| Objective: Organoids use 3D cell culture technology in vitro to simulate the growth environment of cells in vivo.It allows cells to differentiate and develop into organs that contain specific structures and can be passaged and partially simulate physiological functions in the body.Because of its unique structure,organoids have the characteristics of self-renewal,maintaining genomic stability,maintaining individual heterogeneity,reproducing organ functions,studying cell-to-cell interactions,achieving high-throughput research,and low ethical risks.Patient-derived tumor organoids biobanks can be used for drug development research,personalized treatment and regenerative medicine.Pancreatic Ductal Adenocarcinoma(PDAC)is a highly aggressive cancer,accounting for about 85-95% of all pancreatic malignancies,and the majority of tumors are located in the head of the pancreas(60-70%).The early symptoms of pancreatic ductal adenocarcinoma far from the common bile duct are not obvious,and most patients can only be found in the advanced stage,having lost the opportunity of surgical resection,and the 5-year survival rate of patients is only about 4%.For these patients,drug therapy is an effective means to improve their survival rate.Therefore,the establishment of pancreatic ductal carcinoma model is of great significance for the study of pathogenesis and drug screening.Endoscopic ultrasonic-guided Fine-Needle Aspiration(EUS-FNA)is currently the standard method for the diagnosis of pancreatic space-occupying lesions.EUS-FNA examination can obtain sufficient pancreatic samples for subsequent diagnosis and organoid culture.The diagnosis of pancreatic space occupying lesions and the culture of organoids are related to the quality of the puncture sample,which is affected by multiple technical parameters in the EUS-FNA procedure,among them,puncture needle type,negative pressure size and the number of needle moves back and forth are relatively important variables.Most of the current studies have investigated a single variable in the operation parameters.Although a single study has found some rules,it is difficult to clarify the rules by Meta-analysis of large samples.Therefore,this study will use the Response Surface Methodology to conduct a multi-factor and multi-level comprehensive investigation of multiple operating parameters affecting the quality of EUS-FNA samples,construct prediction models of EUS-FNA/B operating parameters on sample quality control,find the key factors affecting the quality of samples,and study the interaction between the factors.Finally,we can determine the operating parameters of EUS-FNA according to the sample requirements.Under the guidance of the prediction model,the key parameters in EUS-FNA/B operation were controlled to obtain suitable tissue samples for the culture of pancreatic cancer organoids,and to provide a method and basis for the efficient construction of pancreatic cancer organoids.Methods: Firstly,BBD(Box-Behnken Design)of Response Surface Methodology was used to automatically generate 17 different experiments.Among the 17 experiments,5 repeated experiments were performed to ensure the stability of the experiments.In order to satisfy the operability of the experiment and reduce experimental error,17 groups of experiments were performed on the pancreases of two Bama miniature pigs.There were 17 sets of experiments,each repeated three times,and the results were averaged.Three groups were randomly selected to verify the model.Design-Expert 10 software was used to analyze the data.The total cell number reflects the cell volume,the total protein in EUSFNA samples reflects the tissue volume,the number of viable cells to total protein reflects the tissue activity,and the amount of hemoglobin reflects the mixed blood volume.The effects of puncture needle type(19 ga,22 ga,25 ga),negative pressure(10-20 ml),and the number of needle moves back and forth(5-15 times)on cell volume,tissue volume,tissue activity and mixed blood volume of puncture samples were investigated comprehensively,and the corresponding prediction models were constructed.Secondly,under the guidance of the prediction model,the key parameters of EUS-FNA operation were controlled to obtain high tissue activity of pancreatic cancer samples,and the technical route of pancreatic cancer organoid culture was established.Finally,the construction of pancreatic cancer organoid came true.Results: 1.The Box-Behnken Design(BBD)was used to regulate and control the operation parameters of EUS-FNA,and the prediction model of cell volume,tissue volume and tissue activity in EUS-FNA samples was successfully constructed.The constructed model was highly reliable.A predictive model for the amount of mixed blood in EUS-FNA samples was not established.(1)According to the feasibility of operation,the operating conditions of EUS-FNA with high cell number obtained by Response Surface Methodology were as follows: needle size 22 ga,negative pressure size 20 ml,and the number of needle moves back and forth 10 times.(2)According to the feasibility of operation,the operating conditions of EUS-FNA with high total protein quality obtained by Response Surface Methodology were as follows: needle size 22 ga,negative pressure size 20 ml,and the number of needle moves back and forth 9 times.2.The key operational parameters affecting the quality of EUS-FNA samples were identified by the predictive model.(1)The quadratic of needle type and the quadratic of pulling times were significant factors affecting the total number of cells.(2)The type of needle and the quadratic of the type of needle were significant factors affecting the total protein.(3)The number of needle moves back and forth,the interaction between the type of needle and the number of needle moves back and forth,and the quadratic of the number of needle moves back and forth were significant factors affecting the number of viable cells to total protein.Pairwise interactions among needle type,negative pressure and the number of needle moves back and forth were identified and described by the prediction model.3.Highly tissue activity EUS-FNA/B samples were obtained according to the constructed prediction model,and the technical route of EUS-FNA/B derived tissue samples to prepare pancreatic cancer organoids was established.Pancreatic cancer organoids were successfully constructed,which could be passaged for 5 times,and frozen pancreatic cancer tissues could be used for the establishment of pancreatic cancer organoids.4.The number of needle moves back and forth may affect the formation of EUS-FNA/ B-derived pancreatic organoids by affecting tissue activity.Conclusion: In this study,the Response Surface Methodology was introduced into the quality evaluation system of EUS-FNA puncture samples for the first time,providing a relatively stable and efficient method for obtaining high-quality EUS-FNA samples.At the same time,under the guidance of the construction model,by controlling the key technical parameters of EUS-FNA/B operation,the obtained samples with high tissue activity can be used for the construction of pancreatic cancer organoids. |
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