Tissue culture medium

Claim 3

A serum composition comprising (a) a serum derived from natural bovine serum (B.S.) which comprises less than 30 mg/dl of total lipids; cholesterol levels within 0-10 mg/dl; triglyceride levels within 0-20 mg/dl; hemoglobin levels less than 20 mg/dl; mycoplasma levels and enveloped virus levels being substantially undetectable and; (b) a natural fetal calf serum (FCS), wherein said FCS is present in 1-99% of said combination, and said B.S. derived serum if present in 99-1% of said composition.

Claim 4

A serum composition comprising (a) a serum derived from natural bovine serum (B.S.) which comprises less than 30 mg/dl of total lipids; cholesterol levels within 0-10 mg/dl; triglyceride levels within 0-20 mg/dl; hemoglobin levels less than 20 mg/dl; mycoplasma levels and enveloped virus levels being substantially undetectable and; (b) a natural fetal calf serum (FCS), wherein said FCS is present in 1-99% of said combination, and said B.S. derived serum if present in 99-1% of said composition.

Claim 5

The serum composition of claim 2 wherein prior to said combination, said B.S. derived serum (a) contains albumin, - globulin and - glubulin levels similar to those of fetal calf serum.

Claim 6

The serum composition of claim 2, wherein prior to said combination said B.S. derived serum (a) has albumin levels within 2-4 g/dl, alpha globulin levels within 0.4-2.0 g/dl, and beta globulin levels within 0.4-2.0 g/dl.

Claim 7

A tissue culture medium comprising the serum of any of claims 2, 3, or 4.

Claim 8

The serum composition of claim 2, 3 or 4 wherein prior to said combination, said B.S. derived serum (a) has gamma-globulin levels within 0.0-1.0 g/dl and endotoxin levels less than 2.0 ng/ml.

Claim 9

The serum composition of claim 2, 3 or 4 wherein prior to said combination, said B.S. derived serum (a) has cortisol levels less than 5 g/ml.

Claim 10

The serum composition of claim 2, 3 or 4 wherein prior to said combination, said B.S. derived serum (a) has (a) gamma-globulin levels within 0.1-1.0 g/dl; (b) cortisol levels less than 5 g/ml; and (c) endotoxin levels less than 2.0 ng/ml.

Claim 11

The serum composition of claims 2 or 3 wherein prior to said combination said B.S. derived serum (a) has gamma-globulin levels within 0.0-0.5 g/dl.

Claim 12

The serum composition of claim 2 wherein said fetal calf serum is present at 5-10% by volume of said combination.

Claim 13

An in vitro cell culture comprising animal or plant cells together with the serum of claim 2.

Claim 14

The cell culture of claim 11 wherein said serum is present in growth promoting amounts.

Claim 15

The cell culture of claim 12 wherein said cells are transformed or non-transformed animal cells.

Claim 16

The cell culture of claim 12 wherein said cells are hybridomas.

Claim 17

The cell culture of claim 11 wherein said fetal calf serum if present at 5-10% by volume of said combination.

Claim 18

A method of culturing animal or plant cells in vitro which comprises contacting said cells with a growth promoting amount of the serum of claim 2.

Claim 19

The method of claim 16 wherein said fetal calf serum is present at 5-10% by volume of said combination.

Claim 20

The method of claim 16 wherein said cells are or are derived from lymphocytes or leukocytes.

Claim 21

The method of claim 16 wherein said cells are used for the production of viruses.

Claim 22

A process for the preparation of a serum which comprises (a) delipidizing natural bovine serum to a concentration of lipids less than 30 mg/dl, (b) precipitating proteins with ammonium sulfate to concentrations of alubumin and β-globulin substantially similar to those of fetal bovine serum, a concentration of α-globulin lower than or similar to that of fetal bovine serum and to γ-globulin levels of up to 1.0 g/dl; and (c) admixing 99-1% of the serum obtained from steps (a) and (b) with 1-99% of natural fetal calf serum.

Claim 23

A process for the preparation of a serum which comprises (a) delipidizing natural bovine serum to a concentration of lipids less than 30 mg/dl, (b) precipitating proteins with ammonium sulfate to concentrations of alubumin and β-globulin substantially similar to those of fetal bovine serum, a concentration of α-globulin lower than or similar to that of fetal bovine serum and to γ-globulin levels of up to 1.0 g/dl; and (c) admixing 99-1% of the serum obtained from steps (a) and (b) with 1-99% of natural fetal calf serum.

Claim 24

The process of claim 20 wherein said delipidization treatment is carried out by contacting said serum with fumed silica.

Claim 25

The process of claim 21 wherein said fumed silica is added batchwise to said serum to about 10-100 g/l.

Claim 26

The process of claim 21 wherein said fumed silica is immobilized in a fibrous matrix.

Claim 27

The process of claim 21 wherein prior to the contact with said silica, said serum is treated with a divalent metal ion to a concentration of 0.01-0.5 M.

Claim 28

The process of claim 20 which further comprises contacting said serum with activated charcoal.

Claim 29

The process of any of claims 24 or 25 wherein said divalent metal ion is calcium ion.

Claim 30

The process of any of claims 20 or 25 which further comprises separating precipitated protein from the supernatant, recovering the supernatant and substantially decreasing the concentration of dissolved salt remaining therein.

Claim 31

The process of claim 20 which comprises (a) adding a divalent metal ion to said serum to a concentration of 0.01-0.5M; then (b) contacting said serum with fumed silica for a time sufficient to bring the total lipid concentration to less than 30 mg/dl; then (c) separating said silica from said serum; (d) precipitating globulins from said serum with 20-35% ammonium sulfate, then (e) separating said precipitated protein from the supernatant and substantially decreasing the concentration of dissolved precipitating salt remaining in said supernatant; then (f) contacting said substantially salt-free supernatant with activated charcoal; separating said serum from said charcoal to obtain a clarified serum; then (g) adjusting the protein concentration in said clarified serum to a range of 3-7 g/dl; and then (h) heat inactivating said serum.

Claim 32

The process of claim 28 which further comprises sterilizing said serum after step (h).

Claim 33

The serum prepared by the process of claim 28.

Claim 34

The serum prepared by the process of claim 20.

Claim 35

A method of enhancing the cell growth promoting ability of natural bovine serum which comprises treating said serum by the process of claim 20.