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Organ and tissue preservation, cryopreservation and reconditioning

Saturday October 28, 2023 - 14:00 to 15:30

Room: Indigo H

338.8 Cryogenic preservation of perinatal stem cells in DMSO-supplemented or DMSO-free GMP-grade conditions

Stefan Bencina, Sweden

Karolinska Institutet


Cryogenic preservation of perinatal stem cells in DMSO-supplemented or DMSO-free GMP-grade conditions

Stefan Bencina1, Fanny Pulcini2, Stacey Donoghue3, Alexia Nitti4, Alice Berti1, Roberto Gramignoli1.

1Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden; 2Dept. of Biotechnological and Applied Clinical Science, University of L’Aquila, L'Aquila, Italy; 3School of Biological, Health and Sports Science, Technological University Dublin, Dublin, Ireland; 4Faculty of Biology and Pharmacy, University of Cagliari, cagliari, Italy

Introduction: Advanced medical treatments are biological products (viable cells) with a limited shelf life. Ultra-low temperature preservation may circumvent issues. Cryopreservation ensures the immediate availability of the medicinal cells and allows time for quality tests and donor-recipient match. However, new ATMP products such as human epithelial or stromal cells present challenges in relation to viability and stability post-thawing. Our group was the first to report stem cell nature in human amnion epithelial cells (AEC) isolated from full-term placentae and validated the GMP-grade cryopreservation procedure. The exposure to standard-of-care cryoprotective agents, typically dimethyl sulfoxide (DMSO), has been proven challenging for clinical use and negatively affecting several cell viability and functions.

Methods: We performed cryogenic storage of human perinatal stem cells, both AEC and Wharton Jelly mesenchymal stromal cells (WJ-MSC), using commercial GMP-grade solutions. We evaluated both standard cooling rate (1 C/min) and higher or lower freezing rates using a controlled rate freezer device. The thawing procedure was performed using a warming rate of 60–80 C/min, by immersion in a water bath or a GMP-grade thawing device.

Results: We tested 20 different AEC and WJ-MSC batches using both formulations supplemented with 10% DMSO, and new DMSO-free cryosolutions. A different outcome in viability and cell recovery was measured in the two perinatal cell populations. Since detrimental mitochondrial activities (e.g., reduced ATP production) have been reported at low temperatures, we coupled viability and recovery measurements with ATP quantification. We analyzed cell adhesion to a substrate upon thawing and proliferation capacity.
Human AEC presented a superior capacity to survive and perform equally well as freshly isolated cells when exposed to DMSO-free cryosolutions. Conversely, WJ-MSCs had a batch-to-batch variability in terms of survival and identity in the presence of DMSO. Cell adhesion and expansion were batch-related with a preference for DMSO-free conditions.

Conclusions: Controlled‐rate freezers or passive freezing devices represent valuable and efficient strategies to control the rate at which cells traverse that temperature difference. DMSO-free cryosolutions showed great efficacy in preserving AEC at low and ultra-low temperatures, for the long term, without loss of energetic characteristics and efficacy. Such a switch into DMSO-free conditions may not serve equally well for perinatal MSC. Post-thawing washes may be appropriate for both ATMPs to provide a stable and safe product to infuse in patients.

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IPITA-IXA-CTRMS Joint Congress • San Diego, CA, USA • October 26-29, 2023
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