Int J Stem Cells.  2015 Nov;8(2):155-169. 10.15283/ijsc.2015.8.2.155.

Cryopreservation of Human Wharton's Jelly-derived Mesenchymal Stem Cells Following Controlled Rate Freezing Protocol Using Different Cryoprotectants; A Comparative Study

• Shivakumar SB
• Bharti D
• Jang SJ
• Hwang SC
• Park JK
• Shin JK
• Byun JH
• Park BW
• Rho GJ

• KMID: 2380810
• DOI: http://doi.org/10.15283/ijsc.2015.8.2.155

Abstract

OBJECTIVES
To compare the effect of three different cryoprotectants on basic stem cell characteristics for the possibility of using well defined, dimethyl sulfoxide (DMSO) and serum free freezing solutions to cryopreserve human Wharton's jelly-derived mesenchymal stem cells (WJMSCs) following controlled rate freezing protocol.
METHODS
The mesenchymal stem cells isolated from human Wharton's jelly were cryopreserved using 10% DMSO, 10% polyvinylpyrrolidone (PVP) and a cocktail solution comprising of 0.05 M glucose, 0.05 M sucrose and 1.5 M ethylene glycol following controlled rate freezing protocol. We investigated the post-thaw cell viability, morphology, proliferation capacity, basic stem cell characteristics, in vitro differentiation potential and apoptosis-related gene expression profile before and after cryopreservation.
RESULTS
The cryoprotectant 10% DMSO has shown higher post-thaw cell viability of 81.2+/-0.58% whereas 10% PVP and cocktail solution have shown 62.87+/-0.35% and 72.2+/-0.23%, respectively at 0 h immediately thawing. The cell viability was further reduced in all the cryopreserved groups at 24 h later post-thaw culture. Further, the complete elimination of FBS in cryoprotectants has resulted in drastic reduction in cell viability. Cryopreservation did not alter the basic stem cell characteristics, plasticity and multipotency except proliferation rate. The expression of pro-apoptotic BAX and p53 genes were higher whilst p21 was lower in all the cryopreserved groups when compare to the control group of WJMSCs.
CONCLUSION
Although 10% DMSO has shown higher post-thaw cell viability compare to 10% PVP and cocktail solution, the present study indicates the feasibility of developing a well-defined DMSO free cryosolution which can improve storage and future broad range applications of WJMSCs in regenerative medicine without losing their basic stem cell characteristics.

Keyword

Apoptosis; Cocktail solution; Controlled rate freezing; Cryopreservation; Wharton's jelly mesenchymal stem cells

MeSH Terms

Apoptosis
Cell Survival
Cryopreservation*
Dimethyl Sulfoxide
Ethylene Glycol
Freezing*
Genes, p53
Glucose
Humans*
Mesenchymal Stromal Cells*
Plastics
Povidone
Regenerative Medicine
Stem Cells
Sucrose
Transcriptome
Wharton Jelly
Dimethyl Sulfoxide
Ethylene Glycol
Glucose
Plastics
Povidone
Sucrose

Figure

• 

  Fig. 1 Adherent, fibroblast-like morphology of WJMSCs from passage 3 on day 3 culture. Where (A)=Control, (B)=Solution A, (C)=Solution B and (D)=Solution C. Scale bar=100 μm.

• 

  Fig. 2 Viability percentage of WJMSCs under Control, Solution A, Solution B, Solution C, Solution D, Solution E and Solution F groups assessed immediately after thawing at 0 h (A), and at 24 h later post-thaw culture (based on the cell numbers obtained at 0 h) (B). Significant difference among groups was considered when p<0.05 and represented by different superscripts (lower case letters).

• 

  Fig. 3 Growth curves for WJMSCs under Control, Solution A, Solution B and Solution C over 14 days of in vitro culture. Cells were cultured in 24-well plates at an initial rate of 2×103 cells/well (A) and Flowcytometric analysis of cell cycle for WJMSCs under different groups. A total of 10,000 cells were counted for each sample in triplicates and the values are expressed as percentage mean±standard error of mean (SEM). Significant difference among groups was considered when p<0.05 and represented by different superscripts (lower case letters) (B).

• 

  Fig. 4 Flowcytometric analysis of the expression of surface markers by WJMSCs under Control, Solution A, Solution B and Solution C groups. WJMSCs were negative for CD34 and CD45 whereas positive for CD73, CD90 and CD105 expression (percentage of expression is also indicated in the figure).

• 

  Fig. 5 In vitro differentiation potential of WJMSCs of Control, Solution A, Solution B and Solution C groups. WJMSCs were stained both before and after adipo/osteo lineage induction. Adipogenesis was indicated by Oil red O staining of lipid globules after induction (A). Confirmation of osteogenesis was done by Alizarin red and Von Kossa staining (B).

• 

  Fig. 6 RT-PCR and western blot analysis. Relative mRNA level of transcription factors such as OCT4, SOX2 and NANOG (A) and their product size (B). Relative mRNA level of apoptosis-related BAX, BCL2, p53 and p21 genes (C) and their product size (D). Different superscripts (lower case letters) represent significant difference (p<0.05) among groups. Western blot analysis of transcription- & apoptosis related proteins (E).

• 

  Fig. 7 RT-PCR analyses on adipogenesis and osteogenesis. Relative mRNA level of adipogenic specific markers (A) and their product size (B). Relative mRNA level of osteogenic specific markers (C) and their product size on agarose gel (D). Significant differences were considered when p<0.05 and represented by different superscripts (lower case letters).

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