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Colorectal cancer remains one of the most commonly diagnosed cancers and a leading cause of cancer-related mortality worldwide¹. To investigate tumor biology and evaluate therapeutic strategies, researchers have traditionally used cancer cell lines and animal models. However, these systems often fall short in capturing the complexity of human tumors^1,2.

Cancer-derived intestinal organoids, also known as patient-derived tumor organoids, offer 3D models that retain the tissue architecture, cellular heterogeneity, and morphology of the original tumor, providing a physiologically relevant experimental system.^2,4. When cultured with IntestiCult™, they provide a robust platform for studying cancer biology, drug response, and signaling pathways^3-5.

Increasingly adopted as part of New Approach Methodologies (NAMs), organoid models offer a complex in vitro system for studying metabolic, transport, and other functional characteristics, enabling their use in preclinical high-throughput drug screening, target validation, and predictive toxicology. They also support immune co-culture systems, allowing the study of tumor-T cell interactions for immunotherapy research^4-7.

In this Technical Bulletin, we describe the culture of Wnt-independent cancer-derived organoids using IntestiCult™ Plus (Catalog #100-1677) or IntestiCult™ OGM Human Basal Medium (Catalog #100-0190).

For complete instructions, use this document in coordination with the Product Information Sheets (PIS) for IntestiCult™ Plus () and IntestiCult™ OGM Human Basal Medium (Document #10000023716), which includes a materials list and instructions for isolating human colonic crypts from biopsy samples, establishing human intestinal organoids from the isolated crypts, as well as expanding and maintaining organoid cultures via passaging.

Protocol

The following protocol was developed for the culture of Wnt-independent colorectal tumor biopsies in IntestiCult™ OGM Human Basal Medium or in IntestiCult™ Plus. Tumors that do not carry an activating mutation of the Wnt pathway cannot be maintained as organoids using this method.

A. Medium Preparation

1. For cultures using IntestiCult™ Plus:
1. Thaw IntestiCult™ Plus Basal Medium at room temperature (15 - 25°C) or at 2 - 8°C overnight. Mix thoroughly. Use immediately or aliquote and store at -20°C for up to 12 months. After thawing aliquots, use immediately. Do not re-freeze.
2. Add desired antibiotics immediately before use (e.g. 50 μg/mL gentamicin).
2. For cultures using IntestiCult™ OGM Human Basal:
1. Thaw IntestiCult™ OGM Human Basal Medium at room temperature (15 - 25°C) or at 2 - 8°C overnight. Mix thoroughly. Use immediately or aliquot and store at −20°C for up to 3 months. After thawing aliquots, use immediately. Do not refreeze.
2. Add 50 mL IntestiCult™ OGM Human Basal Medium to 50 mL DMEM/F-12 with 15 mM HEPES. Mix thoroughly. If not used immediately, store at 2 - 8°C for up to 1 week.
3. Add desired antibiotics immediately before use (e.g. 50 µg/mL gentamicin).

B. Isolation of Tissue from Tumor Biopsies

1. Thaw 100 μL of Matrigel® on ice.
   Note: This is sufficient for plating up to 4 culture domes.
2. Place the following reagents on ice: D-PBS (Without Ca++ and Mg++) and DMEM + 1% BSA (See PIS Document #10000031450 or #10000023716 for preparation).
3. Warm a tissue culture-treated 24-well plate in a 37°C incubator for at least 2 hours.
   Note: If using 24-well Organoid Culture Plates, warming is not necessary.
4. In a 15 mL conical tube, wash the tissue sample with 10 mL of ice-cold PBS. Allow the tissue to settle by gravity (~5 seconds) then aspirate the supernatant.
5. Repeat step 4, leaving 1 mL of supernatant in the tube.
6. Transfer the tissue and remaining supernatant to a 1.5 mL microcentrifuge tube using a 1 mL pipettor.
7. Using sterile scissors, thoroughly mince the tissue into ~5 mm pieces. Transfer the fragments to a new 15 mL conical tube. Rinse the microcentrifuge tube with PBS and add the rinse to the tissue fragments.
8. Allow the tissue to settle (~5 seconds), then aspirate the supernatant.
9. Add 10 mL of Gentle Cell Dissociation Reagent. Incubate at 37°C on a rocking platform set at medium speed (~40 rpm) for 60 minutes.

C. Organoid Culture From Isolated Biopsy Tissue

For complete instructions on plating isolated tissue as organoid cultures, proceed to section B, step 1 in the IntestiCult™ Plus PIS (Document #10000031450) or section B, step 2 in the IntestiCult™ OGM Human Basal PIS (Document #100000237). The growth medium should be replaced every 2 days, and cultures can be passaged every 6 - 12 days.

For complete passaging instructions, refer to section C of the relevant PIS. When passaging organoids derived from cancerous tissue, use the medium prepared in section A (above).

Product Information