A Response Surface Methodology Based Characterization and Optimization of Pseudoislet Cryopreservation
Date
2021-08
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Abstract
Efficient and consistent cryopreservation of human islets would be a useful tool in generating islet biobanks for transplantation and research uses. However, current islet cryopreservation attempts have an array of challenges due to large islet aggregate size and susceptibility to cryopreservation. Centrifugal-force-aggregation pseudoislets (CFA-PI) offer a promising tool to address these challenges as they can be size controlled and additionally modified to survive the stress associated with cryopreservation. In this thesis, the objective was to use a response surface methodology approach to characterize the effects of cryopreservation manipulated variables on CFA-PI survival post-cryopreservation and use this characterization to optimize (maximize) survival. Two separate cryopreservation processes were characterized, controlled 1 ⁰C/minute cooling rate cryopreservation (CCRC) and vitrification (extremely rapid-cooling using liquid nitrogen). Initial peak post-CCRC CFA-PI survival was 26.9% ± 27.1% of initially cryopreserved islet material with increasing islet purity, FBS concentration of 20%, CFA-PI size of 750 cells/aggregate, 5% DMSO, and 0 additional days of CFA-PI culture time increasing survival. In comparison peak post-vitrification CFA-PI survival was 26.9% ± 6.9% with increasing islet purity, incorporation of fibroblasts into CFA-PI, 10% DMSO, and 0 or 100 µM emricasan increasing CFA-PI survival. Due to constraints on islet supply during the Covid-19 pandemic, a third unifying experiment comparing post-cryopreservation survival directly between CCRC and vitrification, with similar experimental variables, was characterized using a model fibroblast cell line. Validation of this model using human CFA-PI resulted in a survival of 41.0 ± 0.723% using CCRC with intermediate CFA-PI size of 500 cells/aggregate, 5% DMSO, 24% FBS, and no emricasan. While islet survival improved using this approach compared to earlier attempts, there was no statistically significant difference in peak islet survival post-cryopreservation across the different methods of cryopreservation in this thesis. Ultimately, while CFA-PI survival post-cryopreservation did not statistically significantly improve, the effect of cryopreservation manipulated variables on survival was characterized as a modeled and manipulatable mathematical relationship. This relationship establishes a framework for optimizing the survival of CFA-PI, when additional islet tissue becomes available after Covid-19.
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Keywords
Cryopreservation, Vitrification, Pseudoislet, Response Surface Methodology, Islet Transplantation
Citation
Sidhu, S. (2021). A response surface methodology based characterization and optimization of pseudoislet cryopreservation (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.