ברצוננו להודות לד&quot;ר פול ווייגל על ​​אונ. אוקלהומה לשימושם של נוגדנים חד שבטיים 30 לגילוי של הקולטן HARE/Stabilin-2 וג&#39;נט ווייגל לקבלת סיוע טכני שלה. מחקר זה מומן על ידי אוניברסיטת נברסקה קרנות מחקר.

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Res. 99, 444-449 (1976).</li><li>Graupera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinusoidal+endothelial+COX-1-derived+prostanoids+modulate+the+hepatic+vascular+tone+of+cirrhotic+rat+livers.">Sinusoidal endothelial COX-1-derived prostanoids modulate the hepatic vascular tone of cirrhotic rat livers.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 288, 763-770 (2005).</li><li>Yannariello-Brown, J., Zhou, B., Ritchie, D., Oka, J. A., Weigel, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+ligand+blot+assay+detects+different+hyaluronan-binding+proteins+in+rat+liver+hepatocytes+and+sinusoidal+endothelial+cells.">A novel ligand blot assay detects different hyaluronan-binding proteins in rat liver hepatocytes and sinusoidal endothelial cells.</a> Biochem. Biophys. Res. 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אין ניגודי אינטרסים הכריז.

הרדמה של החולדה בשיטה טיפה תלוי בו isoflurane הכרה אדי מעוררת היא השיטה המועדפת ללימודים שלנו. מאדה עשוי לשמש גם במקום מזרק עם צמר גפן כדי לשמור על חיים מחוץ לחדר, אך ניתוח זה כל כך מהר, זה לא נדרש. פוליאתילן גליקול מתווספת isoflurane כדי להקטין את לחץ אדים וקצב אידוי. יותר מדי אדי isoflurane תגרום למוות מהר מדי. אם החיה מתה לפני הכבד cannulated והחוויר עם TBS, איגום קריש דם עלול בכבד ולמנוע כביסה יעיל של sinusoids ועיכול עם collagenase. תוספת של הפרין עשוי להיות שימושי כמו קרישה, אך אינו משמש במחקרים אלה שכן אנו משתמשים הפרין שכותרתו בדיקה שלנו. שנאגרו של גיי תמיסת מלח (GBSS) מוחלף לעתים קרובות לטיהור של שניות על ידי מעבדות אחרות. אמנם זה שימושי עבור טיהור-SEC, בענייניםatocytes לא לסבול GBSS וכן Buffers 1-3 ו viabilities אינם בגובה דומה. כאשר מודדים אנדוציטוזה, זהו תרגול טוב כדי למדוד את רמות הרקע האיבר וגם מטוהרים hepatocytes. שיטה זו היא אחת משתי לטיהור יעיל של שניות לאחר זלוף כבד עם collagenase. השיטה האחרת מפרידה הלא parenchymal תאים על ידי צנטריפוגה elutriation 19 במקום שיפוע Percoll. היתרונות לשימוש elutriation על הדרגתיים Percoll הם viabilities תא מעט גבוה יותר ומספרים. החיסרון הוא צנטריפוגה elutriation דורש הרוטור התמחה, צנטריפוגה ייעודי, ומשאבה peristaltic יחד שעולה עשרות אלפי דולרים. שיטה זו רק מחייב שימוש צנטריפוגה העליון בקירור שולחן המשאבה peristaltic אשר תקן במעבדות רבות. הפרדת שניות ו KCs על ידי הדבקה סלקטיבית היא שיטה סטנדרטית20-22. אמנם נכון כי שניות תדבק זכוכית ופלסטיק, נקודת המפתח היא להשתמש חומצה שטף זכוכית נקייה עם לא יותר מ דגירה של 15 דקות בשעה 37 ° C 5% CO 2. KCs תמיד לדבוק שניות מהר יותר וגם רצוי לשטוף בעדינות את KCs עם התקשורת כדי לחלץ כל שנותר שניות שהפכו מחוברת באופן רופף. Pronase ו פרוטאזות אחרים השמיט גם את מערכת העיכול צעדים collagenase בפרוטוקול זה כדי להגדיל את viabilities של התאים. פרוטאזות לעיכול קולטני תאי ועלול לעכב הידבקות הסלולר השלבים לטיהור הסופי. השיטה המוצגת כאן יש מגוון רחב של יישומים. למרות שאנחנו מראים את התוצאה הסופית שבה הפרין נלקח בתחילה ל זלוף אישור, הכבד להשגת hepatocytes העיקרי, KC, שניות, ועל HSCs עשוי להיות שימושי עבור מספר מחקרים שכללו מסלולים מטבוליים, immunoregulation, פעילויות נבלות, ועוד פיזיולוגיים Studies. החלק הקשה ביותר של הליך זה הוא cannulation טוב, עיכול של הכבד, וטיפול בכבד מבלי להחליק צנתר מהעורק הפורטל.

<ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מאגר 1: 142 mM NaCl 6.7 mM KCl, HEPES 10 מ&quot;מ, 1.5% BSA, pH 7.4 </li><li style=";text-align:right;direction:rtl"> מאגר 2 (חיץ זלוף): 67.0 mM NaCl, KCl mM 6.7, 4.8 mM CaCl 2-H2O, HEPES 101.0 מ&quot;מ, BSA 1.5%, pH 7.4. </li><li style=";text-align:right;direction:rtl"> מאגר 3: 137 mM NaCl, KCl mM 4.7, 0.7 mM MgSO 4, 1.2 mM CaCl 2-2H 2 O, HEPES 10 מ&quot;מ, 1.5% BSA, pH 7.4 </li><li style=";text-align:right;direction:rtl"> RPMI / BSA: 15g / L BSA ב RPMI </li><li style=";text-align:right;direction:rtl"> טריס שנאגרו מלוחים (TBS): 154 mM NaCl, 10 mM מלח Trizma Base. </li><li style=";text-align:right;direction:rtl"> Collagenase: collagenase (# 11088785103) של חברת רוש, collagenase Type I (# LS004691) מ וורטינגטון ביוכימיים או collagenase אני (# C0130) או IA (# C9891) מבית Sigma-Aldrich. </li></ul> כל המלחים מן Sigma-Aldrich <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם </td><td> חברה </td><td> דגם / קטלוג # </td><td> הערה </td></tr><tr><td> אמבט מים 20L </td><td> ThermoFisher </td><td> 2231 </td><td> המים על 45 מעלות צלזיוס, כדי לפצות על קירור </td></tr><tr><td></td><td></td><td></td><td> בתוך הצינור. </td></tr><tr><td> Peristaltic משאבה </td><td> קולמן בן הזוג </td><td> 7553-70 </td><td> Masterflex סדרה </td></tr><tr><td> קירור צנטריפוגה </td><td> Sorvall </td><td> מקרא XTR </td><td></td></tr><tr><td> צנתר </td><td> BD Biosciences </td><td> 381444 </td><td></td></tr><tr><td> Dessicator קאמרית </td><td> דיג </td><td> 08595E </td><td> השתמש צלחת פנימי </td></tr><tr><td> Percoll </td><td> סיגמא </td><td> P4937 </td><td></td></tr><tr><td> שור סרום אלבומין </td><td> SeraCare </td><td> AP-4510-01 </td><td></td></tr><tr><td> 100 מיקרומטר רשת </td><td> ספקטרום מעבדות </td><td> 146488 </td><td></td></tr><tr><td> 30 מיקרומטר רשת </td><td> ספקטרום מעבדות </td><td> 146506 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 21870 </td><td></td></tr><tr><td> פוליאתילן גליקול </td><td> ספקטרום מעבדהs </td><td> PO107 </td><td></td></tr></tbody></table>

in vivo liver endocytosis followed by purification of liver cells by liver perfusion

הכבד הוא מרכז חילוף החומרים בגופם של יונקים ומשמש כמסנן עבור בדם. הארכיטקטורה הבסיסית של הכבד מתוארת באיור 1, שבו יותר מ 85% ממסת הכבד מורכב hepatocytes ואת 15% הנותרים של מסת הסלולר מורכב של תאים Kupffer (KCs), בתאי stellate (HSCs), ו לתאי אנדותל סינוסי (שניות). שניות בצורת דפנות כלי הדם בתוך הכבד מכילים מורפולוגיה מיוחד הנקרא fenestrae בתוך בציטופלסמה. אשנוב של הציטופלסמה היא מראה של חורים (~ 100 מיקרומטר) בתוך התאים, כך לפעול שניות כמו מסננת שבה רוב chylomicrons, שרידי chylomicron ו מקרומולקולות, אך לא תאים, עוברים את hepatocytes ו HSCs 1 (איור 1). בשל היעדר קרום במרתף, הפער בין שניות ו hepatocytes בצורת שטח של Disse. HSCs למלא חלל זה ולשחק תפקיד מרכזי בוויסות ותגובה injury, אחסון של החומצה הרטינואית ו immunoregulation 2 של הכבד. שניות הם בין התאים הפעילים ביותר בגוף endocytically הצגת מערך של קולטנים נבלות על פני התא שלהם 3. אלה כוללים SR-A, Stabilin-1 ו Stabilin-2. באופן כללי, חלקיקים colloidal קטן פחות מ 230 ננומטר מקרומולקולות בשלב חיץ נתפסות על ידי שניות, ואילו, חלקיקים גדולים ופסולת הסלולר הוא endocytosed (phagocytosed) על ידי KCs 4. לפיכך, אישור עיקר החומר תאיים כגון glycosaminoglycans מהדם תלויה במידה רבה על הבריאות פונקציות endocytic סעיפים 5,6. לדוגמה, עלייה בדם hyaluronan מעיד על מחלת כבד החל קלה עד צורות חמורות יותר 7. למעט דו&quot;ח אחד 8, אין קווי הנציח SEC תא הקיום. אפילו את שורות התאים הוא הנציח דה differentiated בכך שהוא אינו מבטא קולטנים נבלות שנמצאים שניות הראשי (הנתונים שלנו, לא מוצג). מחקרים ביולוגיים כל תא חייב להתבצע על תאים ראשוניים שהתקבלו טרי מן החיה. לרוע המזל, שניות dedifferentiate בתנאים תרבות סטנדרטי יש להשתמש תוך 1 או 2 ימים על במנותק מן החיה. דיפרנציאציה של שניות מסומן על ידי הביטוי של Stabilin-2 או קולטן, הארי 9, CD31, ואת הנוכחות של אשנוב cytoplasmic 1. דיפרנציאציה של שניות ניתן להאריך על ידי תוספת של VEGF בתקשורת תרבות או על ידי תאים culturing במדיום מותנה hepatocyte 10,11. בדו&quot;ח זה, נדגים את הפעילות endocytic של שניות בתוך איבר שלם באמצעות רדיו שכותרתו הפרין עבור hyaluronan עבור קולטן Stabilin-2-SEC ספציפיים. לאחר מכן אנו לטהר hepatocytes ו שניות מהכבד perfused למדוד אנדוציטוזה.

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In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

המחקר בתאי הכבד סינוסי אנדותל (שניות) חייב להתבצע עם תאים ראשוניים שהתקבלו החיה לא כמו שורות תאים קיימות. שיטה זו מסתמכת על מערכת העיכול הכבד צנטריפוגה ההפרש לטיהור SEC עבור culturing ניסויים עתידיים.

ב אנדוציטוזה כבד vivo ואחריו טיהור בתאי הכבד על ידי זלוף כבד

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

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In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

19.0K Views.  University of Nebraska, Lincoln. המחקר בתאי הכבד סינוסי אנדותל (שניות) חייב להתבצע עם תאים ראשוניים שהתקבלו החיה לא כמו שורות תאים קיימות. שיטה זו מסתמכת על מערכת העיכול הכבד צנטריפוגה ההפרש לטיהור SEC עבור culturing ניסויים עתידיים.

Video: In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

טיהור של Hepatocytes ותאי אנדותל Sinusoidal מן הכבד העכבר זלוף

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Biochemistry

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

בידוד של מסחטות ושלפוחיות רקמה מהכבד

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

הבידוד של Fibroblasts פורטל עכברוש ידי<em&gt; באתרו</em&gt; כבד זלוף

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

בידוד של hepatocytes האנושי על ידי שני שלבים Collagenase זלוף נוהל

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

פרוטוקול הבידוד של hepatocytes האדם הראשי הקביל סרן אוכלוסיות של תאים בכבד ללא parenchymal

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

פרוטוקול יעיל יותר בזמן ובעבודה לבידוד הפטוציטים ראשוני של עכברים

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

בידוד של הפטוציטים ראשוניים של חולדות עם בקרת זלוף רב-פרמטרית

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

בידוד ואפיון של תאי אנדותל סינוסואידליים ראשוניים בכבד של עכבר

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

הדמיה תוך-עינית לא פולשנית in vivo של כדורי כבד מושתלים בעכברים

In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until now, optical imaging of the liver in situ is possible by intravital imaging, which offers high-resolution imaging at the cellular level but cannot be performed multiple times and, therefore, longitudinally in the same animal. Noninvasive imaging methods, such as bioluminescence, allow repeated imaging sessions on the same animal but do not achieve cell resolution. To address this methodology gap, we have developed a platform for noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. In the workflow described in this study, primary mouse liver spheroids are generated in vitro and transplanted into the anterior chamber of the eye of recipient mice, where they engraft on the iris. The cornea acts as a natural body window through which we can image the engrafted spheroids by conventional confocal microscopy. The spheroids survive for months in the eye, during which the cells can be studied in contexts of health and disease, as well as being monitored in response to different stimuli over repeated imaging sessions using appropriate fluorescent probes. In this protocol, we provide a breakdown of the necessary steps to implement this imaging system and explain how to best harness its potential.

מחבר זרקור: רתימת תאי עיניים של עכבר למחקרים לא פולשניים על כדורי כבד

Here, we describe a platform that allows noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. The workflow spans from generating spheroids from primary liver cells to transplantation into the mouse eye and in vivo imaging at cellular resolution by confocal microscopy.

In Vivo הדמיה של כדורי כבד המושתלים בחדר הקדמי של עין העכבר

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until ...

ב אנדוציטוזה כבד vivo ואחריו טיהור בתאי הכבד על ידי זלוף כבד

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Chapters in this video

טיהור של Hepatocytes ותאי אנדותל Sinusoidal מן הכבד העכבר זלוף

בידוד של מסחטות ושלפוחיות רקמה מהכבד

הבידוד של Fibroblasts פורטל עכברוש ידי

בידוד של hepatocytes האנושי על ידי שני שלבים Collagenase זלוף נוהל

פרוטוקול הבידוד של hepatocytes האדם הראשי הקביל סרן אוכלוסיות של תאים בכבד ללא parenchymal

פרוטוקול יעיל יותר בזמן ובעבודה לבידוד הפטוציטים ראשוני של עכברים

בידוד של הפטוציטים ראשוניים של חולדות עם בקרת זלוף רב-פרמטרית

בידוד ואפיון של תאי אנדותל סינוסואידליים ראשוניים בכבד של עכבר

In Vivo הדמיה של כדורי כבד המושתלים בחדר הקדמי של עין העכבר

In Vivo הדמיה של כדורי כבד המושתלים בחדר הקדמי של עין העכבר