PSIX-19 Characterizing Apical-out Chicken Intestinal Organoids Grown in a Microcavity Culture Plate

Abstract Intestinal organoids grown in extracellular matrix exhibit epithelial cell polarity with the apical surface internal to the organoid, limiting its access for functional studies. Apical-out organoids can be formed in matrix-free media (in suspension), which introduces different challenges for organoid manipulation (e.g., loss during media changes or washing) due to their “free-floating” nature. Both techniques result in multiple depths of view during visualization. We aimed to determine whether uniformly sized apical-out organoids from chicken could be grown, visualized, and recovered from microcavity culture plates designed for uniform growth of spheroids. Apical-out organoids were developed from crypts/villi isolated from duodenum/jejunum of embryonic-day 18 chicks (ROSS 308) placed in culture media in 12 wells of a 24-well, round-bottom Elplasia culture plate (Corning) at 37°C, 5% CO2 (Day 0). Each well has ~554 microcavities and was seeded with an estimated 300 crypts/villi. Organoid number and area (μm2) were determined daily using an ECHO Revolve Microscope (4x magnification) on Days 1 to 6 from 7 fields of view per well (~33 microcavities per field of view). On Day 1, mean ± SE organoid area was 3,106 ± 60 μm2 (n = 3,590); mean of 43 ± 1 organoids per field. Some organoids exhibited budding by Day 6 where mean area was 5,208 ± 125 μm2 (n = 2,599); 31 ± 1 organoids per field. Organoids were easily recovered from the microcavities of each well for seeding into a 96-well plate and maintenance for an additional 32 h. Analysis at 16.5, 20, 24, 28, and 32 h using PrestoBlue HS (Invitrogen) and a fluorometric microplate reader indicated organoids remained viable for downstream analysis. In conclusion, microcavity culture plates greatly increased ease of visualization, counting, and manipulation of organoids grown in suspension, but organoid size was highly variable.