CN115449504A - A method for establishing a 2D culture model of porcine small intestine organoids - Google Patents

The application discloses a method for establishing a stable 2D culture model of a porcine small intestine organoid, which comprises the steps of firstly obtaining a passable porcine small intestine organoid, preparing a porcine small intestine organoid tissue suspension, and then culturing to form a porcine small intestine organoid 2D growth model. The application changes the method for obtaining the porcine small intestine organoid monolayer, changes the traditional mechanical dissociation into the enzymatic dissociation and is beneficial to keeping the integrity of cells; 2D two-dimensional culture of cells is utilized to help reduce the influence of interaction between injected substances and apical membranes due to fragments accumulated in an inner cavity in the 3D culture process; the 2D intestinal organoid model enables functional research to better accord with real physiological conditions, can provide simpler and more convenient operation for high-flux nutrient screening and bioactive substance function identification, and solves the problem that the intestinal nutrition and health related research feasibility is poor due to the fact that the intestinal luminal surface of the 3D intestinal organoid cannot be in direct contact with a culture environment.

一种猪小肠类器官2D培养模型的建立方法A method for establishing a 2D culture model of porcine small intestine organoids

技术领域technical field

本发明属于动物组织学技术领域。具体涉及一种猪小肠类器官2D培养模型的建立方法。The invention belongs to the technical field of animal histology. In particular, it relates to a method for establishing a 2D culture model of pig small intestine organoids.

背景技术Background technique

近十年来，类器官技术作为一个整体技术领域系统地在生物学、医学等多个研究学科得以应用，基于其自组织能力，收获具有诱导形成多功能器官能力的干细胞可以体外形成结构和功能与体内对应物相似的3D结构(Juan He，et al.Organoid technology fortissue engineering.Journal of molecular cell biology.2020，12(8)：569-579.)。类器官培养技术在肠道研究中的应用十分广泛。肠道干细胞在合适地体外培养条件下，最初形成具有单个中央内腔的囊状空泡结构，干细胞进一步分裂增殖产生过渡扩增细胞，囊腔也随之向外扩展形成芽状凸起，过渡扩增细胞经过4-5次分裂后产生肠上皮细胞、肠内分泌细胞、杯状细胞、潘氏细胞等多种终末端分化细胞，使囊状结构呈现出芽特征，5-7天后形成典型的包含所有肠道上皮细胞类型和完整隐窝-绒毛结构的类器官结构(Akkerman N，etal.Dawn of the organoid era.Bioessays.2017，39(4).)。已有研究优化了类器官体外培养的方法，论证了体外长期培养的可能性，至少可以连续传代1年半以上仍保持遗传性质的稳定(Sugimoto S，et al.Establishment of 3D Intestinal Organoid Cultures fromIntestinal Stem Cells.Methods in Molecular Biology.2017，1612：97.)。肠道类器官不仅在细胞谱系组成上与肠上皮组织高度相似，其功能特性也与来源组织保持高度一致(FoUlke-AbelJ，et al.Human Enteroids as a Model of Upper Small Intestinal IonTransport Physiology and Pathophysiology.Gastroenterology.2016，150(3)：638-649)。由此可见，小肠类器官这一全新技术的引入，必将深化我们对生物活性物质对肠粘膜屏障功能调控的认识，为研究营养物质的肠道健康改善功能提供新的思路也将为个体化的精准营养及代谢研究奠定理论及实践基础。In the past ten years, organoid technology has been systematically applied in multiple research disciplines such as biology and medicine as a whole technical field. Similar 3D structures to their in vivo counterparts (Juan He, et al. Organoid technology fortissue engineering. Journal of molecular cell biology. 2020, 12(8): 569-579.). Organoid culture technology is widely used in intestinal research. Under appropriate in vitro culture conditions, intestinal stem cells initially form a cystic vacuolar structure with a single central lumen, stem cells further divide and proliferate to produce transitional amplified cells, and the cystic cavity also expands outwards to form bud-like protrusions. After 4-5 divisions of the expanded cells, various terminally differentiated cells such as intestinal epithelial cells, enteroendocrine cells, goblet cells, and Paneth cells are produced, making the cystic structure exhibit the characteristics of budding, and a typical inclusion is formed after 5-7 days Organoid structures of all intestinal epithelial cell types and intact crypt-villus structures (Akkerman N, et al. Dawn of the organoid era. Bioessays. 2017, 39(4).). Studies have optimized the method of in vitro culture of organoids, and demonstrated the possibility of long-term culture in vitro, at least one and a half years of continuous passage can still maintain the stability of genetic properties (Sugimoto S, et al.Establishment of 3D Intestinal Organoid Cultures from Intestinal Stem Cells. Methods in Molecular Biology. 2017, 1612: 97.). Intestinal organoids are not only highly similar to intestinal epithelial tissue in terms of cell lineage composition, but their functional properties are also highly consistent with the source tissue (FoUlke-AbelJ, et al. Human Enteroids as a Model of Upper Small Intestinal IonTransport Physiology and Pathophysiology. Gastroenterology .2016, 150(3):638-649). It can be seen that the introduction of the new technology of small intestinal organoids will surely deepen our understanding of the regulation of intestinal mucosal barrier function by bioactive substances, and provide new ideas for the study of intestinal health improvement functions of nutrients. It lays a theoretical and practical foundation for precise nutrition and metabolism research.

在营养对肠道健康影响领域相关研究中，类器官模型与多个学科交叉应用发挥作用。类器官最显著的特点就是其强大的自我更新能力以及与体内微环境的高度相似性(高云等.肠类器官的研究与应用.国际消化病杂志.2017，37(2)：87-91)。其此类特点的优势，类器官体外培养技术发展迅速。虽然基于啮齿类动物、犬等动物模型开展肠道营养生物学的研究已取得重大进展，但由于上述动物与人类之间在解剖结构和生理特性方面明确差异的存在限制了这些模型的应用(Beumer J，et al.Regulation and plasticity ofintestinal stem cells during homeostasis and regeneration.Development.2016，143(20)：3639-3649)。例如小鼠模型缺乏代表人类胃肠道疾病的关键临床症状和病理变化，从而限制了用小鼠为模型开展临床营养学研究；而犬类也由于社会伦理问题，在相关研究中的使用也被广泛限制(Sara Rahmani，et al.Intestinal organoids：A new paradigmfor engineering intestinal epithelium in vitro.Biomaterials.2019，194)。相比较而言，猪是一种相对可靠的动物模型，其优点在于猪与人类肠道生理特点与解剖特征的相似性(Koopmans，Sietse Jan，Schuurman，Teun.Considerations on pig models forappetite，metabolic syndrome and obese type 2diabetes：From food intake tometabolic disease[J].European Journal of Pharmacology.2015，759：231-239)。因此，猪肠道类器官结合了上述体内外研究模型的优势。目前，已经研究开发出较完整的猪肠道类器官培养方法但该培养方法形成的类器官在一些模拟真实肠道环境的应用中仍然存在一定的不足。In the field of research on the impact of nutrition on intestinal health, organoid models play a role in the interdisciplinary application of multiple disciplines. The most notable feature of organoids is their strong self-renewal ability and high similarity with the microenvironment in vivo (Gao Yun et al. Research and Application of Intestinal Organoids. International Journal of Digestive Diseases. 2017, 37(2): 87-91 ). Due to the advantages of such characteristics, organoid in vitro culture technology has developed rapidly. Although significant progress has been made in the study of intestinal nutritional biology based on animal models such as rodents and dogs, the application of these models is limited by the existence of clear differences in anatomy and physiological properties between these animals and humans (Beumer J, et al. Regulation and plasticity of intestinal stem cells during homeostasis and regeneration. Development. 2016, 143(20): 3639-3649). For example, the mouse model lacks the key clinical symptoms and pathological changes representing human gastrointestinal diseases, which limits the use of mice as a model for clinical nutrition research; and the use of dogs in related research is also restricted due to social and ethical issues. Widely restricted (Sara Rahmani, et al. Intestinal organoids: A new paradigm for engineering intestinal epithelium in vitro. Biomaterials. 2019, 194). In comparison, pig is a kind of relatively reliable animal model, and its advantage is the similarity (Koopmans, Sietse Jan, Schuurman, Teun.Considerations on pig models forappetite, metabolic syndrome and obese type 2diabetes: From food intake tometabolic disease [J]. European Journal of Pharmacology. 2015, 759: 231-239). Thus, porcine intestinal organoids combine the advantages of the aforementioned in vitro and in vivo research models. At present, a relatively complete porcine intestinal organoid culture method has been researched and developed, but the organoids formed by this culture method still have certain shortcomings in some applications that simulate the real intestinal environment.

肠道类器官3D培养模型是研究组织成体干细胞生长分化以及器官形成的新兴体外研究系统，是肠道生物学研究领域里程碑式的研究成果。但是，3D化培养的肠道类器官由于其肠腔面被包裹在球形结构的腔体内侧，阻碍了其与外界环境的接触，导致对肠道营养健康的研究受阻，此外，3D培养类器官的内腔会积聚来自细胞更新的碎片，其可结合或阻碍注射物质与顶膜的相互作用，从而对整个类器官的培养造成影响。所以，优化培养程序并形成更方便操作的体外类器官应用模型具有一定现实意义，也正是本文所解决的技术问题。Intestinal organoid 3D culture model is an emerging in vitro research system for studying the growth and differentiation of tissue adult stem cells and organ formation, and is a milestone research achievement in the field of intestinal biology research. However, the intestinal luminal surface of 3D cultured intestinal organoids is wrapped inside the cavity of the spherical structure, which hinders its contact with the external environment, which hinders the study of intestinal nutritional health. In addition, 3D cultured organoids The lumen of the cell can accumulate debris from cell turnover, which can bind or hinder the interaction of the injected substance with the apical membrane, thereby compromising the culture of the entire organoid. Therefore, optimizing the culture procedure and forming a more convenient in vitro organoid application model has certain practical significance, which is also the technical problem solved in this paper.

发明内容Contents of the invention

本发明的目的是建立一种稳定的猪小肠类器官2D培养模型的建立方法，首先获取可传代的猪小肠类器官、制备猪小肠类器官组织悬液，再培养形成猪小肠类器官2D生长模型。The purpose of the present invention is to establish a method for establishing a stable 2D culture model of porcine small intestine organoids. Firstly, obtain passageable porcine small intestine organoids, prepare porcine small intestine organoid tissue suspension, and then cultivate to form a 2D growth model of porcine small intestine organoids .

本申请是通过以下技术措施来实现的：一种猪小肠类器官2D培养模型的建立方法，包括：获取可传代的猪小肠类器官：无菌采集来自猪空肠中段约5～10cm，经过两次清理至上清液澄清；吸去上清液，将肠段转移至含有30mL 8mM EDTA-PBS溶液中，平放于冰上摇动40min，去除上清液，加入新的30mL 8Mm EDTA-PBS溶液，再次冰上摇动40min，吸去EDTA溶液，用30mL PBS晃动清洗，置于冰上让肠组织块通过重力沉降，去上清，重复此过程1～2次；将沉淀转移至15mL离心管中，于5mL DMEM/F12(含10％FBS，1％三联抗生素)培养基中重悬沉淀，取3-5μL镜检计数隐窝数量；在4℃下500g离心5min后，弃去上清液，将沉淀与基质胶Matrigel均匀混合，按20μL/孔的量点于48孔板中；将48孔细胞培养板转移到二氧化碳培养箱中，37℃、5％CO₂条件下孵育15min，至Matrigel固化；待Matrigel固化后，加入预热的200μL/孔完全培养基；将其置于37℃培养箱中继续培养，每隔2-3天更换一次新鲜培养基；每5-7天根据生长情况进行传代；This application is achieved through the following technical measures: A method for establishing a 2D culture model of porcine small intestine organoids, including: obtaining passageable porcine small intestine organoids: aseptically collecting about 5-10 cm from the middle section of porcine jejunum, after cleaning twice Until the supernatant is clear; suck off the supernatant, transfer the intestinal segment to a solution containing 30mL 8mM EDTA-PBS, place it flat on ice and shake for 40min, remove the supernatant, add a new 30mL 8mM EDTA-PBS solution, and ice again Shake it up for 40 minutes, suck off the EDTA solution, shake and wash with 30mL PBS, put it on ice and let the intestinal tissue pieces settle by gravity, remove the supernatant, repeat this process 1-2 times; transfer the precipitate to a 15mL centrifuge tube, put Resuspend the pellet in DMEM/F12 (containing 10% FBS, 1% triple antibiotics) medium, take 3-5 μL for microscopic examination and count the number of crypts; centrifuge at 500g for 5 min at 4°C, discard the supernatant, and mix the pellet with Mix Matrigel evenly, and place 20 μL/well in a 48-well plate; transfer the 48-well cell culture plate to a carbon dioxide incubator, and incubate for 15 minutes at 37°C and 5% CO ₂ until the Matrigel is solidified; After solidification, add preheated 200 μL/well complete medium; place it in a 37°C incubator to continue culturing, and replace the fresh medium every 2-3 days; pass passage every 5-7 days according to the growth situation;

制备猪小肠类器官组织悬液：将培养的猪类器官在生长至可传代的时候，吸去培养基，每孔加入500μL预冷的PBS，室温下孵育1min；移液枪吸取PBS将肠类器官混匀，转移至15mL离心管中；每孔加1mL PBS冲洗，加入同一15mL离心管中；4℃下200g离心5min，弃去上清液；根据细胞量加入适量TrypL E，37℃水浴5min，期间每隔2min摇晃一次；加入适量DMEM/F12(含10％FBS)培养基终止消化；室温500g离心5min，弃去上清液；加入适量培养基吹打，得到猪小肠类器官组织悬液；Prepare porcine small intestinal organoid tissue suspension: When the cultured porcine organoids grow to the point where they can be passaged, remove the medium, add 500 μL pre-cooled PBS to each well, and incubate at room temperature for 1 min; pipette the PBS to remove the intestinal tissue. Mix the organ and transfer it to a 15mL centrifuge tube; add 1mL PBS to each well and add it to the same 15mL centrifuge tube; centrifuge at 200g for 5min at 4°C, discard the supernatant; add an appropriate amount of TrypL E according to the amount of cells, and bathe in water at 37°C for 5min , shake once every 2 minutes during this period; add an appropriate amount of DMEM/F12 (containing 10% FBS) medium to stop digestion; centrifuge at 500g at room temperature for 5 minutes, discard the supernatant; add an appropriate amount of medium and pipette to obtain a porcine small intestine organoid tissue suspension;

建立猪小肠2D模型：将制备好的猪小肠类器官组织悬液接到铺好胶的细胞培养板表面或加入细胞培养板中，形成猪小肠类器官2D单层。Establish a 2D model of porcine small intestine: connect the prepared porcine small intestinal organoid tissue suspension to the surface of a gel-coated cell culture plate or add it to a cell culture plate to form a 2D monolayer of porcine small intestinal organoids.

作为优选，采用Matrigel铺胶法建立猪小肠2D模型，具体包括：As a preference, the 2D model of pig small intestine is established by using the Matrigel glue-laying method, which specifically includes:

(1)将Matrigel和冰浴PBS按1∶100比例稀释后用于包被48孔细胞培养板；(1) Dilute Matrigel and ice-bathed PBS at a ratio of 1:100 and use it to coat 48-well cell culture plates;

(2)每孔加入100μL基质胶用于孔板的包被；(2) Add 100 μL Matrigel to each well for coating the well plate;

(3)滴入100μLPBS，将其压在Matrigel上层；(3) Drop 100 μL PBS and press it on the upper layer of Matrigel;

(4)在37℃二氧化碳细胞培养箱中静置2h，待基质胶凝固后吸出PBS；(4) Stand in a carbon dioxide cell incubator at 37°C for 2 hours, and suck out the PBS after the matrigel solidifies;

(5)加入PBS，在37℃二氧化碳培养箱中静置5min，重复三次；(5) Add PBS, let it stand in a 37°C carbon dioxide incubator for 5 minutes, and repeat three times;

(6)加100μLPBS到孔中，在37℃二氧化碳培养箱静置备用；(6) Add 100 μL PBS to the well, and let it stand in a 37° C. carbon dioxide incubator for later use;

(7)取适量制备好的猪小肠类器官组织悬液按照约10⁴个/mL的密度接到铺好胶的48孔细胞培养板表面，静置培养于37℃细胞培养箱，最终汇合成2D单层细胞。(7) Take an appropriate amount of prepared porcine small intestinal organoid tissue suspension and connect it to the surface of a 48-well cell culture plate covered with glue at a density of about 10 ⁴ /mL, and culture it statically in a 37°C cell culture incubator, and finally confluent into 2D monolayer cells.

作为优选，所述Matrigel和冰浴PBS的比例为1∶20。Preferably, the ratio of Matrigel to ice-bathed PBS is 1:20.

作为优选，采用Transwell法建立猪小肠2D模型，具体包括：(1)将已经制备好的猪小肠类器官组织悬液按照2×10⁴个/孔加入嵌套Transwell嵌套的24孔细胞培养板中；(2)置于细胞二氧化碳培养箱中培养约3天，每隔24h测量一次电阻并换液，观察电阻值变化情况；(3)培养至猪小肠类器官2D单层培养物形成。As a preference, the Transwell method is used to establish a 2D model of the pig small intestine, which specifically includes: (1) Adding the prepared pig small intestine organoid tissue suspension at 2×10 ⁴ per well to a nested Transwell nested 24-well cell culture plate Medium; (2) cultured in a cell carbon dioxide incubator for about 3 days, measured the resistance and changed the medium every 24 hours, and observed the change of resistance value; (3) cultured until a 2D monolayer culture of porcine small intestinal organoids was formed.

作为优选，所述采用Transwell法建立猪小肠2D培养模型，通过透射电镜观察细胞结构，具体包括：a.将接种了猪小肠类器官并经过3天培养后的Transwell膜用PBS润洗两次，用刀片割下该Transwell膜，置于戊二醛溶液(2.5％)中，4℃固定过夜；b.吸弃掉固定液，加入0.1M磷酸缓冲液(pH7.0)漂洗膜，重复三次，每次15min；c.加入1％的锇酸溶液固定样品1-2h；d.吸出锇酸废液，加入足量的0.1M磷酸缓冲液(pH7.0)漂洗膜三次，每次15min；e.准备浓度分别为30％、50％、70％、80％的乙醇溶液依次浸没样品15min，使样品脱水，此后，再将样品依次浸入90％和95％的丙酮溶液中，分别浸泡15min；最后，将样品置于纯丙酮中20min，重复进行一次；等待过程中，为使其脱水完全，需适度振荡样品；f.将样品置于Spurr包埋剂∶丙酮＝1∶1(体积比)的混合液中浸泡1h；g.将样品置于Spurr包埋剂∶丙酮＝3∶1(体积比)的混合液中浸泡3h；h.将样品置于纯包埋剂中，室温过夜；i.包埋、切片及染色观察：将经过渗透处理的样品包埋起来，70℃加热过夜，即得到包埋好的样品，样品在LEICAEM UC7型超薄切片机中切片，获得70-90nm的切片，切片经柠檬酸铅溶液和醋酸双氧铀50％乙醇饱和溶液各染色5-10min，在Hitachi H-7650型透射电镜中观察。As a preference, the Transwell method is used to establish a 2D culture model of pig small intestine, and the cell structure is observed through a transmission electron microscope, which specifically includes: a. The Transwell membrane inoculated with pig small intestine organoids and cultured for 3 days is rinsed twice with PBS, Cut off the Transwell membrane with a razor blade, place it in glutaraldehyde solution (2.5%), and fix overnight at 4°C; b. Discard the fixative by suction, add 0.1M phosphate buffer (pH7.0) to rinse the membrane, repeat three times, 15 minutes each time; c. Add 1% osmic acid solution to fix the sample for 1-2 hours; d. Aspirate the osmic acid waste solution, add enough 0.1M phosphate buffer (pH7.0) to rinse the membrane three times, 15 minutes each time; e .Prepare ethanol solutions with concentrations of 30%, 50%, 70%, and 80% to immerse the samples in turn for 15 minutes to dehydrate the samples. After that, immerse the samples in 90% and 95% acetone solutions in turn for 15 minutes respectively; finally , place the sample in pure acetone for 20min, repeat once; in the waiting process, in order to make it completely dehydrated, shake the sample moderately; f. Soak in the mixed solution for 1h; g. Soak the sample in the mixed solution of Spurr embedding agent: acetone=3:1 (volume ratio) for 3h; h. Place the sample in pure embedding agent, overnight at room temperature; i. Embedding, sectioning and staining observation: Embed the infiltrated sample and heat it at 70°C overnight to obtain the embedded sample. The sample is sliced in a LEICAEM UC7 ultra-thin microtome to obtain a 70-90nm slice. The sections were stained with lead citrate solution and 50% saturated ethanol solution of uranyl acetate for 5-10 min respectively, and observed in Hitachi H-7650 transmission electron microscope.

作为优选，将所述获取可传代的猪小肠类器官的方法和/或制备猪小肠类器官组织悬液的方法应用于猪肠道类器官的相关研究。Preferably, the method for obtaining passageable porcine small intestinal organoids and/or the method for preparing porcine small intestinal organoid tissue suspensions are applied to related research on porcine intestinal organoids.

作为优选，将所述采用Matrigel铺胶法建立猪小肠2D培养模型和/或采用Transwell法建立猪小肠2D培养模型的方法应用于猪肠道类器官的相关研究。Preferably, the method of establishing a 2D culture model of porcine small intestine by Matrigel method and/or establishing a 2D culture model of porcine small intestine by Transwell method is applied to the related research of porcine intestinal organoids.

本申请的有益效果：本申请改变了猪肠道类器官单层获得方法，将传统的机械解离改变为酶法解离，有利于保持细胞的完整性；利用细胞2D(二维)培养有助于减小3D培养过程中由于内腔积聚的碎片阻碍注射物质与顶膜相互作用的影响；利用2D小肠类器官模型使生物活性物质的功能性研究更加符合真实生理情况，能为高通量的营养素筛选和生物活性物质功能鉴定提供更为简易、方便的操作，弥补了3D肠道类器官肠腔面无法与培养环境直接接触，导致对肠道营养与健康相关研究可行性差的问题。Beneficial effects of this application: This application changes the method for obtaining a monolayer of porcine intestinal organoids, and changes the traditional mechanical dissociation into enzymatic dissociation, which is conducive to maintaining the integrity of cells; using 2D (two-dimensional) culture of cells has It helps to reduce the impact of the debris accumulated in the inner cavity hindering the interaction between the injected substance and the apical membrane during the 3D culture process; the use of the 2D small intestinal organoid model makes the functional research of bioactive substances more in line with the real physiological situation, which can be used for high-throughput Nutrient screening and functional identification of biologically active substances provide simpler and more convenient operations, making up for the problem that the luminal surface of 3D intestinal organoids cannot be in direct contact with the culture environment, resulting in poor feasibility of research on intestinal nutrition and health.

附图说明Description of drawings

附图用来提供对本申请的进一步理解，并且构成说明书的一部分，与本申请的实施例一起用于解释本申请，并不构成对本申请的限制。在附图中：The accompanying drawings are used to provide a further understanding of the present application, and constitute a part of the specification, and are used together with the embodiments of the present application to explain the present application, and do not constitute a limitation to the present application. In the attached picture:

图1为猪小肠类器官接板培养三天的光镜图；Figure 1 is a light microscope image of porcine small intestine organoid cultured on a plate for three days;

图2为透射电镜观察培养三天的猪小肠类器官2D单层微观形态。Figure 2 shows the 2D monolayer microscopic morphology of porcine small intestinal organoids cultured for three days observed by transmission electron microscopy.

具体实施方式detailed description

下面将参照附图更详细地描述本公开的示例性实施例。虽然附图中显示了本公开的示例性实施例，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided for more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

本发明的一种获取可传代的猪小肠类器官，包括如下步骤：A method of obtaining a passageable porcine small intestine organoid of the present invention comprises the following steps:

(1)无菌采集猪空肠中段约5～10cm，置于预冷的PBS(含1％三联抗生素)并置于冰上作为实验肠段一待用；(1) Aseptically collect about 5-10 cm of the middle section of porcine jejunum, put it in pre-cooled PBS (containing 1% triple antibiotics) and put it on ice as the experimental intestinal section-1 for later use;

(2)将实验肠段一转移至含10mL PBS(含1％三联抗生素)的平皿中，去除肠段外表面附着的结缔组织，用10mL注射器清洗肠段内腔，将肠段纵向剥开，用无菌的载玻片刮去肠段中的内容物，再用PBS(含1％三联抗生素)冲洗后转移至超净工作台；(2) Transfer the experimental intestinal segment 1 to a plate containing 10mL PBS (containing 1% triple antibiotics), remove the connective tissue attached to the outer surface of the intestinal segment, clean the inner cavity of the intestinal segment with a 10mL syringe, and peel the intestinal segment longitudinally. Use a sterile glass slide to scrape off the contents of the intestinal segment, rinse with PBS (containing 1% triple antibiotics) and transfer to the ultra-clean workbench;

(3)将肠段转入装有30mL PBS的无菌离心管中，颠倒清洗肠段，待肠组织自然沉降后，吸去上清液，再次加入30mL PBS重复上述步骤，直至上清液澄清；(3) Transfer the intestinal segment into a sterile centrifuge tube filled with 30mL PBS, wash the intestinal segment upside down, after the intestinal tissue settles naturally, absorb the supernatant, add 30mL PBS again and repeat the above steps until the supernatant is clear ;

(4)将肠段转移至盛有10mL PBS的无菌培养皿中，用无菌载玻片轻刮表面去除残留内容物，将肠段剪成长约2mm左右作为实验肠段二；(4) Transfer the intestinal segment to a sterile petri dish filled with 10 mL of PBS, gently scrape the surface with a sterile glass slide to remove the residual content, and cut the intestinal segment to a length of about 2 mm as the experimental intestinal segment 2;

(5)将实验肠段二转移至装有30mL PBS的无菌离心管中，颠倒清洗肠段至上清液澄清；(5) Transfer the experimental intestinal segment 2 to a sterile centrifuge tube filled with 30 mL of PBS, and wash the intestinal segment upside down until the supernatant is clear;

(6)吸去上清液，将肠段二转移至30mL含8mM EDTA的PBS溶液中，平放于冰上摇动40min，去除上清液，加入新的30mL含8mM EDTA的PBS溶液，再次冰上摇动40min，吸去EDTA溶液，用30mL PBS晃动清洗，置于冰上使肠组织块通过重力沉降，去上清，重复此过程1～2次；(6) Aspirate the supernatant, transfer intestinal segment 2 to 30mL PBS solution containing 8mM EDTA, place it flat on ice and shake for 40min, remove the supernatant, add new 30mL PBS solution containing 8mM EDTA, ice again Shake for 40 minutes, absorb the EDTA solution, shake and wash with 30mL PBS, place on ice to allow the intestinal tissue to settle by gravity, remove the supernatant, and repeat this process 1 to 2 times;

(7)将沉淀的肠组织块转移至新的无菌15mL离心管中，于5mL DMEM/F12(含10％FBS，1％三联抗生素)培养基中重悬，取3-5μL镜检计数单个隐窝数量；(7) Transfer the precipitated intestinal tissue pieces to a new sterile 15mL centrifuge tube, resuspend in 5mL DMEM/F12 (containing 10% FBS, 1% triple antibiotics) medium, take 3-5μL microscopic examination and count single number of crypts;

(8)4℃下500g离心5min后，弃去上清液，将沉淀与Matrigel均匀混合，按20μL/孔的量点于48孔细胞培养板中；(8) After centrifuging at 500g for 5min at 4°C, discard the supernatant, mix the precipitate with Matrigel evenly, and put 20μL/well into a 48-well cell culture plate;

(9)将48孔细胞培养板转移到二氧化碳培养箱中，37℃、5％CO₂条件下孵育15min，至基质胶Matrigel固化；(9) Transfer the 48-well cell culture plate to a carbon dioxide incubator, and incubate for 15 minutes at 37° C. and 5% CO ₂ until the Matrigel is solidified;

(10)待Matrigel固化后，按照200μL/孔的量加入预热的200μL/孔完全培养基；将其置于37℃培养箱中继续培养，每隔2-3天更换一次新鲜培养基；每5-7天根据生长情况进行传代。(10) After Matrigel is solidified, add preheated 200 μL/well complete medium according to the amount of 200 μL/well; place it in a 37°C incubator to continue culturing, and replace the fresh medium every 2-3 days; 5-7 days for subculture according to the growth situation.

一种猪小肠类器官组织悬液制备方法，包括如下步骤：A method for preparing porcine small intestine organoid tissue suspension, comprising the steps of:

(1)待培养的猪小肠类器官生长至可传代状态，吸去培养基，每孔加入500μL预冷的PBS，室温下孵育1min；(1) The porcine small intestinal organoids to be cultured grow to a state that can be passaged, the medium is aspirated, and 500 μL of pre-cooled PBS is added to each well, and incubated at room temperature for 1 min;

(2)移液枪吸取PBS将小肠类器官混匀，转移至15mL离心管中；(2) Use a pipette to absorb PBS to mix the small intestinal organoids, and transfer them to a 15mL centrifuge tube;

(3)每孔加1mL PBS冲洗，加入同一15mL离心管中；(3) Add 1mL PBS to each well and add to the same 15mL centrifuge tube;

(4)4℃下200g离心5min，弃去上清液；(4) Centrifuge at 200g for 5min at 4°C, discard the supernatant;

(5)根据细胞量加入适量TrypL E，37℃水浴5min，期间每隔2min摇晃一次；(5) Add an appropriate amount of TrypL E according to the amount of cells, bathe in water at 37°C for 5 minutes, and shake once every 2 minutes during this period;

(6)加入适量DMEM/F12(含10％FBS)培养基终止消化；(6) Add appropriate amount of DMEM/F12 (containing 10% FBS) medium to terminate digestion;

(7)室温500g离心5min，弃去上清液；(7) Centrifuge at 500 g for 5 min at room temperature, and discard the supernatant;

(8)加入适量培养基吹打，得到猪小肠类器官组织悬液。(8) Add an appropriate amount of culture medium and pipette to obtain a porcine small intestine organoid tissue suspension.

本发明的两种猪小肠类器官2D培养模型的建立方法，分别包括如下步骤：The establishment method of two kinds of porcine small intestine organoid 2D culture models of the present invention comprises the following steps respectively:

Matrigel铺胶法：Matrigel Laying Method:

(1)将Matrigel和冰浴PBS按1∶100比例稀释后用于包被48孔细胞培养板；(1) Dilute Matrigel and ice-bathed PBS at a ratio of 1:100 and use it to coat 48-well cell culture plates;

(2)每孔加入100μL基质胶用于孔板的包被；(2) Add 100 μL Matrigel to each well for coating the well plate;

(3)滴入100μL PBS，将其压在Matrigel上层；(3) Drop 100 μL of PBS and press it on the upper layer of Matrigel;

(4)在37℃二氧化碳细胞培养箱中静置2h，待基质胶凝固后吸出PBS；(4) Stand in a carbon dioxide cell incubator at 37°C for 2 hours, and suck out the PBS after the matrigel solidifies;

(5)加入PBS，在37℃二氧化碳培养箱中静置5min，重复三次；(5) Add PBS, let it stand in a 37°C carbon dioxide incubator for 5 minutes, and repeat three times;

(6)加100μL PBS到孔中，在37℃二氧化碳培养箱静置备用；(6) Add 100 μL of PBS to the well, and let it stand in a 37°C carbon dioxide incubator for later use;

(7)取适量制备好的猪小肠类器官组织悬液按照约10⁴个/mL的密度接到铺好胶的48孔细胞培养板表面，静置培养于37℃细胞培养箱，通常约72h可汇合成2D单层细胞。(7) Take an appropriate amount of prepared porcine small intestinal organoid tissue suspension and connect it to the surface of a 48-well cell culture plate covered with glue at a density of about 10 ⁴ /mL, and culture it statically in a 37°C cell culture incubator, usually for about 72 hours Can confluent into 2D monolayer cells.

其中，Matrigel和冷PBS的比例确定：将猪小肠类器官传代后的单细胞悬液接板于三种不同的Matrigel铺胶比例中，分别为1∶10、1∶15、1∶20。Among them, the ratio of Matrigel and cold PBS was determined: the single-cell suspension after subculture of porcine small intestinal organoids was plated in three different Matrigel coating ratios, which were 1:10, 1:15, and 1:20, respectively.

由图1D可知，在肠类器官组织悬液接板三天后，铺胶比例为1∶10的实验组没有形成单层膜结构，而是出现了3D类器官的结构，说明按照1∶10铺胶比例过厚，从而导致细胞趋向于类器官3D生长方式；而铺胶比例为1∶15和1∶20的实验组中铺胶厚度较为合适，培养三天后，均能形成单层上皮样结构，由图1E和图1F可知，在1∶20的铺胶比例下，单层汇合度高于1∶15铺胶时的汇合度。因此，采用Matrigel∶PBS比例为1∶20进行铺胶，采用继续培养三天后汇合形成的猪小肠类器官2D单层培养物进行研究。It can be seen from Figure 1D that three days after the intestinal organoid tissue suspension was plated, the experimental group with a glue spreading ratio of 1:10 did not form a single-layer membrane structure, but a 3D organoid structure appeared, indicating that the 1:10 laying ratio The glue ratio is too thick, which leads to the cells tending to the 3D growth mode of organoids; the thickness of the glue in the experimental group with the ratio of 1:15 and 1:20 is more appropriate, and after three days of culture, a single-layer epithelial-like structure can be formed , it can be seen from Figure 1E and Figure 1F that under the glue laying ratio of 1:20, the confluence of single layer is higher than that of 1:15 glue laying. Therefore, the Matrigel:PBS ratio of 1:20 was used to spread the glue, and the 2D monolayer culture of porcine small intestinal organoids formed after continuous culture for three days was used for research.

Transwell法：Transwell method:

(1)将已经制备好的猪小肠类器官组织悬液按照2×10⁴个/孔加入Transwell嵌套的24孔细胞培养板中；(1) Add the prepared porcine small intestinal organoid tissue suspension into a Transwell nested 24-well cell culture plate at a rate of ² ×104 per well;

(2)置于细胞培养箱中培养约3d，每隔24h测量一次电阻并换液，观察电阻值变化情况；(2) Place it in a cell incubator for about 3 days, measure the resistance and change the liquid every 24 hours, and observe the change of the resistance value;

(3)待猪小肠类器官2D单层培养物形成后即可进行后续鉴定和分析。(3) Subsequent identification and analysis can be carried out after the 2D monolayer culture of porcine small intestinal organoids is formed.

采用透射电镜观察2D单层培养物结构的步骤：Steps to observe the structure of 2D monolayer culture by transmission electron microscope:

①将接种了猪小肠类器官并经过3天培养后的Transwell膜用PBS润洗两次，用刀片割下该Transwell膜，置于戊二醛溶液(V/V＝2.5％)中，4℃固定过夜；① Rinse the Transwell membrane inoculated with porcine small intestine organoids and cultured for 3 days with PBS twice, cut off the Transwell membrane with a razor blade, and place it in glutaraldehyde solution (V/V=2.5%) at 4°C fixed overnight;

②吸弃掉固定液，加入0.1M磷酸缓冲液(pH7.0)漂洗膜，重复三次，每次15min；② Discard the fixative, add 0.1M phosphate buffer (pH7.0) to rinse the membrane, repeat three times, 15min each time;

③加入锇酸溶液(V/V＝1％)固定样品1-2h；③Add osmic acid solution (V/V=1%) to fix the sample for 1-2h;

④吸出锇酸废液，加入足量的0.1M磷酸缓冲液(pH7.0)漂洗样品三次，每次15min；④ Suck out the osmic acid waste solution, add enough 0.1M phosphate buffer (pH7.0) to rinse the sample three times, each time for 15 minutes;

⑤准备浓度分别为30％、50％、70％、80％的乙醇溶液依次浸没样品15min，使样品脱水。此后，再将样品依次浸入90％和95％的丙酮溶液中，分别浸泡15min；最后，将样品置于纯丙酮中20min，重复进行一次；等待过程中，为使其脱水完全，需适度振荡样品；⑤Prepare ethanol solutions with concentrations of 30%, 50%, 70%, and 80%, respectively, to immerse the samples for 15 minutes to dehydrate the samples. After that, immerse the sample in 90% and 95% acetone solution in turn for 15 minutes respectively; finally, place the sample in pure acetone for 20 minutes and repeat it again; during the waiting process, in order to completely dehydrate the sample, it is necessary to shake the sample moderately ;

⑥将样品置于Spurr包埋剂∶丙酮＝1∶1(体积比)的混合液中浸泡1h；⑥ Soak the sample in a mixture of Spurr embedding agent: acetone = 1:1 (volume ratio) for 1 hour;

⑦再将样品置于Spurr包埋剂∶丙酮＝3∶1(体积比)的混合液中浸泡3h；⑦ Soak the sample in a mixture of Spurr embedding agent: acetone = 3:1 (volume ratio) for 3 hours;

⑧将样品置于纯包埋剂中，室温过夜；⑧Put the sample in pure embedding medium and leave overnight at room temperature;

⑨包埋、切片及染色观察：用包埋剂将经过上述渗透处理的样品包埋起来，70℃静置过夜，即可得到包埋好的样品。⑨Embedding, sectioning and staining observation: Embed the above-mentioned infiltrated sample with embedding agent, and let it stand overnight at 70°C to obtain the embedded sample.

利用LEICA EM UC7型超薄切片机对样品进行切片，获得70-90nm的切片，将其用柠檬酸铅溶液和醋酸双氧铀50％乙醇饱和溶液各染色5-10min，后续即可在Hitachi H-7650型透射电镜中观察类器官形成的2D化单层细胞。The samples were sliced with a LEICA EM UC7 ultrathin microtome to obtain 70-90nm slices, which were stained with lead citrate solution and uranyl acetate 50% ethanol saturated solution for 5-10min respectively, and then they could be processed in Hitachi H -7650 type transmission electron microscope to observe the 2D monolayer cells formed by organoids.

观察结果：利用透射电镜观察培养约三天后的猪小肠类器官2D单层培养物的微观形态，结果如图2所示。从图中可以清晰地观察到，猪小肠类器官2D单层已经分化出肠绒毛及紧密连接结构，图中箭头所指的部分即为细胞间的紧密连接结构，而圈出的边缘位置则为肠绒毛结构。Observation results: The microscopic morphology of the 2D monolayer culture of porcine intestinal organoids after about three days of culture was observed by transmission electron microscopy, and the results are shown in Figure 2. It can be clearly observed from the figure that the 2D monolayer of porcine small intestinal organoids has differentiated into intestinal villi and tight junction structures. The part indicated by the arrow in the figure is the tight junction structure between cells, and the circled edge position is Intestinal villi structure.

显然，本领域的技术人员可以对本申请进行各种改动和变型而不脱离本申请的精神和范围。这样，倘若本申请的这些修改和变型属于本申请权利要求及其等同技术的范围之内，则本申请也意图包含这些改动和变型在内。Obviously, those skilled in the art can make various changes and modifications to the application without departing from the spirit and scope of the application. In this way, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application is also intended to include these modifications and variations.