Use of organotypic or three-dimensional systems with basic fibroblast growth factor for in vitro culture of immature collared peccary testicle

Abstract The objective was to evaluate the effects of different culture systems and the addition of fibroblast growth factor (bFGF) during in vitro culture (IVC) of testicular tissue fragments from prepubertal collared peccaries. Testes from five individuals were collected, dissected, and cultured for up to 56 days (34°C and 5% CO2) in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented or not with FGF at 10 ng/mL, in organotypic (ORG) or 3D system culture. Samples were evaluated every 14 days for histomorphology, cell viability, DNA integrity, and proliferative activity. Overall, the ORG system without FGF addition was the best to preserve testicular fragment histomorphology, viability, and DNA integrity during IVC. However, the 3D system, regardless of the presence of FGF, impaired the DNA integrity of testicular cells in all culture periods analyzed. Regarding cell proliferation, at 14 days the ORG group without addition of FGF showed a percentage of Ki-67 positive cells indicative of proliferation similar to the non-cultured group, while the other treatments reduced proliferation. However, at 28 days a reduction in proliferation was observed in this same group and an increase in proliferation in the others. Cell proliferation was reduced in all groups at 42 days (P < 0.05). In summary, we suggest the use of the organotypic system for long-term culture of testicular fragments of prepubertal collared peccaries. In addition, FGF supplementation to the culture medium does not seem to be essential. Lay summary Animals do not produce sperm cells before puberty. However, in case of unexpected death of young individuals carrying critical genes for the diversity and sustainability of an animal population, sperm cells can be obtained by recovering and culturing tissue from the testes in proper laboratory conditions. Resulting sperm cells can then be used to produce embryos using IVF methods. The goal of the present work was to find the best culture conditions to keep pieces of testicular tissue alive for extended periods of time using the collared peccary as a model. Two different methods were tested. The first approach was to place a piece of tissue on top of a gel that was rich in nutrients, similar to the natural supply to the tissue. This method is called organotypic culture. The second approach was to recreate a more natural environment by embedding the tissue inside the gel, which is known as 3D culture. Overall, the organotypic culture was the best way to keep the tissues alive for 56 days. This is a major step forward to allow the production of sperm cells from peccaries in the laboratory.