preparing vascular cells for in vivo matrix gel plug assay

Modifiye Matris Jel Tıkaç Testi ile Anjiyogenez Çalışmalarının Basitleştirilmesi

Angiogenesis, the process of formation of neo-vessels from pre-existing vessels, plays a critical role in many physiological and pathological processes, such as embryonic development, wound healing, atherosclerosis, tumor development, etc.1,2,3,4,5. This dynamic process involves several steps, including the degradation of the matrix, vascular cell proliferation, migration and self-organization to form tubular structures and the stabilization of the neo-vessels6. Promoting angiogenesis has been demonstrated to be critical in the treatment of myocardial infarction, stroke and other kinds of ischemic diseases7 while inhibiting angiogenesis has been considered a promising strategy in the treatment of cancers8 and rheumatoid diseases9. Angiogenesis has been considered an organizing principle for drug discovery10. Thus, the construction of a reliable and convenient method to assess the extent of angiogenesis is critical for mechanical research or drug discovery in angiogenesis-dependent diseases.
Several in vitro and in vivo models have been developed to evaluate angiogenesis11. Among these, two-dimensional (2-D) models, like matrix gel tube formation assay12, cannot form functional tubular structures. The animal models, such as the hind limb ischemia model13,14, can reproduce the angiogenesis process but are complex and require a laser speckle blood flow imaging system. 3D models of vascular morphogenesis, like matrix gel plug assay, provide a simple platform that can mimic the process of angiogenesis in vivo15, but the detection of angiogenesis requires immunohistochemistry or immunofluorescence staining16,17,18, which are variable and poorly visualized.
Here, we describe a protocol for a modified matrix gel plug assay where vascular cells were mixed with matrix gel and subcutaneously injected into the back of mice to form a plug. In the plug, vascular cells need to degrade the matrix, proliferate, migrate, and self-organize to finally form functional vessels with blood flow in the internal environment. Thereafter, fluorescent-labeled dextran is injected via the tail vein, to flow through the plug, and the label is visualized to indicate neo-vessels. The content of angiogenesis can be quantitatively evaluated by the length of the vessels. This method can form functional vessels that cannot be produced in 2-D angiogenesis models12, and does not need complex stain process as in ordinary matrix gel plug assay11. It also does not require expensive specific instruments like laser speckle blood flow imaging system in hind limb ischemia model13,14,19. This method is versatile, low-cost, quantifiable, and easy to perform, and can be used to determine the pro- or anti-angiogenic capability of drugs or be used in mechanical research involved in angiogenesis.

Yazar Spot Işığı: Modifiye Matris Jel Tıkaç Testi ile Anjiyogenez ve Damar Geçirgenliğinin Araştırılması

Anjiyogenez Çalışmaları için Modifiye In Vivo Matrix Jel Plug Testi

Our research deals with angiogenesis, the process of formation of new vessels from preexisting vessels. It plays a critical role in many physiological and pathological processes, such as embryonic development or curing atherosclerosis, tumor development, etc. The construction of a reliable model to evaluate the extent of angiogenesis is critical for myocardial research or the discovery of medicines for angiogenesis-depend diseases.However, most of the existing models are complex, expensive, and require specialized equipment. Here, we present a modified matrix gel plug assay that can be used to investigate angiogenesis and vessel permeability without staining in a simple, more intuitive, as well as efficient manner.

In Vivo Matrix Jel Plug Testi için Vasküler Hücrelerin Hazırlanması

460 mililitre endotel hücre ortamı kullanarak ve içine 50 mililitre FBS, beş mililitre penisilin streptomisin ve beş mililitre endotel hücre büyüme takviyesi ekleyerek tam endotel kültür ortamını hazırlayarak başlayın. Hazırlanan ortam bir ay boyunca dört santigrat derecede saklanabilir. Daha sonra, hazırlanan tam endotel kültür ortamının sekiz mililitresinde 0.1 milyon vasküler hücre içeren 100 milimetrelik bir doku kültürü kabında ve hücreleri 37 santigrat derecede ve% 5 karbondioksitte% 70 akıcılık elde edilene kadar kültürleyin.Ardından, kültür ortamını tabaktan çıkarın ve bağlanmamış hücreleri ve kalıntıları ortadan kaldırmak için hücreleri PBS ile iki kez durulayın. PBS'yi çıkardıktan sonra, hücrelere 2.21 milimolar EDTA içeren üç mililitre% 0.25 tripsin ekleyin ve tabağı bir dakika boyunca 37 santigrat derecede inkübe edin. Hücre ayrılmasını ışık mikroskobu altında 40 kat büyütmede doğrulayın.Tripsini yedi mililitre tam endotel kültürü ortamı ile nötralize edin ve hücreleri kültür kabından nazikçe durulayın. Hücre süspansiyonunu 15 mililitrelik bir tüpte 400G'de 10 dakika boyunca topladıktan sonra santrifüjleyin. Süpernatanı çıkardıktan sonra hücreleri beş mililitre tam endotel kültür ortamında yeniden süspanse edin, daha sonra bir hemo-sitometre kullanarak hücreleri sayın ve 2 milyon hücre içeren hesaplanmış bir süspansiyon hacmini steril 1.5 mililitrelik bir tüpe aktarın.Süpernatanı çıkarmadan önce süspansiyonu 400G'de beş dakika santrifüjleyerek hücreleri peletleyin. Vasküler hücreler artık matris jel tıkacı testi için matris jel ile karıştırılmaya hazırdır.

preparing-vascular-cells-for-in-vivo-matrix-gel-plug-assay

Research

JoVE Journal

Biology

Preparing Vascular Cells for In Vivo Matrix Gel Plug Assay

44 Görüntüleme.  Pingyang Affiliated Hospital of Wenzhou Medical University. 460 mililitre endotel hücre ortamı kullanarak ve içine 50 mililitre FBS, beş mililitre penisilin streptomisin ve beş mililitre endotel hücre büyüme takviyesi ekleyerek tam endotel kültür ortamını hazırlayarak başlayın. Hazırlanan ortam bir ay boyunca dört santigrat derecede saklanabilir. Daha sonra, hazırlanan tam endotel kültür ortamının sekiz mililitresinde 0.1 milyon vasküler hücre içeren 100 milimetrelik bir doku kültürü kabında ve hücreleri 37 santigrat dere...

Video: In Vivo Matrix Jel Plug Testi için Vasküler Hücrelerin Hazırlanması

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...

Biyoloji

Begin by preparing the complete endothelial culture medium using 460 milliliters of endothelial cell medium, and adding into it, 50 milliliters of FBS, five milliliters of penicillin streptomycin, and five milliliters of endothelial cell growth supplement. The prepared media can be stored at four degrees celsius for a month. Next, in a 100 millimeter tissue culture dish containing 0.1 million vascular cells at eight milliliters of the prepared complete endothelial culture medium and culture the cells at 37 degrees Celsius and 5%carbon dioxide until 70%co-fluency is achieved.Then, remove the culture medium from the dish and rinse the cells twice with PBS to eliminate unattached cells and debris. After removing the PBS, add three milliliters of 0.25%trypsin, containing 2.21 millimolar EDTA to the cells and incubate the dish at 37 degrees Celsius for one minute. Verify cell detachment under a light microscope at 40 times magnification.Neutralize the trypsin with seven milliliters of complete endothelial culture medium and gently rinse the cells off the culture dish. Centrifuge the cell suspension after collecting it in a 15 milliliter tube at 400G for 10 minutes. Re-suspend the cells in five milliliters of complete endothelial culture medium after removing the supernatant, then count the cells using a hemo-cytometer, and transfer a calculated volume of suspension containing 2 million cells to a sterile 1.5 milliliter tube.Pellet the cells by centrifuging the suspension at 400G for five minutes before removing the supernatant. The vascular cells are now ready for mixing with the matrix gel for matrix gel plug assay.

Preparing Matrix Gel with Vascular Cells for In Vivo Matrix Gel Plug Assay

To begin, prepare 10 times M199 culture medium by dissolving M199 powder to 10 times concentration in 90 milliliters of deionized water, followed by the addition of 10 milliliters of FBS. Filter the solution through a 0.22 micron filter. The filtered medium can be stored at four degrees Celsius for up to two months.For preparing the matrix gel with vascular cells, use about 2 million vascular cells. Cool the sterile 1.5 milliliter tube containing the appropriately prepared cell pellet in an ice bath. Thaw the matrix gel completely in an ice bath without mixing or vortexing it.Similarly, cool the prepared 10 times M199 culture media along with the test reagent or drug of interest to four degrees Celsius. Next, maintaining a volume ratio of 8.8 to 1 to 0.2, combine the pre-cooled matrix gel with the pre-cooled 10 times M199 media containing 10%FBS, and the pre-cooled test reagent to produce the matrix gel mixture. Finally, re-suspend the pre-cooled cells in 400 microliters of the prepared matrix gel mixture by mixing gently to avoid forming bubbles.Keep the tube containing the cells in matrix gel on ice until the mice are ready for the matrix gel injection.

In Vivo Matrix Jel Plug Testi için Vasküler Hücreli Matrix Jel Hazırlama

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

To begin, place the 30 gauge one milliliter insulin syringes in a four degrees Celsius refrigerator for cooling. Keep the freshly prepared matrix gel mix containing vascular cells on ice until the mouse is ready for the injection. Next, place the properly anesthetized mouse on the operating board in a prone position and secure its limbs onto the board using tape.Load a pre cooled insulin syringe with 300 microliters of the matrix gel mixture, avoiding the formation of bubbles. Disinfect the skin on the back of the mouse using 75%alcohol pads, then subcutaneously inject 300 microliters of the matrix gel mixture in the syringe into one side of the animal's back. Gently remove the needle from the injection site to prevent any leakage of the gel mixture.Ensure the formation of a small hump at the injection site. Position the mouse on a heating pad for two minutes to allow the matrix gel mixture to coagulate and form a plug. Repeat the demonstrated steps to create a plug on the other side of the mouse's back.Mark the edges of the hump using a marker pen. House the animal in a specific pathogen free experimental animal laboratory at 20 to 24 degrees with 12 hour light and 12 hour dark cycles for seven days.

Matrix Jel Fiş Testi için Fareye Matrix Jel Karışımı Enjeksiyonu

Dextran-FITC Injection and Matrix Gel Plug Collection from Mouse for Matrix Gel Plug Assay

To inject the mouse with Dextran, FITC seven days post the matrix gel injection, RESUSPEND 0.5 milligrams of Dextran, FITC in 500 microliters of double distilled water. Thoroughly vortex it to obtain a one microgram per microliter Dextran, FITC solution. Fill a 29 gauge syringe with 50 microliters of the Dextran FITC solution.Secure the mouse on the tail vein injection instrument, sanitize the tail with a 75%alcohol soaked cotton ball before carefully injecting 50 microliters of the Dextran FITC solution in the syringe into the tail vein. Apply pressure to the injection site with the cotton swab for one minute to staunch bleeding. Return the mouse to its cage and let it relax for 30 minutes before collecting the matrix gel plug.To collect the gel plug, cut the skin of the euthanized mouse along the marked border of the plug, using surgical scissors. And remove the skin above the matrix gel plug. Collect the plug and rinse it in a beaker with phosphate buffered saline to wash off any excess blood.Next, cover the bottom of the tissue embedding cassette with about 0.5 milliliters of tissue embedding gel. Then swiftly place the matrix gel plug on the tissue embedding gel in the planned orientation. Embed the plug with additional tissue embedding gel.Solidify the cassettes at minus 80 degrees Celsius for 12 hours. After removing the solidified plug block, cut 12 micron thick sections using a freezing microtome. Then mount the sections onto microscope slides.