Name: Isolation of Mouse Salivary Gland Stem Cells (Video) | JoVE Journal
Uploaded: 2011-02-08T18:00:00.000+00:00
Duration: 9 min 52 s
Description: Learn to isolate mouse salivary gland stem cells using enzymatic and mechanical digestion techniques. This protocol enables culture of salispheres with therapeutic potential for treating Xerostomia and advancing regenerative medicine research.

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5 minutes

1. 	Regent Preparation
<ol>
<li>Buffer: 1 % (w/v) bovine serum albumin in Hank's balanced salt solution</li>
<li>Reconstitute enzymes. Hyaluronidase enzyme: 40mg / mL, dissolved in buffer. Collagenase II: 23mg / mL, dissolved in buffer. Use freshly prepared enzyme solutions fresh for each isolation. When dissolved, store at 4 °C until use for digestion.</li>
<li>50 mM calcium chloride in distilled water. Filter sterilize through a 0.2 uM pore size filter.</li>
<li>Mouse salivary gland (MSG) culture medium: DMEM:F12 with penicillin (100 I.U. / mL), streptomycin (100 μg / mL), glutamax (2 mM), epidermal growth factor-2 (20 ng / mL), fibroblast growth factor-2 (20 ng / mL), N2 supplement (1 %), insulin (10 μg / mL) and dexamethasone (1 μM).</li>
</ol>
2. 	Mechanical and Enzymatic Tissue Digestion
<ol>
<li>Weigh the dissected salivary glands.</li>
<li>Chop glands into a homogenous pulp using sterile curved dissection scissors in a small petri dish.</li>
<li>Collect minced tissue in 14 mL tubes, using 1mL of buffer per 80mg submandibular tissue. Rinse the petri dishes clean of tissue using some of the buffer.</li>
<li>Add another 1 mL of buffer per 80 mg tissue, followed by 25 μL collagenase II enzyme solution, 25 μL hyaluronidase enzyme solution and 250 μL calcium chloride solution per 80 mg tissue. If working with large amounts of tissue, steps 2.4 - 2.9 can be performed in T25 tissue culture flasks for convenience.</li>
<li>Incubate in a shaking waterbath set at 37 °C for 20 minutes. Remove tubes and triturate by pipette to mix enzyme thoroughly through tissue again.</li>
<li>Replace in waterbath for another 20 mins.</li>
<li>Collect tissue by centrifugation at 400 x g, for 8 minutes. Discard supernatant.</li>
<li>Resuspend in 2 mL buffer for each 80 mg tissue, and repeat enzyme and calcium chloride addition as above. Incubate 20 minutes in shaking water bath. Remove tubes and triturate by pipette to mix enzymes thoroughly.</li>
<li>Incubate for final 20 min in shaking water bath. Collect cells by centrifugation as above, discard supernatant.</li>
</ol>
3. 	Washing Steps
<ol>
<li>Resuspend each 80 mg of tissue in 2 mL buffer and pipette to wash tissue free of enzymes.</li>
<li>Centrifuge as previously to collect. Discard supernatant.</li>
<li>Repeat wash using 1 mL buffer per 80 mg tissue. Centrifuge to collect, discard supernatant.</li>
</ol>
4. 	Filtering
<ol>
<li>Resuspend tissue solution in 1 mL buffer per 80 mg tissue.</li>
<li>Add solution to 100 μm pore-size filter placed over 50 mL Falcon tube. Do not apply more than 3 mLs of minced tissue solution per column, as filters may become blocked. Allow to seep through. Remove filtered material hanging on underside of filter by pipette, and add to filtrate.</li>
<li>Use syringe with 26 gauge needle to take filtrate from 50mL tubes and apply to 50 μm pore size filters on 5 mL tubes. Allow to filter through, assisting by loosening lids if necessary.</li>
<li>Centrifuge tubes as previously to collect. Discard supernatant.</li>
</ol>
5. 	Plating and Medium
<ol>
<li>Combine all pellets into one volume. Count using automated cell counter or haemocytometer.</li>
<li>Plate cells at density of 0.4 x 106 cells per well of 12-well plate, or 2.67 x 106 cells per T25 tissue culture flask. Add 1 mL MSG medium to each well or 6 mL to each T25 flask.</li>
<li>Incubate at 37 °C. Spheres should be clearly visible by day 2.</li>
</ol>
6. Representative Results:
After two to three days in culture, small aggregates of cells (salispheres) will be apparent in the cultures. Salispheres will continue to grow in size over a period of ten days in culture. Representative phase contrast microscopy images of salispheres are shown in Figure 1. Proliferating cells expressing stem cell-associated marker proteins can be isolated from these spheres, optimally between days 3-5 post isolation, and are capable of differentiation into functional, saliva producing acinar cells.
<img src="/files/ftp_upload/2484/2484fig1.jpg" alt="Cell growth over 10 days in culture; microscope images day 0, 4, 7, 10; cell cluster formation." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Salisphere formation in vitro. Following mechanical and enzymatic digestion using the present protocol, spheres of increasing size can be found in the floating cultures. Panels are representative phase contrast microscopy images of spheres from days 0 (A), 4 (B), 7 (C) and 10 (D). Scale bar = 50 μm.

<ol><li>Vissink, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oral+Sequelae+of+Head+and+Neck+Radiotherapy%5BTitle%5D)+AND+%22Crit+Rev+Oral+Biol+Med%22%5BJournal%5D)">Oral Sequelae of Head and Neck Radiotherapy.</a> Crit Rev Oral Biol Med. 14, 199-212 (2003).</li>
<li>Nape&ntilde, as, J, J., Brennan, M. T., Fox, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnosis+and+treatment+of+xerostomia+%28dry+mouth%29%5BTitle%5D)+AND+%22Odontology%22%5BJournal%5D)">Diagnosis and treatment of xerostomia (dry mouth).</a> Odontology. 97, 76-83 (2009).</li>
<li>Denny, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parenchymal+cell+proliferation+and+mechanisms+for+maintenance+of+granular+duct+and+acinar+cell+populations+in+adult+male+mouse+submandibular+gland%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Parenchymal cell proliferation and mechanisms for maintenance of granular duct and acinar cell populations in adult male mouse submandibular gland.</a> 235, 475-485 (2005).</li>
<li>Man, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Persistence+of+a+perinatal+cellular+phenotype+in+submandibular+glands+of+adult+rat%5BTitle%5D)+AND+%22J.+Histochem.+Cytochem%22%5BJournal%5D)">Persistence of a perinatal cellular phenotype in submandibular glands of adult rat.</a> J. Histochem. Cytochem. 43, 1203-1215 (1995).</li>
<li>Cotroneo, E., Proctor, G. B., Carpenter, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regeneration+of+acinar+cells+following+ligation+of+rat+submandibular+gland+retraces+the+embryonic-perinatal+pathway+of+cytodifferentiation%5BTitle%5D)+AND+%22Differentiation%22%5BJournal%5D)">Regeneration of acinar cells following ligation of rat submandibular gland retraces the embryonic-perinatal pathway of cytodifferentiation.</a> Differentiation. 79, 120-130 (2010).</li>
<li>Eirew, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+quantifying+normal+human+mammary+epithelial+stem+cells+with+in+vivo+regenerative+ability%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability.</a> Nat Med. 14, 1384-1389 (2008).</li>
<li>Gorjup, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glandular+tissue+from+human+pancreas+and+salivary+gland+yields+similar+stem+cell+populations%5BTitle%5D)+AND+%22European+Journal+of+Cell+Biology%22%5BJournal%5D)">Glandular tissue from human pancreas and salivary gland yields similar stem cell populations.</a> European Journal of Cell Biology. 88, 409-421 (2009).</li>
<li>Alonso, L., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cells+of+the+skin+epithelium%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Stem cells of the skin epithelium.</a> Proc Natl Acad Sci U S A. 100, Suppl 1. 11830-11835 (2003).</li>
<li>Lombaert, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rescue+of+salivary+gland+function+after+stem+cell+transplantation+in+irradiated+glands%5BTitle%5D)+AND+%22Plos+One%22%5BJournal%5D)">Rescue of salivary gland function after stem cell transplantation in irradiated glands.</a> Plos One. 3, e2063-e2063 (2008).</li>
<li>Coppes, R. P., Goot, A. vander, Lombaert, I. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+Cell+Therapy+to+Reduce+Radiation-Induced+Normal+Tissue+Damage%5BTitle%5D)+AND+%22Seminars+in+Radiation+Oncology%22%5BJournal%5D)">Stem Cell Therapy to Reduce Radiation-Induced Normal Tissue Damage.</a> Seminars in Radiation Oncology. 19, 112-121 (2009).</li>
</ol>

No conflicts of interest declared.

The tissue culture method described here represents a reproducible protocol for the isolation of stem cell-containing salispheres from the salivary glands of mice. Studies using cells isolated in this manner have highlighted the regenerative capacity of salivary gland stem cells 9. Transplantation of one hundred of c-Kit+ cells derived from the salispheres induced functional recovery of irradiated mouse salivary glands. These data are exciting and provide a starting point for the investigation of stem-cell based therapy for xerostomia. Many avenues remain to be explored however, including the full marker protein expression profile of the stem cells, the ability of submandibular glands to rescue the function of irradiated parotid salivary glands and vice versa, and the characterisation of the putative in vivo stem cell niche of the cells. Ultimately, the translation of this protocol to human tissue samples and the subsequent potential for the therapy of xerostomia in human patients using the isolated cells is the most exciting application of the described method.

<table><tbody><tr><td>Hyaluronidase</td><td>Sigma-Aldrich</td><td>H3506</td><td>Store at &amp;#8211; 20 &amp;#x00B0;C</td></tr><tr><td>Collagenase II</td><td>GIBCO, by Life Technologies</td><td>17101-015</td><td>Store at 4 &amp;#x00B0;C</td></tr><tr><td>Epidermal Growth Factor-2</td><td>Sigma-Aldrich</td><td>E9644</td><td>Make a stock of 10 &amp;#x03BC;g / mL in phosphate buffered saline (PBS). Store at &amp;#8211; 20 &amp;#x00B0;C in single use aliquots.</td></tr><tr><td>Fibroblast Growth Factor-2</td><td>Sigma-Aldrich</td><td>F0291</td><td>Make stock of 25 &amp;#x03BC;g / mL in PBS. Store at &amp;#8211; 20 &amp;#x00B0;C in single use aliquots.</td></tr><tr><td>N2 supplement</td><td>GIBCO, by Life Technologies</td><td>17502-048</td><td>As manufacturer instructions.</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td><td>Make stock of 2 mg / mL in tap water. Adjust water to pH 2-3 using glacial acetic acid prior to dissolving.</td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td></td></tr><tr><td>100 &amp;mu;M pore-size sterile cell strainers</td><td>BD Biosciences</td><td>352360</td><td></td></tr><tr><td>Polystyrene round-bottomed tubes with cell strainer caps (50 &amp;mu;M pore size)</td><td>BD Biosciences</td><td>352235</td><td></td></tr></tbody></table>

isolation of mouse salivary gland stem cells

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

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.

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Learn to isolate mouse salivary gland stem cells using enzymatic and mechanical digestion techniques. This protocol enables culture of salispheres with therapeutic potential for treating Xerostomia and advancing regenerative medicine research.

Isolation of Mouse Salivary Gland Stem Cells

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