Name: isolation and culture of primary endothelial cells from canine arteries and veins
Uploaded: 2016-11-18T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

The authors would like to acknowledge Hans de Graaf and Tomas Veenendaal for their technical assistance in culturing the ECs.

Ethics statement: Blood vessels used in this study were harvested as surplus material obtained from fresh canine cadavers (n= 4) from healthy dogs euthanized for other unrelated research (University 3R policy). Aberrant blood vessels (intra- and extrahepatic portosystemic shunts, n= 1 each) were harvested post-mortem after informed consent of the owners from dogs presented to the University Clinic for Companion Animals of Utrecht University.
1. Isolation and Culture of Primary Canine Endothelial...

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Heart Fail. 3 (5), 635-642 (2010).</li><li>Gonzalez-Miguel, J., Morchon, R., Siles-Lucas, M., Simon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinolysis+and+proliferative+endarteritis:+two+related+processes+in+chronic+infections?+The+model+of+the+blood-borne+pathogen+Dirofilaria+immitis.">Fibrinolysis and proliferative endarteritis: two related processes in chronic infections? The model of the blood-borne pathogen Dirofilaria immitis.</a> PLoS One. 10 (4), e0124445 (2015).</li><li>Sacks, T., Moldow, C. F., Craddock, P. R., Bowers, T. K., Jacob, H. 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A., 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=MIQE+precis:+Practical+implementation+of+minimum+standard+guidelines+for+fluorescence-based+quantitative+real-time+PCR+experiments.">MIQE precis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments.</a> BMC Mol. Biol. 11, 74 (2010).</li><li>Brinkhof, B., Spee, B., Rothuizen, J., Penning, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+canine+reference+genes+for+accurate+quantification+of+gene+expression.">Development and evaluation of canine reference genes for accurate quantification of gene expression.</a> Anal. 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In studies focusing on canine ECs the CnAOEC primary line is used to model the endothelial lineages of the dog3, 12, 13. In human studies, the HUVEC culture is still considered the gold standard. Clearly, focusing merely on ECs derived from umbilical cord is a firm restriction in cardiovascular research. Endothelial cells have a specific gene expression pattern determining arteriovenous specification. In order to account for these differences in postnatal vessels we present this novel isolation method based on...

Dogs are used as large animal model for cardiovascular disease research and can also suffer from inborn (genetic) vascular abnormalities1, 2. To study these diseases commercial endothelial cell lines are often used to assess endothelial cell (EC) functionality. For dogs there is one commercial endothelial cell line available (CnAOEC), derived from canine aorta. This cell line is mostly used in studies as control normal ECs3-5. In human cardiovascular research the most commonly used endothelial cell lines are Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs) derived from human umbilical cord vein and artery, respectively. HUVECs have been used as the golden standard in vascular research since the 1980s6. They are considered to be the classic model system to study endothelial function and disease adaptation. Endothelial cells isolated from different blood vessels vary in appearance and functionality due to genetic background and exposure to the microenvironment7. In addition, HUVECs and HUAECs are derived from umbilical cord, a developmental vascular structure that might not fully mimic adult blood vessels with respect to the conditions they are exposed to and response to disease. Hence, translating results found in HUVECs and HUAECs to cardiovascular disease in general is inadequate.
When studying adaptation and behavior of adult ECs, primary ECs from the vessel of interest should be used as a more direct approach. To isolate these cells, several methods have been reported. A widely described method, which is also used for HUVECs, is flushing the vessel with an enzymatic digestion solution8. This often results in contamination with non-ECs such as smooth muscle cells and fibroblasts9. Another frequently used method for isolation is enzymatic digestion of minced vessel tissue followed by fluorescence-activated cell sorting (FACS) based on endothelial cell marker Cluster of Differentiation (CD)317, 8. FACS sorting and subsequent cell culture requires relatively large amounts of cells and is therefore not suitable for the isolation of endothelium from small blood vessels. We therefore aimed at developing a new robust method for isolating a pure endothelial cell population from various canine blood vessels with high purity. To test the efficiency of the new isolation method, we isolated and obtained pure Canine Primary Endothelial Cell (CaPEC) cultures from different canine arteries and veins, both large and small. This method also enables the culture of endothelial cells originating from diseased and/or aberrant vessels such as inborn intra- or extra-hepatic portosystemic shunts, a common disease in dogs2. The method allows the isolation of additional relevant cell types of the same vessel such as vascular smooth muscle cells since most of the vessel remains intact during the procedure.

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isolation and culture of primary endothelial cells from canine arteries and veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Different blood vessels were successfully subjected to the described isolation protocol (Figure 2). It was possible to dissect and invert aorta, vena cava, vena porta, and coronary artery from healthy dogs (all vessels from each dog, n= 4). With the same approach ECs were isolated from two congenital portosystemic shunts (extrahepatic and intrahepatic, n= 1 each). Although aorta was easily inverted, thoracic aorta segments were more challenging than abdominal aorta. In th...

Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

The overall goal of this method is to isolate and culture primary endothelial cells from dogs to study cardiovascular disease in vitro. This method can help answer key questions in the field of cardiovascular research such as the behavior of endothelial cells during angiogenesis, tumor angiogenesis, and atherosclerosis. The main advantage of this technique is that a pure population of primary endothelial cells can be obtained and cultured even from very small blood vessels.Generally, individuals new to this method will struggle because the inversion and suturing of the vessel can seem difficult, but are in fact easily learned in practice. To isolate and culture primary canine endothelial cells or CAPECs, use 0.5%gelatin to pre-coat six-well plates and incubate in a humidified chamber at 37 degrees Celsius and 5%CO2 for two hours. After aseptically removing blood vessels of interest from a canine cadaver according to the text protocol, transfer the blood vessels to Petri dishes filled with ice cold HBSS.Then using surgical scissors, remove any adherent tissue and fat from the outside of the vessel making sure the vessel itself is visible at all times when cutting and keeping the vessel itself intact. Use ligatures to close any branches of the vessel and then use surgical scissors or a scalpel to remove the branches. With a curved Halsted mosquito forceps, carefully enter a vessel end, clamp the tip of the forceps at the other end of the vessel and then retract, slowly inverting the vessel until it is completely inside out with the endothelial cell layer on the outside.It is important to keep the blood vessel moistened with HBSS to facilitate the inversion process. Next, place purse string sutures at both ends of the vessel to close it completely to prevent exposure of any non-endothelial vascular tissue to digestion. Then using the ligature ends to manipulate the inverted vessel, transfer the vessel to a 50 milliliter tube and use HBSS to rinse it twice to remove erythrocytes or residual freezing medium in case of thawing.Add 30 milliliters of collagenase type two and dispase in canine endothelial cell growth medium or CECGM and incubate the tube at 37 degrees Celsius with gentle agitation to digest the vessel. Remove the vessel from the tube and centrifuge the cell suspension at 250 times g for five minutes. Then resuspend the cell pellet in CECGM and seed two milliliters of the suspension per well in one to three wells depending on the vessel size.Culture the cells at 37 degrees Celsius in a humidified atmosphere with 5%CO2 in air and change the culture medium twice a week. When the cells reach 70 to 80%confluency, use pre-warmed HBSS to wash away dead or non-attached cells. Then add 200 microliters of recombinant cell dissociation enzyme to each well and place the dish back into the incubator for five minutes or until the cells detach.Transfer the cells to a 15 milliliter tube and stop trypsinization by adding 10 milliliters of CECGM with 10%FCS, centrifuge for five minutes at 250 times g. Then continue the culture in a gelatin pre-coated plate or flask. To characterize canine aortic endothelial cells or CnAOECs, culture the cells in CECGM on a 0.5%gelatin pre-coated T75 flask.When the CnAOECs reach 70 to 80%confluency, trypsinize the cells as demonstrated earlier in this video. Then centrifuge the cell suspension at 250 times g for five minutes. Discard the supernatant and use one milliliter of culture medium to resuspend the cells.Dilute a 10 microliter aliquot of the cell suspension one to one with 0.4%Trypan Blue. Then use an automated cell counter to count the cells and plate four times 10 to the fifth viable cells in a new T75 flask. Add 10 milliliters of pre-warmed CECGM to the flask and culture at 37 degrees Celsius in a humidified atmosphere with 5%CO2.To isolate RNA from passage one of CAPECs and CnAOECs, collect at least one times 10 to the three cells as a pellet. Add 20 microliters of sample preparation reagent and incubate for one minute to lyse the cells. After the incubation, store the sample at negative 70 degrees Celsius.Measure gene expression and assess EC functionality according to the text protocol. Using the protocol demonstrated in this video, it was possible to dissect and invert aorta, vena porta, vena cava, and coronary artery from healthy dogs and two congenital portosystemic shunts. Between one and two weeks post-isolation, adhered endothelial cells were visible in the culture plate.CAPECs from aorta, vena cava, and vena porta had a polygonal shape and grew in patches and reached 80%confluency after 10 days in culture. On average, the cells could be maintained a maximum of eight passages and then stopped growing. As seen here by qPCR, isolated endothelial cells expressed the endothelial cell marker CD31.The expression in endothelial cells derived from aorta, vena cava, and vena porta from four dogs exhibited comparable CD31 expression to a control culture of CnAOECs. This figure shows that CAPECs derived from aorta, vena cava, and vena porta displayed branching six hours after incubation on the angiogenesis slide. Once mastered, this technique can be done in three hours.While attempting this procedure, it's important to remember to work aseptically. Following this procedure, other methods like angiogenesis assays can be performed in order to answer additional questions like whether gene silencing or certain drugs will influence endothelial cell behavior. After watching this video, you should have a good understanding of how to isolate primary endothelial cells from blood vessels, a technique that can also be applied to normal and abhorrent blood vessels from other species.

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Medicine

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.
Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.
Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...