עבודה זו התאפשרה הודות לקרן הרקמה האנושית וחקר תא, מה שהופך את הרקמות אנושיות זמינות למחקר. תמיכה כספית עבור עבודה זו קיבלה מהמשרד הפדרלי לחינוך ולמחקר (שם מענק: כבד רשת וירטואלית, מספר מענק: 0,315,759) וHepacult GmbH. תודתנו גם ללכת לעוזרים הטכניים מבנק רקמות Grosshadern בית החולים לאיסוף הדגימות הכבד והעוזרים הטכניים ממתקן Core בידוד תא לביצוע זלוף הכבד ובידוד הפטוציט. בפרט, אנו רוצים להודות לNatalja Löwen עבור הוכחת בהליך זה בווידאו. לבסוף, ברצוננו להודות לNatalja Löwen וEdeltraud Hanesch ליצירת האיורים לאיור 1 ודמויות בסקירה סכמטית של הווידאו.

1. הכנה של פתרונות זלוף ובידוד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את הפתרונות דרושים לטפטוף של חתיכת הכבד והבידוד של hepatocytes לפי טבלת 1. ניתן לאחסן את פתרונות על 4 מעלות צלזיוס עד לשימוש. </li><li style=";text-align:right;direction:rtl"> סינון סטרילי כל הפתרונות באמצעות מסנן 0.22 מיקרומטר. </li><li style=";text-align:right;direction:rtl"> כל הפתרונות שבאים במגע עם הכבד צריכים להיות סטרילי. </li></ol> 2. הכנת זלוף הציוד ופתרונות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הציוד לטפטוף של פיסת הכבד יש להגדיר כפי שמוצגים באיור 1. </li><li style=";text-align:right;direction:rtl"> אמבט המים צריך להיות מוגדר בטמפרטורה מתאימה, שהוא שונה בכל ניסיוני הגדרה מסוימת, כך שהפתרונים נמצאים בטמפרטורה של C ° 37 כאשר הם מגיעים לחתיכת הכבד. במקרה זה, אמבט המים מוגדר 41 מעלות צלזיוס כדי לחמם את הפתרונות 1, 2 ו -3 וקבל זכוכית במעיל. פתרון 4 יש לחמם עד37 מעלות צלזיוס באמבט מים נפרד לשימוש כדי להפחית את האובדן של פעילות collagenase. </li><li style=";text-align:right;direction:rtl"> זמן קצר לפני זלוף הכבד, להדליק את הרגולטור של מיכל הדלק המכיל 95% O 2/5% CO 2 לגז מנגנון החמצון (איור 1E). </li></ol> 3. טפטוף של הכבד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יש לקבל חתיכת כבד עם כמה שיותר הקפסולה של Glisson שלמה ככל האפשר ואופן אידיאלי עם משטח רק 1 לחתוך מפתולוג לזלוף. </li><li style=";text-align:right;direction:rtl"> מניחים חתיכת הכבד הזה במשפך ביכנר המכיל מסנן דיסק מחורר (איור 1). </li><li style=";text-align:right;direction:rtl"> מערכת זלוף צריכה להיות דרוכה עם פתרון 1. </li><li style=";text-align:right;direction:rtl"> עם קצב זרימה נמוך, cannulae השקיה המעוגל עם טיפים זית צריך להיות מוכנס לתוך כלי דם גדולים יותר על פני השטח החתך של פיסת הכבד. הדם שוטף החוצה מן הכבד, הרקמות הופכת להיות קלות יותר באזורים עם זלוף טוב. מספר cannulae משמש לגדלים שוניםשל כבדים מוצג באיור 2 א. גודל מד נבחר צריך לגרום להתאמה הדוקה שתחזיק cannulae במקום. יש להשאיר כלי הדם הקטנים יותר פתוחים לחיץ זלוף לניקוז מחוץ לחתיכת הכבד. </li><li style=";text-align:right;direction:rtl"> להגדיל את קצב הזרימה במשאבת peristaltic לבין 110-460 מיליליטר / דקה בהתאם לגודל של הכבד (איור 2). התוצאה היא קצב זרימה של 44 ± 16 ממוצע מיליליטר / דקה לצינורית (איור 2 ג). המהירות שנבחרה תלויה בחתיכת הכבד וצריכה לגרום להתפחה עד קלה של חתיכת הכבד. במקרים מסוימים, ייתכן שיהיה צורך להדק את שהחלק מכלי השיט הפתוח עם מלחציים בכלי דם מיקרו על מנת להשיג את ההתפחה הקלה שהוזכר לעיל. זלוף טוב ניתן להבחין כאשר חתיכת הכבד היא צבע בהיר יותר לאורך כל דרך. </li><li style=";text-align:right;direction:rtl"> שמור על פיסה הלחה הכבד במהלך זלוף על ידי כיסוי אותו עם פיסת הגזה הספוגה בתמיסת מלח. </li><li style=";text-align:right;direction:rtl"> Perfuse עם 1 ליטר של פתרון 1 כדי לשטוף את כל remaining דם בחתיכת הכבד. </li><li style=";text-align:right;direction:rtl"> לשנות את נוזל זלוף לפתרון 2 וינקב עבור 10 דקות. </li><li style=";text-align:right;direction:rtl"> לעבור את נוזל זלוף לפתרון 3 וינקב עם 0.5 L. </li><li style=";text-align:right;direction:rtl"> לשנות את נוזל זלוף ל4 פתרון, אשר מכיל .1-0.15% מcollagenase (טבלה 2). </li><li style=";text-align:right;direction:rtl"> לצעד זה, זלוף צריך להתבצע באופן מחזוריות ל9-12 דקות או עד שהכבד מתעכל מספיק; רקמת הכבד אמורה להופיע להתפרק מעט מתחת הכמוסה של Glisson ולהרגיש התרכך כשמשש עם הצד הקהה של אזמל. </li></ol> 4. בידוד של hepatocytes <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כבה את משאבת peristaltic ולהסיר cannulae מחתיכת הכבד. </li><li style=";text-align:right;direction:rtl"> מניחים את חתיכת הכבד בצלחת מתגבשת המכילה 100-200 מיליליטר של פתרון 5. </li><li style=";text-align:right;direction:rtl"> הסר את הקפסולה של Glisson בזהירות ובעדינות לנער את התאים. אם ישנם אזורים שאינם perfused היטב, אזמל יכול לשמש כדי לחתוךאזורים אלה כדי לשחרר את התאים בתוך. הוסף עוד פתרון 5 צורך כבמהלך התהליך. </li><li style=";text-align:right;direction:rtl"> הוסף עוד פתרון 5 עד לנפח סופי של 500 מיליליטר. </li><li style=";text-align:right;direction:rtl"> סנן השעיה תא פעמיים; ראשון דרך רשת 210 ניילון מיקרומטר ואחריו רשת ניילון 70 מיקרומטר. , הבא לשפוך את ההשעיה התא לתוך 200 צינורות צנטריפוגה מיליליטר. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה ההשעיה התא ב72 גרם במשך 5 דקות ב 4 ° C. לשאוב supernatant ותא גלולה בעדינות resuspend בעדינות ב200 מיליליטר של פתרון 5. </li><li style=";text-align:right;direction:rtl"> חזור על מספר שלב הכביסה 4.6 שלוש פעמים. על המדרגה צנטריפוגות הסופית, תאי resuspend בפתרון אחסון קר (ראו רשימה של חומרים). תאים צריכים להיות כ 2-5,000,000 hepatocytes למיליליטר להערכה של תשואה וכדאיות באמצעות assay הרחקת trypan הכחול מבוסס hemocytometer. </li><li style=";text-align:right;direction:rtl"> כדי לבצע את assay הרחקת trypan הכחול, להוסיף 0.1 מיליליטר של תאים בדילול כראוי (2-5,000,000 ≈ / מיליליטר) לצינור microfuge מכיל 0.5 מיליליטר של soluti trypan הכחולב( 0.4% trypan הכחולים מומס בפוספט שנאגרו מלוח (PBS)) ו0.4 מיליליטר של PBS. לאחר ערבוב ההשעיה התא ביסודיות, טען hemocytometer עם ההשעיה ולבחון תחת מיקרוסקופ בהגדלה 100X. תחת מיקרוסקופ, התאים המתים יהיו מוכתמים כחולים ואילו תאי החיים מופיעים בלא כתם. לספור את מספר תאי חיים ומוחלט בכל אחד ממ&quot;מ 2 הרשתות 1 מסומנת על hemocytometer. כדאיות (%), תשואה של תאי חיים (מיליון hepatocytes למיליליטר פתרון אחסון קר (CSS) או מיליון hepatocytes לכבד ז) יכולה להיות מחושבת באמצעות נוסחאות שלהלן. </li></ol><img alt="משוואת 1" fo:src="/files/ftp_upload/50615/50615eq1highres.jpg" src="/files/ftp_upload/50615/50615eq1.jpg" /> הערה: במקרה זה, גורם לדילול trypan הכחול הוא 10 וגורם hemocytometer הוא 10,000.

<ol>
	<li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods in Cell Biology. 13, 29-83 (1976).</li><li>Schneider, W. C., Potter, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assay+of+animal+tissues+for+respiratory+enzymes+II.+Succinic+dehydrogenase+and+cytochrome+oxidase.">The assay of animal tissues for respiratory enzymes II. Succinic dehydrogenase and cytochrome oxidase.</a> J. Biol. Chem. 149, 217-227 (1943).</li><li>Aubin, P. M. G., Bucher, N. L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Study+of+Binucleate+Cell+Counts+in+Resting+and+Regenerating+Rat+Liver+Employing+a+Mechanical+Method+for+the+Separation+of+Liver+Cells.">A Study of Binucleate Cell Counts in Resting and Regenerating Rat Liver Employing a Mechanical Method for the Separation of Liver Cells.</a> Anat. Rec. 112, 797-809 (1952).</li><li>Anderson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mass+Isolation+of+Whole+Cells+from+Rat+Liver.">The Mass Isolation of Whole Cells from Rat Liver.</a> Science. 117, 627-628 (1953).</li><li>Jacob, S. T., Bhargava, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Method+for+Preparation+of+Liver+Cell+Suspensions.">New Method for Preparation of Liver Cell Suspensions.</a> Experimental Cell Research. 27 (62), 453-467 (1962).</li><li>Berry, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Properties+of+Cells+Isolated+from+Adult+Mouse+Liver.">Metabolic Properties of Cells Isolated from Adult Mouse Liver.</a> Journal of Cell Biology. 15, 1-8 (1962).</li><li>Laws, J. O., Stickland, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+Isolated+Liver+Cells.">Metabolism of Isolated Liver Cells.</a> Nature. 178, 309-310 (1038).</li><li>Berry, M. N., Friend, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+preparation+of+isolated+rat+liver+parenchymal+cells:+a+biochemical+and+fine+structural+study.">High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study.</a> The Journal of Cell Biology. 43, 506-520 (1969).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Rat-Liver+Cells+.3.+Enzymatic+Requirements+for+Tissue+Dispersion.">Preparation of Rat-Liver Cells .3. Enzymatic Requirements for Tissue Dispersion.</a> Experimental Cell Research. 82 (73), 391-398 (1973).</li><li>Thasler, W. E., 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=Charitable+State-Controlled+Foundation+Human+Tissue+and+Cell+Research:+Ethic+and+Legal+Aspects+in+the+Supply+of+Surgically+Removed+Human+Tissue+For+Research+in+the+Academic+and+Commercial+Sector+in+Germany.">Charitable State-Controlled Foundation Human Tissue and Cell Research: Ethic and Legal Aspects in the Supply of Surgically Removed Human Tissue For Research in the Academic and Commercial Sector in Germany.</a> Cell and Tissue Banking. 4, 49-56 (2003).</li><li>Queral, A. E., DeAngelo, A. B., Garrett, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+different+collagenases+on+the+isolation+of+viable+hepatocytes+from+rat+liver.">Effect of different collagenases on the isolation of viable hepatocytes from rat liver.</a> Analytical Biochemistry. 138, 235-237 (1984).</li><li>Smedsrod, B., Pertoft, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Pure+Hepatocytes+and+Reticuloendothelial+Cells+in+High-Yield+from+a+Single-Rat+Liver+by+Means+of+Percoll+Centrifugation+and+Selective+Adherence.">Preparation of Pure Hepatocytes and Reticuloendothelial Cells in High-Yield from a Single-Rat Liver by Means of Percoll Centrifugation and Selective Adherence.</a> J. Leukocyte Biol. 38, 213-230 (1985).</li><li>Cantrell, E., Bresnick, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzpyrene+Hydroxylase-Activity+in+Isolated+Parenchymal+and+Nonparenchymal+Cells+of+Rat-Liver.">Benzpyrene Hydroxylase-Activity in Isolated Parenchymal and Nonparenchymal Cells of Rat-Liver.</a> Journal of Cell Biology. 52, 316-321 (1972).</li><li>Weiskirchen, R., Gressner, A. M. <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+culture+of+hepatic+stellate+cells.">Isolation and culture of hepatic stellate cells.</a> Methods in Molecular Medicine. 117, 99-113 (2005).</li><li>Baccarani, U., 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=Steatotic+versus+cirrhotic+livers+as+a+source+for+human+hepatocyte+isolation.">Steatotic versus cirrhotic livers as a source for human hepatocyte isolation.</a> Transplant P. 33, 664-665 (2001).</li><li>Renz, J. F., 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=Utilization+of+extended+donor+criteria+liver+allografts+maximizes+donor+use+and+patient+access+to+liver+transplantation.">Utilization of extended donor criteria liver allografts maximizes donor use and patient access to liver transplantation.</a> Annals of Surgery. 242, 556-563 (2005).</li><li>Schemmer, P., 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=Extended+donor+criteria+have+no+negative+impact+on+early+outcome+after+liver+transplantation:+a+single-center+multivariate+analysis.">Extended donor criteria have no negative impact on early outcome after liver transplantation: a single-center multivariate analysis.</a> Transplant Proc. 39, 529-534 (2007).</li><li>Alexandre, E., 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=Influence+of+pre-,+intra-+and+post-operative+parameters+of+donor+liver+on+the+outcome+of+isolated+human+hepatocytes.">Influence of pre-, intra- and post-operative parameters of donor liver on the outcome of isolated human hepatocytes.</a> Cell and Tissue Banking. 3, 223-233 (2002).</li><li>Spotnitz, W. D., Burks, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-of-the-art+review:+Hemostats,+sealants,+and+adhesives+II:+Update+as+well+as+how+and+when+to+use+the+components+of+the+surgical+toolbox.">State-of-the-art review: Hemostats, sealants, and adhesives II: Update as well as how and when to use the components of the surgical toolbox.</a> Clinical and applied thrombosis/hemostasis : official journal of the International Academy of Clinical and Applied Thrombosis/Hemostasis. 16, 497-514 (2010).</li><li>Spotnitz, W. D., Burks, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemostats,+sealants,+and+adhesives+III:+a+new+update+as+well+as+cost+and+regulatory+considerations+for+components+of+the+surgical+toolbox.">Hemostats, sealants, and adhesives III: a new update as well as cost and regulatory considerations for components of the surgical toolbox.</a> Transfusion. 52, 2243-2255 (2012).</li><li>Toriumi, D. M., Raslan, W. F., Friedman, M., Tardy, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histotoxicity+of+cyanoacrylate+tissue+adhesives.+A+comparative+study.">Histotoxicity of cyanoacrylate tissue adhesives. A comparative study.</a> Archives of otolaryngology--head &amp; neck surgery. 116, 546-550 (1990).</li><li>Vinters, H. V., Galil, K. A., Lundie, M. J., Kaufmann, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histotoxicity+of+cyanoacrylates.+A+selective+review.">The histotoxicity of cyanoacrylates. A selective review.</a> Neuroradiology. 27, 279-291 (1985).</li><li>Bhogal, R. H., 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+of+primary+human+hepatocytes+from+normal+and+diseased+liver+tissue:+a+one+hundred+liver+experience.">Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience.</a> PloS ONE. 6, e18222 (2011).</li><li>Olson, H., 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=Concordance+of+the+toxicity+of+pharmaceuticals+in+humans+and+in+animals.">Concordance of the toxicity of pharmaceuticals in humans and in animals.</a> Regul. Toxicol. Pharm. 32, 56-67 (2000).</li><li>Koebe, H. G., Pahernik, S. A., Sproede, M., Thasler, W. E., Schildberg, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+hepatocytes+from+slaughterhouse+organs.+An+unlimited+resource+for+bioartificial+liver+devices.">Porcine hepatocytes from slaughterhouse organs. An unlimited resource for bioartificial liver devices.</a> ASAIO Journal. 41, 189-193 (1995).</li><li>Russell, W. M. S., Burch, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The principles of humane experimental technique. , (1959).</li></ol>

אופטימיזציה של פרוטוקול זה מומנה בחלקו על ידי מענק מHepacult GmbH. ד&quot;ר וולפגנג Thasler הוא אחד ממייסדי Hepacult GmbH ונשאר אחד מחברי הדירקטוריון בחברה זו. העסקת Maresa Demmel היא באופן חלקי על ידי Hepacult. מריה Hauner הוא מועסק על ידי Hepacult GmbH. Hepacult היא חברת ספין אוף בתחום הביוטכנולוגיה באוניברסיטה, אשר מציעה hepatocytes אדם בהסכמה וגישה פתוחה למטרות מחקר.

פרוטוקול זה גורם לבידוד של hepatocytes האנושי עם כדאיות גבוהה ובטהרה. על מנת להשיג את התוצאות הללו, חשוב להתחיל עם חתיכת הכבד מתאימה. חתיכת הכבד צריכה הכמוסה של Glisson שלמות על כל המשטחים, פרט למשטח לחתוך 1. גורם חשוב נוסף הוא אצווה המסוים של collagenase משמש, כפי שקבוצות שונות יכולו...

A correction was made to: <a href="http://www.jove.com/video/50615/isolation-human-hepatocytes-two-step-collagenase-perfusion">Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure </a>
The changes listed below have been made to Table 1.
1). In the recipe for Solution 2, the Final Concentration of EGTA has been changed from:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>EGTA&#160;</td>
			<td>0.1mM</td>
		</tr>
	</tbody>
</table>
to:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>EGTA&#160;</td>
			<td>1mM</td>
		</tr>
	</tbody>
</table>
2). In the recipes for Solution 3 and 4, the Final Concentration of Calcium chloride dihydrate has been changed from:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Calcium chloride dihydrate&#160;</td>
			<td>0.5&#181;M</td>
		</tr>
	</tbody>
</table>
to:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Calcium chloride dihydrate&#160;</td>
			<td>5mM</td>
		</tr>
	</tbody>
</table>

A correction was made to: Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure.
The Final Concentration of EGTA in Solution 2 and the Final Concentration of Calcium chloride dihydrate in solutions 3 and 4 have been updated in Table 1.

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

השעיה תא כבד ניתן להכין מהכבד בשיטות מכאניות או אנזימטי. שיטות מכאניות המשמשות להכנת תאי כבד שלמים כוללות כפייה של כבד דרך הגזה 2, רועד חתיכת כבד עם חרוזי זכוכית שייקרה קאהן 3, באמצעות homogenizers זכוכית עם עלי רופף 4,5 וכו &#39;במהלך השנים, שיטות מכאניות נפלו מתוך להעדיף בשל הנזק לקרומי תאים ואובדן התפקוד של הפטוציטים המבודדים 6,7. כתוצאה מכך, השימוש בשיטה אנזימטית הוא כיום השיטה העיקרית לבידודה של הפטוציטים. בידוד של hepatocytes באמצעות שיטה האנזימטית השתפר מאוד כאשר ברי והחבר 8 perfused collagenase וhyaluronidase דרך כבד דרך וריד הפורטל בחולדות. תהליך זה מנוצל זלוף כלי הדם כדי לאפשר לאנזימים לבוא במגע קרוב עם רוב של התאים, מה שמוביל לעלייה של פי 6 בתשואה של hepatocytes 8. יתר על כן, שיטה זו הניבה תאים ששמרו על השלמות המבנית שלהם, עם כמעט ללא שינוי של reticulum endoplasmic לתוך שלפוחית ​​מבודדת ולא נגרם נזק המיטוכונדריה 8. שיטה זו שונה על ידי 1 Seglen, שהיה חלוץ הליך זלוף שני שלבים לבידוד בתאים כבד. בהליך זה, בכבד חולדת perfused עם 2 + חיץ חופשי Ca אחרי זלוף עם חיץ collagenase המכיל Ca 2 + 1. ההסרה של Ca 2 + בשלב הראשון מסייעת לשבש דסמוזומים, תוך התוספת של Ca 2 + בשלב השני נדרשת לפעילות collagenase אופטימלית 1,9. בהתחשב בכך שהעבודה שפורסמה שתוארה לעיל בוצעה בחולדות, מאמר זה נועד להדגים הליך שונה שיכול לשמש לבידודה של הפטוציטים עם כדאיות גבוהה מזמזוםכבדים. השימוש בhepatocytes האנושי נשאר חשוב למחקר translational ולאימות ניסויים תוך שימוש במודלים של בעלי חיים. חתיכות הכבד האנושיות השתמשו במחקר זה נרכשו בהסכמה לממשל דרך הקרן לחקר רקמות ותאים אנושיים, קרן ללא כוונת רווח בשליטה ממשלתית 10. לאחר הפתולוג הסיר את מה שנדרש לאבחון, חתיכות כבד נאספו מהרקמות שנותרו. הרקמה מחולק על ידי פתולוג הייתה מורפולוגית רקמה בריאה המתקבלת משולי כריתה לאחר כריתת כבד.

<table><tbody><tr><td>Bubble trap</td><td>Ga&amp;szlig;ner Glastechnik</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Glass jacketed condenser</td><td>Ga&amp;szlig;ner Glastechnik</td><td></td><td>&amp;nbsp;</td></tr><tr><td>41 &amp;deg;C Water bath</td><td>Julabo</td><td>35723-H24/EG</td><td>&amp;nbsp;</td></tr><tr><td>37 &amp;deg;C Water bath</td><td>GFL</td><td>1083</td><td>&amp;nbsp;</td></tr><tr><td>Compressed gas cylinder (95 % O&lt;sub&gt;2&lt;/sub&gt;/5 % CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Linde</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Gas permeable tubing</td><td>Neolab</td><td>2-4440</td><td>&amp;nbsp;</td></tr><tr><td>Peristaltic pump</td><td>Ismatec</td><td>IP65</td><td>&amp;nbsp;</td></tr><tr><td>Scalpel</td><td>Feather</td><td>320010</td><td>&amp;nbsp;</td></tr><tr><td>Forceps</td><td>Omnilab</td><td>5171014</td><td>&amp;nbsp;</td></tr><tr><td>Conical flasks 1 L</td><td>Schott Duran</td><td>2121654</td><td>&amp;nbsp;</td></tr><tr><td>Conical flasks 5 L</td><td>Schott Duran</td><td>2121673</td><td>&amp;nbsp;</td></tr><tr><td>Beakers</td><td>Schott Duran</td><td>2110654</td><td>&amp;nbsp;</td></tr><tr><td>200 ml centrifuge tubes</td><td>Becton Dickinson</td><td>352075</td><td>&amp;nbsp;</td></tr><tr><td>Crystallizing dish</td><td>Omnilab</td><td>5144063</td><td>&amp;nbsp;</td></tr><tr><td>Curved irrigation cannulae with ball tips</td><td>Ernst Kratz GmbH</td><td>1464LL/ 1465LL A+B/ 1472LL</td><td>&amp;nbsp;</td></tr><tr><td>Micro vascular clamps</td><td>Ernst Kratz GmbH</td><td></td><td>&amp;nbsp;</td></tr><tr><td>B&amp;uuml;chner funnel</td><td>Carl Roth</td><td>HT38.1</td><td>&amp;nbsp;</td></tr><tr><td>Nylon mesh 210 &amp;mu;m</td><td>Neolab</td><td>4-1413</td><td>&amp;nbsp;</td></tr><tr><td>Nylon mesh 70 &amp;mu;m</td><td>Neolab</td><td>4-1419</td><td>&amp;nbsp;</td></tr><tr><td>0.22 &amp;mu;m sterile filters</td><td>Peske</td><td>99505</td><td>&amp;nbsp;</td></tr><tr><td>500 ml bottles</td><td>Schott Duran</td><td>2180144</td><td>&amp;nbsp;</td></tr><tr><td>1 L bottles</td><td>Schott Duran</td><td>2180154</td><td>&amp;nbsp;</td></tr><tr><td>Hemocytometer</td><td>Peske</td><td>06-0001</td><td>&amp;nbsp;</td></tr><tr><td>1.5 ml tubes</td><td>Eppendorf</td><td>0030 120,086</td><td>&amp;nbsp;</td></tr><tr><td>50 ml conical tubes</td><td>BD Biosciences</td><td>352070</td><td>&amp;nbsp;</td></tr><tr><td>Ice bucket</td><td>Neolab</td><td>1508454</td><td>&amp;nbsp;</td></tr><tr><td>Sterile Pasteur pipettes</td><td>Brand</td><td>747715</td><td>&amp;nbsp;</td></tr><tr><td>Motorised pipette filler (Pipette boy acu)</td><td>Integra</td><td>155017</td><td>&amp;nbsp;</td></tr><tr><td>Refridgerated centrifuge</td><td>Eppendorf</td><td>5810R</td><td>&amp;nbsp;</td></tr><tr><td>Laminar flow</td><td>Kendro</td><td>Hera safe-KS9</td><td>&amp;nbsp;</td></tr><tr><td>Aspirator (Low-flow surgical suction pump)</td><td>Atmos</td><td>C361</td><td>&amp;nbsp;</td></tr><tr><td>Laboratory Gas Burner</td><td>Integra</td><td>Fire Boy eco</td><td>&amp;nbsp;</td></tr><tr><td>Disposable laboratory coat</td><td>Paperlynen GmbH</td><td>MD0202414</td><td>&amp;nbsp;</td></tr><tr><td>Surgical mask with visor</td><td>Kimberly-Clark</td><td>48247</td><td>&amp;nbsp;</td></tr><tr><td>Surgical hood</td><td>Barrier</td><td>42072</td><td>&amp;nbsp;</td></tr><tr><td>Latex gloves</td><td>Semper Care</td><td>CE0321</td><td>&amp;nbsp;</td></tr><tr><td>Collagenase (Batch number NB 4G)</td><td>Serva</td><td>17465</td><td>&amp;nbsp;</td></tr><tr><td>Calcium chloride dihydrate</td><td>Merck</td><td>2382</td><td>&amp;nbsp;</td></tr><tr><td>EGTA</td><td>Sigma</td><td>E4378</td><td>&amp;nbsp;</td></tr><tr><td>Sodium chloride</td><td>Roth</td><td>9265.2</td><td>&amp;nbsp;</td></tr><tr><td>Hepes</td><td>Roth</td><td>9105.3</td><td>&amp;nbsp;</td></tr><tr><td>Potassium chloride</td><td>Serva</td><td>26868</td><td>&amp;nbsp;</td></tr><tr><td>Albumin</td><td>Biomol</td><td>01400-2</td><td>&amp;nbsp;</td></tr><tr><td>Glucose</td><td>Serva</td><td>22700</td><td>&amp;nbsp;</td></tr><tr><td>Sodium hydrogen carbonate</td><td>Serva</td><td>30180</td><td>&amp;nbsp;</td></tr><tr><td>0.4% Trypan blue solution</td><td>Lonza</td><td>17-942E</td><td>&amp;nbsp;</td></tr><tr><td>Cold storage solution</td><td>Hepacult GmbH</td><td></td><td>&amp;nbsp;</td></tr></tbody></table>

isolation of human hepatocytes by a two-step collagenase perfusion procedure

הכבד, איבר עם יכולת התחדשות יוצאת דופן, מבצע מגוון רחב של פונקציות, כגון סילוק רעלים, חילוף חומרים ועל הומאוסטזיס. ככזה, hepatocytes הוא מודל חשוב למגוון רחב של שאלות מחקר. בפרט, השימוש בhepatocytes האנושי הוא חשוב במיוחד בתחומי פרמקוקינטיקה, טוקסיקולוגיה, התחדשות כבד ומחקר translational. לכן, שיטה זו מציגה גרסה שונה של הליך זלוף collagenase שני שלבים לבודד hepatocytes כפי שתואר על ידי 1 Seglen. בעבר, hepatocytes היה מבודד בשיטות מכאניות. עם זאת, שיטות אנזימטיות הוכחו להיות מעולה כמו hepatocytes לשמור על השלמות המבנית שלהם ותפקוד לאחר בידוד. שיטה זו המוצגת כאן מתאים את עצמו בשיטה שנועדה בעבר לכבדי חולדה לחתיכות כבד אנושיות ותוצאות בתשואה גדולה של הפטוציטים עם כדאיות של 77 10% ±. העיקריהבדל בהליך זה הוא התהליך של cannulization של כלי הדם. יתר על כן, בשיטה שתוארה כאן יכולה להיות מיושמת גם לכבדים ממינים אחרים עם גדלים בכבד או בכלי דם דומים.

התקנת זלוף הציוד הדרוש לזלוף כבד יש להגדיר על פי איור 1. כדאיות ותשואה של hepatocytes אדם מבודד הכדאיות הממוצעת של hepatocytes אדם הבודד הייתה 77 ± 10% והתשואה המ?...

Watch this Scientific Journal Video about Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure at JoVE.com

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

הליך זלוף collagenase שני שלבים שונה לבידוד של hepatocytes אדם מתואר. שיטה זו יכולה להיות מיושמת גם לכבדים של יונקים אחרים. Hepatocytes מבודד זמינים בתשואה גבוהה וכדאיות, מה שהופך אותם המודל מתאים למחקר מדעי בתחומים כגון התחדשות כבד, פרמקוקינטיקה וטוקסיקולוגיה.

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. ...

isolation-human-hepatocytes-two-step-collagenase-perfusion

Research

JoVE Journal

Biology

30.9K Views.  Grosshadern Hospital, Munich.  הליך זלוף collagenase שני שלבים שונה לבידוד של hepatocytes אדם מתואר. שיטה זו יכולה להיות מיושמת גם לכבדים של יונקים אחרים. Hepatocytes מבודד זמינים בתשואה גבוהה וכדאיות, מה שהופך אותם המודל מתאים למחקר מדעי בתחומים כגון התחדשות כבד, פרמקוקינטיקה וטוקסיקו?...

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

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 ...

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsr&#248;d et al. designed for rat liver cell isolation and provides high yield (up to 14 x 106 cells per mouse) and high purity (&#62;95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient.

Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.

בידוד תא Kupffer לNanoparticle בדיקת רעילות

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle ...

Immunology and Infection

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

מדידה של חומצת שומן β-חמצון בהשעיה של הפטוציטים עכברים מבודדים טריים

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

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

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 figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

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

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

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 ...

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 ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

בידוד של הפטוציטים מתחדשים לאחר כריתת כבד חלקית בעכברים

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

Medicine

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

בידוד וכריך קולגן 3D תרבות של העכבר הראשי Hepatocytes כדי ללמוד את התפקיד של שלד ציטוזה היווצרות של המרה בתוך מחוץ לגופית

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

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. ...