Chia-Hsing Pi1, Kathlyn Hornberger2, Peter Dosa3, Allison Hubel4. 1. Departments of Mechanical Engineering, Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, Minnesota, USA. 2. Departments of Biomedical Engineering, Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, Minnesota, USA. 3. Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, Minnesota, USA. 4. Departments of Mechanical Engineering, Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, Minnesota, USA; Departments of Biomedical Engineering, Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, Minnesota, USA. Electronic address: hubel001@umn.edu.
Abstract
BACKGROUND: This study examined the freezing responses of peripheral blood mononuclear cells (PBMCs) and specific white blood cell subsets contained therein when cryopreserved in three combinations of osmolytes composed of sugars, sugar alcohols and amino acids. METHODS: A differential evolution algorithm with multiple objectives was used to optimize cryoprotectant composition and thus the post-thaw recoveries for both helper and cytotoxicity T cells simultaneously. RESULTS: The screening of various formulations using a differential evolution algorithm showed post-thaw recoveries greater than 80% for the two subsets of T cells. The phenotypes and viabilities of PBMC subsets were characterized using flow cytometry. Significant differences between the post-thaw recovery for helper T cells and cytotoxic T cells were observed. Statistical models were used to analyze the importance of individual osmolytes and interactions between post-thaw recoveries of three subsets of T cell including helper T cells, cytotoxic T cells and natural killer T cells. The statistical model indicated that the preferred concentration levels of osmolytes and interaction modes were distinct between the three subsets studied. PBMCs were cultured for 72 h post-thaw to determine the stability of the cells. Because post-thaw apoptosis is a significant concern for lymphocytes, apoptosis of helper T cell and cytotoxic T cells frozen in a DMSO-free cryoprotectant was analyzed immediately post-thaw and 24 h post-thaw. Both cell types showed a decrease in cell viability 24 h post-thaw compared with immediately post-thaw. Helper T cell viability dropped 17%, and cytotoxic T cells had a 10% drop in viability. Immediately post-thaw, both cell types had >30% of cells in early apoptosis, but after 24 h the number of cells in early apoptosis decreased to below 20%. CONCLUSION: This study helped us identify the freezing responses of different human PBMC subsets using combinations of osmolytes.
BACKGROUND: This study examined the freezing responses of peripheral blood mononuclear cells (PBMCs) and specific white blood cell subsets contained therein when cryopreserved in three combinations of osmolytes composed of sugars, sugar alcohols and amino acids. METHODS: A differential evolution algorithm with multiple objectives was used to optimize cryoprotectant composition and thus the post-thaw recoveries for both helper and cytotoxicity T cells simultaneously. RESULTS: The screening of various formulations using a differential evolution algorithm showed post-thaw recoveries greater than 80% for the two subsets of T cells. The phenotypes and viabilities of PBMC subsets were characterized using flow cytometry. Significant differences between the post-thaw recovery for helper T cells and cytotoxic T cells were observed. Statistical models were used to analyze the importance of individual osmolytes and interactions between post-thaw recoveries of three subsets of T cell including helper T cells, cytotoxic T cells and natural killer T cells. The statistical model indicated that the preferred concentration levels of osmolytes and interaction modes were distinct between the three subsets studied. PBMCs were cultured for 72 h post-thaw to determine the stability of the cells. Because post-thaw apoptosis is a significant concern for lymphocytes, apoptosis of helper T cell and cytotoxic T cells frozen in a DMSO-free cryoprotectant was analyzed immediately post-thaw and 24 h post-thaw. Both cell types showed a decrease in cell viability 24 h post-thaw compared with immediately post-thaw. Helper T cell viability dropped 17%, and cytotoxic T cells had a 10% drop in viability. Immediately post-thaw, both cell types had >30% of cells in early apoptosis, but after 24 h the number of cells in early apoptosis decreased to below 20%. CONCLUSION: This study helped us identify the freezing responses of different human PBMC subsets using combinations of osmolytes.
Authors: Sandhya R Panch; Sandeep K Srivastava; Nasha Elavia; Andrew McManus; Shutong Liu; Ping Jin; Steven L Highfill; Xiaobai Li; Pradeep Dagur; James N Kochenderfer; Terry J Fry; Crystal L Mackall; Daniel Lee; Nirali N Shah; David F Stroncek Journal: Mol Ther Date: 2019-05-30 Impact factor: 11.454