Cryopreservation of human mesenchymal stem cells for clinical applications


Mesenchymal stem cells (MSCs) are anchorage-dependent, non-hematopoietic fibroblast-like stem cells with multi‑lineage potential. They can be induced to differentiate into many cell types, including osteoblasts, myoblasts, adipocytes and chondrocytes. Although they were originally detected in bone marrow, they have also been found in dental pulp, adipose tissue and amniotic fluid. More than 500 clinical trials are exploring cellular therapy with MSCs for a wide range of purposes, most commonly with application to bone, cartilage, heart, neurodegenerative and autoimmune diseases (1). The therapeutic properties of MSCs originate from their ability to secrete a wide range of bioactive cytokines that regulate apoptosis, angiogenesis, inflammation, immune response and tissue regeneration. MSCs activate multiple pathways in response to local signals from injured tissue, releasing cytokines and growth factors that enable tissue repair.

Cryopreservation is necessary for MSC cell banking, reduces the need for fresh tissue and allows time for quality control and screening prior to transplantation. For clinical use, MSCs are often frozen in an electrolyte solution with 5% human serum albumin containing cryoprotectant, typically 5–10% dimethyl sulphoxide (DMSO) (1). Standard procedures utilize slow cooling, achieved with either a programmable rate-controlled freezer or immersion in an alcohol bath, which cools at an average rate of 1o C per minute. Samples are warmed rapidly prior to use. Adipose MSCs frozen with an injectable ice-cold solution of 5% DMSO, 5% human albumin, and slow cooling have viability similar to that of fresh samples (2). MSCs stored in this way also retain the ability to differentiate into chondrocytes, adipocytes and osteoblasts (tri-lineage potential).

A variety of studies with a focus on optimizing cell survival have demonstrated that MSCs from different source tissues can be frozen using variations in cryoprotectant, cooling rate and storage temperature without affecting viability or pluripotency (2). Regarding cryopreservation, MSCs are considered robust in comparison to other cell types. Therefore, it is possible to vary freezing conditions depending on need.  Researchers have explored strategies for reducing cellular toxicity caused by the cryoprotectant, using non-animal-derived reagents to eliminate the potential for contamination and minimizing cellular damage induced by freeze/thaw.

Reducing or eliminating DMSO from MSCs intended for clinical use is a primary goal. Patients who have been treated with hematopoietic stem cells containing DMSO have experienced gastrointestinal side effects, headache and hypotension (1). Careful cooling in the absence of cryoprotectant is one option. Lowering DMSO from 10% to 5% has also been successful and does not have an effect on viability. Sugars such as trehalose, lactose and sucrose serve as cryoprotectants without the toxicity of DMSO, and post-thaw survival varies from 60–90% depending on the study (1). It is also highly desirable to eliminate animal serum, which may evoke an immune response as well as transmit pathogens. Human albumin has been used successfully for this purpose. Synthetic cryogenic reagents are also being developed and tested. Although MSC cryopreservation may appear to be a small cog in the stem-cell machine, it is essential for expanding the clinical use of MSCs.

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  1. 1. Marquez-Curtis, L.A. (2015) Mesenchymal stromal cells derived from various tissues: Biological, clinical and cryopreservation aspects. Cryobiology 71, 181.
  2. 2. Minonzio, G. et al. (2014) Frozen adipose-derived mesenchymal stem cells maintain high capability to grow and differentiate. Cryobiology 69, 211.


Ina Scheuerpflug

Ina Scheuerpflug, PhD is the Global Market Director in Discovery Science at QIAGEN. She has written a number of scientific publications and focuses at QIAGEN on gene expression and gene regulation on a global level. She received her PhD at MAX-PLANCK-INSTITUT for Biology in 1996, studying a bacterial pilus adhesion and the interaction to the human host receptor/signaling pathway. Ina's primary interest is in the emerging importance of gene expression studies.

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