Q1：Is it necessary to use serum-free medium for organoid culture?

We recommend avoiding the use of serum when expanding organoids under defined conditions. However, the presence of serum is found in many organoids culture protocols, such as the intestinal organoid culture system established by Professor Hans Clevers' lab. The Wnt3a conditioned medium contains 5% FBS and it has been the most robust way to produce active Wnt3a. But now, alternative sources of serum-free Wnt3a are becoming available, such as Surrogate Wnt^[1][2][3][4].

In addition, in the ovarian organoid established by Professor Joanna E Burdette et al., it was found that the medium supplemented with BSA and FBS had the best growth conditions^[5].

Q2：What are the common biomarkers for intestinal organoids?

Intestinal organoids are composed of different types of cells. Classical crypt–villus-like architecture is regarded as an important feature of successful organoid establishment. The expression of intestinal stem cell marker Lgr5 can analyse the maintenance of intestinal stem cells by real-time quantitative PCR or by imaging using a Lgr5-fluorescent reporter. The intestinal differentiation gene marker CDX2 can also be detected by fluorescent quantitative PCR. Common differentiation lineages can be detected by marker staining including: paneth cells (lysozyme), intestinal cells (villin/EPCAM), goblet cells (mucin 2), enteroendocrine cells (somatostatin, chromogranin A), epidermal cells (E-cadherin), proliferating cells (Ki-67)^[6][7].

Q3：How to assess whether organoids are established successfully?

It is important to evaluate whether cultured organoids recapitulate key aspects of the representative human tissue or organ. It is typically performed by assessing organoid cellular composition, structure, functions and robustness of phenotype^[6].

1) Composition and structure. Both low-throughput gene expression validation and high-throughput whole-genome transcriptome analyses are typically performed, including real-time PCR, western blotting, immunofluorescence and immunohistochemical imaging, scRNA-seq and flow cytometry^[6][8][9].

2) Morphology. Organoid morphology is assessed to determine the presence of structural similarities between cultured organoids and corresponding in vivo tissues or organs^[6].

3) Functionality. Assessment of organoid function includes, but is not limited to, factors such as production of mature cells, formation of vasculature or neuronal networks, accurate response to external stimuli, and efficient secretion of cytokines or hormones^[6].

The establishment of organoid-based culture requires considerations about major components that make up organoid cultures — cells, soluble factors and matrix, physical cues — and the successful integration of these components.

Q4：How can organoids be used in screening?

Numerous studies have emerged showing that organoids provide accurate and reliable drug screening systems. A common protocol for organoid drug screening using refers to PMID: 35036959^[10].

Q5：Is there density dependence in organoid culture?

Yes. Guoliang Sun et al. (2020) showed that organoid growth rate and size is proportional to the number of cells seeded. Cells plated at the right density have enhanced efficiency of organoid culture establishment. But plating too densely will result in increased cell death at the core of the dome^[11].

Q6：Which enzyme should I use to tissue dissociation?

Tissue dissociation primary cells can be obtained from tissue using mechanical dissociation, enzymatic digestion, or a mixture of both methods. Enzymatic dissociation is the process of digesting minced tissue using enzymes, effectively releasing cells from the tissue. Specific enzymes are often more effective with certain tissues, so it is important to determine which enzymes are best suited for the tissue you want to dissociation. Collagenase is an important enzyme for single-cell tissue digestion, and it is an essential step in the preparation of single-cell samples. In addition, common digestive enzymes for tissue dissociation include trypsin, deoxyribonuclease I, hyaluronidase, papain, neutral protease and elastase.

Q7：Are ascites-derived organoid culture protocols different for different cancer patients?

At present, there are few reports on ascites-derived organoids. Md Mynul Hassan et al. (2023) established ascitic fluid-derived organoid from ascites of hispanic ovarian cancer patients^[12]. They referred to the culture of corresponding tumor organoids, adding cortisol and estradiol. The culture conditions of organoids established in colorectal cancer ascites were the same as the conditions of organoids culture from colorectal cancer tissues (I Ubink et al. 2019)^[13]. And the conditions of organoids established in gastric cancer ascites were based on the corresponding cancer organoids (Jie Li et al. 2019)^[14]. But patent CN110129270B described a kind of ascites culture method of ovarian cancer, lung cancer, breast cancer, liver cancer^[15].