Affi gel beads

RS cells from passage 4 were grown in culture with Affi-Gel beads that were incubated overnight at 4°C with SHH protein (25 |μL of a 1 |μg/|μL solution, R&D Systems, Minneapolis, MN), 5E1 SHH inhibitor (388 |μg/mL, Developmental Studies Hybridoma Bank. Iowa City, IA), or PBS (control) for 3–6 days. Concentration of SHH protein and inhibitor was based on previous optimization of tissue response in penis and nerve tissues.^{13 (link),18 (link),19 (link),25 (link)} Matrigel (100 |μL) was placed at four places along the periphery of wells in six-well plates. Affi-Gel beads (extended release vehicle) containing SHH protein, 5E1 SHH inhibitor, or PBS were placed on the Matrigel and were gelled at 37°C for 5 min. RS cells (15 000) were placed in the center of each well and the cells were allowed to adhere for 1 h prior to adding 3.5 mL of DMEM/F12 media containing 10% FBS and antibiotic. Culture plates were placed in an atmosphere- controlled incubator (5% CO₂) at 37°C for 3–6 days. Cells were quantified by hemocytometer at 3 and 6 days. Four wells for each treatment group were quantified with four hemocytometer measurements performed on cells from each well. Experiments were performed in duplicate at each time point and were replicated on cells from three patients.

Once primary culture of human corpora cavernosal cells were established, cells were allowed to grow to passages 3–4, and were plated as described above with Affi-Gel beads that were treated overnight at 4°C with either 10 μg or 25 μg of SHH protein (1 μg/μl solution, R&D), or MSA (control, Sigma). Cells were grown for 3–4 days in the presence of SHH protein or control MSA protein and were quantified as described above.