Prolonged culturing of colonic epithelial organoids derived from healthy individuals and ulcerative colitis patients … – Nature.com

Patient cohort description and pathology classification

The present study was approved by the Kaunas Regional Biomedical Research Ethics Committee (Protocol No. BE-2-31) and written informed consent was obtained from each subject who participated in the study. All research was performed in accordance with relevant guidelines and regulations.This study consisted of three age- and sex-matched groups (Fig.4). It enrolled 13 age- and sex-matched patients (7 males, 6 females, mean age of patients group 41.915.4 years) with a previously established diagnosis of UC based on clinical, endoscopic, and histological examinations, that were scheduled for a colonoscopy either because of a disease flare or for screening purposes. 6 subjects who underwent colonoscopy procedure through colorectal cancer screening program (3 males, 3 females, mean age of control group 53.28.5 years) without inflammatory, oncological, or other gastrointestinal diseases were enrolled as controls. Colon biopsy samples from sigmoid colon, rectum, or descending colon were obtained during standard colonoscopy procedure from control group individuals and subjects with active UC (aUC) or UC in remission (quiescentqUC) who were examined at the Department of Gastroenterology, Hospital of Lithuanian University of Health Sciences. Remission of ulcerative colitis was confirmed in patients with stool frequency3/day, no rectal bleeding, and healed mucosa at endoscopy (Endoscopic Mayo score1). The biopsies for tissue level methylation analysis were immediately flash frozen, whereas biopsies for organoid establishment were placed in DMEM/F-12 medium and processed immediately. Table 2 represents other summarized clinical and demographic data of the study subjects.

Scheme representing experimental study design and workflow (created with BioRender.com).

3D undifferentiated colonic epithelial organoids from adult intestinal stem cells were established and cultured according to the protocol of IntestiCult Organoid Growth Medium (Human) (OGMH) (06010, StemCell Technologies) with slight adjustments. Briefly, colon biopsies were first minced with sterile scalpel and incubated in Gentle Cell Dissociation reagent (1000485, StemCell Technologies) to digest colon tissue. After centrifugation and removal of the supernatant, crypts containing intestinal stem cells were removed from biopsies by vigorous pipetting in cold DMEM/F-12 (supplemented with 1% BSA and 15mM HEPES) medium, passed through a 70m pore filter, and the number of isolated crypts was estimated. After centrifugation and removal of the supernatant, isolated colonic crypts were mixed with basement membrane matrix Matrigel (356231, Corning) and seeded into a 24-well cell culture plate, forming 50l volume domes. Colon epithelial organoids were cultured in OGMH medium containing factors necessary for structure formation and stem cell renewal, supplemented with antibiotics (penicillin/streptomycin (100g/ml) (15140122, Gibco) and the RHO/ROCK signaling pathway inhibitor Y-27632 (for the first two days after seeding). OGMH was changed every 2days. Colonic organoids were incubated at 37C with 5% CO2. The growth of undifferentiated 3D colonic organoids was evaluated microscopically (inverted fluorescent microscope ZEISS Axio Observer 7, ZEISS ZEN 3.1 (blue edition) software). The first splitting of the organoid culture was performed after 714days. Each subsequent passage of organoids was performed once the organoids were mature (710days post-passage) until the fifth passage. A portion of fully formed undifferentiated colonic organoids after passage 0, 1, and 5 and portion of initial samples of isolated colon crypts were cryopreserved using CryoStor CS10 (07930, StemCell Technologies) cell storage reagent. The suspension was transferred to a cryotube and immediately placed at 80C. See Fig.4 for the overview of study design.

The morphology, cellular composition and functional parameters of the formed 3D intestinal epithelial organoids were evaluated by brightfield and immunofluorescence microscopy. Organoid growth dynamics were monitored daily. For immunofluorescence microscopy, undifferentiated organoids were fixed in 4% paraformaldehyde (1.00496.0700, Sigma-Aldrich) solution, incubated for 30min, thus releasing them from the Matrigel matrix. Further organoid cells were permeabilized with 0.5% Triton-X (900293-1, Sigma-Aldrich) solution and blocked with 2% BSA blocking solution. Finally, fluorochrome-conjugated monoclonal antibodies diluted in antibody dilution solution (1:501:500) were added to the prepared organoids and incubated for 60min at RT. Antibodies were applied that are specifically directed against: 1. cell polarity markers (Anti-beta-catenin-Alexa Fluor 488 (53-2567-42, eBioscience), F-actin phalloidin-Alexa Fluor 660 (A22285, Invitrogen)); 2. tight-junction marker (Anti-ZO-1-Alexa Fluor 555 (MA3-39100-A555, Invitrogen)); 3. proliferating cell marker (Anti-ki67-Alexa Fluor 488 (ab206633, Abcam)); 4. markers to identify differentiated/specialized cells (Goblet cells, colonocytes, enteroendocrine cells) (Anti-Mucin2-Alexa Fluor 555 (bs-1993R-A555, Biocompare), anti-Cytokeratin 20-Alexa Fluor 488 (ab275988, Abcam), anti-Chromogranin A-Alexa Fluor 488 (ab199192, Abcam), respectively). Cell nuclei were labeled with the fluorescent dye Hoechst 33342 (R37605, Invitrogen). Both brightfield and immunofluorescence microscopy of the samples were performed with 5, 10and 40objectives using an inverted fluorescence microscope ZEISS Axio Observer with ZEISS ZEN 3.1 (blue edition) software.

DNA from fresh frozen biopsy samples, cryopreserved crypts and organoid specimens was extracted using AllPrep DNA/RNA Mini Kit (80204, Qiagen). Briefly, frozen biopsy samples were lysed on MagNA Lyser (Roche Diagnostics) (6000rpm, twice for 15s with a 15s break) using Lysing Matrix D tubes (116913050-CF, MP Biomedicals) and 350l buffer RLT Plus. Cryopreserved pellets of crypts and organoids in CryoStor CS10 medium (07930, StemCell Technologies) were gently thawed at+4C and centrifuged for 5min at 400g at+4C. Supernatant was removed and pellets was lysed in 350l buffer RLT Plus. The following steps of DNA extraction from the lysates of biopsies, crypts and organoids were performed in line with the manufacturers instructions.

In total 200 ng of the isolated genomic DNA was bisulfite converted using MethylCode Bisulfite Conversion Kit (MECOV50, Applied Biosystems) and applied for 146 bp size LINE-1 region amplification via PCR. Samples containing no template were used for PCR contamination control. Custom-made primers set (F: 5-TTTTGAGTTAGGTGTGGGATATA-3, R: 5-biotin-AAAATCAAAAAATTCCCTTTC-3) (final concentration of each 0.2M) and PyroMark PCR Kit (978703, Qiagen) was used for PCR amplification. Thermal-cycling conditions: 95 C for 15 min; 45 cycles of 94C for 30s, 56C for 30s, 72C for 30 s; 72C for 10 min cycling mode. The specificity of PCR amplicon was verified using 2% agarose gel.

Methylation level of three CpG islands in amplified LINE-1 region was analyzed using PyroMark Q24 (Qiagen) pyrosequencing system. Briefly, 20 l of PCR product was immobilized to Streptavidin Sepharose HP beads (17-5113-01, Cytiva), processed with the PyroMark Q24 Vacuum Workstation and annealed to the sequencing primer 5-AGTTAGGTGTGGGATATAGT-3. Sequence analysis was performed by applying PyroMark Gold Q24 reagents (970802, Qiagen). All samples were analyzed in duplicates. Positive control CpG Methylated Human Genomic DNA (SD1131, Thermo Scientific) and PCR negative control were used in each sequencing run. Methylation level of 60% and higher value was considered as high LINE-1 methylation based on the previous publications25,29,30.

The pyrograms of LINE-1 region were analyzed using the PyroMark Q24 software (v. 2.0.8, Qiagen). Statistical analysis of methylation data results and data visualization were performed using R studio (R version 4.0.3) software and its packages (ggplot2, ggsignif, ggpubr, scales, base, stats, tidyverse). Differences between groups were considered statistically significant when the calculated p-value was equal to or lower than the critical level (p0.05). The distribution of data in groups according to the normal (Gaussian) distribution was assessed by the ShapiroWilk normality test. Since the data in the study groups were not normally distributed, the Wilcoxon signed-rank test was used for statistical analysis.

Read the original:
Prolonged culturing of colonic epithelial organoids derived from healthy individuals and ulcerative colitis patients ... - Nature.com