Mesenchymal stromal/stem cells stem (MSC) have been widely studied due to their great potential for application in tissue engineering and regenerative and translational medicine. In MSC-based therapy for human diseases, cell proliferation is required to obtain a large and adequate number of cells to ensure therapeutic efficacy. During in vitro culture, cells are under an artificial environment and manipulative stress that can affect genetic stability. Several regulatory agencies have established guidelines to ensure greater safety in cell-based regenerative and translational medicine, but there is no specific definition about the maximum number of passages that ensure the lowest possible risk in MSC-based regenerative medicine. In this context, the aim of this study was to analyze DNA damage and chromosome alterations in adipose-derived mesenchymal stromal cells (ADMSC) until the eleventh passage and to provide additional subsidies to regulatory agencies related to number of passages in these cells. Thus, two methods in genetic toxicology were adopted: comet assay and micronucleus test. The comet assay results showed an increase in DNA damage from the fifth passage onwards. The micronucleus test showed a statistically significant increase of micronucleus from the seventh passage onwards, indicating a possible mutagenic effect associated with the increase in the number of passages. Based on these results, it is important to emphasize the need to assess genetic toxicology and inclusion of new guidelines by regulatory agencies to guarantee the safety of MSC-based therapies for human diseases. stromalstem MSC (MSC medicine MSCbased based diseases efficacy culture stability cellbased context adiposederived adipose derived ADMSC (ADMSC Thus adopted onwards

Adipose tissue-derived mesenchymal stromal/stem cells (ASCs) are considered important tools in regenerative medicine and are being tested in several clinical studies. Porcine models are frequently used to obtain adipose tissue, due to the abundance of material and because they have immunological and physiological similarities with humans. However, it is essential to understand the effects and safe application of ASCs from pigs (pASCs) as an alternative therapy for diseases. Although minipigs are easy-to-handle animals that require less food and space, acquiring and maintaining them in a bioterium can be costly. Thus, we present a protocol for the isolation and proliferation of ASCs isolated from adipose tissue of farm pigs. Adipose tissue samples were extracted from the abdominal region of the animals. Because the pigs were not raised in a controlled environment, such as a bioterium, it was necessary to carry out rigorous procedures for disinfection. After this procedure, cells were isolated by mechanical dissociation and enzymatic digestion. A proliferation curve was performed and used to calculate the doubling time of the population. The characterization of pASCs was performed by immunophenotyping and cell differentiation in osteogenic and adipogenic lineages. The described method was efficient for the isolation and cultivation of pASCs, maintaining cellular attributes, such as surface antigens and multipotential differentiation during in vitro proliferation. This protocol presents the isolation and cultivation of ASCs from farm pig as an alternative for the isolation and cultivation of ASCs from minipigs, which require strictly controlled maintenance conditions and a more expensive process.