Primaria dishes

Retinal dissociates from P0‐2 mice were resuspended in REM (Appendix Tables S3 and S4) and plated at a concentration of 3 × 10⁷ cells in 10 ml of REM into 10 cm Primaria™ dishes (353801, BD Falcon, BD Biosciences, Oakville, ON, Canada). Lentiviruses were added to the plate at a titre to achieve > 80% infection efficiency. All cultures were maintained in 5% CO₂‐buffered incubators (50116048, Heracell™ 150i CO₂, Thermo Fisher Scientific, Asheville, NC, USA) at 37°C. After 2  DIV, cells were de‐adhered with Trypsin‐EDTA (0.05% Trypsin with EDTA 4Na) (25300062, Thermo Fisher Scientific, Mississauga, ON, Canada) for 5 min and the enzymatic digestion was stopped with 10% BSA. Cells were washed in Ca²⁺/Mg²⁺ ‐free PBS four times and filtered through a 40‐μm cell strainer. To confirm that GFP expression was not the result of lentivirus carryover in donor cell preparation, for every transplantation experiment we injected 2 μl of the final cell wash to the SRS in three different animals and analysed GFP expression after 21 days. Cells were resuspended at a concentration of 200,000 cells per μl in 0.005% DNAse in EBSS and kept on ice prior to transplantation or cell culture.

To determine the angiogenic potential of PBMNCs, cells derived from patients with MMD (n = 23), patients with MMD-O (n = 7), and controls (n = 23) were cultured in semisolid medium (MethoCult SFBIT; STEMCELL Technologies Inc., Vancouver, British Columbia, Canada) with provasculogenic growth factors/cytokines on 35-mm Primaria dishes (BD Falcon, USA) as reported previously^{10 (link),11 (link)}. EPC colonies that adhered to the dishes were then counted^{9 (link)}. Aliquots of the cells were seeded at 2 × 10⁵ cells/dish (three dishes per PBMNCs/cultured PBMNCs) for the EPC-CFA. The number of adherent colonies per dish was measured using a gridded scoring dish (STEM CELL Technologies, Canada) under a phase-contrast light microscope (Eclipse TE300; Nikon, Tokyo, Japan) 16 to 20 days after the initiation of the culture.
We measured the numbers of small- and large-cell colonies per dish after IL-10 (Pepro Tech, Inc., TX, USA) or anti-IL-10 (Abcam, UK) antibody was added to the freshly isolated PBMNCs or those cultured from patients with MMD (n = 7), patients with MMD-O (n = 3) and controls (n = 6).

Fresh PBMNCs derived from healthy volunteers and corresponding QQMNCs were seeded at a concentration of 2 × 10⁵ cells/dish in 35-mm Primaria dishes (BD Falcon) and cultured for 16–18 days using the EPC-CFA system (Methocult SF^BIT; STEMCELL Technologies Inc., Vancouver, BC, Canada). The semi-solid culture medium was supplemented with a growth factor/cytokine mixture of 66.7 ng/mL SCF, 33.3 ng/mL VEGF, 33.3 ng/mL basic fibroblast growth factor (FGF), 33.3 ng/mL EGF, 33.3 ng/mL insulin-like growth factor 1 (IGF-1), 13.3 ng/mL IL-3 (Peprotech, Rocky Hill, NJ, USA), 1.33 IU/mL heparin (Shimizu Pharmaceutical Co., Shizuoka, Japan), and 30% fetal bovine serum (SAFC Biosciences, St. Louis, MO, USA). After 16–18 days in culture, the colonies in each dish were enumerated under an inverted microscope (Nikon Corporation, Tokyo, Japan) at 40× magnification, as described previously^{10 (link)}. Two types of EPC colony-forming units (EPC-CFU) were observed and determined: primitive EPC-CFU (pEPC-CFU) and definitive EPC-CFU (dEPC-CFU)^{5 (link),12 (link),13 (link)}.