Otimização dos protocolos de criopreservação celular e xenotransplante de cartilagem auricular para conservação de onças-pintadas (Panthera onca)

Abstract

In the pursuit of conserving genetic diversity among jaguar populations, the establishment of genetic material biobanks is gaining prominence. An initial crucial stride involves refining protocols to ensure the viability of genetic material subsequent to cryopreservation. Notably, the cryopreservation of somatic cells in jaguars has exhibited favorable outcomes, particularly with the utilization of the slow freezing curve involving isopropyl alcohol. This research endeavors to ascertain whether the viability of cells can be maintained using rapid freezing as compared to the conventional approach. A key objective encompasses the exploration of an alternative method for cellular storage, alongside the assessment of xenotransplantation feasibility utilizing tissue samples from deceased animals. The study further scrutinizes whether material revitalization is feasible during the initial stages of decomposition. In the primary experiment, cells were cultured until the third passage, assessed for viability, and subsequently cryopreserved using both the traditional slow freezing method (referred to as SF) employing the Mr. Frosty equipment, and the rapid freezing approach (referred to as RF) utilizing nitrogen vapor and stored in 0.25ml straws. Following a defined timeframe, the cells were thawed, viability was assessed through trypan blue dye (TB) and fluorescent probes, including necrosis and apoptosis tests (using acridine orange and propidium iodide - AO), and flow cytometry (employing Hoechst33342 and propidium iodide - CT). Post-assessment, the cells were cultured again, and upon reaching 70-80% confluence, underwent further evaluation. Statistical analysis indicated significant differences (p<0.05) between thawed cells from slow freezing (TB: 86.7±3.7, AO: 79.4±1.9) and fast freezing (TB: 83.8±4.5, AO: 73.0±6.7), as well as at confluence for slow freezing (TB: 99.2±0.5, AO: 92.0±1.4, CT: 94.91±0.61) and fast freezing (TB: 99.6±0.1, AO: 87.8±2.7, CT: 92.73±1.7). Notably, cultured cells demonstrated comparable viability to fresh cells (p>0.05) (TB: 98.24±0.3, AO: 88.7±3.9, CT: 94.24±2.16). Thawed cells exhibited reduced viability, which subsequently improved after reaching confluence. Discrepancies were observed in viability analyses, with the TB marker identifying more cells as non-viable (SF: 86.76±3.68, RF: 83.78±4.55), while AO (SF: 92±1.38, RF: 87.80±2.69) and cytometry results (SF: 94.91±0.61, RF: 92.73±1.7) yielded analogous outcomes. In the subsequent experiment, ear fragments from deceased jaguars were processed, with the skin removed and cartilage cleansed before initiating culture. A portion of the fragments were subjected to fresh culture, while six were vitrified on a solid surface. Post-reheating, fragments were either cultured, fixed for histological analysis, or xenotransplanted into immunodeficient mice – both intraperitoneally (XenoIP) and subcutaneously (XenoSC). Histological evaluation a week post-xenotransplantation revealed varying fibroblast counts. Notably, XenoSC fragments exhibited higher fibroblast counts for certain animals compared to XenoIP and thawed samples. A positive trend was observed for xenotransplantation across all animals, with XenoSC emerging as the most conducive for maintaining cell viability. Importantly, the average fibroblast count obtained after thawing closely aligned with literature values for tissue vitrification on solid surfaces. Overall, this research contributes valuable insights into the viability of cryopreserved jaguar somatic cells and the potential of xenotransplanting post-mortem tissue fragments. The findings underscore the significance of rapid freezing techniques and the feasibility of preserving genetic material and tissue samples to aid in jaguar population conservation efforts.