Name: preparation and in vitro characterization of magnetized mir-modified endothelial cells
Uploaded: 2017-05-02T14:00:00.000+00:00
Duration: 9 min 58 s
Description: Watch this Scientific Journal Video about Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells at JoVE.com

ברצוננו להודות ג פולדה (מרכז מיקרוסקופי אלקטרונים, רוסטוק אונ&#39;, גרמניה) עבור התמיכה הטכנית ברכישת תמונות TEM של חלקיקים פאראמגנטי מסוננים בביצוע ניתוח רנטגן שלהם. העבודה מתבצעת על רוסטוק RTC נתמכה על ידי המשרד הפדרלי לחינוך ולמחקר גרמניה (FKZ 0312138A, FKZ 316,159 ו VIP + 03VP00241) ואת פומרניה מדינת מקלנבורג-מערב עם הקרנות המבניות של האיחוד האירופי (ESF / IV-WM-B34- 0030/10 ו ESF / IV-BM-B35-0010 / 12) ועל ידי DFG (DA 1296-1), ורטיבות-קרן, וקרן הלב גרמנית (F / 01/12). פרנק Wiekhorst נתמכה על ידי תוכנית המחקר FP7 האיחוד האירופי &quot;Nanomag&quot; FP7-NMP-2013-גדול 7.

אין לנו שום אינטרסים כלכליים מתחרים לחשוף.

את ייצור תאי מהונדסים גנטית עמוסה חלקיקים פאראמגנטי לקבלת הנחיות נוספות מגנטית נשלטת שלהם מוצג בפרוטוקול הנוכחי. היישום המוצלח של אסטרטגיה זו מאפשר לפתרון קשיים מסוימים של טיפול בתא, כגון החזקה נמוכה engraftment העני באזור נפצע 2, 3, <s...

ג&#39;ין וטיפול תא הם כלים רבים עצמה יש פוטנציאל לפתור אתגרים נוכחיים בטיפול CVD. למרות העובדה כי הן הגישות הללו נבדקות כעת בניסויים קליניים, הם עדיין אינם מוכנים ליישום קליני רחב 1. יש לציין, גישה משותפת להתמודדות עם האתגרים של גן לבין טיפול בתא היא לפתח וקטורים למסירת גן רבים תכליתיים מתאימים ליישום קליני. היעדר מערכות למסירת הגן בטוחות ויעילות הוא הדאגה העיקרית של ריפוי גנטי. במקביל, ההנדסה הגנטית של מוצרי הסלולר לפני ההשתלה יכולה להתגבר על האתגרים הרציניים של טיפול בתא, כגון יעילות נמוכה (למשל, בתחום הלב, רק ~ 5% של שיפור תפקודי מושגת פוסט-תאי גזע להשתלת 1 ) ושימור / engraftment עני באתר של פציעה (כלומר, החזקת תא יורד מתחת 5 - 10% בתוך דקות עד שעות POst-יישום, ללא קשר לתוואי ממשל 2, 3, 4). נכון להיום, וקטורים ויראליים מאוד יעלו מערכות שאינן ויראליות במונחים של יעילות, אשר הביאה היישום הרחב יותר שלהם בניסויים קליניים (~ 67%) 5. עם זאת, כלי רכב ויראלי לשאת בסיכונים חמורים, כגון חיסוני (ואת התגובה הדלקתית שלאחר מכן, עם סיבוכים חמורים), oncogenicity, ומגבלות בגודל של החומר הגנטי הנישא 6. בשל חששות בטיחות אלה לבין העלויות הגבוהות של ייצור וקטור ויראלי, השימוש במערכות הלא-ויראלית עדיף במקרים מסוימים 7, 8. היא מתאימה במיוחד עבור הפרעות הדורשות תיקון גנטי חולף, כגון הביטוי של גורמי גדילה השליטה אנגיוגנזה (למשל, לטיפול CVD) או deliveר&quot;י של חיסונים. בקבוצה שלנו, מערכת מסירה תוכננה על ידי שילוב מסועף 25-kDa polyethyleneimine (תש&quot;ן) ואת חלקיקי תחמוצת ברזל פאראמגנטי (MNP) קשורים יחדיו על ידי האינטראקציה ביוטין- streptavidin 9. וקטור זה הוא כלי פוטנציאלי עבור הנדסה גנטית של תאי, המאפשר המגנטיזציה שלהם סימולטני לפני ההשתלה. זה האחרון מספק בסיס הדרכה / שימור מגנטי, אשר מבטיחה במיוחד בימינו, כמו טכניקות מיקוד מגנטיות מתקדמות מפותח בהצלחה 10. יתר על כן, התא מגנטי תגובה וכתוצאה מכך יש את הפוטנציאל להיות פולשני במעקב דימות בתהודה מגנטית (MRI) או הדמית חלקיקים מגנטית 11, 12. במקרה של וקטור PEI / MNP, polyamine מבטיח התעבות חומצות גרעין ובכך הגנה מפני גורם משפיל<html> ים, הפנמת וקטור בתאים, ו endosomal להימלט 5. MNPs משלימים את המאפיינים של PEI, לא רק במונחים של הדרכה מגנטי, אלא גם על ידי הפחתת הרעילות 7 הידוע PEI, 13, 14. בעבר, תש&quot;ן / נכסים וקטור MNP הותאמו מבחינת יעילות משלוח (כלומר, pDNA ו מירנה) ובטיחות באמצעות פיברובלסטים ותאי גזע mesenchymal אנושי 15, 16. בכתב היד הזה, פרוטוקול מפורט על היישום של PEI / MNPs עבור הדור של תאי-modified מירנה מתואר 17. לשם כך, HUVECs משמש ומהווה מודל הוקם עבור אנגיוגנזה במבחנה. הם מאתגר transfect והם רגישים השפעה רעילה 18, 19,התחת = &quot;Xref&quot;&gt; 20. בנוסף, אנו מספקים אלגוריתם להעריך תאים כאלה במבחנה, כולל מיקוד שלהם, תקשורת בין-תאית, וזיהוי MRI.

<table><tbody><tr><td>PEI 25 kDa</td><td>Sigma Aldrich</td><td>408727</td><td></td></tr><tr><td>EZ-Link Sulfo-NHS-LC-Biotin</td><td>Thermo Scientific</td><td>21335</td><td></td></tr><tr><td>PD-10 Desalting Columns</td><td>GE Healthcare</td><td>17085101</td><td>Containing Sephadex G-25 Medium</td></tr><tr><td>Ninhydrin Reagent solution 2%</td><td>Sigma Aldrich</td><td>7285</td><td></td></tr><tr><td>Glycine</td><td>Sigma Aldrich</td><td>410225</td><td></td></tr><tr><td>Pierce Biotin Quantitation Kit</td><td>Thermo Scientific</td><td>28005</td><td></td></tr><tr><td>Microplate reader Model 680</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Streptavidin MagneSphere Paramagnetic Particles</td><td>Promega</td><td>Z5481</td><td></td></tr><tr><td>Millex-HV PVDF Filter</td><td>Merck</td><td>SLHV013SL</td><td>0.45 &amp;micro;m</td></tr><tr><td>Libra 120 transmission electron microscope</td><td>Zeiss</td><td></td><td>Acceleration Voltage 120&amp;nbsp;kV</td></tr><tr><td>Sapphire X-ray detector</td><td>EDAX-Amatek</td><td></td><td></td></tr><tr><td>Cell culture plastic</td><td>TPP</td><td></td><td></td></tr><tr><td>NHS-Esther Atto 565</td><td>ATTO-TEC GmbH</td><td>AD 565-31</td><td></td></tr><tr><td>NHS-Esther Atto 488&amp;nbsp;</td><td>ATTO-TEC GmbH</td><td>AD 488-31</td><td></td></tr><tr><td>Cy5 miRNA Label IT kit</td><td>Mirus Bio</td><td>MIR 9650</td><td></td></tr><tr><td>Biotin Atto 565</td><td>ATTO-TEC GmbH</td><td>AD 565-71</td><td></td></tr><tr><td>Collagense Type IV Gibco</td><td>Thermo Scientific</td><td>17104019</td><td></td></tr><tr><td>Endothelial growth medium, EGM-2</td><td>Lonza</td><td>CC-3156 &amp;amp; CC-4176</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Scientific</td><td>15140122</td><td>100 U/mL, 100 &amp;micro;g/mL</td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>356234</td><td></td></tr><tr><td>anti-PECAM-1 antibody</td><td>Santa Cruz</td><td>sc-1506</td><td></td></tr><tr><td>MS MACS columns</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-042-201</td><td></td></tr><tr><td>Near-IR Live/Dead Cell Stain Kit</td><td>Thermo Scientific</td><td>L10119</td><td></td></tr><tr><td>Cy3 Dye-Labeled Pre-miR Negative Control</td><td>Thermo Scientific</td><td>AM17120</td><td>&quot;Cy3-miR&quot; or &quot;Cyanine-miR3&quot; in the manuscript</td></tr><tr><td>Pre-miR miRNA Precursor Molecules - Negative Control&amp;nbsp;</td><td>Thermo Scientific</td><td>AM17110</td><td>&quot;scr-miR&quot; in the manuscript</td></tr><tr><td>Anti-hsa-miR92a-3p synthetic Inhibitor&amp;nbsp;</td><td>Thermo Scientific</td><td>AM10916</td><td></td></tr><tr><td>LSM 780 ELYRA PS.1 system</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>158127</td><td>4% solution in PBS</td></tr><tr><td>DAPI nuclear stain</td><td>Thermo Scientific</td><td>D1306</td><td></td></tr><tr><td>NucleoSpin RNA isolation Kit</td><td>Machery-Nagel</td><td>740955</td><td></td></tr><tr><td>mirVana miRNA Isolation Kit</td><td>Thermo Scientific</td><td>AM1560</td><td></td></tr><tr><td>TaqMan MicroRNA Reverse Transcription Kit</td><td>Thermo Scientific</td><td>4366596</td><td></td></tr><tr><td>StepOnePlus Real-Time PCR System</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Thermo Scientific</td><td>4368814</td><td></td></tr><tr><td>hsa-miR-92a TaqMan assay</td><td>Thermo Scientific</td><td>000431</td><td>Mature miRNA Sequence: UAUUGCACUUGUCCCGGCCUGU</td></tr><tr><td>FastGene Taq Ready Mix</td><td>Nippon Genetics</td><td>LS27</td><td></td></tr><tr><td>ITGA5 TaqMan assay</td><td>Thermo Scientific</td><td>Hs01547673_m1</td><td></td></tr><tr><td>RNU6B TaqMan assay</td><td>Thermo Scientific</td><td>001093</td><td></td></tr><tr><td>18S rRNA Endogenous Control</td><td>Thermo Scientific</td><td>4333760F</td><td></td></tr><tr><td>Gelatin</td><td>Sigma Aldrich</td><td>G7041</td><td></td></tr><tr><td>CellTrace Calcein Red-Orange</td><td>Thermo Scientific</td><td>C34851</td><td></td></tr><tr><td>PBS</td><td>Pan Biotech</td><td>P04-53500</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>MACS buffer</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-091-221</td><td></td></tr><tr><td>Agarose</td><td>Sigma Aldrich</td><td>A9539</td><td></td></tr><tr><td>7.1 Tesla animal MRI system</td><td>Bruker Corporation</td><td>A7906</td><td></td></tr><tr><td>ImageJ software</td><td>National Institutes of Health</td><td></td><td>upgraded with an AngiogenesisAnalyzer (NIH)</td></tr><tr><td>MPS device</td><td>Bruker Biospin</td><td></td><td></td></tr><tr><td>Matlab software</td><td>Mathworks</td><td></td><td></td></tr><tr><td>Ring Neodym Magnet&amp;nbsp;</td><td>magnets4you GmbH</td><td>RM-10x04x05-G</td><td>&amp;oslash; 10 mm; remanescence is ~ 1.3&amp;nbsp;T, coercivity &amp;ge; 955 kA/m</td></tr><tr><td>Click-iT EdU Alexa Fluor 647 Imaging Kit</td><td>Thermo Scientific</td><td>C10340</td><td></td></tr><tr><td>FluorSave Reagent</td><td>Merck</td><td>345789</td><td></td></tr><tr><td>Ultrasonic bath</td><td>Bandelin electronic</td><td>Type: RK 100 SH</td><td></td></tr></tbody></table>

preparation and in vitro characterization of magnetized mir-modified endothelial cells

נכון להיום, הטיפולים כירורגיים תרופתיים הזמינים עבור מחלות לב וכלי דם (CVD) מוגבלים ולעתים קרובות פליאטיבי. במקביל, טיפולי גני תא הם מאוד גישות חלופיות מבטיחות לטיפול CVD. עם זאת, היישום הקליני הרחב של ריפוי גנטי מוגבל מאוד על ידי חוסר מערכות למסירת הגן מתאימות. ההתפתחות של וקטורים למסירת הגן מתאימים יכולה לספק פתרון לאתגרים נוכחיים טיפול בתא. בפרט, חסרונות קיימים, כגון יעילות מוגבלת ושימור תאי נמוך ב האיבר נפצע, ניתן להתגבר על ידי הנדסת תא מתאימה (כלומר, גנטי) לפני ההשתלה. הפרוטוקול המובא מתאר את השינוי החולף היעיל ובטוח של תאי האנדותל באמצעות ננו-חלקיקים מגנטיים פאראמגנטי polyethyleneimine (PEI / MNP) וקטור משלוח מבוסס. כמו כן, האלגוריתם ושיטות לאפיון תאים מוגדרים. Intracellu המוצלחמסירה של microRNA (miR) לתוך האדם ורידים טבורי תאים אנדותל (HUVECs) הושגה מבלי להשפיע על כדאיות התא, פונקציונליות, או תקשורת בין תאיים. יתר על כן, גישה זו הוכחה לגרום השפעה תפקודית חזקה מיר exogenous הציג. חשוב לציין, היישום של וקטור זה מבוסס MNP מבטיח מגנטיזציה התא, עם אפשרויות נלוות של מיקוד מגנטי לא מעקב פולשני MRI. זה עשוי לספק בסיס מגנטי מונחה, מהונדס גנטית תרפויטים תא שניתן לפקח ללא פולשנית עם MRI.

המטרה העיקרית של הפרוטוקול המוצע היא לייצר מגנטי תגובת תאי miR-modified וכדי לנהל האפיון המדויק שלהם (איור 1). כתוצאה מכך, תאי transfected ביעילות, מגיבים בחירה מגנטית והדרכה לזיהוי באמצעות MRI, יש לקבל. <p class="jove_content" fo:keep-together.within-page="1" style=";text-align:right;directi...

Watch this Scientific Journal Video about Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells at JoVE.com

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

כתב היד הזה מתאר את משלוח יעיל, שאינו ויראלי של miR לתאי אנדותל ידי וקטור PEI / MNP ו המגנטיזציה שלהם. לכן, בנוסף שינוי גנטי, גישה זו מאפשרת להדרכת תא מגנטית ויכולת גילוי MRI. הטכניקה יכולה לשמש כדי לשפר את המאפיינים של מוצרי תא טיפוליים.

הכנה<em&gt; במבחנה</em&gt; אפיון ממוגנטים miR-modified לתאי אנדותל

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

preparation-vitro-characterization-magnetized-mir-modified

Research

JoVE Journal

Medicine

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

7.6K Views.  University of Rostock.  כתב היד הזה מתאר את משלוח יעיל, שאינו ויראלי של miR לתאי אנדותל ידי וקטור PEI / MNP ו המגנטיזציה שלהם. לכן, בנוסף שינוי גנטי, גישה זו מאפשרת להדרכת תא מגנטית ויכולת גילוי MRI. הטכניקה יכולה לשמש כדי לשפר את המאפיינים של מוצרי תא טיפוליים. 

Video: Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

דור של קו הונצח Murine מוח כלי דם האנדותל סלולרי כ<em&gt; במבחנה</em&gt; דגם מחסום דם מוח

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Neuroscience

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention.

Bare Metal פרומגנטי סטנט ללכיד תא האנדותל ושייר

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Bioengineering

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

סינתזה של Cationized Magnetoferritin עבור המגנטיזציה אולטרה-מהירה של תאים

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

A Patient-Derived Xenograft Model for Venous Malformation

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7&#8210;9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

We present a detailed protocol to generate a murine xenograft model of venous malformation. This model is based on the subcutaneous injection of patient-derived endothelial cells containing hyper-activating TIE2 and/or PIK3CA gene mutations. Xenograft lesions closely recapitulate the histopathological features of VM patient tissue.

מודל קסנוגרפט נגזר ממטופל למום ורדי

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional ...

Developmental Biology

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

בידוד של תאים הראשוניים Murine מוח כלי הדם האנדותל

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

<em&gt; במבחנה</em&gt; דגם של מחסום דם-מוח באמצעות ספקטרוסקופיית עכבה: התמקדות T Cell-אנדותל אינטראקציה תא

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Immunology and Infection

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

פרוטוקול להעברת MicroRNA לתוך למבוגרים הנגזרות מח עצם Hematopoietic תאי גזע כדי לאפשר הנדסה תא בשילוב עם מיקוד מגנטי

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

בידוד של תאי אנדותל Microvascular ראשי שריר השלד מאתר

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/&#946;-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion.

Here, we describe a protocol, the extended endothelial cell culture method (EECM), that allows differentiation of pluripotent stem cells to brain microvascular endothelial cell (BMEC)-like cells. These cells show endothelial cell adhesion molecule expression and are thus a human blood-brain barrier model suitable to study immune cell interactions in vitro.

התמיינות של תאי גזע פלוריפוטנטיים המושרים על ידי בני אדם לתאים דמויי תאי אנדותל מיקרו-וסקולריים במוח עם פנוטיפ חיסוני בוגר

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous ...

ייצור מגנטי אופטימלי בעל תפוקה גבוהה של ננו-שלפוחיות שמקורן באנדוזום

A Magnetic Separation-Assisted High-Speed Homogenization Method for Large-Scale Production of Endosome-Derived Vesicles

Extracellular vesicles (EVs) have attracted significant attention in physiological and pathological research, disease diagnosis, and treatment; however, their clinical translation has been limited by the lack of scale-up manufacturing approaches. Therefore, this protocol provides a magnetic separation-assisted high-speed homogenization method for the large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) derived from the endosomes, which have about 100-time higher yield than conventional ultracentrifugation method. In this method, magnetic nanoparticles (MNPs) were internalized by parental cells via endocytosis and were subsequently accumulated within their endosomes. Then, MNPs-loaded endosomes were collected and purified by hypotonic treatment and magnetic separation. A high-speed homogenizer was utilized to break MNP-loaded endosomes into monodisperse nanovesicles. The resulting endosome-derived vesicles feature the same biological origin and structure, characterized by nanoparticle tracking analysis, transmission electron microscope, and western blotting. Their morphology and protein composition are similar to native EVs, indicating that EMs may potentially serve as a low-cost and high-yield surrogate of native EVs for clinical translations.

Author Spotlight: פיתוח שיטת ייצור בקנה מידה גדול הניתנת לשחזור עבור מימטיקה אקסוזום באמצעות ננו-חלקיקים מגנטיים 

Here, we describe a magnetic separation-assisted high-speed homogenization method for large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) that share the same biological origin and similar structure, morphology, and protein composition of native extracellular vesicles (EVs).

שיטת הומוגניזציה במהירות גבוהה בסיוע הפרדה מגנטית לייצור בקנה מידה גדול של שלפוחיות שמקורן באנדוזום

Extracellular vesicles (EVs) have attracted significant attention in physiological and pathological research, disease diagnosis, and treatment; however, ...

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דור של קו הונצח Murine מוח כלי דם האנדותל סלולרי כ