Cell Culture

Media Formulation

   Carrier and Transport Proteins
Hormones
Hydrolysates
Lipids and Lipid Carriers
Other Supplements
Carrier and Transport Proteins

 

Cell culture is essential to biotechnology.  Historically, cells have been cultured in serum containing media.  Serum is a source of many nutrients required by cells to proliferate and survive. However, the use of serum is problematic. It is complex, expensive and a potential source of adventitious biological contaminants.

Eliminating the use of serum in cell culture requires an understanding of what it does in in vitro systems. Among other things, serum contains proteins which serve as carriers and protective agents for other molecules.  Albumin is a protein in serum which serves these and other functions.  Albumin binds vitamins such as pyridoxal, fatty acids, such as oleic, linoleic, linolenic, arachidonic, myristic and palmitic acids, and ions such as copper.  It increases the solubility of these molecules, maintains them in chemical states that are unstable in an unbound form, and protects them from oxidation.

Sigma offers a number of albumins for use in formulating cell culture media.  Some albumins are purified using methods that protect the natural association of albumin and the bound molecules. Others are stripped of these molecules so that the may be reconstituted in a defined way.

Product # Product Name Description Application
A 1470 Albumin
Cohn Analog
TM
  • Powder;
  • Purity, minimum 95%;
  • Low endotoxin;
  • Cell culture tested.

Recommended as a growth-enhancing supplement for cell culture. Manufactured by a novel process that maintains the lipid and growth factor characteristics of cold-ethanol precipitated albumins.

Fatty acids which are naturally associated with albumins are important for the growth of many biotechnologically  useful cells lines. Albumin serves as natural delivery molecule for free fatty acids such as oleic, linoleic, linolenic, and arachidonic acid.

A 1595 Albumin Solution (10%)
Fraction V
Bovine
  • Liquid;
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

Recommended as a base for the preparation of a defined, fatty acid supplemented albumin. Can be loaded with up to 4 moles of fatty acids per mole of albumin.

Fatty acids are important for the growth of many biotechnologically  useful cells lines. Albumin serves as natural delivery molecule for free fatty acids such as oleic, linoleic, linolenic, and arachidonic acid.

A 9576 Albumin Solution (30%)
Fraction V
Bovine
  • Liquid;
  • Prepared from A 9418 in D-PBS (D 5652);
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

Recommended as a base for the preparation of a defined, fatty acid supplemented albumin. Can be loaded with up to 4 moles of fatty acids per mole of albumin.

Fatty acids are important for the growth of many biotechnologically  useful cells lines. Albumin serves as natural delivery molecule for free fatty acids such as oleic, linoleic, linolenic, and arachidonic acid.

I 3153 Iron Supplement,
Chemically Defined (1000x)
Animal Component-free.
  • Liquid;
  • Proprietary product;
  • Recommended for use in cell culture applications at 0.5 to 2 ml per liter of medium.
  • Iron content: 222-334 ppm
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

Designed to replace transferrin for iron transport and delivery to cells in a broad range of cell culture applications.

The proper sequestration of iron in media is a critical factor in preventing oxidative stress to cells.  Iron is an essential grow element for all cell systems.

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Hormones

 

Many cells require hormones to modulate their proliferation or differentiation.

Sigma offers a wide range of hormones formulated and tested for use in cell culture.  This table provides a partial list of hormones or hormone formulations that may be particularly useful in biotechnology applications.

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Product # Product Name Description Application
H 6909 Hydrocortisone Solution
Animal Component-free
  • Liquid;
  • Formulated to supplement at 0.2 to 2 ml per liter of medium;
  • Formulated to contain 50 ug/ml in cell culture grade water (W 3500);
  • Sterile filtered
  • Endotoxin tested;
  • Cell culture tested.

Use as a cell culture medium supplement.

I 2767 Insulin,
Human Recombinant,
sodium salt
Escherichia coli
  • Powder;
  • FW: 5749.5;
  • Purity, not less than 90% by HPLC;
  • Soluble in 10 mM HCl (20 mg/ml);
  • Zinc, not more than 0.01%.

A two-chain polypeptide hormone produced by the beta-cells of pancreatic islets. Its molecular weight is approximately 7700 Da. The alpha and beta chains are joined by two inter-chain disulfide bonds, the alpha chain contains an intra-chain disulfide bond.  Insulin regulates the cellular uptake, utilization, and storage of glucose, amino acids and fatty acids and inhibits the breakdown of glycogen, protein and fat.

Recombinant insulin in the sodium salt form for use in medium supplementation. 
Insulin is widely used in media formulations.
Suitable for use in serum-free media formulations when the use of animal-derived proteins is to be avoided.

I 0259 Insulin,
Human Recombinant,
Escherichia coli
  • Powder;
  • FW: 7707.6;
  • Potency: not less than 27.5 USP units/mg on a dry basis;
  • Soluble in 10 mM HCl (20 mg/ml).

A two-chain polypeptide hormone produced by the beta-cells of pancreatic islets. Its molecular weight is approximately 7700 Da. The alpha and beta chains are joined by two inter-chain disulfide bonds, the alpha chain contains an intra-chain disulfide bond.  Insulin regulates the cellular uptake, utilization, and storage of glucose, amino acids and fatty acids and inhibits the breakdown of glycogen, protein and fat.

Insulin is widely used in media formulations. Suitable for use in serum-free media formulations when the use of animal-derived proteins is to be avoided.

I 0516 Insulin Solution,
Bovine derived, natural
  • Liquid;
  • Formulated to supplement at 0.2 to 2 ml per liter of medium;
  • Formulated to contain 50 mg/ml in 25 mM HEPES, pH 8.2;
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

A two-chain polypeptide hormone produced by the beta-cells of pancreatic islets. Its molecular weight is approximately 7700 Da. The alpha and beta chains are joined by two inter-chain disulfide bonds, the alpha chain contains an intra-chain disulfide bond.  Insulin regulates the cellular uptake, utilization, and storage of glucose, amino acids and fatty acids and inhibits the breakdown of glycogen, protein and fat.

Insulin is widely used in media formulations.
Ready to use solution of insulin. 
Suitable for use in serum-free media formulations when animal-derived components are acceptable.

I 9278 Insulin Solution,
Human Recombinant
Escherichia coli
  • Liquid;
  • Formulated to supplement at 0.5 to 1 ml per liter of medium;
  • Formulated to contain 10 mg/ml in 25 mM HEPES, pH 8.2;
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested, CHO, L929, and BHK.

A two-chain polypeptide hormone produced by the beta-cells of pancreatic islets. Its molecular weight is approximately 7700 Da. The alpha and beta chains are joined by two inter-chain disulfide bonds, the alpha chain contains an intra-chain disulfide bond.  Insulin regulates the cellular uptake, utilization, and storage of glucose, amino acids and fatty acids and inhibits the breakdown of glycogen, protein and fat.

Insulin is widely used in media formulations.
Ready to use solution of insulin. Suitable for use in media formulations when the use of animal-derived proteins is to be avoided.

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Hydrolysates

 

Traditionally, media formulations are based upon the addition of individual L-amino acids.  These are often referred to as essential and non-essential amino acids.  It is now understood that cells can also use amino acids that are in the peptide form. Peptides can range in size from two amino acids to many amino acids. The size and make-up of the peptide mixture is believed to affect their availability to cells.  Peptides that are derived by the hydrolysis of proteins provide a supplemental (<) source of amino acids in a form that supports superior growth of a number of cell types.

Sigma offers hydrolysates from various non-mammalian protein sources to provide different mixtures of amino acids in peptide form for use in cell culture.

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Product # Product Name Description Application
H 6784 HyPep 4601TM
Protein Hydrolysate
Wheat gluten derived
Animal component-free.
  • Powder;
  • Formulated to supplement at 1 to 5 gm per liter of medium;
  • Endotoxin tested, by supplier;
  • Cell culture tested.

Recommended for use as a rich source of L-glutamine and other small peptides in cell culture systems when the use of animal-derived proteins is to be avoided.

P 8358 Peptone,
Fish Derived.
  • Powder;
  • Formulated to supplement at 1 to 5 gm per liter of medium;
  • Cell culture tested.

This supplement is useful for delivering additional amino acids, in the form of small peptides, to the cell culture medium. Supplementation of a medium with peptides often results in superior cell proliferation.

P 0521 Peptone, Type IV;
Soybean derived.
(HySoy
TM)
  • Powder.

This is an enzymatic hydrolysate of soybean protein  useful in microbial fermentation and mammalian cell culture.  Supplementation of a medium with peptides often results in superior cell proliferation.

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Lipids and Lipid Carriers

 

Lipids are essential to the proliferation, growth and survival of all cells.  Among the critical building blocks of lipids are cholesterol and the fatty acids.

Cholesterol is a major component of all biological membranes (approximately 25% of the total brain is cholesterol), and it is an important building block used by cells to make other lipids. Free fatty acids are incorporated into a large number of lipid molecules essential to cellular structures such as the cell membrane.

Cholesterol and fatty acids are not very soluble in water.  In addition, unsaturated fatty acids are susceptible to oxidative degradation and free radical generation, which is harmful to cultured cells.  Sigma offers a number of formulations of cholesterol and free fatty acids that are designed to increase their solubility and to protect them from oxidation.

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Product # Product Name Description Application
C 9913 Cholesterol, synthetic
  • Liquid;
  • Purity, not less than 95%;
  • Synthesized from materials of non-animal origin;
  • Molecular  Weight=386.7.

Use as a medium supplement in situations where the use of animal-derived components is to be avoided.

Sigma is the only commercial manufacturer of synthetic cholesterol.

C 4951 Cholesterol,
Water Soluble
  • Powder;
  • Contains approx. 40 mg of cholesterol per gram; balance is methyl-beta-cyclodextrin;
  • Soluble at 200 mg/ml in water;
  • Cell culture tested.

Cholesterol is insoluble in water. This product is formulated with methyl-beta-cyclodextrin to improve its solubility and make it more useful as a medium supplement.

F 7050 Fatty Acid Supplement
  • Liquid;
  • Proprietary mixture of fatty acids;
  • Use at 0.25 to 0.5 ml per liter of medium;
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

This mixture of fatty acids was developed to optimize the growth of and expression of protein by Chinese Hamster Ovary (CHO) cells in serum-free culture. However, it may prove useful for proliferation of a wide range of mammalian cell types.

Animal component-free. May be used as a medium supplement in situations where the use of animal-derived components is to be avoided.

F 7175 Fatty Acid-Albumin Supplement
  • Liquid;
  • Contains 100 mg/ml of bovine serum albumin in DPBS (D 5652);
  • Contains 2 moles of linoleic and 1 mole of oleic acid per mole of albumin;
  • Use at 0.5 to 1.0 ml per liter of medium;
  • Sterile filtered;
  • Cell culture tested, CHO, L929, and BHK.

Useful for the development of serum-free medium. Linoleic and oleic acids are important fatty acid found associated with albumins in nature. Albumin helps to improve the solubility of fatty acids and to protect them from oxidation. Historically, fatty acids have  been added to cell cultures as components of serum.

This supplement is recommended for use with epithelial-derived cells. However, it may prove useful for proliferation of a wide range of mammalian  cell types.

L 8384 Linoleic Acid-Albumin.
  • Powder, lyophilized;
  • Contains 1.1 to 2.3 moles of linoleic acid per mole of bovine serum albumin;
  • Sterilized by gamma irradiation;
  • Cell culture tested.

Useful for the development of serum-free medium. Linoleic acid is an important unsaturated fatty acid that is found associated with albumins in nature. Albumin helps to improve the solubility of linoleic acid and to protect it from oxidation. Historically, it has been added to cell cultures as a component of serum.

L 9655 Linoleic, Oleic,
Acid-Albumin.
  • Liquid;
  • Contains 100 mg/ml of bovine serum albumin in DPBS (D 5652);
  • Contains 2 moles each of linoleic and oleic acids per mole of albumin;
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

Useful for the development of serum-free medium. Linoleic and oleic acids are important unsaturated fatty acids that are found associated with albumins in nature. Albumin helps to improve the solubility of linoleic and oleic acids and to protect them from oxidation. Historically, they have  been added to cell cultures as a components of serum.

L 0163 Linoleic, Oleic, Arachidonic
Acid-Albumin.
  • Liquid;
  • Contains 100 mg/ml of bovine serum albumin in DPBS (D 5652);
  • Contains 2 moles each of linoleic, oleic and arachidonic acids per mole of albumin;
  • Use at 0.5 to 1.0 ml per liter of medium;
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

Useful for the development of serum-free medium. Linoleic, oleic and arachidonic acids are important fatty acids that are found associated with albumins in nature. Albumin helps to improve the solubility of linoleic, oleic and arachidonic acids and to protect them from oxidation. Historically, they have been added to cell cultures as a components of serum.

When evaluating our lipid loaded albumins, we recommend that L 0163 be evaluated first.  It is the most extensively lipid loaded albumin that Sigma offers.

L 0288 Lipid Mixture 1,
Chemically Defined.
  • Liquid;
  • Contains non-animal derived fatty acids;
  • Contains 2 ug/ml arachidonic and 10 ug/ml each linoleic, linolenic, myristic, oleic, palmitic and stearic acids; 0.22 mg/ml cholesterol from New Zealand sheep's wool, 2.2 mg/ml Tween-80, 70 ug/ml tocopherol acetate and 100 mg/ml Pluronic F-68 solubilized in cell culture grade water (W 3500);
  • Use at 1 to 10 ml per liter of medium;
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

This supplement formulation is designed for use in serum-free formulations.

 It was developed to provide a source of fatty acids that are historically delivered into the cell culture system as components of serum. 

Tocopherol, in addition to being an important vitamin for cell proliferation, helps to reduce oxidation of the unsaturated fatty acids.

Tween-80 increases the solubility of the fatty acids and Pluronic F-68 helps to protect fragile cell membranes from disruption during agitation of suspension cultures.

O 3008 Oleic Acid-Albumin
  • Liquid;
  • Contains 100 mg/ml of bovine serum albumin in DPBS (D 5652);
  • Contains 2 moles of oleic acid per mole of albumin;
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

Useful for the development of serum-free medium. Oleic acid is an important fatty acid that is found associated with albumins in nature. Albumin helps to improve the solubility of oleic acid and to protect it from oxidation. Historically, it has been added to cell cultures as a component of serum.

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Other Supplements

 

Sigma offers a wide range of cell culture supplements.  This table is a listing of Sigma products that may be particularly useful in various biotechnology cell culture application and techniques.

     

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Product # Product Name Description Application
A 1345 Anti-oxidant Supplement (1000x)
  • Liquid;
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

Proprietary medium supplement specially formulated to boost cell growth for enhanced protein expression. May be used in combination with Polyamine Supplement (P 8483)

C 6295 Cell Freezing Medium-DMSO
Serum-free
  • Liquid, frozen;
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

Complete ready-to-use medium containing Minimum Essential Medium, dimethyl sulfoxide (8.7%) and methylcellulose. Prepared according to the formulation of Waymouth (TCA Manual 2(1), 311-313 (1976). Use to  cryo-preserve cells.

D 8537 Dulbecco's Phosphate Buffered Saline
Modified
  • Liquid;
  • Without calcium chloride and magnesium chloride;
  • Cell culture tested;
  • Sterile-filtered;
  • Endotoxin tested.

Calcium and magnesium promote cell adhesion. Use this formulation of D-PBS when tissue or cell dissociation or release is the objective.

D 8662 Dulbecco's Phosphate Buffered Saline
  • Liquid;
  • With calcium chloride and magnesium chloride;
  • Cell culture tested;
  • Sterile-filtered;
  • Endotoxin tested;

Known as DPBS, this is a general use, isotonic saline solution for washing cells and tissues.

It contains calcium and magnesium ions which facilitate cell attachment.

G 7513 L-Glutamine Solution, 200 mM
  • Liquid;
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

Prepared in cell culture grade water (W 3500).  Useful for supplementing CHO, hybridoma cell and other mammalian cell media.

H 9394 Hanks' Balanced Salt Solution
Modified
  • Liquid;
  • With sodium bicarbonate;
  • Without calcium chloride, magnesium sulfate;
  • Sterile-filtered;
  • Endotoxin tested;
  • Cell culture tested.

Same as H 9269, except that calcium and magnesium have been removed.

Calcium and magnesium support cell adhesion.

Recommended for washing adherent cells during passage.

P 1300 Pluronic F-68,
Non-ionic Detergent
  • Liquid;
  • Cell culture tested.

A media additive often used to protect cells growing in suspended culture systems from damage which may occur during agitation and aeration.

P 8483 Polyamine Supplement (1000x)
  • Liquid;
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

Proprietary medium supplement formulated to boost cell growth for enhancement of protein expression.

T 3924 Trypsin-EDTA
Solution (1x)
  • Liquid, frozen;
  • Contains 0.5 grams of porcine trypsin and 0.2 grams of EDTA-4Na per liter of Hank's Balanced Salt Solution with phenol red;
  • Trypsin powder is negative for parvovirus by 9 CFR;
  • Sterile filtered;
  • Cell culture tested.

Use to release adherent cells from supporting surfaces during passage.

T 6414 Trypsin Inhibitor Solution (1x)
  • Liquid, frozen;
  • Contains chromatographically purified soybean trypsin inhibitor and iron supplemented calf serum (C 8056);
  • Prepared in Dulbecco's Phosphate Buffered Saline without calcium or magnesium;
  • Sterile filtered;
  • Endotoxin tested;
  • Cell culture tested.

Use to arrest the enzymatic activity of trypsin during passage of adherent cell lines.

W 3500 Water
  • Liquid;
  • Sterile filtered;
  • Endotoxin tested, not more than 1.0 EU/ml;
  • Cell culture tested.

This water is produced by a validated WFI system and is quality controlled for use in cell culture. Use cell culture grade water to solubilize components and supplements for addition to cell culture medium.

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