Supplementary MaterialsS1 Fig: hiPSC (Gibco hiPSC line) expansion in E8 medium, inside a 50 mL spinner flask. Systems Cardiomyocytes Differentiation Kit by plating microcarriers with hiPS cells in low-attachment plates. Beating cell-VtnM aggregates in low-attachment plate were observed at day time 10 of differentiation.(MOV) pone.0151264.s005.mov (6.5M) GUID:?2187E08B-C6CE-4C37-B0B6-1531E04FC693 S3 Video: Human being iPSCs (Gibco hiPSC line) cultured in spinner flasks with E8 medium and VtnB retain their differentiation potential. CM, acquired on VtnM after 10 days of differentiation using Existence Systems Cardiomyocytes Differentiation Kit, were re-plated onto GP and it was observed the presence of contracting colonies.(MOV) pone.0151264.s006.mov (3.2M) GUID:?AA23A202-FBE2-4627-9A6F-F5DCD1947EFD Data Availability StatementAll relevant data are within the paper and its Supporting Information documents. Abstract Human being induced pluripotent stem (hiPS) cell tradition using Essential 8? xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell growth either in coated plates or on polystyrene-coated microcarriers, while keeping hiPS cell features and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation rate, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1 1.4×106 cells/mL after 10 days of culture. After dynamic tradition, hiPS cells managed their standard morphology upon re-plating, exhibited pluripotency-associated marker manifestation as well as tri-lineage differentiation ability, which was verified by inducing their spontaneous differentiation through embryoid body CX-6258 formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, strong and cost-effective xeno-free tradition system was successfully developed and implemented for the scale-up production of hiPS cells. Introduction Human being induced pluripotent CX-6258 stem (hiPS) cells are capable of self renewing indefinitely, and to differentiate into all the cell forms of the body [1]. Because of these characteristics, analogous to human being embryonic stem (hES) cells, hiPS cells are encouraging sources for a number of biomedical applications [2]. However, to fully understand the potential of hiPS cells for cellular therapy, drug testing and disease modelling, the development of standardized and strong scalable processes to produce large numbers of these cells while keeping their critical biological functionality and security are of perfect importance. Typically, hiPS cells are expanded using adherent static cell tradition systems that cannot provide a LIMK2 sufficient number of cells for downstream applications, showing low cell yields and inherent variability of the tradition process and of the final product. Translating cell tradition from static plates to suspension systems is needed to accomplish scalability of the process. Stirred bioreactors are CX-6258 an appropriate tradition system for moderate large-scale cell production given their robustly controlled operation and well-established scale-up protocols [3,4,5]. Several methodologies for human being pluripotent stem (hPS) cell tradition in these systems have been implemented in the last few years, including cultivation of cells encapsulated typically inside hydrogels [6,7], adherent onto microcarriers [8,9], or as 3D aggregates in suspension [10,11]. Microcarrier technology confers unique advantages as it provides homogeneous tradition conditions to the cells, large surface areas for cell adhesion and growth [12,13] and importantly, a large surface/volume percentage. Also, microcarrier tradition on fully controlled bioreactors allows monitoring and controlling of environmental guidelines, and may become scaled up relatively very easily. Nevertheless, despite recent progress on scalable microcarrier hPS cell suspension tradition, most of the methods are based on the.