Fig 1.
Growth dynamics of bovine rectal organoids derived from fresh and cryopreserved tissues.
(A) Phase-contrast microscopy provided representative images showcasing isolated crypts 24 hours post-seeding in Matrigel (D1) and the evolution of organoids during the initial 1–2 weeks of culture in Matrigel (D3-12), across both the first (P0) and third passages (P3). Notably, organoids failed to develop from the flash-frozen tissue samples. (B&C) The surface area of growing organoids was measured on Days 1, 3, and 5 during P0, and on Day 5 during P3. The organoids derived from fresh tissues were significantly larger than those from slow-frozen tissues during P0 (p<0.001), but this size discrepancy was not observed at P3 (p = 0.24) (B). Additionally, the growth rates of organoids during P0, expressed as fold changes relative to their size on Day 1, showed no significant differences between the groups (C). These measurements were based on 4 to 10 randomly selected organoids from a minimum of two biological replicates. The results are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed with Wilcoxon’s signed rank tests. FT, Fresh Tissue; SFT, Slow-Frozen Tissue; FFT, Flash-Frozen Tissue. Bar, 100 μm. *** p<0.001.
Fig 2.
Immunocytochemical characterization of fresh (FT) and slow-frozen tissue (SFT)-derived bovine rectal organoids.
(A) Structural and cellular characteristics of organoids were indifferent between the two groups. Confocal microscopy images demonstrated formation of cystic luminal structure with basolateral epithelial adherens junctions (E-cadherin, yellow), apical brush border (F-actin, cyan) and basal nuclei (DAPI, blue), and presence of multi-cellular populations including stem cells (SOX9, magenta), mucin-producing goblet cells (SNA, magenta), epithelial cells (EpCAM, yellow), and actively proliferating cells (EdU, magenta). (B) The percent positive cells for SOX9, SNA and EdU were evaluated by normalizing the number of positively stained cells by the total numbers of nuclei. No difference was noted between the two groups in all markers using Wilcoxson’s signed rank test (SOX9) or paired t-tests (SNA and EdU). 4 to 10 randomly selected organoids from at least two biological replicates were assessed. The results are presented as mean ± standard error of the mean (SEM). Bar, 50 μm.
Fig 3.
Gene expression profiles of fresh (FT) and slow-frozen tissue (SFT)-derived bovine rectal organoids.
RT-qPCR was performed to determine expression of epithelial cell markers: Leucine rich repeat containing G protein-coupled receptor 5 (LGR5) for stem cells (A), Chromogranin A (CHGA) for enteroendocrine cells (B), Lysozyme C (LYZC) for Paneth cells (C), Mucin 2 (MUC2) for goblet cells (D), and fatty acid-binding protein 2 (FABP2) for enterocytes (E) in organoids at Day 5 of culture following three passaging. Three technical replicates from three biological replicates were used. The gene expression levels of each of the target genes were calculated relative to that of the internal control, which was the mean of GAPDH, RPL0, and ACTB. The results are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed with either paired t-test (LGR5 and CHGA) or Wilcoxon’s signed rank test (LYZC, MUC2, and FABP2) for independent samples. * p<0.05, ** p<0.01.
Fig 4.
Development of stable bovine rectal organoid-derived 2D monolayer.
(A) Representative phase-contrast microscopy images of 2D monolayer at Days 2, 6 and 8 (D2-8) of culture on a cell culture insert. (B) Transepithelial electrical resistance (TEER) and permeability assay using 4 kDa FITC-dextran demonstrated development of stable and functional epithelial barrier integrity by Day 3 of culture. The results are presented as mean ± standard error of the mean (SEM) from a minimum of two biological replicates with at least two technical replicates. (C) Immunofluorescent staining revealed uniform expression of F-actin (Phalloidin, cyan) and basolateral adherens junctions (E-cadherin, yellow), and presence of mucin-producing goblet cells (SNA, magenta). Nuclei are visualized by DAPI staining (blue). Top-down view and z-stack images are shown. Bars, 50 μm. (D) RT-qPCR of the monolayer at Day 6 of culture documented expression of stem and lineage cell marker genes. Two technical replicates from three biological replicates were evaluated. The gene expression levels of each of the target genes were calculated relative to that of the internal control, which was the mean of GAPDH, RPL0, and ACTB. LGR5: Leucine rich repeat containing G protein-coupled receptor 5, CHGA: Chromogranin A, LYZC; Lysozyme C, MUC2: Mucin 2, FABP2: Fatty acid-binding protein 2.
Fig 5.
Electron microscopic characterization of the bovine rectal organoid-derived 2D monolayer.
Representative scanning (A-D) and transmission (E-G) electron microscopy images of 2D monolayer on Day 5 of culture on a cell culture insert. The formation of microvilli on the apical surface of the monolayer was confirmed at low (A) and high (B) power magnifications. A goblet cell was identified on the apical surface of the monolayer at low power magnification (C), where a goblet cell orifice (GO) and microvilli were highlighted at high power magnification (D). (E) A low power magnification image revealed uniformly developed microvilli (MV) on the apical surface and a goblet cell containing multiple mucin granules (MG) in the cytoplasm. N = nucleus. (F&G) A high power magnification images highlight the formation of inter-cellular tight junctions (TJ), desmosome (D) and microvilli (MV) covered with glycocalyx (GLX). Bars, 1 μm.