1. Are organoids composed of a single cell type or are they multicellular structures?
Organoids are three-dimensional (3D) tissue analogues derived from adult stem cells or pluripotent stem cells through in vitro 3D culture. They are not composed of a single cell type. Instead, they originate from stem cells with progenitor properties that undergo proliferation and differentiation, self-assembling into multicellular structures that mimic the morphology, structure, and function of their corresponding in vivo organs.
2. What are the sources of samples for organoid culture?
- Organoids derived from stem cells, including adult stem cells (ASCs), pluripotent stem cells (PSCs), and induced pluripotent stem cells (iPSCs).
- Tissue-derived organoids, commonly established from tumor tissues
3. Can cryopreserved tissues be used for 3D culture in the absence of fresh tissue?
Yes, but the size of the cryopreserved tissue is critical. Moreover, the viability of primary cryopreserved tissues and cells is significantly reduced, which greatly lowers the success rate of subsequent culture.
4. How are organoids cryopreserved and thawed?
Organoids are best cryopreserved at passage 2 to 5 (P2–P5), when their viability and differentiation potential are optimal. Thawing organoids can follow standard cell recovery protocols.
5. Should the size of cultured organoids be controlled? Is larger always better?
Yes, size control is essential. Organoids should ideally be maintained under 500 μm in diameter, as they lack vascular and fluid circulation systems. In larger organoids, cells in the core are deprived of sufficient oxygen and nutrients due to limited diffusion, leading to increased cell death in the central regions.
6. Besides Matrigel, what other materials can be used for organoid culture?
Alternative substrates include:
- Decellularized extracellular matrix (dECM) and derived proteins;
- Synthetic hydrogels;
- Engineered recombinant protein-based gels.
7. How can directed differentiation of organoids be achieved?
The early development of stem cell-derived organoids is regulated by multiple signaling pathways. In vitro, these pathways can be mimicked by adding specific growth factors and cytokines to guide differentiation. For example, Y27632 and Activin A can induce pluripotent stem cells (PSCs/iPSCs) to differentiate into definitive endoderm (DE), followed by Wnt3a, FGF-4, and Noggin to further direct lineage-specific differentiation.
8. How to avoid contamination when collecting clinical samples?
- Perform sterile collection whenever possible.
- Pre-treat tissue with PBS containing double antibiotics (e.g., penicillin/streptomycin): For tissues exposed to external environments (e.g., gastric, intestinal, bladder tumors), soak in PBS with 3–5% antibiotics for 5–10 min; for others, use 1–2% antibiotics for ~5 min.
- Add 1% antibiotics and appropriate primary cell antibiotics to all reagents used during cell isolation.
9. What are the considerations for tumor tissue collection, preservation, and transportation?
Collect as much tumor-rich tissue as possible and minimize the time the sample is exposed to air to reduce contamination risk. Immediately place the tissue in a sterile tube containing specialized preservation solution and transport it rapidly under cold conditions (~4 °C) to the lab (ideally within 2–4 hours post-sampling).
10. Are there differences between organoids derived from tumor lesions and adjacent normal tissues? What are the requirements for tumor tissue sampling?
Yes, differences exist. Tumors are inherently heterogeneous, and organoids from different regions often exhibit morphological and functional variations. Organoids from primary tumor sites typically display more irregular and invasive structures compared to those from adjacent normal tissues. To minimize variability in modeling or drug screening, sample multiple viable regions from the tumor.
11. What types of drugs can be tested in patient-derived organoid (PDO) drug sensitivity assays?
Major categories of anti-cancer drugs suitable for PDO testing include:
- Cytotoxic chemotherapeutics (e.g., paclitaxel, cisplatin/carboplatin, 5-FU);
- Targeted therapies (e.g., inhibitors targeting EGFR, HER2, VEGFR);
- Immunotherapies, particularly immune checkpoint inhibitors (e.g., PD-1/PD-L1 antibodies).
12. What is the success rate of PDO culture?
The success rate varies slightly depending on tissue origin, but generally ranges from 63% to 70%, with some reports reaching up to 90%. Success is highly dependent on tissue viability. Clinical handling procedures and shorter ex vivo times can improve success rates.
13. Can frozen tissues be used for organoid culture?
Cryopreservation is generally not recommended due to significant viability loss. However, if tissues are stored at –80 °C, the optimal window for organoid culture is within 6 weeks. For tissues preserved in liquid nitrogen, longer storage is possible, but culturing within 6 months is advised for best results.
14. During primary cell isolation, fibroblasts are often present. How should they be handled?
- Exploit the weak adhesion of fibroblasts by performing repeated pre-plating to remove most contaminating fibroblasts.
- Use commercially available fibroblast depletion kits, though their impact on organoid formation should be experimentally validated.
15. How much tumor tissue is required for tumor organoid culture? Is biopsy material sufficient?
For surgical specimens, tumor tissue should be larger than 2–3 peas. For core needle biopsies, at least 2–3 biopsy cores are recommended. For endoscopic biopsies, a minimum of 6 tissue fragments should be collected.
16. If the initial tumor tissue is small and the resulting organoids are insufficient for downstream assays, what can be done?
Due to potential phenotypic drift during passaging, extensive expansion is not recommended. Literature suggests limiting passaging to 2–3 generations (maximum 5). If cell numbers remain insufficient after 5 passages, consider alternative detection platforms such as 384-well plates or microfluidic devices to reduce assay volume.
17. Do tumor tissues contain normal cells? How can they be removed?
Yes, tumor tissues may contain a small fraction of normal cells. During dissection, avoid normal tissue as much as possible. After primary cell isolation, magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) can be used to enrich tumor cells before organoid culture. Small amounts of normal cells usually do not significantly affect organoid modeling and may be tolerated.
18. Why are primary cells extracted from tumor tissue sometimes red?
Tumors are highly vascularized, so residual red blood cells (RBCs) are common. Small amounts of RBCs do not interfere with organoid culture. If RBC contamination is heavy, use a red blood cell lysis buffer before culture.
19. Black particles appear during organoid culture. How can they be removed?
Black particles are likely debris or cellular fragments. Two methods can be used:
1. Digest organoids into single cells and wash repeatedly with culture medium to dilute contaminants.
2. Use a sterile scalpel to cut organoids in half, then gently flush the interior with culture medium using a 1 mL syringe.
20. Is there a limit on the number of passages for organoids? How many passages are possible?
The number of passages depends on the source cell type. Most organoids can be passaged up to 10 times (>6 months) in vitro. Culture medium formulation also plays a role—conditioned media often support longer-term expansion than fully defined synthetic media.
21. Can tumor cell lines (e.g., HepG2) be cultured into patient-derived organoids (PDOs)?
No. PDOs are complex, self-organized 3D structures derived from heterogeneous tissue. A 3D spheroid formed from a single immortalized cell line does not qualify as a PDO and should be referred to as a 3D spheroid or tumor spheroid.
22. What are the criteria for passaging organoids?
Passaging depends on organoid development. Typically, organoids are passaged every 5–10 days when they reach 100–200 μm in diameter. Some slower-growing types may require several weeks to reach passaging size.
23. How to count viable organoids?
Add calcein-AM to the culture medium to a final concentration of 0.2 μmol/L and incubate at 37 °C for 60 min. Gently wash with PBS to remove excess dye, then add fresh medium. Image under a fluorescence microscope using 490 nm excitation and 515 nm emission filters. Viable organoids appear green and well-defined. Count organoids with a diameter >20 μm.
24. How is organoid viability calculated?
Viability is calculated as:
X = (N_live / N_total) × 100%
Where:
X = organoid viability (%)
N_live = number of viable organoids
N_total = total number of organoids
25. What methods are used to characterize organoids?
Basic characterization includes light microscopy and H&E staining for morphology. Further validation includes Western blot, qRT-PCR, immunofluorescence, and flow cytometry to detect lineage-specific biomarkers. Genomic sequencing can assess genetic fidelity to the source tissue. Functional assays (e.g., acid secretion in gastric organoids, spontaneous beating in cardiac organoids) provide additional validation.
26. Can normal cells form organoids? How to eliminate normal organoids during tumor organoid culture?
Yes, normal epithelial cells can also form organoids. To enrich for tumor organoids:
- Manual picking under a microscope based on H&E morphology;
- Modify culture medium (e.g., add selective inhibitors) to favor tumor organoid growth;
- Dissociate organoids into single cells and perform FACS or MACS for tumor cell enrichment.
27. Should PDOs be dissociated from Matrigel before drug sensitivity testing?
No. The 3D architecture is essential to recapitulate in vivo responses. Removing Matrigel compromises structural integrity and reduces assay accuracy. Most soluble drugs can diffuse through Matrigel. However, for immune cell co-culture or cytotoxicity assays, Matrigel removal may be necessary.
28. Can PDO models fully replace animal models (e.g., PDX)?
PDOs can partially replace PDX models but cannot fully substitute them. Animal models better recapitulate systemic drug metabolism, tumor microenvironment interactions, immune infiltration, and metastasis—complex processes not fully modeled in vitro.
29. If PDOs exhibit abnormal growth (e.g., shortened cycle, rapid proliferation), what could be the cause?
External factors:
- Contamination by fast-growing cells (e.g., fibroblasts). Histological staining can help identify such contamination.
- Changes in culture medium composition (e.g., addition of growth-promoting factors).
Internal factors:
- Genetic mutations. Perform sequencing and compare with early-passage PDOs to confirm.
30. How to assess drug sensitivity in PDOs?
Common methods include CCK-8, ATP-based viability assays, and live/dead staining. ATP assay is most widely used, as ATP levels reflect metabolic activity and viable cell number. IC50 values (half-maximal inhibitory concentration) are calculated using analysis software to identify the most effective drugs.
31. Is the drug concentration range the same for PDOs and primary tumor cells in drug sensitivity assays?
No. The effective drug concentration for PDOs is typically higher than for 2D primary cells. A pre-experiment (dose-finding assay) is recommended before formal drug testing.
32. At what stage are organoids suitable for drug testing?
Drug testing is best performed using organoids within 5 passages, when genetic stability and biological activity are highest.
33. What defines successful organoid establishment?
- Early morphological changes: formation of cystic, budding, compact, or loose structures.
- Expression of lineage-specific biomarkers confirmed by immunostaining, matching the distribution in the original tissue.
- Further validation via sequencing to assess genetic similarity to the source tissue.
34. What are the key differences between organoid culture and conventional 2D cell culture?
(1) Culture method: Organoids require 3D scaffolds (e.g., Matrigel) to maintain structure; 2D cultures grow on flat surfaces.
(2) Differentiation & complexity: Organoids undergo in vitro differentiation and self-organization, requiring complex media with multiple growth factors. 2D cultures typically involve single cell types and simpler media.
(3) Cell source: Organoids originate from multipotent epithelial stem cells; 2D cultures can use various cell types, including immortalized lines.
35. How to confirm whether a 3D structure is a bona fide organoid matching the target tissue?
Use multimodal validation: H&E staining, immunohistochemistry (IHC), and single-cell RNA sequencing. For tumor organoids, assess expression of known biomarkers. Below are representative markers based on NCCN guidelines and literature:
|
Organoid Type |
Key Biomarkers |
|
Gastric Cancer |
p53, CEACAM1, KRT20, E-Cadherin, KRT7, Ki67 |
|
Breast Cancer |
ER-α, Her2, E-Cadherin, KRT5, KRT14, PR, p63, Cytokeratin 8, P-cadherin, KRT18, Ki67 |
|
Nasopharyngeal Carcinoma |
Ki67, CD133, CD44 |
|
Ovarian Cancer |
p53, Her2, PAX8, ER-α, PR, KRT7, E-Cadherin, CD9, KRT19, EpCAM, ALDH1A1, CD44, Cytokeratin 8, KRT20, Ki67, HE4 |
|
Colorectal Cancer |
Ki67, MSH6, CDX2, KRT20, β-Catenin, P-CK, OLFM4 |
|
Pancreatic Cancer |
Ki67, Synaptophysin, KRT19, Chromogranin A, MUC1 |
|
Lung Cancer |
p63, Napsin A, KRT7, NCAM, Synaptophysin |
|
Liver Cancer |
Ki67, EpCAM, α-fetoprotein, GPC3, β-Catenin, KRT19 |
36. Why might my organoids differ in morphology from those described in the literature?
Variations can arise from:
- Inter-patient heterogeneity and tumor subtypes;
- Differences in growth factor quality or batch;
- Culture conditions.
It is recommended to validate organoid identity using HE, IHC, and sequencing against the source tissue. Morphology may vary between labs and should not be strictly limited to published images.
37. In organoid drug assays, DMSO is used as a solvent. Should its concentration be controlled?
Yes. The final DMSO concentration should generally be kept below 0.5% (v/v) to avoid cytotoxic effects.
38. How to recover organoids from Matrigel?
Recommended: Use commercial organoid recovery solutions, which gently dissociate Matrigel without damaging cells or surface proteins.
Alternative: Chill samples on ice to liquefy Matrigel, then gently pipette to release organoids.
39. During organoid recovery, many organoids stick to the tube walls. How to improve recovery efficiency?
After collection, use a swinging-bucket (horizontal) rotor in the centrifuge. Slightly increase centrifugation force (e.g., ~300 × g, ~1000–1200 rpm) to pellet organoids effectively and minimize wall adhesion.