Oct 22, 2025
Comprehensive Experimental Workflow for Filovirus Infection Studies in iPSC-derived and Primary-derived Human Gut Organoids Part 1
- Elizabeth Flores1,
- Elizabeth Flores2
- 1Center for Regenerative Medicine (CReM), Boston University Chobanian & Avedisian School of Medicine and Boston Medical Center, Boston, MA, USA.;
- 2National Emerging Infectious Diseases Laboratories (NEIDL), Boston University Chobanian & Avedisian School of Medicine, Boston, MA, USA.
- Elizabeth Yvonne Flores
Protocol Citation: Elizabeth Flores, Elizabeth Flores 2025. Comprehensive Experimental Workflow for Filovirus Infection Studies in iPSC-derived and Primary-derived Human Gut Organoids Part 1. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw44p2lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Working / Validated Protocol
Created: October 21, 2025
Last Modified: October 22, 2025
Protocol Integer ID: 230414
Keywords: comprehensive experimental workflow for filovirus infection study, standardized infection of human gut organoid, filovirus infection study, comparative studies of filovirus pathogenesi, filovirus pathogenesi, downstream viral infection, studying ebola virus, derived human gut organoids part, colonic organoid system, filovirus, ebola virus, culture of primary colonic organoid, human gut organoid, colonic organoid, primary colonic organoid, foundational organoid culture platform, standardized infection procedure, derived intestinal, standardized infection, primary human colonic tissue, ebola, infection workflow, marburg virus, organoid differentiation from induced pluripotent stem cell, flow cytometry, organoid differentiation, downstream immunofluorescence, cytometry analysis
Disclaimer
All procedures involving human induced pluripotent stem cells (hiPSCs) were approved by the Boston University Institutional Review Board. Parental iPSC lines, including the BU1CG line harboring a CDX2/GFP reporter (SPC2-ST-B2 clone), were previously described and are available through the CReM iPSC Repository (https://crem.bu.edu/cores-protocols/). The BU1CG line is derived from a healthy male donor and exhibits a normal 46XY karyotype.
Abstract
Experimental Workflow:
This workflow outlines the step-by-step experimental pipeline for studying Ebola virus (EBOV) and Marburg virus (MARV) infection in human intestinal and colonic organoid systems. It integrates organoid differentiation from induced pluripotent stem cells (hiPSCs) and primary human colonic tissue, standardized infection procedures, functional assays, and downstream immunofluorescence and flow cytometry analyses. Each section provides detailed methods and reagents to ensure reproducibility in BSL-4-compatible systems and support comparative studies of filovirus pathogenesis in 3D human models.
Sections:
Protocol 1: Differentiation and Culture of Human iPSC-derived Intestinal and Colonic Organoids and Culture of Primary Colonic Organoids
This step defines the foundational organoid culture platform that supports all downstream viral infection and analysis assays.
Protocol 2: Standardized Infection of Human Gut Organoids with Ebola (EBOV) and Marburg (MARV) Viruses
This describes the infection workflow including inoculation, timing, containment, and MOI setup.
Materials
2. Materials and Reagents
2.1 Cells
- Human iPSC lines (e.g., BU1CG, CDX2/GFP reporter)
- Primary human colonic tissue-derived cells (PCOs)
2.2 Matrices
| Matrix | Source | Catalog # |
| --- | --- | --- |
| Growth Factor Reduced Matrigel | Corning | 356230 |
| Matrigel Basement Membrane Matrix | Corning | 354234 |
2.3.a Small Molecules 26 Growth Factors
| Name | Source | Catalog # | Working Concentration |
| --- | --- | --- | --- |
| SB431542 | Tocris | 1614 | 3 µM |
| Dorsomorphin | Stemgent | 04-0024 | 3 µM |
| CHIR99021 | Tocris | 4423 | 3 µM |
| Recombinant human FGF10 | R 26D Systems | 345-FG-025 | 10 ng/mL |
| Recombinant human KGF | R 26D Systems | 251-KG-010 | 10 ng/mL |
| Recombinant human BMP4 | R 26D Systems | 314-BP | 10 ng/mL |
| Retinoic acid | Sigma | R2625 | 0.1 µM |
| Y-27632 dihydrochloride | Sigma | 1254 | 10 µM |
| Dexamethasone | Sigma | D4902 | 50 nM |
| cAMP | Sigma | B7880 | 0.1 mM |
| IBMX | Sigma | I5879 | 0.1 mM |
| Recombinant Human FGF4 | Thermo Fisher | H3570 | 10 ng/mL |
| Trypsin-EDTA (0.05%) | Thermo Fisher | 25300-120 | as needed |
| Defined Fetal Bovine Serum | NC0652331 | as indicated |
| Recombinant human Noggin | R 26D Systems | 6057NG025 | 500 ng/mL |
| Recombinant human EGF | R 26D Systems | 236EG200 | 100 ng/mL |
| Recombinant Human R-Spondin 1 Protein | R 26D Systems | 4645-RS-025 | 100 ng/mL |
| N2 Supplement | Thermo Fisher | 17502-048 | 1x |
| B27 Supplement | Invitrogen | 12587-010 | 1x |
| GlutaMAX | Invitrogen | 35050061 | 1x |
2.3.b Antibodies 26 Flow Cytometry Reagents
| Target | Vendor | Catalog # |
| --- | --- | --- |
| Anti-human CD47 PerCP/Cy5.5 Conjugate | BioLegend | 323110 |
| Anti-human CD26 PE Conjugate | BioLegend | 302705 |
| Calcein Blue | Life Technologies | C1429 |
2.4 Media Recipes
iPSC-derived Organoid Differentiation Media
| Medium | Components |
| --- | --- |
| IM+CHIR | RPMI-1640 + 2% FBS + CHIR99021 3 µM |
| IM+CK | RPMI-1640 + 2% FBS + CHIR99021 3 µM + KGF 10 ng/mL + FGF4 10 ng/mL |
| Hindgut Medium | IMDM 75% + Ham’s F12 25% + B27 + N2 + Retinoic Acid 0.1 µM |
| cSFDM | DMEM/F12 + HEPES + GlutaMAX + B27 + N2 + CHIR99021 3 µM + KGF/FGF7 10 ng/mL |
| CK+DCI | cSFDM + CHIR99021 3 µM + KGF/FGF7 10 ng/mL + Dexamethasone 50 nM + cAMP 0.1 mM + IBMX 0.1 mM |
Primary-Derived Colonic Organoid Media
| Medium | Components |
| --- | --- |
| Splitting Medium | 500 mL DMEM/F12, 10% FBS, 1% Penicillin/Streptomycin |
| DF20 | 500 mL DMEM/F12, 20% FBS, 1% Penicillin/Streptomycin |
| R-spondin selection medium | 500 mL Advanced DMEM + 50 mL FBS + 5 mL Penicillin/Streptomycin + 5 mL Glutamax + 1.5 mL Zeocin |
| R-spondin collection medium | 500 mL DMEM/F12 + 5 mL Glutamax. Note no Penicillin/Streptomycin |
| R-spondin freezing medium | 5 mL selection medium + 3 mL FBS + 2 mL DMSO |
| WNT selection medium | 500 mL Advanced DMEM + 50 mL FBS + 2 mL G418 (100mg/mL). Note no Penicillin/Streptomycin. |
| WNT collection medium | 500 mL Advanced DMEM + 50 mL FBS. Note no Penicillin/Streptomycin |
| WNT Freezing medium | 5 mL WNT selection medium + 3 mL FBS + 2 mL DMSO |
| Basal Growth Medium (BGM) | DMEM/F12 + Primocin 100 ng/mL + B27 + N2 + Noggin 500 ng/mL + R-Spondin 100 ng/mL + EGF 100 ng/mL |
Materials and Reagents:
| Component | Specification / Source | Purpose / Notes |
| --- | --- | --- |
| Human gut organoids | Distal HCOs, proximal HIOs, and primary-derived gut organoids | Maintained in 12-well plates embedded in Matrigel |
| Matrigel | Basement membrane matrix (Corning, pre-chilled) | 3D scaffold for organoid culture and re-embedding |
| Cell Recovery Solution (CRS) | Corning | 500 µL per well; preserves organoid integrity during harvesting |
| Phosphate-buffered saline (PBS) | Sterile | For rinsing and washing organoids |
| Organoid culture media | CKDCI, CKDI, IMCK, or primary-derived gut organoid media | CKDCI: distal HIOs; CKDI: distal-cAMP HIOs; IMCK: proximal HIOs; primary-derived gut organoid media (see table) |
| EBOV and MARV stocks | Purified from cell culture supernatants via ultracentrifugation through 20% sucrose cushion | Viral titers quantified by TCID_50 assay |
| P1000 pipette tips | Standard, distal end cut | Minimizes mechanical disruption during transfer of Matrigel droplets |
| 1.5 mL microcentrifuge tubes | Sterile | Collection and centrifugation of organoid suspensions |
| Centrifuge | Capable of 200 × g | Pelleting organoids for medium exchange and infection preparation |
Troubleshooting
Before start
1. Culture in CB/RA medium (CHIR99021 + rhBMP4 + Retinoic Acid). Add Retinoic Acid freshly to the medium before use.
2. Medium changes are required every other day.
Protocol 1: Differentiation of hiPSCs into Human Intestinal and Colonic Organoids and Culture of Primary Colonic Organoids
Authors: Elizabeth Yvonne Flores et al.
Abstract
This protocol describes two distinct workflows: A) Differentiation of human induced pluripotent stem cells (hiPSCs) into intestinal (HIO) and colonic (HCO) organoids, including the Day 15 sorting of CDX2-GFP^+^ gut progenitors and maintenance of organoids. B) Thawing, expansion, and maintenance of primary-derived colonic organoids (PCOs) from healthy adult donors. Reproducibility is ensured through standardized media formulations, defined growth factor conditions, and the use of independent biological replicates.
Materials and Reagents
Cells
- Human iPSC lines (e.g., BU1CG, CDX2/GFP reporter)
- Primary human colonic tissue-derived cells (PCOs)
Matrices
| Matrix | Source | Catalog # | |
| Growth Factor Reduced Matrigel | Corning | 356230 | |
| Matrigel Basement Membrane Matrix | Corning | 354234 |
Small Molecules and Growth Factors
| Name | Source | Catalog # | Working Concentration | |
| SB431542 | Tocris | 1614 | 3 μM | |
| Dorsomorphin | Stemgent | 04-0024 | 3 μM | |
| CHIR99021 | Tocris | 4423 | 3 μM | |
| Recombinant human FGF10 | R&D Systems | 345-FG-025 | 10 ng/mL | |
| Recombinant human KGF | R&D Systems | 251-KG-010 | 10 ng/mL | |
| Recombinant human BMP4 | R&D Systems | 314-BP | 10 ng/mL | |
| Retinoic acid | Sigma | R2625 | 0.1 μM | |
| Y-27632 dihydrochloride | Sigma | 1254 | 10 μM | |
| Dexamethasone | Sigma | D4902 | 50 nM | |
| cAMP | Sigma | B7880 | 0.1 mM | |
| IBMX | Sigma | I5879 | 0.1 mM | |
| Recombinant Human FGF4 | Thermo Fisher | H3570 | 10 ng/mL | |
| Trypsin-EDTA (0.05%) | Thermo Fisher | 25300-120 | as needed |
| Defined Fetal Bovine Serum | NC0652331 | as indicated | ||
| Recombinant human Noggin | R&D Systems | 6057NG025 | 500 ng/mL | |
| Recombinant human EGF | R&D Systems | 236EG200 | 100 ng/mL | |
| Recombinant Human R- Spondin 1 Protein | R&D Systems | 4645-RS-025 | 100 ng/mL | |
| N2 Supplement | Thermo Fisher | 17502-048 | 1x | |
| B27 Supplement | Invitrogen | 12587-010 | 1x | |
| GlutaMAX | Invitrogen | 35050061 | 1x | |
| Gentle Cell Dissociation Reagent | StemCell Technologies | 07174 | as per manufacturer | |
| Ascorbic Acid | Sigma | A4403 | 50 μg/mL | |
| Cell Recovery Solution | Corning | 354253 | as per manufacturer | |
| Paraformaldehyde | Electron Microscopy Sciences | 19208 | 4% | |
| ReLeSR | StemCell Technologies | 05873 | as per manufacturer | |
| StemDiff Definitive Endoderm Kit | StemCell Technologies | 05110 | as per manufacturer | |
| Accumax | Sigma | A7089 | as per manufacturer |
Media Recipes
| Medium | Components | |
| IM+CHIR | RPMI-1640 + 2% FBS + CHIR99021 3 μM | |
| IM+CK | RPMI-1640 + 2% FBS + CHIR99021 3 μM + KGF 10ng/mL + FGF4 10 ng/mL | |
| Hindgut Medium | IMDM 75% + Ham’s F12 25% + B27 + N2 + RetinoicAcid 0.1 μM | |
| cSFDM | DMEM/F12 + HEPES + GlutaMAX + B27 + N2 +CHIR99021 3 μM + KGF/FGF7 10 ng/mL | |
| CK+DCI | cSFDM + CHIR99021 3 μM + KGF/FGF7 10 ng/mL +Dexamethasone 50 nM + cAMP 0.1 mM + IBMX 0.1 mM |
Antibodies & Flow Cytometry Reagents
| Target | Vendor | Catalog # | |
| Anti-human CD47 PerCP/Cy5.5 Conjugate | BioLegend | 323110 | |
| Anti-human CD26 PE Conjugate | BioLegend | 302705 | |
| Calcein Blue | Life Technologies | C1429 |
Primary-Derived Colonic Organoid Media
| Composition of Culture Medium for Primary-Derived Gut Organoids | |
| Splitting Medium: 500 mL DMEM/F12, 10% FBS, 1% Penicillin/Streptomycin | |
| DF20: 500 mL DMEM/F12, 20% FBS, 1% Penicillin/Streptomycin | |
| R-spondin selection medium: 500 mL Advanced DMEM + 50 mL FBS + 5 mLPenicillin/Streptomycin + 5 mL Glutamax + 1.5 mL Zeocin | |
| R-spondin collection medium: 500 mL DMEM/F12 + 5 mL Glutamax. Note noPenicillin/Streptomycin | |
| R-spondin freezing medium: 5 mL selection medium + 3 mL FBS + 2 mL DMSO | |
| WNT selection medium: 500 mL Advanced DMEM + 50 mL FBS + 2 mL G418(100mg/mL). Note no Penicillin/Streptomycin. | |
| WNT collection medium: 500 mL Advanced DMEM + 50 mL FBS. Note noPenicillin/Streptomycin | |
| WNT Freezing medium: 5 mL WNT selection medium + 3 mL FBS + 2 mL DMSO |
Equipment
- Tissue culture incubator (37°C, 5% CO₂)
- Class II biosafety cabinet
- Centrifuge with swinging-bucket rotor
- Flow cytometer
Methods
Section A: iPSC-Derived Human Intestinal Organoids (HIOs) and Human Colonic Organoids (HCOs)
4.1 iPSC Maintenance
Culture hiPSCs on growth factor-reduced Matrigel in mTeSR1.
Passage at 70–80% confluency using ReLeSR.
Confirm morphology and pluripotency markers.
4.2 Differentiation to HIOs
Day 0 – Definitive Endoderm Induction
Dissociate hiPSC colonies using Gentle Cell Dissociation Reagent.
Plate 2 × 10^6^ cells/well in mTeSR1 + 5 µM Y-27632 (ROCK inhibitor).
Differentiate using StemDiff Definitive Endoderm Kit.
Confirm CXCR4^+/c-Kit^+^ population via flow cytometry.
Refer to 2.3b Antibodies 6 Flow Cytometry Reagents.
Day 3 – Hindgut Specification
Passage 1:3 onto 2D Matrigel-coated plates.
Treat with DS/SB43 + Y-27632 for 24 h, then DS/SB43 alone for 48 h.
Day 6-15 – Intestinal Patterning
Culture in CB/RA medium (CHIR99021 + rhBMP4 + Retinoic Acid). Add Retinoic Acid freshly to the medium before use.
Medium changes are required every other day.
Day 15: CDX2-GFP^+^ Sorting, Seeding, and Lineage Specification
Cell Preparation and Sorting of CDX2-GFP+ Gut Progenitors: Collect Day 15 cells and dissociate them into single cells or small clusters using 0.05% Trypsin-EDTA for no longer than 5 minutes at 37°C. Immediately quench the enzymatic reaction by adding an equal volume of medium containing fetal bovine serum (FBS). Centrifuge the cell suspension at 200 × g for 5 minutes, aspirate the supernatant, and resuspend the pellet in FACS buffer (e.g., PBS + 2% FBS). Pass the cell suspension through filtered FACS tubes with cell-strainer caps to remove aggregates. Add Calcein Blue AM to identify live cells. Prepare FACS collection tubes containing IM+CHIR medium supplemented with 10 µM Y-27632. Sort CDX2-GFP^+^ gut progenitors by fluorescence-activated cell sorting (FACS) directly into the chilled collection medium.
Centrifuge sorted cells (200 × g, 5 min, 4°C), resuspend in cold Growth Factor Reduced Matrigel (Corning) at 2,000–5,000 cells per 50 µL 3D matrigel dome. Plate domes on pre-warmed 12-well plates, polymerize at 37°C for 30 min, then overlay with appropriate medium:
- Intestinal organoids (HIOs): IM+CK medium + Noggin 500 ng/mL, + R-Spondin 100 ng/mL, + EGF 100 ng/mL + CHIR99021 3 µM.
- Colonic organoids (HCOs): cSFDM medium + CHIR99021 3 µM + KGF/FGF7 10 ng/mL + Dexamethasone 50 nM + cAMP 0.1 mM + IBMX 0.1 mM.
Include Y-27632 for 24 h post-sort, then remove. Feed every 3–4 days.
Day 15 Onward: Long-Term Culture
Maintain HIOs and HCOs in their respective media with medium changes every 3–4 days. Mature organoids for 14–21 days before experimental use. Passage or cryopreserve as needed.
Section B: Primary Cell-Derived Colonic Organoids (PCOs)
PCOs were provided by Dr. Ryan B. Corcoran (Massachusetts General Hospital) and derived from healthy adult donors. Unlike iPSC-derived organoids, PCOs do not undergo differentiation or sorting; they are maintained and expanded directly.
4.4 Thawing and Recovery
Thaw organoids rapidly in a 37°C water bath (1–2 min), transfer to a 15 mL tube with pre-warmed selection medium, centrifuge (200 × g, 5 min), and resuspend in fresh selection medium. Embed in 50 µL 3D Matrigel domes and overlay with Basal Growth Medium (BGM).
4.5 Maintenance and Passaging
Replace medium every 3–4 days. To passage, place plates on ice, add Cell Recovery Solution (CRS, 1 mL/well), incubate 15 min, then collect and incubate on ice for 1 h. Centrifuge (200 × g, 5 min), digest with TrypLE (5 min, 37°C), and mechanically dissociate with a 20G needle. Neutralize with Splitting Medium, centrifuge, resuspend in fresh Matrigel, and replate domes.
4.6 Quality Control and Replication
All organoid derivations were performed in at least three independent differentiations or passages per donor line. Morphology was confirmed microscopically and epithelial identity by immunofluorescence for CDX2, EPCAM, and MUC2. HIOs, HCOs, and PCOs were used for infection experiments only after confirmation of mature epithelial differentiation.
Quality Control
Flow cytometry at definitive endoderm stage (CXCR4^+/c-Kit^+^).
Morphology: uniform cystic structures.
Confirm absence of contamination and consistent differentiation efficiency.
Reproducibility Recommendations
Use ≥2 independent iPSC lines and ≥2 donor-derived PCO lines.
Perform ≥3 independent differentiations or expansions per line.
References
Flores, Elizabeth Y et al. “Human-induced Pluripotent Stem Cell-derived Gut Organoids Recapitulate Regional Specific Genetic Programs and a Role for cAMP in Lineage Specification.” Cellular and molecular gastroenterology and hepatology vol. 19,9 (2025): 101534. doi:10.1016/j.jcmgh.2025.101534
Mithal, Aditya et al. “Generation of mesenchyme free intestinal organoids from human induced pluripotent stem cells.” Nature communications vol. 11,1 215. 10 Jan. 2020, doi:10.1038/s41467-019-13916-6
Protocol 2: Standardized Infection of Human Gut Organoids with Ebola (EBOV) and Marburg (MARV) Viruses
Authors: Elizabeth Yvonne Flores et al.
Abstract
This protocol describes the controlled infection of iPSC-derived human intestinal organoids (HIOs), human colonic organoids (HCO) and primary-derived gut organoids with Ebola virus (EBOV) or Marburg virus (MARV), enabling quantitative and qualitative evaluation of viral replication dynamics and host cellular responses. All infection experiments with EBOV and MARV were conducted under BSL-4 containment in compliance with institutional and federal biosafety regulations. Organoids are first harvested from Matrigel and subjected to gentle dissociation and preparation steps to preserve structural integrity prior to exposure to viral inocula of defined multiplicity of infection (MOI). Following viral exposure, organoids are re-embedded in 3D Matrigel matrices to maintain tissue architecture, facilitating downstream analyses including histological, immunofluorescence, flow cytometry, and transcriptomic assays. Mock-infected controls are processed in parallel to account for any perturbations arising from handling or culture manipulations. This methodology provides a robust and reproducible platform for assessing viral infectivity, investigating dose-dependent responses, and interrogating mechanistic host-pathogen interactions in physiologically relevant 3D human intestinal tissue models.
Materials and Reagents
| Component | Specification / Source | Purpose / Notes | |
| Human gut organoids | Distal HCOs, proximal HIOs, and primary-derived gut organoids | Maintained in 12-well plates embedded in Matrigel | |
| Matrigel | Basement membranematrix (Corning, pre- chilled) | 3D scaffold for organoid culture and re-embedding | |
| Cell Recovery Solution (CRS) | Corning | 500 µL per well; preservesorganoid integrity during harvesting | |
| Phosphate-buffered saline (PBS) | Sterile | For rinsing and washing organoids | |
| Organoid culture media | CKDCI, CKDI, IMCK, orprimary-derived gut organoid media | CKDCI: distal HIOs; CKDI: distal-cAMP HIOs; IMCK: proximal HIOs; primary-derived gut organoid media (see table) | |
| EBOV and MARV stocks | Purified from cell culture supernatants via ultracentrifugation through20% sucrose cushion | Viral titers quantified by TCID₅₀ assay | |
| P1000 pipette tips | Standard, distal end cut | Minimizes mechanical disruption during transfer of Matrigel droplets | |
| 1.5 mL microcentrifuge tubes | Sterile | Collection and centrifugation of organoidsuspensions | |
| Centrifuge | Capable of 200 × g | Pelleting organoids for medium exchange and infection preparation |
Methods
Step 1 — Preparation of Organoids for Infection
- Aspirate culture medium from each well while preserving Matrigel droplet integrity.
- Add 500 µL chilled CRS per well and incubate at 4°C for 30 minutes.
- Transfer Matrigel droplets to 1.5 mL microcentrifuge tubes using a P1000 pipette tip with the distal end removed.
- Rinse wells with 500 µL PBS to recover residual organoids and combine with the initial suspension.
- Centrifuge at 200 × g for 5 minutes. Aspirate supernatant carefully, leaving minimal medium to avoid pellet loss.
- Resuspend organoids in 1 mL of the appropriate culture medium (CKDCI for distal HIOs, CKDI for distal-cAMP HIOs, IMCK for proximal HIOs).
Step 2 — Viral Stock Preparation
7. Purify EBOV and MARV stocks from cell culture supernatants via ultracentrifugation through a 20% sucrose cushion.
8. Determine viral titers using the tissue culture infectious dose 50 (TCID_50) assay.
9. Process mock-infected controls in parallel, including dissociation, incubation, Matrigel re-embedding, media exchange, and inactivation, but without viral exposure.
Step 3 —Determination of Multiplicity of Infection (MOI)
10.Dissociate organoids from three representative wells into single-cell suspensions using enzymatic digestion.
11. Count cells to determine the average cell number per well to normalize for organoid size and density.
Calculate infectious units required: Infectious units = Average cell number per well × MOI × Number of wells
12. Dilute the viral stock in the appropriate organoid medium to achieve the desired MOI.
Standard infections are performed at MOI = 10; additional MOIs (0.1, 10, 50, 100) may be used to evaluate dose-dependent responses.
Step 4 —Viral Infection Procedure
13. Add the calculated viral volume to the organoid suspension and incubate at 37°C for 1 hour.
14. Wash organoids once with 1 mL PBS.
15. Resuspend organoids in fresh 3D Matrigel and replate into 12-well plates.
16. Incubate at 37°C for 30 minutes to allow Matrigel polymerization.
17. Overlay with 1 mL of appropriate organoid medium and return plates to the incubator for downstream analyses.
Quality Control and Reproducibility
| Problem | Possible Cause | Recommended Solution | |
| Organoids dissociate orcollapse during harvesting | Excessive mechanicalstress or high centrifugation speed | Use cut pipette tips, keepsamples on ice, centrifuge at 200 × g | |
| Low infection efficiency | Inaccurate MOI calculation, diffusion barriers | Verify cell counts, optimize viral mixing, consider higher MOI for 3Dorganoids | |
| Matrigel does not polymerize properly | Temperature too low or delayed plating | Keep Matrigel and organoids at 37°C for atleast 30 min before adding medium | |
| High cell death in mock controls | Handling stress or prolonged manipulation | Minimize time on ice, handle droplets gently,reduce pipetting steps |
Protocol references
1. Flores, Elizabeth Y et al. “Human-induced Pluripotent Stem Cell-derived Gut Organoids Recapitulate Regional Specific Genetic Programs and a Role for cAMP in Lineage Specification.” Cellular and molecular gastroenterology and hepatology vol. 19,9 (2025): 101534. doi:10.1016/j.jcmgh.2025.101534
2. Mithal, Aditya et al. “Generation of mesenchyme free intestinal organoids from human induced pluripotent stem cells.” Nature communications vol. 11,1 215. 10 Jan. 2020, doi:10.1038/s41467-019-13916-6
Acknowledgements
PCOs were provided by Dr. Ryan B. Corcoran (Massachusetts General Hospital) and derived from healthy adult donors. Unlike iPSC-derived organoids, PCOs do not undergo differentiation or sorting; they are maintained and expanded directly.
Quality Control and Reproducibility:
| Problem | Possible Cause | Recommended Solution |
| --- | --- | --- |
| Organoids dissociate or collapse during harvesting | Excessive mechanical stress or high centrifugation speed | Use cut pipette tips, keep samples on ice, centrifuge at 200 × g |
| Low infection efficiency | Inaccurate MOI calculation, diffusion barriers | Verify cell counts, optimize viral mixing, consider higher MOI for 3D organoids |
| Matrigel does not polymerize properly | Temperature too low or delayed plating | Keep Matrigel and organoids at 37°C for at least 30 min before adding medium |
| High cell death in mock controls | Handling stress or prolonged manipulation | Minimize time on ice, handle droplets gently, reduce pipetting steps |