CN1193091C - Serumless medium suitable for growth and maintenance of young hamster kidney cell - Google Patents

Abstract

The invention discloses a low-protein serum-free medium suitable for high-density growth and maintenance of baby hamster kidney (BHK) cells. The medium has the characteristics of low protein content, easy separation and purification, and is suitable for industrial production.

Description

A kind of baby hamster kidney cell growth and serum free medium of keeping of being suitable for

Technical field

The present invention relates to a kind of low albumen serum free medium that is suitable for the baby hamster kidney cell high-density growth and keeps.

Background technology

Young hamster kidney (BHK) cell is a kind of mammal cell line that can infinitely go down to posterity, and has been widely used in producing biological technology products such as vaccine, diagnostic reagent and medicine.

Bhk cell is cultivated in blood serum medium is arranged usually, but that the interpolation of serum exists is many unfavorable: the first, and serum is very expensive, and the expense of serum accounts for more than 50% of substratum total expenses, causes production cost to increase substantially; The second, the difference of serum quality can influence the cell growth between criticizing and criticizing, and finally influences quality product; The 3rd, serum is easily by mycoplasma and external reagent contamination; The 4th, serum protein has increased that the downstream cultured products is separated, the difficulty of purifying, and these shortcomings make the use of serum in cell large scale is cultivated be restricted, and cause people to launch the development and application research of serum free medium.

In recent years, the research and development development of serum free medium is very fast, reported the multiple serum-free culture that can support the growth of different sorts cell, Casser et al. (In Vitro Cell.Devel.Biol. for example, 88,21:588 1985) developed a kind of substratum that can support Chinese hamster ovary celI growth.This substratum has added Transferrins,iron complexes, Regular Insulin and Sodium Selenite based on DMEM/F12 (1: 1), (the serum free medium basic components of basic medium+ITS) that Here it is now is widely adopted.United States Patent (USP) U.S.592107 discloses a kind of serum free medium, and this substratum contains high-level amino acid, and has added different somatomedins and trace element, can support Chinese hamster ovary celI high-density suspension culture.But these substratum are limited to hybridoma more and Chinese hamster ovary celI is cultivated, and general protein content is still higher, can reach tens milligrams usually to several grams, cause the difficulty of downstream product separation and purification and cost to increase.Do not appear in the newspapers and relate to the low albumen serum free medium that is suitable for the baby hamster kidney cell long-term cultivation.

Summary of the invention

The present invention discloses a kind of young hamster kidney (BHK) cell high-density growth and low albumen serum free medium of keeping of being suitable for, this substratum have protein content low, be easy to separation and purification and be suitable for the characteristics of suitability for industrialized production.

Design of the present invention:

The recruitment factor kind of alternative serum is a lot, generally includes some human necessary somatomedins, hormone, lipid, conjugated protein and various trace elements etc.For effective alternative serum, the high-density growth of sustenticular cell, the present invention has added following composition on the basis of basic medium DMEM:

(1) Transferrins,iron complexes: a kind of glycoprotein in conjunction with iron, with the single-minded receptor acting of cell surface, transmit iron ion.

(2) Regular Insulin: beta Cell of islet excretory polypeptide hormone can promote the cell growth.

(3) VITAMIN: the moiety of cell is the essential material of cell growth institute, and cell self can't synthesize, and must absorb from the external world.

(4) trace element: as Cu, Fe, Zn, Se, Co, Mo, Sn, Si, V, Mn etc. mainly play the cell physiological process and regulate.

(5) beta-mercaptoethanol: promote the absorption of Gelucystine, can make gsh be in reduced state, thereby the protection cell is avoided the infringement of hydrogen peroxide.

(6) lipid: comprise some lipid acid and lipoid, can promote the cell growth.For example: linolic acid, linolenic acid, oleic acid and cholesterol etc.

Technical scheme of the present invention:

Substratum of the present invention is a kind of composition, forms (unit of additive capacity is mg/L if no special instructions) by basic medium DMEM and following additive:

L-aspartic acid 0-20

L-L-glutamic acid 10-40

Altheine 20-50

L-Serine 0-10

L-glutaminate 0-200

L-Histidine 0-40

L-L-Ala 0-40

L-arginine 50-150

L-methionine(Met) 10-50

L-tryptophane 0-50

L-phenylalanine 10-60

L-leucine 20-60

L-Methionin 40-100

L-proline(Pro) 10-30

NH ₄VO ₃ 0.02-0.1

SnCl ₂2H ₂O 1.00-2.00

Ba(C ₂H ₃O ₂) ₂ 0.01-0.05

AgNO ₃ 0.01-0.05

AlCl ₃6H ₂O 0.3-1.5

3CdSO ₄8H ₂O 0.001-0.005

CoCl ₂6H ₂O 0.03-0.15

(NH ₄) ₆MoO ₂₄4H ₂O 0.01-0.05

NiCl ₂6H ₂O 0.1-0.5

Na ₂SeO ₃ 0.01-0.05

KBr 0-0.0002

NaF 0-0.005

KI 0-0.0005

Na ₂SiO ₃ 0.01-0.05

NaH ₂PO ₃2H ₂O 0-100

FeSO ₄7H ₂O 0.3-1.5

CuSO ₄5H ₂O 0.001-0.005

ZnSO ₄ 0.3-1.5

HEPES 1000-2500

Regular Insulin 2-20

Transferrins,iron complexes 2-20

Sodium.alpha.-ketopropionate 0-150

Primatone 1000-2500

Lipid mixture * 0.1%-0.5% (v/v)

Reduced glutathion 0.3-1.5

Vitamins C 3.5-13.5

Vitamins B ₆0-0.06

Vitamins B ₁₂0-0.68

Choline chloride 60 0-5

Inositol 0-5

Vitamin H (Biotin) 0-0.02

Niacinamide 0-2.02

Riboflavin 0-0.044

VitB1 0-2.17

Xanthoglobulin 0-2

Thioctic Acid 0-0.1

Beta-mercaptoethanol 0-2.5

*: the chemical ingredients of producing for GIBCO determine the lipid concentrated solution (attached composition, mg/L)

Arachidonic acid 2

Cholesterol 220

DL-alpha-tocopherol acetate 70

Linolic acid 10

Linolenic acid 10

Myristic acid 10

Oleic acid 10

Palm diluted acid 10

Palmitinic acid 10

Pluronic?F-68 100000

Stearic acid 10

Tween 80 2200

The above all substances is the commercial goods.

Substratum of the present invention adopts conventional method formulated.

Substratum using method of the present invention is an ordinary method.

Description of drawings

Fig. 1 is the test-results diagram of embodiment 1 and Comparative Examples.

Wherein:

●-the adopt cell density (* 10 of serum free medium ⁵Cells/mL)

▲-the adopt cell density (* 10 that blood serum medium is arranged ⁵Cells/mL)

Specific implementation method

Below will the present invention is further illustrated by embodiment.

Embodiment 1

(comprise basic medium DMEM composition, mg/L) insert bhk cell (seed cell is the serum-free culture cell) then in this substratum, inoculum density is 1.4 * 10 according to following concentration preparation serum free medium ⁵Cells/mL, cultivate 4 days (96 hours) after, cell density can reach 1.4 * 10 ⁶Cells/mL.

First part: amino acid

L-Gelucystine two hydrochloric acid 63

L-aspartic acid 8

L-L-glutamic acid 19

Altheine 31

Glycine 30

L-Serine 44

L-glutaminate 688

L-Histidine 63

L-L-Ala 12

L-arginine 190

L-methionine(Met) 48

L-tryptophane 28

L-phenylalanine 86

Two water L-tyrosine disodiums 104

L-leucine 147

L-Isoleucine 105

L-Methionin 209

L-proline(Pro) 17

L-Threonine 95

L-Xie Ansuan 94

The second section trace element

CaCl ₂ 200

Fe(NO ₃) ₃·9H ₂O 0.1

KCl 400

NCl 6400

NaHCO ₃ 3700

NH ₄VO ₃ 0.092

SnCl ₂2H ₂O 1.556

Ba(C ₂H ₃O ₂) ₂ 0.02

AgNO ₃ 0.02

AlCl ₃·6H ₂O 0.99

3CdSO ₄·8H ₂O 0.003

CoCl ₂·6H ₂O 0.101

MnSO ₄·H ₂O 97.67

(NH ₄) ₆MoO ₂₄·4H ₂?O 0.033

NiCl ₂·6H ₂O 0.35

Na ₂SeO3 0.01

KBr 0.0001

NaF 0.0042

KI 0.0002

Na ₂SiO ₃ 0.01

NaH ₂PO ₃·2H ₂O 193.00

FeSO ₄·7H ₂O 0.834

CuSO ₄·5H ₂O 0.0025

ZnSO ₄ 0.863

Third part: VITAMIN and other composition

HEPES 2000

Regular Insulin 5

Transferrins,iron complexes 5

Phenol red 15

D-glucose 4500

Sodium.alpha.-ketopropionate 193

Lipid mixture * 0.2% (v/v)

Primatone 1250

Reduced glutathion 0.75

Beta-mercaptoethanol 2.5

D-calcium pantothenate 4

Vitamins C 6.25

Vitamins B ₆4.06

Vitamins B ₁₂0.68

Choline chloride 60 9

Inositol 12.2

Folic acid 4.00

Vitamin H (Biotin) 0.02

Niacinamide 6.02

Riboflavin 0.44

VitB1 6.17

Xanthoglobulin 2.00

Thioctic Acid 0.10

Comparative Examples

Adopt the cultural method identical with embodiment 1, used substratum is the DMEM substratum that has added 5% (v/v) foetal calf serum, and cell density is 1.2 * 10 to the maximum ⁶Cells/mL.

Can be found by the above embodiments contrast, cultivate bhk cell in serum free medium of the present invention, the cell growth obviously is better than having the substratum of serum, and complete alternative serum is used for bhk cell and cultivates.

Claims (1)

1. A serum-free medium suitable for the growth and maintenance of young hamster kidney cells, characterized in that the medium is a composition consisting of basal medium DMEM and the following additives, and the unit of additive dosage is as follows: Special instructions are all mg/L: L-Aspartic Acid 0-20 L-glutamic acid 10-40 L-Asparagine 20-50 L-serine 0-10 L-Glutamine 0-200 L-histidine 0-40 L-alanine 0-40 L-Arginine 50-150 L-Methionine 10-50 L-tryptophan 0-50 L-phenylalanine 10-60 L-leucine 20-60 L-Lysine 40-100 L-proline 10-30 NH ₄ VO ₃ 0.02-0.1 SnCl ₂ 2H ₂ O 1.00-2.00 Ba(C ₂ H ₃ O ₂ ) ₂ 0.01-0.05 AgNO ₃ 0.01-0.05 AlCl ₃ 6H ₂ O 0.3-1.5 3CdSO ₄ 8H ₂ O 0.001-0.005 CoCl ₂ 6H ₂ O 0.03-0.15 (NH ₄ ) ₆ MoO ₂₄ 4H ₂ O 0.01-0.05 NiCl ₂ 6H ₂ O 0.1-0.5 Na ₂ SeO ₃ 0.01-0.05 KBr 0-0.0002 NaF 0-0.005 KI 0-0.0005 Na ₂ SiO ₃ 0.01-0.05 NaH ₂ PO ₃ 2H ₂ O 0-100 FeSO ₄ 7H ₂ O 0.3-1.5 CuSO ₄ 5H ₂ O 0.001-0.005 ZnSO ₄ 0.3-1.5 HEPES 1000-2500 Insulin 2-20 Transferrin 2-20 Sodium pyruvate 0-150 Primatone 1000-2500 Lipid blend* 0.1%-0.5% v/v Reduced glutathione 0.3-1.5 Vitamin C 3.5-13.5 Vitamin _B6 0-0.06 Vitamin B ₁₂ 0-0.68 Choline Chloride 0-5 Inositol 0-5 Biotin 0-0.02 Nicotinamide 0-2.02 Riboflavin 0-0.044 Thiamine 0-2.17 Hypoxanthine 0-2 Lipoic acid 0-0.1 β-mercaptoethanol 0-2.5 ^* : Determine the chemical composition of the lipid concentrate produced by GIBCO: the composition is as follows, the unit is mg/L Arachidonic acid 2 Cholesterol 220 DL-Acetate α-Tocopherol 70 Linoleic acid 10 Linolenic acid 10 Myristic acid 10 Oleic acid 10 Palmitic Acid 10 Palmitic Acid 10 Pluronic F-68 100000 Stearic acid 10 Tween 80 2200