The overall goal of this procedure is to demonstrate the isolation of putative stem cells from neuron SIV glands. This is accomplished by first harvesting submandibular sali glands from adult mice. The second step of the procedure is to use curved scissors to process the tissue into a homogenous pulp.
The third step of the procedure is to further digest the tissue using collagenase and higher liase enzymes. Care should be taken not to over digest tissue. Since a complete cell suspension does not yield very many spheres, clusters of two to three cells are optimal.
The final step of the procedure is filtration of the homogenate through 100 micron and 50 micron filters. Ultimately, results can be obtained that show the culture and isolation of siv gland stem cells through the combined use of mechanical and enzymatic digestion of whole SIV glands. The implications of this technique extend towards the therapy of Xerostomia, which is also known as dry mouth, and which can result from radiotherapy treatment.
Saliva gland stem cells may be used to rescue Xerostomia in patients who suffer from head and neck cancers Before beginning this procedure. Prepare buffers, mouse, sali gland culture, media and enzyme solutions. According to the accompanying written protocol, all dissection and tissue culture steps should be performed in a sterile tissue culture hood.
After isolating and dissecting the mouse saliva glands, weigh the sali gland tissue on a balance and record the weight. Use a spatula to transfer the sali glands to a small Petri dish and using curved dissection scissors, chop the sali glands into a homogenous pulp. Cut the end of a P 1000 pipette tip to produce a wider aperture.
Use this to pipette one milliliter of buffer per 80 milligrams of tissue into the Petri dish and transfer the homogenate to a 15 milliliter centrifuge tube pipe. Pat another one, milliliter of buffer per 80 milligrams of tissue onto the Petri dish. To collect any remaining small pieces of tissue, add this solution to the centrifuge tube.
Then quickly add 250 microliters of calcium chloride solution, 25 microliters of hyaluronidase solution, and 25 microliters of collagenase. Two enzyme solution per 80 milligrams of tissue. Place the tube containing the tissue and enzyme mixture into a 37 degree Celsius, shaking water bath and incubate for 20 minutes.
Following incubation, remove the tubes. Use a P 1000 tip with the end removed as before to thoroughly mix the tissue and enzyme solution approximately 10 times. Return the tube to the 37 degrees Celsius water bath for an additional 20 minutes.
After the second incubation period, centrifuge the sample at 400 times gravity for eight minutes following centrifugation, discarded the supinate and carefully resuspend the pellet in two milliliters of buffer per 80 milligrams of starting tissue. Then add a further 250 microliters of calcium chloride solution, 25 microliters of hyaluronidase solution and 25 microliters of collagenase. Two enzyme solution per 80 milligrams of tissue.
And return the tube to the 37 degrees Celsius shaking water bath for 20 minutes. Remove the tissues from the water bath and repeat as previous incubations following incubation. Centrifuge the sample at 400 times gravity for eight minutes.
Once more and then discard the supinate. The tissue is now ready to be washed in preparation for filtering after the previous centrifugation. Pipette two milliliters of buffer into the tube and triturate tore.
Suspend the pellet palt following resus suspension. Centrifuge the sample at 400 times gravity for eight minutes. Then discard the supinate pipette.
One milliliter of buffer per 80 milligrams of tissue into the tube, and again triturate. To resuspend the pellet. Repeat the centrifugation.
Step at 400 times gravity for eight minutes. During the centrifugation, prepare for the filtration step by placing a 100 micron pore size filter over a 50 milliliter centrifuge tube following centrifugation, discard the supinate and pipette a final one milliliter of buffer per 80 milligrams of tissue into the tube. Using a P 1000 tip with the end cutters before pipette up and down to ensure that the pellet is thoroughly resuspended.
Then transfer the homogenate to the 100 micron pore size filter and allow it to seep through the filter into the 50 milliliter centrifuge tube. It is important not to apply more than three milliliters of homogenate as the filter may become blocked. If this volume is exceeded, use a P 1000 pipette to remove any tissue that has collected on the underside of the filter and transfer this tissue to the 50 milliliter centrifuge tube containing the filtrate.
Using a one milliliter syringe fitted with a 26 gauge needle, aspirate the homogenate from the 50 milliliter centrifuge tube and apply it to the 50 micron pore size filter cap fitted over the five milliliter tube. Allow the homogenate to seep through the filter. If filtering is slow, loosen the filter cap as too tight.
A seal can prevent filtering when all of the homogenate has passed through the filter into the tube. Centrifuge the sample at 400 times gravity for eight minutes. Following centrifugation, the pelleted cells are ready to be plated to plate.
The saliva gland cells begin by resus suspending them in one milliliter of MSG medium per 80 milligrams of tissue. Count the cells according to standard protocols. Here, an automated cell counter is used to determine cell number.
Calculate the volume of cell suspension required for 0.4 times 10 to the six cells, and from this, the number of wells that may be seeded. Add one milliliter of MSG medium per well to a 12 well culture plate. Ensure that the cell suspension is well mixed and add the required volume of cell suspension for 0.4 times 10 to the six cells to each of the wells.
Transfer the plates to a 37 degrees Celsius incubator with 5%carbon dioxide. After two days of incubation, examine the cultures for the presence of SLOs spheres and for lack of ace in our cells. This is an example of mouse saliva gland tissue processed as described and visualized by phase contrast microscopy from zero to 10 days in culture, the small aggregates of cells are SLOs spheres, which grow in size throughout this time period.
Shown here is immuno staining of a representative three to five day SLOs sphere culture for the stem cell marker. Protein C kit. As analyzed by flow cytometry, the expression of sea kit confirms the presence of putative stem cells in the SLOs fare cultures three to five day old SLOs spheres can also be differentiated to form a sin and duct like structures with similar morphology to those in the native saliva gland.
After watching this video, you should have a good understanding of how to isolate saliva glands, stem cells from murine saliva glands.
