scholarly journals The Co-Culture of Staphylococcal Biofilm and Fibroblast Cell Line: The Correlation of Biological Phenomena with Metabolic NMR1 Footprint

2021 ◽  
Vol 22 (11) ◽  
pp. 5826
Author(s):  
Joanna Czajkowska ◽  
Adam Junka ◽  
Jakub Hoppe ◽  
Monika Toporkiewicz ◽  
Andrzej Pawlak ◽  
...  

Staphylococcus aureus is one of the most prevalent pathogens associated with several types of biofilm-based infections, including infections of chronic wounds. Mature staphylococcal biofilm is extremely hard to eradicate from a wound and displays a high tendency to induce recurring infections. Therefore, in the present study, we aimed to investigate in vitro the interaction between S. aureus biofilm and fibroblast cells searching for metabolites that could be considered as potential biomarkers of critical colonization and infection. Utilizing advanced microscopy and microbiological methods to examine biofilm formation and the staphylococcal infection process, we were able to distinguish 4 phases of biofilm development. The analysis of staphylococcal biofilm influence on the viability of fibroblasts allowed us to pinpoint the moment of critical colonization—12 h post contamination. Based on the obtained model we performed a metabolomics analysis by 1H NMR spectroscopy to provide new insights into the pathophysiology of infection. We identified a set of metabolites related to the switch to anaerobic metabolism that was characteristic for staphylococcal biofilm co-cultured with fibroblast cells. The data presented in this study may be thus considered a noteworthy but preliminary step in the direction of developing a new, NMR-based tool for rapid diagnosing of infection in a chronic wound.

2009 ◽  
Vol 21 (1) ◽  
pp. 125
Author(s):  
M. Skrzyszowska ◽  
M. Samiec ◽  
W. Mlodawska ◽  
J. Kochan ◽  
A. Okolski ◽  
...  

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.


2013 ◽  
Vol 13 (2) ◽  
pp. 275-293 ◽  
Author(s):  
Marcin Samiec ◽  
Maria Skrzyszowska ◽  
Jolanta Opiela

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.


2021 ◽  
Vol 23 (09) ◽  
pp. 540-555
Author(s):  
Deepak Tom Jose ◽  
◽  
Sivagurunathan, P ◽  
Aswini, B ◽  
Dinesh, MD ◽  
...  

Antimicrobial peptides from Streptomyces sp. and marine fish (Carangoides malabaricus) were extracted and developed as conjugates in the present study. The objective was framed to analyze the ability of conjugate to retard the growth of test bacteria causing diabetic foot ulcers. Fibroblast cell adhesion on AMP conjugates coated mesh samples were recorded using microscopic studies with an aim of developing a novel tissue engineered wound dressing material. Thus developed tissue engineered materials were evaluated for its antibacterial potential against wound pathogens; and to assay the wound healing ability using a standard in vitro wound scratch method. Tissue engineered materials were developed using L929 fibroblast cells. L929 fibroblast cells attachment and its stage wise development on wound dressing mesh materials were microscopically observed. In vitro wound healing assay revealed that the developed conjugates (containing AMPs) exhibited cell migration and proliferation after 12th hour of incubation indicating the wound healing abilities. The results showed that the developed tissue engineered wound dressing material has commercial interest in near future.


2009 ◽  
Vol 03 (02) ◽  
pp. 114-119 ◽  
Author(s):  
Mustafa Ulker ◽  
H. Esra Ulker ◽  
Mustafa Zortuk ◽  
Mehmet Bulbul ◽  
Ali Riza Tuncdemir ◽  
...  

ABSTRACTObjectives: The aim of the present study was to evaluate the cytotoxic effects of three different provisional restoration materials on fibroblasts. Two bis-acrylic based [Tempofit Duomix (Detax), Protemp 3 Garant (3M ESPE)] and one urethan dimethacrylate [Revotek LC (GC Corporation)] based provisional restoration materials used.Methods: Materials were prepared according to the manufacturers’ instructions in standard teflon disks (2×5mm) and four samples were extracted in 7 ml of Basal Medium Eagle with 10% new born calf serum and 100 mg/ml penicillin/streptomycin for 24 hours. The L929 fibroblast cells were plate (25.000 cells/ml) in well plates, and maintained in a CO2 incubator at 37°C for 24h. After 24 hours, the incubation medium was replaced by the immersed medium in which the samples were stored and the L929 fibroblasts were incubated in contact with eluates for 24 hours at 37°C for 24h. The fibroblast cell viability was analyzed by measuring the mitochondrial activity with the methyltetrazolium test (MTT). Twelve well used for each specimen and experiment repeated for two times. The data was statistically analyzed by Mann-Whitney U tests.Results: The results showed that, Revotek LC and Protemp 3 Garant were not cytotoxic for fibroblast cells when compared to control group (P>.05). However, Tempofit duomix was cytotoxic for L929 fibroblasts when compared to control group and other tested materials (P%.05).Conclusions: Taking into consideration the limitations of an in vitro study, our study indicate that provisional restoration materials might have cytotoxic effects on fibroblasts and should be selected carefully for clinical applications. (Eur J Dent 2009;3:114-119)


2005 ◽  
Vol 17 (2) ◽  
pp. 182 ◽  
Author(s):  
M. Skrzyszowska ◽  
M. Samiec

One of the most important factors that determine the developmental potential of mammalian cloned embryos is the structuro-functional quality of nuclear donor cells. Biochemical changes that are some of the earliest symptoms of apoptosis signal transduction are not reflected in the morphological features of somatic cells. Therefore, an appropriate system of cell selection would enable the sorting of donor nuclei with high morphological and biochemical susceptibility to somatic cloning. The aim of our study was to examine the in vitro developmental competencies of porcine nuclear transfer (NT) embryos reconstructed with fetal fibroblast cells that had been analyzed for apoptosis by live-fluorescent labelling. Frozen/thawed fetal fibroblast cells, which had been in vitro-cultured to a confluent state, were used for analysis. To detect the early apoptotic changes in the fibroblast cells, a single cell suspension of nuclear donor cells was subjected to dyeing with live-DNA green fluorochrome YO-PRO-1. The recipient cells were in vitro-matured oocytes. Maternal chromosomes were removed by a chemically assisted microsurgical technique. Then, single nuclear donor cells were inserted into the perivitelline space of enucleated oocytes. Fibroblast cell-ooplast couplets were simultaneously fused and activated with two consecutive DC pulses of 1.2 kV/cm for 60 μs. Reconstructed embryos were in vitro cultured in 50-μL drops of NCSU-23 medium supplemented with 0.4% BSA-V for 6 to 7 days at 38.5°C in a humidified atmosphere of 5% CO2 and 95% air. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. After fluorescent analysis of approximately 50 different random samples collected from the population of fetal fibroblast cells, that had been labelled with YO-PRO-1 dye, it was found that a relatively high proportion of donor cells revealed ultrastructural apoptotic changes. The percentage of late apoptotic cells with advanced morphological transformations was about 40% of the total pool of the fibroblast cells. A total of 262/270 (97.0%) enucleated oocytes were subjected to reconstruction and 141/262 (53.8%) were successfully fused with non-apoptotic nuclear donor cells. Following the simultaneous fusion/activation protocol, reconstituted oocytes were selected for in vitro culture. Out of 262, 133 (50.8%) cultured NT embryos cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 48/133 (36.0%) and 10/133 (7.5%), respectively. In conclusion, morphology is a sufficient selection factor for detection of apoptosis in the cultured (confluent) fetal fibroblast cells to be used for cloning. Moreover, it was found that YO-PRO-1 fluorochrome may be not able to detect the early phases of apoptosis, because only the morphologically abnormal cells emitted the YO-PRO-1-derived fluorescence. This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-KBN-084/P06/2002/4.2 from years 2003 to 2005.


2021 ◽  
Vol 23 (10) ◽  
pp. 206-221
Author(s):  
Deepak Tom Jose ◽  
◽  
Sivagurunathan, P ◽  
Aswini, B, ◽  
Uma, C ◽  
...  

Antimicrobial peptides from Streptomyces sp. and marine fish (Carangoides malabaricus) were extracted and developed as conjugates in the present study. The objective was framed to analyze the ability of conjugate to retard the growth of test bacteria causing diabetic foot ulcers. Fibroblast cell adhesion on AMP conjugates coated mesh samples were recorded using microscopic studies with an aim of developing a novel tissue engineered wound dressing material. Thus developed tissue engineered materials were evaluated for its antibacterial potential against wound pathogens; and to assay the wound healing ability using a standard in vitro wound scratch method. Tissue engineered materials were developed using L929 fibroblast cells. L929 fibroblast cells attachment and its stage wise development on wound dressing mesh materials were microscopically observed. In vitro wound healing assay revealed that the developed conjugates (containing AMPs) exhibited cell migration and proliferation after 12th hour of incubation indicating the wound healing abilities. The results showed that the developed tissue engineered wound dressing material has commercial interest in near future.


2013 ◽  
Vol 13 (4) ◽  
pp. 735-752 ◽  
Author(s):  
Marcin Samiec ◽  
Maria Skrzyszowska ◽  
Michał Bochenek

Abstract The study was undertaken to examine whether various strategies, including contact inhibition and serum starvation, that were used for artificial synchronization of mitotic cycle of porcine fibroblast cell lines affect differently the distribution of cell cycle stage frequencies and the occurrence of apoptotic cell death in the analysed cell samples. In vitro cultured (contact-inhibited or serumstarved) somatic cells were subjected to flow cytometric diagnostics of mitotic cycle together with the detection of late-apoptotic cell fractions with hypodiploid number of nuclear DNA molecules. Moreover, impact of the methods applied to synchronize the cell division cycle of different types of nuclear donor fibroblast cells (adult cutaneous and foetal fibroblasts) on the preimplantation developmental outcomes of cloned pig embryos was investigated. The developmental capabilities of nuclear-transferred (NT) embryos that were reconstituted with contact-inhibited or serum-depleted adult cutaneous fibroblast cells to reach the morula and blastocyst stages remained at the levels of 169/278 (60.8%) and 76/278 (27.3%) or 121/265 (45.7%) and 46/265 (17.4%), respectively. The proportions of NT embryos originating from contact-inhibited or serum-deprived foetal fibroblast cells that completed their development to the morula and blastocyst stages were 223/296 (75.3%) and 108/296 (36.5%) or 165/261 (63.2%) and 67/261 (25.7%), respectively. In conclusion, the flow cytometric analysis of cultured porcine adult cutaneous and foetal fibroblast cells revealed the high efficiency of the artificial synchronization of mitotic cycle at the G0/G1 stages as a consequence of applying the methods of either contact inhibition or serum deprivation. For both types of fibroblast cells used to reconstruct the enucleated oocytes, the strategies that were utilized to synchronize the cell division cycle of nuclear donor cells considerably influenced the in vitro developmental abilities of NT pig embryos. Developmental competencies to reach the morula/blastocyst stages for cloned embryos that had been reconstructed with contact-inhibited or serum-starved foetal fibroblast cell nuclei were significantly higher than those for embryos that had been reconstructed with contact-inhibited or serum-starved adult cutaneous fibroblast cell nuclei.


2020 ◽  
Vol 5 (2) ◽  
pp. 196-199
Author(s):  
Wardiansyah ◽  
Rachmat Hidayat ◽  
Msy Rulan Adnindya

A B S T R A C TIntroduction. A CO 2 incubator is an essential tool for the initiation of theproliferation of primary culture cells or cell lines. In principle, this tool works bykeeping the sample cell line at an optimum temperature of 37 o C and 5% carbondioxide supply. The ability of the CO 2 incubator to maintain temperature and supplyof 5% carbon dioxide are essential points in the development of the CO 2 incubator.This study is an attempt to convince the potential of Sriwijaya CO 2 Incubator inmaintaining the proliferation ability of cultured cells in an in vitro study. Methods.This study is an experimental pre-post test that explores the percentage of viabilityof primary culture cells (fibroblasts) before and after incubation in CO 2 incubators.The object of this study was fibroblast cells obtained from the prepuce of patientswho performed circumcision. Results. Fibroblast cell proliferation in CO 2 incubatorsshows an increase in the number of fibroblast proliferation which can be seen withthe increasing number of cells visualized by inverted microscopy. Conclusion.Sriwijaya CO 2 incubator has the potential to be used in in vitro research to triggerthe growth and proliferation of fibroblast cells.


Author(s):  
Ekayanti M. Kaiin ◽  
Ita Djuwita

The aim of this study was to examine the potency of fibroblast cells transdifferentiated to neuron cells in vitro. Newborn rat neuron conditioned medium (NBRN CM) was collected from neuron cells cultured with mDMEM without serum for 48 hours. Fibroblast cells werecollected from fetal rat muscle treated with trypsin. Fibroblast cells were culture with 3 kind of culture medium: mDMEM + 0.01 mM β-mercaptoethanol; mDMEM + 50% NBRN-CM and mDMEM + 0.01 mM β-mercaptoethanol + 50% NBRN CM . As control, cells was culturedwith mDMEM +10% newborn calf serum (NBCS). The addition of NBRN CM into culture medium resulted in 12.97% newborn cells in fibroblastculture medium passage I. Newborn rat neuron conditioned medium in fibroblast culture medium resulted 12.97% neuron cells at passage 1. Thepercentage was increased (14.60%) when β- mercaptoethanol added into medium. The same result was found at passage 3 (12.67%; 13.17%). Itshowed that fibroblast cells has potency to transdifferentiated into neuron cells when cultured with NBRN CM. Further research is needed toknow the fibroblast transdifferentiation potency.Key words: fibroblast, transdifferentiation, conditioned medium, neuron cells, in vitro 


Author(s):  
Abdulaziz Alqahtani ◽  
London Mena ◽  
Dean Scholl ◽  
Cassandra Kruczek ◽  
Jane A. Colmer-Hamood ◽  
...  

<i>Pseudomonas aeruginosa</i>, a Gram-negative opportunistic pathogen, is one of the major species isolated from infected chronic wounds. The multidrug resistance exhibited by <i>P. aeruginosa</i> plus its ability to form biofilms that are difficult to eradicate, along with rising cost of producing new antibiotics, has necessitated the search for alternatives to standard antibiotics. Pyocins are antimicrobial compounds produced by P. aeruginosa to protect itself from competitors. We synthesized and purified recombinant <i>P. aeruginosa</i> R2 pyocin and used it in aqueous solution (rR2P) or formulated in polyethylene glycol (rR2PC) to treat P. aeruginosa-infected wounds. Clinical strains of <i>P. aeruginosa</i> were found to be sensitive (completely), partially sensitive, or resistant to rR2P. In the in vitro biofilm model, rR2P inhibited biofilm development by rR2P-sensitive isolates; while rR2PC eliminated partial biofilms formed by these strains in the in vitro wound biofilm model. In the murine model of excision wound, and at 24 h post infection, rR2PC application significantly reduced the bioburden of clinical isolate BPI86. Application of rR2PC containing two glycoside hydrolase antibiofilm agents eliminated BPI86 from the infected wound. These results suggest that the topical application of rR2PC is an effective therapy to treat wounds infected with R2P-senstive P. aeruginosa strains.


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