Evaluation of transport conditions for autologous bone marrow-derived mesenchymal stromal cells for therapeutic application in horses

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.

Veterinary Regenerative Medicine for Musculoskeletal Disorders: Can Mesenchymal Stem/Stromal Cells and Their Secretome Be the New Frontier?

“…Regarding cell transportation, many studies have shown which are the shipping criteria that should be considered-for instance, supplementing media, transport temperature, and other variables, such as the type of container for the shipping. The latter did not show meaningful evidence; in detail, the use of plastic rather than glass containers did not show differences in terms of cell viability after 24 h at room temperature (20-22 • C) at several MSC concentrations [39]. Shipping temperature is up to cell storage conditions: frozen cells should be transported in dry ice around −80 • C, inside cryovials often wrapped with precooled material and placed in a leak-proof container to prevent direct contact of samples; otherwise, they can be transported in liquid nitrogen to maintain −196 • C temperature.…”

“…As a result, the majority agreed that MSC transport should be performed at 4 °C, which seems to be the most practical one. According to this, shipping fresh equine MSCs in isotonic saline solution at 4 °C within 24 h is considered ideal for immediate administration, although, in these conditions, MSC viability decreases up to 70% [ 39 , 40 , 43 ]. Only one study considered room temperature as superior in the case of short-duration transport [ 43 ].…”

Peer Review #1 of "Preservation media, durations and cell concentrations of short-term storage affect key features of human adipose-derived mesenchymal stem cells for therapeutic application (v0.1)"

“…Compared to intravenous injection, subcutaneous injection and intramuscular injection require higher concentrated cell products with smaller volumes. As previous study has reported [28], MSCs suspension injected into small tendinous lesions would leak outside the defect and into the peritendinous tissue even the volume of cell suspension was only 1ml. Thus cell products of high cell concentration are needed.…”

“…Although cell viability in 20% HSA and 5% glucose in Ringer's lactate was high (>80%) till 48h, there was no research on the proliferation and immunosuppressive capacity of MSCs [21]. It has been reported that cell concentrations may affect biological properties of hematopoietic stem cells and cell viability of non-MSC cell lines [27,28]. Thus we also evaluated the effects of cell concentrations during short-term storage on the characteristics of MSCs.…”

Effect of Stem Cells and Gene Transfected Stem Cells Therapy on the Pancreas of Experimentally Induced Type 1 Diabetes

“…Isolation and propagation of AMSCs from rats (11,12): Under sterile conditions, rats were euthanized with carbon dioxide (CO2), their abdomens were cut open and the adipose tissues were removed. After the adipose tissues were washed with saline solution, all the tissues were collected and incubated in Dulbecco's modified Eagle's medium (DMEM), (GIBCO/BRL) supplemented with 10% fetal bovine serum (FBS) (GIBCO/BRL).…”