Determination of the Role of the Distal Outflow Pathway Tissue in Glucocorticoid-induced Ocular Hypertension

Introduction Prolonged application of glucocorticoids (GCs) induces ocular hypertension (OHT) and glaucoma. This increased intraocular pressure (IOP) is due to pathological changes in the trabecular meshwork (TM) outflow pathway tissues including impaired cell functions and extracellular matrix deposition. The changes and role of the TM in GC-induced OHT have been well studied. However, the role of the tissues distal to the TM (distal outflow tissues) is unclear. This study aims to further uncover the role of distal outflow tissue in GC-induced OHT using a novel perfusion organ culture (POC) model. Methods Huma corneal rims tissues were attached to 3D printed transparent perfusion plates using a combination of thin and thick glues. The artificial anterior chamber was perfused with DMEM-low glucose medium at 2ul/min to mimic aqueous humor production, and IOP was recorded using pressure transducers and a computerized system. To determine the role of distal tissue in GC-induced IOP changes, the TM tissue was carefully removed from both eyes, and one eye was treated with ethanol (EtOH) and the fellow eye with dexamethasone (DEX). Results The model was validated through a comparison of the IOP and TM stiffness of glue contaminated to non-contaminated corneal rims. The glue contaminated rim showed highly increased IOP and TM stiffness while the non-contaminated rim showed normal values. After validation, the TM was removed from paired corneal rims. One rim was treated with 100nM DEX and the fellow rim with 0.1% EtOH. The DEX treated rim showed increase in IOP while the EtOH control showed little change. Conclusion We created a novel corneal rim perfusion culture model for the study of GC-induced OHT. This model showed promising results of distal outflow involvement in glucocorticoid induced ocular hypertension. Further studies are needed to elucidate the role of distal outflow tissues in GC responsiveness in the eye.

Bonding of Flexible Membranes for Perfusable Vascularized Networks Patch

BACKGROUND: In vitro generation of three-dimensional vessel network is crucial to investigate and possibly improve vascularization after implantation in vivo. This work has the purpose of engineering complex tissue regeneration of a vascular network including multiple cell-type, an extracellular matrix, and perfusability for clinical application. METHODS: The two electrospun membranes bonded with the vascular network shape are cultured with endothelial cells and medium flow through the engineered vascular network. The flexible membranes are bonded by amine-epoxy reaction and examined the perfusability with fluorescent beads. Also, the perfusion culture for 7 days of the endothelial cells is compared with static culture on the engineered vascular network membrane. RESULTS: The engineered membranes are showed perfusability through the vascular network, and the perfused network resulted in more cell proliferation and variation of the shear stress-related genes expression compared to the static culture. Also, for the generation of the complex vascularized network, pericytes are co-cultured with the engineered vascular network, which results in the Collagen I is expressed on the outer surface of the engineered structure. CONCLUSION: This study is showing the perfusable in vitro engineered vascular network with electrospun membrane. In further, the 3D vascularized network module can be expected as a platform for drug screening and regenerative medicine.

Development of a Microfluidic Device to Form a Long Chemical Gradient in a Tissue from Both Ends with an Analysis of Its Appearance and Content

Tissue assays have improved our understanding of cancers in terms of the three-dimensional structures and cellular diversity of the tissue, although they are not yet well-developed. Perfusion culture and active chemical gradient formation in centimeter order are difficult in tissue assays, but they are important for simulating the metabolic functions of tissues. Using microfluidic technology, we developed an H-shaped channel device that could form a long concentration gradient of molecules in a tissue that we could then analyze based on its appearance and content. For demonstration, a cylindrical pork tissue specimen was punched and equipped in the H-shaped channel device, and both ends of the tissue were exposed to flowing distilled and blue-dyed water for 100 h. After perfusion, the tissue was removed from the H-shaped channel device and sectioned. The gradient of the blue intensity along the longitudinal direction of the tissue was measured based on its appearance and content. We confirmed that the measured gradients from the appearance and content were comparable.

Liver ductal organoids reconstruct intrahepatic biliary trees in decellularized liver grafts.

Three-dimensional scaffolds decellularized from native organs are a promising technique to establish engineered liver grafts and overcome the current shortage of donor organs. However, limited sources of bile duct cells and inappropriate cell distribution in bioengineered liver grafts have hindered their practical application. Organoid technology is anticipated to be an excellent tool for the advancement of regenerative medicine. In the present study, we reconstructed intrahepatic bile ducts in a rat decellularized liver graft by recellularization with liver ductal organoids. Using an ex vivo perfusion culture system, we demonstrated the biliary characteristics of repopulated mouse liver organoids, which maintained bile duct markers and reconstructed biliary tree-like networks with luminal structures. We also established a method for the co-recellularization with engineered bile ducts and primary hepatocytes, revealing the appropriate cell distribution to mimic the native liver. We then utilized this model in human organoids to demonstrate the reconstructed bile ducts. Our results show that liver ductal organoids are a potential cell source for bile ducts from bioengineered liver grafts using three-dimensional scaffolds.

Gravity-Based Flow Efficient Perfusion Culture System for Spheroids Mimicking Liver Inflammation

The spheroid culture system provides an efficient method to emulate organ-specific pathophysiology, overcoming the traditional two-dimensional (2D) cell culture limitations. The intervention of microfluidics in the spheroid culture platform has the potential to enhance the capacity of in vitro microphysiological tissues for disease modeling. Conventionally, spheroid culture is carried out in static conditions, making the media nutrient-deficient around the spheroid periphery. The current approach tries to enhance the capacity of the spheroid culture platform by integrating the perfusion channel for dynamic culture conditions. A pro-inflammatory hepatic model was emulated using a coculture of HepG2 cell line, fibroblasts, and endothelial cells for validating the spheroid culture plate with a perfusable channel across the spheroid well. Enhanced proliferation and metabolic capacity of the microphysiological model were observed and further validated by metabolic assays. A comparative analysis of static and dynamic conditions validated the advantage of spheroid culture with dynamic media flow. Hepatic spheroids were found to have improved proliferation in dynamic flow conditions as compared to the static culture platform. The perfusable culture system for spheroids is more physiologically relevant as compared to the static spheroid culture system for disease and drug analysis.

Development of a Hybrid Polymer-Based Microfluidic Platform for Culturing Hepatocytes towards Liver-on-a-Chip Applications

The drug development process can greatly benefit from liver-on-a-chip platforms aiming to recapitulate the physiology, mechanisms, and functionalities of liver cells in an in vitro environment. The liver is the most important organ in drug metabolism investigation. Here, we report the development of a hybrid cyclic olefin copolymer (COC) and polydimethylsiloxane (PDMS) microfluidic (HCP) platform to culture a Huh7 hepatoma cell line in dynamic conditions towards the development of a liver-on-a-chip system. The microfluidic platform is comprised of a COC bottom layer with a microchannel and PDMS-based flat top layer sandwiched together. The HCP device was applied for culturing Huh7 cells grown on a collagen-coated microchannel. A computational fluid dynamics modeling study was conducted for the HCP device design revealing the presence of air volume fraction in the chamber and methods for optimizing experimental handling of the device. The functionality and metabolic activity of perfusion culture were assessed by the secretion rates of albumin, urea, and cell viability visualization. The HCP device hepatic culture remained functional and intact for 24 h, as assessed by resulting levels of biomarkers similar to published studies on other in vitro and 2D cell models. The present results provide a proof-of-concept demonstration of the hybrid COC–PDMS microfluidic chip for successfully culturing a Huh7 hepatoma cell line, thus paving the path towards developing a liver-on-a-chip platform.

Engineering a 3D Vascularized Adipose Tissue Construct Using a Decellularized Lung Matrix

Critically sized defects in subcutaneous white adipose tissue result in extensive disfigurement and dysfunction and remain a reconstructive challenge for surgeons; as larger defect sizes are correlated with higher rates of complications and failure due to insufficient vascularization following implantation. Our study demonstrates, for the first time, a method to engineer perfusable, pre-vascularized, high-density adipose grafts that combine patient-derived adipose cells with a decellularized lung matrix (DLM). The lung is one of the most vascularized organs with high flow, low resistance, and a large blood–alveolar interface separated by a thin basement membrane. For our work, the large volume capacity within the alveolar compartment was repurposed for high-density adipose cell filling, while the acellular vascular bed provided efficient graft perfusion throughout. Both adipocytes and hASCs were successfully delivered and remained in the alveolar space even after weeks of culture. While adipose-derived cells maintained their morphology and functionality in both static and perfusion DLM cultures, perfusion culture offered enhanced outcomes over static culture. Furthermore, we demonstrate that endothelial cells seamlessly integrate into the acellular vascular tree of the DLM with adipocytes. These results support that the DLM is a unique platform for creating vascularized adipose tissue grafts for large defect filling.

The effect of macropore size of hydroxyapatite scaffold on the osteogenic differentiation of bone mesenchymal stem cells under perfusion culture

Previous studies have proved that dynamic culture could facilitate nutrients transport and apply mechanical stimulation to the cells within three-dimensional scaffolds, thus enhancing the differentiation of stem cells towards the osteogenic phenotype. However, the effects of macropore size on osteogenic differentiation of stem cells under dynamic condition are still unclear. Therefore, the objective of this study was to investigate the effects of macropore size of hydroxyapatite (HAp) scaffolds on osteogenic differentiation of bone mesenchymal stem cells under static and perfusion culture conditions. In vitro cell culture results showed that cell proliferation, alkaline phosphate (ALP) activity, mRNA expression of ALP, collagen-I (Col-I), osteocalcin (OCN) and osteopontin (OPN) were enhanced when cultured under perfusion condition in comparison to static culture. Under perfusion culture condition, the ALP activity and the gene expression of ALP, Col-I, OCN and OPN were enhanced with the macropore size decreasing from 1300 to 800 µm. However, with the further decrease in macropore size from 800 to 500 µm, the osteogenic related gene expression and protein secretion were reduced. Computational fluid dynamics analysis showed that the distribution areas of medium- and high-speed flow increased with the decrease in macropore size, accompanied by the increase of the fluid shear stress within the scaffolds. These results confirm the effects of macropore size on fluid flow stimuli and cell differentiation, and also help optimize the macropore size of HAp scaffolds for bone tissue engineering.