Human corneal epithelium coexists with tear fluids and shows its barrier functionality under the dynamic conditions of eye blinking. However, the current in vitro cell culture settings for corneal epithelial cells lack the dynamic flow conditions to recapitulate the shear stress of eye blinking, hindering corneal function evaluation. We developed a microfluidic platform enabling the dynamic culture of the human corneal barrier with recapitulation of eye blinking. The device consisted of upper and lower channels separated by a porous membrane. Human corneal epithelial cells (HCE-T) were seeded on the porous membrane (upper channel) and cultured for ten days. The cells formed a barrier with high expression of zonula occludens 1 (ZO-1) tight junction protein on day seven, and the translocation of fluorescein sodium across the barrier in the microfluidic device was comparable to that in the transwell system, used as a control. Then, bidirectional and unidirectional flows were applied in the upper and lower channels, respectively, and the cells in the upper channels were stimulated with 0.6 dyn s cm⁻² shear stress. After 24 h, while the fluid stimuli did not affect cell adhesion, they facilitated the expression of cytokeratin 19 (CK-19) intermediate filaments in cells, indicating the strengthening of the barrier function. Furthermore, morphological single-cell analysis revealed an increase in the cell body area rather than nuclei. We envision that this multi-corneal barrier-on-a-chip device will unlock new possibilities in ophthalmic drug development and will be useful for studying the effects of eye blinking shear stress on the ocular surface.

In this study, a novel phospholipid-based microbubble formulation containing doxorubicin and perfluoropropane gas (DLMB) was developed. The DLMBs were prepared by mechanical agitation of a phospholipid dispersion in the presence of perfluoropropane (PFP) gas. An anionic phospholipid, distearoyl phosphatidylglycerol (DSPG) was selected to load doxorubicin in the microbubbles by means of electrostatic interaction. The particle size, zeta potential, echogenicity and stability of the DLMBs were measured. Drug loading was ⩾ 92%. The potential of the DLMBs for use as a theranostic modality was evaluated in tumor bearing mice. Gas chromatography analysis of PFP showed significant enhancement of PFP retention when doxorubicin was used at concentrations of 10-82% equivalent to DSPG. The inhibitory effects on the proliferation of B16BL6 melanoma murine cells in vitro were enhanced using a combination of ultrasound (US) irradiation and DLMBs. Moreover, in vivo DLMBs in combination with (US) irradiation significantly inhibited the growth of B16BL6 melanoma tumor in mice. Additionally, US echo imaging showed high contrast enhancement of the DLMBs in the tumor vasculature. These results suggest that DLMBs could serve as US triggered carriers of doxorubicin as well as tumor imaging agents in cancer therapy.

In this study, stable nano-sized bubbles (nanobubbles [NBs]) were produced using the mechanical agitation method in the presence of perfluorocarbon gases. NBs made with perfluoropropane had a smaller size (around 400 nm) compared to that of those made with perfluorobutane or nitrogen gas. The lipid concentration in NBs affected both their initial size and post-formulation stability. NBs formed with a final lipid concentration of 0.5 mg/ml tended to be more stable, having a uniform size distribution for 24 h at room temperature and 50 h at 4 °C. In vitro gene expression revealed that NBs/pDNA in combination with ultrasound (US) irradiation had significantly higher transfection efficacy in colon C26 cells. Moreover, for in vivo gene transfection in mice left limb muscles, there was notable local transfection activity by NBs/pDNA when combined with US irradiation. In addition, the aged NBs kept at room temperature or 4 °C were still functional at enhancing gene transfection in mice. We succeeded in preparing stable NBs for efficient in vivo gene transfection, using the mechanical agitation method.

Human skeletal muscles are characterized by a unique aligned microstructure of myotubes which is important for their function as well as for their homeostasis. Thus, the recapitulation of the aligned microstructure of skeletal muscles is crucial for the construction of an advanced biomimetic model aimed at drug development applications. Here, we have developed a 3D printed micropatterned microfluid device (3D-PMMD) through the employment of a fused deposition modeling (FDM)-based 3D printer and clear filaments made of biocompatible polyethylene terephthalate glycol (PETG). We could fabricate micropatterns through the adjustment of the printing deposition heights of PETG filaments, leading to the generation of aligned half-cylinder-shaped micropatterns in a dimension range from 100 µm to 400 µm in width and from 60 µm to 150 µm in height, respectively. Moreover, we could grow and expand C2C12 mouse myoblast cells on 3D-PMMD where cells could differentiate into aligned bundles of myotubes with respect to the dimension of each micropattern. Furthermore, our platform was applicable with the electrical pulses stimulus (EPS) modality where we noticed an improvement in myotubes maturation under the EPS conditions, indicating the potential use of the 3D-PMMD for biological experiments as well as for myogenic drug development applications in the future.

Dry eye syndrome (DES) is a complex ocular condition characterized by an unstable tear film and inadequate tear production, leading to tissue damage. Despite its common occurrence, there is currently no comprehensive in vitro model that accurately reproduce the cellular characteristics of DES. Here we modified a corneal epithelium-on-a-chip (CEpOC) model to recapitulate DES by subjecting HCE-T human corneal epithelial cells to an air–liquid (AL) interface stimulus. We then assessed the effects of AL stimulation both in the presence and absence of diclofenac (DCF), non-steroidal anti-inflammatory drug. Transcriptomic analysis revealed distinct gene expression changes in response to AL and AL_DCF, affecting pathways related to development, epithelial structure, inflammation, and extracellular matrix remodeling. Both treatments upregulated PIEZO2, linked to corneal damage signaling, while downregulating OCLN, involved in cell–cell junctions. They increased the expression of inflammatory genes (e.g., IL-6) and reduced mucin production genes (e.g., MUC16), reflecting dry eye characteristics. Metabolomic analysis showed increased secretion of metabolites associated with cell damage and inflammation (e.g., methyl-2-oxovaleric acid, 3-methyl-2-oxobutanoic acid, lauroyl-carnitine) in response to AL and even more with AL_DCF, indicating a shift in cellular metabolism. This study showcases the potential use of AL stimulus within the CEpOC to induce cellular characteristics relevant to DES.