Cmrl 1066 medium

Human islets were provided from Tianjin First Central Hospital. All human studies were conducted according to the principles of the Declaration of Helsinki and approved by Ethics Committee of the Tianjin First Central Hospital^{49 (link)}. Written informed consent was obtained from all subjects. High purity islets (>80%) were collected and cultured in CMRL-1066 medium (Corning, Manassas, VA, USA), supplemented with 10% Human Serum Albumin (Baxter, Vienna, Austria), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in 5% CO₂.
Mice islets were isolated by collagenase digestion and enriched using a Histopaque (Sigma Aldrich) density gradient^{50 (link)}. Isolated islets were collected and resuspended in RPMI-1640 medium (glucose: 11.1 mmol/l) containing 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified 5% CO₂ atmosphere. After equilibrating for 3 h, the islets were counted and replanted into 6- or 48-well plates and cultured overnight for further experiments. For pathophysiological concentrations of palmitate, glucose, and proinflammatory cytokines treatment, islets were incubated in modified medium with 0.5% (weight for volume) BSA, various concentrations of glucose (low glucose: 2.5 mmol/l; high glucose: 33.3 mmol/l), palmitate (0.5 mmol/l), IL-1β + TNFα (IL-1β: 5 ng/ml; TNFα: 30 ng/ml).

Islet isolation and transplantation were performed as previously described^{53 (link)}. Briefly, islets were isolated from monkey pancreas using the modified Ricordi method with collagenase MTF C/T (Roche, Mannheim, Germany). For the purification of isolated islets, the discontinuous Ficoll density gradient method was used. Next, purified islets were cultured with CMRL-1066 medium (Corning, NY, USA) containing 10% fetal bovine serum (FBS; Gibco-Thermo Fisher Scientific, Waltham, MA, USA) and 1% antibiotics (Gibco-Thermo Fisher Scientific) in a humidified 5% CO₂ atmosphere at 37 °C.

Human islet isolations from cadaveric human organ donors were performed in the Good Manufacturing Practice facility of the Leiden University Medical Center according to the method used in the center for the procurement of clinical-grade material (Nijhoff et al., 2016 (link)). Islets were isolated from donor pancreas allocated (after anonymization) by Eurotransplant for the clinical islet transplantation program of the Leiden University Medical Center. Islets were used for research only if they could not be used for clinical purposes, and if research consent was obtained according to Dutch national laws.
Islets were cultured in CMRL 1066 medium (Corning, 5.5 mmol/l glucose) containing 10% FCS (Bodinco), 20 mg/ml ciprofloxacin (Fresenius), 50 mg/ml gentamycin (Lonza), 2 mM L-glutamine (Lonza), 10 mM HEPES (pH 7.21, Lonza), and 1.2 mg/ml nicotinamide (prepared by the Leiden University Medical Center pharmacy) in a humidified atmosphere with 5% CO₂. Islets were dispersed into single cells by adding 0.025% trypsin solution containing 10 mg/ml DNase (Pulmozyme, Genentech) at 37°C for 6–8 min.

The rat urogenital ridge bioassay was conducted as previously reported (16 , 17 (link)). Briefly, the female embryonic ridges were dissected from timed pregnant Sprague-Dawley rats (E14.5) and incubated on 2% agarose (wt/vol) at the air/media interface on agarose gel suspended in a CMRL 1066 medium (Corning Life Sciences) supplemented with 10% female fetal bovine serum (Biologos), 1% L-glutamine (Thermo Fisher Scientific), 1% fungizone (Thermo Fisher Scientific), and 5 nM testosterone (Sigma Aldrich). The small-molecule compounds dissolved in DMSO were added into the culture medium for testing and compared to DMSO (0.02% final volume) vehicle control, and rhMIS (10 µg/mL) for positive control. Tissues were incubated for 72 h, fixed, embedded in paraffin, and then sectioned and stained with hematoxylin (DAKO) and eosin (Sigma Aldrich) (H&E). The regression score of the Mullerian duct was evaluated by two experienced blinded observers under a light microscope. The consensus scores were recorded, ranging from grade 0 (no regression) to grade 5 (complete regression). The integrity of the Wolffian duct and the appearance of the tissue were used as an indicator of the toxicity of compounds.