Mammalian Cell Culture

Growing Mammalian Cells In Vitro

Cell growth requires a complex mixture of nutrients including sugars, amino acids, albumin, vitamins, minerals, and growth factors. Cell culture media supply these essential nutrients for in vitro mammalian cell culture. Mammalian cell cultures are grown in incubators that provide a sterile environment for growth at optimum temperature (37 °C) with 5% CO₂ to maintain pH levels similar to that of mammalian blood.  

Except for certain types of blood cells that can be cultured in suspension, most cells derived from mammalian tissues require adherence to a surface for proper cell division and growth. Cell culture surfaces are provided by treated polystyrene or glass flasks, plates, and filters designed for cell attachment. Cell attachment can be facilitated by using synthetic polymers such as poly-D-lysine (PDL) and extracellular matrix proteins including collagen that provide an attachment framework for adhesion.  

Techniques in Mammalian Cell Culture

Aseptic techniques are used to prevent contamination of cells grown in vitro. Mammalian cell cultures require passaging or subculturing – which involves dilution of cells that have reached confluence and replacement of depleted culture media – for cell health and propagation. To passage adherent cell cultures, cells must be dissociated from culture surfaces using enzymatic methods such as trypsinization or mechanical methods such as scraping prior to transfer to new culture flasks or plates for re-attachment. Cell counting methods using hemacytometers and automated cell counters assess cell viability and help to determine seeding densities for passaging and proper cell densities for downstream processes. Cultured cells can be cryopreserved by storage in liquid nitrogen, from which seed stocks can be thawed when working stocks are needed again.