Video 1. How to dissect a mouse, isolate the intestine, and cut into 5 mm pieces

5. Aspirate the supernatant, being careful to avoid the intestinal fragments, and replace with 10 mL of 5 mM EDTA-PBS. Place at 4 °C on a benchtop roller for 10 min (at the speed of 2.5 scale using the ROTATOR RT-50).

6. Repeat step A5 by aspirating the supernatant, replacing it with 10 mL of 5 mM EDTA-PBS, and then place it at 4 °C on a benchtop roller for 30 min.

7. Wait for fragments to settle by gravity on ice, aspirate the supernatant, gently add 10 mL of cold PBS to wash the crypts, and then vigorously shake for 15 s.

8. Collect this 10 mL supernatant fraction in a separate 15 mL tube (Fraction 1).

Note: Fraction 1 is thrown away because it is a villus-rich fraction.

9. Add 10 mL of cold PBS to the crypts again, vigorously shake for 15 s, and then collect this fraction in a separate 15 mL tube (Fraction 2).

10. Repeat step A9 three times to get a total of five fractions (Fractions 3–5).

11. Filter Fractions 2–5 through a 70 μm cell strainer into a 1% BSA-coated 50 mL tube and then spin the filtrate at 300× g for 5 min at 4 °C.

Note: BSA coating is performed by shaking vigorously a 50 mL tube containing 1% PBS-BSA.

12. Aspirate the supernatant.

Note: The tissues and the crypts have to be kept on ice during the whole procedure of crypt isolation (steps A1–12).

B. Dissociation of crypt cells (Figure 1)

1. Resuspend the pelleted crypts in 1.0 mL of TrypLE Express containing 100 μL of DNase I using a 2 mL pipette and move it into a 15 mL tube on ice.

Note: Since TrypLE Express loses its effectiveness in ISC and Paneth cell isolation within 3–4 weeks after opening, it is recommended to prepare a fresh batch regularly.

2. Immediately incubate the suspended crypts in a 37 °C water bath for 5 min and place on ice again.

Note: Incubation for longer times or at higher temperatures could result in damage to the cells.

3. Promptly move to a clean bench and add 12 mL of ice-cold MEMα. Gently mix by pipetting up and down twice using a 10 mL pipette and centrifuge at 200× g for 5 min at 4 °C. Aspirate the supernatant.

Note: Steps B1–3 are particularly critical, as they can directly damage the cells. It is important to minimize any intervals between these steps as much as possible.

4. Resuspend the pellets in 0.5 mL of ice-cold MEMα containing Pacific Blue–conjugated CD24 antibody, APC-conjugated CD31 antibody, and APC-conjugated CD45 antibody (1/500) and incubate on ice for 15 min. While waiting, prepare basal medium.

5. Add 12 mL of MEMα, triturate pellets, and filter through a 70 μm cell strainer into a 50 mL tube.

6. Transfer the content into a 15 mL tube and centrifuge at 200× g for 5 min at 4 °C. Aspirate the supernatant. While waiting, prepare basal media containing 1.5 μM PI.

7. Resuspend the pellets with 1.5 mL of basal medium containing 1.5 μM PI and transfer through a 40 μm cell strainer into a 50 mL tube.

8. Transfer the sample into a FACS tube.

Note: The condition of the Pipet-Aid can influence isolated cell quality throughout section B. Frequent cleaning and replacement of the filter are especially important to minimize cellular damage.

C. Sorting of Paneth cells and ISCs

1. Prepare two collection tubes (1.5 mL tubes) per sample containing 500 μL of basal medium.

Note: Collection tubes are coated with 1% PBS-BSA.

2. Sort Paneth cells as CD24^hi SideScatter^hi Lgr5-EGFP⁻CD31⁻CD45⁻PI⁻ cells and ISCs as Lgr5-EGFP^hi CD24⁻ CD31⁻CD45⁻PI⁻ cells by FACS cell sorter. Gating for single-cell isolation is shown in Figure 2. Approximately 20,000–50,000 ISCs and Paneth cells per mouse are sorted into each collection tube.

Note: Filter the samples through a 40-µm cell strainer again right before sorting, when you start sorting more than 1 h after preparation of dissociated cells.

Figure 2. Gating strategy used for cell sorting by the BD FACS Aria III Cell Sorter. All events were first plotted based on FSC-A and PI to exclude dead cells (P1). Single cells were identified by SSC-H vs. SSC-W (P2) and FSC-H vs. FSC-W (P3). Subsequent gating on FSC-H vs. FSC-A (P4) excluded APC⁺ cells, yielding the APC^- fraction (P5). Within this population, Paneth cells were identified as Pacific Blue⁺ cells (P6). The remaining Pacific Blue^- fraction was further analyzed for GFP expression, and GFP⁺ cells were defined as Lgr5⁺ intestinal stem cells (ISCs, P9). Arrows indicate the sequential gating hierarchy.

D. Co-culture of Paneth cells and ISCs

1. Centrifuge collection tubes at 300× g for 5 min at 4 °C.

2. Aspirate the supernatant. Total volume is adjusted to 30–100 μL depending on the number of sorted cells by basal medium.

3. Pick up 5 μL from a collection tube, put it on a hemocytometer, and count the number of cells under the microscope. Cell concentration should be 1,000–2,000 cells/µL.

4. Put 2,000 Paneth cells with 2,000 ISCs at the bottom of a new 1.5 mL tube on ice.

5. Prepare organoid growth medium.

6. Prepare 75% Matrigel diluted by organoid growth medium. Put JAG-1 (1/1,000: 1 μM) and Y-27632 (1/1,000: 10 μM) into diluted Matrigel.

Notes:

1. The protein concentration of the original Matrigel should be 9–10 mg/mL.

2. JAG-1 in the Matrigel mixture stimulates Notch signaling, which is critical for ISC maintenance via interaction with Paneth cells.

7. Put 25 μL of prepared Matrigel on the mixture of ISCs and Paneth. Gently mix them and put the Matrigel drops with Paneth cells and ISCs on a 48-well plate (Figure 3).

8. Incubate the 48-well plate for 10 min in a 37 °C incubator to allow the Matrigel drops with ISCs and Paneth cells to solidify.

9. Put 250 μL of organoid growth media gently on the solidified Matrigel in each well.

Note: Media does not have to be exchanged from day 1 to day 5 of culture.

10. Quantify the number of colonies with lumen (Figure 4) under the microscope on day 5 of culture.

Note: We recommend setting up more than two technical replicates to give a reliable count.

Figure 3. Schematic diagrams of the co-culture of Paneth cells and ISCs. Sorted cells, containing 2,000 stem cells and 2,000 Paneth cells, were embedded in 25 µL of Matrigel and plated in a dome shape. After 10 min, 250 µL of culture medium was gently overlaid.


Figure 4. Organoid formation from the co-culture of isolated Paneth cells and intestinal stem cells (ISCs). ISCs and Paneth cells were isolated from ad libitum–fed (AD) and calorie-restricted (CR) mice by flow cytometry to more than 90% purity, and 2 × 10³ cells each were co-cultured. In trypan blue staining, the viability of isolated ISCs was 94.9%, whereas that of Paneth cells was 95.6%. The number of colonies was assessed at day 5 (3 wells/ group) (A), and representative images of the organoids (B) are shown. Original magnification: 40×. Scale bar: 100 μm. Values represent the mean ± SEM. Significant differences are denoted by p values (Student’s t-test). Red arrow marks organoids. Calorie restriction enhances the supportive function of the Paneth cell as described previously [5].

Validation of protocol

This protocol has been used and validated in the following research articles:

1. Isotani et al. [8]. Nicotine enhances the stemness and tumorigenicity in intestinal stem cells via Hippo-YAP/TAZ and Notch signal pathway. eLife (Figure 2).

2. Igarashi et al. [7]. NAD+ supplementation rejuvenates aged gut adult stem cells. Aging Cell (Figure 2).

3. Igarashi et al. [5]. mTORC1 and SIRT1 Cooperate to Foster Expansion of Gut Adult Stem Cells during Calorie Restriction. Cell (Figures 3–6).

General notes and troubleshooting

Troubleshooting

Problem: Organoids do not grow, or the organoid size is too small.

Possible cause: ISCs and Paneth cells are damaged in the isolation procedures.

Solution: Replace TrypLE Express or the filter of Pipet-Aid. Make sure that steps B1–3 are performed without stopping. When the amount of crypt pellets is large, cells tend to be damaged in section B (Figure5). Then, adjust the size of the crypt pellets by discarding some of Fractions 2–5 in step A11.

Figure 5. Example of an appropriate pellet size (corresponding to step B3, after centrifugation). Left: Cells in too large crypt pellets tend to be damaged in step B. Right: The example of appropriate size pellet in step B3. The pellet size should be adjusted to the size shown above.

Acknowledgments

We acknowledge the IMSUT FACS Core laboratory for assistance with flow cytometry analysis.

We acknowledge the authors of the previous work [7] from which this protocol was modified.

Competing interests

The authors declare that they have no competing interests.

Ethical considerations

Animal experiment was performed according to the protocol approved by the Committee on the Ethics of Animal Experiments of the University of Tokyo (Permit Number: A2023M043).

References

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2. Barker, N., van Es, J. H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P. J., et al. (2007). Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 449(7165): 1003–1007. https://doi.org/10.1038/nature06196
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5. Igarashi, M. and Guarente, L. (2016). mTORC1 and SIRT1 Cooperate to Foster Expansion of Gut Adult Stem Cells during Calorie Restriction. Cell. 166(2): 436–450. https://doi.org/10.1016/j.cell.2016.05.044
6. Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., van Es, J. H., Abo, A., Kujala, P., Peters, P. J., et al. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 459(7244): 262–265. https://doi.org/10.1038/nature07935
7. Igarashi, M., Miura, M., Williams, E., Jaksch, F., Kadowaki, T., Yamauchi, T. and Guarente, L. (2019). NAD⁺ supplementation rejuvenates aged gut adult stem cells. Aging Cell. 18(3): e12935. https://doi.org/10.1111/acel.12935
8. Isotani, R., Igarashi, M., Miura, M., Naruse, K., Kuranami, S., Katoh, M., Nomura, S. and Yamauchi, T. (2025). Nicotine enhances the stemness and tumorigenicity in intestinal stem cells via Hippo-YAP/TAZ and Notch signal pathway. eLife. 13: e95267. https://doi.org/10.7554/elife.95267