3D Cell Culture And High Throughput Drug Screening Based On Microfluidic Droplet Systems

High-throughput drug screening plays an essential role in drug discovery.However,the existing high-throughput screening systems are mainly based on multi-well plates,which have disadvantages such as single cell culture condition,laborious and time consuming operation,and high consumption of reagents.Moreover,it is difficult to achieve complex drug combination screening.Microfluidic technique,which is able to manipulate and control microfluids in micro-scale channels,has the characteristics of low consumption,high efficiency,high throughput and automation.Therefore,it can overcome the shortcomings of the screening systems based on multi-well plates.Microfluidic systems can be used to control and simulate the micro-environment for cell growth,and 3D cell culture,and have broad application in the fields of cell behavior and response research,as well as drug discovery.In chapter 1,we review the research progress of microfluidic techniques for high-throughput drug screening,including microfluidic drug screening techniques based on 2D and 3D cell culture.In chapter 2,we presented a novel droplet-based 3D cell culture method by using droplet array attached on the sidewall of a PDMS piece.Such an arrangement not only avoids cells from adhering on the chip surface for achieving 3D cell culture in nanoliter-scale droplets,but also facilitates performing multiple operations to cells in droplets,including cell suspension droplet generation,drug treatment,and cell staining with a capillary-based liquid handling system,as well as in-situ observation and direct scanning with a confocal laser scanning microscope.We optimized the system by studying the effects of various conditions to cell culture including droplet volume,cell density and fabrication methods of the PDMS pieces.We have applied this system in the 3D culture of HepG2 cells and the stimulation testing of an anticancer drug,doxorubicin,to 3D cell spheroids.In chapter 3,we designed a microfluidic hanging droplet system for 3D cell culture,by combing a PDMS through-hole array chip with the SODA system capable of generating drug concentration gradient automatically,to realize the 3D cell culture and rapid high-throughput drug stimulation.We studied the responses of two kinds of cell spheroids to doxorubicin,including homotypic cell tumors(SGC-7901)and heterotypic cell tumors(SGC-7901+3T3).The experimental results indicated that different kinds of cell spheroids have different responses to the anticancer drugIn chapter 4,we utilized the SODA system to flexibly perform multi-step operations including liquid metering,mixing,transferring,and dispensation,to form droplet array with concentration gradient up to 4 orders of magnitude.With the present system,drug combination screening for sorafenib,CPT,luteolin,trans-chalcone,indole-3-carbinol,melatonin,and tetrandrine were performed in 576 HepG2 cell droplets.The combinations of sorafenib and trans-chalcone,sorafenib and tetrandrine,CPT and tetrandrine showed synergistic antitumor activity.The results of the first two combinations are consistent with the previous literatures,while the latter is reported for the first time,demonstrating the ability of SODA system to generate large-scale concentration gradients for drug combination screening,and broad application prospect in large-scale complex drug combination screening.