Vitrification for cryopreservation of 2D and 3D stem cells culture using high concentration of cryoprotective agents
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RESEARCH ARTICLE
Open Access
Vitrification for cryopreservation of 2D and 3D stem cells culture using high concentration of cryoprotective agents Young-Hoon Jeong1†, Ukjin Kim2†, Seul-Gi Lee1, Bokyeong Ryu2, Jin Kim2, Artyuhov Igor3, Jong Soo Kim1, Cho-Rok Jung4, Jae-Hak Park2 and C-Yoon Kim1,3*
Abstract Background: Vitrification is the most promising technology for successful cryopreservation of living organisms without ice crystal formation. However, high concentrations (up to ~ 6–8 M) of cryoprotective agents (CPAs) used in stem cell induce osmotic and metabolic injuries. Moreover, the application of conventional slow-freezing methods to cultures of 3-D organoids of stem cells in various studies, is limited by their size. Results: In this study, we evaluated the effect of high concentrations of CPAs including cytotoxicity and characterized human mesenchymal stem cell (MSC) at single cell level. The cell viability, cellular damage, and apoptotic mechanisms as well as the proliferation capacity and multipotency of cells subjected to vitrification were similar to those in the slow-freezing group. Furthermore, we identified the possibility of vitrification of sizecontrolled 3-D spheroids for cryopreservation of organoid with high survivability. Conclusions: Our results demonstrate successful vitrification of both single cell and spheroid using high concentration of CPAs in vitro without cytotoxicity. Keywords: Vitrification, Cryoprotective agent, In vitro, MSC, Spheroid
Background Organ cryopreservation is one of the most promising methods of storage and long-term preservation of transplantable cells and tissues. Cells, tissues, and organs for transplantation at cryogenic temperature (i.e., liquid nitrogen at − 196 °C) can be stored indefinitely, theoretically [1]. Cryogenic technology has the potential to revolutionize the overall status of organ transplantation in the medical field. Recently, organoids mimicking human organs have been widely studied to recapitulate the process of organogenesis in vitro. However, cryoprotective * Correspondence: [email protected] † Young-Hoon Jeong and Ukjin Kim contributed equally to this work. 1 Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 05029, Republic of Korea 3 Kriorus, Klimentovsky Per, 115184 Moscow, Russia Full list of author information is available at the end of the article
agents (CPAs) are hard to permeate organoids due to their strong cell-to-cell junctions and 3D structures without blood vessels compared with tissues or organs [2]. Moreover, ice crystal formation can cause severe cellular damage and destroy complex macroscopic tissues in preserved organs, and therefore, it is difficult to cryo-preserve an intact organoid. Vitrification is a novel approach to cryopreservation that facilitates freezing of living cells without crystallization. Vitrification simplifies and enhances cryopreservation by eliminating mechanical injury induced by ice crystal formation, and provides the optimal range of ‘critical cooling & rewarming rate
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