Abstract
Background. Mesenchymal stromal cells (MSCs) are increasingly used for clinical applications in equine patients. For MSC isolation and expansion, a laboratory step is mandatory, after which the cells are sent back to the attending veterinarian. Preserving the biological properties of MSCs during this transport is paramount. The goal of the study was to compare transport-related parameters (transport container, media, temperature, time, cell concentration) that potentially influence characteristics of culture expanded equine MSCs.Methods. The study was arranged in three parts comparing (I) five different transport containers (cryotube, two types of plastic syringes, glass syringe, CellSeal), (II) seven different transport media, four temperatures (4 °C vs. room temperature; −20 °C vs. −80 °C), four time frames (24 h vs. 48 h; 48 h vs. 72 h), and (III) three MSC concentrations (5 × 106, 10 × 106, 20 × 106 MSC/ml). Cell viability (Trypan Blue exclusion; percent and total number viable cell), proliferation and trilineage differentiation capacity were assessed for each test condition. Further, the recovered volume of the suspension was determined in part I. Each condition was evaluated using samples of six horses (n = 6) and differentiation protocols were performed in duplicates.Results. In part I of the study, no significant differences in any of the parameters were found when comparing transport containers at room temperature. The glass syringe was selected for all subsequent evaluations (highest recoverable volume of cell suspension and cell viability). In part II, media, temperatures, or time frames had also no significant influence on cell viability, likely due to the large number of comparisons and small sample size. Highest cell viability was observed using autologous bone marrow supernatant as transport medium, and “transport” at 4 °C for 24 h (70.6% vs. control group 75.3%); this was not significant. Contrary, viability was unacceptably low (<40%) for all freezing protocols at −20 °C or −80 °C, particularly with bone marrow supernatant or plasma and DMSO. In part III, various cell concentrations also had no significant influence on any of the evaluated parameters. Chondrogenic differentiation showed a trend towards being decreased for all transport conditions, compared to control cells.Discussion. In this study, transport conditions were not found to impact viability, proliferation or ability for trilineage differentiation of MSCs, most likely due to the small sample size and large number of comparisons. The unusual low viability after all freezing protocols is in contrast to previous equine studies. Potential causes are differences in the freezing, but also in thawing method. Also, the selected container (glass syringe) may have impacted viability. Future research may be warranted into the possibly negative effect of transport on chondrogenic differentiation.
Highlights
Mesenchymal stromal cells (MSCs) are increasingly applied for various diseases in humans (Sharma et al, 2014) and animals (Guercio et al, 2012; Smith, Garvican & Fortier, 2014)
Mesenchymal stromal cells can be isolated from multiple body tissues including bone marrow (BM), adipose tissue and blood (Sharma et al, 2014; Smith, Garvican & Fortier, 2014)
Viability assessed by Trypan Blue staining was lower in all transport groups compared to the control group, i.e., viability at time point 0 (76.8% interquartile range (IQR) 14.6%), this was not statistically significant
Summary
Mesenchymal stromal cells (MSCs) are increasingly applied for various diseases in humans (Sharma et al, 2014) and animals (Guercio et al, 2012; Smith, Garvican & Fortier, 2014). The goal of the study was to compare transport-related parameters (transport container, media, temperature, time, cell concentration) that potentially influence characteristics of culture expanded equine MSCs. Methods. The study was arranged in three parts comparing (I) five different transport containers (cryotube, two types of plastic syringes, glass syringe, CellSeal), (II) seven different transport media, four temperatures (4 ◦C vs room temperature; −20 ◦C vs −80 ◦C), four time frames (24 h vs 48 h; 48 h vs 72 h), and (III) three MSC concentrations (5×106, 10×106, 20×106 MSC/ml). In part II, media, temperatures, or time frames had no significant influence on cell viability, likely due to the large number of comparisons and small sample size. Highest cell viability was observed using autologous bone marrow supernatant as transport medium, and ‘‘transport’’ at 4 ◦C for 24 h (70.6% vs control group 75.3%); this was not significant. Transport conditions were not found to impact viability, proliferation or ability for trilineage differentiation of MSCs, most likely due to the
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