Isolatie en Verrijking van Human Adipose-afgeleide stromale cellen voor Enhanced Osteogenesis

The overall goal of this procedure is to isolate subpopulations of human adipose-derived stem cells with enhanced osteogenic potential using flow assisted cytometry to accomplish this. Human adipose-derived stem cells or ASCs are harvested from human lipo aspirate and cultured for 36 hours. The cells are then stained with antibodies selected to label pros osteogenic populations and the desired populations are isolated using flow assisted cytometry.

Ultimately, alkaline phosphatase and azarin staining reveal enhanced osteogenic potential of the isolated cells. The implications of this technique extend towards regenerative medicine and the development of new translational therapies because it describes how to isolate a subpopulation of ASCs with enhanced osteogenesis. The following procedure is performed using human subcutaneous adipose tissue from healthy female patients who have undergone elective lipo aspiration of the abdomen, flank and or thigh region under local or general anesthesia.

Begin with 500 milliliters to one liter of tissue in a suction canister taken directly from the operating room. Immediately upon collection, begin the harvesting procedure. This will maximize the cell viability to obtain the stromal vascular fraction or SVF from the lipo aspirated adipose tissue Begin by adding sterile PBS at a ratio of one to one.

Then gently shake the canister to mix. After mixing. Allow the solution to stand until it separates into three layers.

The top oil layer is clear and yellow. The middle fat layer is a viscous, opaque, and yellow, and the bottom blood layer is red. Carefully using a serological, pipette, aspirate and discard the bottom blood layer, aspirate the top oil layer.

Then repeat this. Wash an additional two times. After preparing the collagenase digestion buffer and fax buffer as ascribed in the accompanying document, place them in the hood along with the red blood cell lysis buffer so that they can warm to room temperature.

Next, transfer the washed adipose tissue to a large flask and to digest it at an equal volume of collagenase digestion buffer. Place the flask securely in a shaking water bath for 60 minutes. At 37 degrees Celsius.

It is best to use a larger volume digestion vessel than required as this allows for maximum digestion during shaking. For example, 250 of collagenase digestion buffer and 250 mls of washed adipose tissue should be placed in a sterile one liter flask. After the incubation, neutralize the enzymatic activity by adding an equal volume of fax buffer and allowing the tissue to sit at room temperature for five minutes.

Next, transfer the digestive tissue to a 250 milliliter disposable conical centrifuge tube and centrifuge at 233 Gs, or 20 minutes at four degrees Celsius. Following the spin, aspirate the supinate and re suspend the pel pellet by adding five to 10 milliliters of traditional growth. Medium, depending on the size of the pellet.

Then pipette the suspension through a 100 micrometer nylon cell strainer into a 50 milliliter conical tube to remove any cellular debris. After discarding the supernatant, the palette should appear as shown. Here, Reese, suspend the palate in 10 milliliters of room temperature.

Red lysis buffer. Allow the solution to sit at room temperature for five minutes and then centrifuge it at 233 Gs for five minutes at room temperature. Next centrifuge at 233 Gs for five minutes at four degrees Celsius, and discard the snat without disturbing the pellet.

Then Resus, suspend the pellet in five to 10 milliliters of traditional growth. Medium, depending on the size of the pellet. After counting the cells, place 2 million cells in a 10 centimeter standard culture dish and establish primary cultures overnight.

Maintain the SVF cells at sub confluent levels to prevent spontaneous differentiation. 36 hours after seeding lift the cells using Accutane according to the manufacturer's protocol. Wash cells once with fax buffer, then centrifuge at 233 Gs for five minutes at four degrees Celsius.

After the spin, discard the snat without disrupting the pellet. Resuspend the cells in 0.5 to one milliliter of effects.Buffer. Count the cells using a hemo cytometer.

Allocate 1.2 million cells to an unsorted group that will be plated separately as a control. For this experiment, transfer 100 microliter quats into one 1.5 milliliter einor tube. For each sample to be analyzed, prepare one unstained sample plus single color controls for each of the antibodies to be used and place them on ice.

Next to label the osteogenic subpopulation dilute anti CD 90 or anti CD 1 0 5 in fax buffer to label the other cell populations such as hematopoetic and endothelial cells dilute anti CD 45, anti CD 34, and or anti CD 31 in fax buffer. After the incubation, wash the cells twice by filling the tubes with fax buffer and centrifuge in at 233 Gs for five minutes at four degrees Celsius. After the spin aspirate, the snat after the second wash resuspend the cells in 400 to 500 microliters of effects.

Buffer filter the samples through a 70 micrometer nylon cell strainer into a centrifuge tube to remove cell clumps. Perform fluorescence activated cell sorting as detailed in the accompanying protocol. Using fact software design analysis plots to examine forward scatter side scatter fico, eryn, Texas Red Pacific blue fluorescent isothiocyanate, allof cyanide, and any other Fluor four used to label cells.

It is important to ensure that facts analysis plots are drawn appropriately so as not to inadvertently exclude the desired cell populations. Sort the labeled cell populations to either 1.5 milliliter centrifuge tubes, or 15 milliliter conical tubes containing culture. Medium manually cease sorting.

Once the required quantity of cells has been obtained for this experiment, approximately 2 million cells are needed. Next, using facts, perform a purity check by analyzing a small fraction about 500 cells of the acquired cell population. Note this step confirms the purity of the sorted cell populations.

Ideally, purity should be greater than 90 to 95%immediately after sorting centrifuge cells at 233 Gs for five minutes at four degrees Celsius. Then resuspend the cells in one milliliter, fresh medium containing 10%FBS plate, the cells on gelatin coated plates to improve cell viability. Depending on the final cell yield plate, 300, 000 cells per well in a six well plate, or 100, 000 cells per well.

In a 12 well plate to isolate human adipose arrives stromal cells with increased osteogenic potential. ASCs were stained with Pacific Blue conjugated anti-human CD 45, Fitz Conjugated anti-human CD 1 0 5 an A PC conjugated anti-human CD 90 and isolated as described in this video as shown here, sorting based on high CD 90 or low CD 1 0 5 expression yielded highly enriched populations of human ASCs to characterize the osteogenic potential of the two subpopulations. CD 90 positive CD 1 0 5, low and unsorted ASCs were cultured in osteogenic differentiation medium for 14 days as can be seen in this image.

Alkaline phosphatase staining, which serves as an early indicator of bone formation, was increased in the culture CD 90 positive population relative to the other groups quantification using image analysis software confirmed that the observed difference was significant. Likewise, at day 14, aller and red staining, which is used to detect extracellular matrix mineralization and serves a metric for end terminal osteogenic differentiation was increased in the CD 90 positive population relative to other groups. Quantification using image analysis software also confirmed that the observed difference was significant.

These data indicate that CD 90 positive ASCs are able to mineralize a significantly greater amount of extracellular matrix than unsorted cells, suggesting that isolated ASCs retain the ability to undergo osteogenic differentiation after sorting. After watching this video, you should have a good understanding of how to isolate a subpopulation of ASCs with enhanced osteogenic potential. When working with human samples as described in this protocol, it's important to always wear appropriate protective clothing.