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

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 25 οC and 4 οC for 6 and 12 hrs. as transportation via SC media groups. Three samples were frozen at three different temperatures (- 20, - 75 and - 196 οC) 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 - 196 οC and two series of - 20 οC was unsuccessful. There was no significant difference between the surface area of colonies in all groups except for - 20 οC. The growth rate of transportation via stem cell media at 25 οC for 6 hrs. was similar to that of the control group. MTT analysis revealed a significant difference between 25 οC 12 hrs. Group and other experimental groups except for control, 4 οC 12 hrs. and - 196 οC group. CONCLUSION: Data have shown freezing at - 75 οC, transportation via stem cell media at 4 οC for 12 hrs. and 25 οC for 6 hrs. are acceptable tissue preservation and transportation methods due to minor effects on MSCs features.

Intra-cerebroventricular Administration of Crocin Attenuates Sleep Deprivation-induced Hyperalgesia in Rats.

INTRODUCTION: Sleep deprivation can cause hyperalgesia and interfere with analgesic treatments. The aim of the present study was to establish an obligatory sleep-abstinence model and also evaluate the effects of Intracerebroventricular (ICV) injection of crocin on pain perception in Wistar rats. METHODS: In this experimental study, 35 adult male Wistar rats were randomly divided into 5 groups (n=7). The intra-ventricular cannulation was done for all rats before sleep deprivation. Sleep deprivation was performed by placing animals on a chamber equipped with an automatic animated conveyor (5 s with an interval of 3 min) for 72 h. Subsequently, the sleep-deprived animals received ICV injection of saline (MOD), Morphine 10 µg (MOR), Crocin 10 ug (Cr10), and Crocin40 µg (Cr40) using a microsyringe. Besides, a non-sleep-deprived group was allocated as a Control Group (NC) and only received an ICV injection of saline. Fifteen minutes after the ICV injections, pain perception was evaluated by the hot plate test (54±0.4°C). RESULTS: Compared with the NC group, latency significantly decreased in the MOD group (6.28±0.48 vs. 4.28± 0.48, P<0.0001). In comparison with the MOD group, both morphine (8.42±1.53) and crocin (7.60±1.45 for Cr10 and 8.14±0.89 for Cr40) could significantly increase latency in the sleep-deprived animals (P<0.0001). There was no statistically significant difference between the Cr10 and Cr40 (P=0.42), Cr10, and MOR (P=0.059) and Cr40 with MOR (P=0.86) groups. CONCLUSION: Our results indicated that crocin could attenuate hyperalgesia induced by sleep deprivation in rats.