CRC Organoid Culture: A Method to Obtain 3D Organoids from Colorectal Cancer Cells

Overview

This video describes the detailed protocol for generating 3D organoids from murine-model derived colorectal cancer cells by propagating them on artificial extracellular matrix coated plates. These CRC organoids can be used as a model to study the primary growth of human tumor cells and metastasis.

Protocol

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Extraction of PDXs into the Cell Suspension for the CRC Organoid Culture

Experimental procedures for dissociation of CRC PDXs are outlined in Figure 1B (step 3).

1. Place the tumor fragments derived from NOD/Shi-scid IL2Rγ null (NOG) mouse on a 10 cm Petri dish.
2. Mince the fragments using sterile razor blades and transfer them into a 15 mL conical tube containing 8 mL of 10% fetal calf serum (FCS)-Dulbecco's Modified Eagle's Medium (DMEM) with 80 µL of collagenase enzyme stock (see Table of Materials).
3. Close the tube tightly and wrap the cap with parafilm to prevent leakage. To dissociate the minced tumor fragments into the cell suspension, agitate it gently for 2 h at 37 °C. Note that there will still be many fragments of undigested tissue remaining in the tube.
4. Centrifuge the tube at 300 x g for 5 min at 4 °C and remove the supernatant. Resolve the cell pellet using 10 mL of 10% FCS-DMEM to make the cell suspension.
5. To eliminate undigested tissue debris, filter the cell suspension twice using the 40 µm cell strainer set on a 50 mL conical tube. Then, centrifuge the flow-through at 300 x g for 5 min at 4 °C. Remove the supernatant and keep the pellet on ice.

2. Generation of the CRC Organoids Cultured on Artificial Extracellular Matrix

Experimental procedures for the CRC organoid culture of the colon PDXs are outlined in Figure 1B (step 4).

1. Establish a culture medium suitable for the PDX-derived CRC organoids (see Table of Materials, "the CRC organoid culture medium").
2. Apply 150 µL of artificial extracellular matrix (see Table of Materials) per well on a 12-well plate on ice and then incubate it for 30-60 min in a 37 °C, 5% CO₂ incubator to solidify the gels.
3. Suspend the cell pellet (as described in 1.5). Remove the supernatant and keep the pellet on ice) in the CRC organoid culture medium (from step 2.1) with 5% FCS to achieve adjustment to 3 x 10⁵ cells/mL. Then, seed 1 mL of the medium onto the artificial extracellular matrix-coated plate prepared in step 1.2 and incubate overnight at 37 °C under 5% CO₂. Use a hemocytometer for counting the number of tumor cells including single cells and multicellular clusters (a group of adherent cells) in the cell suspension.
   NOTE: 5% FCS is used to quench the residual enzymatic activity of collagenase in this study.
4. Carefully collect the culture medium, including floating cells that have detached from the artificial extracellular matrix-coated plate, into a 1.5 mL centrifuge tube on the following day and centrifuge it at 1,400 x g for 5 min. Then, remove the supernatant and resuspend the pellet in 70 µL of artificial extracellular matrix on ice.
5. To increase CRC cell viability, overlay the tumor cell-containing artificial extracellular matrix onto the tumor organoid cells attached to the artificial extracellular matrix-coated plate and incubate for 30 min in a 37 °C, 5% CO₂ incubator to solidify the artificial extracellular matrix coating. Then, incubate it with 1 mL of the CRC organoid cell culture medium with 1% FCS at 37 °C under 5% CO₂.
6. Change the culture medium every second day. As the CRC organoids fill the medium, it may become necessary to change the medium daily.

Representative Results

Figure 1: Schematic representation of generation of metastases by the PDX-derived CRC organoids labeled with GFP lentivirus in NOG mice. (A) Implantation of small pieces of the CRC tissue subcutaneously into NOG mice (step 1). The CRC tissue surgically dissected from the patient was cut into pieces and implanted subcutaneously into NOG mice. s.c.: subcutaneous implantation. (B) Generation of CRC organoids dissociated from PDXs (step 2–4). The developed CRC xenografts were minced (step 2) and transferred into a 15 mL tube containing the culture medium including collagenase (step 3). After incubation with slow agitation, the CRC cell suspension was filtered (step 3). Then, the organoid cell suspension in the CRC organoid medium was seeded onto an artificial extracellular matrix-coated plate and incubated overnight in a CO₂ incubator (step 4). The CRC organoid cells attached to the artificial extracellular matrix were also coated with an additional artificial extracellular matrix and incubated in a CO₂ incubator (step 4). s.c.: subcutaneous implantation. (C) Generation of CRC organoids transduced by GFP lentiviral particles prior to employing injection into recipient mice (step 5–7). The GFP lentiviral particles were generated at a high titer (step 5). The PDX-derived CRC organoids grown on an artificial extracellular matrix were directly harvested with a cell scraper and transferred into a microtube (step 6). After centrifugation, the cell pellet was resuspended in PBS. The cell suspension was centrifuged and the cell pellet was dissociated. Then, the CRC organoid cell suspension was incubated with GFP virus stock in the CRC organoid culture medium on the artificial extracellular matrix-coated plate overnight in a CO₂ incubator (step 6). The CRC organoid cells attached to the artificial extracellular matrix were coated with an additional artificial extracellular matrix and incubated in a CO₂ incubator to solidify the artificial extracellular matrix coating (step 6). The CRC organoids were then cultured for periods of 7–10 days to expand cell growth (step 6). To develop a spontaneous metastasis model, the dissociated 5 x 10⁵ CRC organoid cells labeled with GFP suspended in 50 µL of PBS with 50% artificial extracellular matrix were injected orthotopically into NOG mice (step 7). To generate an experimental metastasis model, 4 x 10⁴ CRC organoid cells labeled with GFP in 50 µL of PBS were injected intrasplenically into NOG mice (step 7). o.t.: orthotopic injection, i.s.: intrasplenic injection.

Materials

Name	Company	Catalog Number	Comments
NOD/Shi-scid IL2Rγ null (NOG) mice	The Central Institute for Experimental Animals,Kanagawa, Japan		Breed 6-week-old male mice under germ-free and specific pathogen free conditions
CRC organoid culture medium with 1% or 5% FCS			DMEM/F-12 with GlutaMAXTM supplement (Gibco #10565018) supplemented with 1% or 5% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 ng/ml hEGF and 10 µM Y27632, a ROCK inhibitor. Store at 4°C. Use within 1 month.
Matrigel basement membrane matrix	Corning	#354234	Store aliquots at -20°C. Place on ice until use
Collagenase type 1	Sigma	#C1030	150 mg/ml collagenase type1 in 1×PBS. Store aliquots at -20°C for up to 1 year
Hemocytometer	Erma	#03-202-1
DMEM/F-12 with GlutaMAXTM	Gibco	#10565018	Store at 4°C. Warm at 37°C before use
50ml conical tube	Sumitomo Bakelite	MS-57500
Microtube	Eppendorf	#0030120086	Autoclave before use
40μm cell strainer	Corning	#352340