Tips and tricks for successfully culturing and adapting human induced pluripotent stem cells

Abstract

Reprogramming somatic cells toward pluripotency became possible over a decade ago. Since then, induced pluripotent stem cells (iPSCs) have served as a versatile and powerful tool not only for basic research but also with the long-term goal of using them in human cell transplantation after differentiation. Nonetheless, downstream applications are frequently blurred by the difficulties that researchers have to face when working with iPSCs, such as trouble with clonal selection, in vitro culture and cryopreservation, adaptation to feeder-free conditions, or expansion of the cells. Therefore, in this article we aim to provide other researchers with practical and detailed information to successfully culture and adapt iPSCs. Specifically, we (1) describe the most common problems when in-vitro culturing iPSCs onto feeder cells as well as its possible troubleshooting, and (2) compare different matrices and culture media for adapting the iPSCs to feeder-free conditions. We believe that the troubleshooting and recommendations provided in this article can be of use to other researchers working with iPSCs and who may be experiencing similar issues, hopefully enhancing the appeal of this promising cell source to be used for biomedical investigations, such as tissue engineering or regenerative medicine applications.

Podocytes derived from human induced pluripotent stem cells: characterization, comparison, and modeling of diabetic kidney disease

“…1 B). We repeated our podocyte differentiation protocol on three independently-derived iPSC lines from two genetic backgrounds: DYR0100 (origin: SCRC-1041 foreskin fibroblast cell line), MAFB:mTagBFP2/GATA3mCherry (origin: CRL-2429 foreskin fibroblast cell line), and LRP2:mTagBFP2 (origin: CRL-2429 foreskin fibroblast cell line) [ 45 – 48 ] (Additional file 1 : Fig. S2).…”

Large-scale smart bioreactor with fully integrated wireless multivariate sensors and electronics for long-term in situ monitoring of stem cell culture

“…After 2 days of biosensor installation, the iPSC colonies continued to grow and expanded by more than threefold. The colonies were compact with well-defined edges, and all cells exhibited a high nucleus/cytoplasm ratio, which indicated the right morphology of iPSCs with no differentiation ( 70 ). On day 4, iPSCs exhibited robust growth, reaching over 80% confluency, indicating that they were ready for passage and storage.…”

Investigating nanoplastics toxicity using advanced stem cell-based intestinal and lung in vitro models

“…The cultivation and differentiation of stem cells into a mature model usually covers time spans of two weeks or more for a single experiment (Kabeya et al, 2018;Naumovska et al, 2020), and is quite expensive in terms of consumption of medium and the multitude of growth factors required for differentiation. The handling of stem cells requires highly trained operators, since wrong handling can easily lead to death or spontaneous differentiation of stem cells, e.g., by suboptimal cell confluence or inappropriate passaging (Kogut et al, 2014;Castro-Viñuelas et al, 2021;Yamamoto et al, 2022), which drastically impairs the robustness of the model itself. This issue of robustness is further supported by the lack of standardized protocols for stem cell-based models in the current literature.…”