Dmem cell culture medium

The hydrogel used in this study was prepared using a method of freeze-thaw cycles (Fig. 1) [19 (link)]. PVA, CS, polysorbate (PS)-80, DMEM cell culture medium, fetal bovine serum, and lyophilizer (solution) were purchased from Sigma Aldrich Ltd., Shanghai, China. 5 g PVA was discharged into ultra-pure water and swelled after heating in 60 °C for 20 min. The system was stirred in the magnetic blender for 3 h in 90 °C to ensure that the PVA was completely dissolved. The PVA solution was prepared with 10% weight ratio concentration. CS powder and 1% acetic acid (1 g: 50 ml) were poured into the beaker. The system was covered, sealed, and then blended on a magnetic blender for 1 h to achieve complete dissolution. The mixture of these two solutions at various proportions was left in the vacuum drier for 1 h and degassed. The mixture solution was added into a sterile 24-well plate, with each well containing less than 2 mL of solution. The plate was sealed and kept in − 20 °C for 20 h and then thawed for 8 h in RT. The freeze-thaw cycle was repeated seven times total. Afterwards, 1% sodium hydroxide (NaOH) solution was added into the 24-well plate to adjust pH to neutral. The mixture was then kept in ultra-pure water.Fig. 1

Diagram of PVA/CS composite hydrogel preparation

HT22 cells were obtained from the Fourth Military Medical University. Sprague-Dawley (SD) rats were purchased from the Laboratory Animal Center of Sun Yat-sen University. The CB1 cannabinoid receptor agonist ACEA, CB1 receptor antagonist AM251 (can penetrate the cell membrane), cell membrane CB1 receptor antagonist hemopressin (Hemo), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), DMEM cell culture medium and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

RPMI-1640 cell culture medium and DMEM cell culture medium were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Fetal bovine serum (FBS) and Plasmocin™ were purchased from Thermo Fisher Scientific (Waltham, MA, USA). LipoFiter^TM Liposomal Transfection Reagent was purchased from Hanbio Biotechnology (Shanghai, China). CDDP was purchased from Selleck Chemicals (Houston, TX, USA). Antibodies used were listed in Table 1. HRP-conjugated goat anti-rabbit and anti-mouse secondary antibodies were from Sigma-Aldrich Inc. (St. Louis, MO, USA). EnVision^TM Ⅲ Detection System (GK500705) was purchased form Gene Tech (Shanghai, China).