Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers
Summary
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.
Abstract
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 Sjögren’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.
Introduction
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 Sjögren'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.'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'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 >50 primary human tissue samples using this methodology. Moreover, the primary salivary cells can easily be cryopreserved for use at a later date.
Protocol
Representative Results
Discussion
Salivary dysfunction represents a concern for the quality of life for those suffering from Sjögren'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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 100 mm culture dishes | Thermo Scientific | 172931 | |
| 15 mL conical tubes | Thermo Scientific | 339651 | |
| 50 mL conical tubes | Thermo Scientific | 339653 | |
| Bronchial Epithelial Cell Growth Media | Lonza | CC-3171 | Add bullet kit as per manufacturer's instructions. Supplement with 20 mL of charcoal stripped serum. |
| Cell strainer 70 µm nylon mesh | Fisher | 22-363-548 | |
| Charcoal stripped fetal bovine serum | Gibco | 12676-029 | |
| Collagenase type III | Worthington | LS004182 | Store at 4 °C. |
| Cryogenic Tube | Fisher | 5000-0020 | |
| Dispase | Cell Applications | 07923 | Dissolve collagenase to make a 0.15% (w/v) stock. Filter sterilize then store at -20 °C. |
| Dissecting scissors | Fisher | 08-940 | |
| Dulbecco phosphate buffered saline | Corning | 55-031-PC | |
| General Chemicals | Sigma | ||
| PathClear Basement membrane extract | Cultrex | 3432-005-01 | Thaw at 4 °C at least 24 hr prior to use. Always handle on ice. |
| Six-well culture dishes | Falcon | 353046 | |
| Surgical forceps | Fisher | 22-079-742 | |
| Trypsin-EDTA solution | Corning | 25-052-CI |
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