Daily, MI orcid.org/0000-0001-8387-1394, Whale, TF orcid.org/0000-0002-1062-2685, Partanen, R et al. (6 more authors) (2020) Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation. Cryobiology, 93. pp. 62-69. ISSN 0011-2240
Abstract
Cryopreservation of mammalian cells has, to date, typically been conducted in cryovials, but there are applications where cryopreservation of primary cells in multiwell plates would be advantageous. However excessive supercooling in the small volumes of liquid in each well of the multiwell plates is inevitable without intervention and tends to result in high and variable cell mortality. Here, we describe a technique for cryopreservation of adhered primary bovine granulosa cells in 96-well plates by controlled rate freezing using controlled nucleation. Inducing ice nucleation at warm supercooled temperatures (less than 5 °C below the melting point) during cryopreservation using a manual seeding technique significantly improved post-thaw recovery from 29.6% (SD = 8.3%) where nucleation was left uncontrolled to 57.7% (9.3%) when averaged over 8 replicate cultures (p < 0.001). Detachment of thawed cells was qualitatively observed to be more prevalent in wells which did not have ice nucleation control which suggests cryopreserved cell monolayer detachment may be a consequence of deep supercooling. Using an infra-red thermography technique we showed that many aliquots of cryoprotectant solution in 96-well plates can supercool to temperatures below −20 °C when nucleation is not controlled, and also that the freezing temperatures observed are highly variable despite stringent attempts to remove contaminants acting as nucleation sites. We conclude that successful cryopreservation of cells in 96-well plates, or any small volume format, requires control of ice nucleation.
Metadata
Item Type: | Article |
---|---|
Authors/Creators: |
|
Copyright, Publisher and Additional Information: | © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) |
Dates: |
|
Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) The University of Leeds > Faculty of Environment (Leeds) > School of Earth and Environment (Leeds) > Inst for Climate & Atmos Science (ICAS) (Leeds) The University of Leeds > Faculty of Medicine and Health (Leeds) > School of Medicine (Leeds) > Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM) > Discovery & Translational Science Dept (Leeds) |
Funding Information: | Funder Grant number EU - European Union 648661 EU - European Union GA 713664 |
Depositing User: | Symplectic Publications |
Date Deposited: | 10 Mar 2020 09:47 |
Last Modified: | 19 Dec 2024 14:16 |
Status: | Published |
Publisher: | Elsevier |
Identification Number: | 10.1016/j.cryobiol.2020.02.008 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:157958 |