Implementação e padronização da cultura de fibroblastos derivados de pele para obtenção de DNA genômico para aplicações em técnicas de sequenciamento e análise de cariótipo

Abstract

Cancer represents a major global public health challenge, with high incidence, morbidity, and mortality, characterized by the accumulation of genetic and epigenetic alterations that compromise genomic integrity and promote malignant transformation. Among the various types of cancer, hematological neoplasms stand out, including leukemias, lymphomas, and myelodysplastic syndromes, associated with mutations in genes involved in the cell cycle and DNA repair. The distinction between germline and somatic variants is essential for diagnosis, prognosis, and clinical management, with Next Generation Sequencing (NGS) being an efficient tool for detecting these alterations. This study aimed to implement and standardize a protocol for fibroblast culture from skin biopsies to obtain high-quality genomic DNA for NGS. This is an experimental and descriptive study using samples from individuals with and without a history of hematological neoplasms, recruited at the Walter Cantídio University Hospital (HUWC), older than 17 years, processed at the Cancer Cytogenomics (LCC), at the Center for Research and Development of Drugs (NPDM). A cell culture protocol adapted to local conditions was standardized. Under a laminar flow hood, tissue fragments were processed in supplemented AmnioMax-II medium (30% SFB and 1% antibiotic), with dermis isolation and culture in T25 flasks at 37 °C and 5% CO2 until adhesion and growth. The medium was renewed until 80% confluence, followed by passaging. Culture conditions were standardized regarding medium, temperature, CO2, and number of passages. Cell morphology, growth curve, doubling time, cytogenetic stability by G-banding, and DNA integrity by gel electrophoresis were evaluated. The sample included 20 participants, 65% female, and eight samples were excluded due to contamination or lack of growth. The median age was 31 years (17–75) and the mean BMI was 24.96 ± 3.66 kg/m2. The mean growth time was 11.50 ± 3.80 days and processing time was 42.92 ± 23.75 days. DNA showed a median of 83.10 ng/μL (spectrophotometry) and 56.60 ng/μL (Qubit), with purity (A260/A280) of 1.91 ± 0.09. Cultures were successfully established with adequate and predictable growth, presenting typical phases of adaptation, exponential growth, and plateau, with maintenance of chromosomal stability even in late passages. Dermal fibroblasts proved suitable for germline variant analysis due to lower influence of somatic and environmental mutations. A t(9;22) translocation was observed in bone marrow from one patient, absent in fibroblasts, reinforcing its somatic nature and the genetic stability of the model. Despite the limited number of samples, the results demonstrate the feasibility of the model as a reliable source of DNA for NGS with potential applications in research and clinical practice, contributing to the

standardization of laboratory workflows in cancer genetics studies, as well as supporting investigations into genetic predisposition.