This work was supported by National Institutes of Health Grant 5KO8DK085136-02 to JY.

1. Obtain Colon Tissue
Mouse
A protocol to perform animal experiments, detailing the rationale and objectives of the research, was submitted and approved by our institutional Office of Animal Research Oversight.
<ol>
	<li>Euthanize the mouse with inhaled isoflurane followed by cervical dislocation.</li>
	<li>Make a ventral midline incision, retract the small bowel away and identify the colon. Retract the bladder and uterus (if present) laterally and identify the pubi...

<ol>
	<li>Armaka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+cell+targeting+by+TNF+as+a+common+pathogenic+principle+in+chronic+inflammatory+joint+and+intestinal+diseases.">Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases.</a> J. Exp. Med. 205, 331-337 (2008).</li><li>Powell, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+cells+important+in+health+and+disease.">Paracrine cells important in health and disease.</a> Am. J. Physiol. 277, C1-C9 (1999).</li><li>Yoo, J., Rodriguez Perez, C. E., Nie, W., Sinnett-Smith, J., Rozengurt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+D1+mediates+synergistic+MMP-3+expression+induced+by+TNF-alpha+and+bradykinin+in+human+colonic+myofibroblasts.">Protein kinase D1 mediates synergistic MMP-3 expression induced by TNF-alpha and bradykinin in human colonic myofibroblasts.</a> Biochem. Biophys. Res. Commun. 413, 30-35 (2011).</li><li>Yoo, J., Chung, C., Slice, L., Sinnett-Smith, J., Rozengurt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+D+mediates+synergistic+expression+of+COX-2+induced+by+TNF-{alpha}+and+bradykinin+in+human+colonic+myofibroblasts.">Protein kinase D mediates synergistic expression of COX-2 induced by TNF-{alpha} and bradykinin in human colonic myofibroblasts.</a> Am. J. Physiol. Cell Physiol. 297, C1576-C1587 (2009).</li><li>Valentich, J. D., Popov, V., Saada, J. I., Powell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+of+an+intestinal+subepithelial+myofibroblast+cell+line.">Phenotypic characterization of an intestinal subepithelial myofibroblast cell line.</a> Am. J. Physiol. 272, C1513-C1524 (1997).</li><li>Rodriguez Perez, C. E., Nie, W., Sinnett-Smith, J., Rozengurt, E., Yoo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-alpha+potentiates+lysophosphatidic+acid-induced+COX-2+expression+via+PKD+in+human+colonic+myofibroblasts.">TNF-alpha potentiates lysophosphatidic acid-induced COX-2 expression via PKD in human colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 300, G637-G646 (2011).</li><li>Yoo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-alpha+induces+upregulation+of+EGFR+expression+and+signaling+in+human+colonic+myofibroblasts.">TNF-alpha induces upregulation of EGFR expression and signaling in human colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 302, G805-G814 (2012).</li><li>Mahida, Y. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+human+colonic+subepithelial+myofibroblasts+express+extracellular+matrix+proteins+and+cyclooxygenase-1+and+-2.">Adult human colonic subepithelial myofibroblasts express extracellular matrix proteins and cyclooxygenase-1 and -2.</a> Am. J. Physiol. 273, G1341-G1348 (1997).</li><li>De Wever, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming+and+potentiation+of+DNA+damage+response+by+fibronectin+in+human+colon+cancer+cells+and+tumor-derived+myofibroblasts.">Priming and potentiation of DNA damage response by fibronectin in human colon cancer cells and tumor-derived myofibroblasts.</a> Int. J. Oncol. 39, 393-400 (2011).</li><li>Edwards, R. A., Smock, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+arachidonate+release+and+PGE2+production+in+Gi+alpha2-deficient+intestinal+and+colonic+subepithelial+myofibroblasts.">Defective arachidonate release and PGE2 production in Gi alpha2-deficient intestinal and colonic subepithelial myofibroblasts.</a> Inflamm. Bowel Dis. 12, 153-165 (2006).</li><li>Hoang, B., Trinh, A., Birnbaumer, L., Edwards, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+MAPK-+and+PGE2-dependent+IL-11+production+in+Gialpha2-/-+colonic+myofibroblasts.">Decreased MAPK- and PGE2-dependent IL-11 production in Gialpha2-/- colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 292, G1511-G1519 (2007).</li><li>Li, Z. B., Kollias, H. D., Wagner, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myostatin+directly+regulates+skeletal+muscle+fibrosis.">Myostatin directly regulates skeletal muscle fibrosis.</a> J. Biol. Chem. 283, 19371-19378 (2008).</li><li>Saada, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subepithelial+myofibroblasts+are+novel+nonprofessional+APCs+in+the+human+colonic+mucosa.">Subepithelial myofibroblasts are novel nonprofessional APCs in the human colonic mucosa.</a> J. Immunol. 177, 5968-5979 (2006).</li><li>Lahar, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+subepithelial+myofibroblasts+support+in+vitro+and+in+vivo+growth+of+human+small+intestinal+epithelium.">Intestinal subepithelial myofibroblasts support in vitro and in vivo growth of human small intestinal epithelium.</a> PLoS One. 6, e26898 (2011).</li></ol>

The general technique is similar when using either mouse or human colon. This technique can also be used to isolate myofibroblasts from the small intestine. Specific details for the isolation of myofibroblasts from 1. mouse and 2. human colon are discussed below.
1. Mouse Colon
Irrigation of the mouse colon is facilitated by attaching a 4 in, 16 G Popper blunt-end needle to one end of the colon. This also helps when cutting the colon lengthwise. You can accordion the c...

The regulation of gastrointestinal (GI) tract function involves complex, dynamic interactions between the mucosal layer and its underlying mesenchyme. These interactions normally serve to maintain homeostasis but may also lead to the development of disease states under pathologic conditions1. Myofibroblasts are a subpopulation of stromal cells located subjacent to the epithelial layer that communicate in a paracrine fashion with surrounding cell subpopulations to regulate a number of critical GI tract processes that include mucosal regeneration, repair, and fibrosis2.
The 18Co cell line is an example of a human colonic myofibroblast cell line that has been widely used to study myofibroblast function because it shares many of the characteristics of primary in situ myofibroblasts. These include the protein expression of a-smooth muscle actin (&#945;-SMA) and vimentin, as well as the expression of a number of cell surface receptors such as the epidermal growth factor receptor, or receptors for lysophosphatidic acid3,4. Because of these shared ultrastructural characteristics, as well as similarities in biologic function5, the 18Co cell line has been used extensively to study myofibroblast function in the context of many disease states such as inflammatory bowel disease or colorectal cancer3,6. However, there are inherent limitations and concerns when using a cell line. These include genotypic instability over time that differentiates cell lines from primary cells, altering the phenotype and biologic function of the cell, including growth rates and interactions with other cell populations. Cell lines also lack the normal components of the GI microenvironment (epithelial, stromal, and vascular components), which is a major limitation to its use. Therefore, the conclusions drawn from experimental cell line research requires further validation to reduce the risk of misinterpretation.
Primary myofibroblasts can be obtained from human and mouse colon tissue to confirm experimental findings identified in a cell line7. The method was originally described by Mahida, et al.8, and our protocol is one of many well-described techniques that can be used to isolate primary myofibroblasts from mouse and human colon9. For any mouse model of colonic disease, this technique can be used to isolate primary myofibroblasts to study how they interact with neighboring cells or contribute to both normal or pathologic GI processes10,11. This technique can be used to study how myofibroblasts contribute to mucosal healing, fibrosis, and the development of colorectal adenomas and carcinomas. Primary myofibroblasts can also be obtained from human colon tissue that has been resected at the time of operation for benign (inflammatory bowel disease, strictures, diverticulitis) or malignant conditions. Isolated primary cells from human and mouse colon tissue can be grown in cell culture and utilized over a limited number of passages.

Table of specific reagents and equipment:
<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </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

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

Once isolated, primary human myofibroblasts can be grown in cell culture and used over a limited number of passages (up to passage 4). These cells are characterized as being a-smooth muscle actin and vimentin positive, and desmin-negative (Figure 1A), consistent with intestinal subepithelial myofibroblasts5,7. They also have a characteristic stellate morphology (Figure 1B). Primary myofibroblasts can then be used to confirm experimental findings identified in a cell line. This...

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

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

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

19.3K vues.  UCLA.  Le myofibroblastes est une cellule influent stromale du tractus gastro-intestinal qui régule les processus physiologiques importants dans les états normaux et pathologiques. Nous décrivons une technique qui permet l'isolement des myofibroblastes primaires à partir de la souris et le tissu du côlon humain, qui peut être utilisé pour l'expérimentation in vitro. 

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

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

isolation of myofibroblasts from mouse and human esophagus

Isolation of Myofibroblasts from Mouse and Human Esophagus

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.

isolation of cd 90+ fibroblast/myofibroblasts from human frozen gastrointestinal specimens

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

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.

three-dimensional culture of murine colonic crypts to study intestinal stem cell function ex vivo

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout...

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

tissue processing and isolation of primary fibroblasts from the human vagina

This protocol demonstrates a reliable and effective technique to isolate primary fibroblasts from either premenopausal or postmenopausal human vaginal tissue. Existing protocols for vaginal fibroblast isolation do not consider the challenges of cell isolation from senescent tissue. Vaginal tissue was obtained from women after pelvic organ prolapse...

Tissue Processing and Isolation of Primary Fibroblasts from the Human Vagina

isolation, culture, and characterization of primary dermal fibroblasts from human keloid tissue

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable...

isolation of primary human colon tumor cells from surgical tissues and culturing them directly on soft elastic substrates for traction cytometry

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

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.

isolation and characterization of adult cardiac fibroblasts and myofibroblasts

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

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...

isolation of human endothelial cells from normal colon and colorectal carcinoma - an improved protocol

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is...

in vitro organoid culture of primary mouse colon tumors

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do...

In vitro Organoid Culture of Primary Mouse Colon Tumors

isolation and culture of primary mouse keratinocytes from neonatal and adult mouse skin

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from...

primary microglia isolation from postnatal mouse brains

Primary Microglia Isolation from Postnatal Mouse Brains

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to...

generation of murine primary colon epithelial monolayers from intestinal crypts

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence...

isolation and culture of primary schwann cells from a human dermis

This video demonstrates a protocol for isolating and culturing Schwann cells from human dermis tissues. The human dermis contains various cell types, including fibroblasts and Schwann cells. After isolation, the cells are treated with an antimitotic agent to eliminate proliferating fibroblasts, resulting in a pure culture of Schwann...

Isolation and Culture of Primary Schwann Cells from a Human Dermis

isolation and differentiation of primary myoblasts from mouse skeletal muscle explants

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

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...

isolation and culture of primary synovial macrophages and fibroblasts from murine arthritis tissue

Author Spotlight: Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue  

The present study provides a modified protocol to isolate synovial macrophages and fibroblasts from murine inflammatory arthritis tissue.

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue

engineering fibrin-based tissue constructs from myofibroblasts and application of constraints and strain to induce cell and collagen reorganization

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific...

Digest the minced dermis with 5 milliliters of collagenase at 37 degrees Celsius for 2 and 1/2 hours, with gentle trituration using a pipette tip every 30 minutes until the dermis dissociates completely. Once digested, filter the cell suspension with a 70-micrometer cell strainer, followed by dilution of the filtered cell suspension with 5 milliliters of complete DMEM medium to stop the enzymatic activity of...

isolation and culture of primary oral keratinocytes from the adult mouse palate

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also...

isolation and functional analysis of mitochondria from cultured cells and mouse tissue

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

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.

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

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

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.

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

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...