Impact of delayed culture initiation on the viability and functionality of precision-cut adipose tissue slices

Details

Supervisors

Debier, Cathy ; Rees, Jean-François

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en sciences et technologies de l'environnement, à finalité spécialisée

Abstract

Adipose tissue is an essential endocrine organ that plays a key role in various biological functions. Its metabolism can be disrupted by various factors such as diseases or exposure to endocrine disruptors, which can eventually affect the entire organism. Our research group has recently developed a novel approach of precision-cut adipose tissue slices (PCATS) to study in vitro adipose function and dysfunction. Adipose tissue is often present in large quantities in pig, which is used as an animal model to perform PCATS, but only a small proportion of it can be used for tissue culture experiments in a time frame adequate for its survival. In addition, the sampling of some wild species, such as polar bears and whales, presents a real challenge for preservation between the time of collection and culture so that the tissue is not damaged. This master thesis aimed at developing a protocol that would allow (i) a long-term preservation of adipose tissue through cryopreservation or (ii) a short-term preservation of adipose tissue through the postponement of the culture onset. Parameters were calibrated in order to adjust the model. We mainly focused on lipolysis to test the in vitro functionality of the tissue and on the release of LDH in the culture media to test the cellular viability. In addition, RNA was extracted for quantification and integrity measurements. The porcine PCATS were exposed to different volumes of the cryoprotectant DMSO (3.3%) + trehalose (7.6%) as well as to 2 intermediate temperatures (-20°C and -80°C). Cryopreservation was tested on both PCATS and whole biopsy cores. Once in culture, different treatments (non-induction of lipolysis, direct induction of lipolysis, delayed induction of lipolysis after 24 hours of culture) were applied on slices in order to evaluate the effects of the types of preservation on tissue functionality and viability. Our results showed a variation in LDH production during the experiment, suggesting that this test is not the most suitable to measure PCATS viability. Therefore, cell viability was mainly studied via lipolysis, an indicator of tissue functionality. Cryopreservation of adipose tissue under the form of PCATS or whole biopsy cores appeared challenging as, in both cases, tissue functionality was lost after freezing/thawing processes. Therefore, a second way of preserving adipose tissue was investigated, this time on a short-term basis via delayed cultures. Whole cores were pre-incubated in sampling medium for 24, 48 and 72 hours at various temperatures (-4°C, room temperature and 37°C). The benefit of oxygen within the sampling medium was also investigated. The results were more promising than for cryopreservation.