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.
Highlights
Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into multiple different tissue lineages [1] making them an attractive prospect for regenerative medicine
Changes in the temperature of ice formation due to the inclusion of an ice nucleator were measured during controlled rate freezing at 1 ◦C/min
Comparisons between the results of that study and the results presented here are difficult due to the relative presentation of the data, the processes and tools presented in this study are less labour intensive
Summary
Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into multiple different tissue lineages [1] making them an attractive prospect for regenerative medicine. MSCs can be isolated from many sources, including bone marrow and adipose tissue [3]. From surplus extracted teeth, is a good source of highly proliferative MSCs without the technical limitations of other tissues. MSCs possess a multi-lineage differentiation capability and immunomodulation effect through production of molecules such as PGE2, IL-6 and nitric oxide. Through these mechanisms, MSCs have demonstrated possible positive effects against human age-related diseases [4] and have already reached the clinic for diseases like Alzheimer’s [5]. Minimal criteria to characterize MSCs were laid out by the International Society for Cellular Therapies (ISCT) in 2006 including, plastic adherence, tri-lineage differentiation (osteoblast, adipocyte, chondroblast) and positivity for surface markers of CD90, CD105 and CD73. CD 14, CD34 and CD45 must be lacking expression in MSCs
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