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Meeting the need for large-scale production of stem cells
July 2012
by Robert Shaw, EMD Millipore  |  Email the author
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As demand for stem cells for both drug discovery and clinical applications grows, effectively translating the promise of stem cells into therapeutic reality will require large-scale "industrialized" production under tightly controlled conditions. Achieving this level of productionówhile meeting rigorous quality and regulatory standardsówill depend on further progress in the areas of cell culture and scale-up, characterization, enrichment, purification and process control to deliver a consistent and reproducible supply of cells in a safe and cost-effective manner.  
 
Large-scale cGMP production
 
Stem cell-based clinical trials require well- characterized cells produced under tightly controlled, consistent, reproducible culture conditions 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 and differentiation. The use of efficient, standardized methods for growing and harvesting cells will ensure consistent cell populations with uniform properties and predictable behaviors.  
 
When used for basic research applications, stem cells are typically grown in small-scale, tissue culture flasks using media and culture supplements (e.g., growth factors) that are not always fully defined or characterized, and in some cases, of animal origin.  Human embryonic stem cell cultures were originally grown on "feeder layers" of mouse fibroblast cells. While the soluble factors secreted by the mouse cells help provide the proper environment for stem cell proliferation, use of feeder layers or co-culture systems have significant drawbacks when culturing stem cells for clinical applications.  Furthermore, the use of undefined matrices used for adherent cells also is undesirable.  
 
As more stem cell-based therapeutics progress towards clinical testing, the consistency, quality and reproducibility of large-scale culture systems become an imperative. When manufactured under cGMP conditions, supplements and cell-binding matrices enabling robust proliferation of stem cells in culture will be required. Use of cGMP supplements contributes to high-quality, consistent and reproducible culture conditions.  
 
Integrated cell 'manufacturing' systems  
 
Because stem cells themselves are the "product," culture systems must minimize variability, effectively control differentiation, enable harvesting and formulation without damaging cells and incorporate processes to ensure cell viability during storage, transport and administration to the patient.   
 
Large-scale, economical production of stem cells will require fully integrated, scalable systems that include:
  • Microcarrier technology or alternative solutions that enable particulate-free culture of stem cells in a bioreactor. When cultured in bioreactors, adherent stem cells must be grown on a solid surface such as microcarriers. However, small particulates, or "fines," are often generated during the microcarrier manufacturing process and can find their way into the culture system. Fines can also result from beads being crushed during the cell harvest process. As stem cell cultures cannot be readily filtered to remove these particulates, any small particles will be co-purified with the cells. The presence of foreign particulate matter such as microcarrier fines is unacceptable for injectable products.
  • Bioreactors optimized for stem cell culture. Existing bioreactor technology is designed primarily to support the production of proteins expressed in the supernatant of cell cultures. They provide an efficient, scalable method for production and allow for direct monitoring and control. Supernatant samples are easily extracted from the reactor for analysis. In the case of stem cell cultures, however, bioreactors must allow rapid sampling of small volumes containing the actual cells. Stem cell cultures need to be well mixed in the bioreactor prior to sampling as they tend to settle quickly. Because the cells are the actual product (in contrast to protein therapeutics produced by cells), the sample size must be small to not waste valuable product and processed rapidly while the cells are still viable.
  • Technology for harvesting and packaging of live cells. Existing centrifugation and filtration technologies are not optimized for the harvest and recovery of live cells. While centrifugation is often used to collect cells for research applications, it is not always practical for collection of large numbers of stem cells. Centrifugation is typically not a closed system, and shear forces can damage cells. Once cells are harvested, they must be rapidly concentrated, the media washed away with buffer solution and packaged into containers for freezing or administration to patients. No systems currently exist to efficiently manage this fill/finish process for stem cells.
In a recent study, EMD Millipore reported on the growth of mesenchymal stem cells (MSCs) in a 3L single-use, stirred tank bioreactor in combination with microcarriers.  MSCs are multipotent with an ability to differentiate into a variety of cell types including osteoblasts, chondrocytes and adipocytes. These cells have been explored for the repair and regeneration of connective tissues such as cartilage and bone and for transfusion therapy in patients following bone marrow or peripheral blood stem cell transplants to reduce complications from life-threatening graft-versus-host disease.  
Clinical demand for MSCs is driving the need for development of robust large-scale production, beyond what can be delivered using 2D tissue culture vessels. The study demonstrated the utility of collagen-coated microcarriers in a 3L single-use bioreactor for the expansion of human bone marrow- derived MSCs. Different microcarrier types were evaluated for their ability to support MSC attachment, growth and viable detachment. 
 
MSCs were able to propagate in the 3L single-use bioreactor for five days, while doubling the working volume, with a greater than five-fold increase in total cell number.  MSCs were capable of growing for multiple passages after being removed from the bioreactor and showed similar levels of gene and protein expression of MSC characterization genes. Comparison to flat culture showed that no differences could be detected using both FACS and gene array analysis. After differentiating to adipocytes, both the cells from the 3L bioreactor and cells grown on gelatin contained lipid vacuoles that stained positively red, confirming successful differentiation. 
 
Investigative toxicity testing  
 
In addition to their direct use in the area of regenerative medicine, stem cells offer unique advantages when incorporated into the small- molecule drug discovery and development process. Stem cells are now being used to elucidate disease mechanisms and pathways, facilitate novel target discovery, assess and optimize lead compounds and improve metabolic profiling and toxicity evaluation. One area that is receiving a great deal of attention is the use of stem cell-derived human hepatocytes in investigative toxicity studies.  
 
Drug-induced liver injury is one of the principal reasons clinical trials are suspended and approved drugs withdrawn from market. In fact, drug-induced liver injury has been the most frequent single cause of safety-related withdrawals of marketed drugs in the United States over the past 50 years.
 
Investigative in-vitro liver toxicity studies are typically conducted using primary human hepatocytes or an immortalized (genetically transformed) liver-derived cell line such as HepG2. Despite routine use for investigative toxicity, both of these options present significant drawbacks:
  • Primary human hepatocytes are derived from fresh liver tissue, typically sourced from cadavers or cancer resections. Supply of these cells can be limited and the tissue can vary widely among donors.  
  • Primary hepatocytes cannot be sustained for more than a few days in culture without losing function. Securing a consistent supply of cells requires repetitive sourcing, which further contributes to variability.
  • Immortalized hepatocyte cell lines can be cultured indefinitely, which addresses the supply and variability issues associated with use of primary human hepatocytes. However, these cells display distinct differences from normal liver cells and may not exhibit normal cell behavior or response. For example, most cytochrome P450 enzymes (responsible for drug metabolism) are expressed only weakly in HepG2 cells compared to normal human hepatocytes.

The challenges of hepatocyte-based, in-vitro toxicity testing have led to reliance on animal models for preclinical metabolism and toxicity testing. But animal models also have limitations. Animal models may not be fully and reliably predictive of human toxicity, are low-throughput and expensive and raise ethical concerns for some. 
 
Cost and throughput often relegate use of animal models to the later stages of preclinical development, after a company has invested significant resources and time in a lead compound. This delayed evaluation of toxicity contributes to the high failure rate of compounds in late-stage preclinical testing, which is extremely costly.  
 
Earlier, more effective assessment of drug candidate toxicity has the potential to reduce the attrition rate of drugs in later stages of development. Differentiation and expansion of human stem cells into hepatocytes for use in investigative toxicity studies could overcome the shortcomings of primary hepatocytes and immortalized cell lines. Use of stem cell-derived hepatocytes (and other cell types commonly used for toxicity studies) offers a number of important advantages to investigative toxicity studies, including availability of a consistent source of cells that more closely match in-vivo phenotype and physiology; elimination of reliance on donor sources which can have sporadic availability; a more standardized, reproducible process for toxicity testing; reduction in the use of animal models and animal tissue; and improvement in the predictive capabilities of early toxicity studies leading to reduction in late-stage attrition of drugs.  
 
More efficient and predictive toxicity studies enabled by stem cell-derived cells can be expected to reduce development costs associated with the late-stage failure of drug candidates. Identification of drug candidates with toxicity issues earlier in the discovery process will result in improved safety for clinical trial participants and patients.
 
Robert Shaw is the commercial director of EMD Millipore's Stem Cell Initiative in Billerica, Mass.


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