Matrigel

Endothelial colony forming cells (ECFCs) cultured as a cell monolayer were treated overnight with apical and basal low‐risk and high‐risk RPE EVs. EVs were derived from cell conditioned media from 200,000 RPE cells (per replicate) to model 1:1 ratio of exposure RPE:ECFCs. Additionally, to assess the functional effect of EVs, dose‐response experiments were carried out at different ratios of RPE EVs – ECFCs (i.e., 1:4 and 1:2). Exosome depleted media was used to harvest EVs secreted by RPE cells over 72 h and EVs were purified using size exclusion chromatography as described in Section 2.2. After treatment, ECFCs were detached and counted. The angiogenesis μ‐Slide (Ibidi) was used to assess 3D tube formation capacity. An ECFC suspension was mixed in a 2:3 ratio with Matrigel (Corning) and dispensed into slide to form a 10 μl Matrigel droplet containing 15,000 cells (final density of 1.5 million ECFCs per ml). angiogenesis μ‐Slides were left in the incubator for 30 min to enable Matrigel polymerisation, and then covered with 50 μl of EGM‐2 media (Lonza). Tube‐like structures were assessed after 48 h, using the EVOS Cell Imaging system (Thermo Fisher). Analysis of the tube area was performed using ImageJ.

GLUTag cells were plated onto 24-well plates precoated with Matrigel (Lonza, O Porriño, Spain) at a density of 200.000 cells/mL 24 h before the secretion study. Cells were washed twice with PBS buffer and treated for 2h at 37 °C with the CP, PFR, PFP, and Pagri samples following intestinal digestion dissolved in HEPES buffer (1.25 mM) at a concentration of 5 mg protein/mL. All the treatments were performed by duplicate in each cell plate and repeated for 3 passages. After the treatment, the medium of each well was collected and stored at −80 °C in aliquots of 25 µL until the determination of total GLP-1 and LDH release assay following the manufacturer’s instructions. Then the cells were lysed with RIPA buffer, lysates were stored at −80 °C, and then used to analyse total protein content using a BCA kit (Pierce, Thermo Fisher Scientific) and for LDH quantification in the cells.

Twenty-four-well culture plates were set up with 300 μl aliquots of Matrigel® (BD Bioscience, CA, USA) and incubated for 30 min at 37 °C to allow polymerization. Freshly isolated MPCs, P2-MSCs, and HUVECs were seeded over Matrigel® thick layers at 3 × 10³ or 5 × 10³ cells/well and cultured in EGM-2 medium (Lonza) at 37 °C, 5% CO₂. After 48 h, phase-contrast microphotographs were taken and processed for image analysis to measure tube lengths and lumen areas.

Human (HUM4198; Lonza) and rhesus macaque (Cat. No. MKR103; Lonza) primary hepatocytes were used for 2D and 3D culture systems. For 2D culture, 24-well plates were coated with collagen for 45 min. After washing the coated wells with phosphate-buffered saline, primary hepatocytes were seeded 400,000 cells/well in complete hepatocyte plating media (HCM kit; Cat. No. CC-3198; Lonza). After 4 h of incubation at 37°C and 5% CO₂, media were removed and gently covered with hepatocyte maintaining media (HCM kit; Cat. No. CC-3198; Lonza) containing 0.25 mg/mL Matrigel (Cat. No. 354234; Corning) and incubated at 37°C for 90 min. The AAV mix was added diluted in hepatocyte basal media (HBM; Cat. No. CC-3199; Lonza). Media were changed daily for 3 days, until harvest using Cell Recovery Solution (Cat. No. 354253; BD) and processing for NGS.
The 3D printed hydrogels containing human and rhesus hepatocytes were generated using a Rastrum Cell Printer (Inventia, Sydney, Australia). The cell printing followed the manufacturer's instructions and previously published protocols.^{38 (link),39 (link)} In brief, hepatocytes were thawed, resuspended in crosslinker solution (Inventia), and printed in 96-well plates at 8,000 cells/well. Cells were maintained in HBM and AAVs were added as described previously. Cells were harvested as whole printed gels and processed for NGS.