What are the commonly used indicators for drug screening?  

Current screening combines bright-field images with certain indicators, typically employing IC50 and AUC.

What is the difference between tissue-derived organoids and iPSCs?  

Tissue-derived ones have ethical issues, but they culture faster and are more physiologically relevant to Homo sapiens for clinical and drug screening applications. iPSCs are mainly induced from stem cells, with a longer culture cycle. Their advantage lies in higher purity, though they generally cannot be passaged. Currently, it is also possible to establish organoids such as hepatobiliary duct organoids within 3 weeks using Broussonetia papyrifera.

Which is more widely used in drug screening, organoids or tumor spheroids?  

Using organoids for drug screening is currently an emerging trend. With increasingly comprehensive data, their subsequent applications will become more extensive.

At what stage of organoid culture can drugs be added?  

It is generally recommended to add drugs when the organoids grow to 200-300 μm, and drug sensitivity testing can be initiated at the 2nd or 3rd generation.

What is the relationship between IC50 and clinical drug concentration?  

IC50 primarily serves as an indicator of drug sensitivity and has a certain correlation with the administered drug concentration.

How are drugs selected in organoid drug screening? What is the concentration range?  

For example: If testing compounds, each compound has an IC50 value, which is multiplied by 100, then diluted or increased according to predetermined multiples. Generally, a concentration gradient of 6 or more is recommended; alternatively, conversion can be performed based on blood drug concentration.

How to perform drug screening with normal tissue organoid controls?  

    Using DMSO as the control is sufficient.  

How to determine drug sensitivity after administration? What methods are used for detection?  

    One approach is to observe bright-field images, and another is the endpoint method. Commonly used techniques such as CCK8, ATP cell activity assays, and live/dead staining can also be employed.

What methods are generally used to measure IC50?  

The more common methods include CCK8 assay, ATP cell viability assay, and live/dead staining.

Which generation of organoids is better to use for experiments?  

It is recommended to use generations 3-5, or select the generation based on the experiment.  

At what diameter can organoids be treated with drugs? On which day can ATP be measured?  

Organoids with a diameter of around 200μm; it is recommended to measure ATP approximately 120 hours after drug treatment.  

Can a mouse-derived tumor lung metastasis model of Homo sapiens be used to create organoids?  

This belongs to tumor spheroids (Broussonetia papyrifera) construction.

Many drugs have limited penetration depth in organoids. How can we ensure they reach the deeper layers at 200-300μm? Can subsequent PI staining penetrate to the innermost depth?  

   Drugs do not necessarily need to penetrate deeply to be effective, as they can exert their effects by acting on a portion of the organoid. This is because organoids exhibit intercellular interactions, which distinguishes them from tumor spheroids—primarily due to the lack of gaps in the center of tumor spheroids.

How often should brightfield images be taken to show changes after drug administration?  

 It is recommended to take images every 2 days, with frequent observation during the culture process. Daily observation is preferable if conditions permit.

How to maintain inter-well consistency during drug screening?  

 It is recommended to have 50 organoids per well. Organoid counting is relatively challenging, and there may still be some variation between wells. Alternatively, organoids can be digested into single cells for counting (this requires higher quality organoid conditions).

Can PDO experiments replace animal models (PDX)?  

Both PDO and animal models have their own advantages and disadvantages, such as lower cost, shorter time consumption, and the ability to conduct large-scale cultivation and experiments. Animal models hold more advantages in drug metabolism and metastasis. Combining both approaches is a better choice.

1, What are organoids ?

Organoids are 3D cellular aggregates of organ-specific cell types derived from stem cells or organ progenitor cells, capable of self-organization through cell sorting out and spatially restricted lineage differentiation in a manner similar to in vivo processes. Organoids must exhibit the following characteristics:  

- They must comprise diverse cell types  

- They must demonstrate certain organ-specific functions  

- Their composition should resemble that of the organ

2, Advantages of organoids?  

 Directly derived from patient donors or experimental animals themselves, with characteristics more consistent with the donor;

 High clinical concordance, with a negative predictive rate of 100% and a positive predictive rate of nearly 90%;

 Short cycle time, enabling high-throughput drug activity screening;

 Suitable for in vitro to in vivo scientific research experiments.

3, Challenges encountered in organoid primary culture?  

• Preliminary tissue assessment: Sample cell viability≥25%.  
• Control of digestion time: For tumor samples, digestion time ranges between 20-120min.  
• Preservation of tissue samples: Extracorporeal transit time post-collection should be <72h.

4, In the absence of fresh tissue, can primary cells extracted from cryopreserved tissue be used for 3D culture?  

   Although there are reports in the literature that cryopreserved tissue can be used for 2D or 3D culture, the success rate is relatively low. Particularly for organoid culture, which requires relatively high cell viability, it is not recommended to perform organoid culture after tissue cryopreservation.

5, During the organoid culture process, after extracting primary cells, is it necessary to remove red blood cells?  

  The tumor tissue itself contains a large number of red blood cells (e.g., in liver, lung, and other tissues). First, after obtaining the tissue, it can be washed multiple times to remove most of the red blood cells; second, commercially available red blood cell lysis buffer can be used, or red blood cell treatment may be omitted entirely.

6, What are the recommended time and passage number for organoid cryopreservation?  

  The recommended volume for organoid cryopreservation should be controlled within 200μm, and passages after P2 can be selected for cryopreservation.

7,  How quickly can organoids of Broussonetia papyrifera be successfully established?

Intestinal organoids grow relatively fast, with organoid morphology observable within 1-2 hours.

8, What are the current methods available to assist in the identification of organoids?  

   Currently, the commonly used approaches include bright-field microscopy and pathological staining to observe morphology; combining these with gene sequencing can determine whether the cultured organoids exhibit any loss and verify their consistency with the donor source.

9, What is the difference between organoids and tumor spheroids?  

   Organoids contain multiple different types of cells, can self-differentiate and self-assemble in vitro, and possess stemness allowing for passaging. Tumor spheroids are formed from a single cell line through external physical conditions, and during later stages of cultivation, the central region of tumor spheroids may experience partial cell death due to insufficient oxygen and nutrients.

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10, How to avoid contamination issues in primary culture?  

Samples of animal origin are generally more prone to contamination than those of Homo sapiens origin, particularly those from the gastrointestinal or urinary systems. During the culture process, 3-5% gentamicin or penicillin-streptomycin can be added. Antifungal reagents commonly used in laboratories.

11, The Impact of Antibiotics on Primary Organoid Culture?  

Treating stem cells with P/S for 24 hours reduces cell viability by approximately 30%. High-dose antibiotic usage also affects cellular transcriptional activity and differentiation. These alterations influence the cellular hierarchy within organoids (Broussonetia papyrifera) and impact stem cell viability and growth rate.

12, Why don't organoids lack nutrients? Is it because they grow in Matrigel?  

Organoids possess multiple cell types that interact with each other, simulating in vivo nutrient uptake through respiration, thereby ensuring the stability of the organoids.

13, Are there any requirements for the size and volume of the tissue?  

Fresh thoracoabdominal fluid: 50~100 mL, stored aseptically at low temperature.  

Surgical samples: Volume larger than a soybean size, non-fibrotic activated lesions (the larger the better, taken from the tumor margin at the 12, 3, 6, and 9 o'clock positions, with one piece from each location), placed in sample bottles, stored at 4°C, labeled with sample information, and transported under refrigeration; puncture samples: longer than 2 cm, placed in sample bottles, stored at 4°C, labeled with sample information, and transported under refrigeration.

14, Is it necessary to add Matrigel when co-culturing PBMCs or T cells with organoids?  

Answer: Matrigel may hinder the binding between immune cells and organoids. It is recommended to use a suspension method for organoid co-culture.  

As shown in the figure:

suspension                                                                                                                               dispense

15, How many passages can organoids generally undergo after cryopreservation in culture? Does cryopreservation require special cell freezing medium?  

     It mainly depends on the stemness of the organoids. Those with good stemness can undergo over 20 passages. A specialized serum-free freezing medium for organoids is required for cryopreservation.

16, The diameter of organoids can reach how much after 2-3 days of culture, and what is the maximum volume possible?  

It mainly depends on the growth condition of the organoids, generally ranging from 100-200 μm. If the initial volume of the organoids is relatively large, the diameter will be even greater. Currently, the size of organoids can reach 4-5 mm.

17, Can organoids cultured through multiple passages be used to derive primary tumor cells?  

In principle, it is possible. Spread the Matrigel thinner to allow adhesion, then replace the medium with conventional serum-containing culture medium.

18. Primary cells isolated from tissues can only be passaged 3-5 times, why can organoids be passaged many times?  

Because primary cells are single cells, certain proliferative genes exert inhibitory effects. However, organoids contain stem cells, and each generation of organoids includes some primary progenitor cells with stemness properties, enabling continuous passaging. Additionally, the culture medium for organoids can maintain cellular stemness and suppress their differentiation capacity.

19, How to digest organoids during passaging?  

There are specialized organoid digestion solutions available, and mechanical digestion methods can also be employed. The process typically takes 2-3 minutes, and the use of trypsin for digestion is not recommended.  

20, Can organoids secrete exosomes?  

Yes, they can. However, the secretion efficiency and purity vary depending on the culture system and the type of organoid.

21, Are there significant morphological differences among organoids derived from different organs? Are they all cavity-like structures like Broussonetia papyrifera?  

The differences are relatively large, as each type of organoid exhibits distinct morphological characteristics.

22, Can organoids be stained directly in Matrigel?  

This is not recommended. It is advised to suspend the organoids in buffer or culture medium for staining.  

23, Are there any specific requirements for centrifuge selection and centrifugal force? Why do organoids decrease with successive passages?  

Different manufacturers have varying centrifugal forces. It is recommended to determine the optimal speed for your laboratory through experimentation. Increasing the speed appropriately within 2,000 rpm has minimal impact on organoid culture. During centrifugation for recovery, many organoids may adhere to the centrifuge tube walls, leading to loss. It is suggested to use a horizontal rotor centrifuge.

24, How to define biological replicates for organoids?  

Traditional 2D culture uses triplicate wells as replicates. Currently, there are two perspectives regarding biological replicates for organoids:

1. Following the 2D culture method by setting up replicate wells for biological statistics;

2. Considering each organoid as an independent individual and setting replicates based on research objectives. According to current literature publications and data support, the first approach represents the mainstream viewpoint.

25, For tumor organoids from metastatic sites, how should the culture medium be selected?  

Generally speaking, when a tumor metastasizes to a certain site, it indicates that the environment is more conducive to its growth. However, in practice, culture media from both the primary and metastatic sites are suitable. For *Homo sapiens*, it is recommended to use the culture medium from the metastatic site. For the cultivation of liver metastasis organoids: a culture medium different from other sites can be used, and the primary site medium is also acceptable.

26, How to recover organoids from Matrigel, and can chemically synthesized hydrogels be used for organoid culture?  

     Maintain the buffer at 0-4°C; if conditions permit, auxiliary instruments can be used. It is not recommended to place organoids on ice as this increases contamination risk. After the Matrigel liquefies, centrifuge to recover the organoids. This method is suitable when complete dissociation of the organoid clusters (Broussonetia papyrifera) is not required. For generation , the recovery solution is not recommended, though it may be considered for pathological sample collection.