmaceutical composition using novel human cell line |
| JP4745750B2 (en) * | 2005-08-01 | 2011-08-10 | 株式会社ツーセル | Serum-free medium for animal stem cell culture |
| EP3640326A1 (en) * | 2007-04-18 | 2020-04-22 | Hadasit Medical Research Services & Development Limited | Stem cell-derived retinal pigment epithelial cells |
| JP2011177140A (en) * | 2010-03-03 | 2011-09-15 | Nippon Dental Univ | Serum-free medium for stem cell culture |
-
1992
- 1992-06-12 DE DE4219250A patent/DE4219250A1/en not_active Withdrawn
-
1993
- 1993-05-15 EP EP19930107948 patent/EP0573812A2/en not_active Withdrawn
- 1993-06-11 CA CA 2098255 patent/CA2098255A1/en not_active Abandoned
- 1993-06-11 JP JP14022793A patent/JPH0678759A/en active Pending
- 1993-06-11 AU AU40183/93A patent/AU4018393A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| DE4219250A1 (en) | 1993-12-16 |
| EP0573812A2 (en) | 1993-12-15 |
| AU4018393A (en) | 1993-12-16 |
| JPH0678759A (en) | 1994-03-22 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |