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.