Construction of multiple concentration gradients for single-cell level drug screening

Drug screening is a critical but expensive and time-consuming process. Cellular heterogeneity, even within genetically identical populations, significantly impacts drug efficacy and resistance. Single-cell analysis offers a powerful approach to understand this heterogeneity, but existing methods often lack the ability to study multiple drug concentrations simultaneously. Microfluidic devices provide advantages for single-cell studies due to their low reagent consumption, precise control, and high throughput. Microfluidic-based concentration gradient generators have been developed, but most focus on whole-cell populations and do not enable the study of multiple drug combinations at the single-cell level. This research aimed to address this gap by creating a microfluidic platform integrating a multi-concentration gradient generator with a single-cell capture array to enable comprehensive single-cell drug screening and analysis of drug interactions.

The introduction extensively reviews existing literature on drug screening challenges, the importance of single-cell analysis in overcoming these challenges, and the advantages of microfluidic technology for single-cell studies. It highlights limitations of previous microfluidic approaches, particularly the lack of multi-concentration gradient generation capabilities for single-cell analysis and the inability to simultaneously study the effects of multiple drugs on single cells. The authors cite numerous publications supporting the need for their proposed approach and the relevance of studying single-cell heterogeneity in drug response.

The study involved designing and fabricating a microfluidic device composed of a concentration gradient generator and a single-cell capture array. The gradient generator utilizes Tai Chi-spiral mixers to efficiently create three independent concentration gradients. The device fabrication employed soft lithography techniques using PDMS and a silicon master mold. Computational fluid dynamics (CFD) simulations were used to model fluid flow and concentration gradients within the device, verifying the design's effectiveness. Human breast carcinoma (MCF-7) and human hepatoma (HepG2) cells were used to assess the device's performance. Cells were captured in the array, exposed to different drug concentrations (5-fluorouracil and cisplatin), and their viability assessed using microscopy. Image analysis was performed to quantify drug effects and analyze correlations between cell biomechanics and drug resistance.

Numerical simulations and fluorescein experiments validated the generation of three stable and symmetrical concentration gradients in the microfluidic device. The Tai Chi-spiral mixers effectively enhanced mixing, achieving consistent gradients across various flow rates. Single-cell drug screening experiments demonstrated the efficacy of both 5-fluorouracil and cisplatin in inhibiting cancer cell growth. A synergistic effect was observed when the drugs were used in combination, particularly for HepG2 cells. A correlation analysis revealed that smaller and/or more deformable cells were more resistant to the drugs compared to larger and/or less deformable cells. This finding highlights the importance of considering single-cell heterogeneity in cancer drug response.

The findings demonstrate the successful construction of a robust microfluidic platform for single-cell drug screening with multiple concentration gradients. The device's ability to generate and control precise drug concentrations, combined with its single-cell capture capabilities, provides a powerful tool for studying drug efficacy and identifying optimal drug combinations. The observed correlation between cell biomechanics and drug resistance suggests potential targets for improving cancer therapy. This platform's high throughput and reduced reagent consumption offer significant advantages over traditional methods, facilitating efficient high-throughput drug screening.

This research successfully developed a novel microfluidic platform capable of generating multiple drug concentration gradients and performing single-cell level drug screening. The device offers a significant advancement in single-cell drug analysis by enabling the simultaneous study of drug combinations and facilitating the identification of optimal dosages. The correlation found between cell biomechanics and drug resistance opens avenues for future research on personalized cancer treatments. Future studies could explore the device's application to a wider range of drugs and cancer cell lines, and investigate the underlying mechanisms of drug resistance at the single-cell level.

The current study used only two specific anticancer drugs and two cell lines. Further research is needed to validate the platform’s generalizability to other drug combinations and cancer types. The device's design and operation could be further optimized to increase throughput and reduce experimental time. The analysis of cell biomechanics was limited, and more detailed investigations could provide deeper insights into the mechanisms of drug resistance.