The biological and therapeutic assessment of a P(3HB-co-4HB)-bioactive glass-graphene composite biomaterial for tissue regeneration.

An ideal wound dressing should create a healing environment that relieves pain, protects against infections, maintains moisture, removes debris, and speeds up wound closure and repair. However, conventional options like gauze often fall short in fulfilling these requirements, especially for chronic or nonhealing wounds. Hence there is a critical need for inventive formulations that offer efficient, cost-effective, and eco-friendly alternatives. This study focuses on assessing the innovative formulation based on a microbial-derived copolymer known as poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB) bioactive glass and graphene particles, and exploring their biological response in vitro and in vivo-to find the best combination that promotes cell adhesion and enhances wound healing. The formulation optimized at concentration of bioactive glass (1 w/w%) and graphene (0.01 w/w%) showed accelerated degradation and enhanced blood vessel formation. Meanwhile biocompatibility was evaluated using murine osteoblasts, human dermal fibroblasts, and standard cell culture assays, demonstrating no adverse effects after 7 days of culture and well-regulated inflammatory kinetics. Whole thickness skin defect using mice indicated the feasibility of the biocomposites for a faster wound closure and reduced inflammation. Overall, this biocomposite appears promising as an ideal wound dressing material and positively influencing wound healing rates.

Radiation-induced DNA damage by proton, helium and carbon ions in human fibroblast cell: Geant4-DNA and MCDS-based study.

Background. Radiation-induced DNA damages such as Single Strand Break (SSB), Double Strand Break (DSB) and Complex DSB (cDSB) are critical aspects of radiobiology with implications in radiotherapy and radiation protection applications.Materials and Methods. This study presents a thorough investigation into the effects of protons (0.1-100 MeV/u), helium ions (0.13-100 MeV/u) and carbon ions (0.5-480 MeV/u) on DNA of human fibroblast cells using Geant4-DNA track structure code coupled with DBSCAN algorithm and Monte Carlo Damage Simulations (MCDS) code. Geant4-DNA-based simulations consider 1µm × 1µm × 0.5µm water box as the target to calculate energy deposition on event-by-event basis and the three-dimensional coordinates of the interaction location, and then DBSCAN algorithm is used to calculate yields of SSB, DSB and cDSB in human fibroblast cell. The study investigated the influence of Linear Energy Transfer (LET) of protons, helium ions and carbon ions on the yields of DNA damages. Influence of cellular oxygenation on DNA damage patterns is investigated using MCDS code.Results. The study shows that DSB and SSB yields are influenced by the LET of the particles, with distinct trends observed for different particles. The cellular oxygenation is a key factor, with anoxic cells exhibiting reduced SSB and DSB yields, underscoring the intricate relationship between cellular oxygen levels and DNA damage. The study introduced DSB/SSB ratio as an informative metric for evaluating the severity of radiation-induced DNA damage, particularly in higher LET regions.Conclusions. The study highlights the importance of considering particle type, LET, and cellular oxygenation in assessing the biological effects of ionizing radiation.

When Chromatin Decondensation Affects Nuclear γH2AX Foci Pattern and Kinetics and Biases the Assessment of DNA Double-Strand Breaks by Immunofluorescence.

Immunofluorescence with antibodies against phosphorylated forms of H2AX (γH2AX) is revolutionizing our understanding of repair and signaling of DNA double-strand breaks (DSBs). Unfortunately, the pattern of γH2AX foci depends upon a number of parameters (nature of stress, number of foci, radiation dose, repair time, cell cycle phase, gene mutations, etc…) whose one of the common points is chromatin condensation/decondensation. Here, we endeavored to demonstrate how chromatin conformation affects γH2AX foci pattern and influences immunofluorescence signal. DSBs induced in non-transformed human fibroblasts were analyzed by γH2AX immunofluorescence with sodium butyrate treatment of chromatin applied after the irradiation that decondenses chromatin but does not induce DNA breaks. Our data showed that the pattern of γH2AX foci may drastically change with the experimental protocols in terms of size and brightness. Notably, some γH2AX minifoci resulting from the dispersion of the main signal due to chromatin decondensation may bias the quantification of the number of DSBs. We proposed a model called "Christmas light models" to tentatively explain this diversity of γH2AX foci pattern that may also be considered for any DNA damage marker that relocalizes as nuclear foci.

In Vitro Assessment of Cardiac Fibroblast Activation at Physiologic Stiffness.

Cardiac fibroblasts (CF) are an essential cell type in cardiac physiology, playing diverse roles in maintaining structural integrity, extracellular matrix (ECM) synthesis, and tissue repair. Under normal conditions, these cells reside in the interstitium in a quiescent state poised to sense and respond to injury by synthesizing and secreting collagen, vimentin, hyaluronan, and other ECM components. In response to mechanical and chemical stimuli, these "resident" fibroblasts can undergo a transformation through a continuum of activation states into what is commonly known as a "myofibroblast," in a process critical for injury response. Despite progress in understanding the contribution of fibroblasts to cardiac health and disease, much remains unknown about the signaling mediating this activation, in part owing to technical challenges in evaluating CF function and activation status in vitro. Given their role in monitoring the ECM, CFs are acutely sensitive to stiffness and pressure. High basal activation of isolated CFs is common due to the super-physiologic stiffness of traditional cell culture substrates, making assays dependent on quiescent cells challenging. To overcome this problem, cell culture parameters must be tightly controlled, and the use of dishes coated with biocompatible reduced-stiffness substrates, such as 8-kPa polydimethylsiloxane (PDMS), has shown promise in reducing basal activation of fibroblasts. Here, we describe cell culture protocol for maintaining CF quiescence in vitro to enable a dynamic range for the assessment of activation status in response to fibrogenic stimuli using PDMS-coated coverslips. Our protocol provides a cost-effective tool to study fibroblast signaling and activity, allowing researchers to better understand the underlying mechanisms involved in cardiac fibrosis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 8-kPa polydimethylsiloxane (PDMS)/gelatin-coated coverslips for cardiac fibroblast cell culture Basic Protocol 2: Isolation of adult cardiac fibroblasts and plating onto PDMS coverslips Basic Protocol 3: Assessment of cardiac fibroblast activation by α smooth muscle actin (αSMA) immunocytochemistry.

3D Bioprinted Human Skin Model Recapitulating Native-Like Tissue Maturation and Immunocompetence as an Advanced Platform for Skin Sensitization Assessment.

Physiologically-relevant in vitro skin models hold the utmost importance for efficacy assessments of pharmaceutical and cosmeceutical formulations, offering valuable alternatives to animal testing. Here, an advanced immunocompetent 3D bioprinted human skin model is presented to assess skin sensitization. Initially, a photopolymerizable bioink is formulated using silk fibroin methacrylate, gelatin methacrylate, and photoactivated human platelet releasate. The developed bioink shows desirable physicochemical and rheological attributes for microextrusion bioprinting. The tunable physical and mechanical properties of bioink are modulated through variable photocuring time for optimization. Thereafter, the bioink is utilized to 3D bioprint "sandwich type" skin construct where an artificial basement membrane supports a biomimetic epidermal layer on one side and a printed pre-vascularized dermal layer on the other side within a transwell system. The printed construct is further cultured in the air-liquid interface for maturation. Immunofluorescence staining demonstrated a differentiated keratinocyte layer and dermal extracellular matrix (ECM)-remodeling by fibroblasts and endothelial cells. The biochemical estimations and gene-expression analysis validate the maturation of the printed model. The incorporation of macrophages further enhances the physiological relevance of the model. This model effectively classifies skin irritative and non-irritative substances, thus establishing itself as a suitable pre-clinical screening platform for sensitization tests.

In-vivo assessment of application of folinic acid and botulinum toxin A in cleft lip surgical defects.

INTRODUCTION: Folinic acid and botulinum toxin A have shown promising results in wound healing in different studies. This study aimed to compare the effects of these approaches on wound healing after simulating cleft lip surgery in rats. METHODS: In this experimental animal study, after creating lip defects, 30 rats were randomly divided into three groups and received normal saline (CTL), botulinum toxin A (BOT), and folinic acid (FOL). Biopsy from the skin wounds was performed after 14- and 28-days. These samples were stained with haematoxylin and eosin and Masson trichrome staining. Finally, each pathological parameter of wound healing was rated in this study. RESULTS: While the inflammatory response was not different among the study groups, fibroblast proliferation and collagen deposition were significantly higher in FOL group compared to BOT group. Moreover, both BOT and FOL facilitated epithelial healing and 14-day angiogenesis as compared with normal saline. CONCLUSIONS: Improved wound healing was observed using both botulinum toxin A and folinic acid in rat animal models. However, the application of botulinum toxin A caused less fibroblast proliferation and collagen deposition which can potentially lead to less scar formation, which can be particularly important in the aesthetic zone.

Viability and Proliferation Assessment of Gingival Fibroblasts Cultured on Silver Nanoparticle-Doped Ti-6Al-4V Surfaces.

PURPOSE: To investigate the biocompatibility of silver nanoparticle (AgNP)-doped Ti-6Al-4V surfaces by evaluating the viability and proliferation rate of human gingival fibroblasts (HGFs)-as the dominant cells of peri-implant soft tissues-seeded on the modified surfaces. MATERIALS AND METHODS: AgNPs (sizes 8 nm and 30 nm) were incorporated onto Ti-6Al-4V specimen surfaces via electrochemical deposition, using colloid silver dispersions with increasing AgNP concentrations of 100 ppm, 200 ppm, and 300 ppm. One control and six experimental groups were included in the study: (1) control (Ti-6Al-4V), (2) 8 nm/100 ppm, (3) 8 nm/200 ppm, (4) 8 nm/300 ppm, (5) 30 nm/100 ppm, (6) 30 nm/200 ppm, and (7) 30 nm/300 ppm. HGF cell primary cultures were isolated from periodontally healthy donor patients and cultured in direct contact with the group specimens for 24 and 72 hours. The cytotoxicity of AgNP-doped Ti-6Al-4V specimens toward HGF was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and BrdU (5-bromo-2'-deoxyuridine) assay tests. Calcein AM and ethidium homodimer (EthD-1) fluorescent stains were used to determine the live and dead cells. The morphology and attachment properties of the HGFs were determined via scanning electron microscopy (SEM). RESULTS: Energy dispersive x-ray (EDX) analysis confirmed the presence of AgNPs on the specimens. The MTT test revealed that AgNPs of both sizes and all concentrations presented a decreased cellular metabolic activity compared to the control discs. All concentrations of both sizes of AgNPs affected the cell proliferation rate compared to the control group, as revealed by the BrdU assay. Overall, cytotoxicity of the modified Ti-6Al-4V surfaces depended on cell exposure time. Observation via confocal microscopy confirmed the results of the MTT and BrdU assay tests. Specifically, most cells remained alive throughout the 72-hour culture period. SEM images revealed that adjacent cells form bonds with each other, creating confluent layers of conjugated cells. CONCLUSIONS: The findings of the present study indicate that Ti-6Al-4V surfaces modified with 8 nm and 30 nm AgNPs at concentrations of 100 ppm, 200 ppm, and 300 ppm do not produce any serious cytotoxicity toward HGFs. The initial arrest of the HGF proliferation rate recovered at 72 hours. These results on the antibacterial activity against common periodontal pathogens, in combination with the results found in a previous study by the same research group, suggest that AgNP-doped Ti-6Al-4V surfaces are potential candidates for use in implant abutments for preventing peri-implant diseases.

Plants as a cost-effective source for customizable photosynthetic wound dressings: A proof of concept study.

Oxygen is essential for tissue regeneration, playing a crucial role in several processes, including cell metabolism and immune response. Therefore, the delivery of oxygen to wounds is an active field of research, and recent studies have highlighted the potential use of photosynthetic biomaterials as alternative oxygenation approach. However, while plants have traditionally been used to enhance tissue regeneration, their potential to produce and deliver local oxygen to wounds has not yet been explored. Hence, in this work we studied the oxygen-releasing capacity of Marchantia polymorpha explants, showing their capacity to release oxygen under different illumination settings and temperatures. Moreover, co-culture experiments revealed that the presence of these explants had no adverse effects on the viability and morphology of fibroblasts in vitro, nor on the viability of zebrafish larvae in vivo. Furthermore, oxygraphy assays demonstrate that these explants could fulfill the oxygen metabolic requirements of zebrafish larvae and freshly isolated skin biopsies ex vivo. Finally, the biocompatibility of explants was confirmed through a human skin irritation test conducted in healthy volunteers following the ISO-10993-10-2010. This proof-of-concept study provides valuable scientific insights, proposing the potential use of freshly isolated plants as biocompatible low-cost oxygen delivery systems for wound healing and tissue regeneration.

Assessment of mesh shrinkage using fibroblast-populated collagen matrices: a proof of concept for in vitro hernia mesh testing.

PURPOSE: This study uses free-floating contractile fibroblast-populated collagen matrices (FPCMs) to test the shrinkage of different hernia mesh products. We hope to present this model as a proof of concept for the development of in vitro hernia mesh testing-a novel technology with interesting potential. METHODS: FPCMs were formed by seeding Human Dermal Fibroblasts into collagen gels. FPCMs were seeded with three different cell densities and cast at a volume of 500 µl into 24-well plates. Five different mesh products were embedded within the collagen constructs. Gels were left to float freely within culture media and contract over 5 days. Photographs were taken daily and the area of the collagen gel and mesh were measured. Media samples were taken at days 2 and 4 for the purposes of measuring MMP-9 release. After 5 days, dehydrated FPCMs were also examined under light and fluorescence microscopy to assess cell morphology. RESULTS: Two mesh products-the mosquito net and large pore lightweight mesh were found to shrink notably more than others. This pattern persisted across all three cell densities. There were no appreciable differences observed in MMP-9 release between products. CONCLUSIONS: This study has successfully demonstrated that commercial mesh products can be successfully integrated into free-floating contractile FPCMs. Not only this, but FPCMs are capable of applying a contractile force upon those mesh products-eliciting different levels of contraction between mesh products. Such findings demonstrate this technique as a useful proof of concept for future development of in vitro hernia mesh testing.

In vitro Safety Assessment of Extracts and Compounds From Plants as Sunscreen Ingredients.

This work investigated the safety of extracts obtained from plants growing in Colombia, which have previously shown UV-filter/antigenotoxic properties. The compounds in plant extracts obtained by the supercritical fluid (CO2) extraction method were identified using gas chromatography coupled to mass spectrometry (GC/MS) analysis. Cytotoxicity measured as cytotoxic concentration 50% (CC50) and genotoxicity of the plant extracts and some compounds were studied in human fibroblasts using the trypan blue exclusion assay and the Comet assay, respectively. The extracts from Pipper eriopodon and Salvia aratocensis species and the compound trans-ß-caryophyllene were clearly cytotoxic to human fibroblasts. Conversely, Achyrocline satureioides, Chromolaena pellia, and Lippia origanoides extracts were relatively less cytotoxic with CC50 values of 173, 184, and 89 µg/mL, respectively. The C. pellia and L. origanoides extracts produced some degree of DNA breaks at cytotoxic concentrations. The cytotoxicity of the studied compounds was as follows, with lower CC50 values representing the most cytotoxic compounds: resveratrol (91 µM) > pinocembrin (144 µM) > quercetin (222 µM) > titanium dioxide (704 µM). Quercetin was unique among the compounds assayed in being genotoxic to human fibroblasts. Our work indicates that phytochemicals can be cytotoxic and genotoxic, demonstrating the need to establish safe concentrations of these extracts for their potential use in cosmetics.

Surface treatment of PET multifilament textile for biomedical applications: roughness modification and fibroblast viability assessment.

OBJECTIVES: The aim of this study was to investigate the potential of tuning the topography of textile surfaces for biomedical applications towards modified cell-substrate interactions. METHODS: For that purpose, a supercritical Nitrogen N2 jet was used to spray glass particles on multi-filament polyethylene terephthalate (PET) yarns and on woven fabrics. The influence of the jet projection parameters such as the jet pressure (P) and the standoff distance (SoD) on the roughness was investigated. RESULTS: The impact of the particles created local filament ruptures on the treated surfaces towards hairiness increase. The results show that the treatment increases the roughness by up to 17 % at P 300 bars and SoD 300 mm while the strength of the material is slightly decreased. The biological study brings out that proliferation can be slightly limited on a more hairy surface, and is increased when the surface is more flat. After 10 days of fibroblast culture, the cells covered the entire surface of the fabrics and had mainly grown unidirectionally, forming cell clusters oriented along the longitudinal axis of the textile yarns. Clusters were generated at yarn crossings. CONCLUSIONS: This approach revealed that the particle projection technology can help tuning the cell proliferation on a textile surface.

Performance of [18F]FDG PET/CT versus FAPI PET/CT for lung cancer assessment: a systematic review and meta-analysis.

PURPOSE: This article aims to compare the diagnostic performance of 18-fluorodeoxyglucose ([18F]FDG) PET/CT and fibroblast activating protein inhibitor (FAPI) PET/CT in the assessment of primary tumors, lymph nodes, and distant metastases in lung cancer patients. METHODS: A systematic search was conducted on the Cochrane Library, Embase, and PubMed/MEDLINE databases from inception until November 1, 2022. Included studies assessed the use of FAPI PET/CT and [18F]FDG PET/CT in patients with lung cancer. The Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool was used to evaluate the risk of bias. A random variable model was used to analyze the diagnostic tests of the two imaging modalities. RESULTS: The sensitivity of FAPI PET/CT in detecting primary lung cancer lesions was 0.98 (95% CI: 0.88-1.00), while the sensitivity of [18F]FDG PET/CT was 0.99 (95% CI: 0.74-1.00). For the detection of metastatic lesions (lymph node metastases and distant metastases), FAPI PET/CT had a sensitivity of 0.99 (95% CI: 0.90-1.00), while the sensitivity of [18F]FDG PET/CT was 0.77 (95% CI: 0.66-0.85). However, the specificity of the two imaging modalities could not be assessed due to the lack of sufficient information on pertinent true negatives. CONCLUSION: In the diagnosis of metastatic lung cancer lesions, FAPI PET/CT demonstrated a higher sensitivity compared to [18F]FDG PET/CT. Therefore, FAPI PET/CT may be considered an alternative imaging modality for the assessment of primary lung cancer tumors, lymph node metastases, and distant metastases. CLINICAL RELEVANCE STATEMENT: FAPI may be an alternative to [18F]FDG in the assessment of primary lung cancer tumors, lymph node metastases, and distant metastases, which plays a very important role in treatment. KEY POINTS: • This article is to compare the performance of [18F]FDG PET/CT with FAPI PET/CT in the assessment of primary tumors, lymph nodes, and distant metastases in lung cancer. • However, FAPI PET/CT has a higher sensitivity for the diagnostic assessment of metastatic lung cancer lesions.

Mutagenicity assessment of high-power 1.6-THz pulse laser radiation.

The effect of terahertz (THz) radiation has been studied in medicine. However, there is a lack of scientific information regarding its possible mutagenicity. Therefore, the present study aimed to assess the mutagenicity of 1.6 THz laser irradiation. The Ames test was conducted using five bacterial tester strains. The bacteria were subjected to (i) 1.6 THz laser irradiation at 3.8 mW/cm2 for 60 min using a tabletop THz pulse laser system, (ii) ultraviolet irradiation, (iii) treatment with positive control chemicals (positive control) or (iv) treatment with the solvent used in the positive control (negative control). After treatment, the bacterial suspensions were cultured on minimal glucose agar to determine the number of revertant colonies. In addition, the comet assay was performed using fibroblasts (V79) to assess possible DNA damage caused by the THz laser irradiation. The Ames test demonstrated that the THz laser irradiation did not increase the number of revertant colonies compared to that in the negative control group, whereas the ultraviolet irradiation and positive control treatment increased the number of revertant colonies. Thus, 1.6 THz laser irradiation is unlikely to be mutagenic. The comet assay additionally suggests that the THz laser irradiation unlikely induce cellular DNA damage.

Expression Profile of Microenvironmental Factors in the Interface Zone of Colorectal Cancer: Histological-Stromal Biomarkers and Cancer Cell-Cancer-Associated Fibroblast-Related Proteins Combined for the Assessment of Tumor Progression.

INTRODUCTION: The characterization of tumor microenvironment (TME) related factors and their impact on tumor progression have attracted much interest. We investigated cancer cells and cancer-associated fibroblasts (CAFs) to evaluate biomarkers that are associated with neoplastic progression, observing them in different interface zones of colorectal cancer. METHODS: On 357 CRC tissue microarrays, using immunohistochemistry, we examined the associations of podoplanin and α-SMA expressed in cancer cells and CAFs and evaluated them in different areas: tumor core, invasive front, tumor budding, tumor-stroma ratio (TSR) scoring, and desmoplastic stroma. RESULTS: CAFs expressing α-SMA were found in more than 90% of the cases. Podoplanin+ was detected in cancer cells and CAFs, with positivities of 38.6% and 70%, respectively. Higher α-SMA+ CAFs and podoplanin+ cancer cells were observed predominantly at the TSR score area: 94.3% and 64.3% of cases, respectively. The status of podoplanin in CAFs+ was higher in the desmoplastic area (71.6%). Stroma-high tumors showed increased expression of α-SMA and podoplanin in comparison with stroma-low tumors. The status of podoplanin in cancer cells was observed in association with lymphatic invasion and distant metastasis. CONCLUSION: The substance of the CRC was composed predominantly of the surrounding stroma-α-SMA+ CAFs. Podoplanin expressed in the prognosticator zones was associated with unfavorable pathological features. The combination of histologic and protein-related biomarkers can result in a tool for the stratification of patients with CRC.

68 Ga-FAPI-04 PET/CT in Assessment of Fibroblast Activation in Keloids : A Prospective Pilot Study.

PURPOSE: Keloids are benign fibroproliferative disorders characterized by the massive proliferation of fibroblasts. Fibroblast activation plays a key role in the invasive growth of keloids. Therefore, a prospective pilot study was conducted to explore the value of 68 Ga-FAPI-04 PET/CT in the assessment of keloids activity. PATIENTS AND METHODS: Twenty-five patients with keloid were enrolled to conduct 68 Ga-FAPI-04 PET/CT. All patients accepted surgery to remove part of the lesions within 1 week. SUV mean and SUV max were measured for semiquantitative analysis and compared with the Vancouver Scar Scale, Laser Speckle Contrast Imaging, pathology, and immunohistochemical stains. RESULTS: A total of 123 lesions were detected in 25 patients, most of which were distributed in the anterior chest wall. The 68 Ga-FAPI-04 uptake was significantly different at different sites ( P < 0.0001). There was uptake heterogeneity within the keloid lesions, and a significant difference was found between the edge and center of some large lesions. The SUV max of 68 Ga-FAPI-04 showed significantly correlation with the Vancouver Scar Scale ( r = 0.565, P < 0.0001) moderately and the Laser Speckle Contrast Imaging parameters mildly. The SUV max of 68 Ga-FAPI-04 had a moderate correlation with FAPI expression ( r = 0.520, P = 0.022). Moreover, collagen, fibroblast activator protein, and Ki-67 expression were found higher at the edges of keloid tissue than in the center. CONCLUSIONS: 68 Ga-FAPI-04 PET/CT can reflect the distribution characteristics of activated fibroblasts in keloid tissue and may provide a novel method for keloid evaluation for further fibroblast-related therapies.

In Vitro Assessment of Gold Nanoparticles on Telomerase Activity and Telomere Length in Human Fibroblasts.

Telomerase activity coincides with lengthening of the ends of chromosomes known as telomeres. Telomere length is used as a marker for cellular aging. Telomeres shorten over time as cells divide, and certain bioactive compounds such as gold nanoparticles (AuNPs) may slow the shortening of telomeres by increasing telomerase activity. The objective of the present study is to assess the effect of AuNPs on telomerase activity and telomere length in human fibroblasts. Telomerase activity was measured using enzyme-linked immunosorbent assay (ELISA) in primary human lung fibroblasts (IMR90) and using quantitative PCR-based telomeric repeat amplification protocol (Q-TRAP) in primary human dermal fibroblasts, neonatal (HDFn). Telomere length was determined by Telomere Analysis Technology (TAT®)assay in HDFn. In IMR90, all AuNP treatments showed significant increases in telomerase activity when compared to earlier passages. HDFn treated with AuNPs at 0 ppm, 0.05 ppm, 0.5 ppm, or 5 ppm did not show significant differences in telomerase activity compared to the control group. Significant differences in telomere length in HDFn were observed at 2 weeks of 0.05 and 0.5 ppm AuNPs under oxidative culture conditions as compared to the control group. The study showed preliminary evidence that AuNPs may increase telomerase activity and decelerate the shortening of telomeres in human fibroblasts, suggesting its potential anti-aging effects, which warrants further investigation.

Cell culture media dependent in vitro dynamics and culture characteristics of adult caprine dermal fibroblast cells.

The enhanced availability of functional fibroblasts from precious tissue samples requires an ideal cell-culture system. Therefore, this study was designed to investigate the performance of caprine adult fibroblast cells (cadFibroblast) when cultivated in different culture media. The cadFibroblast cell lines from adult Barbari (Capra hircus) bucks were established and the effect of different media viz. DMEM/F-12 [with low-glucose (5.5 mM; DL) and high-glucose (30 mM; DH)], α-MEM [with low-glucose (5.5 mM; ML) and with high-glucose (30 mM; MH)], and fibroblast growth medium (FGM) were evaluated. Cells were then compared for growth characteristics and in-vitro dynamics through cellular morphology, proliferation, population-doubling time, double-immunocytochemistry, colony-forming units, wound healing, transwell migration, and differential expression of fibroblast-specific markers (FSP-1 and vimentin). The results of immunocytochemistry, transwell migration/invasion, and wound healing assays showed the superiority of DH over DL and other media tested. Whereas, similar effects of glucose supplementation and expression of FSP-1 were not observed in α-MEM. Transwell migration was significantly (p < 0.05) lower in FGM compared with other media tested. Overall, our results illustrate the media-dependent deviation in in-vitro dynamics and culture characteristics of cadFibroblasts that may be useful to develop strategies to cultivate these cells efficiently for research and downstream applications.

Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion.

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different types of feeder cells for maintenance in an undifferentiated state in long-term culture. This approach has been supplanted to a large extent by feeder-free culture protocols, but these involve more costly reagents and can promote a transition to a primed state, which restricts the cells' differentiation capacity. In both feeder and feeder-free conditions, the harvesting of hESC or hiPSC colonies for passaging is a necessary procedure for expanding the cultures. To provide an easy and high-yield procedure for passaging hESCs/hiPSCs cultured on feeder cells, we have established a harvesting method using dis-adhesion elicited by the calcium chelator ethylenediaminetetraacetic acid (EDTA). We have assessed the yield and quality of the resultant passaged cells by comparing this approach to the original mechanical harvesting approach, in which colonies are isolated with a scalpel under a microscope (mechanical harvesting was chosen as a comparator to avoid the reagent variability associated with enzymatic harvesting). In one set of experiments, two different hESC lines were maintained on a feeder cell layer of human foreskin fibroblasts. Each line was subjected to multiple passages using EDTA-based or mechanical harvesting and assessed for colony size and morphology, cell density, stemness marker expression, differentiation to the three germ layers in embryoid bodies, and genomic aberrations. In another set of experiments, we used EDTA-based harvesting on two different hiPSC lines and obtained similar results. EDTA-induced dis-adhesion saved time and gave a higher yield of colonies of a more favorable size and more uniform morphology compared to mechanical harvesting. It was also faster than enzymatic harvesting and not prone to enzyme batch variability. The EDTA-induced dis-adhesion method also facilitates the transfer of hESC/hiPSC lines from feeder cell-based culture to feeder-free conditions if desired for downstream use and analysis.

Comparative assessment of primary cancer cell culture techniques and cellular composition analysis in non-small cell lung cancer.

Preclinical models are required to study individual therapy responses to improve all cancer treatments, particularly non-small cell lung cancer (NSCLC). Patient-derived explants (PDEs) culture model is of great importance in terms of the possibility of tumor cell culture with the microenvironment, and the development of molecular mechanisms and personalized treatment methods. In our study, primary tumor culture with microenvironment was performed using different methods from tumor tissues obtained from 51 patients with NSCLC. To identify the most efficient method, mechanical, enzymatic, and tumor fluid techniques were applied. While the malignant cell rate was > 95% in 3 of these cases, the cancer-associated fibroblasts (CAF) microenvironment was high in 46 (80-94%) and low in 2 (1-79%). Subtyping of cells obtained from culture was performed using the light microscope and, if necessary, additional immunohistochemical markers. Consequently, using different techniques, here we successfully performed primary cell culture from patients with NSCLC with microenvironment. Depending on the cell type and culture conditions, proliferation rate was shown to be altered.

Dynamic assessment of the relationship between oxidative stress and apoptotic pathway in embryonic fibroblast cells exposed to glycidamide: possible protective role of hesperidin.

Worldwide research is being conducted to determine the level of acrylamide (ACR) that humans are exposed to from food and environmental sources. Glycidamide (GA) is an important epoxide metabolite of ACR, and its cytotoxicity is stronger than ACR. In this study, it was aimed to elucidate the effects and underlying mechanisms of GA on the induction of apoptosis in embryonic fibroblast cells. The toxicogenomic profile of GA was studied in terms of both apoptotic and oxidative stress. Embryonic fibroblast cells were exposed to GA (1 and 1000 µM) in the presence and absence of hesperidin (Hes) (20 µM) or vitamin C (VitC) (50 µM) for 24 h. Cell viability, cytotoxicity, lipid peroxidation, hydroxyl radicals, hydrogen peroxide, antioxidant enzyme levels and gene expressions, apoptotic, and oxidative stress-related gene expressions were measured in embryonic fibroblast cells. The results showed that GA induced cytotoxicity and diminished the expression levels of apoptotic genes. Furthermore, GA increased the levels of oxidative stress markers and significantly changed the oxidative stress-related gene expression. It has been determined that antioxidant molecules are considerably suppressed in GA-induced toxicity at both gene and enzyme levels. In addition to these results, when VitC, which is known to have strong antioxidant properties in eliminating the toxic effects of GA, is taken as reference, it has been proven that Hes has stronger antioxidant properties compared to VitC. Finally, GA-induced apoptosis in embryonic fibroblast cells is associated with nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent oxidative stress and Hes has antioxidant properties with strong effects.