Therapeutic and industrial applications of pluripotent stem cells and their derivatives require large cell quantities generated in defined conditions. and ability of cells to differentiate into derivates of all three germ layers was managed, Gedatolisib underlining practical power of this new process. The offered data provide important actions toward scalable mass growth of human iPS and ES cells thereby enabling translation of stem cell research to (pre)clinical application in relevant large animal models and useful assays for drug development and affirmation as well. Introduction Human pluripotent stem cells (hPSCs; including human induced pluripotent stem cells (hiPS) and human embryonic stem cells (hESC)) and their progenies are considered excellent research tools to elucidate cellular mechanisms of stemness and differentiation, and to investigate molecular disease pathways as well. Induction of pluripotency in somatic cells further stimulated consideration of such cells for ARPC1B cellular therapies.1,2 Estimations suggest that billions of cells per single patient will be required to replace substantial, irreversible cell loss induced by metabolic, inflammatory, or other disorders, such as neurodegeneration, cardiovascular disease, or diabetes.3,4 More immediately, equivalent cell numbers are mandatory to establish and optimize preclinical efficiency studies in physiologically relevant large animal models such as pigs, dogs, or primates.5,6 Both applications, assays and novel regenerative therapies, will require large cell numbers that cannot be produced by traditional two-dimensional (2D) culture as adherent colonies on mitotically inactivated feeder cells or other supportive substrates.7C10 In the field of vaccines and recombinant protein production, cultivation of mammalian cell lines in several 100C1,000?L dimensions has been thoroughly established in suspension culture bioreactors.11 Given this knowhow, suspension culture (3D cultivation) is the method of choice to generate stem cells and their progenies at a scale that deems feasible for their envisioned, high cell number demanding applications. Initial reports aiming at adapting matrix-attached hESC cultivation to suspension culture focused on microcarriers.12C14 These spherical particles are kept in suspension by stirring or by other mixing techniques and provide an enlarged attachment surface in a relatively small reactor volume due to their high surface area to volume ratio. Microcarriers, which exist in a plethora of shapes and sizes, have been previously used in conventional cell culture for production of vaccines, recombinant proteins, or other mammalian cell-derived products.15,16 Despite published proof-of-concept for hPSC cultivation on microcarrieres12,13 critical assessment of these reports reveals a number of issues. Particularly, the tendency of undifferentiated hPSCs to preferentially stick to each other rather than to thoroughly prescreened types of microcarriers might induce additional levels of culture heterogeneity.12,13 This includes only partial and uncontrolled cell-substrate versus cell-cell attachment and subsequently bold heterogeneity of cell-particle and cell-cell clusters sizes that might further increase in stirred, dynamic Gedatolisib systems. The approach would also require potentially cumbersome removal of microcarriers from clinical-grade cell preparations prior to clinical application. Recently, we and others have demonstrated expansion of undifferentiated human ES and iPS cells as cell-only-aggregates in suspension culture.17C20 While the group of Itskovitz-Eldor has established culture conditions based on aggregate-passaging in an interleukin-supplemented medium, 17 we have shown highly reproducible suspension cultures of several human ESC, human iPSC, and a cynomolgus monkey ESC line applying other conditions.19C21 Key features of the technology include (i) a fully defined serum-free culture media22 (ii) the use of Gedatolisib a Rho-associated coiled-coil kinase (ROCK) inhibitor (RI)23 enabling defined, single cell-based culture inoculation, and (iii) significant long-term expansion of pluripotent hES/hiPS cells in scalable suspension culture independent of any extracellular matrices or scaffolds. In contrast to previously reported feeder-free culture systems,24 our technology does not require preadaptation (i.e., preselection) of cells prior to initiation of expansion culture. Initial adaptation to dynamic culture was also tested employing stirred spinners or rotated Erlenmeyer flasks.19,20 Notably, robust expansion rates observed in.