Ultra low cluster 96 well plate

ECM-mimetic hydrogels (50 μl) were generated from a HEPES solution (20 mM, 150 nM NaCl, pH 7.4) containing 8 mg/ml human fibrinogen (Enzyme Research Laboratories), 1 mg/ml human plasma fibronectin (Sigma), 500 μg/ml human vitronectin (Peprotech), 50 ug/ml human tenascin C (R&D Systems), 50 μg/ml heparan sulfate (Sigma) and 500 ng/ml of PDGF-BB or IL-1Ra variants. Matrices were polymerised in Ultra-Low Cluster 96-well plate (Corning) at 37 °C for 2 h with 10 U/ml bovine thrombin (Sigma) and 5 mM CaCl₂. Then, matrices were transferred to Ultra-Low Cluster 24-well plate (Corning) containing 500 μl of buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% BSA, pH 7.4). Control wells that served as 100% released control contained only PDGF-BB and IL-1Ra variants in 500 μl of the buffer. Every 24 h, buffers were removed from wells and kept at −20 °C. Wells were replenished with fresh release buffer. For the 100% release control well, 20 μl of buffer was taken out every day and stored at −20 °C. After 7 days, the cumulative release of PDGF-BB and IL-1Ra variants was quantified by ELISA using the 100% released control as reference (PDGF-BB DuoSet, IL-1Ra/IL-1F3 DuoSet; R&D Systems). For release assays with plasmin, the same method was used except that the release buffer contained 100 μU/ml of plasmin (Roche).

The Ultra-Low Cluster 96-well plate (Corning, NY, USA) was applied with 180 μL of complete medium mixed with 5000 TtT/GF cells and 20 μL of collagen (cat: 40125ES10, Yeasen, Shanghai, China) per well, then the 96-well plate was centrifuged at 4 °C and 1000× g for 10 min to form the cell sphere. Photographs were taken after 1 day and 5 days.