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Updated: Apr 18, 2026
Author Spotlight: Development of an Efficient and Feasible Method of Retinal Pigment Epithelial Cell Freezing
Published on: November 3, 2023
Efficient recovery of undifferentiated human embryonic stem cell cryopreserved with hydroxyethyl starch, dimethyl
Maristela Delgado Orellana1, Gil Cunha De Santis2, Kuruvilla Joseph Abraham3
1Hemotherapy Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Brazil; Department of Clinical Medicine, Ribeirão Preto Medical School, University of São Paulo, Brazil.
Background:
The therapeutic use of human embryonic stem cells (hESCs) is dependent on an efficient cryopreservation protocol for long-term storage. The aim of this study was to determine whether the combination of three cryoprotecting reagents using two freezing systems might improve hESC recovery rates with maintenance of hESC pluripotency properties for potential cell therapy application.
Methods:
Recovery rates of hESC colonies which were frozen in three cryoprotective solutions: Me2SO/HES/SR medium, Defined-medium® and Me2SO/SFB in medium solution were evaluated in ultra-slow programmable freezing system (USPF) and a slow-rate freezing system (SRF). The hESC pluripotency properties after freezing-thawing were evaluated.
Results:
We estimated the distribution frequency of survival colonies and observed that independent of the freezing system used (USPF or SRF) the best results were obtained with Me2SO/HES/SR as cryopreservation medium. We showed a significant hESC recovery colonies rate after thawing in Me2SO/HES/SR medium were 3.88 and 2.9 in USPF and SRF, respectively. The recovery colonies rate with Defined-medium® were 1.05 and 1.07 however in classical Me2SO medium were 0.5 and 0.86 in USPF and SRF, respectively. We showed significant difference between Me2SO/HES/SR medium×Defined-medium® and between Me2SO/HES/SR medium×Me2SO medium, for two cryopreservation systems (P<0.05).
Conclusion:
We developed an in house protocol using the combination of Me2SO/HES/SR medium and ultra-slow programmable freezing system which resulted in hESC colonies that remain undifferentiated, maintain their in vitro and in vivo pluripotency properties and genetic stability. This approach may be suitable for cell therapy studies.
