Banca de DEFESA: Cecibel María León Félix

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : Cecibel María León Félix
DATE: 31/08/2023
TIME: 14:00
LOCAL: Meio virtual
TITLE:

 Obtaining decellularized extracellular matrix from bovine ovarian cortex and its characterization

KEY WORDS:

ovaries, SDS, decellularization, matrisome, proteomics

PAGES: 123
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

The decellularized extracellular matrix (ECM) of various tissues (heart, lung, kidney, uterus and testis) is allowing the regeneration and recovery of native cells and tissue functions. An alternative to maintain fertility in women diagnosed with cancer, who would become infertile due to chemotherapy and/or radiotherapy, is the development of a transplantable artificial ovary. Decellularized ECM of ovarian tissue was obtained using sodium dodecyl sulfate (SDS), usually at a concentration of 1% for 24 h. However, SDS can leave residues in the tissue, which can be toxic to seeded cells. Therefore, the objective of the present study was to obtain a decellularized extracellular matrix from bovine ovarian tissue with SDS, but with lower concentration and shorter incubation time. In addition, to identify the molecules and functions of the native and decellularized ECM. For this, bovine ovaries from slaughterhouses were transported to the laboratory in saline solution at 37 °C. In the laboratory, antral follicles were punctured with a needle, and the capsule and medullary region of the ovary were removed. Slices (10 mm x 5 mm x 1 mm) were obtained from the cortical region of the ovary, weighed and distributed among the treatment groups. The SDS stock solution was prepared with 1% SDS (Sigma-Aldrich, Brazil) and 0.2M NaOH in distilled water and then diluted to the desired concentrations. The SDS concentrations used were 1%, 0.5%, 0.1%, 0.05% and 0.01% and incubation times of 24 h, 12 h and 6 h. The ovary slices were individually submerged in 10 mL of each mixture, under constant agitation (100 rpm), at room temperature and for the period defined for each treatment. Then, they were washed in 50 ml of distilled water for 6 hours, changing the distilled water every 30 minutes. The tissue obtained from each treatment was analyzed by histology, by electrophoresis to assess the size of the DNA fragments remaining in the ovarian slices, and by DNA quantification using the Qubit® 2.0 Fluorometer (Thermo Fisher Scientific). Histological analysis confirmed decellularization and staining with Mallory's trichrome showed the conservation of collagen and elastin fibers in all samples after each treatment. In addition, the lowest concentration of SDS that did not show remaining DNA in the electrophoresis analysis was 0.1%, when incubated for 24 h and 12 h, but not for 6 h. Cell viability showed that MEC decellularized by incubation in 0.1% SDS for 12 h was not toxic to ovarian cells during in vitro cultivation for 24 and 72 h. Also, the molecules and functions of the native and decellularized ECM by 0.1% SDS for 12 h were identified through proteomics analysis. Thus, 155 proteins were identified in native ECM (87 core proteins and 68 matrisome-associated proteins) and 145 proteins in decellularized ECM (84 core proteins and 61 matrisome-associated proteins). The biological and cellular functions of native and decellularized ECM proteins were also identified. Thus, we conclude that the 0.1% SDS protocol for 12 h of incubation decellularizes the bovine ovarian tissue, generating a non-toxic decellularized ECM for ovarian cells and preserves the decellularized ECM molecules as similar as possible to the native ECM.

COMMITTEE MEMBERS:
Presidente - 2374060 - CAROLINA MADEIRA LUCCI
Externa à Instituição - ELLEN CRISTINA RIVAS LEONEL - UFG
Externa ao Programa - 3088161 - JULIANA LOTT DE CARVALHO - nullExterna à Instituição - MARGOT ALVES NUNES DODE - EMBRAPA
Externo à Instituição - MAURÍCIO MACHAIM FRANCO - EMBRAPA