【作者】 陈辉；

【导师】 国世上； 郭峰；

【作者基本信息】 武汉大学 ， 材料物理与化学， 2022， 博士

【摘要】 3D生物打印由于其优异的组织结构构建能力和生物材料的操作性,在组织工程和再生医学工程中得到了广泛的应用。生物打印已面向构建结构,但在生物机制研究中的应用较少。声表面波3D打印器件可实现声液滴打印,单细胞操控,多材料,高粘度液体打印以及实现精细尺度的构建。声表面波3D打印技术可用于解决生物医学工程中的各种紧迫问题,例如疾病模型的建立、个性化治疗研究。本论文正是在开发的声表面波(SAW)3D打印器件的基础上,挖掘声表面波3D打印器件在生物医学工程中的应用。针对现有癌症个性化治疗中存在的问题,如缺少机理研究工具、忽视患者肿瘤侵袭能力的影响、特定癌种的治疗方式以及难以重现肿瘤真实环境,耐药性分析不足等问题,本文进行了针对性研究。首先进行了器件可行性验证,通过声学3D打印构建肿瘤球-成纤维细胞(CAFs)侵袭模型,并对模型进行观察,证明了声学3D打印器件可实现单细胞球的精细操作以及生物模型的构建;其次,结合类器培养技术进行了声学3D打印类器官精细排布用于低位直肠癌括约肌间切除术(ISR)辅助治疗;最后,针对现有肿瘤个性化治疗中的不足点,通过声学3D打印器件构建同一患者来源的正常类器官包围癌类器官的微组织模型,重构肿瘤真实结构,使试药更精准,用于结直肠癌个性化治疗。与现有3D打印技术相比较,声表面波3D打印技术有着更精准的单细胞液滴(肿瘤球或类器官)操控能力,以满足精细模型的建立,用于机理研究和临床辅助治疗。研究内容如下:1.利用声表面波3D打印精准操控肿瘤球和细胞的特性,构建了单颗肿瘤球-成纤维细胞(CAFs)模型用于研究结直肠癌中CAFs与肿瘤侵袭机理。通过悬滴法将患者癌组织来源细胞快速(36 h)培养成肿瘤球,anti-Ep CAM免疫磁珠完成同源患者CAFs的阴选分离培养。利用声打印器件开腔结构,可以实现微量液体的精细操作和高粘度材料的无阻塞打印。将单颗肿瘤球打印在接收板上,随后将含甲基丙烯酸酯化明胶(Gel MA)的CAFs打印在肿瘤球周围,构建结直肠肿瘤球-CAFs侵袭模型,观察肿瘤球和CAFs的侵袭过程、判断肿瘤侵袭能力。随后,用临床药物5-氟尿嘧啶(5-FU)对模型进行试药,观察模型中各种蛋白质的免疫荧光变化。根据结果,对患者的临床预防提出建议。声表面波3D打印器件建模验证和侵袭研究,证明了声表面波3D打印侵袭模型的可行性以及侵袭模型具有一定临床指导意义。2.利用声表面波3D打印器件能够构建肿瘤模型的特性,结合开发的类器官培养技术,培养直肠癌患者癌组织衍生的癌类器官以及患者癌旁1-2cm处组织衍生的正常类器官,对同一患者来源的癌类器官和正常类器官进行打印排布,构建类器官侵袭模型,模拟低位直肠癌肿瘤空间布局,观察癌类器官侵袭过程,评估其侵袭能力,结合低位直肠癌患者临床核磁共振成像(MRI)和错配修复(MMR)蛋白检测对患者肿瘤位置和浸润性,辅助判断类器官来源患者在手术治疗时是否需要进行括约肌间切除术(ISR)。通过实验证明,该方法可用于低位直肠癌极限保肛的辅助工具。3.声表面波3D打印器件构建类器官肿瘤微组织模型(PDMs)用于患者个性化治疗。我们结合前面开发的声学打印技术与类器官培养技术相结合,并对类器官进行了DNA全外显子测序(DNA WES),将患者的结肠类器官精确定位到肿瘤类器官周围,形成中心是癌类器官、周围是正常类器官结构,称为类器官肿瘤微组织(PDMs),再现真实肿瘤结构。对PDMs进行了5-FU试药和侵袭性研究,通过肿瘤-结肠类器官相互作用动力学的延时成像分析肿瘤微组织生长状况。通过蛋白免疫荧光分析PDMs对5-FU的敏感性。此外,通过RNA转录组测序(RNA-SEQ)对5-FU处理和对照组的PDMs从差异基因上进行了研究,分析PDMs模型对5-FU的敏感性。结合70例患者样本,证明了声表面波3D打印器件构建类器官肿瘤微组织在临床个性化治疗中具有应用前景。更多还原

【Abstract】 3D bioprinting has been widely used in tissue engineering and biomedical engineering due to its excellent tissue structure construction and manipulability of biomaterials.Bioprinting has been oriented towards building structures and has less application in the study of biological mechanisms.SAW 3D printed devices enable acoustic droplet printing,single-cell manipulation,multi-material,high-viscosity liquid printing,and fine-scale construction.SAW 3D printing technology can be used to solve various pressing problems in biomedical engineering,such as the establishment of disease models,personalized treatment research.In this paper,the applications of surface acoustic wave(SAW)3D printing devices in biomedical engineering are explored based on the developed SAW 3D printing devices.In view of the problems existing in the current personalized cancer treatment,such as the lack of mechanism research tools,ignoring the impact of patients’ tumor invasion ability,specific cancer treatment methods,difficult to reproduce the real tumor environment,drug resistance analysis,etc.,this paper conducted a targeted study.Firstly,the feasibility of the device was verified.The tumor sphere-fibroblast(CAFs)invasion model was constructed by acoustic 3D printing,and the observation of the model proved that the acoustic 3D printing device could realize the fine operation of single cell spheres and the construction of biological model.Secondly,acoustic 3D printing of organoid fine arrangement combined with organoid culture technology was performed for the adjuvant treatment of low rectal cancer intersphincter resection(ISR).Finally,aiming at the shortcomings of the existing personalized cancer treatment,the acoustic 3D printing device is used to construct the microtissue model of normal organoids from the same patient surrounding the cancer organoids,reconstruct the real tumor structure,and make the drug test more accurate,which can be used for personalized treatment of colorectal cancer.Compared with the existing 3D printing technology,the surface acoustic wave 3D printing technology has more accurate manipulation ability of single-cell droplet(tumor ball or organoid),so as to meet the establishment of fine model,and be used for mechanism research and clinical adjuvant therapy.1.Using surface acoustic wave 3D printing to precisely control the characteristics of tumor spheres and cells,a single tumor sphere-fibroblast(CAFs)model was constructed to study the mechanism of CAFs and tumor invasion in colorectal cancer.The patient’s cancer tissue-derived cells were quickly(36 h)cultured into tumor spheres by the hanging drop method,and anti-Ep CAM immunomagnetic beads were used to complete the negative separation and culture of homologous patient CAFs.Using the open-cavity structure of acoustic printing devices,fine manipulation of trace amounts of liquids and non-blocking printing of high-viscosity materials can be achieved.A single tumor sphere was printed on the receiving plate,and then CAFs containing methacrylated gelatin(Gel MA)were printed around the tumor sphere to construct a colorectal tumor sphere-CAFs invasion model to observe the invasion process of tumor spheres and CAFs to determine the tumor invasive ability.Subsequently,the model was tested with the clinical drug 5-fluorouracil(5-FU),and the immunofluorescence changes of various proteins in the model were observed.Based on the results,recommendations are made for clinical prevention of the patient.As the modeling verification and invasion research of SAW 3D printing device,it proves the feasibility of SAW 3D printing invasion model and the invasion model has certain clinical guiding significance.2.Using the characteristics of surface acoustic wave 3D printing devices to build tumor models,combined with the developed organoid culture technology,the cancer organoids derived from the cancer tissue of patients with rectal cancer and the normal organoids derived from the tissue 1-2cm adjacent to the cancer of the patient were cultured,Using the advantages of acoustic 3D printing devices to build fine structures,we can print and arrange cancer organoids and normal organoids from the same patient,build an organoid invasion model,simulate the spatial layout of low rectal cancer tumors,and observe the invasion process of cancer organoids.To evaluate its invasive ability,combined with clinical Magnetic resonance imaging(MRI)and mismatch repair(MMR)proteins detection in patients with low rectal cancer,to determine the tumor location and infiltration of patients,and to help determine whether intersphincterectomy(ISR)is required for patients with organoids derived from surgical treatment.3.SAW 3D printing devices to construct organoid tumor microtissue models(PDMs)for personalized treatment of patients.We combined the previously developed acoustic printing technology with the organoid culture technology to precisely position the patient’s colon organoids around the tumor organoids to form a cancer organoid in the center and a normal organoid structure around it,which is called an organoid tumor microstructure tissues(PDMs),reproducing the real tumor structure.5-FU reagent and invasiveness studies were performed on PDMs,and tumor microtissue growth was analyzed by time-lapse imaging of tumor-colon organoid interaction kinetics.The sensitivity of PDMs to 5-FU was analyzed by protein immunofluorescence.In addition,the 5-FU-treated and control group PDMs were differentially studied by RNA transcriptome sequencing(RNA-SEQ)to analyze the sensitivity of the PDMs model to5-FU.DNA whole exome sequencing(DNA WES)was performed on the organoids.Combined with 70 patient samples,it is proved that the surface acoustic wave 3D printing device to construct organoid tumor microtissue has a wide range of applications in clinical personalized treatment.更多还原