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.

New Carbon-Mineral Sorbent Based on Aluminum Oxide, Polydimethylsiloxane, and Single-Wall Carbon Nanotubes: Assessment of the Effect on Erythrocytes In Vitro.

A comparative study of the effect of a sorbent with nanotubes (Al2O3@ WCNT-PDMS) and a carbon-mineral sorbent (Al2O3@C) on the parameters of human erythrocytes was carried out. Using scanning flow cytometry, the morphological and functional parameters of venous blood erythrocytes as well as drainage blood after its perfusion through columns with sorbents were determined. The compared samples Al2O3@SWCNT-PDMS and Al2O3@C are similar by their effect on the morphological and functional parameters of erythrocytes. The maximum membrane extensibility increased to a greatest extent after contact with Al2O3@C, the amount of hemoglobin in erythrocytes decreased to the greatest extent after perfusion through a column with Al2O3@SWCNT-PDMS sorbent. The scanning flow cytometry is promising for assessing the effect on erythrocytes of new sorption materials intended for blood detoxification. Changes in the parameters of erythrocytes of blood collected in a sterile drainage system for subsequent reinfusion were revealed.

GuttaFlow® Bioseal Cytotoxicity Assessment: In Vitro Study.

The sealers used for root canal treatment should be biocompatible for the peri-radicular tissues, to evaluate the cytotoxic effects of GuttaFlow® bioseal sealer and to compare them with AH26® epoxy resin. Culture media were conditioned with the GuttaFlow® bioseal and AH26® pellets. MDPC-23 odontoblast cell cultures were treated with conditioned medium and serial dilutions. To evaluate the metabolic activity and cellular viability, the MTT and SRB assays were performed. To determine the production of reactive oxygen species, the DHE and DCF-DA probes were used. Cell cycle and cell-death types were assessed by cytometry, and to evaluate the mineralization capacity, the Alizarin Red S coloration was used. Statistical analysis was performed using analysis of variance (ANOVA) when normality was found and Kruskal-Wallis on the opposite case. For the comparison with normality values, the Student t-test was used. Cells exposed to the GuttaFlow® bioseal conditioned medium maintained high metabolic activities, except at higher concentrations. Likewise, viability was maintained, but a significant decrease was observed after exposure to the highest concentration (p < 0.001), associated with cell death by late apoptosis and necrosis. When cell cultures were exposed to AH26®, metabolic activity was highly compromised, resulting in cell death. An imbalance in the production of peroxides and superoxide anion was observed. GuttaFlow® bioseal showed higher biocompatibility than AH26®.

Assessment of antifouling efficacy of polyhedral oligomeric silsesquioxane based poly (urea-urethane-imide) hybrid membranes.

UNLABELLED: A series of polyhedral oligomeric silsesquioxane (POSS) based poly(urea-urethane-imide) (PUUI-POSS) membranes were synthesized by varying the proportions of imide using 2,4-toluene diisocyanate (TDI) and bis(aminopropyl) terminated polydimethylsiloxane (PDMS). The molecular structures of poly(urea-urethane-imide)s were characterized by Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic technique. Incorporation of imide domain and its influence on surface roughness was investigated by atomic force microscopy (AFM). Hydrophobicity of polymeric membrane surfaces was determined by contact angle measurement. The thermal properties of the polymers were studied by thermogravimetric analysis. The antimicrobial activities and inhibition of bacterial attachment of these polymeric membranes were studied on Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) by the disc-diffusion method. SIGNIFICANCE AND IMPACT OF THE STUDY: The antifouling performance has been evaluated for the polymeric membranes against two bacteria (Staphylococcus aureus (ATCC 6538)) (Escherichia coli (ATCC 8739)). The polymeric membranes were incorporated with imide moiety to improve thermal stability of the polymeric materials. The synthesized polymeric membranes have shown good morphological properties for better antifouling activities. This study found that these membranes are capable of preventing micro-organisms besides offering excellent bio-fouling resistance.

Biological implications of polymeric microdevices for live cell assays.

Lab-on-a-chip technologies have the potential to deliver significant technological advances in modern biomedicine, through the ability to provide appropriate low-cost microenvironments for screening cells. However, to date, few studies have investigated the suitability of poly(dimethylsiloxane) (PDMS) for live cell culture. Here, we describe an inexpensive method for production of reusable, optical-grade PDMS microculture chips which provide a static and self-contained microwell system analogous to conventional polystyrene multiwell plates. We use these structures to probe the effects of PDMS upon live cell culture bioassays, using time-lapse fluorescence imaging to explore the toxicity of the substrate. We use three model systems to explore the efficacy of the microstructured devices: (i) live cell culture, (ii) adenoviral gene delivery to mammalian cells, and (iii) gravity enforced formation of multicellular tumor spheroids (MCTS). Results show that PDMS is nontoxic to cells, as their viability and growth characteristic in PDMS-based platforms is comparable to that of their polystyrene counterparts.

Assessment of viability and proliferation of in vivo silicone-primed lymphocytes after in vitro re-exposure to silicone.

The functional response of peripheral blood lymphocytes isolated from 22 patients with silicone gel-filled breast implants was assessed after in vitro re-exposure to silicone. Using cell culture test methods to quantify proliferation and viability and/or activation of lymphocyte microcultures, i.e., the uptake of tritiated thymidine (3H-TdR uptake test) and the reduction of formazan salts (MTT assay), interesting data were obtained. Peripheral blood lymphocytes purified from patients wearing silicone gel-filled breast implants react in vitro to silicone showing a statistically significant increase of both proliferation and viability, while healthy subjects do not respond on in vitro exposure to silicone. Differences resulted even more statistically significant when patients were divided into two groups depending on the type of surgery they underwent: patients with breast augmentation for aesthetic reasons seem to have an increased responsiveness in vitro to silicone compared to patients who experienced a reconstructive surgery of the breast. Although they are still preliminary, being referred to a limited population, these results suggest that the lymphocytes of patients with silicone gel-filled breast implants could be sensitized in vivo toward silicone; the re-exposure of these cells to silicone leads to a higher functional response which could be looked for by using quantitative in vitro test methods.

The use of radiotelemetry techniques for the in vivo assessment of antacids.

The use of the pH radio pill for the assessment of antacid effect has been evaluated. The correlation between the pH pill and a conventional pH electrode system was high (r2 = 0.997). The records of in vivo pH against time allow unique measurements of antacid efficacy (defined as the integrated area under the pH against time curve) and duration of action to be obtained. Using a free pill, antacid assessments were performed in 13 subjects in the left lateral position. The effects of repeated antacid administrations were studied in five subjects using a pH pill which was prevented from leaving the stomach by tethering it to the teeth. These longer studies were performed in the sitting position. The efficacy (p less than 0.02) and duration of action (p less than 0.02) of sodium citrate 0.3 M were less in those subjects in whom the pill was tethered. The differences between the results of the studies using either a free or a tethered pill can be attributed to posture. The overall duration of action of sodium citrate 15 ml was short, the mean (SEM) value being 42.4 (4.5) minutes in the left lateral position and 21.2 (4.0) minutes in the upright position.