This protocol provides a simple cost effective way to isolate ASCs without the use of harsh enzymes or centrifugation steps, which can alter the cell phenotype and behavior. The lack of harsh steps yields less manipulated cells than those isolated using enzymes and centrifugation, minimizing questions as to whether experimental observations are an artifact of the isolation method. This explant method can generate clinical relevant ASCs to be introduced into a patient to treat a variety of diseases or expand in the life afford research applications.
It is difficult to full explain the mincing method in words. The visual demonstration can serve other scientists the hassles of learning to break apart the tissue themselves. Similar explant methods can be used to isolate other populations of MSCs, such as those from umbilical tissues.
To mince the abdominoplasty tissue samples, first transfer the tissue to the lid of a sterile 100 millimeter plate. Then use a sterile needle to hold a piece of tissue in place and use a sterile scalpel to cut the tissue into less than one millimeter pieces. Transfer the tissue to a fresh tube and invert or shake the sample five to six times to ensure mixing of all layers.
Then, with a sterile spatula, transfer 2.5 to five milliliters of tissue to a 100 millimeter vacuum gas plasma treated tissue culture plate. Measure the volume of the sample by pouring into a fresh centrifuge tube. Next, add an equivalent volume of tissue culture media to the plate and gently swirl the plate to mix the contents.
Incubate the plate at 37 degrees Celsius in five percent carbon dioxide until a sufficient number of cells are seen on the base of the plate. Once a sufficient number of cells are noted on the plate or after seven days, whichever is sooner, remove all remaining pieces of tissue. Next, culture the ASCs in tissue culture media until they reach 70%confluency or until the clusters become dense at which point the cells are to be passaged.
Gently rinse the plate with 1X phosphate buffered saline. To trypsinize the adherent cells, aspirate the phosphate buffered saline, add one milliliter of trypsin supplemented with 0.25%EDTA to each plate and incubate at 37 degrees Celsius for five minutes. Add a small amount of media to the plate to deactivate the trypsin.
Then gently pipette the media trypsin from the top of the plate downwards, loosing any weakly attached cells from the plate. To seed the cells, first add media to the plate. Then, add the cells in a dropwise manner and then swirl the plate to ensure even distribution across the plate.
After three passages, proceed to flow cytometry and differentiation assays. After three passages, isolated ASCs from lipoaspirate and abdominoplasty samples were found to have an MSC morphology and phenotype, similar to isolated ASCs following an enzymatic digestion. Like other MSCs, the explant isolated ASCs are negative for CD45 and positive for CD73, CD90, and CD105.
When mincing the tissue, the surface area of each piece must be sufficiently large for the ASCs to migrate out of the tissue. These are fundamental stem cells that can be used for a broad range of applications. The isolated ASCs can be used for any downstream application, in vivo or in vitro.
