Generation of human colon organoids from healthy and inflammatory bowel disease mucosa

Abstract

Ulcerative colitis and Crohn's disease are chronic inflammatory bowel diseases (IBD) of unknown cause characterized by a relapsing-remitting behavior. Growing evidence supports the idea that the epithelial barrier plays a central role in the pathogenesis of IBD as well as in its evolution over time, thus representing a potential target for novel therapeutic options. In the last decade, the introduction of 3D epithelial cultures from ex vivo-expanded intestinal adult stem cells (ASCs) has impacted our ability to study the function of the epithelium in several gastrointestinal disorders, including IBD. Here, we describe in detail a reproducible protocol to generate Matrigel-embedded epithelial organoids from ASCs of non-IBD and IBD donors using small colonic biopsies, including steps for its optimization. A slightly modified version of this protocol is also provided in case surgical samples are used. With this method, epithelial organoids can be expanded over several passages, thereby generating a large quantity of viable cells that can be used in multiple downstream analyses including genetic, transcriptional, proteomic and/or functional studies. In addition, 3D cultures generated using our protocol are suitable for the establishment of 2D cultures, which can model relevant cell-to-cell interactions that occur in IBD mucosa.

Fig 1. Schematic representation of the experimental workflow from sample collection to 3D organoid generation and differentiation.

Running the first round of expansion (i.e., from crypts to organoids and their differentiation) takes 13 days on average. Step 3 can be repeated several times (↻) depending on the experimental plan. Once generated, both stem cell-enriched and differentiated cultures can be used for several downstream applications, including transcriptional analysis of any kind (from qPCR to single cell RNAseq), imaging, and protein analysis (e.g., Western Blot or ELISA assay). This figure was created using www.biorender.com.

Fig 2. Pie chart indicating the success rate of generating organoids from non-IBD and IBD (UC and CD) mucosa using the described protocol.

Data were retrieved from a cohort of biopsy samples collected over a 5-year period (2016–2021) and which were processed according to the described protocol. Biopsy samples in non-IBD, UC and CD groups are from pediatric and adult subjects (representing the 23.2% and the 76.8% of the total number of biopsies, respectively), and were obtained from different parts of the colon (i.e., ascending, descending and sigmoid colon). The percentages refer to the successful generation (“Expansion”) of viable organoids after one passage (from crypts culture to organoids), with success being defined as the generation of a growing culture of 3D organoids after 5 days of expansion. In our hands, failure (“No expansion”) primarily stemmed from crypt culture contamination due to microorganisms or from undetermined causes. For UC and CD patient cohorts, samples were derived from non-inflamed/mildly inflamed involved colonic mucosa. Total number of biopsy samples processed: non-IBD samples, n = 74; UC samples, n = 49; CD samples, n = 66.

Fig 3. Representative histological sections stained with hematoxylin and eosin of colonic biopsies from non-IBD and IBD donors.

The images show the marked histological alterations of the epithelial layer due to inflammation in active IBD mucosa, compared to the healthy and non-inflamed IBD (i.e., uninvolved or previously affected) mucosa. (A) Healthy sigmoid mucosa from a non-IBD subject; (B) Non-inflamed ascending colonic mucosa from a UC patient; (C) Inflamed sigmoid colonic mucosa from a UC patient; (D) Non-inflamed sigmoid colonic mucosa from a CD patient; (E) Inflamed ascending colonic mucosa from a CD patient. All samples are from adult donors. Scale bar: 200 μm.

Fig 4. Swelling of colonic crypts embedded in Matrigel and cultured in organoid growth medium for 24–48 hours.

If the adult stem cell niche is preserved, swelling of the lumen will be observed in most viable crypts. (A) Swelling of an intact crypt derived from a non-inflamed sigmoid mucosa of a pediatric CD patient; (B) Swelling of a broken crypt derived from a non-inflamed sigmoid mucosa of an adult CD patient. Scale bar: 200 μm.

Fig 5. 3D expansion of colonic ASCs derived from the dissociation of organoid cultures generated from non-IBD and IBD donors.

Once properly dissociated (day 0), organoid cultures can be expanded for 5–6 days before further organoid dilution is required. Samples were derived from the healthy sigmoid colon of a non-IBD donor, the non-inflamed transverse colon of a UC patient, and the not inflamed sigmoid colon of a CD patient. The organoid cultures shown in the figure, all generated from adult subjects, had been previously expanded for two passages. Scale bar: 200 μm.

Fig 6. Differentiation of organoids generated from non-IBD and IBD donors.

The image shows the morphological heterogeneity that can be observed within the differentiated cultures, regardless if there were derived from non-IBD or IBD subjects. “Stem”: organoid cultures enriched in the stem cell population on day 5 after passaging; “Diff”: organoid cultures on day 5 after differentiation. Samples consist of biopsies obtained from the healthy sigmoid colon of a non-IBD donor, the non-inflamed sigmoid colon of a UC patient, and the mildly inflamed sigmoid colon of a CD patient.