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Serum-free non-toxic freezing solution for cryopreservation of human adipose tissue-derived mesenchymal stem cells

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Abstract

Objective

To develop a cost-effective, non-toxic and xeno-free freezing solution for the preservation of adipose tissue-derived stem cells (hADSC) with a long shelf-life.

Results

The potential of various hydrocolloids and organic osmolytes as cryoprotectants and individual components of phosphate buffered saline (PBS) as carrier media were evaluated to formulate a freezing solution for the cryopreservation of hADSCs. Among the hydrocolloids, the highest viability, 55 %, was achieved with post-thawed (after 48 h storage at −80 °C) hADSCs cryopreserved in 10 % (v/v) polyvinylpyrrolidone (PVP) using PBS as carrier media. 0.9 % NaCl was a superior carrier medium resulting an enhanced cell viability (70 %) when used in 10 % PVP than other components of PBS. A higher cell viability (81 %) was achieved when 10 % PVP/0.9 % NaCl was supplemented with 60 mM ectoin. The cryopreserved cells retained normal cytoskeletal distribution pattern and adipogenic and osteogenic differentiation ability during 14 and 21 days of incubation.

Conclusion

A serum-free and non-toxic 10 % PVP/0.9 % NaCl/60 mM ectoin freezing solution was developed for cryopreservation of hADSC for application in tissue engineering and regenerative medicine.

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Acknowledgments

The authors acknowledge to the Department of Biotechnology (DBT) and Department of Science and Technology, Ministry of Science and Technology, Government of India for providing research facility by sanctioning Program Support on Tissue Engineering Research (sanction no. BT/01/COE/09/1309062010) and research project (sanction no. SR/FTP/ETA-0106/2011) respectively.

Supporting Information

Supplementary Fig. 1—Phase contrast microscopic images shows a round cell morphology within 8 h of culture in DMEM containing 10 % FBS (a) and cell proliferation on 6th and 12th day (b, c). The cells reached confluency on 12th day of culture (scale bar 100 µm).

Supplementary Fig. 2—Flowcytometric analysis showing the expression of positive CD90 (99 %), CD73 (89 %), and CD105 (98 %) markers and negative HLA-DR (0.5 %), CD34 (1.2 %) and CD45 (2 %) markers representing the cells are mesenchymal stem cells.

Author information

Authors and Affiliations

  1. Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, 769008, India

    Sirsendu Sekhar Ray & Krishna Pramanik

  2. Department of Mechanical Engineering, National Institute of Technology, Rourkela, 769008, India

    Sunil Kumar Sarangi

  3. Jeevan Memorial Hospital, Raipur, India

    Nirved Jain

Authors
  1. Sirsendu Sekhar Ray
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  2. Krishna Pramanik
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  3. Sunil Kumar Sarangi
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  4. Nirved Jain
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Corresponding author

Correspondence to Krishna Pramanik.

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Conflict of interest

The authors declare there is no conflict of interest.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

10529_2016_2111_MOESM1_ESM.tif

Supplementary material 1 (TIF 62 kb) Supplementary Fig 1. Phase contrast microscopic images shows a round cell morphology within 8 h of culture in DMEM containing 10% FBS (a) and cell proliferation on 6th and 12th day (b, c). The cells reached confluency on 12th day of culture. (Scale bar 100 µm).

Below is the link to the electronic supplementary material.

10529_2016_2111_MOESM2_ESM.tif

Supplementary material 1 (TIF 278 kb) Supplementary Fig 2. Flowcytometric analysis showing the expression of positive CD90 (99%), CD73 (89%), and CD105 (98%) markers and negative HLA-DR (0.5%), CD34 (1.2%) and CD45 (2%) markers representing the cells are mesenchymal stem cells.

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Ray, S.S., Pramanik, K., Sarangi, S.K. et al. Serum-free non-toxic freezing solution for cryopreservation of human adipose tissue-derived mesenchymal stem cells. Biotechnol Lett 38, 1397–1404 (2016). https://doi.org/10.1007/s10529-016-2111-6

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  • DOI: https://doi.org/10.1007/s10529-016-2111-6

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