To retrieve and culture NPCs, embryos were removed under deep anesthesia by cesarean section at days E13.5 and E15, and were stored in sterile Hank's balanced salt solution (HBSS; PAA Laboratories, Pasching, Germany) with 1% PenStrep (Sigma-Aldrich, Hamburg, Germany), on ice until dissection. The brains were removed and homogenized mechanically. The homogenate then was treated with 0.1% trypsin in HBSS to obtain single cells. The suspension was transferred into a culture medium comprising Dulbecco's modified Eagle's medium–Ham's (DMEM-F12; PAA Laboratories) supplemented with 1% PenStrep, 1% N2 Supplement (PAA Laboratories), 10 ng/μL recombinant human basic fibroblast growth factor (bFGF; RD Systems, Minneapolis, MN), and 10 μL insulin (2.5 mg/mL; PromoCell, Heidelberg, Germany). The suspension was triturated gently and 4.5 × 106 cells were plated at a density of 200,000 cells/mL on noncoated Advanced TC Petri dishes (Greiner Bio-One, Frickenhausen, Germany) to inhibit further differentiation. The cells were incubated at 37°C in a humidified atmosphere of 5% CO2. All cell harvesting steps were performed under sterile conditions.
Rat crybb2 was cloned into pIRES-ACGFP (Clontech, Palo Alto, CA) so that rat crybb2 and green fluorescent protein (GFP) could be translated from a single bicistronic mRNA. For transfection of NPCs, cultured cells that formed neurospheres were dissociated mechanically into single cells and centrifuged for 5 minutes at 400 rpm (4°C). The culture medium was removed and approximately 5 × 10
6 cells were resuspended in 100 μL Amaxa Rat NSC Nucleofector Kit (Lonza Cologne, Cologne, Germany). Vector (3.5 μg DNA) was added, the cells and DNA were mixed gently, and then the cells were electroporated using the Nucleofector device (Lonza Cologne). The transfection protocol used was very similar to that described by the manufacturer, thus resulting in the highest transfection efficacies.
5,27 Cells were used for a maximum of 2 weeks after preparation. After finishing this procedure, crybb2-overexpressing NPCs (crybb2-NPC) were placed into the culture medium and cultured for up to 7 days, and the efficacy of transfection was monitored using GFP fluorescence.
Transfected crybb2-NPC were prepared for injection by first washing the cell suspensions with HBSS. Cell numbers then were counted using the trypan-blue exclusion method, and then the cells were transferred to the transplantation medium, HBSS. Surgery and cell injection were performed within 1 hour of cell preparation.