CryoDefend Your Stem Cells

G. Herr, Y. Tang, M. Andersen, R. Cross, W. Johnson, D.J. Owens, S. Tousey, & J. Aho

ABSTRACT

Efficient cryopreservation of stem cells is essential for the maintenance of consistent stem cell stocks. Many of the existing formulations of cryopreservation media rely on high percentages of poorly defined serum or albumin. Here, we demonstrate the use of CryoDefend-Stem Cells (Catalog # CCM018) media for defined, xeno-free, and protein-free cryopreservation of mesenchymal (MSC), pluripotent, and neural stem cells. Following cryopreservation in CryoDefend-Stem Cells media, viability was assessed post-thaw. Cells were then characterized by verification of stem cell marker expression via immunocytochemistry and flow cytometry as well as functional verification via in vitro directed differentiation. CryoDefend-Stem Cells media was found to have superior or equal performance to both traditional serum/albumin-containing cryopreservation media and existing commercially available defined media.

RESULTS

Mesenchymal Stem Cells
Equivalent recovery of human MSCs from CryoDefend-Stem Cells and control cryopreservation media
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Figure 1: Equivalent recovery of human MSCs from CryoDefend-Stem Cells and control cryopreservation media. Human MSCs were frozen (1 x 106 cells/vial) in control (20% FBS/10% DMSO) or CryoDefend-Stem Cells media. Cells from each sample were thawed and viability was assessed immediately (orange bars). MSCs were then re-plated (0.25 x 106 cells/well) in StemXVivo™ Mesenchymal Stem Cell Expansion Media (Catalog # CCM004), cultured for 5 days, and assessed for viability (red bars). The recovery of viable cells stored in CryoDefend-Stem Cells or control media was not significantly different at thaw (p = 0.923) or after 5 days in culture (p = 0.831). Error bars indicate the standard deviation of triplicate samples.
CryoDefend-Stem Cells-frozen human MSCs retain multipotencyspecific marker expression
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Figure 2: CryoDefend-Stem Cells-frozen human MSCs retain multipotencyspecific marker expression. Human MSCs frozen in CryoDefend-Stem Cells media were thawed and cultured for 5 days in StemXVivo Mesenchymal Stem Cell Expansion Media (Catalog # CCM004). MSC-specific marker expression was assessed by flow cytometry using the Mesenchymal Stem Cell Verification Flow Kit (Catalog # FMC020). As expected, human MSCs stored in CryoDefend-Stem Cells media stained positive for CD73, CD105, and CD90. No staining was observed on MSCs for antibodies included in the kit’s negative marker cocktail. Marker expression for cells cryopreserved in CryoDefend-Stem Cells was similar to those in control media (data not shown). The quadrants were determined by isotype control staining.
CryoDefend-Stem Cells preserves human MSC multipotency
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Figure 3: CryoDefend-Stem Cells preserves human MSC multipotency. Human MSCs frozen in CryoDefend-Stem Cells cryopreservation media were thawed and passaged 3 times in StemXVivo Mesenchymal Stem Cell Expansion Media (Catalog # CCM004). Multipotency was assessed using the Human Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC006). MSC differentiation into adipocytes, osteoblasts, or chondrocytes was confirmed by immunocytochemistry using the lineage-specific antibodies included in the kit (red). The nuclei were counterstained with DAPI (blue).
Pluripotent Stem Cells
Superior human pluripotent stem cell recovery using CryoDefend-Stem Cells
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Figure 4: Superior human pluripotent stem cell recovery using CryoDefend-Stem Cells. BG01V human embryonic stem cells were frozen (1 x 106 cells/vial) in control (90% FBS/10% DMSO), competitor, or CryoDefend-Stem Cells media. A) Cell viability was assessed at the time of thaw (orange bars) and after 4 days in culture (red bars) with Mouse Embryonic Fibroblast Conditioned Media (Catalog # AR005). At thaw, the recovery of viable cells stored in CryoDefend-Stem Cells media was significantly enhanced over cells frozen in competitor media (p = 0.004) but not over cells in control media (p = 0.535). However, after 4 days in culture, the number of viable cells recovered from CryoDefend-Stem Cells was significantly increased compared to both control (p =0.002) and competitor media (p < 0.001). Error bars indicate the standard deviation of triplicate samples. B) Morphology of cells in control, competitor, or CryoDefend-Stem Cells media was assessed by brightfield microscopy after 3 days in culture.
CryoDefend-Stem Cells-frozen human pluripotent stem cells retain pluripotency-specific marker expression
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Figure 5: CryoDefend-Stem Cells-frozen human pluripotent stem cells retain pluripotency-specific marker expression. BG01V human embryonic stem cells frozen in control, competitor, or CryoDefend-Stem Cells media were thawed and cultured for 4 days. Pluripotent marker expression was analyzed by flow cytometry. The cells were stained with an APC-conjugated Mouse Anti-Human Oct-4A Monoclonal Antibody (Catalog # IC6344A) and a PE-conjugated Mouse Anti- Human/Mouse SSEA-4 Monoclonal Antibody (Catalog # FAB1435P). The quadrants were determined by isotype control staining.
CryoDefend-Stem Cells preserves pluripotency of human pluripotent cells
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Figure 6: CryoDefend-Stem Cells preserves pluripotency of human pluripotent cells. BG01V human embryonic stem cells frozen in CryoDefend-Stem Cells media were thawed and expanded for 1 passage. Pluripotency was assessed by differentiation into ectoderm, mesoderm, and endoderm using the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027). Differentiated cells were stained for the indicated lineage-specific markers using fluorochrome-conjugated antibodies included in the Human Three-Germ Layer 3-Color Immunocytochemistry Kit (Catalog # SC022). The nuclei were counterstained with DAPI (blue).
Neural Stem Cells
Superior recovery of rat neural progenitor cells from CryoDefend-Stem Cells media compared to competitor cryopreservation media
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Figure 7: Superior recovery of rat neural progenitor cells from CryoDefend-Stem Cells media compared to competitor cryopreservation media. Rat cortical stem cells (Catalog # NSC001) were frozen (1 x 106 cells/vial) in control (10% BSA/10% DMSO), competitor, or CryoDefend-Stem Cells media. Cell viability was assessed at the time of thaw (orange bars) and after 5 days of culture (red bars) in DMEM/F12 media supplemented with N-2 MAX (Catalog # AR009) and Recombinant Human FGF basic (20 ng/mL; Catalog # 4114-TC). At thaw, recovery of viable cells stored in CryoDefend-Stem Cells media was not significantly different from cells frozen in either competitor (p = 0.106) or control media (p = 0.072). After 5 days in culture the number of viable cells recovered from cryopreservation in CryoDefend-Stem Cells was significantly increased compared to competitor (p < 0.001) media but not control (p = 0.637) media. Error bars indicate the standard deviation of duplicate samples. The asterisks (**) indicate low recovery yield.
CryoDefend-Stem Cells media preserves rat neural progenitor cell expression of multipotency-specific markers
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Figure 8: CryoDefend-Stem Cells media preserves rat neural progenitor cell expression of multipotency-specific markers. Rat cortical stem cells frozen in control, competitor, or CryoDefend-Stem Cells media were thawed and cultured for 5 days. Stem cell-specific marker expression was assessed by flow cytometry. The cells were stained with a PE-conjugated Mouse Anti-Mouse/Rat Nestin Monoclonal Antibody (pink; Catalog # IC2736P), a PE-conjugated Mouse Anti- Human/Mouse SOX2 Monoclonal Antibody (blue; Catalog # IC2018P), or a PEconjugated Mouse IgG2A isotype control (grey; Catalog # IC003P).
CryoDefend-Stem Cells media preserves multipotency of neural progenitor cells
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Figure 9: CryoDefend-Stem Cells media preserves multipotency of neural progenitor cells. Rat cortical stem cells frozen in CryoDefend-Stem Cells media were thawed and cultured for 1 passage. Multipotency was then assessed by functional differentiation into astrocytes, oligodendrocytes, and neurons using the Human/Mouse/Rat Neural Lineage Functional Identification Kit (Catalog # SC028). Differentiated cells were stained with cell-specific antibodies included in the Human/Mouse/Rat Neural 3-Color Immunocytochemistry Kit (Catalog # SC024).

CONCLUSIONS

CryoDefend-Stem Cells media

  • Cryopreserves multiple stem cell types with better or equal efficiency than conventional or commercially available defined media.
  • Preserves stem cell-specific marker expression and differentiation capability.
  • Eliminates experimental variations associated with serum or albumin additives.

For research use only. Not for use in diagnostic procedures.

BG01V human embryonic stem cells are licensed from ViaCyte, Inc.

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