VIABILITY AND DISTRIBUTION EVALUATION OF MESENCHYMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE MARKED BY NANOCRYSTALS IN HORSES WITH CHRONIC LAMINITIS 24 HOURS AFTER REGIONAL INFUSION - PRELIMINARY STUDY

Background The objective of this study was to evaluate the viability, distribution and setting of previously characterized and differentiated equine Ad-MSC labeled with the Qtracker cell labeling kit after infusion into the forelimb of horses with chronic laminitis. Methods Subcutaneous fat from healthy horse was collected, and Ad-MSC were isolated and cultured. In the third pass, Ad-MSC were marked with fluorescent nanocrystals. Labeling was carried out according to the manufacturer‘s instructions. Briefly, two million Ad-MSC were marked by adding 2ul of nanocrystals, 2ul of PBS and 200ul of DMEM medium, followed by incubation at 37°C and 5% CO2 for 1 hour. Cell viability was determined by staining with trypan blue before staining and at 1 and 24 hours after staining. 20 million allogeneic Ad-MSC marked according to the manufacturer‘s standards and suspended with 20 ml of PBS were infused in 2 horses with chronic laminitis. The infusion was performed only in one member (treated), previously drawn, by regional limb perfusion (RLP) and the tourniquet was maintained for 30 minutes. In the contralateral limb, only PBS was infused. Lamellar tissue biopsies were performed 24 hours later. The absorption of nanocrystals in the cell was observed through fluorescence microscopy in samples of labeled cells and lamellar tissue obtained by biopsy. Results Cytoplasmic absorption of nanocrystals was observed 1 and 24 hours after staining. The viability of Ad-MSC in culture before labeling was 91%. After 1 hour of incubation with nanocrystals, viability decreased slightly to 85% followed by 83% viable cells 24 hours after incubation. The results of this study demonstrate that Qdot nanocrystals can be successfully introduced into equine Ad-MSC with minimal cytotoxic effect on these cells. The cytoplasmic absorption of the nanocrystals was observed in the form of cell niches in the secondary laminae in both animals, but in greater quantity in the samples of the treated limb. Although lamellar tissue autofluorescence was observed in both limbs, there was a wide predominance of labeled Ad-MSC in the treated limbs. Conclusion The cell labeling protocol was successful, and the labeled cells remained in the lamellar tissue of horses with chronic laminitis 24 hours after the RLP. The preliminary findings of this study indicate that the cells remained in the limb that received the RLP, demonstrating therapeutic potential in the use of this technique.