The overall goal of this procedure is to isolate adipose-derived stem cells from the fat obtained in the form of a lipo aspirate. This is accomplished by first removing the surgical solution and washing the aspirate of contaminating red blood cells. The second step is to release the stem cells via enzymatic digestion of the washed lipo aspirate with collagenase.
Next, the released cells in the form of a stromal vascular fraction or SVF are collected by centrifugation. The final step is to remove the supernatant and plate, the SVF cells for selection and culture of the adherent ASCs. Ultimately in vitro induction of these cultured ASCs towards the bone, fat and cartilage.
Lineages can be used to confirm the presence of these cells within the culture in addition to confirming their multi potentiality. Generally, individuals new to this method will struggle because of the logistics of working with large volumes of lipo aspirin Prior to the cell harvest. Prepare sterile collagenase Type one a solution and sterile control medium.
Collect the lippo aspirate in an aspiration container. Carefully decant the lippo aspirate into a sterile one liter glass speaker. When the lippo aspirate layers have separated by gravity, aspirate the top oil layer if present.
Also, remove the bottom inate. Assess the volume of the resulting adipose tissue layer. Add an equal volume of sterile one XPBS and stir with the pipette used for aspiration.
When the two layers have settled, aspirate the inate using a 10 milliliter serological pipette. Continue to wash the adipose tissue fraction with PBS until the adipose layer has a yellow gold color. Aspirate the inate one last time, leaving the adipose tissue fraction.
In the glass speaker. Allow the lippo aspirate layers to settle for five minutes after the last wash and before the final aspiration. In a filter unit, prepare sterile collagenase one a solution.
Add a volume equal to that of the adipose fraction and sterile filter. Then carefully pour the washed adipose fraction into the collagenase solution and tightly close the bottle. Then incubate the collagenase adipose mixture in a 37 degrees Celsius water bath for 30 minutes with gentle swirling every five to 10 minutes, evaluate the adipose tissue layer for a smoother appearance as the digestion proceeds and if necessary, increase reaction time to dissolve any solid pieces of fat.
Sterilize the filter unit containing the digested collagenase adipose mixture with 70%ethanol and place it back into the biosafety cabinet. Now pipette 25 milliliter aliquots of the inate containing the stromal vascular fraction into sterile 50 milliliter centrifuge tubes. Add 25 milliliters of control media to each tube and incubated room temperature for five minutes to inactivate the collagenase.
Then centrifuge for 10 minutes at 1, 200 times G to collect the stromal vascular fraction as a pellet in the biosafety cabinet. Aspirate the supernatant from each tube. Ensure the top oil layer and any floating adipocytes are aspirated with this supernatant.
Next, combine the SVF pellets into one centrifuge tube using 30 milliliters of conditioned media and divide equally over two new 50 milliliter centrifuge tubes after centrifugation aspirate the supernatants from the two pellets. If RBC lysis is desired, these two pellets can each be resuspended in 10 milliliters of an RBC lysis buffer, and incubated at room temperature for 10 minutes. After centrifugation, aspirate the supernatants to yield pellets of the stromal vascular fraction.
Combine these two pellets into one new centrifuge tube. Using five to 10 milliliters of control media, large tissue particles may be filtered out using a 100 micron mesh filter placed on top of a new 50 milliliter centrifuge tube. Now aliquot the filtered fraction containing the adipose stem cells onto tissue culture dishes.
Then supplement each dish with an appropriate volume of control. Media culture. The cells for three to four days without change of media aspirate the culture, media and wash cells once with sterile PBS to remove any contaminating red blood cells, then replenish the media and continue to culture when using this manual enzymatic for the isolation of a stromal vascular fraction from a large volume lippo aspirate sample.
The resulting cultured A SC population is relatively homogenous in appearance and comprised of fibroblasts like cells as the most popular means of validation. In vitro induction of the A SC population using defined culture conditions, results in their differentiation into mesodermal, ectodermal, and endodermal lineages. While in vitro tri germ layer potential has resulted in the use of ASCs for a wide variety of regenerative model systems.
Differentiation into adipogenic, osteogenic, and congenic cells is generally sufficient to identify the A SC and confirm its multi potency. In addition, ASCs may also be differentiated into skeletal myogenic and smooth myogenic lineages cells of the ectodermal lineage or endodermal hepatic cell lineage. After watching this video, you should have a good understanding of how to isolate a stromal vascular fraction from Lipos.
For the purpose of Turing, its a SC component.
