Name: isolation of salivary epithelial cells from human salivary glands for in vitro growth as salispheres or monolayers
Uploaded: 2019-07-15T13:00:00.000+00:00
Duration: 8 min 3 s
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
<ol>
	<li>Bronchial epithelial cell growth medium (BEGM)
	<ol>
		<li>Add components from the kit provided by manufacturer (see the Table of Materials).</li>
		<li>Wash each tube once with the base media to ensure full transfer of components. Then add charcoal stripped fetal bovine serum to make a final concentration of 4% serum.</li>
	</ol>
	</li>
	<li>Red blood cell (RBC) lysis buffer
	<ol>
		<li>To make 10x stock, add 40.15 g of NH4Cl, 5.0 g of NaHCO3, and 0.186 g of ethylenediaminetetraacetic acid (EDTA) and bring up to a final volume of 200 mL using dH2O. Then filter sterilize and store at 4 &#176;C.</li>
		<li>Dilute to a working concentration of 1x in sterile dH2O prior to use.</li>
	</ol>
	</li>
	<li>Dissociation solution
	<ol>
		<li>To a 100 mL bottle of dispase solution (see the Table of Materials) add 0.15 g of collagenase type III. After collagenase has fully dissolved, filter sterilize the new solution and aliquot. Store at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Tissue Digestion
<ol>
	<li>Using a 100 mm tissue culture plate, mechanicallymince the tissue into fine pieces ~1-2 mm in size using autoclave-sterilized dissecting scissors and surgical forceps (Figure 1B,C). Connective tissue between pieces should be cut to ensure proper separation and ease in further steps.</li>
	<li>Add 6 mL of the dispase/collagenase solution to the minced tissue. Then incubate at 37 &#176;C for approximately 30 min to 1 h.</li>
	<li>Disrupt the tissue by pipetting with a 5 mL serological pipette 15-20 times.</li>
	<li>Incubate at 37 &#176;C for another 30 min to 1 h.</li>
	<li>Repeat steps 2.3 and 2.4 two-three more times or until the tissue resembles a slurry and can easily pass through the pipette opening (Figure 1D). 
	NOTE: The starting size of the tissue specimen and how fineness of the tissue mincing will determine how many times one needs to repeat steps 2.3 and 2.4. Typically, we receive tissue approximately 1 cm x 1 cm in size. Additionally, one can monitor tissue dissociation using a standard inverted tissue culture microscope to track the progress of cell dissociation from the tissue. Small clusters of cells are ideal for initial outgrowth of salivary cells as salispheres.</li>
</ol>
3. Coating wells with basement membrane matrix
NOTE: Basement membrane matrix (BMM) should be thawed overnight at 4 &#176;C the day before it will be used. Once thawed, BMM should be constantly maintained on ice or at 4 &#176;C as it will rapidly solidify (i.e., form a gel) at warmer temperatures. Additionally, care should be taken if samples have been chilled on ice prior to plating on BMM as cold samples will &#34;melt&#34; the BMM and expose cells to the plastic dish.
<ol>
	<li>Slowly pipette BMM (see the Table of Materials) into each well to be coated. Evenly and slowly distribute the BMM over the well. 
	&#8203;NOTE: Generally, we use 500 &#181;L per each well of a six-well plate, but this volume can be adjusted for use on other variants of plates such as 12-well or 24-well, or as desired for thicker or thinner hydrogels.</li>
	<li>Incubate the coated plates at 37 &#176;C for at least 15 min prior to use to allow the BMM time to solidify.</li>
</ol>
4. Filtering of undigested tissue, RBC lysis, and cell plating
<ol>
	<li>Transfer tissue homogenate from step 2.5 to a 15 mL conical tube. Wash plate once with 6 mL of Dulbecco&#39;s phosphate buffered saline (DPBS) to transfer remaining cells.</li>
	<li>Filter the homogenate into a 50 mL conical tube through a 70 &#181;m nylon mesh cell strainer to remove undigested tissue. Centrifuge strained cells for 5 min at 500 x g.</li>
	<li>Dilute 10x RBC lysis buffer in sterile dH2O to make a 1x working solution.</li>
	<li>Aspirate the supernatant. Then resuspend the cell pellet in 10 mL of 1x RBC lysis buffer. Incubate for 5 min at 37 &#176;C.</li>
	<li>Add 20-25 mL of DPBS to neutralize the RBC lysis buffer and minimize lysis of salivary cells. Centrifuge for 5 min at 500 x g. 
	NOTE: After treatment with RBC lysis buffer, the cell pellet should be white. Red color in the pellet indicates that not all RBC have been fully lysed. Repeat steps 4.4&#160;through 4.5&#160;if needed for complete lysis of all RBC.</li>
	<li>Aspirate the supernatant. Resuspend the pellet in 1-2 mL of BEGM per well then place resuspended cells onto BMM-coated wells. Incubate at 37 &#176;C.</li>
	<li>Once plated on BMM, cells will form into spherical structures or &#34;salispheres&#34; over a 2-3-day period. Cells can be maintained as salispheres for about 5-7 days before the BMM begins to degrade allowing the cells to access and adhere to the plastic and grow as a monolayer.</li>
</ol>
5. Subculturing the salispheres and maintenance on BMM
<ol>
	<li>Aspirate media from wells, then add 1 mL of dispase/collagenase solution to each well and incubate at 37 &#176;C for ~15 min or until BMM has mostly dissolved.</li>
	<li>Transfer cells to a 15 mL conical tube and wash wells once with DPBS to obtain remaining cells. Centrifuge for 5 min at 500 x g.</li>
	<li>Aspirate the supernatant. Resuspend the pellet in 2 mL of trypsin then incubate at 37 &#176;C for 15 min.</li>
	<li>Neutralize the trypsin using any complete media containing 10% serum. Centrifuge for 5 min at 500 x g.</li>
	<li>Aspirate the supernatant. Resuspend the cells in DPBS to remove residual media, trypsin, and serum. Centrifuge for 5 min at 500 x g.</li>
	<li>Aspirate the supernatant. Resuspend in BEGM and plate the resuspended cells onto solidified BMM-coated culture dishes. Resuspended cells can also be plated directly onto cell culture treated dishes for proliferation as a monolayer. For more information regarding growth of salivary cells as a monolayer, see section 6. Cells grown on BMM or on plastic should be fed with fresh BEGM every 2&#8211;3 days.</li>
	<li>Culture the cells in a humidified incubator maintained at 37 &#176;C with 5% CO2.</li>
</ol>
6. Subculturing the salispheres on treated plastic tissue culture dishes
NOTE: If maintaining cells as a monolayer on plastic, start at this step. The following protocol applies to cells growing in a 100 mm dish.
<ol>
	<li>Aspirate the media. Wash once with DPBS to remove residual media.</li>
	<li>Add 2 mL of trypsin then incubate at 37 &#176;C for 15 min, or until cells have fully detached.</li>
	<li>Neutralize trypsin using 4 mL of any complete media containing 10% serum. Transfer cells to a 15 mL conical tube.</li>
	<li>Wash the plate once with approximately 5 mL of DPBS to collect remaining cells. Centrifuge for 5 min at 500 x g.</li>
	<li>Aspirate the supernatant. Resuspend cells in 6-10 mL of DPBS to remove residual media.</li>
	<li>Centrifuge for 5 min at 500 x g.</li>
	<li>Aspirate the supernatant and resuspend in BEGM. Plate cells onto treated plastic tissue culture dishes. Feed with fresh BEGM every 2-3 days.</li>
	<li>Culture the cells in a humidified incubator maintained at 37 &#176;C with 5% CO2.</li>
</ol>
7. Cryopreservation of cells
NOTE: Cryopreservation of cells can be accomplished from a single cell suspension originating from either salisphere or monolayer grown cells.
<ol>
	<li>Count the cells on a hematocytometer to calculate total amount of cells present.</li>
	<li>Centrifuge for 5 min at 500 x g.</li>
	<li>Aspirate the supernatant. Resuspend the pellet in BEGM with 10% dimethyl sulfoxide (DMSO). Concentration of cells should be 2 x 106 cells/mL to 10 x 106 cells/mL.</li>
	<li>Aliquot cells into sterile cryogenic storage tubes. Place the tubes in an insulated foam rack and incubate at -80 &#176;C overnight to slowly freeze the cells.</li>
	<li>On the next day, place tubes in liquid nitrogen for long term storage. 
	NOTE: Cells should be rapidly thawed at 37 &#176;C. Upon thawing, cells should be pelleted at 500 x g and the DMSO-containing media removed prior to plating.</li>
</ol>

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The authors report nothing to disclose.

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 affected tissue9. Additionally, salivary glands are often a site for persistence and transmission of human pathogens. For example, human herpesviruses such as human cytomegalovirus (HCMV) are known to infect and use the salivary glands and saliva to transmit virus to new hosts7,8,17. The molecular mechanisms underlying viral persistence in the salivary gland and movement to saliva remain unknown. The development of in vitro salivary cell systems will provide an essential model system to explore this mechanistic information.
We report here a robust protocol for generating primary salivary &#34;salispheres&#34; that can be grown in vitro either as clusters on BMM or as monolayers on plastic. The ability to culture and propagate primary human salivary gland epithelial cells represents an important platform on which researchers can (1) potentially develop replacement therapies for patients with salivary gland deficiencies for whom no other options exist; (2) better understand the basic physiology underlying salivary gland development, proliferation, secretion, etc.; and (3) define mechanisms used by pathogens to replicate and spread to new hosts.
Using these salivary cell cultures, we have reported that HCMV requires the pentameric glycoprotein complex for primary infection and also that the virus persists for an extended period, reminiscent of what occurs with HCMV in vivo7. Moreover, our functional studies revealed that the salivary cultures do not contain contaminating fibroblasts as fibroblast tropic, lab adapted viruses fail to infect and replicate in the primary salivary derived cells7. While we have used this protocol to generate cells for the evaluation of HCMV replication and spread in a primary salivary system, this protocol could readily be adapted to generate salivary cells useful for the wide range of research topics mentioned above. Additionally, this protocol could be adapted to suit the needs and budget of each individual researcher. While we have not validated other conditions ourselves, others have reported success growing salivary epithelial cells under different media conditions. For example, Dulbecco&#39;s modified Eagle&#39;s medium (DMEM):F12 mixture supplemented with epidermal growth factor and fibroblast growth factor-2 has been reported to promote robust growth of salivary cells18. Alternatively, Nam et al. use minimal essential media supplemented with fetal bovine serum before transferring the cells to a serum-free version of a keratinocyte growth media and have reported robust growth of salivary cells on plastic15. Thus, it does not appear that the salivary epithelial cells exhibit an absolute requirement for one particular media type, but rather appear to grow and proliferate on a number of commercially available media types.
In addition to media formulation, different basement membrane formulations could be evaluated for their abilities to support robust salivary epithelial cell growth. Growth of cells on commercially available basement membrane-containing matrixes readily yield salispheres and provides a simple, albeit expensive culturing system7,19. Similar to that discussed above concerning choice of media for cell growth, researchers have reported growth and development of salispheres on a number of different matrices and hydrogel systems. Hydrogels made from hyaluronic acid have the added benefit in creating an extracellular matrix crosslinked with desired chemical constituents to study their interactions and effects on cells. Using primary human tissue derived salivary cells, Srinivasan et al. reported success in growth and maintenance of a salivary progenitor cell population in hyaluronic acid hydrogels; increased progenitor cell growth was further observed when the hydrogels were incorporated with peptides derived from the basement membrane proteins such as perlecan and laminin20. Another system recently developed by Foraida et al. report using &#34;electrospinning&#34; to develop a nanofiber scaffold made of poly(lactic-co-glycolic acid) (PLGA). The PLGA hydrogels with added elastin were found to readily promote the development of a polarized submandibular cell system, which may be useful in ascertaining how cell polarization influences salivary biology and biochemistry21.
In this protocol, we have presented a simple method for the isolation and growth of primary salivary gland-derived epithelial cells. This protocol results in the rapid outgrowth of an epithelial cell population that can be maintained on average for 5-8 passages. It is critical to monitor the tissue during treatment with dispase/collagenase to ensure appropriate digestion as mentioned in the protocol. Incomplete digestion will severely diminish the number of salispheres that survive. It is also important to monitor the cultures for the outgrowth of fibroblasts that have an elongated shape and are very distinct from the polygonal epithelial cells that typically grow in discrete patches or clumps. We have used this protocol for the isolation of epithelial cells from humans and mice, but it seems likely that it could be adapted for use with other mammalian systems. Moreover, this protocol could be further modified by using additional purification steps based on cell surface marker expression and flow cytometry that could lead to the generation of more homogenous initial cell populations. Similarly, this protocol could be used to determine how different media or matrix/hydrogels formulation lead to the outgrowth of specific cell types when first generating the salispheres. In conclusion, this protocol should serve as a robust starting platform for the isolation and growth of primary salivary cells which can readily be adapted for a wide variety of experimental protocols and research areas.

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><tbody><tr><td>100 mm culture dishes</td><td>Thermo Scientific</td><td>172931</td><td></td></tr><tr><td>15 mL conical tubes</td><td>Thermo Scientific</td><td>339651</td><td></td></tr><tr><td>50 mL conical tubes</td><td>Thermo Scientific</td><td>339653</td><td></td></tr><tr><td>Bronchial Epithelial Cell Growth Media</td><td>Lonza</td><td>CC-3171</td><td>Add bullet kit as per manufacturer&#039;s instructions. Supplement with 20 mL of charcoal stripped serum.</td></tr><tr><td>Cell strainer 70 &amp;micro;m nylon mesh</td><td>Fisher</td><td>22-363-548</td><td></td></tr><tr><td>Charcoal stripped fetal bovine serum</td><td>Gibco</td><td>12676-029</td><td></td></tr><tr><td>Collagenase type III</td><td>Worthington</td><td>LS004182</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Cryogenic Tube</td><td>Fisher</td><td>5000-0020</td><td></td></tr><tr><td>Dispase</td><td>Cell Applications</td><td>07923</td><td>Dissolve collagenase to make a 0.15% (w/v) stock. Filter sterilize then store at -20 &amp;deg;C.</td></tr><tr><td>Dissecting scissors</td><td>Fisher</td><td>08-940</td><td></td></tr><tr><td>Dulbecco phosphate buffered saline</td><td>Corning</td><td>55-031-PC</td><td></td></tr><tr><td>General Chemicals</td><td>Sigma</td><td></td><td></td></tr><tr><td>PathClear Basement membrane extract</td><td>Cultrex</td><td>3432-005-01</td><td>Thaw at 4 &amp;deg;C at least 24 hr prior to use. Always handle on ice.</td></tr><tr><td>Six-well culture dishes</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>Surgical forceps</td><td>Fisher</td><td>22-079-742</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Corning</td><td>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 breakdown of the BMM, cells will become attached to the underlying plastic and begin to proliferate as a monolayer. Salispheres grown on BMM will exhibit variability in size and structural complexity. Salispheres can be maintained by passing them onto freshly prepared BMM as described in the protocol. Within 2-3 days after passing onto fresh BMM, the cells will reform into spheres. Salisphere cells can also be plated onto cell culture-treated plastic and grown as a monolayer where they exhibit morphology consistent with cells of epithelial origin as shown in Figure 2B. While the salisphere cells grown on BMM as salispheres are likely to maintain a phenotype more characteristic of salivary tissue, the cells grown as a monolayer can be advantageous for some experimental protocols. More extensive characterization will need to be performed to determine to what extent the cells maintain a salivary phenotype when grown on a monolayer as compared to cells grown as salispheres.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59868/59868fig1.jpg" /> 
Figure 1: Initial processing of human submandibular glands for salisphere preparation. Excised salivary gland tissue approximately 1 cm in diameter (A) is mechanically minced into smaller pieces using sharp scissors (B) until the gland is separated into digestible pieces of approximately 1-2 mm in size (C). The glandular pieces are then incubated in the dispase/collagenase solution for 30-90 min with periodic passage through serological pipettes until the sample is digested to mostly single cells or small clumps of cells (D). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59868/59868fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59868/59868fig2.jpg" /> 
Figure 2: Primary human salisphere cells can be cultured as spheres on the basement membrane matrix or as a monolayer on treated cell culture dishes. Following initial digestion of salivary tissue, the digested glands are plated directly onto BMM and cultured for 3-10 days. Cells are fed every 2-3 days to provide fresh nutrients. Once the primary salispheres develop, the matrices can be digested in dispase/collagenase solution, trypsinized to generate single cells, and re-plated onto fresh BMM where they reform into spheres (A) or plated onto cell culture dishes where they readily form a cobblestone morphology typical of an epithelial monolayer (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59868/59868fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Anatomy and Physiology

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Tissues of the Human Body

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8.8K Views.  University of Cincinnati College of Medicine. Het genereren van primaire cellulaire modellen of organoïde-achtige structuren is de sleutel tot het beantwoorden van veel hedendaagse vragen in de biologie. We zijn bijvoorbeeld al geruime tijd geïnteresseerd in de mechanismen die door menselijke virussen worden gebruikt om replicatie in de speekselklier te vergemakkelijken, omdat het een belangrijke plaats is voor horizontale transmissie. De ontwikkeling van het in dit protocol beschreven salisfeergebaseerde systeem is een cruciale voorui...

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

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Developmental Biology

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 ...

Biology

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

Bioengineering

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50&#8211;90 &#956;m thick salivary gland sections prior to and following exposure to ionizing radiation.

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with ...

Medicine

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...

Protocol for Isolating Mammary Epithelial Cells from Human Tissue

Isolation and Culture of Primary Human Mammary Epithelial Cells

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

Author Spotlight: Integrating Eastern and Western Medicine for Treatment of Granulomatous Mastitis

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...

Culturing Salispheres from Salivary Gland Tissue: A Method to Develop Salispheres from Human Salivary Gland Derived Epithelial Cells

Source: Beucler, M. J., Miller, W. E. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59868" target="_blank">Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers.</a> J. Vis. Exp. (2019)
This video presents the protocol for culturing salispheres, the three-dimensional spheroids derived from salivary glands. The salispheres can be further used to assess the impact of head and neck cancer on saliva production from salivary glands of affected human patients.

Source: Beucler, M. J., Miller, W. E. Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers. ...

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

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