Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges

Abstract

Mesenchymal stem cells (MSCs) hold many advantages over embryonic stem cells (ESCs) and other somatic cells in clinical applications. MSCs are multipotent cells with strong immunosuppressive properties. They can be harvested from various locations in the human body (e.g., bone marrow and adipose tissues). Cryopreservation represents an efficient method for the preservation and pooling of MSCs, to obtain the cell counts required for clinical applications, such as cell-based therapies and regenerative medicine. Upon cryopreservation, it is important to preserve MSCs functional properties including immunomodulatory properties and multilineage differentiation ability. Further, a biosafety evaluation of cryopreserved MSCs is essential prior to their clinical applications. However, the existing cryopreservation methods for MSCs are associated with notable limitations, leading to a need for new or improved methods to be established for a more efficient application of cryopreserved MSCs in stem cell-based therapies. We review the important parameters for cryopreservation of MSCs and the existing cryopreservation methods for MSCs. Further, we also discuss the challenges to be addressed in order to preserve MSCs effectively for clinical applications.

Cryopreservation and its clinical applications

“…Slow freezing first substitutes the water within the cytoplasm with CPAs which reduces cell damage and adjusts the cooling rate in accordance with the permeability of the cell membrane. Slow-cooling protocols involve a typical cooling rate of about 1 °C/min in the presence of less than 1.0M of CPA, with use of a high-cost controlled-rate freezer or a benchtop portable freezing container 8, 9. The advantages of slow freezing are that it has a low risk of contamination during the procedures and does not demand high manipulation skills.…”

“…1 and Table 1). 5 Cryopreservation processes can generally be grouped into the following types: (1) slow freezing8, 9; (2) vitrification, which involves the solidification of the aqueous milieu of the cell or tissue into a noncrystalline glassy phase 10 ; (3) subzero nonfreezing storage; and (4) preservation in the dry state 11 . Generally, the storage of mammalian cells in the dry state is not readily possible because of difficulties in introducing the disaccharide trehalose (disaccharide of glucose, 342 Da) 12 and amino acids (used as preservatives in plants) into the intracellular region 13 .…”

“…Adult stem cells are capable of differentiating into multiple types of specific cells and can be obtained from various locations other than bone marrow, including fat tissue, the periosteum, amniotic fluid, and umbilical cord blood 9 . Stem cells can be subdivided into embryonic stem cells, mesenchymal stromal cells,29, 36, 48 and hematopoietic stem cells, all of which are considered as goldmines for potential application in regenerative medicine 28, 29, 49, 50.…”

Preservation of cell-based immunotherapies for clinical trials

“…Mesenchymal stromal cells (MSCs) are another cell type involved in a large number of clinical trials, where they are used for their immunomodulatory effect. Recent reviews have already described methods of preserving MSCs [1,2], and thus this cell type has been omitted.…”

Local and targeted drug delivery for bone regeneration

“…With MSCs as an example, the International Society for Cellular Therapy (ISCT) issued three criteria for identifying MSCs: plastic adherence, specific cell surface marker expression, and tri-lineage potential [7]. Additional gains in uniformity have been achieved through GMP-compliant isolation procedures [8], culture conditions [9,10], and cryopreservation techniques [11,12]. There are now Food and Drug Administration GMP-approved facilities (the NIH BMSC Transplantation Center and the Upstate New York Stem Cell cGMP Facility) that generate MSCs for clinical investigations [13 • ].…”