وقدم هذا العمل ممكن من الأنسجة والخلايا مؤسسة البحوث الإنسان، مما يجعل الأنسجة البشرية المتاحة للبحوث. وكان في استقبال الدعم المالي لهذا العمل من الوزارة الاتحادية للتعليم والبحوث (اسم المنحة: شبكة الكبد الظاهري، عدد المنح: 0315759) وHepacult GmbH المزيد. نتوجه بالشكر أيضا إلى المساعدين التقنيين من البنك الأنسجة مستشفى Großhadern لللجمع عينات الكبد والمساعدين التقنيين من مرفق الأساسية عزل خلية لتنفيذ نضح الكبد والكبدية العزلة. على وجه الخصوص، نود أن نشكر Natalja فندق Löwen لإثبات هذا الإجراء في شريط الفيديو. أخيرا، نود أن نشكر Natalja فندق Löwen وEdeltraud Hanesch لخلق الرسوم التوضيحية لالشكل 1 والأرقام في نظرة عامة التخطيطي للفيديو.

1. إعداد الإرواء وعزل حلول <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد الحلول اللازمة لنضح من قطعة الكبد وعزل خلايا الكبد وفقا للجدول 1. ويمكن تخزين الحلول في 4 درجات مئوية حتى الاستخدام. </li><li style=";text-align:right;direction:rtl"> تصفية العقيمة جميع الحلول باستخدام فلتر 0.22 ميكرون. </li><li style=";text-align:right;direction:rtl"> ينبغي لجميع الحلول التي تتلامس مع الكبد تكون معقمة. </li></ol> 2. إعداد معدات الإرواء وحلول <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يجب تعيين المعدات اللازمة لنضح من الكبد حتى قطعة كما هو مبين في الشكل 1. </li><li style=";text-align:right;direction:rtl"> يجب تعيين حمام الماء في درجة حرارة مناسبة، والتي تختلف في كل وجه الخصوص التجريبية مجموعة المتابعة، ان هذه الحلول هي في درجة حرارة 37 درجة مئوية عندما تصل قطعة الكبد. في هذه الحالة، يتم تعيين حمام الماء عند 41 درجة مئوية لتدفئة حلول 1 و 2 و 3 و المكثف الزجاج تغلف. يجب أن تكون درجة حرارة تصل إلى 4 الحل37 درجة مئوية في حمام مائي منفصلة للاستخدام للحد من فقدان النشاط كولاجيناز. </li><li style=";text-align:right;direction:rtl"> قبل فترة وجيزة من نضح الكبد، بدوره على منظم من خزان الغاز التي تحتوي على 95٪ O 2/5٪ CO 2 إلى الغاز جهاز الأوكسجين (الشكل 1E). </li></ol> 3. نضح من الكبد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قطعة الكبد مع قدر سليمة محفظة غليسون وقت ممكن وبشكل مثالي مع قطع فقط 1 السطح ينبغي الحصول عليها من الطبيب الشرعي للالارواء. </li><li style=";text-align:right;direction:rtl"> وضع هذه القطعة الكبد على قمع بوخنر الذي يحتوي على قرص مرشح مثقب (الشكل 1B). </li><li style=";text-align:right;direction:rtl"> يجب أن تستعد نظام نضح مع الحل 1. </li><li style=";text-align:right;direction:rtl"> مع معدل تدفق منخفض، يجب أن يتم إدراج منحني حقنة عادية الري مع نصائح الزيتون في الأوعية الدموية الكبيرة على سطح قطع من قطعة الكبد. كما تمسح الدم يخرج من الكبد، ويصبح أخف الأنسجة في المناطق ذات التروية الجيدة. عدد القنيات المستخدمة في مختلف الأحجاممن كبد هو مبين في الشكل 2A. حجم مقياس اختيار ينبغي أن يؤدي إلى نوبة دافئ من شأنها أن عقد حقنة عادية في المكان. ينبغي أن يترك الأوعية الدموية الصغيرة مفتوحة لنضح عازلة لاستنزاف من قطعة الكبد. </li><li style=";text-align:right;direction:rtl"> زيادة معدل التدفق على مضخة تحوي ما بين 110-460 مل / دقيقة اعتمادا على حجم الكبد (الشكل 2B). هذه النتائج في معدل تدفق 44 ± 16 مل / دقيقة لكل قنية (الشكل 2C). سرعة اختيار يعتمد على قطعة الكبد وينبغي أن يؤدي إلى النفش حتى طفيف من قطعة الكبد. في بعض الحالات، قد يكون من الضروري اتخاذ اجراءات اغلاق بعض السفن مفتوحة مع المشابك الأوعية الدموية الصغيرة لتحقيق النفش طفيف المذكورة أعلاه. ونضح جيدة يمكن ملاحظتها عندما قطعة الكبد هو لون أخف وزنا طوال الوقت. </li><li style=";text-align:right;direction:rtl"> الحفاظ على قطعة رطبة خلال الكبد نضح من خلال تغطية بقطعة من الشاش غارقة في المياه المالحة. </li><li style=";text-align:right;direction:rtl"> يروي مع 1 لتر من محلول 1 إلى طرد أي صemaining الدم في قطعة الكبد. </li><li style=";text-align:right;direction:rtl"> تغيير السائل نضح إلى الحل 2 ويروي لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> تبديل السائل نضح إلى الحل 3 ويروي مع 0.5 L. </li><li style=";text-align:right;direction:rtl"> تغيير السائل نضح إلى الحل 4، والذي يحتوي على 0.1-0.15٪ من كولاجيناز (الجدول 2). </li><li style=";text-align:right;direction:rtl"> لهذه الخطوة، ينبغي أن يتم نضح بطريقة إعادة تدوير ل9-12 دقيقة أو حتى يتم هضمها الكبد بما فيه الكفاية، ويجب أن تظهر أنسجة الكبد لتحطيم قليلا تحت الكبسولة في غليسون ويشعر خففت عند بحثها مع الجانب صريحا ل مشرط. </li></ol> 4. عزل خلايا الكبد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إيقاف المضخة تحوي وإزالة حقنة عادية من قطعة الكبد. </li><li style=";text-align:right;direction:rtl"> وضع قطعة الكبد في طبق بلورة تحتوي على 100-200 مل من محلول 5. </li><li style=";text-align:right;direction:rtl"> إزالة كبسولة من غليسون بعناية وبلطف نفض الخلايا. إذا كانت هناك مناطق لا و perfused جيدا، مشرط يمكن استخدامها لقطع طريقهذه المناطق للافراج عن الخلايا الموجودة داخل. إضافة المزيد من الحلول 5 حسب الحاجة أثناء العملية. </li><li style=";text-align:right;direction:rtl"> إضافة المزيد من الحلول 5 حتى يتم التوصل إلى الحجم النهائي من 500 مل. </li><li style=";text-align:right;direction:rtl"> تصفية تعليق الخلية مرتين، أولا من خلال شبكة 210 ميكرون النايلون تليها 70 ميكرومتر شبكة النايلون. المقبل، صب تعليق خلية في 200 مل أنابيب الطرد المركزي. </li><li style=";text-align:right;direction:rtl"> الطرد المركزي تعليق خلية في 72 جرام لمدة 5 دقائق في 4 درجات مئوية. طاف نضح وبيليه خلية في resuspend بلطف بلطف في 200 مل من محلول 5. </li><li style=";text-align:right;direction:rtl"> تكرار غسل الخطوة رقم 4.6 ثلاث مرات. على الخطوة النهائية أجهزة الطرد المركزي، وخلايا resuspend في حلول التخزين البارد (انظر قائمة المواد). يجب أن تكون الخلايا تقريبا 2-5000000 خلايا الكبد في الملليمتر الواحد لتقييم العائد والجدوى باستخدام التريبان الأزرق الاستبعاد مقايسة القائم على عدادة الكريات. </li><li style=";text-align:right;direction:rtl"> لإجراء التريبان الأزرق الاستبعاد مقايسة، إضافة 0.1 مل من الخلايا تضعف بشكل مناسب (2-5000000 ≈ / مل) إلى أنبوب microfuge تحتوي على 0.5 مل من soluti الأزرق التريبانعلى (0.4٪ التريبان الأزرق الذائبة في الفوسفات مخزنة المالحة (PBS)) و 0.4 مل من برنامج تلفزيوني. بعد خلط تعليق خلية بدقة، تحميل عدادة الكريات مع تعليق ودراسة تحت المجهر في التكبير 100X. تحت المجهر، وسوف تكون ملطخة الخلايا الميتة الأزرق في حين تظهر الخلايا الحية غير ملوثين. حساب عدد الخلايا الحية وإجمالي في كل من 1 مم 2 شبكات ملحوظ على عدادة الكريات. الجدوى (٪)، العائد من الخلايا الحية (خلايا الكبد مليون لكل مل حل التخزين البارد (CSS) أو خلايا الكبد مليون في الكبد ز) يمكن حسابها باستخدام الصيغ أدناه. </li></ol><img alt="المعادلة 1" fo:src="/files/ftp_upload/50615/50615eq1highres.jpg" src="/files/ftp_upload/50615/50615eq1.jpg" /> ملاحظة: في هذه الحالة، فإن عامل التخفيف التريبان الأزرق هو 10 وعامل عدادة الكريات هو 10،000.

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وقد تم تمويل الاستفادة المثلى من هذا البروتوكول جزئيا عن طريق منحة من Hepacult GmbH المزيد. الدكتور وولفغانغ Thasler هو واحد من مؤسسي Hepacult GmbH المزيد ويظل واحدا من أعضاء المجلس في هذه الشركة. توظيف Maresa Demmel هو جزئيا Hepacult. يعمل بواسطة ماريا Hauner Hepacult GmbH المزيد. Hepacult هو العرضية شركة التكنولوجيا الحيوية من جامعة، والتي تقدم خلايا الكبد البشرية بموافقة والوصول المفتوح لأغراض البحث.

النتائج هذا البروتوكول في عزلة خلايا الكبد البشرية مع قابلية عالية ونقاء. من أجل تحقيق هذه النتائج، فمن المهم أن تبدأ مع قطعة مناسبة من الكبد. قطعة من الكبد وينبغي أن يكون الكبسولة سليمة غليسون على جميع الأسطح باستثناء 1 قطع السطح. عامل مهم آخر هو دفعة معينة من كولاجين...

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

خلية التعليق الكبد يمكن إعدادها من الكبد عن طريق الطرق الميكانيكية أو الأنزيمية. وتشمل الطرق الميكانيكية المستخدمة في إعداد خلايا الكبد كله يجبر الكبد من خلال القماش القطني 2، والهز قطعة الكبد مع الخرز الزجاجي في شاكر خان 3، وذلك باستخدام المجانسة الزجاج مع المطاحن فضفاضة 4،5 الخ على مر السنين، انخفضت الطرق الميكانيكية من صالح بسبب الأضرار التي لحقت أغشية الخلايا وفقدان وظيفة خلايا الكبد من 6،7 معزولة. وبالتالي فإن استخدام طريقة الأنزيمية هو حاليا الوسيلة الرئيسية لعزل خلايا الكبد. عزل خلايا الكبد باستخدام أسلوب الأنزيمية وقد تحسنت كثيرا عندما بيري وصديق 8 perfused كولاجيناز وهيالورونيداز من خلال الكبد عبر الوريد البابي في الفئران. تستخدم هذه العملية نضح الأوعية الدموية للسماح للالانزيمات لتتلامس الوثيق مع الغالبية العظمى من الخلايا، مما يؤدي إلىزيادة 6 أضعاف في العائد من خلايا الكبد 8. علاوة على ذلك، حققت هذه الطريقة الخلايا التي احتفظت سلامتها الهيكلية، مع عمليا أي التحول من الشبكة الإندوبلازمية في حويصلات معزولة وعدم وجود ضرر الميتوكوندريا 8. تم تعديل هذه الطريقة من قبل Seglen 1، الذي كان رائدا إجراء نضح من خطوتين لعزل خلايا الكبد. في هذا الإجراء، يتم perfused في كبد الفئران مع كا 2 + عازلة خالية يليه نضح مع العازلة كولاجيناز التي تحتوي على الكالسيوم 2 + 1. إزالة كا 2 + في الخطوة الأولى يساعد على تعطيل desmosomes، في حين أن المطلوب إضافة كا 2 + في الخطوة الثانية للنشاط كولاجيناز الأمثل 1،9. بالنظر إلى أن الأعمال المنشورة المذكورة أعلاه قد تم تنفيذ في الفئران، وتهدف هذه المقالة لشرح إجراء تعديل والتي يمكن استخدامها لعزل خلايا الكبد مع سلامة عالية من همهمةوالأكباد. استخدام خلايا الكبد البشرية يظل من المهم للبحوث ومتعدية للتحقق من صحة التجارب باستخدام نماذج حيوانية. تم الحصول على قطعة كبد الإنسان المستخدمة في هذه الدراسة مع الموافقة على الحكم من خلال مؤسسة أبحاث الأنسجة البشرية والمحمولة، والتي تسيطر عليها الدولة غير ربحية الأساس 10. بعد إزالة الطبيب الشرعي ما هو مطلوب للتشخيص، تم جمع قطع الكبد من الأنسجة المتبقية. كان الأنسجة مقطوع من قبل الطبيب الشرعي شكليا الأنسجة السليمة التي تم الحصول عليها من هوامش استئصال بعد استئصال الكبد.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Bubble trap</td>
			<td>Ga&szlig;ner Glastechnik</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass jacketed condenser</td>
			<td>Ga&szlig;ner Glastechnik</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>41 &deg;C Water bath</td>
			<td>Julabo</td>
			<td>35723-H24/EG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37 &deg;C Water bath</td>
			<td>GFL</td>
			<td>1083</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compressed gas cylinder (95 % O2/5 % CO2)</td>
			<td>Linde</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gas permeable tubing</td>
			<td>Neolab</td>
			<td>2-4440</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Ismatec</td>
			<td>IP65</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather</td>
			<td>320010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Omnilab</td>
			<td>5171014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Conical flasks 1 L</td>
			<td>Schott Duran</td>
			<td>2121654</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Conical flasks 5 L</td>
			<td>Schott Duran</td>
			<td>2121673</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Beakers</td>
			<td>Schott Duran</td>
			<td>2110654</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>200 ml centrifuge tubes</td>
			<td>Becton Dickinson</td>
			<td>352075</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Crystallizing dish</td>
			<td>Omnilab</td>
			<td>5144063</td>
			<td>&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>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro vascular clamps</td>
			<td>Ernst Kratz GmbH</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B&uuml;chner funnel</td>
			<td>Carl Roth</td>
			<td>HT38.1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nylon mesh 210 &mu;m</td>
			<td>Neolab</td>
			<td>4-1413</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nylon mesh 70 &mu;m</td>
			<td>Neolab</td>
			<td>4-1419</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.22 &mu;m sterile filters</td>
			<td>Peske</td>
			<td>99505</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>500 ml bottles</td>
			<td>Schott Duran</td>
			<td>2180144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 L bottles</td>
			<td>Schott Duran</td>
			<td>2180154</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Peske</td>
			<td>06-0001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.5 ml tubes</td>
			<td>Eppendorf</td>
			<td>0030 120,086</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>BD Biosciences</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>Neolab</td>
			<td>1508454</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile Pasteur pipettes</td>
			<td>Brand</td>
			<td>747715</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Motorised pipette filler (Pipette boy acu)</td>
			<td>Integra</td>
			<td>155017</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Refridgerated centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminar flow</td>
			<td>Kendro</td>
			<td>Hera safe-KS9</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aspirator (Low-flow surgical suction pump)</td>
			<td>Atmos</td>
			<td>C361</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laboratory Gas Burner</td>
			<td>Integra</td>
			<td>Fire Boy eco</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Disposable laboratory coat</td>
			<td>Paperlynen GmbH</td>
			<td>MD0202414</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical mask with visor</td>
			<td>Kimberly-Clark</td>
			<td>48247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical hood</td>
			<td>Barrier</td>
			<td>42072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Latex gloves</td>
			<td>Semper Care</td>
			<td>CE0321</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase (Batch number NB 4G)</td>
			<td>Serva</td>
			<td>17465</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Merck</td>
			<td>2382</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Roth</td>
			<td>9265.2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Roth</td>
			<td>9105.3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Serva</td>
			<td>26868</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Biomol</td>
			<td>01400-2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Serva</td>
			<td>22700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>Serva</td>
			<td>30180</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.4% Trypan blue solution</td>
			<td>Lonza</td>
			<td>17-942E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cold storage solution</td>
			<td>Hepacult GmbH</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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

الكبد، والجهاز مع قدرة استثنائية التجديد، ويحمل مجموعة واسعة من الوظائف، مثل إزالة السموم، والتمثيل الغذائي والتوازن. على هذا النحو، هي خلايا الكبد نموذجا هاما لمجموعة كبيرة ومتنوعة من الأسئلة البحثية. على وجه الخصوص، واستخدام خلايا الكبد البشرية أهمية خاصة في مجالات الدوائية، وعلم السموم، وتجديد الكبد والبحوث متعدية. وبالتالي، فإن هذا الأسلوب نسخة معدلة من على بعد خطوتين الإجراء كولاجيناز نضح لعزل خلايا الكبد كما وصفها Seglen 1. سابقا، وقد تم عزل خلايا الكبد عن طريق الطرق الميكانيكية. ومع ذلك، فقد ثبت أساليب الأنزيمية لتكون متفوقة كما تحتفظ خلايا الكبد سلامتها الهيكلية وظيفة بعد العزلة. هذه الطريقة المعروضة هنا تتكيف مع أسلوب تصميم من قبل لكبد الفئران إلى الإنسان قطعة الكبد ويؤدي إلى تحقيق عائد كبير من خلايا الكبد مع بقاء 77 ± 10٪. الرئيسيةالفرق في هذا الإجراء هو عملية إقناء؛ إدخال القنية في الأوعية الدموية. علاوة على ذلك، الطريقة الموصوفة هنا يمكن أيضا أن تطبق على كبد من الأنواع الأخرى مع الكبد أو الأوعية الدموية أحجام قابلة للمقارنة.

الإعداد التروية يجب تعيين المعدات اللازمة لنضح الكبد حتى وفقا لالشكل 1. الجدوى والعائد من خلايا الكبد البشرية المعزولة كان متوسط ​​بقاء خلايا الك...

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

يوصف A-خطوتين الإجراء كولاجيناز نضح تعديل لعزل خلايا الكبد البشرية. ويمكن أيضا أن هذه الطريقة يمكن تطبيقها على كبد الثدييات الأخرى. هي خلايا الكبد معزولة المتاحة في ارتفاع العائد والجدوى، مما يجعلها نموذجا مناسبة للبحث العلمي في مجالات مثل تجديد الكبد، الدوائية والسمية.

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. ...

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

Research

JoVE Journal

Biology

30.8K Views.  Grosshadern Hospital, Munich.  يوصف A-خطوتين الإجراء كولاجيناز نضح تعديل لعزل خلايا الكبد البشرية. ويمكن أيضا أن هذه الطريقة يمكن تطبيقها على كبد الثدييات الأخرى. هي خلايا الكبد معزولة المتاحة في ارتفاع العائد والجدوى، مما يجعلها نموذجا مناسبة للبحث العلمي في مجالات مث?...

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

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

اشتقاق الخلايا الجذعية الجنينية البشرية التي Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

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 ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

الماوس جزيرة من العزلة انجرهانز باستخدام مزيج من كولاجيناز تنقية والبروتياز متعادلة

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

GST-صاحب تنقية: A خطوتين تقارب بروتوكول تنقية استسلام كامل طول البروتينات تنقية

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

فحص<em&gt; ذبابة الفاكهة</em&gt; اليرقات الخلايا الطرفية الرغامي بواسطة المجهر الضوئي

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

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.

بروتوكول لعزل خلايا الكبد الإنسان الابتدائية والموافق السكان الرئيسية للخلايا الكبد غير متني

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

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

تحليل الكائنات المجهرية باستخدام ثنائي الخطوة PCR والجيل التالي من 16S rRNA التسلسل الجيني

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

استراتيجية من خطوتين تجمع بين التعديل اللاجيني والإشارات الميكانيكية الحيوية لتوليد خلايا الثدييات متعددة القدرات

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

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.

عزل خلايا كبد الفئران الأولية مع التحكم في التروية متعدد المعلمات

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

تحسين كمية البروتين باستخدام عزل الميتوكوندريا بخطوتين

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

تسليط الضوء على المؤلف: عزل الميتوكوندريا بخطوتين بدون علامات لتحسين اكتشاف البروتين وقياسه  

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.