Dnase solution

LGs were decellularized as described previously.²⁶ (link) In brief, LGs were cut into pieces 3 mm in diameter and washed in cold PBS (Sigma-Aldrich, St. Louis, MO, USA) containing 5% penicillin/streptomycin (P/S; Sigma-Aldrich) overnight. Cellular components were removed by incubation in a 1% (w/v) solution of sodium deoxycholate monohydrate (Sigma-Aldrich) for 36 hours with three changes, followed by DNase solution (200 U/mL in PBS; Roche, Basel, Switzerland) for 24 hours, and then washing in PBS + 5% P/S for an additional 24 hours. All incubation steps were performed at 4°C under continuous agitation with interposed washing in PBS + 5% P/S. The decellularized LGs were stored at −80°C until further use.

Female NOG mice 7 weeks of age were obtained from Taconic, Denmark. 1,5 million human high-grade serous ovarian cancer OVC316 cells were injected into the uppermost mammary fat pads of the mice, after which tumor diameters were measured twice a week. When the diameters reached 12 mm, the mice were sacrificed, and the tumors were collected. Next, they were chopped into pieces and homogenized using mechanical force. The resulting pieces were digested with trypsin for 10 min in a 37^oC tissue culture incubator while shaking, after which they were incubated for 1 h in a collagenase solution (10 mg/ml, Sigma-Aldrich) in organoid growth medium (DMEM/MEGM; GIBCO/Lonza) supplemented with 5 % FBS (GIBCO), 0.25 % penicillin and streptomycin solution (GIBCO) and 5 µg/ml insulin (Sigma-Aldrich). Next, pieces were pelleted by centrifugation for 10 min at 1500 rpm and digested in a DNAse solution (10 µg/ml, Roche) in DMEM/MEGM for 5 min at RT. Single cells were separated from organoids by differential centrifugation.

Postnatal days 1–2 neonates were sacrificed, and the cerebral hippocampus was removed. Tissues were incubated at 37°C for 15 min with DNase solution (Roche, Basel, Switzerland) and 0.25% trypsin (Gibco, Massachusetts, United States), dissociated and resuspended in DMEM containing 10% FBS (Gibco). Astrocytes were cultivated in T75 flasks (Falcon, Corning, United States) and purified by shaking for 24–48 h from 10–13 days in vitro (DIV 10–13). Astrocytes at DIV 16 were transduced with shRNAmiR AAV for ex vivo experiments. For in vitro experiments, 200,000 astrocytes were cultivated on top of sterile coverslips in 12-well plates (Thermo Fisher Scientific).