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Cryopreservable three-dimensional spheroid culture for ready-to-use systems

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Abstract

Three-dimensional (3D) spheroid culture has applications in many fields as spheroids closely recapitulate physiological conditions. However, spheroid culture and maintenance are time-consuming and unsuitable for urgent situations; therefore, appropriate cryopreservation methods for spheroids are required for their use in an on-demand and ready-to-use manner. We hypothesized that the feasibility of a ready-to-use system relies on diffusion of the preservation solution within spheroids; we thus evaluated the effects of spheroid-forming parameters, such as cell number and culture period, on spheroid viability and functionality. Long-term spheroid culture for seven days interfered with penetration of the cryopreservation solution as it caused cell condensation and extracellular matrix (ECM) secretion, as well as low viability and migratory activity upon replating after storage. However, ready-to-use spheroids, which were cultured for one day and then cryopreserved, showed viability and migration similar to those of non-cryopreserved spheroids, confirming that a short incubation period was suitable for this system. The chondrocyte-based ready-to-use spheroid system designed in this study can be easily applied to regenerative medicine applications that require a large number of cells in the future and can provide information for applying the ready-to-use spheroid system to various cell types.

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Acknowledgement

This research was supported by the Basic Science Research Program (NRF-2020R1A2C2100794 and NRF-2022R1A2C1010161) of the National Research Foundation funded by the Korean government and by the Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Health & Welfare) (KFRM 22A0105L1-11). This research was supported by the Korea Institute of Toxicology, Republic of Korea (1711159823).

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Author notes

  1. These authors contributed equally to this study as the first authors.

Authors and Affiliations

  1. Research Group for Biomimetic Advanced Technology, Korea Institute of Toxicology (KIT), Daejeon, 34114, Korea

    Thuy Trang Truong, Kyoung Hwan Park, Hye-Eun Shim, Jin Jung Song & Sun-Woong Kang

  2. Department of Polymer Science and Engineering, Chungnam National University, Daejeon, 34134, Korea

    Thuy Trang Truong, Kyoung Hwan Park, Hye-Eun Shim, Jin Jung Song & Kang Moo Huh

  3. Department of Advanced Toxicology Research, Korea Institute of Toxicology, Daejeon, 34114, Korea

    Yu Bin Lee

  4. Animal Model Research Group, Jeonbuk Branch Institute, Korea Institute of Toxicology, Jeongeup, 53212, Korea

    Hyung-Sun Kim & Jeong Ho Hwang

  5. Human and Environmental Toxicology Program, University of Science and Technology, Daejeon, 34114, Korea

    Sun-Woong Kang

Authors
  1. Thuy Trang Truong
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  2. Yu Bin Lee
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  3. Kyoung Hwan Park
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  4. Hye-Eun Shim
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  5. Jin Jung Song
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  6. Hyung-Sun Kim
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  7. Jeong Ho Hwang
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  8. Sun-Woong Kang
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  9. Kang Moo Huh
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Corresponding authors

Correspondence to Sun-Woong Kang or Kang Moo Huh.

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Author Contributions

T.T.T and S.W.K, conceived and planned the experiments. T.T.T and K.H.P contributed to sample preparation. T.T.T carried out the experiments, process the experiment data, draft the manuscript and designed the figure. T.T.T, S.H.E, K.H.P and Y.B.L were involved in analyzing data. J.J.S contributed to the histological evaluation. T.T.T and K.H.P performed the measurement, Y.B.L wrote the manuscript with input from all authors. S.W.K and K.M.H supervised the project.

Conflict of Interest

The authors declare no competing interests.

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Truong, T.T., Lee, Y.B., Park, K.H. et al. Cryopreservable three-dimensional spheroid culture for ready-to-use systems. Korean J. Chem. Eng. 40, 390–397 (2023). https://doi.org/10.1007/s11814-022-1279-9

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  • DOI: https://doi.org/10.1007/s11814-022-1279-9

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