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. 2021 Feb 3;2(1):100213.
doi: 10.1016/j.xpro.2020.100213. eCollection 2021 Mar 19.

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

Davi Marco Lyra-Leite  1   2 , Hananeh Fonoudi  1   2 , Mennat Gharib  1   2 , Paul W Burridge  1   2
Affiliations

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

Davi Marco Lyra-Leite et al. STAR Protoc. .

Abstract

The protocol provided here describes methodologies for making a highly cost-effective, chemically defined medium for culturing hiPSCs we call B8 medium. The typical cost of B8 medium is US$10 per liter, which with modifications included here is more affordable than standard media. We provide simple protocols for making B8 supplement aliquots, making the basal media DMEM/F12, Matrigel-coated plates, thawing, passaging, culturing, and cryopreserving hiPSCs. 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).

Keywords: Cell Culture; Stem Cells.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Dissociation with EDTA and plating of hiPSCs using B8T Phase-contrast images of hiPSCs: (A) during EDTA treatment; (B) in suspension in B8T immediately after dissociation; (C) 10 min; and (D) 30 min post seeding in a Matrigel-coated plate. Scale bar, 100 μm.
Figure 2
Figure 2
Representative images of hiPSCs maintained in B8 medium stained for pluripotency markers These cells were generated and expanded in B8 medium for 94 passages, and were cultured in B8 medium for 3 days before fixing and staining. Scale bar, 100 μm.
Figure 3
Figure 3
Representative phase-contrast images of hiPSCs kept in B8 at different time points post plating (A) 4 h, (B) 24 h, (C) 48 h, (D) 72 h; (E) 90 h. Scale bar, 100 μm.
Figure 4
Figure 4
hiPSC-derived cardiomyocytes obtained from hiPSCs cultured in B8 following the protocols described here (A) Phase-contrast image of day 15 cardiomyocytes. (B) hiPSC-CMs stained for TNNT2 (red) and DAPI (blue). Scale bar, 100 μm. (C) Flow cytometry plot indicating the purity of our hiPSC-CMs.
Figure 5
Figure 5
Visual assesment of quality of Matrigel-coated plates Representative images of (A) Matrigel layer in good condition; (B) Matrigel layer that has dried out. Bottom row: zoomed in versions of inserts in the top images. Scale bar, 100 μm.
See this image and copyright information in PMC

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