Dissociating enzymes and attachment factors

A common observation is that end users will specify a cell culture environment entirely free of animal-origin medium constituents, yet persist in dislodging adherent cells from the substratum using porcine pancreatic trypsin. Despite certified vendor processing by gamma irradiation, porcine trypsin represents a significant potential source of parvovirus contamination. We recently investigated and subsequently commercialized a recombinant trypsin-like protease with comparable cell removal kinetics and biological performance to animal origin trypsin, but with superior purity and stability (Nestler et al. 2004) (see also Section 31.5).

Like growth factors (noted above), the most widely used attachment factors in cell culture were historically derived from animal blood fractions (e.g. fibronectin, vitronectin). The response to animal origin concerns has been least successful for adherent cells. There exist multiple nutrient formulations that can support production applications of cells already attached in serum-supplemented media to harvest a target protein or vaccine for human or veterinary applications. Modifications to the basal medium may preferentially support cell attachment or synthesis of extracellular matrix elements. Substrata may be modified by charge density or impregnation of synthetic recognition sequences to enhance cell surface protein recognition and initial attachment. However, development of a stable, cost-effective serum-free medium for extended serial passaging of adherent cells without diminished performance has been elusive.

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