scholarly journals Особливості клітинного циклу мезенхімальних стовбурових клітин з жирової тканини собаки за різних пасажів культивування

2017 ◽  
Vol 19 (78) ◽  
pp. 36-40
Author(s):  
L.V. Kladnytska
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Culture Medium ◽  
Fetal Bovine Serum ◽  
Inverted Microscope ◽  
Proliferative Pool ◽  
Fetal Bovine ◽  
Diploid Cells

The features of the cell cycle of culture of adipose-derived mesenchymal stem cells from the for different cultivating passages were studied. Mesenchymal stem cells were obtained from the adipose tissue of the dog under a laminar flow hood by an explant method in our modification. Cell cultivation was carried out at 37 °C, 100% moisture and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic. The culture medium was changed 2–3 times per week and the cells were selected by their capacity to attach to the flask surface. When culture flasks became 80% confluence, cells were detached with 0.25% trypsin containing 1 mmol/L EDTA and subsequently replayed at a concentration of 104 cells/cm2 for next passaging. A cells culture of adipose derived mesenchymal stem cells was obtained on the 2nd, 7th and 12th passages. The method of flow cytometry determined the level of aneuploid cells and the distribution in the cell cycle phases. The morphology of cells of different passages was studied using an inverted microscope Axiovert 40. It was investigated that the culture of mesenchymal stem cells from adipose tissue in the 2nd passage contains a significant number of the proliferative pool (S + G2/M) cells and it was 29.51 ± 3.56% of the total number of diploid cells. The number of aneuploid cells was 1.55 ± 0.43%. All cells had fibroblast-like morphology. It was established that in the middle passages (7th) in the culture of mesenchymal stem cells from the adipose tissue of the dog no significant changes were found in the distribution of cells in the phases of the cell cycle. The number of diploid cells of the proliferative pool S + G2/M and the G0/G1 pre-synthetic period remains unchanged. The level of aneuploidy increases only within the tendency. Morphologically, cells had fibroblast-like form. It was determined on 12th passage of cultivation, a significant decrease in the number of cells of the proliferative pool (S+G2/M), which was 18.93 ± 0.66% of the total number of diploid cells compared to the 2nd passage. The number of aneuploid cells increased and it was 3.49 ± 0.38%. Morphologically, separate cells had processes. The indicator of the effect of cells cultivation on the content of diploid cells of the proliferative pool (S+G2/M) in culture is ɳ2x = 70% (P < 0.05). So, first characteristic properties of the aging of the culture of canine adipose-derived mesenchymal stem cells appear on the 12th passage of cultivation.

227 MESENCHYMAL STEM CELL ISOLATION AND CULTURE FROM ADIPOSE TISSUE OF A DEAD DOG

2016 ◽  
Vol 28 (2) ◽  
pp. 245
Author(s):  
S. Saini ◽  
V. Sharma ◽  
H. N. Malik ◽  
S. K. Guha ◽  
D. Malakar
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Blue Staining ◽  
Marker Genes ◽  
Specific Marker ◽  
The Future ◽  
Fetal Bovine

Isolation of cells or stem cells from clinically dead animals may serve applications such as revival of the animal through somatic cell nuclear transfer (SCNT) or cryopreservation of their cells for a long period so that cells can be used in the future. Thus, combining isolation of cells from clinically dead animals and SCNT of germplasm of elite animals could benefit research into endangered or extinct species. In the present study, we tried to isolate and culture adipose-derived mesenchymal stem cells (ADSC) from a clinically dead dog. Adipose tissues were collected surgically from the abdomen of a dead dog after 3 h and processed tissues within 10 h of death. The isolated tissues were washed in 70% ethanol for 30 s and washed 5 times in Dulbecco’s PBS supplemented with 50 µg mL–1 gentamicin. These fat tissues were minced to very small pieces and washed in DMEM by centrifugation at 800 rpm for 3 min. The tissue pellet was subjected to enzymatic digestion (collagenase 1 mg mL–1 of Dulbecco’s PBS) at 37°C in CO2 incubator for 1 h, with intermittent shaking after every 10 min. The digestive enzyme was inactivated by equal volume of DMEM/F-12 supplemented with fetal bovine serum (20%) and centrifuged at 1000 rpm for 10 min. The pellet was resuspended in DMEM/F-12 with 10% fetal bovine serum and cultured at 1 × 106 cells mL–1 in 25-cm2 tissue culture flasks. The medium was changed after every 48 h. Mesenchymal stem cells (MSC) were observed under an inverted microscope after 6 days. These cells were subcultured and a confluent monolayer was obtained. We have already standardized the protocol of MSC culture and characterisation as we are treating wounded and paralysed dogs using these MSC in a pet clinic. Characterisation of MSC was performed with specific surface marker genes of CD44, CD29, and CD166 in PCR and by immunocytochemistry of MSC-specific marker of CD44. Differentiation of these MSC into osteogenesis and chondrogenesis were observed after 3 weeks. Chondrogenic differentiation was confirmed by positive expression of chondrocyte-specific marker genes Aggrecan F-TTGGACTTTGGCAGAATACC and R-CTTCCACCAATGTCGTATCC and Collagen II F-AACCCTGGAACTGACGGAAT and R-CTCACCCGTTTGACCTTTCG primer in PCR. The MSC were cryopreserved after 80% confluency was reached. The monolayer cells were scraped out from the culture flask and pelleted down. The pellet was resuspended in DMEM containing 10% DMSO and 20% fetal bovine serum. The number of cells was determined by trypan blue staining using an automatic cell counter and 105 cells mL–1 were added to a 2-mL cryogenic vial. The cryogenic vials were kept in a cryobox at –80°C for slow cooling. Then these vials were transferred to liquid nitrogen tanks after 12 h for long-term storage. We conclude that ADSC were successfully cultured from adipose tissue of a dog within 10 h of death and further subcultured under in vitro conditions. The cells could be used for SCNT to revive the dead animal and cryopreserve these cells for use in the future.

scholarly journals INVESTIGATING COOLING RATE AND FETAL BOVINE SERUM CONCENTRATION ON SURVIVAL AND STEMNESS OF MESENCHYMAL STEM CELLS AFTER CRYOPRESERVATION

2009 ◽  
Vol 12 (9) ◽  
pp. 12-22
Author(s):  
Phuc Van Pham ◽  
Tam Thanh Nguyen ◽  
Nhung Thi Hong Vuong ◽  
Tuyet Thi Bach Duong ◽  
Ngoc Kim Phan
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Serum Concentration ◽  
Cord Blood ◽  
Umbilical Cord Blood ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Fetal Bovine Serum Concentration ◽  
Fetal Bovine ◽  
Different Sources

Mesenchymal stem cells (MSCs) can be derived from many different sources. Umbilical cord blood is a rich source of MSCs. The cryopreservation of MSCs that MSCs are still alive and differentiate into many different kinds of functional cells is very important. The aims of this research are to identify ratio of alive and dead cells as well as stemness of them after thaw. The results showed that the stemness was not affected by cryopreservative protocols or media. All cells being alive after thaw could form colonies and differentiate into adipocytes and osteoblasts. Ratio of alive and dead cells was affected very much by cryopreservative protocols and media.

Histone Deacetylase Inhibitor (Trapoxin A) Enhances Stemness Properties in Adipose Tissue Derived Mesenchymal Stem Cells

Drug Research ◽  
2018 ◽  
Vol 68 (08) ◽  
pp. 450-456 ◽  
Author(s):  
Leila Mousazadeh ◽  
Effat Alizadeh ◽  
Nosratollah Zarghami ◽  
Shahryar Hashemzadeh ◽  
Sedigheh Aval ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitor ◽  
Ex Vivo ◽  
Osteogenic Potential ◽  
Differentiation Potential ◽  
Deacetylase Inhibitor

Abstract Back ground Adipose tissue derived mesenchymal stem cells (ASCs) have unique potential for regenerative cell therapies. However, during ex-vivo cultivation, they undergo considerable quality loss regarding their phenotypic properties, stemness genes expression and differentiation potential. Recent studies reported that the loss of stemness properties of MSCs is a result of chromatin histone deacetylations through in-vitro cultivation. The present work aimed to study the effect of Trapoxin A (TPX) as a histone deacetylase inhibitor (HDACi) on overall stemness properties of ASCs. Methods First, the effects of TPX treatments on ASCs viability and proliferation were evaluated using MTT assay. Second, the desired doses of TPX supporting ASCs proliferation were determined and the lack of their negative effects was confirmed by DAPI staining. In addition, the influence of TPX on cell cycle of ASCs and the mRNA levels of stemness genes were measured by flowcytometry and qPCR, respectively. Finally, the effect of TPX treatment on osteogenic potential of ASCs was studied. Results The results indicated that short time TPX treatment (nM concentrations) caused stimulation of proliferation and considerable percentage of ASCs entered to S-phase of cell cycle (p<0.05). Moreover, the findings demonstrated significant up-regulation of stemness markers genes (Oct-4, Sox-2, Nanog, TERT, Klf-4, Rex-1) (p<0.05) and enhanced osteogenic differentiation potential of ASC after TPX treatment. Conclusion The addition of low dose of TPX to the expansion medium could possibly enhance the stemness properties and prevent the quality decline of ex-vivo cultured ASCs.

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