The development of robust and well-characterized methods of production of cell therapies has become increasingly important as therapies advance through clinical trials toward approval. A successful cell therapy will be a consistent, safe, and effective cell product, regardless of the cell type or application. Process development strategies can be developed to gain efficiency while maintaining or improving safety and quality profiles. This review presents an introduction to the process development challenges of cell therapies and describes some of the tools available to address production issues. This article will provide a summary of what should be considered to efficiently advance a cellular therapy from the research stage through clinical trials and finally toward commercialization. The identification of the basic questions that affect process development is summarized in the target product profile, and considerations for process optimization are discussed. The goal is to identify potential manufacturing concerns early in the process so they may be addressed effectively and thus increase the probability that a therapy will be successful. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:1155-1163 SIGNIFICANCEThe present study contributes to the field of cell therapy by providing a resource for those transitioning a potential therapy from the research stage to clinical and commercial applications. It provides the necessary steps that, when followed, can result in successful therapies from both a clinical and commercial perspective.
Engineering functional activity of liver cell cultures requires the modulation of specific cell-cell interactions. We have investigated the quantitative role of systematically varied presentation of the cell-cell adhesion molecule, E-cadherin, on the differentiated function of cocultured parenchymal liver cells, hepatocytes. Specifically, we incorporated different proportions of E-cadherin transfected L-929 chaperone cells and untransfected chaperone cells, within cultures of primary rat hepatocytes on a collagen substrate. By using a strongly adhesive substrate that restricted cadherin-induced variations in cell spreading and growth-arresting chaperone cells, we could carefully isolate the potential role of cell-cell adhesion on cell differentiation. Using immunofluorescence microscopy, we confirmed that cadherins expressed at hepatocyte-hepatocyte contacts as well as hepatocyte-chaperone contacts were crossreactive. However, hepatocytes cocultured with cadherin-presenting chaperone cells had a 55-65% increase in longterm function over hepatocytes cocultured with control, nonpresenting chaperone cells. Notably, the cadherin-induced increase in function occurred over and above the basal, coculture-induced functional elevation. Further, we quantified the stoichiometric importance of cadherin contacts by comparing established markers of hepatocyte functional activity across a graded range of E-cadherin presentation. At low levels of cadherin-mediated contacts, the induction of differentiated function was weak, while high levels of contacts elicited a marked increase in function. Thus, hepatocyte biochemical functions (albumin and urea secretion) were biphasically governed by the degree of cadherin-based contacts presented during culture. Overall, our results demonstrate the unequivocal role of cell-cell adhesion molecules in hepatocyte functional engineering, through the graded use of cadherin presentation from functionally incompetent, heterotypic chaperone cells.
The successful development of bioartificial and cell-based liver support systems relies on the identification of molecular mechanisms controlling the balance between hepatocellular proliferation and differentiation. Although a definitive function-inductive role for the cell-cell adhesion molecule, E-cadherin, was established through lateral cadherin-cadherin engagement in hepatocyte cocultures (Brevia, T.A., and Moghe, P.V. Biotechnol. Bioeng. 76, 295, 2001), the roles of other modes of cadherin presentation are not well understood. Further, alternative cadherin display configurations promoting cell growth/proliferative pathways, a major requisite for sustainable engineered tissues, remain to be identified. In this report, we employed protein A-functionalized polymeric microsphere substrates that specifically bound self-dimerizing cadherin-IgG/Fc fusion chimeras via their Fc regions, thereby orienting them outward for active adhesion, and presented the E-cadherin chimeras basally to cultured rat hepatocytes to study the effects of cadherin display on cell proliferative potential and differentiated function. In contrast to the previously documented function-inductive roles of laterally expressed cadherin, basal acellular cadherin presentation resulted in an increase in hepatocyte DNA synthesis and cell divisions, accompanied by a decrease in the expression of a key marker of liver-specific function, albumin message levels. Next, we probed the relative effect of basal exogenous display of acellular cadherins on the inductive phase of differentiation within hepatocyte cocultures with cadherin-expressing L929 cells. When acellular cadherins were applied to hepatocyte cocultures involving chaperone cell-mediated cadherin presentation, the previously reported function-inductive effects of cadherins on hepatocyte function were reversed, resulting in lower levels of albumin and urea secretion indicating a dominance of acellular cadherin effects. Our results demonstrate that cadherins are important regulators of the balance between hepatocyte differentiation and proliferation, and furthermore that the direction of balance shift may be dependent on the method of cadherin presentation. Thus, the geometric display of cadherin could be a potential parameter to switch hepatocyte functional-proliferative balance and may aid in customizing scaffolds for regulating hepatic tissue dynamics.
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