Incucyte s5 live cell imaging system

We have used a human tumor sample to establish pancreatic and ovarian organoids. And for lung organoids, we have used tumor samples from an autochthonous mouse model (Kras^G12D+/, p53^R172H+/—, AdCre).^{80 (link)} Tumor samples were chopped and digested enzymatically using digestion media containing collagenase II, Dispase, and 1% FBS. After digestion, the cells were embedded in Matrigel and incubated in the incubator for 15 to 20 min before adding organoids-specific media.^{22 (link),81 (link),82 (link)}Three independent replicates of human ovarian and pancreatic organoids and murine lung organoids were transfected with siRNA (for PAF1/PD2), followed by treatment with cisplatin or gemcitabine alone or in combination with RAD52 inhibitor D-I03. Real-time images of the organoids were captured every 3 h for 48–72 h using the IncuCyte-S5 live-cell imaging system (Sartorius). The kinetic data (organoid average growth and darkness) were analyzed and graphically represented using IncuCyte software (Sartorius). RNA was collected from organoids at the experimental endpoint to perform further analysis.

Human PDAC organoid was generated from fresh tumor tissue of patients who underwent PDAC resection. We used human PDAC tumor samples for the establishment of pancreatic organoids according to the previously described protocol [24 (link), 25 (link)]. In brief, tumor samples were chopped and digested enzymatically using digestion media containing collagenase II (Millipore Sigma, C7657), Dispase (Thermo Fisher, 17105041), and 1% FBS. After digestion, the cells were embedded in matrigel (Corning, 356255) and incubated in the incubator for 15 to 20 mins before adding organoid-specific media [22 (link)]. Three independent replicates of organoids were treated with Gemcitabine or 5FU alone or in combination with Bosutinib. Real-time images of the organoids were captured every 3 hours for 72 hours using the IncuCyte-S5 live-cell imaging system (Sartorius). The kinetic data (organoid average growth) were analyzed and graphically represented using IncuCyte software (Sartorius).