Negligible-Cost and Weekend-Free Chemically Defined Human iPSC Culture

Abstract

Human induced pluripotent stem cell (hiPSC) culture has become routine, yet the cost of pluripotent cell media, frequent medium changes, and the reproducibility of differentiation have remained restrictive. Here, we describe the formulation of a hiPSC culture medium (B8) as a result of the exhaustive optimization of medium constituents and concentrations, establishing the necessity and relative contributions of each component to the pluripotent state and cell proliferation. The reagents in B8 represent only 3% of the costs of commercial media, made possible primarily by the in-lab generation of three E. coli-expressed, codon-optimized recombinant proteins: fibroblast growth factor 2, transforming growth factor b3, and neuregulin 1. We demonstrate the derivation and culture of 34 hiPSC lines in B8 as well as the maintenance of pluripotency long term (over 100 passages). This formula also allows a weekend-free feeding schedule without sacrificing capacity for differentiation.

Cell‐Based Therapy for Canavan Disease Using Human iPSC‐Derived NPCs and OPCs

“…From the immunological point of view, autologous transplantation is ideal for cell therapy because these cells may avoid any potential immune‐mediated complications. It is anticipated that the cost of iPSC‐based cell therapy manufacturing will be reduced with the availability of low‐cost reagents, [ 38 ] and derisking of GMP manufacturing through the development of GMP‐compatible processes as described in this study that are cost‐effective and easily transferrable to GMP.…”

An updated protocol for the cost-effective and weekend-free culture of human induced pluripotent stem cells

“…Changing the number of days of culture based on visual inspection is not appropriate, rather cells should be split on schedule and plated at different ratios, typically 1:15 to 1:20 until they typically reach 70%–80% confluent and rhythm has been established. The protocol provided here uses the “low-cost, easier to make” formula variant of B8 medium with lower levels of insulin and transferrin described in ( Kuo et al., 2020 ) (Figure S5C). NRG1 and TGFB3 are used at very low concentrations in this formula, and we have found it not to be cost-effective to make these proteins in house due to comparative poor yield when generating these ourselves.…”

“… Timing: 30 min Note: The use of a low concentration Matrigel (a 1:800 dilution) was optimized and found to work successfully without sacrificing cell proliferation or differentiation capacity ( Kuo et al., 2020 ). Dilutions up to 1:1,000 are still suitable for hiPSC culture, however 1:800 is preferred to avoid batch-to-batch variability in the supplier concentrations and operator error when filling the wells.…”

“…We show typical differentiation results and provide a comprehensive troubleshooting guide. For complete details on the use and execution of this protocol, please refer to Kuo et al. (2020) .…”

Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research

“…Most time‐consuming are the quality checks at the MCB level (Table 3); thus, it is critical to preselect hiPSC lines based on the parameters specified in Table 2. We recommend freezing only fast‐growing hiPSCs, as an accepted indicator for pluripotency (Kuo et al., 2020), and only cells with excellent morphology. Stringently discard lines that show evidence of spontaneous differentiation during expansion for MCB (Fig.…”