Industrialized Production of Human iPSC-Derived Cardiomyocytes for Use in Drug Discovery and Toxicity Testing - The authors describe an industrialized process for the manufacture of iPSC-derived human

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Industrialized Production of Human iPSC-Derived Cardiomyocytes for Use in Drug Discovery and Toxicity Testing
The authors describe an industrialized process for the manufacture of iPSC-derived human cardiomyocytes.


BioPharm International
Volume 24, Issue 3, pp. 58-67

MANUFACTURE OF HUMAN IPSCS

iPSCs are developed from human fibroblasts, skin, blood, or other somatic cell types collected from individuals. The purified somatic cells are exposed to reprogramming agents that stimulate de-differentiation into iPSCs. Typically, reprogramming somatic cells is a slow and complicated process to which many investigators have contributed different methodologies (8). Originally, the reprogramming process required genetic modification of the source material with viral vectors that permanently integrate into seemingly random locations within the host DNA. Although the reprogrammed cells exhibited the properties of stem cells, the integrated vectors limited the usefulness of the cells to clinical or drug testing applications because of the perceived potential mutagenic or oncogenic effects of the integrated DNA. New reprogramming methodologies have been developed that largely overcome this barrier (9, 10) so that reprogrammed cells do not have foreign DNA, vector or otherwise, integrated into the genome, thus creating iPSC lines with potential applications in clinical as well as discovery settings.


Figure 1. Basic steps in human iPSC-derived cardiomyocyte production.
The key to the mass production of iPSCs is to develop a process that is both scalable and standardizable (see Figure 1). iPSCs, by their nature, are highly proliferative and have the potential to greatly expand their numbers under cell culture conditions. However, they are also very sensitive to manipulation and thus require special treatment and expertise to prevent entry into various non-directed differentiation pathways, events which greatly reduce the ability of the cell population to undergo directed differentiation and reduce the health of the relatively few terminally differentiated cells that are produced.

To prevent entry of iPSC populations cultured under standard conditions into non-directed differentiation pathways, such differentiated cells must be removed and "weeded out" of the population. This weeding step is subjective, labor-intensive, and highly dependent on the skill and attention of the technician. Therefore, to manufacture the necessary number of iPSCs needed to produce terminally differentiated cells for use by the pharmaceutical industry, we have simplified the process to enable standardization and assembled it into a highly parallel structure.

The major production constraint of cell weeding was eliminated by developing a proprietary culture system that a) used standard single-cell splitting techniques to eliminate the need for periodic weeding, and b) added small molecules to the cell cultures to promote survival and proliferation.

Scalability was incorporated into the process by building the cell culture system in a highly parallel nature that enabled the production of billions of iPSCs through the use of CellSTACK culture chambers (Corning, Lowell, MA) rather than standard tissue culture or T-flasks. The large surface area to footprint ratio of the CellSTACK system enabled parallel culturing of iPSCs and resulted in a significant expansion in iPSC production. Currently, this "industrialized" process can generate 100 billion or more iPSCs per month with a small team of manufacturing technicians. Because manufacturing is standardized, production levels can be increased through the addition of additional cell-culture manufacturing lines.

CARDIOMYOCYTE DIFFERENTIATION AND PURIFICATION


Figure 2. Purification of cardiomyocytes. The top row illustrates cardiomyocyte aggregates prior to antibiotic selection (13.39% purity). The bottom row shows cardiomyocytes after antibiotic selection (98.05% purity).
Another barrier to the use of iPSC-derived cell types for drug discovery is the production of highly purified terminally differentiated cell types. Random in vitro differentiation, for example through the embryoid body method, is inherently inefficient. Our proprietary directed differentiation method has been able to increase the efficiency of cardiomyocyte differentiation by orders of magnitude (see Figure 2). However, the final cell product requires greater cell purity in order to ensure that an observed experimental response is due to an effect on the target cell type and not other contaminating cells.

CDI has achieved greater cell purity with the following procedure. Prior to iPSC clonal expansion, genes encoding antibiotic resistance and red fluorescent protein (RFP) under control of a pan-cardiac promoter are introduced into the iPSCs through homologous recombination. The use of homologous recombination can target a location on the host chromosome and insert an element of choice, ensuring minimal disruption of endogenous genes. After curation and quality control, the iPSC clone carrying the selectable marker is expanded in multiple CellSTACKs to produce sufficient iPSCs for the cardiomyocyte differentiation. Once expanded, iPSCs are harvested and seeded into spinner flasks with the presence of small molecules and growth factors. Beating aggregates of cardiomyocytes are observed within days after withdrawal of the growth factor cocktail.

Through this method alone, we have been able to routinely achieve cardiomyocyte purities greater than 50% (see Figure 2). As the cardiomyocytes contain the exogenous antibiotic resistance gene while "contaminating" non-cardiomyocytes do not, the population purity is subsequently increased to approximately 100% through exposure to antibiotic. RFP expression confirms the increase in purity. Purified cardiomyocytes are matured in cardiomyocyte maintenance medium prior to cryopreservation. Following re-animation from the cryopreserved state, the high level of cardiomyocyte purity is maintained by culturing the cells in a proprietary medium developed by CDI that eliminates the growth of proliferating (i.e. non-cardiomyocyte) cells and allows the end-user to maintain pure cardiomyocyte cultures in vitro for weeks.


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