Organoid Passaging and Mechanical Expansion Protocols
Materials Required
Principle
Organoid passaging by mechanical expansion transfers established 3D epithelial organoids from an extracellular matrix dome into fresh matrix after physical fragmentation. The readout is successful re-formation and expansion of organoid fragments into new organoids, reflecting survival of organoid-forming epithelial stem/progenitor cells and continued self-organization in a 3D matrix[1][2][3].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use established organoid cultures, ice-cold AdvDMEM+++, basement membrane extract or Matrigel/BME for 3D embedding, and organoid expansion medium matched to the tissue type.
• BME/Matrigel provides a basement-membrane-like 3D scaffold, while expansion medium supplies niche factors required for continued organoid growth[1][2][4].
• Morphology by bright-field microscopy is the reported routine assessment during expansion.
• Use sterile culture plates, a P1000 pipette, 15-ml tubes, a refrigerated centrifuge, a 37°C/5% CO2 incubator, and a sterile plugged glass Pasteur pipette fitted with a sterile 10-µl pipette tip for mechanical fragmentation[1].
• BME/Matrigel provides a basement-membrane-like 3D scaffold, while expansion medium supplies niche factors required for continued organoid growth[1][2][4].
• Morphology by bright-field microscopy is the reported routine assessment during expansion.
• Use sterile culture plates, a P1000 pipette, 15-ml tubes, a refrigerated centrifuge, a 37°C/5% CO2 incubator, and a sterile plugged glass Pasteur pipette fitted with a sterile 10-µl pipette tip for mechanical fragmentation[1].
Experimental Procedure
• Prepare established organoid cultures for passage when they have expanded sufficiently; in human intestinal organoids, about 7 days is used as a standard estimate, but the optimal interval varies by line.
Passaging too early can reduce expansion efficiency, while passaging too late can lead to spontaneous differentiation, reduced viability, and suboptimal expansion[1].
• Prewarm culture plates before plating, keep AdvDMEM+++ ice-cold during organoid recovery, and prepare fresh BME/Matrigel for replating.
Reported BME volumes are 200 µl per well in 6-well plates, 100 µl per well in 12-well plates, 50 µl per well in 24-well plates, 25 µl per well in 48-well plates, and 5-10 µl per well in 96-well plates[1].
• Remove culture medium from established organoid wells, flush organoids with 1 ml ice-cold AdvDMEM+++ using a P1000 pipette to disrupt BME droplets, and transfer the recovered organoids into a 15-ml tube containing 4 ml ice-cold AdvDMEM+++[1].
• Centrifuge organoids for 5 min at 450 × g and 4°C, remove the supernatant, and remove as much residual BME as possible without losing organoid material.
If organoids remain associated with BME, resuspend the pellet and BME mesh in AdvDMEM+++, incubate on ice for 10 min, and repeat recovery[1].
• Resuspend the pellet in 1 ml ice-cold AdvDMEM+++, attach a sterile 10-µl pipette tip to a glass Pasteur pipette, and pass the organoids through the narrowed opening 5-10 times until large clumps are no longer visible.
A fire-polished glass pipette narrowed to approximately 0.5-1 mm is also reported as an alternative fragmentation tool[1].
• Add 4 ml ice-cold AdvDMEM+++, centrifuge for 5 min at 450 × g and 4°C, remove the supernatant, and resuspend the organoid fragments in 300-400 µl BME using a P1000 pipette.
The final BME concentration should be approximately 75-100%, and bubble formation should be avoided because it destabilizes BME droplets[1].
• Plate 100 µl organoid-fragment suspension per well as multiple approximately 15-µl droplets in a prewarmed 12-well plate, flip the plate upside down, and incubate at 37°C with 5% CO2 for 20 min to allow domes to solidify.
Add 1 ml expansion medium per well and refresh medium every 2-3 days[1].
• For a fully grown culture, the reported split ratio is typically 1:3 to 1:4 by BME volume, and further expansion can be performed after approximately 7 days when the culture has regrown sufficiently[1].
• Assess passaging success by bright-field microscopy after replating; reported images track organoid cultures at days 0, 1, 2, and 4 after mechanical split, with healthy organoids showing renewed growth rather than thick-walled differentiated morphology[1].
• For experimental consistency, record passage number, days since previous split, split ratio, plate format, dome volume, medium-change interval, and morphology before and after passaging.
Published organoid studies use serial passaging and maintenance of growth, morphology, and tissue-associated features as evidence that organoid lines remain expandable over time[1][2][3][5].
Passaging too early can reduce expansion efficiency, while passaging too late can lead to spontaneous differentiation, reduced viability, and suboptimal expansion[1].
• Prewarm culture plates before plating, keep AdvDMEM+++ ice-cold during organoid recovery, and prepare fresh BME/Matrigel for replating.
Reported BME volumes are 200 µl per well in 6-well plates, 100 µl per well in 12-well plates, 50 µl per well in 24-well plates, 25 µl per well in 48-well plates, and 5-10 µl per well in 96-well plates[1].
• Remove culture medium from established organoid wells, flush organoids with 1 ml ice-cold AdvDMEM+++ using a P1000 pipette to disrupt BME droplets, and transfer the recovered organoids into a 15-ml tube containing 4 ml ice-cold AdvDMEM+++[1].
• Centrifuge organoids for 5 min at 450 × g and 4°C, remove the supernatant, and remove as much residual BME as possible without losing organoid material.
If organoids remain associated with BME, resuspend the pellet and BME mesh in AdvDMEM+++, incubate on ice for 10 min, and repeat recovery[1].
• Resuspend the pellet in 1 ml ice-cold AdvDMEM+++, attach a sterile 10-µl pipette tip to a glass Pasteur pipette, and pass the organoids through the narrowed opening 5-10 times until large clumps are no longer visible.
A fire-polished glass pipette narrowed to approximately 0.5-1 mm is also reported as an alternative fragmentation tool[1].
• Add 4 ml ice-cold AdvDMEM+++, centrifuge for 5 min at 450 × g and 4°C, remove the supernatant, and resuspend the organoid fragments in 300-400 µl BME using a P1000 pipette.
The final BME concentration should be approximately 75-100%, and bubble formation should be avoided because it destabilizes BME droplets[1].
• Plate 100 µl organoid-fragment suspension per well as multiple approximately 15-µl droplets in a prewarmed 12-well plate, flip the plate upside down, and incubate at 37°C with 5% CO2 for 20 min to allow domes to solidify.
Add 1 ml expansion medium per well and refresh medium every 2-3 days[1].
• For a fully grown culture, the reported split ratio is typically 1:3 to 1:4 by BME volume, and further expansion can be performed after approximately 7 days when the culture has regrown sufficiently[1].
• Assess passaging success by bright-field microscopy after replating; reported images track organoid cultures at days 0, 1, 2, and 4 after mechanical split, with healthy organoids showing renewed growth rather than thick-walled differentiated morphology[1].
• For experimental consistency, record passage number, days since previous split, split ratio, plate format, dome volume, medium-change interval, and morphology before and after passaging.
Published organoid studies use serial passaging and maintenance of growth, morphology, and tissue-associated features as evidence that organoid lines remain expandable over time[1][2][3][5].
Troubleshooting
Problem: Organoids remain trapped in BME after centrifugation.
• Possible Cause: Undissolved BME can accumulate as a cloudy mesh above the pellet.• Literature-supported Solution: Resuspend the pellet and BME mesh in AdvDMEM+++, incubate on ice for 10 min, and repeat the recovery step while removing BME without aspirating organoids[1].
Problem: Loss of cells during washing.
• Possible Cause: Foam formation during pipetting can cause cell loss.• Literature-supported Solution: Pipette the cell suspension against the middle part of the tube wall to reduce foam formation[1].
Problem: BME droplets disrupt before splitting time.
• Possible Cause: Bubble formation during BME resuspension can destabilize droplets.• Literature-supported Solution: Resuspend organoid fragments carefully in BME and avoid bubbles before plating domes[1].
Problem: Poor expansion after passage.
• Possible Cause: Passaging too early or too late can reduce expansion efficiency, and late passaging can be associated with differentiation and reduced viability.• Literature-supported Solution: Determine the optimal passage interval empirically for each organoid line rather than relying only on a fixed schedule[1].
References:
- [1]. Pleguezuelos-Manzano C, et al. Establishment and culture of human intestinal organoids derived from adult stem cells. Curr Protoc Immunol. 2020;130(1):e106. [Content Brief]
- [2]. Sato T, Stange DE, Ferrante M, Vries RGJ, van Es JH, van den Brink S, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology. 2011;141(5):1762-1772. [Content Brief]
- [3]. Boj SF, Hwang CI, Baker LA, Chio IIC, Engle DD, Corbo V, et al. Organoid models of human and mouse ductal pancreatic cancer. Cell. 2015;160(1-2):324-338. [Content Brief]
- [4]. Hughes CS, et al. Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics. 2010;10(9):1886-1890. [Content Brief]
- [5]. Zhao Z, Chen X, Dowbaj AM, Sljukic A, Bratlie K, Lin L, et al. Organoids. Nat Rev Methods Primers. 2022;2:94. [Content Brief]
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