As demand for stem cells for both drug discovery andclinical applications grows, effectively translating the promise of stem cellsinto therapeutic reality will require large-scale "industrialized" productionunder tightly controlled conditions. Achieving this level of production—whilemeeting rigorous quality and regulatory standards—will depend on furtherprogress in the areas of cell culture and scale-up, characterization,enrichment, purification and process control to deliver a consistent andreproducible supply of cells in a safe and cost-effective manner.
Stem cell-based clinical trials require well-characterizedcells produced under tightly controlled, consistent, reproducible cultureconditions that adhere to Current Good Manufacturing Practice (cGMP) standards.
cGMP stem-cell culture systems will need well-defined,optimized media and supplements to support stem cell expansion anddifferentiation. The use of efficient, standardized methods for growing andharvesting cells will ensure consistent cell populations with uniformproperties and predictable behaviors.
When used for basic research applications, stem cells aretypically grown in small-scale, tissue culture flasks using media and culturesupplements (e.g., growth factors)that are not always fully defined or characterized, and in some cases, ofanimal origin. Human embryonic stem cell cultures were originally grown on"feeder layers" of mouse fibroblast cells. While the soluble factors secretedby the mouse cells help provide the proper environment for stem cellproliferation, use of feeder layers or co-culture systems have significantdrawbacks when culturing stem cells for clinical applications. Furthermore, the use of undefined matricesused for adherent cells also is undesirable.
As more stem cell-based therapeutics progress towardsclinical testing, the consistency, quality and reproducibility of large-scaleculture systems become an imperative. When manufactured under cGMP conditions,supplements and cell-binding matrices enabling robust proliferation of stemcells in culture will be required. Use of cGMP supplements contributes tohigh-quality, consistent and reproducible culture conditions.
Integrated cell'manufacturing' systems
Because stem cells themselves are the "product," culturesystems must minimize variability, effectively control differentiation, enableharvesting and formulation without damaging cells and incorporate processes toensure cell viability during storage, transport and administration to thepatient.
Large-scale, economical production of stem cells willrequire fully integrated, scalable systems that include:
- Microcarriertechnology or alternative solutions that enable particulate-free culture ofstem cells in a bioreactor. When cultured in bioreactors, adherent stemcells must be grown on a solid surface such as microcarriers. However, smallparticulates, or "fines," are often generated during the microcarriermanufacturing process and can find their way into the culture system. Fines canalso result from beads being crushed during the cell harvest process. As stemcell cultures cannot be readily filtered to remove these particulates, anysmall particles will be co-purified with the cells. The presence of foreignparticulate matter such as microcarrier fines is unacceptable for injectableproducts.
- Bioreactorsoptimized for stem cell culture. Existing bioreactor technology is designedprimarily to support the production of proteins expressed in the supernatant ofcell cultures. They provide an efficient, scalable method for production andallow for direct monitoring and control. Supernatant samples are easilyextracted from the reactor for analysis. In the case of stem cell cultures,however, bioreactors must allow rapid sampling of small volumes containing theactual cells. Stem cell cultures need to be well mixed in the bioreactor priorto sampling as they tend to settle quickly. Because the cells are the actualproduct (in contrast to protein therapeutics produced by cells), the samplesize must be small to not waste valuable product and processed rapidly whilethe cells are still viable.
- Technologyfor harvesting and packaging of live cells. Existing centrifugation andfiltration technologies are not optimized for the harvest and recovery of livecells. While centrifugation is often used to collect cells for researchapplications, it is not always practical for collection of large numbers ofstem cells. Centrifugation is typically not a closed system, and shear forcescan damage cells. Once cells are harvested, they must be rapidly concentrated,the media washed away with buffer solution and packaged into containers forfreezing or administration to patients. No systems currently exist toefficiently manage this fill/finish process for stem cells.
In a recent study, EMD Millipore reported on the growth ofmesenchymal stem cells (MSCs) in a 3L single-use, stirred tank bioreactor incombination with microcarriers. MSCs aremultipotent with an ability to differentiate into a variety of cell typesincluding osteoblasts, chondrocytes and adipocytes. These cells have beenexplored for the repair and regeneration of connective tissues such ascartilage and bone and for transfusion therapy in patients following bonemarrow or peripheral blood stem cell transplants to reduce complications fromlife-threatening graft-versus-host disease.
Clinical demand for MSCs is driving the need for developmentof robust large-scale production, beyond what can be delivered using 2D tissueculture vessels. The study demonstrated the utility of collagen-coatedmicrocarriers in a 3L single-use bioreactor for the expansion of human bonemarrow-derived MSCs. Different microcarrier types were evaluated for theirability to support MSC attachment, growth and viable detachment.
MSCs were able to propagate in the 3L single-use bioreactorfor five days, while doubling the working volume, with a greater than five-foldincrease in total cell number. MSCs werecapable of growing for multiple passages after being removed from thebioreactor and showed similar levels of gene and protein expression of MSCcharacterization genes. Comparison to flat culture showed that no differencescould be detected using both FACS and gene array analysis. Afterdifferentiating to adipocytes, both the cells from the 3L bioreactor and cellsgrown on gelatin contained lipid vacuoles that stained positively red,confirming successful differentiation.
In addition to their direct use in the area of regenerativemedicine, stem cells offer unique advantages when incorporated into thesmall-molecule drug discovery and development process. Stem cells are now beingused to elucidate disease mechanisms and pathways, facilitate novel targetdiscovery, assess and optimize lead compounds and improve metabolic profilingand toxicity evaluation. One area that is receiving a great deal of attentionis the use of stem cell-derived human hepatocytes in investigative toxicitystudies.
Drug-induced liver injury is one of the principal reasonsclinical trials are suspended and approved drugs withdrawn from market. Infact, drug-induced liver injury has been the most frequent single cause ofsafety-related withdrawals of marketed drugs in the United States over the past50 years.
Investigative in-vitroliver toxicity studies are typically conducted using primary humanhepatocytes or an immortalized (genetically transformed) liver-derived cellline such as HepG2. Despite routine use for investigative toxicity, both ofthese options present significant drawbacks:
- Primary human hepatocytes are derived from freshliver tissue, typically sourced from cadavers or cancer resections. Supply ofthese cells can be limited and the tissue can vary widely among donors.
- Primary hepatocytes cannot be sustained for morethan a few days in culture without losing function. Securing a consistentsupply of cells requires repetitive sourcing, which further contributes tovariability.
- Immortalized hepatocyte cell lines can becultured indefinitely, which addresses the supply and variability issuesassociated with use of primary human hepatocytes. However, these cells displaydistinct differences from normal liver cells and may not exhibit normal cellbehavior or response. For example, most cytochrome P450 enzymes (responsiblefor drug metabolism) are expressed only weakly in HepG2 cells compared tonormal human hepatocytes.
The challenges of hepatocyte-based, in-vitro toxicity testing have led to reliance on animal models forpreclinical metabolism and toxicity testing. But animal models also havelimitations. Animal models may not be fully and reliably predictive of humantoxicity, are low-throughput and expensive and raise ethical concerns for some.
Cost and throughput often relegate use of animal models tothe later stages of preclinical development, after a company has investedsignificant resources and time in a lead compound. This delayed evaluation oftoxicity contributes to the high failure rate of compounds in late-stagepreclinical testing, which is extremely costly.
Earlier, more effective assessment of drug candidatetoxicity has the potential to reduce the attrition rate of drugs in laterstages of development.
Differentiation and expansion of human stem cells intohepatocytes for use in investigative toxicity studies could overcome theshortcomings of primary hepatocytes and immortalized cell lines. Use of stemcell-derived hepatocytes (and other cell types commonly used for toxicitystudies) offers a number of important advantages to investigative toxicitystudies, including availability of a consistent source of cells that moreclosely match in-vivo phenotype andphysiology; elimination of reliance on donor sources which can have sporadicavailability; a more standardized, reproducible process for toxicity testing;reduction in the use of animal models and animal tissue; and improvement in thepredictive capabilities of early toxicity studies leading to reduction inlate-stage attrition of drugs.
More efficient and predictive toxicity studies enabled bystem cell-derived cells can be expected to reduce development costs associatedwith the late-stage failure of drug candidates. Identification of drugcandidates with toxicity issues earlier in the discovery process will result inimproved safety for clinical trial participants and patients.