CryoDefend-Stem Cells (5 x 10 mL)
R&D Systems, part of Bio-Techne | Catalog # CCM018
Key Product Details
Summary for CryoDefend-Stem Cells (5 x 10 mL)
Kit Summary
Media for defined, protein-free cryopreservation of stem cells.
Key Benefits
- Reduce experimental variability by using fully defined media
- Supports post-thaw cell viability better than conventional freezing media
- Optimized for cryopreservation of stem cells
- Suitable for all stem cell types
Why use fully defined, protein-free media to cryopreserve stem cells?
Cryopreservation is essential for the banking of valuable stem cell cultures. The viability of the recovered cells depends on cell density, the type of cryoprotectant used, the method of cooling, and the amount of the cryoprotectant that remains in the culture after the cells have been thawed.
Additional considerations for stem cell researchers include the effect of the cryopreservative on stem cell potency and the presence of any undefined factors which could limit t he use of recovered cells in preclinical and clinical studies. These additional considerations emphasize the importance of using cryopreservation media that has been specifically optimized for the cryopreservation of stem cells.
R&D Systems offers CryoDefend® Stem Cells Media, a defined, protein-free media, which has been optimized for stem cell cryopreservation. This specially formulated media increases the recovery and viability of healthy undifferentiated cells compared to conventional freezing media.
Kit Contents
Components
- 5 x 10 mL vials of CryoDefend® Stem Cells Media
Stability and Storage
Upon receipt, the CryoDefend® Stem Cells Media should be stored at = -20 °C in a manual defrost freezer. The media can be thawed at 2 °C to 8 °C or at room temperature. Thawed media can be aliquoted and stored at = -20 °C in a manual defrost freezer for up to 3 months. Thaw a fresh aliquot for each use. Avoid repeated freeze-thaw cycles.
Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of pre-implantation embryos. Induced pluripotent stem (iPS) cells can be generated by somatic cell reprogramming following the exogenous expression of specific transcription factors (Oct-3/4, KLF4, SOX2, and c-Myc). These cell types are capable of unlimited, undifferentiated proliferation in vitro and still maintain the capacity to differentiate into a wide variety of somatic cells. In this capacity, pluripotent stem cells have widespread clinical potential for the treatments of heart disease, diabetes, spinal cord injury, and a variety of neurodegenerative disorders.
R&D Systems offers a wide range of products to support pluripotent stem cell culture and differentiation. Mouse embryonic fibroblasts may be used to maintain and expand pluripotent stem cells in an undifferentiated state. We also offer defined culture media, which are specifically optimized for use with human or rodent pluripotent stem cells. In addition, R&D Systems offers a variety of products to assess differentiation status and identify specific stem cell types of interest, including panels of marker antibodies, primer pairs, multi-color flow cytometry kits, and specialized verification kits.
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Human Embryonic Stem Cell Viability Following Cryopreservation in CryoDefend® Stem Cells Media or Cryopreservation Media from Two Competitors. Stem Cells Media or Cryopreservation Media from Two Competitors.BG01V human embryonic stem cells were frozen in cryopreservation media from two different competitors or CryoDefend®Stem Cells Media (Catalog # CCM018) at 1 x 106cells/cryovial and stored in liquid nitrogen for one week. BG01V human embryonic stem cells were then thawed, counted (blue bars), and resuspended in Mouse Embryonic Fibroblast (MEF) Conditioned Media (Catalog # AR005) containing Recombinant Human FGF basic (Catalog # 4114-TC). The resuspended cells were plated into one well of a 6-well plate and after four days in culture, the cells were harvested and counted (red bars). The error bars indicate the standard deviation of triplicate samples. |
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Morphology of Human Embryonic Stem Cells Following Cryopreservation in CryoDefend® Stem Cells Media, Conventional Freezing Media, or Freezing Media from a Competitor. BG01V human embryonic stem cells were frozen in CryoDefend®Stem Cells Media (Catalog # CCM018), conventional freezing media (90% FBS/10% DMSO) or cryopreservation media from a competitor. After thawing, the cells were cultured for 3 days in Mouse Embryonic Fibroblast Conditioned Media (Catalog # AR005) containing FGF basic (Catalog # 4114-TC) and imaged using brightfield microscopy. |
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Expression of Pluripotency Markers in BG01V Human Embryonic Stem Cells Frozen in CryoDefend® Stem Cells Media or Cryopreservation Media from Two Different Competitors. Cells that were cryopreserved in CryoDefend®Stem Cells Media (Catalog # CCM018) or cryopreservation media from one of two competitors CryoDefend®Stem Cells Media (Catalog # CCM018) were thawed and cultured for four days in Mouse Embryonic Fibroblast Conditioned Media (Catalog # AR005). The cells were then assessed for pluripotency marker expression by flow cytometry. The cells were stained with APC-conjugated Mouse Anti-Human Oct-4A (Catalog # IC6344A) and PE-conjugated Mouse Anti-Human/Mouse SSEA-4 (Catalog # FAB1435P). The quadrants were set based on isotype controls. The percent of double positive cells are indicated in the upper right quadrant. |
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Human Embryonic Stem Cells Maintain Pluripotency Following Cryopreservation in CryoDefend® Stem Cells Media. BG01V human embryonic stem cells that were frozen in CryoDefend®Stem Cells Media (Catalog # CCM018) were thawed and cultured for four days before the cells were replated for differentiation into each of the three germ layers according to the insert instructions for the Human Pluripotent Stem Cell Functional ID Kit (Catalog # SC027). Differentiated cells were then stained for markers of each germ layer as indicated in the images using the fluorochrome-conjugated antibodies included in the Human Three-Germ Layer 3-Color Immunocytochemistry Kit (Catalog # SC022). The nuclei were counterstained with DAPI (blue). |
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Recovery and Marker Expression of Rat Mesenchymal Stem Cells Cryopreserved in CryoDefend® Stem Cells Media or Conventional Freezing Media. Rat Mesenchymal Stem Cells (Catalog # PSC003) were cryopreserved in CryoDefend®Stem Cells Media (Catalog # CCM018) or conventional freezing media (95% FBS/5% DMSO) at 0.7x106cells/vial. The cryopreserved cells were thawed and cultured for four days before assessing cell recovery and MSC marker expression. A) The number of cryopreserved cells recovered after four days in culture. B) Expression of positive MSC marker CD90 and negative MSC marker CD45 after the thawed cryopreserved cells were cultured for four days. CD90 was detected with a RPE-conjugated Anti-Rat CD90/Thy1 Antibody and CD45 was detected with 687-conjugated Anti-Rat CD45 Antibody. |
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Rat Cortical Stem Cell Viability Following Cryopreservation in Control Cyropreservation Media or CryoDefend® Stem Cells Media. Rat cortical stem cells (Catalog # NSC001) were frozen in control cryopreservation media (DMEM/F12 supplemented with N2-MAX Media Supplement,10% BSA, and 10% DMSO) or in CryoDefend®Stem Cells Media (Catalog # CCM018) at 1 x 106cells per cryovial and stored in liquid nitrogen for one week. After thawing in DMEM/F12 containing N2-MAX Media Supplement (Catalog # AR009) and 20 ng/mL of FGF basic (Catalog # 4114-TC), the cells were counted (light blue bars) and plated onto Poly-L-ornithine/Fibronectin-coated plates. The cells were cultured for four days prior to passage (Passage 1) at which time the cells were counted (dark blue bars). The error bars indicate the standard deviation of triplicate samples. |
Assay Procedure
Refer to the product datasheet for complete product details.
- Transfer detached cells to a conical tube and centrifuge
- Remove supernatant and resuspend in CryoDefend® Stem Cells Media
- Transfer cells to a cryovial and freeze at -80 °C overnight
- Transfer the frozen cryovial to liquid nitrogen
Reagents supplied in the CryoDefend® Stem Cells Media (Catalog # CCM018):
- 5 x 10 mL vials of CryoDefend® Stem Cells Media
Reagents
- Mouse Embryonic Fibroblast (MEF) Conditioned Media (Catalog # AR005)
Materials
- Stem cells
- Cryovials
- 15 mL centrifuge tubes
- Serological pipettes
- Pipette and pipette tips
Equipment
- 37 °C and 5% CO2 incubator
- Centrifuge
Cryopreservation of Stem Cells
Thaw CryoDefend® Stem Cells Media.
Detach the cells from the cell culture dish.
Transfer the cells to a 15 mL conical tube.
Centrifuge at 200 x g for 5 minutes.
Remove and discard the supernatant.
Resuspend the cells in CryoDefend® Stem Cells Media at 0.5-1.0 x 106 cells/mL.
Transfer the cells to a cryovial.
Freeze the cryovial at -80 °C overnight.
Transfer the frozen cryovial to liquid nitrogen for storage.
Thawing of Cryopreserved Stem Cells
Warm appropriate cell culture media to 37 °C.
Add pre-warmed culture media to the cryovial containing cryopreserved cells.
Pipette up and down and as cells thaw, transfer the thawed cells to a 15 mL conical tube containing pre-warmed expansion media.
Centrifuge at 200 x g for 5 minutes.
Resuspend the cells in pre-warmed expansion media.
Plate the stem cells at the desired density.
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