Genes & Cells

BACKGROUND: The assessment of inhalation toxicity of natural and artificial aerosols and the efficacy of aerosolized drugs is an important practical task. However, in numerous in vitro studies, the test substance is dissolved in a liquid that completely covers the lung tissue cells. For inhalation therapy, this method substantially differs from the physiological scenario of aerosol interaction with the pulmonary epithelium.

AIM: The aim is to develop a platform to study the interaction of inhaled aerosol particles with the pulmonary epithelium on the internal surface of the lung alveoli at the liquid–air interface capable of simulating scenarios of periodic variation of liquid levels during breathing.

METHODS: The A549 human adenocarcinoma cell line was used as a model culture of pulmonary epithelial cells. Cell viability was analyzed using fluorescent microscopy under various exposure conditions at the liquid–air interface and during the deposition of different aerosol particles.

RESULTS: A device has been designed, built, and tested to simulate the internal surface of pulmonary alveoli. It consists of a cell layer located at the air–liquid interface. The cells are cultured on a porous polymer membrane on the surface of a reservoir with culture medium. The hydration level of the cells is controlled by altering the liquid pressure beneath the membrane and recorded by an optical sensor that measures the scattering of laser beam reflected from the surface of the cell layer. The membrane with the cell layer is placed in a chamber allowing to create a directed electric field normal to the aerosol flow passing over the cell layer, which is connected to one electrode. The electric field significantly accelerates the delivery of aerosol particles with an electric charge onto the cell surface. We determined the parameters of static exposure of the model cell monolayer at the air–liquid interface, allowing to maintain high cell viability. The study showed that cyclic variation of cell hydration simulating the respiratory cycle is effective to maintain cell viability for an extended period (60 min). The study showed that doxorubicin nanoaerosol is effective when deposited on the surface of human adenocarcinoma tumor cells located at the liquid–air phase boundary. A model aerosol of a non-toxic substance (glucose) does not have toxic effects under similar conditions.

CONCLUSION: The proposed lung-on-a-chip model is a comprehensive platform to study the inhalation toxicity of natural and artificial aerosols and test the safety and efficacy of aerosolized drugs in situ.

BACKGROUND: The development of a conjunctival equivalent capable of repairing tissue defects and preventing pathological scarring is a critical goal in biomaterial design for reconstructive procedures, in which collagen is frequently employed. Evaluating the biocompatibility and biomechanical properties of such materials supports the advancement of effective surgical approaches for conjunctival reconstruction.

AIM: The work aimed to evaluate the biocompatibility of human conjunctival epithelial cells (hCECs) with a collagen membrane (CM) developed using an original technique in vitro; to confirm its noncytotoxicity toward hCECs; to characterize hCEC morphology and expression of specific proteins on CM; and to compare the biomechanical properties of CM with native conjunctiva.

METHODS: A primary culture of hCECs was obtained using the explant method and verified through staining for the markers cytokeratin 7 and mucin 5AC. Two CM formulations (collagen concentration 10 mg/mL and 30 mg/mL) were tested. Cytotoxicity of the 30 mg/mL CM was evaluated by seeding hCECs at 50,000 cells/cm² followed by MTS assay and viability testing with calcein-AM and Hoechst staining. hCECs morphology was analyzed using phase-contrast images of the cell culture after seeding on CM and immunocytochemical staining for cytokeratin 7 and mucin 5AC. Biomechanical properties of the 30 mg/mL CM and conjunctiva were assessed using static and dynamic tests on an indentation machine.

RESULTS: Phase-contrast microscopy revealed that within 1 week after seeding, hCECs migrated into the inner layers of the 10 mg/mL CM, whereas on the 30 mg/mL CM, the cells formed a confluent monolayer on its surface. The metabolic activity assay revealed no significant difference between the control groups and 30 mg/mL CM. In the live/dead assay, 95% of cells in both groups stained positive with calcein. The immunocytochemical profile of hCECs remained unchanged: as in the control group, cells cultured on the CM expressed conjunctiva-specific cytokeratin 7 and mucin 5AC. The Young’s modulus values for the CM and bulbar conjunctiva were comparable: 0.0008739 ± 0.0004332 GPa and 0.0009472 ± 0.001323 GPa, respectively (p = 0.0549). The hardness of the CM was significantly higher than that of the conjunctiva (p < 0.0001), while its viscosity was significantly lower compared to native tissue (p < 0.0001).

CONCLUSION: hCECs remained viable after seeding on the tested CM, confirming its noncytotoxicity. The migration of hCECs into the matrix indicates the material’s ability to create a favorable microenvironment for conjunctival epithelialization and to undergo resorption following cell colonization. Preservation of the molecular profile of hCECs indicates high biocompatibility of the CM with conjunctival epithelium. The 30 mg/mL CM exhibits elasticity comparable to that of conjunctiva, with higher hardness and lower viscosity. Optimization of CM biomechanical properties for specific clinical needs holds promise.

BACKGROUND: Mesenchymal stem cells of the skin and subcutaneous adipose tissue play a critical role in epithelial regeneration by proliferating and differentiating into skin cells to replace damaged or dead tissue. In addition, they act via autocrine and paracrine signaling to promote tissue repair and wound healing.

AIM: The work aimed to investigate the in vitro effects of serotonin on the regenerative potential—namely, proliferation, migration, and cell death—of dermal fibroblasts (DFs) and subcutaneous adipose tissue-derived mesenchymal stromal cells (SAT-MSCs).

METHODS: Primary DF and SAT-MSC cultures were obtained from Wistar rats and divided into the following groups: DF/SAT-MSCs cultured in standard medium and DF/SAT-MSCs cultured with serotonin supplementation. Cell morphology and proliferation were assessed microscopically using an Axio Vert.A1 microscope (Carl Zeiss, Germany). Cell migration dynamics were studied in a scratch assay. Cell death resulting from apoptosis and/or necrosis was evaluated using fluorescence microscopy with Annexin V-FITC/PI staining (ServiceBio, China).

RESULTS: Both DFs and SAT-MSCs responded to serotonin supplementation in standard culture medium with increased proliferation. Quantification of migrated cells revealed an increase in the conditional migration speed of SAT-MSCs under conditions of serotonin supplementation. Additionally, serotonin-treated cultures demonstrated reduced levels of apoptotic and necrotic cells.

CONCLUSION: Activation of serotonin signaling mechanisms plays an important role in wound healing following various skin and subcutaneous tissue injuries by enhancing cell viability, DF proliferation, and both proliferation and migration of SAT-MSCs, as demonstrated in our study. These findings suggest that serotonin or 5-hydroxytryptamine receptor agonists may serve as promising candidates for promoting skin repair in patients with injuries. Importantly, the cellular response to serotonin signaling is tissue-specific and depends on the receptor subtype and intracellular signaling pathways mediating secondary messenger activation.