How to maintain and transport equine adipose tissue for isolating mesenchymal stem cells?

Abstract

BACKGROUND: Adipose tissue (AT) is one of the most important mesenchymal stem cell (MSC) sources because of its high quantities, availability and ease of collection. After being collected samples, they should be transported to a laboratory for stem cell (SC) isolation, culture and expansion for future clinical application. Usually, laboratories are distant from animal husbandry centers; therefore, it is necessary to provide suitable conditions for adipose tissue transportation, such that adipose-derived MSCs are minimally affected. In the current study, the impact of tissue maintenance under different conditions on MSCs derived from these tissues was evaluated. We aimed at finding suitable and practical transportation methods in which ASCs go through the slightest changes. RESULTS: In the current study, after being collected, equine AT was randomized into eight groups: four samples were maintained in stem cell culture media at 25C and 4C for 6 and 12 hrs. as transportation via SC media groups. Three samples were frozen at three different temperatures (- 20, - 75 and - 196C) as cryopreserved groups; these samples were defrosted 1 week after cryopreservation. Fresh and unfrozen AT was evaluated as a control group. The tissue samples were then initiated into enzymatic digestion, isolation and the culturing of SCs. Cells at passage three were used to evaluate the ability to form colonies, proliferation rate, plotting of the cell growth curve, and viability rate. All experiments were performed in triplicate. Stem cell isolation was successful in all groups, although purification of SCs from the first series of cryopreservation at - 196C and two series of - 20C was unsuccessful. There was no significant difference between the surface area of colonies in all groups except for - 20C. The growth rate of transportation via stem cell media at 25C for 6 hrs. was similar to that of the control group. MTT analysis revealed a significant difference between 25C 12 hrs. Group and other experimental groups except for control, 4C 12 hrs. and - 196C group. CONCLUSION: Data have shown freezing at - 75C, transportation via stem cell media at 4C for 12 hrs. and 25C for 6 hrs. are acceptable tissue preservation and transportation methods due to minor effects on MSCs features.