The overall goal of this procedure is to generate an in vivo human vascular network. This is accomplished by first culturing human blood derived endothelial colony forming cells and human bone marrow derived mesenchymal stem cells. Both cell types are then mixed into a collagen based gel, which is injected subcutaneously into an immunodeficient mouse.
Seven days later, the implant is harvested and evaluated for the formation of a human vascular network. Ultimately, the presence of human specific microvessels inside the implants can be verified through his histology and immunohistochemistry. The main advantage of this technology over a acid method like surgical implantation of construct is LED, is simple and easy to perform, and it doesn require surgery.
Begin by thawing one vial of E CFCs and immediately dilute the cells in a 15 milliliter conical containing 10 milliliters of DMEM. Spin down the cell suspension at 1200 RPM for five minutes and Resus suspend the cell pellet in 10 milliliters of warm EGM two medium. Then transfer the cells to a 100 millimeter tissue culture plate coated by 1%gelatin and incubate the cells overnight in a humidified incubator.
The following morning, gently aspirate out the medium and any unbound cells. Then feed the bound cells with 10 milliliters of fresh E GM two medium. Continue this process over the next few days until a confluent monolayer has formed.
Then wash the cells with 10 milliliters of PBS. Replace the PBS with two milliliters of tripsin EDTA and return the plate to the incubator for five minutes. After five minutes under a microscope, gently tap the plate and check if the cells have detached.
If not, repeat the brief incubations with gentle rocking. Once the cells have completely detached, add eight milliliters of EGM two medium to the cells and transfer the cells to a 15 milliliter conical. Count the cells and spin them down at 1200 RPM for five minutes.
Now plate the cells on tissue culture plates coated with 1%gelatin at a seating density of 5, 000 cells per square centimeter. In EGM two medium, incubate the cells and feed them every two to three days with EGM two medium. Repeat this procedure for subsequent passages between passages four and eight.
The E CFCs will be ready to use. This protocol can also be used to grow up MSCs. Just replace E GM two medium with M-S-C-G-M medium.
Use an uncoated culture plate. Begin subculture at 80%confluence and subculture at a seeding density of 10, 000 cells per square centimeter. This experiment requires 800, 000 E CFCs and 1, 200, 000 MSCs per implant.
Generally five implants are prepared simultaneously. To begin aspirate off a cell medium from the culture plates, and wash each plate of cells with 10 milliliters of PBS. Replace the PBS with two milliliters of trypsin EDTA and incubate the plates with gentle rocking.
Check the cells under a microscope every two to five minutes until the cells have all detached. Then add eight milliliters of DMEM and collect the cell solution. In a 15 milliliter conical, count the cells and transfer enough cells for five implants into a single 15 milliliter conical.
Then spin down the cells at 1200 RPM for five minutes and remove the snat. Carefully prepare a mixture of fibrinogen and collagen fibronectin on ice. Then very gently resuspend the cell pellet in the ice cold mixture.
Avoiding the formation of air bubbles, load the cell suspension into a one milliliter syringe and attach a capped 26 gauge needle. Keep the loaded syringe on ice until the cell mixture is injected. Anesthetized four six week old athymic nude immunodeficient mice using an ISO fluorine chamber.
Once the mice are anesthetized and unresponsive to toe pinch, proceed with the injections for each mouse. Use a 30 gauge needle to inject 50 microliters of thrombin solutions subcutaneously into the upper dorsal region. At the same location, inject 200 microliters of the cell mixture.
Using a 26 gauge needle, the injected macromolecules will gel and form a small but appreciable bump under the skin. After the injection, place the mice on a layer of gauze for comfort and warmth. Observe them until they become ambulatory.
Over the next three days, make a daily observation of the animal's general health. One week after the injections, euthanize the mice with carbon dioxide and surgically remove the gel plug. Once retrieved, photograph the gel plugs alongside of a ruler, then place each gel plug into a histological cassette and submerged them in 10%neutral buffered formin overnight at room temperature.
The next day, wash the 10%neutral buffered formin away with distilled water. Store the cassettes in PBS at four degrees Celsius until they're evaluated for histological evaluation. Make seven micron sections of the implant embedded in paraffin.
Then quantify the density of micro vessels in the middle part of the implants. With the aid of an H and e stain score the number of vessels per square millimeter to demonstrate the human nature of the microvascular vessels. Stain the sections with a commercially available anti-human CD 31 antibody at a one to 100 dilution.
This phase contrast image shows the characteristic cobblestone morphology of endothelial cells in a typical confluent monolayer of cord blood derived e CFCs. This image shows the typical spindle shape morphology of human bone marrow derived MSCs. After a week long incubation in the mouse host, the implant marked by a yellow dashed line looked very different from the host's tissues at higher magnification.
Multiple microvessels were revealed their luminal structures containing red blood cells and are highlighted by the yellow arrowheads. When the plugs were immunohistochemical examined, the lumen of the microvessels were reactive to a monoclonal antibody to human CD 31. The cell nuclei were counterstain with hematin.
Once master, this technology can be done within one ounce if it's performed properly.
