Experimental model of obtaining tissue adipose, mesenchymal stem cells isolation and distribution in surgery flaps in rats (original) (raw)
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2019
Adipose-derived stem cells are being investigated for tissue repair because of their multidifferentiation potential and secreted growth factors as well as cytokines. Currently, there is a lot of preclinical and clinical studies that use these cells to determine the clinical effect in skin wounds, spinal cord injuries, myocardial infarction among others with promising results. In this study, a small sample of adipose tissue was extracted from mice inguinal pad and the stromal vascular fraction was obtained. Then, it was cultivated in vitro in order to promote ASC adhesion and expansion. Growth kinetics, stem cell markers expression and differentiation potential were analyzed. The isolated stem cells showed similar growth kinetics as reported in literature. Thirty percent of analyzed cells were positive for the stem cell markers and they were able to differentiate in adipogenic, chondrogenic and osteogenic lineages. The protocol proven in this work allowed to isolate adipose-derived s...
Assessment of Experimental Models for Obtaining Adipose-Derived Mesenchymal Stem Cells
2020
Creating experimental models for obtaining stem cells from adipose tissue is necessary to elucidate their peculiar features. Objective: This study proposed a reliable reproducible and consistent experimental model for obtaining mesenchymal stem cells from adipose tissue. Material and Method: Lines of New Zealand rabbits, Wistar rats and CaviaPorcellus guinea pigs (4 animals per species) were used. Fatty tissue mesenchymal stem cells were removed from dorsal, epididymal and inguinal regions. Percentage viable cells and percentage cells expanded and submitted to chondrogenic differentiation were compared by animal species and collection site. Results: Chondrogenic differentiation occurred in a similar manner across all samples, independently of animal species or collection site. Among samples assessed, the inguinal region of rats yielded the highest percentage of viable and expanded cells. Conclusion: A reliable, reproducible and consistent model for obtaining mesenchymal stem cells was produced. Of the several variables analysed, the best results were obtained from the inguinal region of the rat.
Qualitative and quantitative analysis of rabbit's fat mesenchymal stem cells
Acta Cirurgica Brasileira, 2010
PURPOSE: To present an experimental model of qualitative and quantitative analysis of mesenchymal stem cells from fat of rabbits obtained by lipectomy. The fat could be a great source for obtaining mesenchymal stem cells and to create conditions for repairing injured tissues by bioengineering. METHODS: New Zealand rabbits (n= 10) adipose panicle (2-3 cm) were removed by lipectomy, fragmented and washed with PBS and enzymatically dissociated with trypsin/EDTA. Lately, these cells were incubated in culture medium DMEM and after 20 days, was performed quantitative analysis of the accession of first and second mesenchymal cells in cell culture bottles. RESULTS: The fat total cells (CTF) were 1.62 x10(6) cells/mL and presented 98% of viability. These cells were taken for cultivation and after 20 days were counted 2.88 x10(6) cells/mL MSC. The same was done and after 20 days we quantified 4.28 x10(6) cells/mL MSC. CONCLUSION: The lipectomy of adipose panicule is a very satisfactory method...
Turkish Journal of Biology, 2012
In this study, our aim was to develop a new simple technique for isolation of mesenchymal stem cells from adipose tissue. For this purpose, mesenchymal stem cells were isolated from rat adipose tissue by using the primary explant culture technique. When the cells became confluent, they were passaged 4 times by using the standard trypsinization method with trypsin/EDTA solution. Cells at second passage were characterized by using immunofluorescence staining against CD13 and CD29 markers. The results showed that these cultured cells were positive for CD13 and CD29 markers. Flow cytometry analysis was also done against CD29, CD90, CD54, MHC Class I, CD45, CD106, and MHC Class II for characterization of mesenchymal stem cells. The results of flow cytometry analysis showed that these cells were mesenchymal stem cells. Half of the cells were cryopreserved at all passages for future applications. It is thought that these mesenchymal stem cells can be used in therapy of cardiovascular diseases as an alternative technique in the near future.
Mesenchymal stem cells from rat visceral fat exhibit multipotential differentiation in vitro
The Anatomical Record, 2003
Human subcutaneous fat-derived stem cells were recently shown to have the potential to differentiate in vitro into a variety of cell types, including adipocytes, osteoblasts, chondrocytes, and myoblasts (Zuk et al., Tissue Eng. 2001;7:211-228). Subcutaneous adipose tissue may therefore prove to be an easily acquired and abundant source of stem cells. Presently it is unclear whether mammals such as rats (which possess small or nonexistent subcutaneous fat pads) contain mesenchymal stem cells within the visceral fat of the abdominal cavity, or whether the visceral fat of any species contains stem cells. In this study we isolated and expanded a pool of mesenchymal cells from visceral fat of adult Sprague-Dawley rats and induced their differentiation in vitro into adipocytes, osteoblasts, neural cells, and chondrocytes. The differentiated phenotypes were verified by morphology as well as detection and expression of tissue-specific protein and mRNA. We conclude that despite well-documented differences in the metabolic and biochemical properties among anatomically distinct depots of fat, the visceral fat of rats contains adult mesenchymal stem cells with developmental potential similar to those isolated from subcutaneous fat in humans. Therefore, animals such as rats provide both a source of fatderived stem cells and an immunocompetent, autologous host animal in which to investigate the capacity of the fat-derived cells to differentiate and form tissues in vivo.
Mesenchymal Stem Cells - Emphasis in Adipose Tissue
The study of stem cells has evolved rapidly in recent decades. The importance is given to the concept that these cells are potentially able to become any cell type and have the power of self-renewal throughout the life of the organism. Mesenchymal stem cells (MSCs) can be isolated from various organs of the body such as bone marrow, adipose tissue, synovium, muscle and dermis, deciduous teeth, umbilical cord, placenta, liver, spleen and thymus. After their isolation in vitro, mesenchymal stem cells have the capacity to differentiate into various mesenchymal lineages and various tissues after the use of appropriate cultures. Studies have reported that mesenchymal stem cells from adipose tissue have the potential to differentiate themselves, like the cells commonly studied bone marrow. Adipose tissue is attractive due to its easy access, rapid expansion in vitro and only one collects the large amount of tissue. This review intends to show the protocols for isolation, cell culture and means of commercial cellular differentiation most widely used with emphasis on adipose tissue.
Cytotherapy, 2006
Background Adipose tissue contains a stromal vascular fraction that can be easily isolated and provides a rich source of adipose tissue-derived mesenchymal stem cells (ASC). These ASC are a potential source of cells for tissue engineering. We studied whether the yield and growth characteristics of ASC were affected by the type of surgical procedure used for adipose tissue harvesting, i.e. resection, tumescent liposuction and ultrasound-assisted liposuction. Methods Frequencies of ASC in the stromal vascular fraction were assessed in limiting dilution assays. The phenotypical marker profile of ASC was determined, using flow cytometry, and growth kinetics were investigated in culture. ASC were cultured under chondrogenic and osteogenic conditions to confirm their differentiation potential. Results The number of viable cells in the stromal vascular fraction was affected by neither the type of surgical procedure nor the anatomical site of the body from where the adipose tissue was harvested. After all three surgical procedures, cultured ASC did express a CD34 ' CD31 (CD105 ' CD166 ' CD45 (CD90 ' ASC phenotype. However, ultrasound-assisted liposuction resulted in a lower frequency of proliferating ASC, as well as a longer population doubling time of ASC, compared with resection. ASC demonstrated chondrogenic and osteogenic differentiation potential. Discussion We conclude that yield and growth characteristics of ASC are affected by the type of surgical procedure used for adipose tissue harvesting. Resection and tumescent liposuction seem to be preferable above ultrasound-assisted liposuction for tissue-engineering purposes.
Isolation of Mesenchymal Stem Cells from Adipose Tissue
The Indonesian Biomedical Journal, 2015
BACKGROUND: In searching for the best source of stem cells, researcher found adipose stem cells as one of the ideal source due to its easiness in harvesting and its potential for differentiating into other cell lineage.METHODS: We isolated stem cells from adipose tissue, cultured and confirmed its immunophenotype using polymerase chain reaction.RESULTS: Cluster of differentiation (CD)44, CD73, CD90, CD105 were expressed, which represent immunophenotype of mesenchymal stem cells. CONCLUSION: Mesenchymal stem cells can be isolated from adipose tissue. KEYWORDS: adipose, mesenchymal stem cells, isolation, immunophenotype
Anatomia, histologia, embryologia, 2017
In this study, mesenchymal stem cells were isolated from rat adipose tissue (AD-MSCs) to characterize and differentiate them into endothelial-like cells. AD-MSCs were isolated by mechanical and enzymatic treatments, and their identity was verified by colony-forming units (CFU) test and by differentiation into cells of mesodermal lineages. The endothelial differentiation was induced by plating another aliquot of cells in EGM-2 medium, enriched with specific endothelial growth factors. Five subcultures were performed. The expression of stemness genes (OCT4, SOX2 and NANOG) was investigated. The presence of CD90 and the absence of the CD45 were evaluated by flow cytometry. The endothelial-like cells were characterized by the evaluation of morphological changes and gene expression analysis for endothelial markers (CD31, CD144, CD146). Characterization of AD-MSCs showed their ability to form clones, to differentiate in vitro and the OCT-4, SOX-2, NANOG genes expression. Immunophenotypic ...
Cell Proliferation, 2012
Background: Mesenchymal stem cells are able to undergo adipogenic differentiation and present a possible alternative cell source for regeneration and replacement of adipose tissue. The human infrapatellar fat pad is a promising source of mesenchymal stem cells with many source advantages over from bone marrow. It is important to determine whether a potential mesenchymal stem-cell exhibits tri-lineage differentiation potential and is able to maintain its proliferation potential and cell-surface characterization on expansion in tissue culture. We have previously shown that mesenchymal stem cells derived from the fat pad can undergo chondrogenic and osteogenic differentiation, and we characterized these cells at early passage. In the study described here, proliferation potential and characterization of fat pad-derived mesenchymal stem cells were assessed at higher passages, and cells were allowed to undergo adipogenic differentiation. Materials and methods: Infrapatellar fat pad tissue was obtained from six patients undergoing total knee replacement. Cells isolated were expanded to passage 18 and proliferation rates were measured. Passage 10 and 18 cells were characterized for cell-surface epitopes using a range of markers. Passage 2 cells were allowed to undergo differentiation in adipogenic medium. Results: The cells maintained their population doubling rates up to passage 18. Cells at passage 10 and passage 18 had cell-surface epitope expression similar to other mesenchymal stem cells previously described. By staining it was revealed that they highly expressed CD13, CD29, CD44, CD90 and CD105, and did not express CD34 or CD56, they were also negative for LNGFR and STRO1. 3G5 positive cells were noted in cells from both passages. These fat pad-derived cells had adipogenic differentiation when assessed using gene expression for peroxisome proliferator-activated receptor c2 and lipoprotein lipase, and oil red O staining. Discussion: These results indicate that the cells maintained their proliferation rate, and continued expressing mesenchymal stem-cell markers and pericyte marker 3G5 at late passages. These results also show that the cells were capable of adipogenic differentiation and thus could be a promising source for regeneration and replacement of adipose tissue in reconstructive surgery.