With the advancement of in vitro 3D culture technology, patient-derived organoids (PDOs) have demonstrated significant value in maintaining the genetic heterogeneity of the original tumors and play an increasingly important role in disease disease model construction and drug screening.
Conventional PDO culture methods still face some technical challenges:
(1) Poor reproducibility and significant differences in activity, size and shape of PDO make it difficult to ensure the reliability of experimental results;
(2) Low throughput and limited sample size make it difficult to perform high-throughput drug screening;
(3) Inadequate tissue mimicry and lack of a co-culture system for vascular cells and tumor-infiltrating immune cells limit applications in anti-vascularization and immunotherapeutic drug screening.
Organoids-on-a-chip are the expansion of organoids in the field of biotechnology, and can effectively address the shortcomings of traditional culture techniques.
The technology is centered on a microfluidic chip, which promotes bionic structural and functional maturation of organoids at the microenvironmental level by controlling fluid perfusion, retractive forces and chemical gradients.
It has the advantages of controllability, high throughput and dynamic monitoring, which provides the possibility of scale-up and automated standardized culture of PDO.
This will significantly improve the success rate of organoid model construction, the accuracy of drug screening, and expand the application of organoids.
1. Advantages of microfluidics-based organoid chips
1) Improvement of organoid stability
The instability of conventional organoid culture techniques in terms of cell density control and physical flow conditions has resulted in culture success rates ranging from only 20% to 95% and variations in the size, morphology, and number of PDOs.
The introduction of microfluidics significantly improved the consistency and stability of the PDO model.
It has been shown that microfluidics is capable of handling and manipulating tiny fluids through microchannels that produce a variety of autocrine or paracrine cytokines by modulating shear stress, interstitial fluid flow, and cell density, resulting in different matrix properties.
These substrate properties have potential effects on physiological states such as morphology, growth rate, and metabolic activity of organoids.
2) Highly simulate the tumor microenvironment
Tumor microenvironment (TME) is an important factor affecting the accuracy of drug response prediction and has become a hotspot at the forefront of drug development for chemotherapy and immunotherapy in lung cancer.
Most models of PDOs contain only tumor cells, while key microenvironmental factors such as immune cells, stromal cells, and cytokines are often lost in culture, which limits the functional testing of PDOs in chemotherapy and targeted drug screening.
In contrast to static co-culture systems, microfluidics enables precise control of physical and chemical parameters in the device at the nanoscale, thereby remodeling the physicochemical properties of the TME and simulating complex interactions between cells.
Thus, organoid microarrays are able to co-culture patient-derived tumor cells with immune cells, overcoming the limitations of traditional functional testing of PDOs.
Organoid microarrays can also exogenously establish vascular membrane layers, enhance the interaction between immune cells and blood vessels in tissues, and can highly reproduce the tumor angiogenesis microenvironment and the dynamic interactions between cells in vitro, thus contributing to the study of related molecular mechanisms and drug analysis.
3) Dynamic monitoring of drug reactions
In drug efficacy assessment, traditional methods usually require staining of PDOs and then determining the activity and status of the cells by fluorescence microscopy.
This method is not only complicated to operate, but also may affect the subsequent experiments, and it is difficult to realize the dynamic real-time monitoring of class organs.
Microfluidics has its origins in the fabrication of integrated circuit chips, which, in combination with the application of sensors, allows for the dynamic and continuous monitoring of organoid responses to drugs without intervention or disruption.
Microfluidics-based organoids are expected to enable dynamic multi-parameter assays for more comprehensive and precise assessment of drug efficacy or toxicity.
For example, connecting a light-addressable potentiometric sensor or using a fluorescent probe allows continuous observation of a wide range of parameters, such as protein macromolecules, mRNA, pH and electrochemical changes, with high sensitivity and without disturbing the organoid culture.
The ease of real-time and the precision and reliability of this technology will accelerate the process of medical research.
4) Realize high-throughput drug screening
The limited availability of tissues through surgery or small sample biopsies poses a challenge for the use of organoids in preclinical drug screening or personalized therapies, especially in providing an adequate source of cells for high-throughput drug screening.
Traditional organoid culture systems are cumbersome to operate manually, difficult to mimic the common periodic drug regimen in the clinic, and are still facing the dilemma of achieving automation and high-throughput drug screening.
Compared to traditional multiwell plates, organoid chips have nanoscale culture capacity and multi-channel parallel design, significantly reducing the number of cells required for PDOs culture.
The chip allows for rapid reactions in microtubules or microculture chambers, consumes low amounts of reagents, and is easy to manipulate manually, resulting in significant cost reductions.
Mechanically automated organoid microarrays reduce time-consuming and laborious pipetting steps, minimize human error and reduce failure rates compared to traditional culture methods that require extensive manual drug delivery.
The device also makes it easy to combine multiple drugs at different concentrations, enabling a variety of functions such as large-scale, multi-drug processing and identification of potential drug combinations.
2. Application of organoid microarrays in precision treatment of lung cancer
1) Personalized drug screening for lung cancer
A study using surgically resected lung cancer tissues on constructed organoid microarrays for full gene sequence analysis of PDOs showed 90% concordance of somatic variants between lung cancer PDOs and specimen sections, which provides a basis for individualized treatment of lung cancer.
The research team developed a superhydrophobic microporous array chip based on lung cancer PDOs capable of generating hundreds of organoids in a very short period of time and performing high-throughput chemotherapeutic drug sensitivity testing.
Notably, this is the first time that lung cancer PDOs were introduced into the chip and screened in high throughput with different concentrations in 1 week, which significantly shortens the time for organoid culture and drug testing, solves an important challenge in precision diagnosis and treatment, and greatly improves the efficiency of organoids in clinical translation.
Recently, some scholars have successfully realized automatic structural digestion of organoids and oriented multi-level heterogeneity analysis at the single-cell level.
This technology makes it possible to achieve single-cell distribution and banking on a chip, and to achieve clustering of drug response differences and classification of single-cell types based on analysis of sequencing data.
2) Exploring complex interactions in TME for lung cancer
Targeting the tumor microenvironment (TME) for anti-tumor metastasis has been a difficult and hot research topic.
Microfluidics has become a new means of constructing lung cancer metastasis models, and the main studies include single-cell analysis, endothelial cell migration and neovascularization.
For example, the researchers designed a simple microfluidic chip to mimic the invasive microenvironment of lung cancer and cell-matrix interactions.
In this device, cultured lung cancer cells are able to form three-dimensional spheres and exhibit features of epithelial-mesenchymal transition (e.g., altered expression of E-cadherin, N-cadherin, Snail1, and Snail2), which in turn is used to assess the potential of tumor cells to invade distant organs (e.g., the brain, bone, and liver).
The dynamic process of tumor-mesenchymal cell interaction, angiogenesis and tumor metastasis in TME is reproduced by organoid microarrays, which helps to study the related molecular mechanism and drug analysis, and further promotes the precision treatment of lung cancer.
3) Constructing organoid models based on tumor circulating cells (CTCs)
Circulating tumor cells (CTCs) are a biomarker in liquid biopsy that refers to tumor cells that are shed from solid tumors and enter the peripheral blood circulation.
Due to their easy and less invasive sampling, CTCs play an important role in early diagnosis, predicting tumor progression, and guiding individualized treatment.
Because the concentration of CTCs in peripheral blood is lower than that of other blood cells, and the ratio of the two is about 1:109, the currently commonly used liquid histology methods (such as density gradient centrifugation, filtration, and immunomagnetic sorting) are difficult to efficiently amplify a very small number of CTCs in vitro, limiting their clinical application.
Using whole blood from 19 patients with early-stage lung cancer, the researchers successfully constructed an organoid microarray model co-cultured with fibroblasts by capturing CTCs on a microfluidic chip.
The model establishes a highly specific affinity between the recognition molecule and the cellular target molecule by molecularly modifying the surface of the chip substrate, thus enabling effective isolation of CTCs.
The model is capable of in vitro amplification of CTCs within 14 days, supporting analysis of various aspects of genes, proteins and functions, which is of great clinical significance for precision tumor medicine.
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