Name: isolation and primary culture of mouse aortic endothelial cells
Uploaded: 2016-12-19T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

This study is supported by American Heart Association Scientist Development Grant 13SDG16930098 and the National Science Foundation of China Youth Award 81300240 (PI: Wang). We thank Roberto Mendez from Wayne State University for assisting in the preparation of the manuscript.

1. Isolation of Aorta from Mice
All the procedures described here were approved by the Institutional Animal Care and Use Committee of Wayne State University.
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	<li>Put the mouse into the induction chamber of the anesthesia machine. Set the isoflurane flow to 4% in conjunction with 25% fresh air and 75% O2. Anesthetize the mouse by inhalation of isoflurane (4% for induction, &#177; 1.0% for maintenance) in conjunction with 25% fresh air and 75% O2 via induct...

<ol>
	<li>Simionescu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+early+structural-functional+changes+in+the+endothelium+for+vascular+disease.">Implications of early structural-functional changes in the endothelium for vascular disease.</a> Arteriosclerosis, thrombosis, and vascular biology. 27, 266-274 (2007).</li><li>Cid, M. C., Segarra, M., Garcia-Martinez, A., Hernandez-Rodriguez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells,+antineutrophil+cytoplasmic+antibodies,+and+cytokines+in+the+pathogenesis+of+systemic+vasculitis.">Endothelial cells, antineutrophil cytoplasmic antibodies, and cytokines in the pathogenesis of systemic vasculitis.</a> Current rheumatology reports. 6, 184-194 (2004).</li><li>Wang, J. 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S., 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=Isolation+and+characterisation+of+human+pulmonary+microvascular+endothelial+cells+from+patients+with+severe+emphysema.">Isolation and characterisation of human pulmonary microvascular endothelial cells from patients with severe emphysema.</a> Respiratory research. 14, 23 (2013).</li><li>Marelli-Berg, F. M., Peek, E., Lidington, E. A., Stauss, H. J., Lechler, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+murine+tissue.">Isolation of endothelial cells from murine tissue.</a> Journal of immunological methods. 244, 205-215 (2000).</li><li>Bowden, R. 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=Role+of+alpha4+integrin+and+VCAM-1+in+CD18-independent+neutrophil+migration+across+mouse+cardiac+endothelium.">Role of alpha4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium.</a> Circulation research. 90, 562-569 (2002).</li><li>Lu, Y., 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=Palmitate+induces+apoptosis+in+mouse+aortic+endothelial+cells+and+endothelial+dysfunction+in+mice+fed+high-calorie+and+high-cholesterol+diets.">Palmitate induces apoptosis in mouse aortic endothelial cells and endothelial dysfunction in mice fed high-calorie and high-cholesterol diets.</a> Life sciences. 92, 1165-1173 (2013).</li><li>Atkins, G. B., Jain, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+differentiation:+molecular+mechanisms+of+specification+and+heterogeneity.">Endothelial differentiation: molecular mechanisms of specification and heterogeneity.</a> Arteriosclerosis, thrombosis, and vascular biology. 31, 1476-1484 (2011).</li><li>Aitsebaomo, J., Portbury, A. L., Schisler, J. C., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brothers+and+sisters:+molecular+insights+into+arterial-venous+heterogeneity.">Brothers and sisters: molecular insights into arterial-venous heterogeneity.</a> Circulation research. 108, 929-939 (2008).</li><li>Shimamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+and+foods+on+contraction+of+endothelial+cells+as+a+key+mechanism+in+atherogenesis+and+treatment+of+atherosclerosis+with+endothelial-cell+relaxants+(cyclic+AMP+phosphodiesterase+inhibitors).">Drugs and foods on contraction of endothelial cells as a key mechanism in atherogenesis and treatment of atherosclerosis with endothelial-cell relaxants (cyclic AMP phosphodiesterase inhibitors).</a> Advances in experimental medicine and biology. 60, 77-105 (1975).</li><li>Chiu, J. J., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+disturbed+flow+on+vascular+endothelium:+pathophysiological+basis+and+clinical+perspectives.">Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives.</a> Physiological reviews. 91, 327-387 (2011).</li><li>Gimbrone, M. A., Topper, J. N., Nagel, T., Anderson, K. R., Garcia-Cardena, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+dysfunction,+hemodynamic+forces,+and+atherogenesis.">Endothelial dysfunction, hemodynamic forces, and atherogenesis.</a> Annals of the New York Academy of Sciences. 902, 239-240 (2000).</li><li>Rostgaard, J., Qvortrup, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+demonstrations+of+filamentous+molecular+sieve+plugs+in+capillary+fenestrae.">Electron microscopic demonstrations of filamentous molecular sieve plugs in capillary fenestrae.</a> Microvascular research. 53, 1-13 (1997).</li><li>Brutsaert, D. 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This study demonstrates a simple method to isolate and culture endothelial cells from a mouse aorta without any special equipment. The immunofluorescence staining indicated that the majority of cells were endothelial cells after the second passage. It is suggested that cells at second to third passage are suitable for in vitro and in vivo experiments to study endothelial biology.
The Key Notes from the Present Protocol
There are fi...

The vascular endothelium is not only a barrier layer that separates blood and tissue, it is considered a vast endocrine gland that stretches over the entire vascular tree with a surface area of 400 square meters1. The well-being of the endothelium is essential to vascular homeostasis. The dysfunctional endothelium participates in various cardiovascular disorders, including atherosclerosis, vasculitis and ischemia/reperfusion injuries, etc. 2-4. To date, the specific cellular and molecular mechanisms involved in these disease settings are not well understood due to the diffused anatomic nature of endothelium.
The mouse is an important model for research because genetic manipulation techniques are more developed in mice than in any other mammalian species. However, the isolation of primary murine aortic endothelial cells is considered particularly difficult because the small size of the aorta makes enzymatic digestion of endothelium impractical. Some reported procedures to isolate and purify ECs require 5-7.
The goal of this protocol is to use a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. In this protocol, the freshly isolated aorta is cut into small segments and seeded onto a matrix with the endothelium facing down to allow for endothelial sprouting. After segments are removed, endothelial cells are expanded in endothelium-favored medium and are ready for experiments after two or three passages. The advantages of the described method are that: 1) considerably high numbers of endothelial cells are harvested from a single aorta; 2) cell viability is well preserved; and 3) no special equipment or technique is needed. It provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell pathophysiology. For those who are interested in studying primary cultured endothelial cells from either gene knock-out mice, gene knock-in mice, or a murine disease model, this protocol is very useful and easy to practice.

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			<td>4- or 6-week-old mice (Jackson Laboratory, #000664).</td>
		</tr>
		<tr>
			<td>Sterile 1X phosphate-buffered saline (PBS, Gibco, #10010-023).</td>
		</tr>
		<tr>
			<td>Sterile 1X PBS containing 1,000 U/ml of heparin (Sigma Aldrich, H3149).</td>
		</tr>
		<tr>
			<td>Endothelial cell growth medium (Dulbeccos&#8217; Modified Eagle&#8217;s Medium[DMEM] with 25mM HEPES[Gibco, #12320-032 ], supplemented with 100&#956;g/ml endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma,#2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000 U/ml heparin [Sigma Aldrich, H3149], 10,000U/ml penicillin and 10mg/ml streptomycin [Gibco, #15140-122]).</td>
		</tr>
		<tr>
			<td>Growth factor reduced matrix (BD Biosciences, #356231).</td>
		</tr>
		<tr>
			<td>Neutral proteinase (Dispase, 1U/ml, Fisher Scientific #CB-40235) and D-Val medium (D-Valine, 0.034g/L[Sigma, #1255], in Dulbeccos&#8217; Modified Eagle&#8217;s Medium, low glucose[Gibco, #12320-032 ]).&#160;</td>
		</tr>
		<tr>
			<td>Gelatin (100&#8209;200 &#956;g/cm2, Sigma, #G1393).</td>
		</tr>
		<tr>
			<td>1,1`-dioctadecyl-3,3,3`,3`- tetramethylindo- carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL, Life Technologies, #L3484),&#160; FITC-labeled Ulex europeus agglutinin (Ulex-Lectin, Sigma, #L9006),&#160; Anti-mouse CD31-FITC conjugated antibody (BD Biosciences, # 553372).</td>
		</tr>
		<tr>
			<td>Anti-mouse vascular endothelial growth factor receptor 2 antibody (Cell signaling, #9698), anti-mouse endothelial nitric oxide synthase (Abcam, #ab5589), anti-mouse vascular endothelium-cadherin (Abcam, #33168), anti-mouse calponin (Abcam, #700), FITC-conjugated anti-rabbit IgG (Sigma, #F6005)</td>
		</tr>
		<tr>
			<td>One ml syringe fitted with 25-G needle (Fisher Scientific, #50-900-04222).&#160;</td>
		</tr>
		<tr>
			<td>100mm Peri dishes (Fisher Scientific,&#160; #07-202-516).</td>
		</tr>
		<tr>
			<td>Six-well cell culture plates (Fisher Scientific, #08-772-1B).</td>
		</tr>
		<tr>
			<td>T12.5 cultuer flask (Fisher Scientific, #50-202-076)</td>
		</tr>
		<tr>
			<td>Scissors, forceps, microdissection scissors and forceps, Scalpel blade (Fine Science Tools, Inc)</td>
		</tr>
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			<td>Anesthesia machine with isoflurane (Webster Veterianary Supply, #07-806-3204), heating lamp</td>
		</tr>
		<tr>
			<td>Centrifuge machine.</td>
		</tr>
		<tr>
			<td>Inverted phase-contrast microscope.</td>
		</tr>
		<tr>
			<td>inverted fluorescence microscope.</td>
		</tr>
	</tbody>
</table>

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.

Endothelial Cell Sprouting
Spontaneous endothelial cell sprouting started from a mouse aorta segment. The mouse aorta segment was allowed to grow on a growth factor-reduced matrix then in endothelial cell growth medium for 4 days. The endothelial cell sprouting usually appears in 2 - 4 days. Photomicrographs were taken on day 4 (Figure 1). As shown in the pictures, numerous endothelial cells migrate away from the segment. The newly formed sprouts continue to e...

Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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 overall goal of this procedure is to use a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Other methods used to isolate endothelial cells are mostly applied to the heart or the lung, not the aorta. This technique for the aorta provides considerably higher yield, high cell viability, and requires no special equipment or techniques.While those apply into the studying of primary endothelial cells from either doing knockout or murine mice are from most disease models. This protocol is very useful and easy to practice. Start this protocol at the beginning of the day.After anesthetizing a mouse and confirming a lack of reflex, place the mouse supine on a surgical pad and position a heating lamp over it to maintain its body temperature. Then sanitize the abdomen by spraying it down with 70%ethanol. Next, with dissection scissors, open the abdomen along the midline to expose the abdominal aorta.Then, open the chest cavity to expose the heart and lungs. Now, release the blood from the mouse by dissecting along the center line of the aorta. Then, into the left ventricle, inject 1000 units of heparin and a milliliter of PBS, thus perfusing the aorta.After the perfusion, push aside impeding organs, quickly remove the thoracic aorta, and transfer it to ice cold PBS. Under a laminar airflow hood, gently flush the aorta with ice cold PBS to remove any remaining blood using a 25-gauge needle and syringe. Then, using microdissection forceps, remove as much attached adipose tissue and lateral vessels as possible.The time between cardiac arrest and the seeding of the aortic segment onto the matrix is critical to the endothelium's viability. So, while removing the periarterial adipose tissue and connective tissue, avoiding stretching the aorta, and limit the time spent on this step, too. At most, 15 minutes.Then, transfer the cleaned-off aorta to endothelial growth medium and cut the aorta into one-millimeter rings with a scalpel. One aorta yields up to 10 rings. Then, open each ring with microdissection scissors.To begin, put a six-well plate on ice and coat it with one milliliter of matrix solution without pouring out any air bubbles. Then, transfer the plate to an incubator for 20 minutes so the matrix solidifies. Once solidified, the aorta pieces can be placed on the matrix, lumen side down.Do so without touching the endothelium. Position three to four segments in each well. Then, pour just enough growth medium over the segments to keep them wet.Next, transfer them to an incubator with 5%carbon dioxide for about four to six hours. At the end of the day, add more medium so the segments are covered. Over the next several days, the aortic segments will periodically sprout.Monitor the sprouting by phase contrast microscopy every day. Adjust the level of medium at the same time. On the fourth day, carefully remove the medium, and gently remove the aortic segments.Do not disturb the cells growing on the matrix. Then, add two milliliters of fresh medium to the endothelial cells on the matrix, and continue the culturing for two to three days. The timing to remove aortic segments from the matrix is critical to the purity of the endothelial cells.The segment must be removed before the development of the tube network, or the cells will be contaminated with fibroblasts or smooth muscle cells. To begin, prepare a T12.5 flask with 0.1%gelatin, and incubate it for 30 minutes. Next, wash the matrix plates carefully with warmed PBS, then add 100 units of neutral protease to each well, and incubate the plates at room temperature on a platform rocker with occasional shaking.When the cells appear detached under phase contrast, add two milliliters of d-Val to each well to end the reaction. Examine the wells, and then collect the supernatants in a 15-milliliter tube. Then, centrifuge the tube at 900 gs for five minutes, and re-suspend the pellet of cells in four milliliters of medium.Plate the suspension in the prepared flask, and incubate the cells for two hours. Then, replace the medium, and continue the incubation for three to four days until the cells are 85-90%confluent. When the cells are ready, prepare two more T12.5 flasks to passage the cells as before.Then, wash the cells with warmed PBS, and use about half a milliliter of Trypsin-EDTA to detach the majority of them within a minute of incubation. Next, end this reaction with two milliliters of media. Now, flow the suspension through a pipette several times, and transfer it to a 15-milliliter tube.Next, centrifuge the cells at 900 gs for five minutes, and re-suspend the pellet in four milliliters of medium. Then, split the suspension between the two prepared flasks, and let them incubate for two hours. Then, replace the medium, and continue growing the cells to confluency as before.After two to three such passages, characterize the cells. Using the described protocol, spontaneous endothelial cell sprouting was observed for mouse aorta segments after two days in culture. After four days, numerous endothelial cells migrated away from the segment, and newly form sprouts continued to extend from the segment and the branch.After culturing these cells and passaging them once, the cells were spindle-shaped and cobblestone-like in appearance. The attached cells were then labeled with Dil-ac-LDL and Ulex Lectin for an hour. Nuclei were also stained.Most of the cells were double positive for Dil-ac-LDL uptake and Ulex binding. After the second passage, more than 95%of the cells were positive for platelet endothelial cell adhesion molecule one. The cells were also positive for VEGFR2, VE-Cadherin, and eNOS.Also, the cells were mostly negative for the smooth muscle marker calponin. This protocol provides a great opportunity to study the endothelial-specific activities of targeted molecules, and can be done in the knockout and transgenic mouse models, making it very useful in the field of cardiovascular research.

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Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

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

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 ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

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

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 ...