**1. Introduction**

Development of restorative therapies for salivary gland dysfunction has been significantly hampered by a lack of accessible and pertinent cell culture models. Salivary glands are made of several cell types, including the saliva-producing acinar cells, ductal cells modifying the saliva as it travels through the lumen, and myoepithelial cells mediating acinus contraction. Acinar cells commonly fail to regenerate following high-dose radiation therapy, aging, or from autoimmune exocrinopathy such as primary Sjögren's syndrome (pSS) [1–3]. pSS is a chronic autoimmune disease characterized by a loss of salivary acinar cell function and a decline in acinar progenitor cell populations exacerbated by lymphocytic glandular infiltration.

Dysfunction of the salivary glands can lead to a reduction in salivary flow, thereby altering saliva composition, pH, and buffering capacity, causing the sensation of dry mouth (xerostomia) [2]. Cell culture models for pSS and xerostomia have been limited, leading researchers to rely on cancer cell lines or short-term cultures of primary salivary gland epithelial cells (SGECs) [4,5]. Moreover, the cell

lines extensively used in salivary research (e.g., HSG, A253, NS-SV-AC) are of male origin [4,6–8]. These cell lines may not recapitulate the pathological processes occurring in pSS, a disease with a higher incidence in female patients [9].

Isolated SGECs have been shown to share ductal and pro-acinar characteristics and exhibit a moderate cell division capacity when grown in serum-free low-calcium media [5,10–13]. As with any primary cell culture model, their limited growth potential can impede reproducibility and applications including, but not limited to, high-throughput drug screening assays. In addition, pSS patient derived SGECs show a markedly reduced proliferation capacity in vitro [3]. The reduced growth capacity of pSS SGECs is mirrored in affected patients' salivary glands where narrowed pools of progenitor cells are found with markedly shortened telomeres [3]. Overall, suboptimal cell culture models combined with the reduced growth ability of pSS SGECs have proven to be major obstacles in salivary research.

Diagnosis of pSS may require a labial salivary gland (LSG) biopsy for histologic analysis of lymphocytic aggregates within the tissue [14]. LSG biopsy tissue is a common explant source for culturing SGECs, but due to limited availability based on clinical access, their potential as a universal model is restricted. Culture of SGECs is typically performed using a dual media approach, where the initial explant medium contains 2.5% fetal bovine serum and is replaced with a second serum-free media to sustain passaging and growth [5,11]. Minor differences in culture conditions, including FBS concentration and time spent in explant medium can have a profound effect on salisphere (i.e., 3D spheroid) formation and passage number, as previously shown for parotid gland progenitor cells isolated from mice [15].

Primary SGEC cultures are comprised of cells with pro-acinar and/or ductal cell-like features depending on the growth substrate, presence of serum in medium, and the specificity of characterization markers used [5,10–13]. The previous work of Fujita-Yoshigaki et al. demonstrated the dedifferentiation of primary parotid acinar cells occurs in culture, which could explain the variety of ductal and acinar cell features expressed [16,17]. In addition, Jang et al. revealed that primary SGECs (known as phmSG) of ductal origin exhibited trans-epithelial resistance, expressed several acinar and epithelial cell markers (i.e., AQP5, SLC12A2, AMY1A), and secreted α-amylase upon β-adrenergic stimulation [5]. Overall, SGECs represent a suitable cell culture model to investigate salivary gland dysfunction in vitro but lack the unlimited growth potential needed for widespread use.

To our knowledge no reliable immortalized human primary SGEC lines derived from female patient's LSG biopsies have been developed or made readily available to researchers. To address this gap, the objective of our study was to generate an in vitro cell culture model for the investigation of the pathophysiology of salivary gland disorders. Using an SV40 Large-T (SV40Lt) lentiviral vector, we generated and characterized immortalized SGECs isolated from LSG biopsies of pSS and xerostomic (sicca) patients. The SV40Lt antigen subunit inhibits both cell cycle regulators, pRB and p53, and prevent telomere shortening-induced cell senescence in some cell types [18,19]. We characterized immortalized SGECs (iSGEC) lines from two non-Sjögren's syndrome (nSS) female patients (referred to as iSGEC-nSS1 and iSGEC-nSS2) and one pSS (referred to as iSGEC-pSS1) female patient, based on the expression of pro-acinar and epithelial cell markers using various molecular methods. iSGECs were grown on matrigel-coated plates to determine three-dimensional (3D) spheroid forming ability, along with the extent to which spheroids recapitulated acinus characteristics, such as differentiated myoepithelial and acinar cells.

## **2. Materials and Methods**
