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. 2018 Oct 9:5:31.
doi: 10.21037/sci.2018.09.01. eCollection 2018.

Equine bone marrow-derived mesenchymal stem cells: optimization of cell density in primary culture

Morteza Zahedi  1 , Abbas Parham  1   2 , Hesam Dehghani  1   2 , Hossein Kazemi Mehrjerdi  3
Affiliations

Equine bone marrow-derived mesenchymal stem cells: optimization of cell density in primary culture

Morteza Zahedi et al. Stem Cell Investig. .

Abstract

Background: The primary cell seeding density of bone marrow-derived mononuclear cells (BM-MNCs) affects several cellular behaviors, including attachment to the culture dish, proliferation, and differentiation.

Methods: The aim of this study was to determine the best density of equine BM-MNCs in primary culture (P0) for obtaining the maximum bone marrow-derived mesenchymal stem cell (BM-MSC) yields at the end of P0. Bone marrow samples of two healthy mares were aspirated. The MNCs were isolated and cultured at different densities (1×105, 2×105, 4×105, 8×105, and 1×106 cells/cm2). Within the 7th and 14th days after seeding, the colonies containing more than 15 cells were counted and the percentage of confluency and the number of cells were calculated on day 21.

Results: The lowest density of MNCs was associated with the least number of colonies, number of adherent cells, and confluency percentage, whereas the highest density was associated with the maximum number of colonies and confluency percentage (P<0.05). However, the maximum number of cells at the end of P0 was associated with the intermediate (4×105 cells/cm2) and the highest concentration (P<0.05).

Conclusions: The maximum number of MSCs at the end of P0 was obtained at the densities of 1×106 and, especially, at 4×105 cells/cm2.

Keywords: Cell count; cell yield; horses; mesenchymal stromal cells; mononuclear cells (MNCs).

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Conflict of interest statement

Conflicts of Interest: The authors declare that there is no conflict of interest.

Figures

Figure 1
Figure 1
Experimental procedure timeline. P3: Cells at passage 3.
Figure 2
Figure 2
A colony with more than 15 cells.
Figure 3
Figure 3
Initial seeding of bone marrow-derived mononuclear cells in flasks. Density: 4×105 cells/cm2. Magnification, ×40.
Figure 4
Figure 4
Overlapping and almost multilayer cell proliferation in the colonies of MSCs in the flask seeded with 4×105 cells/cm2 at passage 0. The arrows represent the overlapping zones (A and B). Monolayer growth of MSCs in passage 1 (C) and in passage 3 (D).
Figure 5
Figure 5
Gene expression analysis for GAPDH, CD29, CD34, CD44, CD90 and MHC-II, of BM-MSCs at passage 3. Gel electrophoresis of PCR products showed the expression of GAPDH (183 bp), CD29 (234 bp), CD44 (193 bp) and CD90 (155 bp). No expression of CD34 and MHC-II was detected. RT had mRNA instead of cDNA template in PCR to exclude DNA contamination. RT: RNA template; NTC: non-template (negative) control.
Figure 6
Figure 6
Tri-lineage in vitro differentiation assays of equine BM-MSCs at passage 3. Calcium deposition was stained by Alizarin Red S in the osteogenic treatment group (A). Intracellular neutral lipid droplets were detected by Oil Red O in the adipogenic treatment group (B). Alician blue was used to stain proteoglycans in the extracellular matrix of the chondrogenic differentiation group (C). (D,E,F) are the control group for osteogenic, adipogenic and chondrogenic, respectively.
See this image and copyright information in PMC

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