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完全悬浮培养人诱导多能干细胞，辅以抑制自发分化的物质。

Complete suspension culture of human induced pluripotent stem cells supplemented with suppressors of spontaneous differentiation.

机构信息

iPS Cell Advanced Characterization and Development Team, RIKEN BioResource Research Center, Ibaraki, Japan.

Regenerative Medicine and Cell Therapy Laboratories, KANEKA CORPORATION, Kobe, Japan.

出版信息

Elife. 2024 Nov 12;12:RP89724. doi: 10.7554/eLife.89724.

DOI:10.7554/eLife.89724
PMID:39529479
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11556790/
Abstract

Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. Adding PKCβ and Wnt signaling pathway inhibitors in the suspension conditions suppressed the spontaneous differentiation of hiPSCs into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed the culture processes of hiPSCs, including the generation of hiPSCs from peripheral blood mononuclear cells with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single-cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our results demonstrate that precise control of the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.

摘要

人类诱导多能干细胞（hiPSCs）是再生医学中产生各种类型组织的有前途的资源；然而，需要改进可精确控制 hiPSCs 细胞状态的可扩展培养系统。利用无微载体或特殊材料的悬浮培养允许在工业化再生医学中进行大规模生产、自动化、具有成本效益和保证安全性。在这里，我们发现与传统的贴壁培养条件相比，在无微载体或细胞外基质成分的连续搅拌的悬浮条件下培养的 hiPSCs 更容易自发分化。在悬浮条件下添加 PKCβ 和 Wnt 信号通路抑制剂分别抑制 hiPSCs 自发分化为外胚层和中胚层。在这些条件下，我们成功完成了 hiPSCs 的培养过程，包括从外周血单核细胞中生成 hiPSCs，通过批量培养和单细胞分选克隆进行扩增，具有强大自我更新特性的长期培养，单细胞克隆，从悬浮培养直接进行冷冻保存及其成功恢复，以及高效大规模生产临床级 hiPSC 系。我们的结果表明，精确控制悬浮培养条件下的细胞状态为其稳定和自动化的临床应用铺平了道路。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/d4d57c10f3b1/elife-89724-fig5-figsupp2.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/c0b7ff43e2e6/elife-89724-fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/11f8f38e3d11/elife-89724-fig4-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/7197ace6174f/elife-89724-fig4-figsupp2.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/d4d57c10f3b1/elife-89724-fig5-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/fe31f157fe60/elife-89724-fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/db1d5942d008/elife-89724-fig1-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/d466facb309d/elife-89724-fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/e1440f722ff3/elife-89724-fig2-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/7dc69bab56a8/elife-89724-fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/ee2d22e54c17/elife-89724-fig3-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/6f5a2a390449/elife-89724-fig3-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/c0b7ff43e2e6/elife-89724-fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/11f8f38e3d11/elife-89724-fig4-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/7197ace6174f/elife-89724-fig4-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/795fe672515b/elife-89724-fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/70c6ce7ae22c/elife-89724-fig5-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d002/11556790/d4d57c10f3b1/elife-89724-fig5-figsupp2.jpg

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