Laminin l2020

Human induced pluripotent stem cell (IPSC)-derived neural progenitor cells (NPCs) (Ax0015; Axol, Cambridge, United Kingdom) were cultured in neural maintenance basal medium with supplements (Ax0031; Axol) according to the manufacturer’s specification. Human IPSC-derived NPCs were grown on plates coated with 20 µg/ml laminin (L2020; Sigma-Aldrich). Human neuroblastoma SK-N-SH and human glioblastoma U87-MG cells were purchased from Sigma-Aldrich and grown in Eagle’s minimum essential medium (EMEM) with Earle’s balanced salt solution (EBSS [Lonza, Breda, The Netherlands) containing 10% heat-inactivated fetal bovine serum (HI-FBS [Lonza]), 100 U penicillin (Gibco Life Sciences, USA), 100 µg/ml streptomycin (Gibco), 2 mM l-glutamine (Lonza), 1% nonessential amino acids (Lonza), 1 mM sodium pyruvate (Gibco), and 1.5 mg/ml sodium bicarbonate (Lonza). Both immortalized cell lines SK-N-SH and U87-MG were used below passage 25. Vero cells (ATCC, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% HI-FBS, 100 µg/ml streptomycin, 100 U penicillin, 2 mM l-glutamine, 1% sodium bicarbonate, and 1% HEPES buffer (all from Gibco). Human NPCs are primary cells, while the other cells are from immortalized cell lines. All cells used in this study tested negative for Mycoplasma sp.

AMG232 and RG7112 were purchased from APExBIO Technology. Anti-p21 (#2947), anti-PARP (#9542), and anti-ZEB1 (#3396) were purchased from Cell Signaling, anti-p53 (sc-6243), anti-MDM2 (sc-813), and anti-Nestin (sc-23927) were purchased from Santa Cruz Biotechnology and anti-beta-actin (ab8227) was purchased from Abcam. Alexa Fluor 488 anti-rabbit secondary antibody (A11008) was purchased from Thermo Fisher Scientific. Laminin (L2020) was purchased from Sigma and Hoechest33342 (H3570) was purchased from Life Technologies.

The myoblasts were isolated and cultured according to the protocol described by Fauconneau and Paboeuf (2000) [103 (link)]. The fast-twitch muscles were collected from the epaxial region and mechanically dissociated with scalpels. To release the muscle cells, the fragments were enzymatically digested with 0.2% collagenase type I (C9891) and 0.1% trypsin (T4799) (Sigma-Aldrich, St. Louis, MO, USA). The cell suspension was filtered in cell strainers (Corning, New York, NY, USA), allowing for the removal of debris, centrifuged, and the cell pellet was resuspended in DMEM medium (DMEM (D7777), 9 mM NaHCO₃ (S5761), 20 mM HEPES (H3375), pH 7.4), with 1% antibiotics (A5955) and 10% fetal bovine serum (F7524) (Sigma-Aldrich, St. Louis, MO, USA). The cells were diluted at a concentration of 2 × 10⁶ cells/mL and plated in 6-wells plates, previously treated with poly-L-lysine (P6282) and laminin (L2020) (Sigma-Aldrich, St. Louis, MO, USA), which have high affinity for the myoblasts. The myoblasts were incubated at 28 °C, with the medium changed every day, and the myoblasts morphology was monitored regularly under a microscope (Olympus, Tokyo, Japan). The results were achieved from 3 independent cell cultures.

The assays were performed as described previously with following modifications [17] (link). Tissue culture plates were coated for 2 hours at 37°C with 10 µg/ml collagen I (C7661), laminin (L2020), fibronectin (all from Sigma, St. Louis, MO, USA) or PBS. For cell adhesion assay, after saturation of wells with DMEM +1% BSA, R3/1-pLXSN or R3/1-FL-RAGE cells (5×10⁴) were resuspended in DMEM +10% FCS and seeded on coated 96-well plates in triplicate for 15 or 45 minutes at 37°C under 5% CO₂. Cells were extensively washed with PBS, fixed with 4% paraformaldehyde, stained with crystal violet for 10 min, lysed with 2% SDS and read on an ELISA plate reader at 550 nm. For cell spreading assay, R3/1-pLXSN or R3/1-FL-RAGE cells (5×10⁴) were resuspended in DMEM +10% FCS and seeded on 12-well coated plates in triplicate for 90 minutes at 37°C under 5% CO₂. Cells were then washed with PBS and fixed with 4% paraformaldehyde. Photographs were taken at 40× magnification in phase contrast (Leica DM IRB). Cell surface area (µm², 25–50 cells) was quantified using Axiovision Software™ Rel 4.7 (Zeiss).

A transwell system (8 μm pore size, Corning Co., NY, United States) was used for co-culture of endothelial cells (ECs) with macrophages. A density of 200 μl of 10⁵ cells/ml was used for each chamber. A microfluidic chamber system was used for assessing innervation of spinal cord explant into area of co-culture of skeletal myocytes and macrophages, as described (Gluska et al., 2014 (link)). Briefly, the explant was well positioned, after which the device was attached to a 60-mm plastic dish with margins sealed with polydimethylsilxane (PDMS) at 60°C for 30 min to prevent the chamber from detaching. Muscle channels were then coated with Matrigel diluted 1:10 with DMEM. The explant well and channel were sequentially filled with 150 ml of 1.5 ng/ml polyornithine (P-8638, Sigma-Aldrich) in PBS and 150 ml laminin (L-2020, Sigma-Aldrich) each for 12 h, respectively. The co-culture was then started afterwards after replacement with corresponding media.

Polydimethylsilxane MFCs were designed and cast as described previously (Ionescu et al., 2016 (link)). After the wells were punched, a small “cave” was created in the explant well near the grooves using a 25G needle, keeping the explant in place. Microfluidic devices were cleaned of surface particles using adhesive tape and were sterilized in 70% ethanol for 15 min. Devices were completely dried under sterile conditions using UV radiation and then attached to a sterile 60 mm plastic dish (Nunc) with gentle pressure, and the margins were sealed with polydimethylsilxane before incubation at 70°C for 30 min to prevent the detachment of the chamber. Muscle channels were coated with Matrigel diluted 1:10 with DMEM containing 2.5% penicillin-streptomycin-nystatin (PSN) for 30 min at 37°C, before filling the muscle wells with 150 μl of Bioamf-2 medium. The explant well and channel were filled with 150 μl of 1.5 ng/ml poly-dl-ornithine (P-8638, Sigma-Aldrich) in PBS overnight and then replaced with 150 μl laminin (L-2020, Sigma-Aldrich), 1:333 in deionized distilled water overnight. One day before plating the SC explant, laminin was replaced with explant medium containing Neurobasal (Invitrogen) supplemented with 2% B27 (Invitrogen), 1% penicillin-streptomycin (Biological Industries), 1% Glutamax (Invitrogen), and 25 ng/ml BDNF (Alomone Labs), until the day on which coculturing began.