Chapter 4 136 Cryopreservation Organoids were cryopreserved on day 50, 100 and 150 of the differentiation protocol. Cryopreservation was performed by fixation of PBS washed spheroids in 4% PFA for 15 minutes followed by thorough PBS wash. Then, organoids were allowed to dehydrate overnight in 30% sucrose (in PBS) at 4°C. Dehydrated spheroids were transferred to embedding moulds with embedding medium (Bio-Optica, 05-9801V) and snap frozen using dry ice and isopropanol. Frozen samples were stored at -80°C until cryo-sectioning. Cryo- sections of 20 µm were made using a standard cryostat and transferred to Superfrost® Plus adhesive microscope slides (Thermo Scientific). Samples were stored at -80°C until immunofluorescence staining. Immunofluorescence To perform immunofluorescence staining, cryosectioned organoids were rehydrated by washing in PBS. After, sections were incubated for 1 hour with blocking solution, PBS containing 0.3% TritonX-100 (Sigma) and 10% Normal Goat Serum (NGS, Sigma-Aldrich, #g9023-10). Primary antibodies made up in blocking solution were incubated overnight at 4°C (See Supplementary Table II). The next day, slides were washed 3 times with PBS prior to 2 hour room temperature incubation with secondary antibodies (Supplementary Table III) made up in PBS with 1% NGS. After incubation, the slides were washed with PBS 3 times. Nuclear visualization was achieved by 10 minutes incubation with Hoechst (1:1000, in PBS). Final wash with PBS was performed before mounting coverslips using FluorSave mounting medium (WR, 345789-20). Images were acquired at the Advanced Light and Electron Microscopy Bioimaging Center (ALEMBIC) at IRCCS San Raffaele Scientific Institute. A Mavig RS-G4 confocal microscope and a 40x oil immersion objective were used. Image analysis and quantification were performed using ImageJ and Imaris software (NIH, Bethesda, MD) to obtain the percentage of positive area of the staining over the DAPI area. For each donor at each timepoint an average was generated by analysing at least 8 up to 29 sections from various organoids. Statistical analysis was then performed using RM two-way ANOVA with Geisser-Greenhouse correction (GraphPad Prism 10.2.0). A full model was fitted. When significant Uncorrected Fisher’s LSD or Tukey’s multiple comparisons test were performed to determine which groups were different. Electron microscopy Organoids were fixed using 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight at 4°C. Samples were post-fixed for 1 hour in a reduced osmium solution (1.5%
RkJQdWJsaXNoZXIy MTk4NDMw