这项研究得到补助YMS从美国国家卫生研究院（CA148828），密歇根胃肠肽中心大学，杰弗里A.科尔比结肠癌研究的美国密歇根大学综合癌症中心的汤姆刘纪念基金。

1。结肠癌的分离和细胞分离<ol><li>肠道肿瘤或治疗引起的任何零星结肠癌模型可以隔绝。应的小鼠安乐死与CO 2。冒号然后收集，用冷的磷酸盐缓冲盐水（PBS）冲洗，并沿纵向打开。识别区，其中包含肿瘤，利用体视显微镜，解剖用一把剪刀，用冷PBS洗。 </li><li>孵育肠片段:含有肿瘤EDTA螯合缓冲液（2 mM的EDTA，5.6毫摩尔/ L的Na 2 HPO 4，8.0毫摩尔/ L KH 2 PO 4，96.2毫摩尔/ L的NaCl，1.6毫摩尔/ L的KCl，43.4毫摩尔/ L蔗糖， 54.9 mmol / L的D-山梨糖醇，0.5毫摩尔/ L的DL-二硫苏糖醇在蒸馏水中）在冰上60分钟。 </li><li>螯合作用后，正常的肠上皮细胞脱落，而肿瘤细胞的间充质细胞将保持附着。螯合缓冲吸含有正常的上皮细胞洗净残余肿瘤碎片一个更多的时间与5毫升冷螯合缓冲。 </li><li>吸螯合缓冲区，用5毫升冷1X PBS洗涤肿瘤碎片。 </li><li>吸出的1倍PBS，孵育肿瘤片段消化缓冲液中（2.5％胎牛血清，1单位/ ml青霉素，1微克/毫升链霉素，和2.5毫微克/毫升的两性霉素B，200 U / ml的Ⅳ型胶原酶， 125微克/毫升分散酶II型的Dulbecco改良的Eagle培养基）2小时，在37℃下</li><li>允许肿瘤片段来解决正常重力下1分钟，收集上清液在15毫升猎鹰管。颗粒的单细胞瘤悬浮液上清，通过离心分离，在200×g离心3分钟，并以200×g离心3分钟，用5ml PBS洗一次。 </li></ol> 2。肠道肿瘤文化<ol><li>用500μlPBS重悬肿瘤细胞沉淀，孤立的单个肿瘤细胞使用血球计数。 </li><li>颗粒肿瘤细胞占rifuging 3分钟后，以200×g，并重新悬浮在5 mg / ml的基底膜上的冰和板在24孔培养板中，每孔15000个细胞每50μl的基底膜。 </li><li>让基底膜在37℃下聚合反应15分钟，并加入500微升/孔的基础培养基（1单位/ ml青霉素，1微克/毫升链霉素，和2.5毫微克/毫升两性霉素B，10 mmol / L的HEPES N2补充2mm GLUTAMAX的，1个，1个B27的补充，1 mmol / L的N-乙酰半胱氨酸高级贝科的修改鹰Medium/F12）含有50毫微克/毫升鼠表皮生长因子。 </li></ol> 3。维护既定的组织体<ol><li>改变基底含有表皮生长因子的培养基中每星期一次，每2天及通过类器官1:5。 </li><li>传代，更换新鲜的基础培养基培养基。机械破坏的类器官和基底膜使用P1000的吸管切断与技巧，并转移到15毫升猎鹰管。进一步的机械分离是通过使用火抛光的巴斯德管TTE。 </li><li>洗离解类器官的基础培养基中，并在200×g离心2分钟离心，用5毫升。 </li><li>弃去上清液，将沉淀重悬于与基底膜和如上所述的培养基中添加。 </li></ol> 4。存储和恢复既定的组织体<ol><li>对于长期储存，冻结液体N 2类器官的稳定，至少2年。对于冻结类器官，扰乱使用P1000的吸管与技巧切断，并转移到15毫升猎鹰管。 </li><li>洗离解类器官的基础培养基中，并在200×g离心2分钟离心，用5毫升。 </li><li>弃去上清液，将沉淀重悬于含20％胎牛血清（FBS）和10％二甲亚砜（DMSO）的Dulbecco改进的Eagle培养基（DMEM）。 </li><li>传输到1.5毫升冻存管细胞，然后把管一个的Nalgene雾先生冷冻集装箱和储存在-80℃的冰箱中实现了冷却速率以-1℃/分钟。过夜培养后，转成液态N 2管。 </li><li>对于恢复，迅速解冻冷冻类器官在37°C的热水浴，用基础培养基，降速，重悬在基底膜和文化与上述条件。 </li></ol> 5。提取RNA，蛋白质的提取及免疫组化<ol><li> RNA的提取:收集细胞，按上述通路。 RNA分离与PicoPure TM RNA提取试剂盒，根据制造商的指示。 </li><li>蛋白质提取方法:如上所述，并溶解在100μl放射免疫沉淀法测定缓冲液（RIPA，收集细胞沉淀，50 mmol / L的pH为7.5的Tris-HCl，150毫摩尔/ L的NaCl，2毫摩尔/ L的EDTA，1％NP-40，0.1 ％SDS）中与1倍的蛋白酶抑制剂。 </li><li>免疫组化:吸细胞培养液中，P1000移液器转移肿瘤组织体切断成低温模具的提示，并立即冻结那朵用干冰冷冻介质（OCT）提起诉讼。在7μm切片，切片冷冻染色按如前所述，26。 </li></ol>

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我们什么都没有透露。

在这个协议中描述的实验程序将允许分离培养初级小鼠结肠肿瘤。该协议是改编自做开创性的工作由博士Clevers组1,24,27。我们优化的消化时间和胶原酶浓度的肿瘤组织体，以获得更好的收益率。关键步骤包括肿瘤细胞消化成单个细胞，基底膜再悬浮，选择性培养。对于肿瘤细胞的消化中，为了获得有效的结肠肿瘤的离解，并维持其生存能力，胶原酶的消化和浓度应根据经验得出和优化。例如，当使用胶原酶在75 U / ml和孵化时间为30分钟，我们没有获得单个肿瘤细胞悬浮液和无类器官在这种条件下形成的，在我们手中。维持细胞-细胞接触，据报道，培养癌细胞28的关键，在这里使用胶原酶300 U / ml和incubation次也观察到3个小时，基质细胞污染。同样，基底膜再悬浮，浓度和凝固时间也可以根据经验来确定。类器官的肿瘤上皮细胞进行准确的评估。肠道肿瘤成立的组织体能够长期培养。我们已供建立类器官数个月，没有明显的差异，增殖和分化。所建立的类器官可以击倒和过度表达基因操纵。此外，肿瘤组织体可以产生从人类肿瘤活检样品，因此，该培养技术可用于研究在患者体内的具体方式的基本机制。 Wnt信号通路是肠胚胎发育的关键。成人小肠的Wnts都正常增殖的关键中转放大细胞和Wnt信号通路的失调是至关重要的结肠肿瘤29。选择性培养无线个EGF是足以维持结肠肿瘤类器官的生长。然而，当与Wnt3a的补充，类器官更有效地增长。因此，细胞是响应Wnt配体的作用，不同于几个结肠衍生的细胞系的生长。 组织体更紧密地模仿比肠上皮细胞建立结肠癌衍生系，我们相信这将提供更准确和生理相关的数据。如上所述，这种技术是有用的调查结肠癌的起始和进展的机制。此外，肿瘤类器官结肠癌治疗的疗效评估时，将提供更准确的数据。

肠上皮细胞增殖，并以惊人的速度，超过了所有其他组织在脊椎动物体内2,3。分裂的细胞，包括肠干细胞（ISC）和中转放大细胞分化（高脚杯，潘氏和肠内分泌）分为分泌细胞或肠3。国际学习中心坐落在该基地的地穴。潘氏细胞向下移动到隐窝底部，寿命长，而其他谱系向上迁移的绒毛3,4。在这里，细胞暴露于肠道内容包括微生物群，并通过的脱落凋亡诱导的细胞凋亡的机制从绒毛提示脱落。虽然结肠没有绒毛和帕内特细胞，能维持体内平衡机制，是相似图4。 有牵连的Wnt信号通路发挥了至关重要的作用在肠道增殖和ISC维护4。删除THê转录因子TCF4，Wnt信号通路的下游效应，导致肠道干细胞的损失和随后的崩溃组织5。同样，转基因的表达，Wnt信号抑制剂DKK1时间上皮细胞增殖和耗尽分泌细胞谱系6。相反，过度的Wnt受体激动剂R-脊椎蛋白1诱导强有力的和快速的肠隐窝细胞增殖的7。 鉴于Wnt信号肠道动态平衡的重要性，Wnt信号通路的突变常常可以观察到在结肠癌8。结肠癌是第三大死因，癌症在美国9。红肉和酒精，过量饮食摄入量与体力活动减少，并继承和体细胞突变被认为是结肠癌的危险因素10,11。腺瘤性息肉病（APC）基因，一个关键Wnt信号因子，在大多数PA突变患者家族性的，零星的，和结肠炎相关性结肠癌12,13。其他因素参与Wnt信号通路包括AXIN2和β-连环蛋白的突变也观察到在结肠癌14,15。但是，确切的机制和有效的治疗结肠癌仍然缺乏。为方便调查为结肠癌的分子机制已经建立，代表不同的癌的进展阶段，从良性积极的细胞类型的人类结肠癌细胞株16-18。小鼠结肠癌细胞株具有不同转移特性也19,20。然而，原代细胞或优于类器官文化转化的细胞系，因为它们紧密地模仿体内状态，并产生更多的生理相关数据21。最受欢迎的结肠癌衍生细胞系生长附着板的单分子层，或作为细胞悬浮液，缺位克顶端基底取向和细胞间紧密连接。此外，正常和肿瘤的肠上皮细胞在体内经过一个自发的形式，被称为细胞凋亡脱落凋亡，分化的细胞到达绒毛的提示和脱落22。这些功能是困难的，扼要重述在细胞系，但结肠癌23的发展过程中是很重要的。这些初级类器官的功能得到维护。此外，肿瘤类器官培养提供了一个有效的系统，以评估肿瘤的自主功能的基因相比， 在体内研究。遗传操纵体内的肠道是一个耗时的过程，主要是通过建立转基因和/或基因敲除小鼠肠道特定的驱动程序。然而，肿瘤类器官易于病毒介导的遗传操作和一个伟大的工具，用于评估精确的分子机制。原发性小肠肿瘤类器官培养甲肝已经证明是一个可行的和强大的技术。原发性肠细胞培养可以建立肠道功能的类器官体外隐窝绒毛结构从单一的成人LGR5 +干细胞24。这些类器官可移植和嫁接到受损的结肠组织再生25。进一步适应的文化条件作出了类似的上皮组织体小鼠结肠和人类小肠和结肠可行1。主正常结肠上皮细胞培养，基础培养基以及生长因子EGF，Noggin与中，R-脊椎和Wnt3a的是必不可少的，而基础培养基和EGF是足够的增长的主要小鼠结肠肿瘤类器官1。在这里，我们描述了一个详细的协议，分离，培养，并产生结肠肿瘤类器官。

<table><tbody><tr><td>Matrigel Basement Membrane Matrix</td><td>BD Biosciences</td><td>356234</td><td>5 mg/ml</td></tr><tr><td>Collagenase Type IV</td><td>Worthington</td><td>LS004188</td><td>375U/mg</td></tr><tr><td>Dispase</td><td>Gibco</td><td>17105-041</td><td>1.8U/mg</td></tr><tr><td>Advanced DMEM/F12</td><td>Invitrogen</td><td>12634010</td><td></td></tr><tr><td>Epidermal Growth Factor (EGF), Murine, Natural</td><td>Invitrogen</td><td>53003-018</td><td></td></tr><tr><td>N2 Supplement</td><td>Invitrogen</td><td>17502-048</td><td>100 x</td></tr><tr><td>B27 Supplement</td><td>Invitrogen</td><td>17504-044</td><td>50 x</td></tr><tr><td>Glutamax-I</td><td>Gibco</td><td>35050-079</td><td>100x</td></tr><tr><td>N-Acetylcysteine</td><td>Sigma</td><td>A9165-5G</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Invitrogen</td><td>11965-092</td><td></td></tr><tr><td>PicoPureTM RNA Isolation Kit</td><td>Invitrogen</td><td>KIT0204</td><td></td></tr></tbody></table>

in vitro organoid culture of primary mouse colon tumors

几个人类和小鼠结肠癌细胞系已经建立，结肠肿瘤，如多细胞层，基底顶端极性，分化能力和失巢凋亡的生理完整性不保持在结肠癌细胞系。目前的研究表明原代培养小鼠结肠肿瘤类器官改编自佐藤T 等人 ，保留了重要的生理功能，结肠肿瘤的方法。这种方法包括小鼠结肠肿瘤组织收集，相邻的正常结肠上皮解离，结肠肿瘤细胞消化成单个细胞，基底膜嵌入结肠肿瘤细胞，选择性培养的原则基础上，肿瘤细胞保持增长限制比较正常营养条件上皮细胞。 原发肿瘤类器官，如果从转基因小鼠中分离，提供了非常有用的系统，来评估肿瘤自治区此功能特定基因的离子。此外，肿瘤类器官适合病毒预谋基因传递的遗传操作，也可以被广泛的研究，因此在结肠肿瘤发生的信号通路参与过表达或击倒。原发肿瘤类器官培养提供了有关的生理和可行的手段来研究结肠肿瘤的发生机制和治疗方式。

从3个月大的APC 分钟/ +鼠标如图1所示的结肠肿瘤类器官形成的时间过程。单细胞在第0天，可以观察到几个小时后镀（ 图1A）。在第1天，存活结肠癌上皮细胞难治核可以观察到。在第3天，细胞的大小增加一倍。在第6天，类器官的大小扩大超过10倍，表现出细胞凋亡在中间。在第14天，的orgnoids会生长成不规则的车厢（ 图1B）。在这里，我们不得不39.33±22.05类器官/ 200 U / ml的胶原酶2小时的消化条件下形成的，比较没有在这项研究中30分钟（ 图1C）75 U / ml的胶原酶消化条件下形成的类器官。 为了显示这一类器官培养模型可用于功能研究，免疫荧光染色β-美食<html>年Wnt信号通路的一个组成部分， 如图2所示。 β-连环蛋白的阳性染色的细胞为位于输出层的类器官，这证实的增长模式与中央管腔提出的博士Clevers组24。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/50210/50210fig1highres.jpg" src="/files/ftp_upload/50210/50210fig1.jpg" /> 图1。一位代表类器官形成从3个月大的APC 分钟/ +小鼠结肠肿瘤来源于培养（A）0，1，3，6，（B）14天。 （C）类器官形成两个不同的胶原酶消化条件下成功率。 50210fig2highres.jpg"SRC ="/ files/ftp_upload/50210/50210fig2.jpg"/&gt; 图2。从3个月大的APC 分钟/ +鼠标从结肠肿瘤派生的类器官的β-连环蛋白免疫荧光染色。使用4&#39;，6 -二脒-2 -苯基吲哚二盐酸盐（DAPI）染色细胞核。

Watch this Scientific Journal Video about In vitro Organoid Culture of Primary Mouse Colon Tumors at JoVE.com

In vitro Organoid Culture of Primary Mouse Colon Tumors

一个简单的方法来建立初级小鼠结肠肿瘤类器官。这种方法利用的功能，结肠肿瘤细胞含有生长因子有限的媒体生存和发展成类器官，而正常结肠上皮不。

在体外化培养基层小鼠结肠肿瘤

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

in-vitro-organoid-culture-of-primary-mouse-colon-tumors

Nanyang Technological University

Research

JoVE Journal

Medicine

In vitro Organoid Culture of Primary Mouse Colon Tumors

34.9K 次观看.  University of Michigan. 一个简单的方法来建立初级小鼠结肠肿瘤类器官。这种方法利用的功能，结肠肿瘤细胞含有生长因子有限的媒体生存和发展成类器官，而正常结肠上皮不。

视频: In vitro Organoid Culture of Primary Mouse Colon Tumors

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

科研

癌症研究

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

淋巴结慢病毒北疆-转基因 Organoids 培养的大鼠结肠肿瘤的高灵敏度检测

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

结直肠癌原位小鼠模型

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

生物学

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

遗传学

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

小鼠膀胱肿瘤器官的培养、操作和正交移植

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

使用磁纳米粒子转导病毒载体进行冷冻切片的原小鼠肠道器官的遗传工程

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

发育生物学

来自新鲜组织的胰腺癌来源的肿瘤类器官和成纤维细胞

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

作者聚焦:从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

该<em&gt;离体</em&gt;文化与模式识别小鼠肠道组织体受体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

在体外化培养基层小鼠结肠肿瘤

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