StemXVivo Serum-Free MSC Freezing Media
StemXVivo Serum-Free MSC Freezing Media Summary
Freezing media for human, mouse, or rat MSC cryopreservation.
- All components were selected and optimized for MSC cryopreservation
- Ready to use
- Recover up to 50% more viable MSCs compared to conventional cryopreservatives
Why use a Defined Media for MSC Cryopreservation?
Mesenchymal stem/stromal cells (MSCs) are a rare population of bone marrow-derived cells. Given their rarity, MSCs are most often expanded in vitro under specific culture conditions
StemXVivo® Serum-Free MSC Freezing Media:
- Is defined to support reproducible MSC cryopreservation.
- Is compatible with defined, serum-free MSC culture conditions.
- Has been developed and optimized for use with human, mouse, and rat MSCs.
The term ‘mesenchymal stromal cells’ is commonly used to describe a heterogeneous population of cultured cells that are adherent to plastic, have a distinct morphology, and express a specific set of marker proteins. Within this heterogeneous population are cells referred to as ‘mesenchymal stem cells.’
Mesenchymal stem cells are multipotent, self-renewing cells that have the ability to differentiate into adipocytes, chondrocytes, and osteoblasts when cultured in vitro. Read More about MSC Nomenclature
StemXVivo® MSC Freezing Media Components
Supplied in a 50 mL volume, this media contains defined, high quality factors to support MSC cryopreservation.
- Does not contain antibiotics.
- Contains 10% dimethylsulfoxide (DMSO).
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MSC Viability Following Cryopreservation in StemXVivo® Serum-Free MSC Freezing Media. Human MSCs were frozen in either conventional, serum-containing freezing media or StemXVivo® Serum-Free MSC Freezing Media (Catalog # CCM016) at 2 x 105 cells/cryovial and stored in liquid nitrogen for one week. The MSCs were thawed, resuspended in StemXVivo® Serum-Free Human MSC Expansion Media (Catalog # CCM014), and cultured in 6-well plates coated with Recombinant Human Fibronectin (Catalog # 4305-FN). After one day in culture, the MSCs were harvested in triplicate, stained with Trypan blue, and viable cells were counted using a hemocytometer. A greater percentage of viable MSCs were recovered following cryopreservation in StemXVivo® MSC Freezing Media compared to conventional, serum-containing freezing media..
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MSC Identification Following Cryopreservation in StemXVivo® Serum-Free MSC Freezing Media. Human MSCs were frozen in StemXVivo® Serum-Free MSC Freezing Media (Catalog # CCM016) at 2 x 105 cells/cryovial and stored in liquid nitrogen for one week. The MSCs were thawed, resuspended in StemXVivo® Serum-Free Human MSC Expansion Media (Catalog # CCM014), and cultured in 6-well plates coated with Recombinant Human Fibronectin (Catalog # 4305–FN). After one day in culture, the MSCs were harvested and incubated with a PE-conjugated Mouse Anti-Human CD45 Monoclonal Antibody (Catalog # FAB1430P; filled histogram) and an APC-conjugated Mouse Anti-Human CD90/Thy1 Monoclonal Antibody (Catalog # FAB2067A; filled histogram), or a Mouse IgG1 or IgG2A Isotype Control (Catalog # IC002P, or IC003A, respectively; open histograms). Thawed MSCs were positive for the MSC multipotency marker, CD90/Thy1 and negative for the hematopoietic marker, CD45.
2006 Proposed Change to MSC Nomenclature
Although mesenchymal stromal cells were once referred to as ‘mesenchymal stem cells’, a change to ‘mesenchymal stromal cells’ was proposed by the International Society for Cellular Therapy in 2006.1
The change in nomenclature originates from two important factors:
- Methods used to isolate mesenchymal stem cells yield a heterogeneous population of cells with only a fraction of these cells demonstrating multipotency.
- The absence of direct evidence that mesenchymal stem cells can self-renew and differentiate in vivo.
Use of Mesenchymal Stem and Stromal Cell Terminology
Data supporting MSC self-renewal and multipotency have been obtained using in vitro conditions, which does not adequately reflect the in vivo environment. The lack of in vivo data has led some researchers to question the validity of the term ‘mesenchymal stem cell’ providing further support for the use of ‘mesenchymal stromal cells’ to describe MSCs.2 While ‘mesenchymal stromal cells’ may be the more scientifically accurate term for MSCs, the two terms are often used interchangeably in the literature. R&D Systems recognizes the use of both mesenchymal stem cells and mesenchymal stromal cells and uses ‘MSC’ to indicate mesenchymal stem/stromal cells to account for both designations.
Definitions of Mesenchymal Stromal Cells and Mesenchymal Stem Cells
- Mesenchymal Stromal Cells – A heterogeneous population of cultured cells with similar characteristics such as the ability to adhere to plastic and the expression of specific marker proteins.
- Mesenchymal Stem Cells – A subpopulation of mesenchymal stromal cells that have the capacity to self-renew and differentiate into mesodermal lineages when cultured in vitro. The capacity to self-renew and differentiate in vivo has yet to be clearly demonstrated for mesenchymal stem cells.
- Dominici, M. et al. (2006) Cytotherapy 8:315.
- Keating, A. (2012) Cell Stem Cell 10:709.
Preparation and Storage
Background: Mesenchymal Stem Cells
The term 'mesenchymal stem cells' (MSCs) is most commonly used to describe multipotent self-renewing cells that can be differentiated in vitro to generate adipocytes, chondrocytes, and osteoblasts. However, because these biological properties and hierarchical relationships remain to be clearly demonstrated in vivo, the term 'multipotent mesenchymal stromal cells' is often used to distinguish cultured cells from their in vivo precursors. Originally discovered in mouse bone marrow, multipotent mesenchymal stromal cells cultured from a variety of species and tissue types, have been shown to differentiate into progeny of additional lineages including, cardiomyocytes, endothelial cells, hepatocytes, and neural cells. Again, the physiological relevance of these findings remains to be determined.
Refer to the product datasheet for complete product details.
Briefly, MSCs can be cryopreserved for long-term storage using the following protocol:
- MSCs are detached from the culture dish
- MSCs are resuspended in StemXVivo® Serum-Free MSC Freezing Media
- Frozen, cryopreserved MSCs are stored in liquid nitrogen
Reagents supplied in the Human StemXVivo® Serum-Free Mesenchymal Stem Cell Freezing Media (Catalog # CCM016):
- 50 mL of serum-free MSC freezing media
- Human StemXVivo® Serum-Free MSC Expansion Media (Catalog # CCM014 or equivalent)
- TrypLE™ Express (Invitrogen® or equivalent)
- Human MSCs
- Tissue culture flasks
- 15 mL centrifuge tubes
- Cryogenic storage vials
- Pipettes and pipette tips
- Serological pipettes
- 37 °C and 5% CO2 incubator
- -80 °C freezer
- Liquid nitrogen storage
- Inverted microscope
- 37 °C water bath
This protocol has been tested using bone marrow- and/or adipose tissue-derived MSCs. If using a different tissue source or cell line, the protocol below may need to be modified.
Detach the MSCs from the culture dish using a dissociation solution.
Transfer the dissociated cells to a 15 mL conical tube.
Centrifuge the MSCs at 200 x g for 5 minutes.
Resuspend the cell pellet in cold StemXVivo® Serum-Free MSC Freezing Media.
Transfer the MSC suspension to a cryovial immediately.
Place the cryovial in a cryogenic container at -80 °C overnight.
Store the frozen cryovial in liquid nitrogen.
Thawing of Cryopreserved Stem Cells
Remove the frozen vials from the liquid nitrogen, and rapidly thaw in a 37 °C water bath.
Transfer the cells into a 15 mL centrifuge tube containing pre-warmed StemXVivo® Mesenchymal Stem Cell Expansion Media.
Centrifuge the MSCs at 200 x g for 5 minutes.
Resuspend the cells in serum-free complete growth medium and plate the MSCs in desired culture conditions.
Can Mesenchymal Stem Cells (MSCs) grown in StemXVivo® MSC Expansion Media (Catalog # CCM004) be cryopreserved?
MSCs grown in StemXVivo® MSC Expansion Media can be cryopreserved using appropriate cryopreservation media, such as StemXVivo® Serum-Free MSC Freezing Media (Catalog # CCM016) or equivalent.
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