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 therapy^3,4,5,6. 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 saliva^7,8. 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 glands^9,10. 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 size^1,2,11. 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 procedures^13,14. 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

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 <…

Representative Results

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…

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 glands^3,4,5,16. 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…

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