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Expansion of mouse primitive hematopoietic cells in three-dimensional cultures on chemically fixed stromal cell layers

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Abstract

To establish a practical and convenient method to expand hematopoietic cells (HCs), we applied chemically-fixed stromal cell layers formed within three-dimensional (3D) scaffolds to feeder of HC cultures. The HCs were expanded using two successive cultures. First, stromal cells were cultured within porous polymer scaffolds and formed tissue-engineered constructs (TECs); the scaffolds containing stromal cells, were fixed using aldehyde (formaldehyde or glutaraldehyde) or organic solvents (acetone, methanol or ethanol). Second, mouse fetal liver cells (FLCs), as a source of HCs, were cultured on the TECs for 2 weeks, and the effects of fixative solutions on expansion of primitive HCs (c-kit+ and CD34+ cells) were examined. In the cultures on aldehyde-fixed TECs, primitive HCs were expanded 2.5- to 5.1-fold in the cultures on TECs fixed with glutaraldehyde, whereas no expansions were detected in those fixed with formaldehyde. However, we achieved expansion of primitive HCs > fivefold in the cultures using TECs fixed with organic solvents. Among these solvents, the highest expansions—of roughly tenfold—were obtained using acetone fixation. Ethanol-fixed TECs also supported the expansion of the primitive HCs well (6.6- to 8.0-fold). In addition to these sufficient expansions, the procedure and storage of fixed TECs is fairly easy. Thus, HC expansion on chemically-fixed TECs may be a practical method for expanding primitive HCs.

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Acknowledgements

We thank Professor Osamu Ohneda (Faculty of Medicine, University of Tsukuba) for providing DAS 104-8 stromal cell line. We thank Mr. Thomas Mayers (Medical English Communications Center, University of Tsukuba) for his editorial assistance.

Funding

This work was supported by a Grant-in-Aid for Scientific Research (No. 20K12592) from the Japan Society for the Promotion of Science.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan

    Hirotoshi Miyoshi, Yuichiro Shimizu, Yutaka Yasui & Satoshi Sugiyama

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  1. Hirotoshi Miyoshi
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  2. Yuichiro Shimizu
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  3. Yutaka Yasui
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  4. Satoshi Sugiyama
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Correspondence to Hirotoshi Miyoshi.

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Miyoshi, H., Shimizu, Y., Yasui, Y. et al. Expansion of mouse primitive hematopoietic cells in three-dimensional cultures on chemically fixed stromal cell layers. Cytotechnology 72, 741–750 (2020). https://doi.org/10.1007/s10616-020-00417-4

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