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Comparison of different cooling rates for fibroblast and keratinocyte cryopreservation

journal contribution
posted on 26.02.2015 by Yahaira Naaldijk, Annett Friedrich-Stockigt, Sebastian Sethe, Alexandra Stolzing
Easy, cost-effective and reliable cryopreservation protocols are crucial for the successful and effective application of tissue engineering. Several different protocols are in use, but no comprehensive comparisons across different machine-based and manual methods have been made. Here, we compare the effects of different cooling rates on the post-thaw survival and proliferative capacity of two basic cell lines for skin tissue engineering fibroblasts and keratinocytes, cultured and frozen in suspension or as a monolayer. We demonstrate that effectiveness of cryopreservation cannot be reliably determined immediately after thawing: the results at this stage were not indicative of cell growth in culture 3days post-thaw. Cryopreservation of fibroblasts in an adherent state greatly diminishes their subsequent growth potential. This was not observed when freezing in suspension. In keratinocytes, however, adherent freezing is as effective as freezing in suspension, which could lead to significant cost and labour savings in a tissue-engineering environment. The 'optimal' cryopreservation protocol depends on cell type and intended use. Where time, ease and cost are dominant factors, the direct freezing into a nitrogen tank (straight freeze) approach remains a viable method. The most effective solution across the board, as measured by viability 3days post-thaw, was the commonly used, freezing container method. Where machine-controlled cryopreservation is deemed important for tissue-engineering Good Manufacturing Practice, we present results using a portfolio of different cooling rates, identifying the 'optimal' protocol depending on cell type and culture method.

Funding

This study was supported in part by Serumwerke Bernburg, a manufacturer of hydroxyethyl starch, and the project ‘Automated Tissue Engineering on Demand’ from the Stiftungsprojekt of the Fraunhofer Society.

History

School

  • Mechanical, Electrical and Manufacturing Engineering

Published in

Journal of Tissue Engineering and Regenerative Medicine

Volume

10

Issue

10

Pages

E354–E364

Citation

NAALDIJK, Y. ... et al., 2016. Comparison of different cooling rates for fibroblast and keratinocyte cryopreservation. Journal of Tissue Engineering and Regenerative Medicine, 10 (10) October 2016, pp. E354–E364

Publisher

© John Wiley & Sons, Ltd.

Version

VoR (Version of Record)

Publisher statement

This work is made available according to the conditions of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) licence. Full details of this licence are available at: https://creativecommons.org/licenses/by-nc-nd/4.0/

Publication date

2013-08-21

Notes

Closed access.

ISSN

1932-6254

eISSN

1932-7005

Language

en

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