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Successful isolation of equine mesenchymal stromal cells from cryopreserved umbilical cord blood-derived mononuclear cell fractions

Catharina De Schauwer (UGent) , Gerlinde Van de Walle (UGent) , Sofie Piepers (UGent) , Maarten Hoogewijs (UGent) , Jan Govaere (UGent) , Evelyne Meyer (UGent) and Ann Van Soom (UGent)
(2013) EQUINE VETERINARY JOURNAL. 45(4). p.518-522
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Abstract
Reasons for performing study: The therapeutic potential of mesenchymal stromal cells for cellular therapy has generated increasing interest in human as well as veterinary medicine. Considerable research has been performed on the cryopreservation of expanded mesenchymal stromal cells, but little information is available on the cryopreservation of the original mononuclear cell fraction. Objectives: The present study describes a protocol to expand equine mesenchymal stromal cells after cryopreserving the mononuclear cells of umbilical cord blood. Methods: To this end, mononuclear cells were isolated from 7 umbilical cord blood samples and cryopreserved at a concentration of 1-2 x 109 cells/l cold freezing solution. Cells were cryopreserved and kept frozen for at least 6 months before thawing. Frozen cryotubes were thawed in a 37 degrees C water bath. Putative equine mesenchymal stromal cells were immunophenotyped using multicolour flow cytometry based on a selected 9 marker panel. Results: Average cell viability upon thawing was 98.7 +/- 0.6%. In 6 out of 7 samples, adherent spindle-shaped cell colonies were observed within 9.0 +/- 2.6 days and attained 80% confluency at 12.3 +/- 3.9 days. After 3 passages, putative equine mesenchymal stromal cells were successfully immunophenotyped as CD29, CD44 and CD90 positive, and CD45, CD73, CD79, CD105, MHC II and monocyte-marker negative. Conclusions and potential relevance: Equine mesenchymal stromal cells can be cultured after cryopreservation of the isolated mononuclear cells, a time- as well as cost-efficient approach in equine regenerative medicine.
Keywords
mesenchymal stromal cells, horse, cryopreservation, mononuclear cell fraction, MULTICOLOR FLOW-CYTOMETRY, SOMATIC STEM-CELLS, BONE-MARROW, CRITERIA, CULTURE, MEDIA

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Chicago
De Schauwer, Catharina, Gerlinde Van de Walle, Sofie Piepers, Maarten Hoogewijs, Jan Govaere, Evelyne Meyer, and Ann Van Soom. 2013. “Successful Isolation of Equine Mesenchymal Stromal Cells from Cryopreserved Umbilical Cord Blood-derived Mononuclear Cell Fractions.” Equine Veterinary Journal 45 (4): 518–522.
APA
De Schauwer, C., Van de Walle, G., Piepers, S., Hoogewijs, M., Govaere, J., Meyer, E., & Van Soom, A. (2013). Successful isolation of equine mesenchymal stromal cells from cryopreserved umbilical cord blood-derived mononuclear cell fractions. EQUINE VETERINARY JOURNAL, 45(4), 518–522.
Vancouver
1.
De Schauwer C, Van de Walle G, Piepers S, Hoogewijs M, Govaere J, Meyer E, et al. Successful isolation of equine mesenchymal stromal cells from cryopreserved umbilical cord blood-derived mononuclear cell fractions. EQUINE VETERINARY JOURNAL. 2013;45(4):518–22.
MLA
De Schauwer, Catharina, Gerlinde Van de Walle, Sofie Piepers, et al. “Successful Isolation of Equine Mesenchymal Stromal Cells from Cryopreserved Umbilical Cord Blood-derived Mononuclear Cell Fractions.” EQUINE VETERINARY JOURNAL 45.4 (2013): 518–522. Print.
@article{4117368,
  abstract     = {Reasons for performing study: The therapeutic potential of mesenchymal stromal cells for cellular therapy has generated increasing interest in human as well as veterinary medicine. Considerable research has been performed on the cryopreservation of expanded mesenchymal stromal cells, but little information is available on the cryopreservation of the original mononuclear cell fraction.
Objectives: The present study describes a protocol to expand equine mesenchymal stromal cells after cryopreserving the mononuclear cells of umbilical cord blood.
Methods: To this end, mononuclear cells were isolated from 7 umbilical cord blood samples and cryopreserved at a concentration of 1-2 x 109 cells/l cold freezing solution. Cells were cryopreserved and kept frozen for at least 6 months before thawing. Frozen cryotubes were thawed in a 37 degrees C water bath. Putative equine mesenchymal stromal cells were immunophenotyped using multicolour flow cytometry based on a selected 9 marker panel.
Results: Average cell viability upon thawing was 98.7 +/- 0.6\%. In 6 out of 7 samples, adherent spindle-shaped cell colonies were observed within 9.0 +/- 2.6 days and attained 80\% confluency at 12.3 +/- 3.9 days. After 3 passages, putative equine mesenchymal stromal cells were successfully immunophenotyped as CD29, CD44 and CD90 positive, and CD45, CD73, CD79, CD105, MHC II and monocyte-marker negative.
Conclusions and potential relevance: Equine mesenchymal stromal cells can be cultured after cryopreservation of the isolated mononuclear cells, a time- as well as cost-efficient approach in equine regenerative medicine.},
  author       = {De Schauwer, Catharina and Van de Walle, Gerlinde and Piepers, Sofie and Hoogewijs, Maarten and Govaere, Jan and Meyer, Evelyne and Van Soom, Ann},
  issn         = {0425-1644},
  journal      = {EQUINE VETERINARY JOURNAL},
  keyword      = {mesenchymal stromal cells,horse,cryopreservation,mononuclear cell fraction,MULTICOLOR FLOW-CYTOMETRY,SOMATIC STEM-CELLS,BONE-MARROW,CRITERIA,CULTURE,MEDIA},
  language     = {eng},
  number       = {4},
  pages        = {518--522},
  title        = {Successful isolation of equine mesenchymal stromal cells from cryopreserved umbilical cord blood-derived mononuclear cell fractions},
  url          = {http://dx.doi.org/10.1111/evj.12003},
  volume       = {45},
  year         = {2013},
}

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