Liquid–liquid interface (LLI) culture provides an alternative platform for the growth and organization of anchorage-dependent cells. Although fluorinated oils are widely used to generate LLIs, their repeated reuse for epithelial tissue engineering has not been sufficiently investigated. In this study, we evaluated whether the fluorinated oil HFE-7500 retains its ability to support epithelial cell culture after repeated recovery and reuse. HFE-7500 was recovered by filtration and reused for up to 20 cycles. The effects of repeated reuse on Madin–Darby canine kidney (MDCK) cell proliferation, lactate dehydrogenase release, epithelial organization, and microdroplet-based culture were evaluated. MDCK cells cultured at LLIs prepared with reused HFE-7500 maintained proliferative activity without a detectable increase in cytotoxicity. Quantitative analysis of nuclear distribution and ZO-2 localization revealed no significant differences in epithelial organization or maturation between LLIs prepared with fresh and reused HFE-7500. In addition, microdroplet interfaces generated using reused HFE-7500 supported continuous fibronectin organization and apical ZO-2 localization. These findings indicate that HFE-7500 retains the biological functionality required to support epithelial cell culture after up to 20 reuse cycles under the conditions examined. Repeated recovery and reuse of HFE-7500 may provide a resource-efficient approach to LLI culture and microdroplet-based epithelial tissue engineering.
Myocardial ischemia-reperfusion injury (IRI) remains a major cause of cardiomyocyte loss despite timely restoration of blood flow. Biomaterials capable of locally supplying oxygen represent a promising strategy to mitigate ischemic damage while limiting the detrimental effects of reperfusion. Here, isolated spinach thylakoid membranes are investigated as a cell-free, light-responsive biomaterial for oxygen generation in a human in vitro model of oxygen-glucose deprivation/reperfusion (OGD/R). Across three independent thylakoid preparations, illuminated membranes sustained oxygen evolution for more than 1.5 h at 25 °C, with an initial rate of approximately 60 nmol O₂ × mL−1 × min−1 at 100 μg chlorophyll × mL−1 and cumulative oxygen production of about 800 nmol O₂ × mL−1. Because photosynthetic activity rapidly declined at 37 °C, a spatially separated platform was developed in which thylakoids and AC16 human cardiomyocytes were maintained in independent compartments connected through a shared gaseous phase, enabling remote oxygen delivery during ischemia. Photosynthetic oxygen supplementation markedly attenuated reperfusion-induced apoptosis, reducing the fraction of cleaved caspase-3-positive cells from 71.52 ± 12.37% in untreated OGD/R cultures to 15.79 ± 3.94%, the latter comparable to normoxic controls. These findings demonstrate that isolated thylakoid membranes preserve the essential functionality of oxygenic photosynthesis, providing a simple, cell-free platform for oxygen supplementation. Although improved thermal stability will be required for clinical translation, this work establishes a proof of concept that a plant-derived platform can modulate IRI, presenting thylakoid membranes as biomaterials for light-triggered oxygen delivery.
