Animal-Origin-Free Serum Substitute

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• Heat Inactivation of Fetal Bovine Serum (FBS)Heat inactivation of fetal bovine serum is a controlled 30-minute incubation at 56 degrees C that destroys the heat-labile proteins of the complement cascade before the serum is added to culture medium. The serum is thawed, brought to 37 degrees C, transferred to a 56 degrees C water bath, held for exactly 30 minutes from the moment the serum itself reaches temperature with gentle swirling every 5-10 minutes, then cooled immediately and aliquoted. For routine culture of established cell lines it is usually unnecessary, because complement activity in fetal serum is low and heating also degrades labile growth factors; it remains standard practice for immunological assays and complement-sensitive cells.
• Recombinant Human Albumin in Cell CultureRecombinant human serum albumin (rHSA) is a 585-amino-acid, 66.5 kDa non-glycosylated protein produced in yeast such as *Pichia pastoris* or in transgenic rice rather than purified from human plasma, and it is used as an animal-origin-free supplement in serum-free and chemically defined culture media. In culture it works as a carrier for fatty acids, lipids, hormones and trace elements, as an antioxidant through its free cysteine-34 thiol, as a scavenger that sequesters toxic metals and excess free fatty acids, and as a shear-protective surface-active protein in stirred and sparged culture. Typical working concentrations are 0.1-10 g/L, most often 0.5-5 g/L, and it is commonly supplied as a 200 mg/mL (20%) solution. Its performance depends heavily on what is bound to it, so fatty-acid-loaded and fatty-acid-free preparations behave quite differently.