大動脈リング共培養アッセイ:体外間葉系間質細胞の血管新生の可能性を評価するために便利なツール

The overall goal of this pre-clinical functional assay is to observe and quantitatively evaluate the angiogenic potential of mesenchymal stem cells intended for regenerative therapy. This method can help answer key questions in the field of vascular targeting regenerative medicine, such as the mode of action and the efficiency of cell therapy candidates. The main advantage of this techniques is that it provides key elements of the in vivo environment while providing control and easy assessment characteristics to in vitro assays.

The implications of this novel technique extend toward cellular therapy of ischemic diseases, as the field is really missing reliable and easy-to-use functional assays. Demonstrating this procedure will be Farwah Iqbal, a PhD student in my laboratory, as well as Peter Szaraz, a research associate in my laboratory. After isolating a rat aorta and preparing media according to the text protocol, coat 12 well tissue culture plates by first placing the plates on a cold surface such as an ice pack.

Using refrigerated pipettes, add 200 microliters per well of freshly thawed basal membrane extract or BME to the wells of the plate. Then, to avoid uneven polymerization of the BME, quickly swirl the BME to ensure even coating. Placing the coated plates into a humidified incubator for 30 minutes.

Return the 1, 000 microliter pipette tips back into the minus 20 degrees Celsius freezer. To reduce variability between ring units caused by residual tissue, place the aorta in a 10 centimeter dish with 10 milliliters of fresh HBSS and use two sets of forceps to carefully separate the excess connective and adipose tissue. Use surgical scissors to separate the remaining branches of the intercostal arteries connected to the aorta and remove any residual coagulated blood from the inside of the aorta.

Place a ruler or grid under the 10 centimeter dish and use a sterile scalpel and forceps to precisely cut one to two millimeter wide sections of the aorta. To embed the aortic rings in BME after removing the BME coated plates from the incubator, use forceps to carefully pick up individual aortic rings and place each one into the middle of a BME coated well. Use chilled 1, 000 microliter pipette tips to add 300 microliters of BME on top of each aortic ring and evenly distribute the BME around the well.

When all of the aortic rings are embedded, return them to the humidified incubator for 30 minutes. Next, following the incubation, add 1, 000 microliters of previously prepared endothelial growth medium or EGM by placing the pipette tip against the wall of the culture well and slowly expelling the solution. Then incubate the tissue for 24 hours.

While replacing the medium with EGM containing FBS every 48 hours, use bright-field microscopy to follow the development of the aortic ring endothelial network. To prepare aortic ring assay MSC co-cultures after using bright-field microscopy to image the rat endothelial networks at baseline, seed 10 to the fourth MSCs onto each embedded aortic ring. Maintain at least three wells of aortic rings without MSCs for a control group.

Next, visualize the fluorescently labeled MSCs in the aortic ring assay. Following a 24 hour incubation of the aortic ring assay MSC co-cultures, use fluorescence microscopy to visualize MSC homing, elongation, and integration with the endothelial networks. Overlap the fluorescence images of MSCs with bright-field images of endothelial cells to observe the co-localization of both cell types.

On days three, five, and seven of the co-cultures with bright-field microscopy, image the aortic ring networks. Use the images to quantify the net effect of MSC co-cultures on endothelial network development. After one week of aortic ring assay MSC co-culture, remove the culture medium and slowly add one milliliter of PBS to each well.

Incubate the co-cultures for three minutes and then remove the buffer before repeating the wash two more times. Add 800 microliters of pre-warmed Dispase to each well and incubate the cultures in the humidified incubators for 15 minutes. Following the incubation, pipette the Dispase five to 10 times to break up the VME, and transfer the contents of each well into separate 15 milliliter tubes.

Add 800 microliters of fresh pre-warmed Dispase to the same wells and pipette until no residual BME is observed in the culture well. Transfer Dispase cell suspensions to 15 milliliter tubes and use pipette tips to remove the aortic rings floating in the cell suspension. Then add one volume of PBS supplemented with 3%FBS to the Dispase cell suspension to inactivate the Dispase.

Spin the cell suspensions at 400 times g for five minutes. Carefully remove the supernatant, leaving three milliliters of cell suspension in the 15 milliliter tube. Add three milliliters of pre-warmed 0.5%trypsin to the cell suspension and vigorously re-suspend the cells.

Incubate the trypsinized cell solutions in the humidified incubators for 10 minutes. Following the incubation, add six milliliters of PBS supplemented with 3%FBS, and re-suspend a few times. Then spin the cell suspensions again.

Carefully remove all the supernatant, leaving the cell pellet in the tube and use one milliliter of PBS supplemented with 3%FBS to re-suspend the cells. The basal membrane extract can make it challenging to recover the embedded cells. However, Dispase treatment followed by trypsin provides a high yield of viable cells.

Using a 70 micron cell strainer, filter the one milliliter cell suspension to remove aggregated cells and residual BME from the cell suspension. Then with a cell counter, count the cells in one milliliter of cell suspension. Carry out flow cytometry and qPCR according to the text protocol.

As shown here, three to five days after aortic rings are embedded in ECM, there is a region characterized by high cell proliferation but low structural organization in the vicinity of the aortic tissue. Developed endothelial networks with high structural organization are fully established and predominantly composed of closed loops, even prior to the addition of MSCs. Developing networks are located at the distal areas of the endothelial cultures and are sites of endothelial cell migration and new structure development.

After 24 hours in co-cultures, labeled FTM HUCPVCs were found at the periphery of the developing endothelial networks, displayed elongated cell morphologies, and contributed to the further development of endothelial networks. By comparison, BMSC is home to the developing networks while displaying less interaction with endothelial cells. High magnification images at 72 hours showed that FTM HUCPVCs maintained high coverage and stabilization of endothelial networks, while BMSCs and co-cultures, presented with spherical morphologies with limited integration into endothelial networks.

Finally, in these images, pre-labeled FTM HUCPVCs are shown adhering with unstained endothelial cells and provide structural support by serving as an axis and attachment surface between nodes. Once mastered, this technique can be done in four hours of hands-on time if it is performed properly. While attempting this procedure, it is important to treat all samples in the same manner and to keep timing stringent.

This applies to both the basal membrane extract and the aortic tissue. Following this procedure, other methods like lymphocyte assays can be performed in order to answer additional questions like whether the tested cells changed their mutagenicity in a microvascular environment. After its development, this technique enables researchers in the field of mesenchymal stem cell therapy to explore the angiogenic potential of cell therapy candidates in a multiplex and quantitative way.

After watching this video, you should have a good understanding on how to establish the rat aortic ring assay so you can observe and evaluate the behavior of therapeutic cells in developing microvasculature. Don't forget that working with primary animal and human tissue can be hazardous, and precautions such as aseptic technique and personal protective equipment should always be worn while performing this procedure.