Cryopreservation of Mesenchymal Stem Cells Using Medical Grade Ice Nucleation Inducer

Abstract

Mesenchymal stem cells (MSCs) can differentiate into multiple different tissue lineages and have favourable immunogenic potential making them an attractive prospect for regenerative medicine. As an essential part of the manufacturing process, preservation of these cells whilst maintaining potential is of critical importance. An uncontrolled area of storage remains the rate of change of temperature during freezing and thawing. Controlled-rate freezers attempted to rectify this; however, the change of phase from liquid to solid introduces two extreme phenomena; a rapid rise and a rapid fall in temperature in addition to the intended cooling rate (normally −1 °C/min) as a part of the supercooling event in cryopreservation. Nucleation events are well known to initiate the freezing transition although their active use in the form of ice nucleation devices (IND) are in their infancy in cryopreservation. This study sought to better understand the effects of ice nucleation and its active instigation with the use of an IND in both a standard cryotube with MSCs in suspension and a high-throughput adhered MSC 96-well plate set-up. A potential threshold nucleation temperature for best recovery of dental pulp MSCs may occur around −10 °C and for larger volume cell storage, IND and fast thaw creates the most stable process. For adhered cells, an IND with a slow thaw enables greatest metabolic activity post-thaw. This demonstrates a necessity for a medical grade IND to be used in future regenerative medicine manufacturing with the parameters discussed in this study to create stable products for clinical cellular therapies.

Pollen derived macromolecules serve as a new class of ice-nucleating cryoprotectants

“…A small number of chemical nucleators, which are capable of nucleating ice near the melting point via heterogeneous nucleation, have also been deployed. These include Snomax™ 20 , 21 , which is derived from the bacterial plant pathogen Pseudomonas syringae , cholesterol crystals 22 , encapsulated silver iodide 23 , biologically inert minerals 24 and sand 25 . However, these existing chemical nucleators are usually insoluble and difficult to sterilise, or else are difficult to introduce into cryopreservation volumes in a reproducible fashion 11 .…”

Nonfreezing Low Temperature Maintains the Viability of Menstrual Blood–Derived Endometrial Stem Cells Under Oxygen–Glucose Deprivation Through the Sustained Release of Autophagy-Produced Energy

“…However, challenges faced during the storage and shipping of therapeutic MSC products from commercial cell preparation companies to hospitals have drawn significant attention in emerging regenerative medicine. To date, methods for the long-term cryopreservation of MSCs in liquid or gaseous nitrogen under the protection of various kinds of cryoprotective agents have been extensively established and tested 14 , 15 . The challenges related to short-term storage and transportation of MSC products before in vivo transplantation remain poorly studied, because of the existence of a time interval between the completion of MSC production and the actual clinical application following the quality control and transport of MSC products.…”

“…However, unlike long-term cryopreservation of MSCs, which has been a concern for the majority of studies 14 , 15 , with MSCs intended for clinical application, there is a time interval between the completion of fresh MSC preparation and transplantation into the patient for actual clinical application. The most basic quality control of MSC products needs to be performed during this period, including but not limited to endotoxin, mycoplasma, and cell surface marker detection 16 , 17 .…”

Impact of Cryopreservation and Freeze-Thawing on Therapeutic Properties of Mesenchymal Stromal/Stem Cells and Other Common Cellular Therapeutics

“…Another crucial aspect is the cryocontainer itself. Wragg et al studied the reduction of ice nucleation effects on MSCs during freezing by using an Ice Nucleation Device (IND) [ 115 ]. The highest cell recovery was accomplished by using the faster thawing rate in combination with the utilization of IND and suitable cryovials, but lower thawing rates in combination with IND produced the best recovery when using micro-well plates.…”