Viabilidade celular da fração mononuclear da medula óssea e fração vascular estromal do tecido adiposo de equinos após o processo de congelamento e descongelamento

Ribeiro, G.; Massoco, Cristina de Oliveira; Neto, J. C. Lacerda

doi:10.1590/s0100-736x2012001300020

Cited by 7 publications

(5 citation statements)

References 25 publications

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“…No significant differences in cell viability were detected between the five passages (94.27±0.07%, p = ns). Similar results were reported by Heimburg et al (2004) and Ribeiro et al (2012), who found high cell viability in human preadipocytes cultured from excised or aspirated adipose tissue (94%) and fatty gluteal tissue of horses (96%), respectively.…”

Section: Resultssupporting

confidence: 89%

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Oxidative stress and viability of adipose tissue-derived mesenchymal cell cultures from the omentum of rabbits

Filho

Rosa

Treichel

et al. 2018

SCA

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Mesenchymal stem cells are a population of somatic cells found in several tissues of an adult organism, including adipose tissue. Reactive oxygen species (ROS) can cause cellular alterations, including mutagenesis and genomic instability and the development of diseases. Thus, it is important to understand ROS-induced damage to cell macromolecules such as DNA, proteins, and lipids. In this study, we investigated oxidative stress rates and viability of adipose tissue-derived mesenchymal stem cells (ADSCs) from the greater omentum of rabbits. Cell cultures were analyzed at different passages (1-5) using the dichlorofluorescein acetate assay for measuring ROS production and cell viability tests. ROS levels were highest at passage 2 and cell viability was highest at passage 4.

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Section: Resultssupporting

confidence: 89%

“…Rabbits C1, C2, and C3 weighed 3.67, 3.12, and 3.38 kg, respectively, at the time of surgery, indicating that the amount of omental tissue excised was not affected by animals' body weights. Conversely, Ribeiro et al (2012) reported that the amount of fat tissue excised varied among horses (mean = 13.44 g of fat tissue per animal).…”

Section: Resultsmentioning

confidence: 99%

Oxidative stress and viability of adipose tissue-derived mesenchymal cell cultures from the omentum of rabbits

Filho

Rosa

Treichel

et al. 2018

SCA

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“…Innumerous discrepancies have been observed, such as the positive identification of the STRO-1 marker in stromal MSC [31,32] and absence of this marker in the adipogenic MSC [33,34]. The conflict persists among studies with MSC isolated from the same anatomic niche and has been identified as CD 10 positive by [32] and negative by [35] and [36]. The most suitable identification method for MSC is prolonged proliferation in culture with morphology maintenance and differentiation capacity in at least two cell lines [28,31,30].…”

Section: Discussionmentioning

confidence: 99%

Isolation, Expansion, Differentiation and Growth Kinetics Essay in Mesenchymal Stem Cells Culture from the Bone Marrow of Collared Peccaries (Tayassu tajacu)

Neto

Feitosa

Sousa

et al. 2016

Acta Scientiae. Vet.

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Background: There are few studies on stem cell isolation in wild animals that provide isolation and culture protocols of these cells in vitro. Among the wild species studied, we present the collared peccary (Tayassu tajacu) as a model with potential to obtain and use MSC in preclinical studies. These animals are phylogenetically close to the domestic pig, popularly known as peccaries and found naturally in South America, Central America and the South of the United States. The aim of the present study was to establish a protocol for the isolation, in vitro cell expansion, differentiation and assessment of the stromal MSC growth curve before and after thawing.Materials, Methods & Results: Mesenchymal stem cells (MSC) from collared peccary bone marrow (Tayassu tajacu) were isolated and expanded by centrifuge in Ficoll® solution and cultured in DMEM® High Glucose medium. The culture was assessed by assays of colony forming units CFU-F and growth curve by saturation (GCS). Cultures in the third passage, with 70% confluence, were replicated at 105 cells/mL concentration in the culture media to induce osteogenic cell differentiation and adipogenic cell differentiation, respectively. The MSC were frozen in nitrogen for 40 days, thawed and re-assessed for cell viability and GCS.Discussion: The bone marrow collected presented high mononuclear cellularity, with a mean variability of 94.5% and 60.83 ± 4.27 UFC were identified in the samples and cells with fibroblast-like-cell morphology were observed. When they were expanded, the mean cell viability was 95%, the mean cell concentration obtained was 233.31 ± 20.04 cells per 25cm2 bottle and the culture reached the growth plateau in GCS between the 13th and 16th day. The osteoblastic cell differentiation assay showed after 18 days, morphology similar to osteoblasts, with irregular cytoplasm limits, cell prolongation formation and flattened appearance. After staining with Alizarin Red, the nucleus presented a wine red coloring and the cytoplasm, more basophilic and well-defined, with calcium deposits inside the cells. The cultures submitted to adipogenic differentiation were large, hexagonal, irregular and presented birrefringent cytoplasm granules after the third week of culture. When stained with Oil Red it was observed that the cytoplasm granules were scattered small fat vacuoles and stained maroon. The viability after thawing was 78% and the mean cell concentration obtained in GCS was 199.71 ± 14.72 cells per 25 cm2 bottle. The curves reached the saturation plateau early, on the eighth day of observation. From then onwards the cultures entered became exhausted and the cell concentration of the samples decreased progressively until minimum values. These results showed the presence of a well-defined MSC population in the collared peccary bone marrow with a high rate of replication in vitro and potential for differentiation confirmed by the adipogenic and osteogenic lines. The cryopreservation technique adopted presented satisfactory results, but indicated a significant cell stress after thawing that justifies investigation of the apoptosis rates induced post thawing in the species. Furthermore, the bone marrow collection did not harm the animals and the facility of stromal MSC isolation and culture qualifies the collared peccary as a viable alternative model to obtain MSC and for studies in the area of cell therapy.

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“…The methods used to collect equine bone marrow and adipose tissue, as well as the techniques used for the isolation of cells from the bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction were considered simple and easy, and had already been described in detail (Ribeiro et al 2012).…”

Section: Discussionmentioning

confidence: 99%

Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

2013

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Pesq. Vet. Bras. 33(Supl.1):20-24, dezembro 2013 20 RESUMO.-[Cultivo de células da fração mononuclear da medulla óssea e da fração vascular stromal do tecido adipose de equinos em diferentes meios.] O objetivo deste estudo foi avaliar o cultivo de células da fração mononuclear da medula óssea e da fração vascular estromal do tecido adiposo de equinos em dois diferentes meios. Cinco cavalos foram submetidos à aspiração da medula óssea do esterno e à coleta de tecido adiposo da região glútea, pró-xima à inserção da cauda. A fração mononuclear e a fração vascular estromal foram obtidas das amostras e cultivadas em meio DMEM suplementado com soro fetal bovino a 10% ou em meio AIM-V. As culturas foram observadas uma vez por semana com um microscópio de luz invertida, com o intuito de se realizar uma análise qualitativa das características morfológicas das células, bem como do aspecto geral do cultivo celular. As unidades formadoras de colônia (CFU) foram contadas nos dias 5, 15 e 25 do cultivo celular. Durante a primeira semana, foram observadas diferenças entre amostras obtidas de mesma origem mantidas em diferentes meios. O número de colônias foi significativamente maior nas amostras de medula óssea em relação às amostras de tecido adiposo.TERMOS DE INDEXAÇÃO: Cavalos, medula óssea, tecido adiposo, meios de cultura. INTRODUCTIONThe bone marrow aspirate concentrate (BMAC) has been used to induce bone formation in humans with osseous defects, nonunion, or osteonecrosis. In horses BMAC has been used to improve cartilage, bone, and tendon repair, The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU) were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

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Viabilidade celular da fração mononuclear da medula óssea e fração vascular estromal do tecido adiposo de equinos após o processo de congelamento e descongelamento

Cited by 7 publications

References 25 publications

Oxidative stress and viability of adipose tissue-derived mesenchymal cell cultures from the omentum of rabbits

Oxidative stress and viability of adipose tissue-derived mesenchymal cell cultures from the omentum of rabbits

Isolation, Expansion, Differentiation and Growth Kinetics Essay in Mesenchymal Stem Cells Culture from the Bone Marrow of Collared Peccaries (Tayassu tajacu)

Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

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