1. Class organs
An organoid is a three-dimensional miniature cellular structure formed by the culture and differentiation of stem cells in vitro, derived from embryonic stem cells, induced pluripotent stem cells, adult stem cells, or tumor stem cells.
Under specific conditions, these stem cells self-organize into cell populations with specific functions, forming tissues and genetic characteristics similar to those of the corresponding organs.
Although organoids are not true complete organs, they can recapitulate complex cellular heterogeneity and spatial structure, and mimic the function of organs in vivo with long-term stable passaging culture.
Microfluidics meets the needs of organoid research in terms of precise manipulation of microfluids and high throughput capabilities.
By modulating the physical and chemical parameters of the culture environment, such as nutrient concentration and waste discharge, microfluidics can dynamically mimic the in vivo environment and improve the realism of organoid reflection of physiological and pathological processes.
In addition, microfluidic technology supports continuous generation of independent organoid and high-throughput drug screening, accelerating the process of drug testing and disease research and improving experimental efficiency.
2. Microfluidic high-throughput organoid culture
Microfluidic organ cultures differ from conventional well plate formats in that high throughput stand-alone culture units are usually constructed, and these high throughput technologies can be categorized into 3 types in terms of specific implementations: microfluidic droplets, microtiter wells, and microcolumns.
1) Microfluidic droplet organoid culture technology
Microfluidic multiphase flow droplet technology provides fast and ultra-high throughput droplet generation by manipulating two or more immiscible liquids in microchannels to generate droplets in a confluent or entrained flow.
This technique effectively reduces reagent consumption and guarantees the consistency of organoid culture. In the water-in-oil droplets, the cell suspension acts as the aqueous phase and the oil phase, to which surfactants have been added, acts as the continuous encapsulated phase. By adjusting the flow rate of the two phases, droplets of controllable sizes are generated in a high-throughput manner, and organoids can be obtained by subsequent culturing.
However, this method results in droplets surrounded by oil, which limits nutrient delivery and waste removal and makes it difficult to achieve continuous long-term culture.
To this end, improved hydrogel organoid culture methods were developed, using a hydrogel cell solution as the encapsulated aqueous phase, with cells pre-suspended in the hydrogel and hydrogel droplets generated by microfluidic entrainment.
The hydrogel cures to form a solid-phase cell gel sphere, which is cultured in the aqueous phase to form organoids. The aqueous solution outside the gel sphere can continuously provide nutrients to the cells and carry away metabolic wastes, realizing the long-term culture of organoids.
Currently, the gel materials used include alginate, chitosan, agarose and polyethylene glycol.
2) Microfluidic Bioprinting of Organoid Cultures
Microfluidic bioprinting creates bioinks by suspending target cells in biodegradable hydrogels, often with scaffolding materials and growth factors. The technique allows for flexible control of how biomaterials and cells are spatially stacked to create complex tumor models.
Bioinks can be printed as planar arrays of complexes on open substrates or dispensed into multiwell plates, generating arrays of organoid droplets with highly controllable positions, enabling high-throughput drug screening.
An organoid can be generated in a matter of seconds, and studies have shown a high degree of reproducibility between parallel printed organoids.
Combining the 3D printing techniques of multiphase flow droplet generation and matrix gel droplets, the speed of organoid generation was increased, with 1,000 organoids generated in 10 minutes and inoculated into the corresponding wells at a rate of 1 per second.
Thanks to the high cell density in the matrix gel droplets, the maturation time of the organoids was shortened, while the consistency between the organoids was significantly improved.
3) Microfluidic Microtiter Array Organoid Culture Technology
Microporous arrays are commonly used for microfluidic cell capture, aggregation, and 3D culture, and by modulating microstructural features, it is possible to control the size of organoids and obtain homogeneous organoids.
The application of scaled-up microwell arrays enables high-throughput organoid culture. Microporous arrays can be prepared by molding method, direct milling, or 3D printing, and the different shapes and materials of micropores contribute to the uniform aggregation of cells and the formation of organoids.
The system does not use matrix gel and enables real-time analysis of organoids in microtiter arrays. The aggregation of the same number of cells within the microtiter wells accelerates the generation of organoids and synchronizes the growth of heterogeneous cells within the population, improving the consistency of batch culture.
The single class of organs grown within each microtiter well facilitates continuous dynamic morphologic monitoring and also supports subsequent analyses such as immunohistochemistry, genomic and transcriptomic.
4) Microfluidic Microcolumn Array Organoid Culture Technology
Microcolumn arrays are equally suitable for cell capture, aggregation and spatial separation for organoid culture. Cells are trapped in the micropillar gaps and undergo organoid differentiation and development in the array.
The micropillar gap facilitates solution flow and is therefore often combined with perfusion chips to support long-term culture of organoids. The system can also be used to study the effects of dynamic fluids on organoid development and function. Perfusion culture solution not only avoids frequent fluid change operations, but also realizes effective delivery of nutrients and drainage of wastes.
Compared with static culture, organoids on the perfusion microcolumn platform exhibited better cell viability and higher organ-specific gene expression. In addition, another microcolumn strategy is to culture organoids on the end face of each microcolumn, and batch organoid culture can be achieved by immobilizing cell-containing gel droplets on the end face of the microcolumn.
This method allows for one-time fluid exchange of organoids in microcolumn arrays, supports long-term cultures, and dramatically reduces reagent consumption.
3. Application of microfluidics to organoid drug screening
Organoid drug screening is categorized into two main groups depending on the export of the application:
One of them is the screening of clinical oncology drugs, i.e., drug sensitivity testing of tumor organoids. The source of organoid cells is a puncture or surgical resection sample from a patient, and the cultured organoids are called patient-derived organoids (PDOs);
The second is high-throughput screening of new drugs, where organoid cells are sourced from stem cells and tumor cell lines, and further cultured into various tissues and tumor-like organs for efficacy and toxicology screening of new drugs.
1) Patient-derived tumor-like organ drug sensitivity testing
Superhydrophobic microporous array chip enables nanoliter-scale liquid manipulation for applications in organoid culture. The system was able to complete drug sensitivity testing of tumor-like organs within 1 week, which was highly consistent with clinical data, showing its potential in predicting patient response to therapy.
At the same time, the technology makes organoid culture of biopsy samples possible. Thousands of micro-organospheres were generated from a small amount of tissue using multiphase flow droplet microfluidics, enabling rapid prediction of tumor drug response.
Constructing PDOs with complex microenvironments remains challenging, especially in mimicking the tumor microenvironment (TME).
Co-culture of MSCs, peripheral blood immune mononuclear cells and hepatocellular carcinoma cells by microfluidics successfully mimicked the TME of hepatocellular carcinoma-like organs and improved the prediction accuracy of the immunotherapy response, demonstrating the importance of microfluidics for the prediction of immunotherapy effects in PDO.
2) Screening of new stem cell-derived organoid drugs
The organoid is obtained by differentiation and culture of human stem cells, and possesses a spatial organization highly similar to that of the corresponding organ, which is capable of realistically reproducing the organ function and drug response.
Therefore, organoid replacement of 2D cell and animal experiments for drug screening can achieve higher clinical consistency with the advantages of speed, high throughput and low cost.
In addition, organoids provide a rich resource for deep sequencing, functional assays, and phenotyping.
The high-throughput drug screening platform based on conventional cell lines uses pipetting robots and cell culture flasks and multiwell plates to automate the process of cell culture and differentiation, and is capable of rapidly standardizing the production of billions of iPSCs to support high-throughput drug screening.
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