Sukki, L. | Search bioRxiv

bioRxiv2025

Biomimetic hydrogel platform reveals active force transduction from retinal pigment epithelium to photoreceptors

In the eye, the retinal pigment epithelium (RPE) maintains the functionality and welfare of retinal photoreceptors and forms a tight, interlocked structure with photoreceptor outer segments (POSs). The RPE-retina interaction is difficult to recapitulate in vitro, limiting the studies addressing the retinal maintenance functions of the RPE. To overcome this challenge, we constructed a retina-mimicking structure using a soft polyacrylamide hydrogel coated with Matrigel. This structure was introduced to RPE cells apical side to model the RPE-retina interface in vitro. As a result, RPE cells attached to the hydrogels during culture, enabling further studies of cell adhesion and force transduction between the RPE-hydrogel with rheology and traction force microscopy. These methods were applied to a critical interactive process between the retina and the RPE: phagocytosis of the aged tips of POSs enabling their renewal. During phagocytosis, RPE cells imposed considerable traction forces to the POS particles. The force generation was actin-dependent, and the forces were significantly reduced by the disruption of RPEs actin cytoskeleton. These results add another layer to the diverse interaction mechanisms between the RPE and the neural retina and pave the way for further studies of the RPE-retina interplay.

biophysics↗

bioRxiv2025

Human iPSC-based coculture model reveals neuroinflammatory crosstalk between microglia and astrocytes

BackgroundMicroglia and astrocytes have been implicated as central mediators of neuroinflammatory processes in several neurodegenerative diseases. However, their intricate crosstalk and contributions to pathogenesis remain elusive, highlighting the need for innovative in vitro approaches for investigating glial interactions in neuroinflammation. The aim of this study was to develop advanced human-based glial coculture models to explore the inflammatory roles and interactions of microglia and astrocytes in vitro. MethodsWe utilized human induced pluripotent stem cell (iPSC)-derived microglia and astrocytes cultured both in conventional culture dishes and in a compartmentalized microfluidic chip coculture platform. This novel platform features separate compartments for both cell types, enabling the creation of fluidically isolated microenvironments with spontaneous migration of microglia toward astrocytes through interconnecting microtunnels. To induce inflammatory activation, glial cultures were stimulated with lipopolysaccharide (LPS), a combination of tumor necrosis factor- (TNF-) and interleukin-1{beta} (IL-1{beta}), or interferon-{gamma} (IFN-{gamma}) for 24 hours. The glial activation and crosstalk were analyzed with immunocytochemistry, the secretion of inflammatory factors from the culture media was measured, and microglial migration was quantified. ResultsMicroglia-astrocyte cocultures were successfully generated in both conventional cultures and the microfluidic chip platform. Inflammatory stimulation with LPS and TNF-/IL-1{beta} elicited cell type-specific responses in microglia and astrocytes, respectively. Notably, the levels of secreted inflammatory mediators were altered under coculture conditions, revealing significant glial crosstalk. Utilization of our microfluidic coculture platform facilitated the study of microglial migration and glial activation within distinct inflammatory microenvironments. Microglia migrated efficiently toward the astrocyte compartment, and the chemoattractant adenosine diphosphate (ADP) notably increased microglial migration within this platform. Furthermore, inflammatory stimulation of the microfluidic chip cocultures successfully recapitulated glial crosstalk, revealing unique responses. This crosstalk was associated with elevated levels of complement component C3 in the cocultures, emphasizing the intricate interplay between microglia and astrocytes under inflammatory conditions. ConclusionsOur results depict an elaborate molecular crosstalk between inflammatory microglia and astrocytes, providing evidence of how glial cells orchestrate responses during neuroinflammation. Importantly, we demonstrate that the microfluidic coculture platform developed in this study for microglia and astrocytes provides a more functional and enhanced setup for investigating inflammatory glial interactions in vitro.

neuroscience↗