The Suppression of Medium Acidosis Improves the Maintenance and Differentiation of Human Pluripotent Stem Cells at High Density in Defined Cell Culture Medium

Liu, Weiwei; Ren, Zhili; Lü, Kai; Song, Chengcheng; Cheung, Edwin; Zhou, Zhou; Chen, Guokai

doi:10.7150/ijbs.24681

Cited by 30 publications

(22 citation statements)

References 38 publications

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“…The DMEM/F12 basal medium we use from Corning contains higher levels of sodium bicarbonate ($29 mM or 2,438 mg L À1 ) compared with DMEM/F12 from other manufacturers ( Figure S1C). Supplementation of Gibco DMEM/F12, which contains 14 mM sodium bicarbonate, with 20 mM additional sodium bicarbonate has recently been demonstrated to be advantageous to hiPSC growth rate by suppressing acidosis of the medium (Liu et al, 2018). In our hands, the standard 29 mM sodium bicarbonate was sufficient ( Figure 2F).…”

Section: Optimization Of Additional Medium Componentsmentioning

confidence: 57%

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

et al. 2020

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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.

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Section: Optimization Of Additional Medium Componentsmentioning

confidence: 57%

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

et al. 2020

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“…Supplementation of Gibco DMEM/F12, which contains 14 mM of sodium bicarbonate, with 20 mM of additional sodium bicarbonate has recently been demonstrated to be advantageous to hiPSC growth rate by suppressing acidosis of the medium (Liu et al, 2018). In our hands, the standard 29 mM of sodium bicarbonate was optimal ( Figure 2F).…”

Section: Optimization Of Additional Media Componentsmentioning

confidence: 57%

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

Kuo¹,

Gao²,

DeKeyser³

et al. 2019

Preprint

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Human induced pluripotent stem cell (hiPSC) culture has become routine, yet pluripotent cell media costs, frequent media changes, and reproducibility of differentiation have remained restrictive, limiting the potential for large-scale projects. Here, we describe the formulation of a novel 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. B8 eliminates 97% of the costs of commercial media, made possible primarily by the in-lab generation of three E. coli-expressed, codon-optimized recombinant proteins: an engineered form of fibroblast growth factor 2 (FGF2) with improved thermostability (FGF2-G3); transforming growth factor β3 (TGFβ3) -a more potent TGFβ able to be expressed in E. coli; and a derivative of neuregulin 1 (NRG1) containing the EGFlike domain. The B8 formula is specifically optimized for fast growth and robustness at low seeding densities. We demonstrated the derivation and culture of 34 hiPSC lines in B8 as well as maintenance of pluripotency long-term (over 100 passages). This formula also allows a weekendfree feeding schedule without sacrificing growth rate or capacity for differentiation. Thus, this simple, cost-effective, and open source B8 media, will enable large hiPSC disease modeling projects such as those being performed in pharmacogenomics and large-scale cell production required for regenerative medicine.

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“…Recently, media acidosis was shown to negatively impact hPSC, with low pH resulting in decreased glucose consumption, cell cycle arrest, and cell death. 34 Therefore, sodium L-lactate was used as the lactate source to avoid pH shifts due to the lactate addition and to decouple the effects of pH and lactate on K3 iPSC phenotype. An equal molar concentration of sodium chloride was added to the non-lactate cultures to balance osmolarity, as described in the materials and method section.…”

Section: Cell Growth and Parallel Labeling Experimentsmentioning

confidence: 99%

Induced pluripotent stem cells can utilize lactate as a metabolic substrate to support proliferation

Odenwelder

Harcum

2020

Biotechnology Progress

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Human-induced pluripotent stem cells (iPSCs) hold the promise to improve cell-based therapies. Yet, to meet rising demands and become clinically impactful, sufficient high-quality iPSCs in quantity must be generated, a task that exceeds current capabilities. In this study, K3 iPSCs cultures were examined using parallel-labeling metabolic flux analysis ( 13 C-MFA) to quantify intracellular fluxes at relevant bioprocessing stages: glucose concentrations representative of initial media concentrations and high lactate concentrations representative of fed-batch culture conditions, prior to and after bolus glucose feeds. The glucose and lactate concentrations are also representative of concentrations that might be encountered at different locations within 3D cell aggregates. Furthermore, a novel method was developed to allow the isotopic tracer [U-13 C 3 ] lactate to be used in the 13 C-MFA model. The results indicated that high extracellular lactate concentrations decreased glucose consumption and lactate production, while glucose concentrations alone did not affect rates of aerobic glycolysis. Moreover, for the high lactate cultures, lactate was used as a metabolic substrate to support oxidative mitochondrial metabolism. These results demonstrate that iPSCs have metabolic flexibility and possess the capacity to metabolize lactate to support exponential growth, and that high lactate concentrations alone do not adversely impact iPSC proliferation.

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The Suppression of Medium Acidosis Improves the Maintenance and Differentiation of Human Pluripotent Stem Cells at High Density in Defined Cell Culture Medium

Cited by 30 publications

References 38 publications

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

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

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

Induced pluripotent stem cells can utilize lactate as a metabolic substrate to support proliferation

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