这项工作是由卫生部资助5KO8DK085136-02 JY国家机构的支持。

1。获得结肠组织鼠标的协议进行动物实验，详细介绍了基本原理和研究的目的，已提交并经我们的机构动物研究监督处。 <ol><li>安乐死鼠标与吸入异氟醚后颈椎脱位。 </li><li>使下腹正中切口，收回小肠路程，确定结肠。收回膀胱和子宫（如果存在的话）横向和识别耻骨，切细剪刀，露出直肠。 </li><li>解剖直肠背侧肠系膜断结肠，动员结肠从肛门至盲肠，并横切到断开小肠结肠。 </li><li>将样品在冰冷的磷酸盐缓冲盐水（PBS）。 </li><li>填有10毫升注射器用冰冷的PBS冲洗和结肠腔数次以清除其内容，再切结肠氧化聚乙烯Ñ​​长度方向用剪刀。 </li><li>将结肠在50ml锥形管中加入20ml冰冷的PBS。结肠并没有被放置在任何特定的方位。通过更换PBS洗结肠直至油液清晰，没有颗粒物。 </li></ol>人的一个协议获得人体组织从外科病人，详细介绍了获得该组织以及潜在的风险和收益进行评估的重要性，已提交并经本机构的人类研究保护计划办公室。 <ol><li>与结直肠外科医生的研究合作是必要的，以便能够获得人体组织对你的研究。一个协议，以获得人类结肠组织应提交并批准您的机构审查委员会。 </li><li>书面知情同意从患者得到的外科手术之前。 </li><li>外科医生将在展台进行结肠切除术ARD时尚。一旦结肠已经从患者体内取出，将试样立即送往病理。中的不需要的病理评价结肠壁全层的截面A 0.5提供，并立即放置在冰冷的PBS。这个标本不应包括结肠肠系膜和网膜附属物应删除为好，因为脂肪的存在会影响消化过程。 </li></ol> 2。剥夺上皮细胞<ol><li>孵育整个小鼠结肠中在50ml锥形管中加入25ml 5mM EDTA的HBSS中（室温）。将锥形管在37℃下振荡空气浴（250 rpm）离心15分钟。 15分钟后，小心地倒出液体并重新填充该管，用25ml的5毫摩尔EDTA，重复，共5次洗涤。对于人类结肠癌，获得的结肠壁一个半英寸带从病理学家后，用剪刀剪结肠分成四个相等大小的块。放置两片到一个50ml锥形管中，并将其余两块结肠组织到一个独立的50ml锥形管中。然后孵化用25ml 5mM EDTA的HBSS中，并执行清洗，如上所述。 </li><li>在20ml的冰冷的PBS漂洗两次结肠。 </li><li>将小鼠结肠组织中一个新的50毫升锥形管含有20ml的RPMI-5，10单位分散酶，及2000ü胶原酶的D（室温）。对于人类结肠组织，用中性蛋白酶和胶原酶的两倍（20 U分散酶，4000ü胶原酶D）。 </li><li>将管在37℃下振荡空气浴垂直取向（如果水平放置，有太多的曝气和细胞的损伤）。摇在250转长达60分钟。暴露在分散酶和胶原酶后D，结肠组织将开始消化和结肠组织将开始寻找粘性。这通常需要大约30分钟。当结肠有这个样子，从酶去除结肠。 </li><li>摇各管上下2-4次，打破了组织。 P埃利特组织在200×g离心在台式离心机（4℃）5分钟。小心倒出上清液并丢弃。 </li></ol> 3。肌成纤维细胞的分离与培养<ol><li>通过加入10ml的ACK裂解缓冲液（47℃）重悬每个粒料。再次离心200×g离心5分钟（4℃），倒出并丢弃上清液。 </li><li>通过加入10ml的RPMI-5用移液管重悬浮每个粒料。 </li><li>通过用10毫升吸管成100毫米TC-处理的培养皿一个70微米的筛网过滤器传递一半的细胞悬浮液（5毫升）。然后添加一个额外的15毫升RPMI-5。用其余的5 ml细胞悬液重复插入不同百毫米TC-处理的培养皿中。因此，人们小鼠结肠将产生两根100 mm TC处理的菜肴。人结肠癌组织中的一个半英寸长条将产生共4 100毫米TC-处理的培养皿。 </li><li>放置在组织培养皿中在10％CO 2培养箱中在37℃下培养条件是相同的f或小鼠和人原代细胞。 </li><li> 3小时后，轻轻洗掉非贴壁细胞用HBSS的两种变化。碎片漂浮应该轻轻洗掉。贴壁细胞组成的上皮细胞，巨噬细胞和成肌纤维细胞。一周后播种，只有隔膜肌成纤维细胞，巨噬细胞和上皮细胞中的第一通道后衰老。加入新鲜RPMI-5和成长的细胞在细胞培养。 </li><li>细胞通过胰蛋白酶消化传代5分钟，被分割为2:1。 </li></ol>缩略语 HBSS:汉克平衡盐溶液，RPMI-5:罗斯韦尔公园纪念研究所媒体，ACK​​:氯化铵，钾，FCS:胎牛血清，TC处理:组织培养处理解决方案 ACK裂解缓冲液- （4.15克氯化铵 0.5克KHCO 3，18.6毫克的Na 2 EDTA，加入400ml H 2 O，调节pH值至7.2〜7.4，用1N HCl）。过滤器sterilize到0.2微米的过滤器，并储存于4℃ 的RPMI-5 - （使500毫升:454.5毫升RPMI，将25ml的FCS，加入5ml 200mM的L-谷氨酰胺，将5ml 1M的HEPES，pH为7.4，加入5ml 100 1mM丙酮酸钠，5毫升100X青霉素 - 链霉素，加入500ml的50mM乙-ME的PBS中）。

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作者什么都没有透露。

无论是使用鼠标或结肠癌时，通用的技术是相似的。这种技术也可以用于从小肠分离成肌纤维细胞。具体细节，肌纤维母细胞从1隔离。小鼠和2。人结肠癌在下面讨论。 1。小鼠结肠癌小鼠结肠灌溉是通过附加一个4，为16G波普尔钝头针头至结肠的一端提供便利。这也有助于纵向切割结肠时。您可以手风琴结肠组织到针，然后将剪一个尖与尖底起来，滑过剪?...

胃肠道（GI）道功能的调节涉及粘膜层与下伏间质之间复杂的，动态的相互作用。这些相互作用通常用来维持内环境稳定，但也可能导致疾病状态的发展病理条件下，1。肌成纤维细胞是位于下方的所在，以旁分泌方式进行通信及邻近细胞亚群调节若干关键胃肠道工序，包括粘膜再生，修复和纤维化2的上皮细胞层基质细胞亚群。 的18 CO细胞株是已被广泛用于研究肌成纤维细胞功能的人结肠肌成纤维细胞系的一个例子，因为它有许多共同的初级原位成肌纤维细胞的特性。这些包括的α-平滑肌肌动蛋白的蛋白表达（α-SMA）和波形蛋白，以及一个数的表达式细胞表面受体如表皮生长因子受体，或受体的溶血磷脂酸3,4。因为这些共享超微结构特征，以及在生物功能5的相似性，则18 CO细胞系已被广泛用于研究肌成纤维细胞的功能在许多疾病状态如炎性肠疾病或结肠直肠癌3,6的范围内。但是，也有使用的细胞系时固有的局限性和关注。这些包括基因型不稳定随着时间的推移区别于原代细胞的细胞系，从而改变细胞的表型和生物学功能，包括生长速率，并与其他细胞群的相互作用。细胞系也缺乏GI微环境（上皮，基质和血管成分），这是一个主要的限制其使用的正常组成部分。因此，从实验细胞株的研究得出的结论需要进一步的验证，以降低RISK误解。 初级肌成纤维细胞可以从人类和小鼠的结肠组织得到证实在细胞系7识别的实验结果。该方法最初是由马希达， 等。8描述的，和我们的协议是可以被用来从小鼠和人结肠9隔离初级肌成纤维细胞的许多良好描述的技术之一。对于结肠疾病的任何小鼠模型，这种技术可以用来隔离初级肌成纤维细胞，研究它们与邻近的细胞是如何相互作用或促使正常或病理性的GI处理10,11。这种技术可以用来研究肌纤维母细胞如何促进黏膜愈合，纤维化和结直肠腺瘤和癌的发展。初级肌成纤维细胞还可以从已经在切除操作的时间为良性（炎性肠病，狭窄，憩室炎）或恶性Ç人结肠组织获得onditions。来自人类和小鼠的结肠组织分离的原代细胞可以生长在细胞培养物和利用多通道的数量有限。

<table><tbody><tr><td>Name of the reagent</td><td>Company</td><td>Catalogue number</td><td>Comments (optional)</td></tr><tr><td>Dispase I</td><td>Invitrogen/GIBCO</td><td>#17105-041</td><td></td></tr><tr><td>Collagenase D</td><td>Roche</td><td>#1 088 858</td><td></td></tr><tr><td>4 in, 16 G Popper blunt-end needle</td><td>Fisher</td><td>#14-825-16K</td><td></td></tr></tbody></table>

isolation of primary myofibroblasts from mouse and human colon tissue

的肌成纤维细胞是胃肠（GI）道，已获得相当大的关注在许多胃肠功能的关键作用的基质细胞。虽然一些肌成纤维细胞的细胞系是可商购的，研究这些细胞在体外 ，离暴露实验的细胞培养条件的细胞系的研究成果具有因工作过于简单化的性质固有的局限性。使用初级肌纤维母细胞提供了在确认细胞系鉴定实验结果方面有很大的优势。从动物模型隔离初级肌纤维母细胞可为肌成纤维细胞的研究更紧密地模仿所研究的疾病状态条件下。从人结肠组织主要肌纤维母细胞的分离提供了可以说是最相关的实验数据，由于细胞直接来自患者的基础疾病。我们描述了一个行之有效的技术，可以为utilized从小鼠和人的结肠组织隔离初级肌成纤维细胞。这些分离的细胞已被鉴定为α-平滑肌肌动蛋白和波形蛋白阳性的，和结蛋白阴性的，与皮下肠肌成纤维细胞是一致的。初级肌成纤维细胞的细胞可以生长在细胞培养物和用作实验过通道的数量有限。

一旦分离，原代人成肌纤维细胞可以生长在细胞培养物和使用过的通道的一个有限数量（最多通道4）。这些细胞的特征在α- ​​平滑肌肌动蛋白和波形蛋白阳性，结蛋白阴性的（ 图1A），与肠上皮下成肌纤维细胞5,7一致。他们也有一个特点星状形态（ 图1B）。初级成肌纤维细胞随后可以用来确认在细胞系中发现的实验结果。这种方法被用来证明促炎细胞因子TNF-?...

Watch this Scientific Journal Video about Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue at JoVE.com

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

肌成纤维细胞是胃肠道，调节重要生理过程的正常和疾病状态的一个有影响力的基质细胞。我们描述了一种技术，它允许主肌纤维母细胞从小鼠和人结肠组织，它可以用于在体外实验的隔离。

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

isolation-of-primary-myofibroblasts-from-mouse-and-human-colon-tissue

Research

JoVE Journal

Biology

19.6K 次观看.  UCLA. 肌成纤维细胞是胃肠道，调节重要生理过程的正常和疾病状态的一个有影响力的基质细胞。我们描述了一种技术，它允许主肌纤维母细胞从小鼠和人结肠组织，它可以用于在体外实验的隔离。 

视频: Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, &#945;-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.

从人类冷冻胃肠标本分离CD 90+成纤维细胞/肌成纤维细胞


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

生物工程

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

小鼠骨骼肌肉外泄原发性肌基器的分离和分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

使用胶原酶E从毛里结肠分离拉米纳普里亚单核细胞

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

免疫与感染

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

超声波增强原成人成纤维细胞隔离

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

医学

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

从小鼠和人类食管隔离肌纤维母细胞

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

生物学

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Cryosectioning方法小鼠结肠黏膜显微切割

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

小鼠肠间充质分离导致端细胞高产量

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...