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.

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.