Ab222510

Cell culture supernatant samples from mcRPE (30DPS) were obtained by incubating volumes of microcarriers containing approximately 0.8 cm² in 48-well plates with 0.5 mL/well of fresh CTS AIM-V medium for 24 h, ±1 h at 37 °C and 5% CO₂. Brightfield whole-well imaging was performed using a Celigo Image Cytometer (Nexcelom Bioscience, Lawrence, MA, USA), and the number of microcarriers per well was counted using the ImageJ Cell Counter plugin (mean ± s.d., 580 ± 132 microcarriers/well). Samples were frozen at −80 °C until the time of processing. Sandwich ELISAs for Pigment Epithelium-Derived Factor (PEDF) and Vascular Endothelial Growth Factor A (VEGF) were performed using commercially available kits according to the manufacturer’s instructions (PEDF: Abcam (Cambridge, UK) #ab246535, VEGF: Abcam #ab222510). Sample dilutions of 80-fold for the PEDF ELISA and 5-fold for the VEGF ELISA were performed to ensure that all results remained in the linear detection range for each assay. Surface area normalization was based on a mean surface area of 0.091 mm² per microcarrier for Cytodex 1 and 0.071 mm² per microcarrier for Cytodex 3 based on diameters of 170 µm and 150 µm, respectively, with spherical surface area $A=4\pi r^2$ [21 ].

An enzyme-linked immunosorbent assay (ELISA) was used to analyze the levels of fibroblast growth factor-2 (FGF-2, ab246531, Abcam, Cambridge, MA, USA), bone morphogenetic protein 2 (BMP-2, EHBMP2, Invitrogen, Grand Island, NY, USA) and vascular endothelial growth factor (VEGF, ab222510, Abcam, Cambridge, MA, USA) in the medium of the scaffolds after a specified duration of culture. The ELISA kit (Invitrogen, Grand Island, NY, USA) was used according to the manufacturer’s instructions and was further correlated to a standard curve. Three individual tests were conducted for each sample. All the cultivation runs were performed three times in the experiments.

Enzyme-linked immunosorbent assays (ELISAs) were performed with an ELISA kit (ab222510, Abcam) to measure the VEGF in the samples. VEGF concentrations were determined in the culture supernatants of stable IL-1RA-overexpressing Eca109 cells and KYSE410 cells using the manufacturer’s provided protocol.