Comparability of automated human induced pluripotent stem cell culture: a pilot study

Consistent and robust manufacturing is essential for the translation of cell therapies, and the utilisation automation throughout the manufacturing process may allow for improvements in quality control, scalability, reproducibility and economics of the process. The aim of this study was to measure and establish the comparability between alternative process steps for the culture of hiPSCs. Consequently, the effects of manual centrifugation and automated non-centrifugation process steps, performed using TAP Biosystems’ CompacT SelecT automated cell culture platform, upon the culture of a human induced pluripotent stem cell (hiPSC) line (VAX001024c07) were compared. This study, has demonstrated that comparable morphologies and cell diameters were observed in hiPSCs cultured using either manual or automated process steps. However, non-centrifugation hiPSC populations exhibited greater cell yields, greater aggregate rates, increased pluripotency marker expression, and decreased differentiation marker expression compared to centrifugation hiPSCs. A trend for decreased variability in cell yield was also observed after the utilisation of the automated process step. This study also highlights the detrimental effect of the cryopreservation and thawing processes upon the growth and characteristics of hiPSC cultures, and demonstrates that automated hiPSC manufacturing protocols can be successfully transferred between independent laboratories

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

A novel antiangiogenic and vascular normalization therapy targeted against human CD160 receptor

A monoclonal anti-CD160 antibody inhibits the growth of new vessels in pathological ocular and tumor neoangiogenesis but not in healthy tissues

Développement de nouveaux modèles in vivo pour l'étude de deux processus majeurs de la tumorigenèse (La métastase et l'apoptose)

La tumorigenèse est un processus composé de multiples étapes reflétant diverses altérations du génome susceptibles de perturber les voies de prolifération, de différenciation et de mort cellulaire. D'un point de vue clinique, l'émergence des métastases constitue l'étape critique de ce processus. Notre objectif a donc été de tenter de caractériser les cellules tumorales à un stade "pré-métastatique", afin d'identifier de nouveaux marqueurs utilisables en diagnostic précoce ou pour la thérapie. Nous avons étudié le processus métastatique par l'intermédiaire d'un modèle in ovo (membrane chorioallantoïde d'œuf embryonné de poule), lequel nous a permis de caractériser l'efficacité d'intravasation de diverses lignées cellulaires (myélomes et hybridomes de souris syngéniques). Nous avons pu montrer que ces lignées présentent des capacités métastatiques différentes. L'étude a été complétée par des méthodologies in vitro. Une analyse comparative de l'expression de 1176 gènes associés au cancer nous a conduit par ailleurs à mettre en évidence plusieurs voies cellulaires dont la dérégulation pourraient être associée à un phénotype métastatique précoce. Chez les métazoaires, la machinerie apoptotique s'oppose in vivo aux dérives prolifératives liées à l'émergence des métastases. La mitochondrie est fortement impliquée dans ce processus. La caractérisation des mécanismes de mort cellulaire programmée (MCP), est donc un élément clé dans la stratégie anti-cancéreuse. Afin de tenter d'ouvrir de nouvelles voies d'approches dans ce domaine, les phénomènes de MCP ont été étudiés chez certains organismes dépourvus de mitochondries. Nous avons ainsi pu mettre en évidence chez T. vaginalis ou G. intestinalis, une forme de MCP distincte de la nécrose, similaire mais non identique à celles décrites pour les organismes pourvus de mitochondries. Cette découverte ouvre d'intéressantes perspectives tant au niveau moléculaire qu'au niveau évolutif.COMPIEGNE-BU (601592101) / SudocSudocFranceF

The Future of Regenerative Medicine: Cell Therapy Using Pluripotent Stem Cells and Acellular Therapies Based on Extracellular Vesicles

The rapid progress in the field of stem cell research has laid strong foundations for their use in regenerative medicine applications of injured or diseased tissues. Growing evidences indicate that some observed therapeutic outcomes of stem cell-based therapy are due to paracrine effects rather than long-term engraftment and survival of transplanted cells. Given their ability to cross biological barriers and mediate intercellular information transfer of bioactive molecules, extracellular vesicles are being explored as potential cell-free therapeutic agents. In this review, we first discuss the state of the art of regenerative medicine and its current limitations and challenges, with particular attention on pluripotent stem cell-derived products to repair organs like the eye, heart, skeletal muscle and skin. We then focus on emerging beneficial roles of extracellular vesicles to alleviate these pathological conditions and address hurdles and operational issues of this acellular strategy. Finally, we discuss future directions and examine how careful integration of different approaches presented in this review could help to potentiate therapeutic results in preclinical models and their good manufacturing practice (GMP) implementation for future clinical trials

Supplementary Information Files for 'Distributed automated manufacturing of pluripotent stem cell products'

Supplementary Information Files for 'Distributed automated manufacturing of pluripotent stem cell products'Abstract: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.</div