F12 glutamax

Commercially available cells, human dermal fibroblasts (HDFas, Gibco, Billings, MT, USA) and dental pulp stem cells (DPSCs, Lonza, Basel, Switzerland), were used in the work. The cells were cultured in DMEM: F12 + GlutaMAX (Gibco) commercial culture medium supplemented with 10% fetal bovine serum (FBS, Biosera, Cholet, France) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin, Biosera). The cells were grown in a humid atmosphere at 37 °C and 5% CO₂. After reaching an 80–90% confluence, the cells were detached using TrypLE Express Enzyme (Gibco) and seeded at a 5000 cells/cm² density. In this study, cells from passage no. 4 were used. Over the entire course of the experiment, the morphology of the cells was monitored by light microscopy (Optica).

HEK293 cells stably expressing the Ca_v1.2 subunits α_1C, β_2b, and α₂δ₁ under a tetracycline-inducible promoter (HEK293-Ca_v1.2) were purchased from B’SYS (Switzerland). Cells were cultured at 37 °C at 5% CO₂, in Dulbecco’s Modified Eagle Medium/F12 GlutaMAX (Gibco) supplemented with 10% fetal bovine serum, 1× penicillin-streptomycin, 100 μg/ml Hygromycin B, 15 μg/ml Blasticidin, 0.4 μg/ml Puromycin, and 100 μg/ml Zeocin. Cells at approximately 40% confluency were transfected with 1 μg of pHIV- CaM-EGFP variants using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s guidelines. To induce expression of Ca_v1.2, 2.5 μg/ml tetracycline was applied to cells 24 h prior to experimental use.

SUM149PT cell line was purchased from the Anschutz Cell Technologies Shared Resource and short-tandem repeat (STR)-profiled and mycoplasma-tested before use. Cells were maintained in F12/Glutamax (Gibco) medium supplemented with 5 % fetal bovine serum (FBS), 10 mM HEPES, 1 μg/mL hydrocortisone and 5 μg/mL insulin and kept in a humidified incubator at 37 °C and 5 % CO₂.

The generation of hEROs was according to Kuwahara’s inducing protocol and was with slight modifications of the procedure described in our previous studies (Kuwahara et al., 2015 (link); Gong et al., 2019 ; Zou et al., 2019 (link)). In detail, cloned hESCs were digested into single-cell suspensions in TrypLE Express (Gibco) containing 0.05 mg/ml DNaseI (Roche) and 20 μM Y-27632 (Merck). On day 0, 100 μl of cell suspensions (1.2 × 10⁴ cells) with the retinal differentiation medium were forced aggregation in each well (low-cell-adhesion V-bottom 96-well plates, Sumitomo Bakelite) with 5% CO₂/37°C environment. The retinal differentiation medium consisted of 45% IMDM (Gibco), 45% F12-Glutamax (Gibco), 450 μM monothioglycerol (Sigma-Aldrich), and 1% Chemically Defined Lipid Concentrate (Gibco) supplemented with 10% knockout serum replacement (KSR, Gibco) and 20 μM Y-27632. On day 6, the medium was completely exchanged for retinal differentiation medium supplemented with 1.5 nM bone morphogenetic protein 4 (BMP4, Peprotech). Since then, half of the medium was replaced every 3 days. On day 18, hEROs were transferred into low-cell-adhesion dishes (Greiner) with long-term culture medium containing Dulbecco’s modified Eagle’s medium (DMEM)/F12-Glutamax (Gibco) with 1% N2 supplement (Gibco), 10% fetal bovine serum (FBS, Gibco), 0.5 μM RA (Sigma) and 0.1 mM taurine (Sigma).

The PET cell lines used in this study corresponded to BON, CM, QGP1 and U2OS. The BON-1 cell line was cultured in 1:1 mixture of DMEM and F-12 glutamax mediums (Gibco, Massachusetts, USA). The CM and QGP1 were cultured in RPMI glutamax (Gibco). The U2OS was cultured in DMEM medium (Gibco). All the mediums were prepared with 10% fetal bovine serum (Gibco), 1% PenStrep (Gibco) and 0.5% Fungizone (Gibco).

For the production of CM, both cellular populations were plated at 6000 cells/ cm², in triplicates, and cultured in standard culture medium (αMEM 10% FBS) until approximately 80% confluence was reached. Then, the cultures were washed to remove any trace of FBS and other supplements and placed in plain DMEM/F12 GlutaMAX^™ (10565018, Gibco^®) for 24 and 48 hours. Conditioned media samples were collected at both time-points, centrifuged 1800 xg for 10 min to remove any cellular debris and preserved at -80°C until analysis.

Murine (C57BL/6) BM mesenchymal stem cells (GIBCO, S1502-100, Thermo Fisher Scientific, Bleiswijk, The Netherlands) were used for all experiments. Cells were cultured in MSC medium containing DMEM: F12 GlutaMax (31331093, Fisher Scientific, Landsmeer, The Netherlands), 10% Fetal Calf Serum (FCS, 10270106, Fisher Scientific), 0.05% gentamycin (15710064, Fisher Scientific) and 1% penicillin/streptomycin (P/S, 15140163, Fisher Scientific) in T75 flasks (353110, Corning Life Sciences, Amsterdam, The Netherlands). MSCs were passaged once before in vitro and in vivo use, according to the manufacturer’s instructions. Cells were cultured at 37 °C, 5% CO₂ and 90% humidity. For PKH26⁺ labelling, MSCs were labelled with the PKH26 Red fluorescent cell linker kit according to manufacturer’s protocol prior to administration (PKH26GL, Merck KGaA).