Compartmentalized microfluidic device for in vitro co-culture of retinal cells.

The investigation is focused on the development of a compartmentalized microfluidic device for coculturing the cells of crucial retinal cellular layers and assessing cell-to-cell interactions. A perfusion-based microfluidic co-culture device was employed and computationally validated for determining the pressure drop and fluid flow rate within the device microchannels. Fabrication was performed using PDMS polymer and coating of fibronectin and collagen facilitated adherence of the cells over the glass surface. Microfluidic device successfully supported cell proliferation, under continuous perfusion of 1 µl min-1 flow rate. The barrier integrity of this coculture was confirmed by evaluating the permeability of fluorescently labeled molecules. The coculture expressed characteristic phenotypic protein markers like recoverin, PAX6, for retinal precursor cells, and RPE65 for retinal epithelial cells. The coculture also exhibited basal expression of TNF-α under normal conditions. Differentiated photoreceptor cells positively expressed rhod inherently possess sensitivity toward violet/blue light, which was validated in R28 cells by exposure to light having a wavelength of 405 nm, which significantly decreased cell viability via increased TNF-α production and reduced rhodopsin expression. This proof-of-concept investigation proved the functionality of the retinal coculture, which may be used as an appropriate perfusion-based, preclinical tool for the evaluation of novel retinal drugs and delivery systems.

Three-dimensional spheroids of choroid-retinal vascular endothelial cells as an in-vitro model for diabetic retinopathy: Proof-of-concept investigation.

Diabetic retinopathy (DR) is a primary microvascular complication of diabetes mellitus and a vision-threatening condition. Vascular endothelial growth factor (VEGF) induces neovascularization and causes metabolic damage to the retinal and choroidal vasculature in diabetic patients. Existing drug screening models and treatment strategies for DR need to be refined through the establishment of relevant pre-clinical models, which may enable development of effective and safe therapies. The present study discusses the development of an in-vitro three-dimensional (3D) spheroid model, using RF/6A choroid-retinal vascular endothelial cells, to closely mimic the in-vivo disease condition. Compact, reproducibly-sized, viable and proliferating RF/6A spheroids were fabricated, as confirmed by microscopy, live/dead assay, cell proliferation assay and histological staining. In-vitro angiogenesis was studied by evaluating individual effects of VEGF and an anti-VEGF monoclonal antibody, Bevacizumab, and their combination on cellular proliferation and 3D endothelial sprout formation. VEGF stimulated angiogenic sprouting while Bevacizumab demonstrated a dose-dependent anti-angiogenic effect, as determined from the cellular proliferation observed and extent and length of sprouting. These investigations validated the potential of RF/6A spheroids in providing an alternative-to-animal, pathophysiologically-relevant model to facilitate pre-clinical and biomedical research related to DR.

Macromolecular cryoprotectants for the preservation of mammalian cell culture: lessons from crowding, overview and perspectives.

Cryopreservation is a process used for the storage of mammalian cells at a very low temperature, in a state of 'suspended animation.' Highly effective and safe macromolecular cryoprotectants (CPAs) have gained significant attention as they obviate the toxicity of conventional CPAs like dimethyl sulfoxide (DMSO) and reduce the risks involved in the storage of cultures at liquid nitrogen temperatures. These agents provide cryoprotection through multiple mechanisms, involving extracellular and intracellular macromolecular crowding, thereby impacting the biophysical and biochemical dynamics of the freezing medium and the cryopreserved cells. These CPAs vary in their structures and physicochemical properties, which influence their cryoprotective activities. Moreover, the introduction of polymeric crowders in the cryopreservation media enables serum-free storage at low-DMSO concentrations and high-temperature vitrification of frozen cultures (-80 °C). This review highlights the need for macromolecular CPAs and describes their mechanisms of cryopreservation, by elucidating the role of crowding effects. It also classifies the macromolecules based on their chemistry and their structure-activity relationships. Furthermore, this article provides perspectives on the factors that may influence the outcomes of the cell freezing process or may help in designing and evaluating prospective macromolecules. This manuscript also includes case studies about cellular investigations that have been conducted to demonstrate the cryoprotective potential of macromolecular CPAs. Ultimately, this review provides essential directives that will further improve the cell cryopreservation process and may encourage the use of macromolecular CPAs to fortify basic, applied, and translational research.