BIOLOGICAL PECULIARITIES OF ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS AT DIFFERENT PASSAGES OF CULTIVATION

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
L. V. Kladnytska ◽  
◽  
A. Y. Mazurkevych ◽  
S. V. Velychko ◽  
L. V. Garmanchuk ◽  
...  
Keyword(s):  
Stem Cells ◽  
Adipose Tissue ◽  
Golgi Complex ◽  
Trypan Blue ◽  
Morphological Characteristics ◽  
Morphometric Parameters ◽  
Blue Dye ◽  
Serum Free ◽  
Laminar Box ◽  
Cytoplasmic Processes

The studies were conducted on 2–3-months-old males of C57BL/6 mice weighing 20–24 g. Obtaining and operating with adipose tissue-derived mesenchymal stem cell (MSC) culture was performed in a sterile laminar box under conditions of asepsis and antiseptics. The adipose tissue-derived MSC of the 2, 4, 7 and 12 passages were analyzed. Morphometric analysis was performed using a light microscopy. Morphometric parameters such as cell and nucleus area or nuclear-cytoplasmic ratio were calculated using the Axiovision light microscope (Carl Zeiss, Germany) and Image J 1.45 software. Trypan blue dye used for investigation of the viability of MSC. The morphological characteristics of adipose tissue-derived MSC during the process of cultivation changes: at the first passages of cultivation, the cells are spindle-shaped with two, at least three, long cytoplasmic processes, which are located bipolar. Near the nucleus, the Golgi complex is clearly visible – a sign of active cells. At later passages, cells have a small cytoplasmic processes and the bipolar arrangement of processes changes by stellar arrangement. Golgi complex is also clearly visualized. The indicator of the nuclear-cytoplasmic ratio in MSC from adipose tissue is significantly reduced at the 7th passage to 0.2189 ± 0.0122 (P < 0.01), and at the 12th passage to 0.1111 ± 0.0086 (P < 0.001) compared to the 2nd passage. The coefficient of proliferation of adipose tissue-derived MSC is significantly reduced at 12th passage. The viability of MSC from adipose tissue with an increasing of a number of passages significantly reduces and at the 12th passage of cultivation reaches 84.67 ± 1.36 (P < 0.05). The content of apoptotic cells that exhibited sensitivity to serum-free cultivation significantly increased at the 7th and 12th passages and was 21.33 ± 1.36 (P < 0.05) and 23.67 ± 0.97% (P < 0.05), respectively.

scholarly journals Morphological peculiarites and functional activity of adipose-derived mesenchimal stem cells during in vitro cultivation conditions

2018 ◽  
Vol 20 (92) ◽  
pp. 79-82
Author(s):  
L. V. Kladnytska ◽  
A. I. Mazurkevych ◽  
V. T. Khomych ◽  
T. A. Mazurkevych ◽  
Z. G. Stegney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Golgi Complex ◽  
Morphological Characteristics ◽  
Blue Dye ◽  
In Vitro Cultivation ◽  
Cultivation Conditions ◽  
Cytoplasmic Processes

The studies were conducted on 2-3-months-old males of C57BL/6 mice weighing 20–24 g. Obtaining and cultivating of adipose-derived mesenchimal stem cells (AD MSCs) were carried out in a sterile laminar box with compliance of conditions of asepsis and antiseptics. AD MSCs of the 2, 4, 7 and 12 passages were analyzed. Morphometric analysis was performed using a light microscopy. Morphometric parameters such as cell and nucleus area or nuclear-cytoplasmic ratio (NCR) were calculated using the Axiovision light microscope (Carl Zeiss, Germany) and ImageJ 1.45 software. Trypan blue dye used for investigation of the viability of MSC. The morphological characteristics of mesenchymal stem cells from adipose tissue during the process of cultivation changes: at the first passages of cultivation, the cells are spindle-shaped with two, at least three, long long cytoplasmic processes, located bipolar. Near the nucleus the Golgi complex is clearly visible – a sign of active cells. At later passages cells have a small cytoplasmic processes and the bipolar arrangement of processes changes by stellar arrangement. Golgi complex is also clearly visualized. The indicator of the nuclear-cytoplasmic ratio in MSC from adipose tissue is significantly reduced at 7 passage to 0.2189 ± 0.0122 (P < 0.01), and at 12 passage to 0.1111 ± 0.0086 (P < 0.001) compared to the 2 passage. The coefficient of proliferation of MSC from adipose tissue is significantly reduced at 12th passage. The viability of mesenchymal stem cells from adipose tissue with an increasing of a number of passages significantly reduces and at the 12th passage of cultivation reaches 84,67 ± 1,36* (P < 0.05). The content of apoptotic cells that exhibited sensitivity to serum-free significantly increased at 7 and 12 passages and was respectively 21.33 ± 1.36 (P < 0.05) and 23.67 ± 0.97% (P < 0.05).

Comprehensive characterization of human adipose tissue-derived stem cells expanded in vitro

Biologia ◽  
2013 ◽  
Vol 68 (4) ◽  
Author(s):  
Ľuboš Danišovič ◽  
Marcela Kuniaková ◽  
Zuzana Varchulová-Nováková ◽  
Martin Boháč ◽  
Ivan Varga ◽  
...  
Keyword(s):  
Stem Cells ◽  
Adipose Tissue ◽  
Growth Curve ◽  
Morphological Characteristics ◽  
Human Adipose Tissue ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Tem Analysis ◽  
Hla Dr

AbstractAdipose tissue seems to be a rich and safe source of mesenchymal stem cells (MSCs). The present study was aimed to investigate the biological and morphological characteristics of human adipose tissue-derived stem cells (ATSCs). Light and transmission electron microscopy were used. Course of proliferation was analyzed by growth curve. Expression of surface antigens was assessed by flow cytometry. Chondrogenic potential was assessed by immunohistochemistry. Obtained results showed morphology typical of fibroblastoid cells. TEM analysis proved ultrastructural morphology similar to MSCs from other sources. ATSCs reflected their proteosynthetic and metabolic activity. Each cell had irregular shape of nucleus with noticeable nucleoli. Abundant cisterns of rough endoplasmic reticulum were present in their cytoplasm. Karyotype mapping showed normal count of human chromosomes (46,XX). The growth curve revealed high capability for proliferation and population doubling time was 27.36 hours. ATSCs were positive for CD13, CD29, CD44, CD73, CD90, CD105 and CD106, but did not express CD14, CD34, CD45 and HLA-DR. It was also proved that ATSCs underwent chondrogenic differentiation in vitro. On the basis of obtained results it should be emphasized that ATSCs are typical MSCs and after further investigations they may be used in tissue engineering and regenerative medicine.

Serum-free non-toxic freezing solution for cryopreservation of human adipose tissue-derived mesenchymal stem cells

2016 ◽  
Vol 38 (8) ◽  
pp. 1397-1404 ◽  
Author(s):  
Sirsendu Sekhar Ray ◽  
Krishna Pramanik ◽  
Sunil Kumar Sarangi ◽  
Nirved Jain
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Human Adipose Tissue ◽  
Serum Free

Efficiently differentiating vascular endothelial cells from adipose tissue-derived mesenchymal stem cells in serum-free culture

2010 ◽  
Vol 400 (4) ◽  
pp. 461-465 ◽  
Author(s):  
Masamitsu Konno ◽  
Tatsuo S. Hamazaki ◽  
Satsuki Fukuda ◽  
Makoto Tokuhara ◽  
Hideho Uchiyama ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Serum Free

Serum-free isolation and culture system to enhance the proliferation and bone regeneration of adipose tissue-derived mesenchymal stem cells

2015 ◽  
Vol 51 (5) ◽  
pp. 515-529 ◽  
Author(s):  
Kazutoshi Sato ◽  
Takehiro Itoh ◽  
Toshiki Kato ◽  
Yukiko Kitamura ◽  
Sunil C. Kaul ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Bone Regeneration ◽  
Culture System ◽  
Serum Free

Potentiated Activation of VLA-4 and VLA-5 Accelerates Proplatelet-Like Formation In Megakaryocytes.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2585-2585
Author(s):  
Takuya Matsunaga ◽  
Fumio Fukai ◽  
Takuro Kameda ◽  
Kotaro Shide ◽  
Haruko Shimoda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture System ◽  
Dependent Manner ◽  
Adhesive Interaction ◽  
Hematopoietic Stem ◽  
Serum Free ◽  
Amino Terminal ◽  
Three Phase ◽  
Cytoplasmic Processes

Abstract Abstract 2585 Several lines of reports have suggested that mature magakaryocytes (MKs) form long cytoplasmic processes containing platelets (PLT) organelles from which PLT break off due to blood flow pressures in bone marrow (BM). These cytoplasmic processes were termed ‘proplatelet'. MKs differentiated from hematopoietic stem cells by in vitro culture also develop similar processes, referred to as ‘proplatelet-like formation (PPF)'. It has been already reported that fibronectin (FN) and phorbol 12-myristate 13-acetate (PMA) are essential for inducing PPF in MKs using CHRF-288 human megakaryoblastic cell line (Jiang F et al. Blood 99, 2002). FN plays important roles in megakaryocytopoiesis through the FN-receptors. The role of adhesive interactions with FN in BM stroma and FN-receptor beta1-integrins has been reported in proliferation, differentiation and maintenance of megakaryocytic lineage cells. However, the substantial role of these FN-receptors and their functional assignment in PPF are not yet fully understood. We first investigated the effects of beta1-integrins on PPF using CHRF-288 cells, which express alpha4beta1-integrin (VLA-4) and alpha5beta1-integrin (VLA-5) as FN-receptors. When the cells were cultured on FN for 3 days, PMA prompted PPF in a dose-dependent manner. While nearly 15% of the cells displayed PPF with PMA (100 ng/mL), no cells cultured with FN alone or PMA alone exhibited PPF. PPF induced by FN plus PMA combination (FN/PMA) was abrogated by addition of anti-alpha4-integrin monoclonal antibodies (mAb) plus anti-alpha5-integrin mAb combination, but not by the addition of anti-alpha4-integrin mAb alone or anti-alpha5-integrin mAb alone. Thus, the adhesive interaction with FN via VLA-4 and VLA-5 were responsible for PPF. We next investigated the effect of TNIIIA2, which enhances the adhesive interaction between FN and beta1-integrins, in PPF induced by FN/PMA. TNIIIA2 (RSTDLPGLKAATHYTITIRGVC) is a 22-mer peptide derived from the 14th FN type III-like (FNIII) repeat in tenascin (TN)-C molecule which we found recently, and it induces the conformational change necessary for functional activation of beta1-integrins (Fukai F et al. J Biol Chem 282, 2007; J Biol Chem 284, 2009). The PPF induced by FN/PMA was highly accelerated when CHRF-288 cells were enforced adhering to FN by treatment with TNIIIA2 (25 microg/mL). More than 45% of the cells displayed PPF with FN/PMA plus TNIIIA2 combination (FN/PMA/TNIIIA2). Blocking experiments using anti-beta1-integrin mAbs indicated that adhesive interaction with FN via VLA-4 and VLA-5 was also responsible for acceleration of PPF induced by FN/PMA/TNIIIA2. On the other hand, control peptide, TNIIIA2mutant (RSTDLPGLKAATHYTATARGVC) did not accelerate PPF induced by PMA/FN. The calculated yield of the cells with PPF induced by FN/PMA/TNIIIA2 was 2.5-fold more than that induced by FN/PMA. We have previously established ‘a three-phase serum-free culture system' to generate large amount of PLT from human cord blood CD34+ cells (Matsunaga T et al. Stem cells 24, 2006). A study on the effect of TNIIIA2 on our ‘three-phase serum-free culture system' is now underway. Finally, we investigated signal transduction pathways responsible for PPF induced by FN/PMA. While FN/PMA induced activation of extracellular signal-regulated protein kinase 1 (ERK1/2), FN alone or PMA alone did not induce ERK1/2 activation. The results was in accordance with the data previously reported by Jiang et at (Blood 99, 2002). TNIIIA2 strongly enhanced activation of ERK1/2 by FN/PMA. However, c-Jun amino-terminal kinase 1 (JNK1), p38 and phosphoinositide-3 kinase (PI3K)/Akt were not stimulated by FN/PMA even in the presence of TNIIIA2. Thus, enhanced activation of ERK1/2 by FN/PMA/TNIIIA2 was responsible for acceleration of PPF by FN/PMA. Disclosures: No relevant conflicts of interest to declare.

Culture and expansion of Adipose derived Mesenchymal stem cells in Ovine

2017 ◽  
Author(s):  
T. A. Kannan ◽  
J. Violet Beaulah ◽  
S. Ushakumary ◽  
B. Justin William ◽  
Geetha M. Ramesh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Trypan Blue ◽  
Povidone Iodine ◽  
Sterile Condition ◽  
Trypan Blue Exclusion ◽  
Tissue Samples ◽  
Trypan Blue Exclusion Test ◽  
Slaughter House

Culture and expansion of Adipose derived Mesenchymal stem cells (ADMSCs) in Ovine was carried out in this study. Ovine adipose tissue samples were collected from Chennai Corporation slaughter house under sterile condition in normal saline with five per cent povidone iodine, antibiotic and antimycotic solutions. Collected tissue samples were weighed and digested using collagenase enzyme to isolate adipose tissue derived mesenchymal stem cells (ADMSCs). Cell yield and viability of the cells were calculated by using trypan blue exclusion test. The cells were seeded at the density of one million cells in one T25 culture flask in Dulbecco’s modified Eagle’s medium-high glucose (DMEM-HG). On the day of seeding, the cells showed spherical morphology. Plastic adherence was noticed 24 hrs after seeding. Cell expansion was observed after 3 days. At P0 level, 70% confluency was attained on day 14 and the time taken to reach 70% confluency was reduced to 3-4 days in subsequent passages.

A novel cyclic RGD-containing peptide polymer improves serum-free adhesion of adipose tissue-derived mesenchymal stem cells to bone implant surfaces

2012 ◽  
Vol 24 (2) ◽  
pp. 479-488 ◽  
Author(s):  
Péter Tátrai ◽  
Bernadett Sági ◽  
Anna Szigeti ◽  
Áron Szepesi ◽  
Ildikó Szabó ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Bone Implant ◽  
Serum Free

scholarly journals Cannabinoid-Induced Autophagy and Heme Oxygenase-1 Determine the Fate of Adipose Tissue-Derived Mesenchymal Stem Cells under Stressful Conditions

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2298
Author(s):  
Katharina Bublitz ◽  
Sabine Böckmann ◽  
Kirsten Peters ◽  
Burkhard Hinz
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Heme Oxygenase ◽  
Metabolic Activity ◽  
Therapeutic Option ◽  
Protoporphyrin Ix ◽  
Heme Oxygenase 1 ◽  
Serum Free ◽  
Serum Free Conditions

The administration of adipose tissue-derived mesenchymal stem cells (ADMSCs) represents a promising therapeutic option after myocardial ischemia or myocardial infarction. However, their potential is reduced due to the high post-transplant cell mortality probably caused by oxidative stress and mitogen-deficient microenvironments. To identify protection strategies for ADMSCs, this study investigated the influence of the non-psychoactive phytocannabinoid cannabidiol (CBD) and the endocannabinoid analogue R(+)-methanandamide (MA) on the induction of heme oxygenase-1 (HO-1) and autophagy under serum-free conditions. At a concentration of 3 µM, CBD induced an upregulation of HO-1 mRNA and protein within 6 h, whereas for MA only a late and comparatively lower increase in the HO-1 protein could be detected after 48 h. In addition, both cannabinoids induced time- and concentration-dependent increases in LC3A/B-II protein, a marker of autophagy, and in metabolic activity. A participation of several cannabinoid-binding receptors in the effect on metabolic activity and HO-1 was excluded. Similarly, knockdown of HO-1 by siRNA or inhibition of HO-1 activity by tin protoporphyrin IX (SnPPIX) had no effect on CBD-induced autophagy and metabolic activity. On the other hand, the inhibition of autophagy by bafilomycin A1 led to a significant decrease in cannabinoid-induced metabolic activity and to an increase in apoptosis. Under these circumstances, a significant induction of HO-1 expression after 24 h could also be demonstrated for MA. Remarkably, inhibition of HO-1 by SnPPIX under conditions of autophagy deficit led to a significant reversal of apoptosis in cannabinoid-treated cells. In conclusion, the investigated cannabinoids increase metabolic viability of ADMSCs under serum-free conditions by inducing HO-1-independent autophagy but contribute to apoptosis under conditions of additional autophagy deficit via an HO-1-dependent pathway.

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