scholarly journals Enhancement Of Dermal Fibroblast Isolation Method

2015 ◽  
Vol 16 (1) ◽  
pp. 65-69
Author(s):  
Milan Zaric ◽  
Ivana Nikolic ◽  
Ivanka Zelen ◽  
Marina Mitrovic ◽  
Zoran Milosavljevic
Keyword(s):  
Dermal Fibroblast ◽  
Petri Dish ◽  
Culture Conditions ◽  
Dermal Fibroblasts ◽  
Enzyme Digestion ◽  
Digestion Method ◽  
Primary Isolation ◽  
Donor Cells ◽  
Cell Culture Conditions

ABSTRACTCultivated fibroblasts have been widely used in a large number of in vitro studies. Although they readily proliferate under cell culture conditions, improvements in methods for their isolation are necessary. Here, we present our modified enzyme digestion method and compare its efficiency with commonly used techniques.Three foreskin samples from young, middle-aged and old donors were used. The classical explant, standard enzyme digestion method with collagenase and our improved enzyme digestion method were compared for efficiency of fibroblast isolation and the time needed to achieve 95% confluence in a 30-mm Petri dish.The explant method was the slowest to achieve fibroblast confluence, especially with the tissues from the older donors (up to 23 days). With the standard enzyme digestion method, the skin tissue was partially digested, but the fibroblasts reached confluence much faster (the younger donor cells needed approximately 7 days to reach confluence). Our modified “mixed” enzyme digestion method was the fastest (the fibroblasts from the younger donors needed up to 5 days to reach confluence).For studies requiring the primary isolation and cultivation of dermal fibroblasts, the best method to achieve this goal is the tissue digestion method with the multiple enzyme solution.

scholarly journals Mitochondrial and metabolic remodeling in human skin fibroblasts in response to glucose availability

2021 ◽  
Author(s):  
Cláudio F. Costa ◽  
Sónia A. Pinho ◽  
Sonia L.C. Pinho ◽  
Inês Miranda-Santos ◽  
Olivia Bagshaw ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Model ◽  
Complete Removal ◽  
Culture Conditions ◽  
Dermal Fibroblasts ◽  
Physiological Level ◽  
Atp Production ◽  
Metabolic Remodeling ◽  
Cell Culture Conditions

AbstractCell culture conditions highly influence cell metabolism in vitro. This is relevant for preclinical assays, for which fibroblasts are an interesting cell model, with applications in regenerative medicine, diagnostics and therapeutic development for personalized medicine as well as in the validation of ingredients for cosmetics. Given these cells’ short lifespan in culture, we aimed to identify the best cell culture conditions and promising markers to study mitochondrial health and stress in Normal Human Dermal Fibroblasts (NHDF). We tested the effect of reducing glucose concentration in the cell medium from high glucose (HGm) to a more physiological level (LGm), or its complete removal and replacement by galactose (OXPHOSm), always in the presence of glutamine and pyruvate. We have demonstrated that only with OXPHOSm it was possible to observe the selective inhibition of mitochondrial ATP production. This reliance on mitochondrial ATP was accompanied by changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), oxidation of citric acid cycle substrates, fatty acids, lactate and other substrates, mitochondrial network extension and polarization and changes in several key transcripts related to energy metabolism. We also evaluated the relevance of galactose, glutamine and pyruvate for OXPHOS stimulation, by comparing OCR and ECAR in the presence or absence of these substrates. Galactose and pyruvate seem to be important, but redundant, to promote OXPHOS, whereas glutamine was essential. We concluded that LGm does not promote significant metabolic changes but the short-term adaptation to OXPHOSm is ideal for studying mitochondrial health and stress in NHDF.Author ContributionsCC, SAP, SLCP and IMS performed experiments. TCO and PJO designed research and acquired funding. JS, and OB analyzed data. CC and TCO analyzed data and wrote the paper. All authors contributed to the final version of the manuscript.

scholarly journals A Phytocomplex Consisting of Tropaeolum majus L. and Salvia officinalis L. Extracts Alleviates the Inflammatory Response of Dermal Fibroblasts to Bacterial Lipopolysaccharides

2020 ◽  
Vol 2020 ◽  
pp. 1-14 ◽  
Author(s):  
Tunde Jurca ◽  
Ioana Baldea ◽  
Gabriela Adriana Filip ◽  
Diana Olteanu ◽  
Simona Clichici ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Inflammatory Response ◽  
Dermal Fibroblast ◽  
Physicochemical Characterization ◽  
Antibacterial Activities ◽  
Dermal Fibroblasts ◽  
High Dose ◽  
Cytokine Levels ◽  
Bacterial Lipopolysaccharides

Background. The antimicrobial activity and effects of a phytocomplex consisting of Tropaeolum flos (T) and Salviae folium (S) extracts on the cytokine levels and transcription factors on dermal fibroblast BJ exposed to bacterial lipopolysaccharides were examined. Methods. In order to select the most optimal combination ratio of the two extracts for using in vitro, the physicochemical characterization of vegetal extract mixtures was performed and the antioxidant and antibacterial activities were evaluated on five different formulations of T : S, namely, 1 : 1, 1 : 2, 2 : 1, 3 : 1, and 1 : 3. The best combination of bioactive compounds with regard to antioxidant and antibacterial activities (T : S 1 : 2) was selected for in vitro evaluation of the anti-inflammatory effect. Human dermal fibroblast BJ cells were treated with two doses of the extract mixture and then exposed to bacterial lipopolysaccharides (LPS). The levels of the cytokines involved in inflammatory response, namely, interleukin- (IL-) 6, tumor necrosis factor- (TNF-) α, IL-31, and IL-33, were quantified by ELISA, and the expression of transcription factors, namely, signal transducer and activator of transcription (STAT) 3, nuclear factor kappa B (NFκB), and phosphorylated NFκB (pNFκB), were evaluated by western blot analysis. Results. The results have shown that the mixture of T : S 1 : 2 exhibited significant antibacterial effects on Staphylococcus aureus ATCC 25923. LPS exposure increased the cytokine levels in BJ cells and enhanced the NFκB expression. The pretreatment of BF cells exposed to LPS with the two doses of the extract mixture markedly inhibited the increase of IL-33 and TNF-α levels and amplified the NFκB expression and its activation, especially with the high dose. The low doses of the extract reduced NFκB expression but increased its activation. Conclusions. These experimental findings suggest that the mixture of T : S 1 : 2 can exert some protection against bacterial infections and inflammation induced by LPS in BJ cells being a good therapeutic option in related conditions associated with inflammation.

scholarly journals Experimental Study on Cell-free Approach for Articular Cartilage Treatment

2019 ◽  
Vol 5 (1) ◽  
pp. 171-174 ◽  
Author(s):  
Gözde Dursun ◽  
Bernd Markert ◽  
Marcus Stoffel
Keyword(s):  
Articular Cartilage ◽  
Petri Dish ◽  
Treatment Method ◽  
Culture Conditions ◽  
Cyclic Compression ◽  
A Cell ◽  
Static Cultivation ◽  
Promising Treatment ◽  
Bioreactor System

AbstractCell-free based approaches are introduced as a promising treatment method for articular cartilage. The success of this method requires cell colonisation from resident tissue into cell-free implants. The objective of our study is to promote the cell colonisation into cell-free collagen I based implants by mechanical stimulation. Therefore, a new in vitro cellular model consisting chondrocyte-seeded matrix and cell-free implants was developed in a polydimethylsiloxan (PDMS) mold. These constructs were cultured under dynamic and static culture conditions. For the dynamic culture, we have developed an in-house bioreactor system where both the load and the deformation applied to the specimen are recorded. Cyclic compression, with a strain of 5% and frequency of 0.1 Hz, was applied to constructs without any break. At the end of three days of dynamic and static cultivation, the cell-free implants were seperated from cell-seeded matrix and cultured in a petri dish three days long. Afterwards, they were analysed using fluoresence dyes. The microscopic assessment indicated that there was a cell migration into the cell-free implants which were cultured dynamically.

51 DEVELOPMENT OF EQUINE CLONED EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES RECEIVING ADULT DERMAL FIBROBLAST CELL NUCLEI

2009 ◽  
Vol 21 (1) ◽  
pp. 125
Author(s):  
M. Skrzyszowska ◽  
M. Samiec ◽  
W. Mlodawska ◽  
J. Kochan ◽  
A. Okolski ◽  
...  
Keyword(s):  
Dermal Fibroblast ◽  
Fibroblast Cell ◽  
Vero Cells ◽  
Contact Inhibition ◽  
Fibroblast Cells ◽  
Cell Nuclei ◽  
Metaphase Chromosomes ◽  
Condensed Chromosome ◽  
Donor Cells

The purpose of our study was to determine the in vitro developmental competences of equine NT embryos reconstructed with adult dermal fibroblast cells. Frozen/thawed fibroblast cells, whose mitotic cycle had been synchronized at G1/G0 stages through a contact inhibition of their migration and proliferative activity under total confluency, were used as a source of nuclear donor cells in the somatic cell cloning procedure. In vitro-matured oocytes were used as recipient cells for fibroblast cell nuclei. The compact cumulus–oocyte complexes (cpCOCs) were collected from abattoir-derived mare ovaries and selected for in vitro maturation. The cpCOCs were cultured in TC-199 medium supplemented with 5 mU mL–1 follicle-stimulating hormone (FSH), 10% fetal bovine serum (FBS) and 75 μg mL–1 kanamycin monosulfate (kanamycin A) for 30 h at 38.2°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Cumulus-denuded in vitro-matured oocytes were incubated in the maturation medium supplemented with 0.4 μg mL–1 demecolcine for 40 min. The treated oocytes were subsequently transferred into TC-199 medium containing 4 mg mL–1 BSA-V and 5 μg mL–1 cytochalasin B. Metaphase chromosomes, which had been allocated into the chemically-induced protrusion of the plasma membrane, were removed microsurgically. The chemically-assisted enucleation was accomplished by gently aspirating the ooplasmic cone, which contained the condensed chromosome mass, with the aid of a beveled micropipette. The single nuclear donor cells were inserted into perivitelline space of previously enucleated oocytes. Fibroblast cell-ooplast couplets were fused with two consecutive DC pulses of 2.4 kV cm–1 for 30 μs. After a 1.5-h delay, nuclear transfer-derived oocytes were chemically activated by exposure to 5 μm L–1 calcium ionomycin for 5 to 7 min, followed by their incubation in B2 medium with addition of 2 mm L–1 6-dimethylaminopurine (6-DMAP) for 4 h. Reconstructed embryos were in vitro cultured in B2 medium for 2 days. Afterwards, cleaved embryos were co-cultured with Vero cells in B2 medium supplemented with 10% FBS for 5 to 6 days up to morula/blastocyst stages. From among 88 in vitro cultured cpCOCs, 55 (62.5%) acquired meiotic nuclear and cytoplasmic maturity state after reaching the Metaphase II stage. A total of 55 enucleated oocytes underwent reconstruction and 44/55 (80.0%) were successfully fused with nuclear donor cells. Out of 44 cultured NT embryos, 21 (47.7%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 6/44 (13.6%) and 3/44 (6.8%), respectively. In conclusion, the cell nuclei of in vitro cultured adult dermal fibroblast cells, which had undergone the contact inhibition, were able to direct the preimplantation development of equine cloned embryos to morula and blastocyst stages. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

scholarly journals Tissue Specific Differentiation of Human Chondrocytes Depends on Cell Microenvironment and Serum Selection

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 934 ◽  
Author(s):  
Ecke ◽  
Lutter ◽  
Scholka ◽  
Hansch ◽  
Becker ◽  
...  
Keyword(s):  
Articular Chondrocytes ◽  
Culture Conditions ◽  
Serum Supplement ◽  
3 Dimensional ◽  
The Matrix ◽  
Differentiation Degree ◽  
Overlay Technique ◽  
Fetal Bovine ◽  
Cell Culture Conditions

Therapeutic options to cure osteoarthritis (OA) are not yet available, although cell-based therapies for the treatment of traumatic defects of cartilage have already been developed using, e.g., articular chondrocytes. In order to adapt cell-based therapies to treat OA, appropriate cell culture conditions are necessary. Chondrocytes require a 3-dimensional (3D) environment for redifferentiation after 2-dimensional (2D) expansion. Fetal bovine serum (FBS) is commonly used as a medium supplement, although the usage of a xenogeneic serum could mask the intrinsic behavior of human cells in vitro. The aim of this study was to compare human articular chondrocytes cultivated as monolayers (2D) and the development of microtissues (3D) in the presence of FBS with those cultivated with human serum (HS). Evaluation of the expression of various markers via immunocytochemistry on monolayer cells revealed a higher dedifferentiation degree of chondrocytes cultivated with HS. Scaffold-free microtissues were generated using the agar overlay technique, and their differentiation level was evaluated via histochemistry and immunohistochemistry. Microtissues cultivated in the medium with FBS showed a higher redifferentiation level. This was evidenced by bigger microtissues and a more cartilage-like composition of the matrix with not any/less positivity for cartilage-specific markers in HS versus moderate-to-high positivity in FBS-cultured microtissues. The present study showed that the differentiation degree of chondrocytes depends both on the microenvironment of the cells and the serum type with FBS achieving the best results. However, HS should be preferred for the engineering of cartilage-like microtissues, as it rather enables a "human-based" situation in vitro. Hence, cultivation conditions might be further optimized to gain an even more adequate and donor-independent redifferentiation of chondrocytes in microtissues, e.g., designing a suitable chemically-defined serum supplement.

scholarly journals Creation of cloned pig embryos using contact-inhibited or serum-starved fibroblast cells analysed intravitam for apoptosis occurrence / Uzyskiwanie klonalnych zarodków świni z wykorzystaniem komórek fibroblastycznych poddanych inhibicji kontaktowej lub deprywacji troficznej oraz analizowanych przyżyciowo w kierunku apoptozy

2013 ◽  
Vol 13 (2) ◽  
pp. 275-293 ◽  
Author(s):  
Marcin Samiec ◽  
Maria Skrzyszowska ◽  
Jolanta Opiela
Keyword(s):  
Dermal Fibroblast ◽  
Somatic Cells ◽  
Fibroblast Cell ◽  
Serum Starvation ◽  
Fibroblast Cells ◽  
Blastocyst Formation ◽  
Cell Nuclei ◽  
Donor Cells ◽  
Cloned Pig

Abstract Somatic cell cloning efficiency is determined by many factors. One of the most important factors is the structure-functional quality of nuclear donor cells. Morphologic criteria that have been used to date for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Biochemical and biophysical changes that are one of the earliest symptoms in the transduction of apoptotic signal may be not reflected in the morphologic changes of somatic cells. For this reason, adult cutaneous or foetal fibroblast cells that, in our experiments, provided the source of genomic DNA for the cloning procedure had been previously analysed for biochemical and biophysical proapoptotic alterations with the use of live-DNA (YO-PRO-1) and plasma membrane (Annexin V-eGFP) fluorescent markers. In Groups IA and IB, the generation of nucleartransferred (NT) embryos using non-apoptotic/non-necrotic contact-inhibited or serum-starved adult cutaneous fibroblast cells yielded the morula and blastocyst formation rates of 125/231 (54.1%) and 68/231 (29.4%) or 99/237 (41.8%) and 43/237 (18.1%), respectively. In Groups IIA and IIB, the frequencies of embryos reconstituted with non-apoptotic/non-necrotic contact-inhibited or serum-starved foetal fibroblast cell nuclei that reached the morula and blastocyst stages were 171/245 (69.8%) and 97/245 (39.6%) or 132/227 (58.1%) and 63/227 (27.8%), respectively. In conclusion, contact inhibition of migration and proliferative activity among the subpopulations of adult dermal fibroblast cells and foetal fibroblast cells resulted in considerably higher morula and blastocyst formation rates of in vitro cultured cloned pig embryos compared to serum starvation of either type of fibroblast cell line. Moreover, irrespective of the methods applied to artificially synchronize the mitotic cycle of nuclear donor cells at the G0/G1 phases, developmental abilities to reach the morula/blastocyst stages were significantly higher for porcine NT embryos that had been reconstructed with non-apoptotic/non-necrotic foetal fibroblast cells than those for NT embryos that had been reconstructed with non-apoptotic/non-necrotic adult dermal fibroblast cells. To our knowledge, the generation of cloned pig embryos using abattoir-derived oocytes receiving cell nuclei descended from contact-inhibited or serum-deprived somatic cells undergoing comprehensive vital diagnostics for the absence of biochemical and biophysical proapoptotic alterations within their plasmalemmas has not been reported so far.

scholarly journals Defining cell culture conditions to improve human norovirus infectivity assays

2013 ◽  
Vol 67 (4) ◽  
pp. 863-868 ◽  
Author(s):  
T. M. Straub ◽  
J. R. Hutchison ◽  
R. A. Bartholomew ◽  
C. O. Valdez ◽  
N. B. Valentine ◽  
...  
Keyword(s):  
Cell Line ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Qrt Pcr ◽  
Infectivity Assay ◽  
Polymerase Chain ◽  
Single Laboratory ◽  
Cell Culture Conditions ◽  
Human Intestinal Cells

Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus.

scholarly journals Direct repression of Nanog and Oct4 by OTX2 modulates contribution of epiblast-derived cells to germline and somatic lineage

Development ◽  
2021 ◽  
Author(s):  
Luca Giovanni Di Giovannantonio ◽  
Dario Acampora ◽  
Daniela Omodei ◽  
Vincenzo Nigro ◽  
Pasquale Barba ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Regulatory Network ◽  
Primordial Germ Cells ◽  
Culture Conditions ◽  
Specific Cell ◽  
Temporal Window ◽  
Pluripotency Gene ◽  
Gene Regulatory ◽  
Cell Culture Conditions

In mammals the pre-gastrula proximal epiblast gives rise to Primordial Germ Cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work showed that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here we show that OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs occurs also in mutant embryos. We propose that the OTX2 repressive control of Oct4 and Nanog is at the basis of the mechanism determining epiblast contribution to germline and somatic lineage.

55 LIVE PLASMA MEMBRANE ANALYSIS OF EARLY APOPTOTIC CELL DEATH IN PORCINE ADULT DERMAL FIBROBLASTS PRIOR TO SOMATIC CELL CLONING

2008 ◽  
Vol 20 (1) ◽  
pp. 108
Author(s):  
M. Skrzyszowska ◽  
M. Samiec
Keyword(s):  
Plasma Membrane ◽  
Somatic Cell ◽  
Dermal Fibroblast ◽  
Chemical Activation ◽  
Annexin V ◽  
Fibroblast Cells ◽  
Apoptotic Cells ◽  
Cell Cloning ◽  
Donor Cells

The aim of our study was to determine the in vitro developmental capability of porcine nuclear-transferred (NT) embryos reconstructed with adult dermal fibroblast cells, which had been analyzed for apoptosis by live plasma membrane fluorescent labelling. Frozen/thawed fibroblasts, which had been in vitro cultured to confluency, were used for analysis. To detect the early apoptotic changes in the plasma membrane involving the externalization of phosphatidylserine molecules and the subsequent loss of lipid composition asymmetry, the fibroblasts were tagged using a conjugate of annexinV with enhanced green fluorescent protein (eGFP). In the somatic cell cloning procedure, enucleated in vitro-matured oocytes were reconstituted with non-apoptotic dermal fibroblast cell nuclei. Afterwards, NT-derived oocytes were stimulated with a combination of electrical and chemical activation. Simultaneous fusion and electrical activation were induced by application of two successive DC pulses of 1.2 kV cm–1 for 60 �s. A two-step chemical activation procedure was initiated after a 1.5–2 h delay. The cybrids were exposed to 15 µm calcium ionomycin for 5 to 7 min and then incubated in the culture medium supplemented with 10 µg mL–1 cycloheximide for 3 h. Reconstructed embryos were in vitro cultured in NCSU-23 medium for 6–7 days. Fluorescence analysis of the adult dermal fibroblast cells revealed that a relatively high proportion of donor cells exhibited proapoptotic changes in the plasma membrane. The percentage of late apoptotic cells with advanced morphological changes did not exceed 30%. Moreover, an extremely low rate (ranging from 0 to 2%) of early apoptotic cells, with a morphologically normal, i.e., smooth (non-corrugated) and intact (non-blebbing), plasmolemma but which emitted the green eGFP-derived chemiluminescence, was detected. A total of 219 enucleated oocytes were subjected to reconstruction and 185/219 (84.5%) were successfully fused with non-apoptotic nuclear donor cells. Out of 185 cultured NT embryos, 108 (58.4%) cleaved. The frequencies of cloned embryos, that reached the morula and blastocyst stages, were 84/185 (45.4%) and 26/185 (14.0%), respectively. In conclusion, annexin V-eGFP is a sensitive method able to detect the early phases of apoptosis in cultured adult dermal fibroblast cells, because it identified that very small proportion of morphologically normal cells (without shrinkage of the plasmolemma) that also emitted the annexin V-eGFP-derived biochemiluminescence. Nonetheless, the probability of their random erroneous selection for somatic cell cloning appears to be extremely low. It was also found that the preimplantation developmental potential of NT embryos originating from non-apoptotic adult dermal fibroblast cells is relatively high. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

Sign in / Sign up

Export Citation Format

Share Document