Geltrex ldev free

Immediately after collection, the surplus liver biopsy was washed three times in 1X Dulbecco’s PBS (DPBS) and cut in pieces of 0.3–0.5 mm. The tissue samples were then partially digested by incubation with trypsin-EDTA (0.05%, ref. 25300054 Thermo Fisher Scientific) at 37°C for 15 minutes and vortexing every 5 minutes. The trypsin-EDTA solution was carefully removed by aspiration, and the liver biopsy pieces were washed twice with complete medium (S1 File). The samples were then transferred to 6-well culture dishes coated with reduced growth factor basement membrane matrix (ref. A1413301, Geltrex LDEV-Free, Thermo Fisher Scientific) and covered by complete medium (S1 File). Twenty-four hours later, complete medium was replaced by basal medium (S1 File). Tissue samples were left to adhere to the culture dishes for 48 hours before changing the medium for the first time, and non-adherent biopsy pieces were discarded. PLC outgrowth from the adherent biopsy pieces was observed after 7 to 10 days in culture. After 15–20 days of culturing, when the PLC monolayer encompassed about 300–500 cells, the biopsy pieces were removed and discarded. The adherent PLCs (passage 1 (P1)) were then trypsinized and either frozen or passaged into a new coated 6-well culture dish. In the P2 culture, the PLCs were expanded to 80%-90% confluence (~50,000 cells/well).

The NSC induction was conducted from iPSCs obtained from Kangstem Biotech (Seoul, Korea) using a neural induction medium (#A1647801, Gibco). We plated NSCs on geltrex LDEV-Free (#A1413302, Thermo Fisher)-coated plates. For neural differentiation, NSCs were cultured in a neurobasal medium (#21103049, Gibco) with 2% supplement B27 (#17504044, Gibco) and 1% Glutamax (#35050061, Gibco). For astrocytic differentiation, NSCs were incubated in high-glucose DMEM (#SH30022.01, Hyclone) containing 1% N-2 supplement (#17502048, Gibco), 1% FBS, and 1% Glutamax (#35050–061, Gibco). After 5 days of astrocytic differentiation, astrocytes were cultured on neural differentiation media for 48 h with cortisol, and ACM were harvested.
Cultures of mouse primary hippocampal astrocytes were performed as described in the previous protocol [17 (link)]. Hippocampal astrocytes from postnatal day 1 (P1) mice were acquired in compliance and approval with the Institutional Animal Care and Use Committee of Seoul National University (SNU-190523–1-1). In brief, hippocampal astrocytes cultured on poly- _D-lysine coated six-well plates were incubated in MEM (Hyclone) supplemented with glucose (0.6% wt/vol), 10% horse serum (vol/vol), and 1% antibiotic–antimycotic mixture.

HUVECs (Life Technologies, pool of 10 donors) were expanded in basal medium (BM, 300 mOsm) consisting of Medium 200 supplemented with low serum growth factors (LSGS, Life Technologies) and 1% P/S until 80% confluency. A 24‐well plates were coated with 120 μl/well Geltrex LDEV‐free (Thermo Fisher Scientific, Breda, the Netherlands), after which HUVECs at passage two were seeded at 80,000 cells per well. Cells were incubated for 24 h at 37°C and 5% CO₂ in three different medium conditions: BM, NCCM with the same supplements as BM or NCM added to BM (n = 5 biological replicates for each condition). The NCM concentration in medium was adjusted to obtain similar GAG content to NCCM. After 24 h, cells were incubated for 15 min with 10 μM Calcein AM and 10 μM propidium iodide. Per well, three pictures were taken at 4× magnification using a fluorescence microscope (Zeiss Axiovert, Zeiss, Jena, Germany). Per sample, the images were taken at approximately the same locations, covering the largest part of the well without overlap. To assess whether effects of NCCM or NCM may be dependent on HUVEC donor variation, the same experiment was repeated with HUVECs (used at passage 2) from two separate additional donors (Cell Applications, San Diego, CA, n = 2 biological replicates each).