Distributed automated manufacturing of pluripotent stem cell products

Establishing how to effectively manufacture cell therapies is an industry-level problem. Decentralised manufacturing is of increasing importance, and its challenges are recognised by healthcare regulators with deviations and comparability issues receiving specific attention from them. This paper is the first to report the deviations and other risks encountered when implementing the expansion of human pluripotent stem cells (hPSCs) in an automated three international site–decentralised manufacturing setting. An experimental demonstrator project expanded a human embryonal carcinoma cell line (2102Ep) at three development sites in France, Germany and the UK using the CompacT SelecT (Sartorius Stedim, Royston, UK) automated cell culture platform. Anticipated variations between sites spanned material input, features of the process itself and production system details including different quality management systems and personnel. Where possible, these were pre-addressed by implementing strategies including standardisation, cell bank mycoplasma testing and specific engineering and process improvements. However, despite such measures, unexpected deviations occurred between sites including software incompatibility and machine/process errors together with uncharacteristic contaminations. Many only became apparent during process proving or during the process run. Further, parameters including growth rate and viability discrepancies could only be determined post-run, preventing ‘live’ corrective measures. The work confirms the critical nature of approaches usually taken in Good Manufacturing Practice (GMP) manufacturing settings and especially emphasises the requirement for monitoring steps to be included within the production system. Real-time process monitoring coupled with carefully structured quality systems is essential for multiple site working including clarity of decision-making roles. Additionally, an over-reliance upon post-process visual microscopic comparisons has major limitations; it is difficult for non-experts to detect deleterious culture changes and such detection is slow.

Studies on Connexin 43, a Gap-Junction Protein, in P19 Embryonal Carcinoma Cells after Culture on an Apatite Fiber Scaffold

We previously showed enhanced osteoblast differentiation by culturing cells in apatite-fiber scaffold (AFS). The well-developed a-surface of the apatite fibers provides favorable structural features and chemical interactions with the cells, and the scaffold appears to affect cell differentiation. AFS was used here to study its utility for soft-tissue engineering. An embryonal carcinoma cell line, P19.CL6, was cultured in AFS, and the expression and phosphorylation of a gap-junction protein, connexin 43 (Cx43), during cell proliferation and differentiation was examined. We show that treatment with dimethyl sulfoxide appears to induce a change in the isoform composition of Cx43 under the control condition, but not in AFS. We also show that serum starvation induces the phosphorylation of Ser 368 of Cx43 only in functionally mature cells.

Effective and Steady Differentiation of a Clonal Derivative of P19CL6 Embryonal Carcinoma Cell Line into Beating Cardiomyocytes

The P19CL6 cell line is a useful model to study cardiac differentiation in vitro. However, large variations were noticed in the differentiation rates among previous reports as well as our individual experiments. To overcome the unstable differentiation, we established P19CL6-A1, a new clonal derivative of P19CL6 that could differentiate into cardiomyocytes more efficiently and stably than the parent using the double stimulation with 5-Aza and DMSO based on the previous report. We also introduced a new software, Visorhythm, that can analyze the temporal variations in the beating rhythms and can chart correlograms displaying the oscillated rhythms. Using P19CL6-A1-derived cardiomyocytes and the software, we demonstrated that the correlograms could clearly display the enhancement of beating rates by cardiotonic reagents. These indicate that a combination of P19CL6-A1 and Visorhythm is a useful tool that can provide invaluable assistance in inotropic drug discovery, drug screening, and toxicity testing.

Involvement of ubiquitous and tale transcription factors, as well as liganded RXRα, in the regulation of human SOX2 gene expression in the NT2/D1 embryonal carcinoma cell line

SOX2 is a key transcription factor in embryonic development representing a universal marker of pluripotent stem cells. Based on the functional redundancy and overlapping expression patterns of SOXB1 subgroup members during development, the goal of this study has been to analyze if some aspects of regulation of expression are preserved between human SOX2 and SOX3 genes. Thus, we have tested several transcription factors previously demonstrated to play roles in controlling SOX3 gene activity for potential participation in the regulation of SOX2 gene expression in NT2/D1 cells. Here we report on the activation of SOX2 expression by ubiquitous transcription factors (NF-Y, Sp1 and MAZ), TALE family members (Pbx1 and Meis1), as well as liganded RXR?. Elucidating components involved in the regulation of SOX gene expression represent a valuable contribution in unraveling the regulatory networks operating in pluripotent embryonic cells.