D hank s balanced salt solution

Primary NPCs were isolated from the brains of embryos at embryonic day (E) 14.5 as described previously.³², ³³ Briefly, embryonic brains were extracted from mice and placed in D‐Hank's balanced salt solution (Gibco, Grand Island, NY). The hippocampus was dissected by gentle pipetting followed by seeding into DMEM/F‐12 media containing B‐27 Supplement (2%), bFGF (20 ng/mL), and EGF (20 ng/mL) in 100‐mm cell culture dishes (NEST Biotechnology, Wuxi, China). The cells were then cultured in a humidified 37°C, 5% CO₂ incubator to form neurospheres, and half of the medium was replaced with fresh medium after 3 days. The neurospheres were passaged every 6 days with a final density of 5 × 10⁵/mL. Further analyses were performed using the cells at passage three.

Poly(ε-caprolactone) (PCL, MW = 45,000) and polyvinyl alcohol (PVA, MW = 13,000–23,000, 87–89% alcoholised) were supplied by Sigma–Aldrich (St. Louis, MO, USA). Methoxy PEG-Poly(ε-caprolactone) (mPEG-PCL) copolymers with different mPEG and PCL chain lengths (mPEG_5k-PCL_45k, mPEG_2k-PCL_45k) were synthesised according to previous procedures [26 (link),27 (link)] and were kindly provided by Professor Zhiyong Qian of Sichuan University, Chengdu, China. The fluorescent probe P2 (λ_abs/λ_em = 708/732 nm) was synthesised in our lab according to previous procedures [28 (link),29 (link)]. Dichloromethane (DCM was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Roswell Park Memorial Institute (RPMI)-1640, Dulbecco’s modified Eagle’s medium (DMEM), penicillin streptomycin (PenStrep), 0.25% trypsin–0.02% ethylenediamine tetraacetic acid (EDTA), and D-Hank’s balanced salt solution were purchased from Gibco (USA). Foetal bovine serum (FBS) was supplied by Invitrogen (Carlsbad, CA, USA). Purified water was prepared using a Milli-Q purification system (Molsheim, France). All other reagents used in this study were of analytical grade and used as received.

Cells were passaged when the culture reached 80% to 90% confluence. The cell culture solution was aspirated, and the flask was rinsed twice with 3 to 4 mL of D-Hank's balanced salt solution (Gibco). A mixture of 0.25% pancreatin and 0.05% EDTA (1 : 1) was added to the flask, and the cells were digested for 1 to 2 min at room temperature. The cells were then observed under an inverted microscope to detect changes in their morphologies. The pancreatin solution was removed immediately after the cytoplasm began to shrink, the space between cells began to increase, and the cells were becoming rounded. A 1 to 2 mL aliquot of media containing serum was added to stop the digestion. The culture solution containing pancreatin was removed, and 4 mL DMEM culture solution was added. The cells were gently triturated until the cells were released from the flask wall and formed a cell suspension. The cells were then inoculated into a new culture flask for passaging at a ratio of 1 : 2.
For subculturing, cells were released from the flask walls and suspended at a density of 1 × 10⁶ cells/mL for inoculation. The cells were passaged at a ratio of 1 : 2, and cells adhered to the walls of the new flasks over the course of approximately 2 h. Passaging was performed once every 7 to 14 days.

The materials and instruments used for this study were seventeen 2-week-old healthy guinea pigs (female and male without eye disease); a concave lens (self-designed PMMA lens made by Shanghai Freshkon, lens diameter: 1.0 mm, optical diameter: 9.5 mm, primitive arc: 9.0, and both diopters: −10.00 D); a model BME-210 ophthalmic A/B-type ultrasonic diagnostic apparatus developed by the Chinese Academy of Medical Sciences; a streak retinoscope; newborn calf and fetal calf serum; DMEM-F12 (Gibco, USA); D-Hank's balanced salt solution (Gibco, USA); pancreatin (Gibco, USA); rabbit anti-rat vimentin antibodies; anti-desmin, keratin, and S-1001 antibodies, and immunological composite agent (Wuhan Boster); and recombinant TGF-β2 (Gibco, USA).