low protein serum free cell culture medium
A culture medium and serum-free technology, applied in cell culture medium, cell culture active agent, culture process, etc., can solve problems such as unclear components, pollution risks, separation and purification of unfavorable target products, etc., to achieve clear components and strong versatility , the effect of convenient preparation
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Embodiment 1
[0066] Screening and optimization of serum-free medium components.
[0067] The hybridoma cell line 1D7 was cultured in a six-well plate with a 3 mL culture system, and the viable cell density in the culture system was measured by sampling at 72 hours of culture. Utilize the L16(45) orthogonal test method, consult relevant literature and materials, and after screening, determine 5 types of additives that can promote cell culture. The 5 types of additives that can promote cell culture are: A, 8 kinds of lipids; B , 8 kinds of vitamins; C, 3 kinds of proteins; D, 19 kinds of amino acids; E, 7 kinds of inorganic salts.
[0068] The test used DMEM / F12 (v / v, 1:1) as the basal medium, adding 6 μg / L selenite, 1.5 g / L sodium bicarbonate, 3.8 mg / L Hepes, and 1300 mg / L Pluronic F-68 in advance. Orthogonal test method was used to optimize the selected five additives, and each factor (A, B, C, D, E) was set at four different concentration levels (1, 2, 3, 4, the concentration of the addi...
Embodiment 2
[0095] Serum-free medium preparation and cell culture
[0096] 1. Culture Medium Preparation
[0097] Add the following substances to DMEM / F12 (v / v, 1:1) as the base medium:
[0098] Bovine serum albumin 15.0mg / L, transferrin 5mg / L, insulin 10mg / L, arginine 48mg / L, cystine 10mg / L, histidine 12mg / L, isoleucine 19mg / L, Leucine 18mg / L, Lysine 18mg / L, Methionine 7mg / L, Phenylalanine 11mg / L, Threonine 19mg / L, Tryptophan 4mg / L, Tyrosine 14mg / L , Valine 20mg / L, Glycine 5mg / L, Alanine 7mg / L, Asparagine 10mg / L, Aspartic Acid 10mg / L, Glutamic Acid 10mg / L, Proline 6mg / L, Serine 6mg / L, choline chloride 1.2mg / L, calcium pantothenate 1.2mg / L, folic acid 1.2mg / L, nicotinamide 1.2mg / L, pyridoxal hydrochloride 1.2mg / L, riboflavin 1.2mg / L, Thiamine 1.2mg / L, Inositol 1.2mg / L, Ferric Citrate 12mg / L, Sodium Pyruvate 50mg / L, Calcium Chloride 4mg / L, Magnesium Sulfate 3mg / L, Potassium Chloride 7mg / L, Chlorine Sodium Chloride 100mg / L, Sodium Dihydrogen Phosphate 120mg / L, Arachidonic Acid 25μg / L, C...
Embodiment 3
[0105] Preparation of Monoclonal Antibody by Fed-Batch Culture of Hybridoma Cell Lines
[0106] The serum-free medium of the present invention is used for hybridoma cell lines DF92 and 1A4 for suspension culture in a fermenter, and the formulation of the medium is the same as that in Example 2. DF92 uses a 14L bioreactor from Guangzhou Qizhi Biological Company, the initial culture volume is 2L, and the control conditions are: Temp 37±0.5°C, DO50%±5%, pH 6.9±0.1, Stir speed 80rpm, 1A4 uses an Applikon 2L bioreactor , the initial culture volume is 2L, the control conditions are: Temp 37±0.5℃, DO 50%±5%, pH 6.9±0.1, Stir speed 80rpm, during the suspension culture process, the fed-batch culture method is adopted, Sampling and monitoring relevant data at intervals of about 24 hours, these data include glucose concentration in the culture medium, cell density, cell viability and monoclonal antibody expression. The culture results of the two kinds of cells are as follows: figure 2 ...
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