Application Of Organoids-on-a-chip In Drug Sensitivity Analysis Of Individualized Chemotherapy For Bladder Cancer

BackgroundBladder cancer is one of the most common tumors of the urinary system.Clinically,bladder cancer is divided into muscle invasive bladder cancer(MIBC)and non-muscle invasive bladder cancer(NMIBC),and NMIBC accounts for 75% to 85%.On the way of surgical methods,muscle invasive bladder cancer is mainly treated by radical cystectomy,while non-muscular invasive bladder cancer is treated by transurethral cystectomy(TURBT).At the same time,further perfusion chemotherapy is needed after urethral resection for high grade bladder cancer.Bladder cancer has a high recurrence rate after surgery and how to prevent the recurrence of bladder cancer and improve the effectiveness of postoperative chemotherapy has been the focus over the years.There are dozens of drugs currently used in the treatment of bladder cancer.The cisplatin-based chemotherapy regimen has remained unchanged for nearly 30 years,but nearly 50% of patients are resistant to the regimen.Therefore,individualized chemotherapy for bladder cancer is of great significance to patients and urgently needs to be developed.Organoids are a kind of in vitro tissue that can be grown independently by taking samples from human tissues and growing in vitro by simulating the in vivo environment.By simulating the human environment,various culture conditions are set to obtain organoids that are closest to the functional indicators and physical and chemical properties of human organs.Compared with cell line experiments and animal experiments,studies on organoids have better parental similarity and individual characteristics,and have a wider application prospect in various clinical mechanism studies and drug screening studies.With the progress of polymer materials and microsystem processing technology,the application value of microfluidic technology represented by PDMS microfluidic chip in biomedicine has been paid more and more attention in recent years.The PDMS microfluidic chip can achieve precise control of various influencing factors through microchannels because of its better hydrophobic permeability and biocompatibility.And it can simulate various microenvironments of human body,providing a suitable carrier for in vitro culture of various human tissues and organs.Although many kinds of organoids based on microfluidic chips have emerged in recent years,their functions either focus on the simulation of human microenvironment but ignore the main role of organ-like function,or focus on its precise control but ignore its clinical application value.Therefore,most current microfluidic chips cannot be used for clinical treatment in the short term due to their complex and advanced design.Our project hopes to explore the process of microfluidic culture of bladder cancer to construct a kind of organoids-on-a-chip,and apply it to the individualized analysis of chemotherapy drugs for bladder cancer while considering the timeliness and economy,so as to improve the pertinence and effectiveness of drug treatment and provide a better solution to the difficulties in clinical treatment.Objectives1.To explore the main process of in vitro culture and verification of bladder cancer organoids.2.To investigate the design and processing technology of microfluidic chips suitable for the growth of bladder cancer organoids.3.To study the fabrication process of bladder cancer organoids based on microfluidic chips and its application in clinical chemotherapy drug screening.Methods1.The specimens of bladder cancer after transurethral resection or radical cystectomy were processed and cultured in vitro to obtain individual bladder cancer organoids with genetic stability and long-term survival.Then the organoids were verified by immunohistochemistry and chromosome sequencing.2.Based on hydrodynamics and probability principles,we designed microfluidic chips using Auto CAD2019 and SOLIDWORKS.And then,the simulation analysis of flow rate,pressure and drug concentration diffusion in the chips was conducted through COMSOL Multiphysics to select the best design scheme.Finally,the verified design models was made into qualified microfluidic chips through lithography,casting,bonding and other steps and then applied to organoids culture.3.The prepared bladder cancer organoids were introduced into the microfluidic chip.Through the control of the import,culture and chip structure,the pre-cultured bladder cancer organoids were imported into the microfluidic chips to construct a organoids-on-a-chip of bladder cancer that could be applied clinically.The organoid chips were then applied to the screening of clinical chemotherapy drugs.Results1.The results showed that the organoids of bladder cancer could be cultured in vitro for a long time after optimizing the treatment process and conditions.After immunohistochemistry and chromosome sequencing,the organoids had good genetic stability and parental similarity,and could be used for drug screening of individual patients.2.The two-dimensional and three-dimensional models of the microfluidic chip were obtained through software design,and then the microfluidic chip was optimized repeatedly through simulation analysis results.In the process of chip preparation,we overcome the technical difficulties such as degum and pollution,and finally get qualified and stable microfluidic chips.3.The organoids-on-a-chip was successfully constructed by introducing bladder cancer organoids into microfluidic chip.The organoids in the chips can be normally cultured and show different characteristics and the results showed that the application of high-throughput organoids-on-a-chip in the screening of chemotherapy drugs had a higher success rate.ConclusionThis project is a study combining clinical application with basic research.In the early stage,we explored the design and manufacture of microfluidic chips and the in vitro culture of bladder cancer organoids,and developed a reliable organoids-on-a-chip system.Secondly,the organoids-on-a-chips were applied to the sensitivity analysis of clinical chemotherapy drugs.The results indicated that organoids-on-a-chip of bladder cancer showed obvious advantages in sensitivity analysis of clinical chemotherapy drug and had a broad application prospect in the near future.