Name: isolation of salivary epithelial cells from human salivary glands for in vitro growth as salispheres or monolayers
Uploaded: 2019-07-15T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers at JoVE.com

We would like to thank James P. Bridges for providing significant guidance on the methodologies involved for culturing primary cells. Matthew J. Beucler was supported by National Institutes of Health Training Grant T32-ES007250. This work was also supported by National Institutes of Health grants R01-AI121028 and R21-DE026267 awarded to William E. Miller.

These protocols and studies have been reviewed and approved by the Institutional Review Board at the University of Cincinnati (Federal-wide Assurance #00003152, IRB protocol 2016-4183). Salivary tissue is commonly resected in many head and neck surgical procedures and is usually uninvolved by the malignant process. Typically, freshly resected salivary gland tissue is used within 2-4 h after removal from the patient (Figure 1A).
1. Reagent Preparation
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	<li>Kessler, A. T., Bhatt, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+Major+and+Minor+Salivary+Glands,+Part+1:+Anatomy,+Infectious,+and+Inflammatory+Processes.">Review of the Major and Minor Salivary Glands, Part 1: Anatomy, Infectious, and Inflammatory Processes.</a> Journal of Clinical Imaging Science. 8, 47 (2018).</li><li>Holmberg, K. V., Hoffman, M. P., Ligtenberg, A. J. M., Veerman, E. C. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-contamination+of+the+human+salivary+gland+HSG+cell+line+with+HeLa+cells:+A+STR+analysis+study.">Cross-contamination of the human salivary gland HSG cell line with HeLa cells: A STR analysis study.</a> Oral Diseases. 24, 1477-1483 (2018).</li><li>Tran, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Culture+of+Polarized+Human+Salivary+Epithelial+Cells+for+Use+in+Developing+an+Artificial+Salivary+Gland.">Primary Culture of Polarized Human Salivary Epithelial Cells for Use in Developing an Artificial Salivary Gland.</a> Tissue Engineering. 11, 172-181 (2005).</li><li>Pringle, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Mouse+Salivary+Gland+Stem+Cells.">Isolation of Mouse Salivary Gland Stem Cells.</a> Journal of Visualized Experiments. , (2011).</li><li>Nam, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Primary+Epithelial+Cells+Derived+from+Human+Salivary+Gland+Contributing+to+in+vivo+Formation+of+Acini-like+Structures.">Characterization of Primary Epithelial Cells Derived from Human Salivary Gland Contributing to in vivo Formation of Acini-like Structures.</a> Molecules and Cells. 41, 515-522 (2018).</li><li>Vissink, A., Jansma, J., Spijkervet, F. 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Salivary dysfunction represents a concern for the quality of life for those suffering from Sj&#246;gren&#39;s syndrome as well as those undergoing radiation therapy for cancers adjacent to the salivary glands3,4,5,16. One proposed therapy to treat these patients is to grow functional salivary stem cells or organoids in vitro, which can then be inserted into damaged salivary gland to replace aff...

In mammals the salivary glands are organized into 3 pairs of major salivary glands: the parotid, submandibular, and sublingual glands. There also exists a series of minor glands located on the tongue and scattered throughout the oral cavity1. The major glands are responsible for the bulk volume of saliva produced2. In addition to providing antimicrobial protection through secreted factors, the saliva is important in the process of chewing and lubricating food as it passes through the esophagus2. As such, salivary gland dysfunction represents a medical issue where sufferers who are unable to produce enough saliva are more prone to developing maladies such as dental cavities or oral candidiasis3. Additionally, reduced saliva results in greater difficulty eating and digesting food having a significant impact on quality of life.
Salivary gland dysfunction is primarily found in patients suffering from Sj&#246;gren&#39;s syndrome, an autoimmune disorder, or in patients with head and neck cancer who have undergone irradiation therapy3,4,5,6. Damaged secretory acini within the glands as a result of autoimmunity or radiation therapy do not undergo self-renewal, which results in a patient living with irreversible damage6. The study of salivary glands has also garnered the attention or researchers interested in mechanisms of viral pathogenesis. Some pathogens, such as human cytomegalovirus (HCMV), persistently replicate within the salivary glands, which then allows for transmission of nascent virus to a new host via saliva7,8. Thus, there is an unmet need to develop robust and reproducible in vitro models of salivary gland tissue to tackle and understand a diverse array of medical problems.
Several models of the murine salivary gland system have been used as a starting point to understand and characterize the different epithelial cells that form the salivary glands9,10. There exist obvious and major differences between human and murine salivary glands11. Most notably the human parotid gland is larger in relation to the submandibular gland whereas the mouse submandibular and parotid are much similar in size1,2,11. While immortalized human submandibular cell lines exists, there are major concerns that the immortalized cells do not accurately maintain the phenotype of the primary tissue and secondary concerns that the immortalized cells are not actually derived from salivary epithelium itself12. For these reasons there is an interest and a need to study primary salivary tissue from humans.
Here we describe a protocol to isolate primary epithelial cells from human salivary glands for tissue culture. Our protocol is based on the works of Pringle et al. and Tran et al. with modifications made to generally simplify their procedures13,14. First, while Tran et al.&#39;s protocol calls for a long five-hour incubation for enzymatically digesting the salivary tissue, our modified protocol has been successful with as little as one-hour incubation, similar to that in Pringle et al. Second, we use different enzymes for tissue digestion, most notably we don&#39;t use trypsin as is used in the original Tran et al. protocol, but instead use a mixture of dispase and collagenase. We prefer to avoid trypsin in the initial digestion steps as a recent study suggested that initial digestion of the salivary tissue by trypsin results in reduced cell viability, possibly by the loss of a rare stem cell population15. Lastly, we culture the salispheres on the surface of basement membrane matrix (BMM) using a commercially available media originally formulated for the propagation of bronchial epithelial cells. Our protocol can be used to isolate salivary cells from parotid, submandibular, and sublingual glands. These cells express salivary amylase and other salivary specific genes indicating that they maintain a phenotype similar to that of salivary acinar epithelial cells7. We have successfully generated salivary cultures from &#62;50 primary human tissue samples using this methodology. Moreover, the primary salivary cells can easily be cryopreserved for use at a later date.

<table border="0" cellpadding="0" cellspacing="0" style="width:736px;" width="736">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">100 mm culture dishes</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:123px;">172931</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">15 mL conical tubes</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:123px;">339651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">50 mL conical tubes</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:123px;">339653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Bronchial Epithelial Cell Growth Media</td>
			<td style="width:132px;">Lonza</td>
			<td style="width:123px;">CC-3171</td>
			<td>Add bullet kit as per manufacturer&#39;s instructions. Supplement with 20 mL of charcoal stripped serum.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Cell strainer 70 &#181;m nylon mesh</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:123px;">22-363-548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Charcoal stripped fetal bovine serum</td>
			<td style="width:132px;">Gibco</td>
			<td style="width:123px;">12676-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Collagenase type III</td>
			<td style="width:132px;">Worthington</td>
			<td style="width:123px;">LS004182</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Cryogenic Tube</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:123px;">5000-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Dispase</td>
			<td style="width:132px;">Cell Applications</td>
			<td style="width:123px;">07923</td>
			<td>Dissolve collagenase to make a 0.15% (w/v) stock. Filter sterilize then store at -20 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Dissecting scissors</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:123px;">08-940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Dulbecco phosphate buffered saline</td>
			<td style="width:132px;">Corning</td>
			<td style="width:123px;">55-031-PC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">General Chemicals</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">PathClear Basement membrane extract</td>
			<td style="width:132px;">Cultrex</td>
			<td style="width:123px;">3432-005-01</td>
			<td>Thaw at 4 &#176;C at least 24 hr prior to use. Always handle on ice.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Six-well culture dishes</td>
			<td style="width:132px;">Falcon</td>
			<td style="width:123px;">353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Surgical forceps</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:123px;">22-079-742</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Trypsin-EDTA solution</td>
			<td style="width:132px;">Corning</td>
			<td style="width:123px;">25-052-CI</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of salivary epithelial cells from human salivary glands for in vitro growth as salispheres or monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

Two-three days after plating cells from digested tissue onto BMM, cells will readily form small clusters that will continue to expand in size up to 15-20 cells per cluster (Figure 2A). Cellular debris and detached dead cells are typically seen and should be removed by aspirating and replenishing with fresh media. Cells will continue to proliferate as salispheres for about 3-10 days, or as long as the BMM layer remains intact. Occasionally, due to partial brea...

Watch this Scientific Journal Video about Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers at JoVE.com

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Generating primary cellular models or organoid-like structures is key to answering many contemporary questions in biology. For example, we've been interested for quite some time in the mechanisms used by human viruses to facilitate replication in the salivary gland as it is an important site for horizontal transmission. The development of the salisphere-based system described in this protocol is a crucial advancement in our quest to answer these types of questions.This technique allows us to generate primary cells from a variety of different patient samples. The cellular models are more reminiscent of the in vivo type situation as the cells are not modeled or transformed like many cell types commonly used for in vitro experiments. We've used this protocol to generate similar cells from mouse primary submandibular gland and the protocol would likely be useful for other organs with similar cellular structures.Within two to four hours after removal from the patient, on a culture plate, use autoclaved sterilized dissecting scissors and surgical forceps to mince the salivary gland tissue into fine pieces at the size of one to two millimeters. Add six milliliters of the Dispase/Collagenase solution to the minced tissue. Then incubate at 37 degrees Celsius for approximately 30 minutes to one hour.Disrupt the tissue by pipetting with a five milliliter serological pipette 15 to 20 times. Incubate at 37 degrees Celsius for another 30 minutes to one hour. Repeat the pipetting and incubation step two to three more times or until the tissue resembles a slurry and can easily pass through the pipette opening.To coat wells with BMM, slowly pipette 500 microliters of overnight thawed BMM into each well of a six well plate. Slowly swirl the plate to evenly distribute the BMM over the wells. Then incubate the coated plates at 37 degrees Celsius for at least 15 minutes prior to use to allow the BMM to solidify.First, transfer the Dispase/Collagenase solution containing homogenized tissue to a 15 milliliter conical tube. Wash the wall of the plate once with six milliliters of DPBS to transfer the remaining cells into the conical tube. Then through a 70 micrometer nylon mesh cell strainer, filter the homogenate into a new 50 milliliter conical tube to remove undigested tissue.Centrifuge strained cells for five minutes at 500 times g. Aspirate the supernatant. Then resuspend the cell pellet in 10 milliliters of 1X RBC lysis buffer.Incubate for five minutes at 37 degrees Celsius. Then add 20 to 25 milliliters of DPBS to neutralize the RBC lysis buffer and minimize lysis of salivary cells. Centrifuge for five minutes at 500 times g.White cell pellet indicates complete lysis of all RBC. If the pellet has red color, repeat treatment with RBC lysis buffer. Aspirate the supernatant and resuspend the pellet in one to two milliliters of BEGM and then transfer the resuspended cells onto BMM coated wells.Incubate at 37 degrees Celsius. Once plated on BMM, cells will form into spherical structures or salispheres over a two to three-day period. Cells can be maintained as salispheres for about five to seven days before the BMM begins to degrade allowing the cells to access and adhere to the plastic and grow as a monolayer.First, aspirate the media from the wells and add one milliliter of Dispase/Collagenase solution to each well. Incubate at 37 degrees Celsius for approximately 15 minutes or until BMM has mostly dissolved. Then transfer the cells to a 15 milliliter conical tube and wash wells once with one to three milliliters of DPBS to obtain the remaining cells.Centrifuge for five minutes at 500 times g. Aspirate the supernatant and resuspend the pellet in two milliliters of Trypsin. Incubate again at 37 degrees Celsius for 15 minutes.Next, add in to the tube any complete media containing 10%serum to neutralize the Trypsin. Centrifuge for five minutes at 500 times g. Aspirate the supernatant to remove residual media, Trypsin and serum and then resuspend the cells in DPBS.Centrifuge for five minutes at 500 times g. Aspirate the supernatant and resuspend the pellet in BEGM. To maintain the pellet as spheres, plate the resuspended cells onto solidified BMM coated culture dishes.Culture all cells in a humidified incubator maintained at 37 degrees Celsius with 5%carbon dioxide for five to seven days prior to subculturing. Feed with fresh BEGM every two to three days. To proliferate the cells as a monolayer onto plastic culture dishes, aspirate the BEGM media from the dish and wash once with DBPS to remove residual media.Add two milliliters of Trypsin and incubate at 37 degrees Celsius for 15 minutes or until cells have fully detached. Neutralize Trypsin using four milliliters of any complete media containing 10%serum. Then gently tilt the dish and transfer cells to a 15 milliliter conical tube.Wash the plate once with approximately five milliliters of DPBS to collect the remaining cells. Centrifuge for five minutes at 500 times g. Aspirate the supernatant.To remove residual media, resuspend cells in six to 10 milliliters of DPBS. Centrifuge for five minutes at 500 times g. Aspirate the supernatant and resuspend in BEGM.Then plate the cells onto the treated plastic tissue culture dishes. Culture the cells in a humidified incubator maintained at 37 degrees Celsius with 5%carbon dioxide for five to seven days prior to subculturing. Feed with fresh BEGM every two to three days.In this protocol, after plating cells from the digested tissue onto BMM for two to three days, cells readily formed small clusters that continued to expand in size up to 15 to 20 cells per cluster. Salisphere cells were plated onto cell cultured treated plastic and grown as a monolayer where they exhibit morphology consistent with cells of epithelial origin. Proper aseptic technique and care is needed to avoid bacterial or fungal contamination.No amount of antibiotics or antimycotics will save a culture swarming with microbes. We are answering questions about viral pathogenesis and transmission. But for others, this provides a way to study human salivary cells for any number of applications such as tissue regeneration therapies.For us who study human Cytomegalovirus, this technique has allowed us to begin exploring the molecular mechanisms the virus utilizes to infect and ultimately transmit to new hosts. Nothing used in this protocol is inherently hazardous but caution should always be taken when handling human tissue and any sort of chemical reagents.

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Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...