نود أن نشكر الدكتور بول يغل في جامعة. أوكلاهوما لاستخدام ريتوكسيماب 30 لاكتشاف مستقبلات HARE/Stabilin-2 ويجل جانيت للحصول على المساعدة التقنية لها. ويتم تمويل هذا البحث من جامعة نبراسكا لصناديق البحوث.

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الإعلان عن أي تضارب في المصالح.

التخدير من الفئران من خلال طريقة قطرة شنقا في اللاوعي التي isoflurane يدفع البخار هو الأسلوب المفضل لدراستنا. ويمكن أيضا أن تستخدم المرذاذ بدلا من الحقنة مع القطن للحفاظ على الحيوانات خارج الغرفة ، ولكن العمليات الجراحية يتم ذلك بسرعة ، ليست مطلوبة. إضافة إلى البولي ايثيلين جلايكول isoflurane لخفض ضغط البخار ومعدل التبخر. كثيرا جدا سوف بخار isoflurane لحث الموت بسرعة كبيرة جدا. إذا مات الحيوان قبل مقنى الكبد ومتبيض مع TBS ، وتجميع تجلط الدم قد في الكبد ومنع غسل الفعال للsinusoids والهضم مع كولاجيناز. بالإضافة إلى ذلك قد يكون من المفيد الهيبارين كمضاد للتخثر ولكنه لا يستخدم في هذه الدراسات منذ نستخدم الهيبارين وصفت بأنها التحقيق لدينا. غالبا ما يتم استبدال حل Gey مخزنة في المياه المالحة (GBSS) من قبل مختبرات أخرى لتنقية ثانية. في حين أنه مفيد لتنقية المجلس الأعلى للتعليم ، التهاب الكبدatocytes لا تتسامح مع GBSS فضلا عن المخازن 1-3 و viabilities لا يصل تقريبا. عند قياس الإلتقام ، فمن الممارسة السليمة لقياس مستويات الخلفية في الجهاز وتنقيته في خلايا الكبد. هذا الأسلوب هو واحد من اثنين لتنقية كفاءة SECS بعد نضح الكبد مع كولاجيناز. الأسلوب الآخر يفصل الخلايا غير متني بواسطة الطرد المركزي تصويل 19 بدلا من التدرج Percoll. مزايا لاستخدام أكثر من التدرجات تصويل Percoll viabilities هي الخلية أعلى قليلا والأرقام. الجانب السلبي هو أن الطرد المركزي تصويل يتطلب الدوار المتخصصة ، وأجهزة الطرد المركزي المخصصة ، ومضخة تمعجية معا والتي تكلف عشرات الآلاف من الدولارات. هذا الأسلوب يتطلب سوى استخدام جهاز طرد مركزي طاولة المبردة ومضخة تمعجية الذي هو المعيار في مختبرات عديدة. فصل SECS وKCS بواسطة التصاق الانتقائي هو أسلوب قياسي20-22. ولئن كان صحيحا أن SECS ستلتزم الزجاج والبلاستيك ، والنقطة الرئيسية هي استخدام حمض غسل الزجاج نظيفة مع عدم وجود أكثر من 15 دقيقة الحضانة عند 37 درجة مئوية في 5 ٪ CO 2. KCS سوف تلتزم دائما أسرع من SECS ويستحسن أن يغسل بلطف KCS مع وسائل الإعلام لاستخراج أي SECS المتبقية التي أصبحت فضفاضة يعلق. كما يتم حذف Pronase البروتياز وغيرها من الخطوات الهضم كولاجيناز في هذا البروتوكول لزيادة viabilities الخلايا. البروتياز هضم مستقبلات خارج الخلية ، وربما تحول دون التصاق الخلوية في الخطوات تنقية النهائي. الطريقة المعروضة هنا لديها تشكيلة واسعة من التطبيقات. على الرغم من أننا تظهر النتيجة النهائية التي اتخذت في البداية للحصول على الهيبارين التروية ، وإزالة الكبد للحصول على خلايا الكبد الأولية ، قد KC ، SECS ، وHSCs يكون مفيدا لعدد من الدراسات التي تنطوي على المسارات الأيضية ، مناعي ، والأنشطة زبال ، وغيرها الفسيولوجية ستوديوفاق. أصعب جزء من هذا الإجراء هو جيد إقناء ؛ إدخال القنية ، والهضم للكبد ، والتعامل مع الكبد دون الحاجة لقسطرة زلة من الوريد البابي.

<ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> العازلة 1 : 142 مم كلوريد الصوديوم 6.7 ملي بوكل ، 10 HEPES مم ، 1.5 ٪ BSA ، ودرجة الحموضة 7.4 </li><li style=";text-align:right;direction:rtl"> العازلة 2 (العازلة التروية) : 67،0 مم كلوريد الصوديوم ، 6.7 ملي بوكل ، 4.8 ملي CaCl 2 - H2O ، 101.0 ملي HEPES ، BSA 1.5 ٪ ، ودرجة الحموضة 7.4. </li><li style=";text-align:right;direction:rtl"> العازلة 3 : 137 مم كلوريد الصوديوم و 4.7 ملي بوكل ، MgSO 4 0.7 مم ، 1.2 مم 2 - CaCl 2H 2 O ، 10 HEPES مم ، 1.5 ٪ BSA ، ودرجة الحموضة 7.4 </li><li style=";text-align:right;direction:rtl"> RPMI / BSA : 15G / L في جيش صرب البوسنة RPMI </li><li style=";text-align:right;direction:rtl"> TRIS - مخزنة المالحة (TBS) : 154 مم كلوريد الصوديوم ، و 10 قاعدة Trizma ملي الملح. </li><li style=";text-align:right;direction:rtl"> كولاجيناز : كولاجيناز A (# 11088785103) من شركة روش ، كولاجيناز النوع الأول (# LS004691) من رثينجتون بيوكيميائية أو أنا كولاجيناز (# C0130) أو IA (# C9891) من سيغما الدريخ. </li></ul> جميع أملاح هم من سيغما الدريخ <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم </td><td> شركة </td><td> نموذج / كتالوج # </td><td> تعليق </td></tr><tr><td> 20L ماء الحمام </td><td> ThermoFisher </td><td> 2231 </td><td> المياه حوالي 45 درجة مئوية للتعويض عن التبريد </td></tr><tr><td></td><td></td><td></td><td> داخل الأنابيب. </td></tr><tr><td> مضخة تمعجية </td><td> الغمد Parmer </td><td> 7553-70 </td><td> سلسلة Masterflex </td></tr><tr><td> المبردة الطرد المركزي </td><td> Sorvall </td><td> أسطورة XTR </td><td></td></tr><tr><td> القسطرة </td><td> العلوم البيولوجية دينار بحريني </td><td> 381444 </td><td></td></tr><tr><td> Dessicator الغرفة </td><td> الصياد </td><td> 08595E </td><td> استخدام لوحة داخلية </td></tr><tr><td> Percoll </td><td> سيغما </td><td> P4937 </td><td></td></tr><tr><td> مصل بقري الزلال </td><td> SeraCare </td><td> AP - 4510-01 </td><td></td></tr><tr><td> شبكة 100 ميكرون </td><td> مختبرات الطيف </td><td> 146488 </td><td></td></tr><tr><td> 30 ميكرومتر شبكة </td><td> مختبرات الطيف </td><td> 146506 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 21870 </td><td></td></tr><tr><td> البولي ايثيلين جلايكول </td><td> معمل الطيفق </td><td> PO107 </td><td></td></tr></tbody></table>

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

الكبد هو مركز الأيضية للجسم الثدييات وبمثابة تصفية الدم. ويتضح في البنية الأساسية للكبد في الشكل (1) الذي يتكون أكثر من 85 ٪ من كتلة الكبد من خلايا الكبد ، وتتكون النسبة المتبقية البالغة 15 ٪ من الكتلة الخلوية لخلايا كوبفر (KCS) ، والخلايا النجمية (HSCs) ، و الخلايا البطانية الجيبية (ثانية). SECS تشكل جدران الأوعية الدموية داخل الكبد وتحتوي على التشكل داخل متخصصة تسمى نافذي في السيتوبلازم. نوفذة من السيتوبلازم هو مظهر من الثقوب (~ 100 ميكرون) داخل الخلايا بحيث الفعل SECS بمثابة غربال فيه معظم كيلومكرونات ، وبقايا الكيلومكرونات والجزيئات ، ولكن ليس الخلايا ، بالمرور إلى خلايا الكبد وHSCs 1 (الشكل 1). نظرا لعدم وجود الغشاء القاعدي ، والفجوة بين SECS وتشكيل خلايا الكبد من Disse الفضاء. HSCs تشغل هذه المساحة وتلعب دورا بارزا في التنظيم وردا على طnjury ، وتخزين حمض الريتينويك ومناعي لل2 الكبد. SECS هي من بين الأكثر نشاطا endocytically خلايا الجسم تعرض فيها مجموعة من المستقبلات زبال في كل خلية 3 السطح. وتشمل هذه SR - A ، وStabilin 1 - 2 - Stabilin. عموما ، يتم أخذ الجسيمات الغروية صغيرة أقل من 230 نانومتر ، والجزيئات في المرحلة العازلة بنسبة SECS ، بينما هو endocytosed الجزيئات الكبيرة والحطام الخلوية (phagocytosed) بواسطة KCS 4. وبالتالي ، فإن إزالة القسم الأكبر من المواد خارج الخلية مثل الجليكوزامينوجليكان من الدم يعتمد بشكل كبير على صحة وظائف التقامي SECS من 5،6. على سبيل المثال ، زيادة في مستويات hyaluronan الدم يدل على مرض الكبد تتراوح ما بين خفيفة إلى أشكال أكثر خطورة 7. باستثناء تقرير واحد 8 ، لا توجد خطوط الخلية SEC خلدت في الوجود. حتى هذا الخط هو خلد الخلية دي التفاضليةated في أنه لا يعبر عن مستقبلات زبال التي موجودة على SECS الابتدائية (البيانات المتوفرة لدينا ، لا يظهر). يجب إجراء جميع الدراسات الخلية البيولوجية على الخلايا الأولية التي تم الحصول عليها حديثا من الحيوان. للأسف ، في ظل ظروف SECS dedifferentiate ثقافة موحدة ويجب أن تستخدم في غضون أيام 1 أو 2 على بمعزل عن الحيوان. ويتميز تمايز SECS عن طريق التعبير عن Stabilin - 2 أو مستقبلات هير 9 ، CD31 ، ووجود نوفذة حشوية 1. ويمكن تمديد تمايز SECS بإضافة عامل نمو بطانة الاوعية في وسائل الإعلام أو الثقافة أو عن طريق زرع الخلايا الكبدية في المتوسط ​​10،11 مكيفة. في هذا التقرير ، سوف ندلل من النشاط التقامي SECS في الجهاز سليمة باستخدام الراديو المسمى الهيبارين لhyaluronan لمستقبلات Stabilin SEC - 2 - محددة. ثم سنقوم تنقية الكبد وSECS من الكبد perfused لقياس الإلتقام.

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

يجب إجراء دراسة خلايا الكبد البطانية الجيبية (ثانية) مع الخلايا الأولية التي تم الحصول عليها من الحيوان كما لا توجد خطوط الخلايا. هذا الأسلوب يعتمد على الهضم والكبد الطرد المركزي المغاير لتنقية ثانية لزراعة اللاحقة والتجريب.

في الإلتقام الكبد المجراة تلاه تنقية خلايا الكبد الكبد الإرواء

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Biology

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

18.9K Views.  University of Nebraska, Lincoln. يجب إجراء دراسة خلايا الكبد البطانية الجيبية (ثانية) مع الخلايا الأولية التي تم الحصول عليها من الحيوان كما لا توجد خطوط الخلايا. هذا الأسلوب يعتمد على الهضم والكبد الطرد المركزي المغاير لتنقية ثانية لزراعة اللاحقة والتجريب.

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

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

عزل CD133 خلايا + الجذعية الكبد للتوسع نسيلي

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. 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.

عزل الخلايا الليفية بوابة الجرذ من قبل<em&gt; في الموقع</em&gt; الكبد الإرواء

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

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

افعل ذلك بنفسك جهاز لاستخراج العينات Cryopreserved سقطت سهوا في صهاريج التخزين المبردة

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

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.

عزل خلايا الكبد البشرية من قبل خطوتين كولاجيناز الإرواء الداخلي

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

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

في فيفو تتبع 4 الأبعاد للالجذعية المكونة للدم والسلف الكبار في خلايا الفأر قبي نخاع العظم

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

طريقة موحدة لتحليل خلايا الكبد خاص بالمنحنى البطانية وFenestrations من خلال المجهر الإلكتروني

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

تحديد سمات تعبير الجينات الخاص بنوع الخلية في كبد الماوس

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

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.

عزل وتوصيف الخلايا البطانية الجيبية الأولية للكبد الفأر

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

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is often not detected until drugs have reached clinical stages, risking patients' safety and leading to substantial losses for the pharma industry. Taking into account that standard 2D models have limitations in detecting DILI it is essential to develop in vitro models that are more predictive to improve data translatability. To understand causality and mechanistic aspects of DILI in detail, a human liver MPS consisting of human primary liver parenchymal and non-parenchymal cells (NPCs) and cultured in 3D microtissues on an engineered scaffold under perfusion has been developed. Cryopreserved primary human hepatocytes (PHHs) and Kupffer cells (HKCs) were cocultured as microtissues in the MPS platform for up to two weeks, and each compound of interest was repeatably dosed onto liver microtissues at seven test concentrations for up to four days. Functional liver-specific endpoints were analyzed (including clinical biomarkers such as alanine aminotransferase, ALT) to evaluate liver function. Acute and chronic exposure to compounds of various DILI severities can be assessed by comparing responses to single and multi-dosed microtissues. The methodology has been validated with a broad set of severe and mildly hepatotoxic compounds. Here we show the data for pioglitazone and troglitazone, well-known hepatotoxic compounds withdrawn from the market for causing liver failures. Overall, it has been shown that the liver MPS model can be a useful tool to assess DILI and its association with changes in hepatic function. The model can additionally be used to assess how novel compounds behave in distinct patient subsets and how toxicity profiles may be affected by liver disease states (e.g., viral hepatitis, fatty liver disease).

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

Drug-induced liver injury (DILI) is a major cause of drug failure. A protocol has been developed to accurately predict the DILI liability of a compound using a liver microphysiological system (MPS). The liver model uses the coculture of primary hepatic cells and translationally relevant endpoints to assess cellular responses to treatment.

النظام الفيزيولوجي المجهري للكبد البشري لتقييم سمية الكبد التي يسببها الدواء في المختبر

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is ...

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective treatments. Considerable limiting factors hindering the timely and accurate diagnosis (including grading) and monitoring of NAFLD, as well as the development of potential therapies, are the current inadequacies in the characterization of the hepatic microenvironment structure and the scoring of the disease stage in a spatiotemporal and non-invasive manner. Using a diet-induced NAFLD mouse model, we investigated the use of in vivo micro-computed tomography (CT) imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation. This imaging methodology allows for longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with other ways of characterizing the disease progression (steatosis, inflammation, fibrosis) using the proposed method can pave the way toward the establishment of new, more efficient, and reproducible approaches for NAFLD research in mice. This protocol is also expected to upgrade the value of preclinical animal models for investigating&#160;the development of novel therapies against disease progression.

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation.