The overall goal of this procedure is to harvest multi potent stromal cells from adipose tissue for the purpose of osteogenic differentiation, using an in vivo calvarial defect model in a mouse. This is accomplished by first isolating progenitor cells from the stromal vascular layer of adipose tissue, which can be obtained following a liposuction procedure. The next step is to prepare a three-dimensional backbone and then seed the cells onto that scaffold.
The final step is the creation of a critical sized calvarial defect in a rine model into which the scaffolding and cells are placed. Ultimately, the results show that multipotent stromal cells isolated from adipose tissue have the potential for skeletal regeneration in vivo. The applications of this technique has a broad spectrum of possible clinical applications because of the shortage of tissue available for skeletal regeneration.
While autologous bone graft are the gold standard. A lack of donor sites and donor site morbidity limit this option. Allografts and foreign materials can be used but are limited by cost, poor integration and potential infection.
The possible use of adipose or fat tissue for the isolation of progenitor cells for skeletal tissue engineering is exciting. Demonstrating the procedure will be myself and Michael Chung from the long anchor laboratory. First, obtain human subcutaneous adipose tissue from elective lipo aspiration procedures.
And note the two layers in the lipo aspirate. The supernatant contains the vast majority of the process cellular material. The bottom layer is mostly injected saline.
Adipose derived stromal cells can be harvested from either layer, but the yield is much greater from the supernatant. To isolate the stromal vascular fraction or SVF wash, the lipo aspirate extensively with equal volumes of one x phosphate buffered saline or PBS containing 2.5%Betadine aspirate equal volume of one XPBS without Betadine. Allow the wash to precipitate.
Be sure to maintain sterile technique throughout the process to avoid contamination with processed human tissue. Next, aspirate the bottom layer and discard After each wash, then Eloqua 15 milliliters of adipose tissue into 50 milliliter Falcon conical tubes. Then digest the adipose tissue in 15 milliliter filtered 0.075%filtered.
Type two collagenase in Hank's balance salt solution or HBSS at 37 degrees Celsius in a water bath for 60 minutes under constant agitation, be sure to ventilate the tubes every 15 minutes. Then neutralize enzyme activity with 15 milliliters PBS containing 10%fetal bovine serum or FBS and centrifuge. To obtain a high density SVF pellet, the pellet will be a mix of adipose derived stem cells and erythrocytes.
Discard the supernatant fat layer first, and then discard the supernatant liquid layer without disturbing the pellet. Resuspend the pellet in 10 milliliters of traditional growth media. Then combine three tubes into one clean 50 milliliter conical tube through a 100 micron filter and centrifuge.
Discard the supernatant without disrupting the pellet, and then resuspend the pellet. In five milliliters of traditional growth media, combine two 50 milliliter conical tubes per one 10 centimeter plate, or five five milliliter conical tubes for one 15 centimeter plate. Establish primary cultures overnight at 37 degrees Celsius with 21%oxygen and 5%carbon dioxide.
After incubating overnight, wash the plates with PBS to remove residual non-adherent red blood cells. The resulting cell populations are adipose derived stromal cells. Maintain the cells at sub confluent levels to prevent spontaneous differentiation.
At 37 degrees Celsius, 21%oxygen, and 5%carbon dioxide. In growth media, the cells generally need to be split every three to four days in regular growth media. When plated at 50%confluence, prepare the scaffold as explained in the accompanying manuscript.
When the cells reach sub confluence levels, wash the cells with PBS twice and trypsin eyes. Count the cells to quantify them For seeding, prepare to seed the cells onto precut. Four millimeter diameter scaffolds with 1.5 times 10 to the fifth cells.
Place an individual scaffold into one well of a 96. Well plate then resuspend cells in regular growth media with a concentration of roughly 1.5 times 10 to the fifth cells per 20 microliters of media. Seed the scaffold by pipetting 20 microliters of the solution directly into the scaffold and place this in the cell culture incubator for 30 minutes.
After 30 minutes, add 200 microliters of media and allow the cells to incubate on the scaffold overnight. Prior to surgery, it is not necessary to ensure adhesion of cells onto the scaffold as seeding. A sufficient number of cells will ensure that a majority will attach onto the scaffold.
After anesthetizing an adult 60 day old CD one nude mouse, sterilize the surgical site with Betadine and alcohol three times and place lubricant over the eyes of the mice. Then make a midline sagittal incision in the scalp of the mouse to expose the right parietal bone. Remove the perran from the right parietal bone with blunt scraping.
Next, create a unilateral four millimeter full thickness defect in the right non suture associated parietal bone. Using a diamond coated tine drill bit, be extremely careful not to disturb the underlying dura mater as the mouse calvarial thickness is less than 0.3 millimeters before implantation. Rinse the scaffolds with sterile PBS to prevent transfer of medium derived growth factors.
Place the scaffold into the defect. Finally, suture the skin closed and monitor the animal per established postoperative protocols. Perform micro CT on the mouse and obtain a 3D reconstructed image using microview software.
Use Adobe Photoshop to size the images to a standard height. Typically, we measure for calvarial regeneration every two weeks. The mice are anesthetized during the scan for long-term follow-up and ultimately euthanized.
For histological analysis, use the magic wand feature to measure pixels of the calvarial defect. Determine percentage healing of the defect by subsequent CT scans. Measuring the calvarial defect area and determining the pixel number and dividing it by the original defects Pixel number, stain multiple sections along the span of the defect using histological stains like annal in blue and pen aro to denote osteo formation.
Use adobe Photoshop to crop out all areas that are not in the calvarial defect and use the magic wand to determine pixel numbers of de novo bone formation in the area of the defect, and compare this to controls or other variables.Shown. Here is an overview of the harvesting and application of lipo aspirate from the isolation of adipose-derived stromal cells to their expansion differentiation and use in vitro and in vivo. Here is a micro CT showing in vivo healing of the critical size calvarial defect with the application of ASCs through a hydroxyapatite scaffold seen in the bottom row.
Controls, which are seen in the top row include no scaffold and defect. The middle row shows defect with placement of the scaffold without cells. LS shown here is the quantification of osseous healing from the micro ct, which shows significantly increased healing in the A SC group.
This is a histologic image showing increased osteo formation of the A SC group through andal. In blue and pen AROM staining for al. In blue, the osteoid stains dark blue for pen arom.
The osteoid stains yellow shown here are human ASCs labeled with GFP, which were seeded onto a scaffold and sacrificed at two weeks. Staining was done with a GFP labeled antibody to show the human cells contributing to regeneration in the area of the defect. Shown here is human nuclear antigen immunochemistry showing prevalence of human cells in the area of the defect at two weeks.
Once mastered, processing the adipose tissue can be done within four hours, and the surgical procedure can take about 10 minutes per mouse. It's important to remember that the parietal bone is round while the drills flat, so cure has to be taken to get through the entire circumference of the four millimeter defect. Remember to go slowly to avoid injuring the dura mater in the brain.
After watching this video, you should have a good understanding of how to isolate the progenitor shells from the adipose tissue. See these cells onto a scaffold and place them into the mouse.
