A stable immortalized cell model is crucial for clinical research. However, normal somatic cells cultured in vitro have a finite lifespan, known as the Hayflick limit. This phenomenon refers to the limited number of divisions normal cells can undergo before entering senescence and ceasing to divide [24,25,26]. The limit results from progressive telomere shortening with each cell division. Cancerous cells, conversely, maintain their telomeres through various mechanisms, bypassing this limit. To overcome the Hayflick limit and establish immortalized cell lines, strategies are utilized to enhance telomere maintenance (e.g., human telomerase reverse transcriptase [hTERT], SV40 large T antigen, human papillomavirus oncoproteins [HPV], and the myelocytomatosis viral oncogene [Myc]) [27, 28]. However, uncertainty remains regarding consistent expression of target genes in primary cells and potential impacts of these interventions, indicating a need for further validation.

Surgical treatment of VCL is typically performed using a support laryngoscope. The narrow operating space often yields inadequate tissue sample sizes, thereby limiting the utility of primary culture with conventional methods. The pathological types of VCL are diverse, including squamous epithelial hyperplasia, mild, moderate, and severe dysplasia, and carcinoma in situ [7, 8]. Except for carcinoma in situ, other pathological types of VCL cannot be immortalized in vitro due to the Hayflick limit, further complicating primary culture and immortalization. Additionally, VCL tissue comprises both epithelial cells and fibroblasts. Inadequate separation of epithelial cells results in their suppression by fibroblasts [29]. Our team previously established primary VCL epithelial cells [23] without immortalization. Primary VCL epithelial cells tend to undergo senescence after approximately 10 passages, limiting their utility for further experiments and increasing research costs.

Common protocols for primary culture include tissue explant and enzyme digestion methods [30, 31]. However, due to the small size of VCL tissue samples, the tissue explant method often fails to ensure adequate cell viability. The enzyme digestion method uses enzymes such as trypsin, dispase, and collagenase. Non-cancerous cells typically display lower viability, and trypsin can harm primary cells. Dispase, derived from Paenibacillus polymyxa, acts as a neutral protease that effectively isolates epithelial cells while maintaining their viability [32]. Collagenase aids in breaking down collagen within tissues, facilitating the isolation of primary epithelial cells [33, 34]. Therefore, we selected the gentle enzymes dispase II and collagenase I. Because epithelial cells reside on the tissue surface, they are isolated first. Prolonged enzyme digestion can lead to inadvertent isolation of fibroblasts from the tissue. We established three critical points for primary culture of VCL epithelial cells. First, tissue sterility should be ensured using PBS with 1% penicillin-streptomycin. Second, the duration of enzyme digestion should be strictly controlled, ideally between 60 and 120 min, with an optimal digestion time of 100–120 min. Third, the enzyme dosage should be adjusted according to the protocol. Furthermore, we confirmed a stable primary culture protocol: 3 ml Gibco Advanced DMEM/F12 supplemented with 1 ml dispase II (8 U/ml), 80 μl collagenase I (2 μg/μl), 80 μl FBS, and 1% penicillin-streptomycin.

The reduction of fibroblast contamination is crucial when establishing primary cultures of epithelial cells. In our study, we utilized serum-free culture medium to suppress fibroblast growth, supported by single-cell RNA sequencing analysis to confirm effective epithelial cell purification in later stages of serum-free culture. Serum contains numerous growth factors (e.g., fibroblast growth factor and platelet-derived growth factor) that promote fibroblast growth and inhibit epithelial cell growth [35, 36]. Moreover, serum introduces variability, cost, and ethical concerns [37, 38]. Contaminated serum may compromise cell culture experiments. The present study indicated that the addition of 10% FBS led to cell aging and slower growth rates, highlighting the need to minimize the serum concentration [39] or adopt serum-free media for VCL epithelial cell culture [40]. Tracheobronchial and laryngeal mucosal epithelia share similarities, suggesting commonalities in growth factor requirements for in vitro culture. PneumaCult Ex medium, designed for the in vitro expansion of bronchial epithelial cells [41, 42], is serum-free and contains essential growth factors, insulin, retinoic acid, and nutrients that promote epithelial cell proliferation while inhibiting fibroblast growth. This medium helps to prevent fibroblast contamination during the culture of VCL epithelial cells. Additionally, for cryopreservation, we transitioned to a solution comprising 90% PneumaCult Ex medium and 10% DMSO or a commercial serum-free cryopreservation solution.

Previous studies shown that a crisis phase in cell growth can occur during the process of immortalization induced by SV40 LT antigen transfection [43]. In cell culture, the crisis phase is characterized by a halt or slowdown in cell growth, marked by a significant decline in the proliferative capacity of cells. In fact, during the establishment and culture of hVCL-MSDEP01 in this study, we observed a similar crisis phase phenomenon around P8. In the early stages following transfection with the immortalization virus, hVCL-MSDEP01 exhibited a consistent growth rate, allowing for passaging approximately every three days. During the crisis phase, hVCL-MSDEP01 exhibited a slower growth rate and slight changes in cell morphology. However, the cells were still able to be passaged approximately every five days. After around the 10th passage, the growth rate increased and stabilized, allowing for passaging approximately every three days. We guess that hVCL-MSDEP01 may have undergone a crisis phase during cultivation. The subsequent increase and stabilization in growth rate indicate that the cell line successfully overcame the crisis phase and achieved immortalization in vitro.

VCL primarily involves epithelial cells within the vocal cord mucosa, characterized by markers such as keratin and E-cadherin. Our findings confirm that hVCL-MSDEP01 cells maintain stable expression of proteins including E-cadherin and keratin 17/19, whereas they lack vimentin expression. This expression profile suggests that hVCL-MSDEP01 is a reliable in vitro model for VCL. Furthermore, the STR analysis confirmed that hVCL-MSDEP01 exclusively originated from the patient donor without contamination from other cell lines, establishing it as a novel and distinct cell line. The single-cell RNA sequencing results further support these findings, revealing that each cell cluster within both Pri-hVCL and hVCL-MSDEP01 cell lines expresses epithelial cell–specific genes. Our results confirm that the new cell lines are derived from epithelial cells. Additionally, hVCL-MSDEP01 showed higher proportion of apoptosis and necrotic cells than SNU-899. We inferred that the higher apoptosis and necrosis rates in hVCL-MSDEP01 may be due to the reason that this cell line originated from noncancerous tissue. In contrast, SNU-899 is derived from LSCC and still retains the biological characteristics of tumor cells, namely the ability to proliferate indefinitely in vitro. If the cell line needs to proliferate rapidly and expand, it is crucial to have lower apoptosis and necrosis rates. However, despite the immortalization of hVCL-MSDEP01 by the introduction of SV40 LT protein, it still originates from non-tumor tissue. As a non-tumor tissue, its epithelial cells may have a higher rate of apoptosis and necrosis. This higher rate of apoptosis and necrosis gives hVCL-MSDEP01 distinct growth characteristics, differentiating it from tumor cell lines like SNU-899, which facilitates further in-depth research. Experiments for tumor formation in nude mice showed that hVCL-MSDEP01 cells produced no tumors suggesting that our SV40 LT antigen immortalized cell line was not tumorigenic despite the expression of the SV40 LT antigen. The occurrence and development of tumors is a complex and multifactorial process. Although the SV40 LT antigen prolonged the growth cycle of noncancerous cell lines in vitro, such as VCL, it may not have altered their fundamental characteristics. Our experimental results, along with scRNA-seq data and STR analysis, further support this finding.

In previous studies, SV40 LT antigen has been shown to bind to intracellular cell cycle regulatory proteins, such as p53 and p62, thereby exerting its effects on cell cycle regulation [17, 18]. In Pri-hVCL and hVCL-MSDEP01, p53(TP53), p62(SQSTM1) and RB(RB1) are expressed to a certain extent, and no significant differences in their expression levels were observed between the two cell lines. Previous studies have shown that the dysfunction and accumulation of these proteins in VCL tissues can exacerbate the degree of dysplasia, promoting progression toward LSCC [10, 44, 45]. The VCL tissues used in this study exhibit moderate to severe dysplasia, a stage at which VCL is more likely to progress to LSCC in the future. Therefore, it is plausible that these cell cycle regulatory genes maintain a certain level of expression activity in both Pri-hVCL and hVCL-MSDEP01. However, the expression of the TERT gene is very weak, with detectable expression only in a very small number of cells. We guess that this result is related to the fact that SV40 LT antigen does not directly interact with the TERT gene. And this also suggests that there may be some differences in the mechanisms underlying the development and progression of VCL and oral leukoplakia. The potential interactions and effects between SV40 LT antigen and TERT should be further explored and studied in future research.

The immortalized VCL cell line provides an ideal cell model in the research field. Our team have achieved primary culture of VCL epithelial cell which has limited cell cycle. However, primary cells without immortalization increased the cost in research field, such as time and funds. In addition, characterization of epithelial cells and size of VCL tissue made the primary culture of VCL epithelial cell line much more difficult. In this research, our protocol solved the previously existing problems. Thus, it is necessary to develop more epithelial cell lines with pathological types for research use, as well as to expand the knowledge of cancerous progress between VCL and LSCC. Future in vivo xenograft studies of VCL immortalized cell lines such as hVCL-MSDEP01 as well as studies of their in vitro behavior under different conditions, such as under hypoxic conditions and acidic conditions, are needed. Furthermore, the former study proved that heterogeneity still exists in cell lines [46]. Therefore, the heterogeneity among different VCL primary and immortalized epithelial cell lines also need to be analyzed by using single-cell RNA sequencing in the future study.

Some limitations of our study come from the limited number of immortalized VCL epithelial cell lines, which might not include all pathological types. In the future, we will try to develop more VCL epithelial cell lines with different pathological types to increase the diversity of VCL in cell lines in the research field.

In summary, we successfully established the first immortalized VCL epithelial cell line using SV40 lentiviral gene transduction technology. Our cell line was validated in vitro with respect to protein expression, cell growth dynamics, cell cycle regulation, and apoptosis susceptibility. Single-cell RNA sequencing results confirmed the origin of the cell line. Additionally, STR analysis demonstrated that this cell line is a novel entity derived solely from the patient donor. This immortalized VCL epithelial cell line, named hVCL-MSDEP01 and deposited at the China Center for Type Culture Collection at Wuhan University (CCTCC no. 202432), represents a valuable model for investigating the mechanisms that underlie precancerous lesions of the vocal cords. Future research utilizing immortalized cell lines that represent different VCL histological types can further explore in vitro carcinogenesis mechanisms.