Serum-free, synthetic, completely chemically defined tissue culture media

• US 4,657,866 A
• Filed: 09/13/1984
• Issued: 04/14/1987
• Est. Priority Date: 12/21/1982
• Status: Expired due to Fees

• Current Assignee: Sudhir Kumar
• Original Assignee: Sudhir Kumar
• Inventors: Kumar, Sudhir
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): Tarcza, John E.
• Application Number:
  US06/769,708
• Time in Patent Office: 943 Days
• Field of Search: 435/240, 435/241, 435/948
• US Class Current: 435/383
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/40 Nucleotides, nucleosides or bases
• CPC Class Codes: C12N 2500/95 Protein-free medium and culture conditions
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 5/005 Protein-free medium
• CPC Class Codes: C12N 5/0618 Cells of the nervous system
• CPC Class Codes: Y10S 435/948 using viruses or cell lines

Abstract

A serum-free, synthetic tissue culture media is described which is completely defined chemically. The two media described can be used for growing all types of human or animal cell lines in tissue culture without addition of any protein, amino acids, hormones, sources of energy, salts, vitamins, etc. with normally used procedures and methods. The media do not require any supplementation with fetal calf serum to support growth of neural cells.

Claims

1. (a) A cell culturing medium comprising an amino acid composition containing:

space="preserve" listing-type="tabular">______________________________________ Item mg/liter ______________________________________ Alanine 60 Alpha-Amino-n-butyric Acid 5 Arginine 150 Asparagine 5 Aspartic Acid 75 Cysteine-HCl 100 Cystine 25 Ergothionine 10 Glutamic Acid 500 Glutamine 800 Glycine 500 Histidine, HCl 50 Homocysteine 20 Isoleucine 75 Leucine 300 Lysine HCl 300 Methionine 50 Ornithine HCl 10 Phenylalanine 100 Proline 100 Serine 75 Taurine 50 Threonine 100 Tryptophane 50 Tyrosine 100 and Valine 200, ______________________________________

(b) a vitamin mixture containing;

space="preserve" listing-type="tabular">______________________________________ Item mg/liter ______________________________________ DL-Alpha-Tocopherol 2.5 Ascorbic Acid 25 Biotin 20 Calciferol 2.5 Calcium-pentothenate 25 Choline Chloride 25 Cocarboxylase 20 Folic Acid 20 i-Inositol 15 Menadione 2.5 Niacin 5.0 Niacinamide 20 p-Aminobenzoic Acid 1.0 Pyridoxal HCl 20 Pyridoxine HCl 5.0 Riboflavin 20 Thiamine HCl 20 Vitamin B12 50 Vitamin A 10 and Citric Acid 20, ______________________________________

(c) other components containing;

space="preserve" listing-type="tabular">______________________________________ Item mg/liter ______________________________________ Na--Acetate, 3 H.sub.2 O 100 ATP (Sodium Salt) 20 Estradiol 20 Cholate 25 Deoxyadenosine 15 Deoxyguanosine 15 Dextrose 1500 Flavin Adenine Dinucleotide 20 Galactosamine 120 Glucose-1-phosphate 25 Glucuronolactone 25 Hypoxanthine 100 5-Methyl-deoxycytidylic Acid 5 Phenol Red 15 Thioactic Acid 2 Thyroxine 20 UTP 20 PRPP 50 Acetylcholine 25 Adenosine-5'"'"'-phosphate 5 Cholesterol 5 Coenzyme A 50 Deoxycytidine HCl 15 Deoxyribose 50 DPN 10 Fructose-1-6-diphosphate 120 Glucosamine 50 Glucuronate 25 Glutathione 20 Guanine 50 D-Ribose 5 Sodium pyruvate 150 Thymidine 25 TPN 20 Xanthine 25 Glycogen 250 Calcium Chloride 150 Potassium Chloride 500 Magnesium Sulfate 250 Ferric Ammonium Sulfate 20 KH2PO4 75 Sodium Chloride 6500 Sodium Hydrogen Phosphate, disodium 15 Magnesium Chloride 500 L-Lecithin 150 and Sphingomyelin 75, ______________________________________

and Sodium bicarbonate--upto 1500 mg/liter, sufficient to adjust final pH to 7.4.

2. A culture medium as defined in claim 1, in which the concentrations of the vitamin mixture identified in subsection (b) are in the following range:

space="preserve" listing-type="tabular">______________________________________ Item mg/liter ______________________________________ DL-Alpha-Tocopherol 2.0 to 3.0 Ascorbic Acid 20.0 to 30.0 Biotin 10.0 to 25.0 Calciferol 1.0 to 2.8 Calcium-pentothenate 15.0 to 25.0 Choline Chloride 15.0 to 25.0 Cocarboxylase 10.0 to 20.0 Folic Acid 10.0 to 20.0 i-Inositol 10.0 to 20.0 Menadione 2.0 to 3.0 Niacin 4.0 to 5.0 Niacinamide 4.0 to 20.0 p-Aminobenzoic Acid 0.1 to 1.0 Pyridoxal HCl 5.0 to 20.0 Pyridoxine HCl 2.0 to 5.0 Riboflavin 5.0 to 35.0 Thiamine HCl 5.0 to 35.0 Vitamin B12 10.0 to 50.0 Vitamin A 5.0 to 20.0 and Citric Acid 10.0 to 20.0 ______________________________________

3. A culture media, as defined in claim 2, further comprising:

Gentamycin;

5.0 mg/liter.

4. A culture media, as defined in claim 3, further comprising:

space="preserve" listing-type="tabular">______________________________________ Item mg/liter ______________________________________ Colchicine 0.0025 Colcemid 0.0025 EDTA 0.0050 and Phytohemagglutinin 5.0 ml/liter ______________________________________

5. A culture media, as defined in claim 1, further comprising:

Gentamycin;

5.0 mg/liter.

6. A culture media, as defined in claim 3, further comprising:

space="preserve" listing-type="tabular">______________________________________ Item mg/liter ______________________________________ Colchicine 0.0025 Colcemid 0.0025 EDTA 0.0050 and Phytohemagglutinin 5.0 ml/liter ______________________________________

9. A culture media, as defined in any one of the claims 1 thru 8, wherein said medium is free of cell-damaging qualities of proteolytic enzymes.

10. A method for growing human amniotic fluid cells comprising culturing human amniotic fluid cells in the culture media as defined in any one of claims 1 to 8.

11. A method for growing human or animal neural tissues comprising culturing human or animal neural cells in the culture media defined in anyone of claims 1 to 8.

7. An aqueous culture media, for use in cell growth comprising:

space="preserve" listing-type="tabular">______________________________________ Item mg/liter ______________________________________ Alanine 60 Alpha-Amino-n-butyric Acid 5 Arginine 150 Asparagine 5 Aspartic Acid 75 Cysteine-HCl 100 Cystine 25 Ergothionine 10 Glutamic Acid 500 Glutamine 800 Glycine 500 Histidine, HCl 50 Homocysteine 20 Isoleucine 75 Leucine 300 Lysine HCl 300 Methionine 50 Ornithine HCl 10 Phenylalanine 100 Proline 100 Serine 75 Taurine 50 Threonine 100 Tryptophane 50 Tyrosine 100 Valine 200 DL-Alpha-Tocopherol 2.5 Ascorbic Acid 25 Biotin 20 Calciferol 2.5 Calcium-pentothenate 25 Choline Chloride 25 Cocarboxylase 20 Folic Acid 20 i-Inositol 15 Menadione 2.5 Niacin 5.0 Niacinamide 20 p-Aminobenzoic Acid 1.0 Pyridoxal HCl 20 Pyridoxine HCl 5.0 Riboflavin 20 Thiamine HCl 20 Vitamin B12 50 Vitamin A 10 Citric Acid 20 Na--Acetate, 3 H2O 100 ATP (Sodium salt) 20 Estradiol 20 Cholate 25 Deoxyadenosine 15 Deoxyguanosine 15 Dextrose 1500 Flavin Adenine Dinucleotide 20 Galactosamine 120 Glucose-1-phosphate 25 Glucuronolactone 25 Hypoxanthine 100 5-Methyl-deoxycytidylic Acid 5 Phenol Red 15 Thioactic Acid 2 Thyroxine 20 UTP 20 PRPP 50 Acetylcholine 25 Adenosine-5'"'"'-phosphate 5 Cholesterol 5 Coenzyme A 50 Deoxycytidine HCl 15 Deoxyribose 50 DPN 10 Fructose-1-6-diphosphate 120 Glucosamine 50 Glucuronate 25 Glutathione 20 Guanine 50 D-Ribose 5 Sodium pyruvate 150 Thymidine 25 TPN 20 Xanthine 25 Glycogen 250 Calcium Chloride 150 Potassium Chloride 500 Magnesium Sulfate 250 Ferric Ammonium Sulfate 20 KH2PO4 75 Sodium Chloride 6500 Sodium Hydrogen Phosphate, disodium 15 Magnesium Chloride 500 L-Lecithin 150 Sphingomyelin 75 ______________________________________ Sodium bicarbonate up to 1500 mg/liter, sufficient to adjust final pH to 7.4

and Sodium bicarbonate--upto 1500 mg/liter, sufficient to adjust final pH to 7.4.

8. An aqueous culture media as defined in claim 7, wherein the vitamin concentrations are as follows:

space="preserve" listing-type="tabular">______________________________________ Item mg/liter ______________________________________ DL-Alpha-Tocopherol 2.0 to 3.0 Ascorbic Acid 20.0 to 30.0 Biotin 10.0 to 25.0 Calciferol 1.0 to 2.8 Calcium-pentothenate 15.0 to 25.0 Choline Chloride 15.0 to 25.0 Cocarboxylase 10.0 to 20.0 Folic Acid 10.0 to 20.0 i-Inositol 10.0 to 20.0 Menadione 2.0 to 3.0 Niacin 4.0 to 5.0 Niacinamide 4.0 to 20.0 p-Aminobenzoic Acid 0.1 to 1.0 Pyridoxal HCl 5.0 to 20.0 Pyridoxine HCl 2.0 to 5.0 Riboflavin 5.0 to 35.0 Thamie HCl 5.0 to 35.0 Vitamin B12 10.0 to 50.0 Vitamin A 5.0 to 20.0 and Citric Acid 10.0 to 20.0. ______________________________________