Specialty Media and Ingredients
Culture media to support human embryonic stem cell (hESC) research and therapy is one of the hottest topics in biotech. Media must be exquisitely tuned to maintain cells in their desired stage, or to promote their proliferation or differentiation as desired.
A good deal of hESC work was carried out in murine-conditioned media that contained components of mouse embryonic fibroblasts (MEF). The use of murine ingredients to support hESCs has been “imperative” for the evolution of stem cell work, says David C. Hay, Ph.D., principal investigator at the MRC Centre for Regenerative Medicine in Edinburgh, Scotland.
However, murine-conditioned stem cell media suffer from many of the same limitations as serum-based media in biomanufacturing. “These include a general lack of definition, batch-to-batch variation, labor-intensive production, and their xenobiotic nature.”
According to Dr. Hay, fully characterized and defined media will be essential for hESC culture to progress to cGMP quality for therapeutic applications. Taking two significant steps in this direction, Dr. Hay’s group has begun standardizing hESC culture with two serum-free media: mTeSR (Stem Cell Technologies) and STEMPRO® (Life Technologies). Both media are serum-free and promote maintenance and differentiation of hESCs. Until now, stem cells were propagated in serum-free media but differentiated and maintained in conditioned (serum-containing) media.
“We’ve taken an incremental step forward toward defining and understanding culture conditions required for large-scale manufacture,” Dr. Hay reports.
Initial results have been encouraging. Either medium maintains cells in a pluripotent (but undifferentiated state) for more than 30 passages, and produces volumes of human hepatic tissue equal to those generated from cells maintained and propagated in conditioned media.
Dr. Hay is also looking to incorporate CELLstart™ (Life Technologies), a completely humanized defined substrate, in his stem cell work. CELLstart is meant to be used with STEMPRO as a substrate for stem cell attachment under serum-free conditions.
Protein hydrolysates have become standard cell culture media additives, replacing serum as the “magic,” undefined ingredient in many cell cultures. Hydrolysates, which are produced from yeast, soy, and other nonanimal sources, are complex products containing numerous components in sometimes widely disparate concentrations. The principal hydrolysate components are peptides and amino acids, but they also contain fatty acids, trace elements, vitamins, and carbohydrates.
“Benefits of hydrolysates can vary dramatically based on the raw material source, choice of hydrolytic enzyme, and process parameters,” says Christopher Wilcox, Ph.D., R&D director at Sheffield Bio-Science.
“Enhancement to cell growth and productivity is subject to the additive effect of hydrolysate components native to the basal medium, and those added through the hydrolysate,” Dr. Wilcox says. “You may not see the full benefit of hydrolysates unless you do up-front work to determine whether you’re duplicating ingredients or overdosing.”
Since cells thrive on unique concentrations of media ingredients, processors must fully understand, before they turn to hydrolysates, if their serum-free basal media already contains components that will be introduced through hydrolysates as well.
Dr. Wilcox views hydrolysates not as another source, like serum, of inconsistency and unpredictability, but as a vital step for the industry as a whole in its quest for 100% defined media. He notes that within a particular class of hydrolysate Sheffield can provide numerous variants depending on the extent or chemical specificity of the hydrolysis, removal of certain components, and other factors.