Choongseong Hana,c, Geun Ho Ana, Dong-Hun Wooa, Jong-Hoon Kimb, Hee-Kyung Parkc,⁎
A B S T R A C T
Hyposalivation because of curative radiation therapy in patients with head and neck cancer is a major concern.At present, there is no effective treatment for hyposalivation, highlighting the importance of cell therapy as a new therapeutic approach. To provide functional cells for cell replacement therapy, it is important to overcome the limitations of current in vitro culture methods for isolated salivary gland cells. Here, we suggest an improved culture condition method for the cultivation of isolated salivary gland cells. The dissociated submandibular salivary gland cells of mice were seeded and treated with Rho-associated kinase (ROCK) inhibitor (Y-27632), which resulted in an increase in their cell adhesion, viability, migration, and proliferation. In particular, ROCK inhibitor treatment maintained the expression of α-amylase in the primary cultured salivary gland cells for a longtime as compared with untreated cells. The expression of C-Met, a ductal cell marker, was increased in cells treated with ROCK inhibitor. This modified culture condition may serve as an easy and convenient tool for culturing primary salivary gland cells for their application in hyposalivation therapy.
Keywords:ROCK inhibitor,Y-27632,Hyposalivation,Submandibular salivary gland,Ductal cells,Primary cell culture
1.Introduction
Hyposalivation is the most predictable side-effect of radiation therapy in patients with head and neck cancer, attributable to the ir- reversible damage caused to salivary glands. Prolonged reduction in the saliva by hyposalivation may cause several problems, including xer- ostomia, halitosis,dental caries, mucosal infections, burning mouth syndrome, and dysphagia (Vissink et al., 2003a, 2003b). Although several patients undergoing irradiation therapy exhibit hyposalivation or salivary gland atrophy, there are no satisfactory treatments to combat these issues. Therefore, clinical and experimental approaches that may replace or regenerate the impaired salivary gland tissue are desirable (Aframian and Palmon, 2008; Jensen et al., 2014).The application of primary cultured salivary gland cells in re- generative medicines for hyposalivation treatment requires modifica- tions in the current cell culture conditions, as acinar cells that produce and secrete saliva in the salivary gland are rapidly degenerated and lose their secretory functions during the first 24-48 h in in vitro cultures (Wigley and Franks, 1976). Although several reports show that the modulation of the extracellular matrix or basal membrane gel may improve the culture conditions and functional properties of isolated salivary gland cells in vitro (Durban, 1990; Maria et al., 2011; Fujita- Yoshigaki et al., 2005; Oliver et al., 1987), isolation and culture of salivary gland epithelial cells is still difficult.
The enzyme Rho-kinase (ROCK) mediates various important cellular functions such as cell shape, viability, secretion, proliferation, and gene expression (Liao et al., 2007). Modulation of ROCK expression in cul- tures affects the cellular functions of various cell types. ROCK inhibition suppresses proliferation and downregulates migration of vascular smooth muscle cells (Loirand et al., 2006; Kiian et al., 2003). On the other hand, a ROCK inhibitor has been shown to remarkably improve the proliferation and migration of osteoblasts on hydrophobic surfaces (Tian et al., 2009; Yang et al., 2011). In human embryonic stem cells (hESCs), ROCK inhibition resulted in anti-apoptotic effects and treat- ment with the ROCK inhibitor significantly increased the survival rate of hESCs (Watanabe et al., 2007). In osteoblastic cells, ROCK inhibition may reverse the common problems of osteoblast culture, including those related with low adhesion, proliferation, and migration of
Fig. 1. Survival and growth of salivary gland cells by ROCK inhibition. (a) Representative morphological changes in isolated salivary gland cells during culture periods with or without Y-27632 treatment. (b) Calcein-AM/PI staining (live and dead cell assay) for isolated salivary gland cells during culture periods with or without Y-27632 treatment. R(+): cultured cells with Y-27632, R(−): cultured cells without Y-27632. (c) Number of isolated salivary gland cells during culture periods with or without Y-27632 treatment. (d) Numbers of PI-positive dying cells during culture periods with or without Y-27632 treatment. Scale bar = 200 μm. *P < 0.05, **P < 0.01 versus R (-).osteoblasts on hydrophobic surfaces (Tian et al., 2009). Furthermore, ROCK inhibition significantly enhances the migration of human corneal endothelial cells but fails to induce proliferation of human corneal endothelial cells (Pipparelli et al., 2013). As seen in various reports, the modulation of ROCK activity showed various effects on cell cultures; however, the effect of ROCK modulation on salivary gland cells is still unknown. Therefore, in this study, we evaluated the effects of ROCK inhibitor on the survival, proliferation, migration, and functions of primary cultured salivary gland cells.
2. Materials and methods
2.1. Preparation of primary submandibular salivary gland cells from mouse and in vitro culture
Submandibular gland tissues were harvested from 8-week-old C57/
Fig. 2. Proliferation of salivary gland cells by ROCK inhibition. (a) EdU staining of isolated salivary gland cells cultured with or without Y-27632 treatment. (b)pellet was resuspended in DMEM/F12 medium and filtered through a 100 μm cellstrainer (BD Falcon, Bedford, MA, USA). The purified cells were collected by centrifugation at 1000 rpm for 5 min and the cell pellet was resuspended in the culture medium. The cells were plated at a density of 5 × 104 cells/well in a four-well plate in DMEM (Gibco) medium supplemented with 100 U/mL penicillin G and 100 μg strep- tomycin, 20 ng/mL epidermal growth factor (Sigma-Aldrich), 20 ng/mL basic fibroblast growth factor (PeproTech, Rocky Hill, NJ), 1/100 N2 supplement (Gibco), 10 μg insulin-transferrin-selenium (Gibco), and 1 μM dexamethasone (Sigma-Aldrich) with/without 10 μM ROCK in- hibitor Y-27632 (Calbiochem, Merck, Rockland, MA, USA) and in- cubated for 24, 48, 72, and 96 h in 5% CO2 at 37 °C. The plate was pretreated with 20 mg Matrigel (BD Falcon) for 2 h before use.
2.2. Measurement of the anti-apoptotic efect of Y-27632 on submandibular salivary gland cells
Analysis of the survival rate of submandibular salivary gland cells was performed in two different conditions. Two days after cell seeding, cells were treated with fresh medium with or without 10 μMY-27632 at different time points (24, 48, 72, and 96 h). The analysis of cell pro- liferation was performed by determining the kinetic values at each time point using a microscope (Nikon, Tokyo, Japan)
2.3.Cell proliferation assay
To confirm the proliferation of primary mouse salivary gland cells during in vitro culture at each time point (24, 48, 72, and 96 h), cells were incubated with 10 μM 5-ethynyl-2′-deoxyuridine(EdU, Invitrogen, Grand Island, NY, USA) for 24 h and their proliferation evaluated using Click-iT® EdU imaging Kit (Invitrogen), as per the manufacturer’s instructions. The nucleus was stained with 5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and cells were visualized under confocal laser scanning microscope (Carl Zeiss, Thornwood, NY, USA).
2.4. In vitro live-dead assay
Cell viability assay was performed to study the effects of Y-27632 on the in vitro culture of primary mouse salivary gland cells using acet- oxymethyl diacetyl ester of calcein-AM (Sigma-Aldrich) and propidium iodide (PI, Sigma-Aldrich). Cells were incubated with or without 10 μMY-27632 in culture media for 24, 48, 72, and 96 h, followed by their treatment with 2 μM calcein-AM and 10 μM PI to differentiate between live and dead cells. The nucleus was stained with 10 μM Hoechst 33342 (Invitrogen) and cells were visualized under a confocal laser scanning microscope.
2.5. In vitro scratch assay
Quantification of numbers of Edu-positive cells in (a). R(+): cultured cells with Y-27632 (n = 10 at each time point), R(−): cultured cells without Y-27632 (n = 10 at each time point). Scale bar = 200 μm. * P < 0.05 versus R(-).BL6 mice (Raon bio co. Ltd., Korea). The tissues were dissociated from cervical fascia and connective tissues under a dissecting microscope and gently collected in phosphate-buffered saline (PBS; Welgene, Daegu, Korea). Freshly dissociated tissues were washed twice with Dulbecco’s modified Eagle’s medium and Ham’s F-12 mixture (DMEM/F12 medium, 1:1, Gibco, Grand Island, NY, USA) supplemented with 100 U/ mL penicillin G and 100 μg streptomycin (Gibco), minced with oph- thalmic curved scissors, and incubated with DMEM/F12 medium con- taining 4 mg/mL collagenase type IV (Sigma-Aldrich, St Louis, Mo) at 37 °C for 30 min. Dissociated cells were centrifuged at 1000 rpm for 1 min and washed twice with DMEM/F12 medium. The collected cellAfter seeding in a six-well plate, cells were cultured in the medium supplemented with Y-27632, as mentioned above. Upon confluence, cells were washed twice with PBS and scratched using a 200 μL mi- cropipette tip. The cells were washed with PBS and incubated in the medium with or without Y-27632. The results were observed every 5 min until 24 h using a time-lapse live cell movie analyzer, JuLi™ Br (NanoEnTek Inc, Seoul, Korea) in three independent expreiments, then, calculated the percentage of confluence and cell migration rate by the JuLi ™ Br program.
2.6. Immunofluorescence staining
Cells were fixed with cold 4% paraformaldehyde (Sigma-Aldrich) in PBS. After blocking and permeabilization in 0.3% Triton X100 (Sigma- Aldrich) and 10% donkey serum (Sigma-Aldrich) in 0.1% bovine serum albumin (Sigma-Aldrich)/PBS, the cells were overnight incubated at
Fig.3. Migration of salivary gland cells by ROCK inhibition. (a) Representative images from the scratch assay of isolated salivary gland cells with or without Y-27632 treatment. (b and c) Comparison of average confluency rate and cell migration rate of isolated salivary gland cells, calculated with JuLi™ Br. R(+): cultured cells with Y-27632, R(−): cultured cells without Y-27632 treatment in three in- dependent experiments. * P < 0.05 versus R (-).4 °C with primary antibodies, followed by 1 h incubation at room temperature with secondary antibodies. The primary antibodies used were anti-c-amylase (1:50, Santa Cruz Biotechnology) and anti-C-Met (1:200, Santa Cruz Biotechnology), while secondary antibodies in- cluded Alexa Fluor 488 anti-mouse, 594 anti-mouse, and 594 anti- rabbit (1:400, Invitrogen). Apotome-Axiovert 200 M fluorescence mi- croscope (Carl Zeiss) and confocal laser scanning microscope (Carl Zeiss) were used to visualize cells after counterstaining with 5 μg/mL DAPI.
2.7. Western blotting
Cells were harvested in a radioimmunoprecipitation assay (RIPA) lysis buffer (Upstate, Charlottesville, VA, USA) supplemented with a cocktail tablet of protease inhibitors (Roche, Mannheim, Germany). Equal amounts of proteins (50 μg/lane) were electroporated on NuPage™ 4-12% Bis-Tris polyacrylamide gels (Invitrogen) and trans- ferred onto polyvinylidene difluoride (PVDF) membranes (Invitrogen). The membranes were overnight incubated with various primary anti- bodies at 4 °C, followed by their treatment with secondary antibodies for 1.5 h at room temperature. The primary antibodies used were anti- C-Met (1:200, Santa Cruz Biotechnology) and anti-β-actin (1:50, Sigma- Aldrich), while the secondary antibodies used included donkey anti- mouse IgG-horseradish peroxidase (HRP; 1:10000, Santa Cruz Biotechnology), donkey anti-rabbit IgG-HRP (1:10000, Santa Cruz Biotechnology), and donkey anti-goat IgG-HRP (1:10000, Santa Cruz Biotechnology). The proteins were visualized with the enhanced che- miluminescence (ECL), Supersignal® West Pico Chemiluminescence substrate (Thermo, Prod #34,077).
2.8. Statistical analysis
All data were expressed as means ± SD from at least three in- dependent experiments. t-tests and two-way analysis of variance were completed using Sigma Plot. P values less than 0.05 were judged to be statistically significant.
3. Results
3.1. ROCK inhibitor treatment enhances the growth and survival of isolated submandibular gland cells
To test the effect of ROCK inhibition on the growth of salivary gland cells, the submandibular gland tissue was dissociated from mice and the isolated cells were cultured in the presence or absence of Y-27632. After 24 h, cells were attached to the surface of the plates both with or without Y-27632. However, the cell attachment was better in the pre- sence of Y-27632; most attached cells showed three-dimensional ag- gregates in both conditions (Fig. 1a). After 48 h, Intra-abdominal infection a significant increase in the number of attached cells was observed in Y-27632 treatment group and the cells started to spread out from the aggregates. Furthermore, the growth of cells dramatically increased following treatment with Y- 27632 for 72 hand confluent cells were observed at 96 h (Fig. 1a and c). In the absence of Y-27632, most cells maintained their aggregated states at 48 h and exhibited slow growth compared to Y-27632 treated cells (Fig. 1c); several acellular areas were observed at 96 h (Fig. 1a).We assessed the live/dead cell population of cultured cells following their treatment with Y-27632 using fluorescence-based calcein-AM (green) and PI (red) staining. Although PI-positive dead cells (red) were observed at all time points, the number of dead cells was significantly reduced in the presence of Y-27632 (Fig. 1b and d). Therefore, these data indicate that ROCK inhibition enhances the survival and growth of primary cultured salivary gland cells.
3.2. ROCK inhibition increases the proliferation of salivary gland cells
Following the observation of the increased survival and growth of salivary gland cells with ROCK inhibition, we assessed the proliferation of cultured cells with or without Y-27632 by EdU incorporation assay at 24, 48, 72, and 96 h. We counted the number of cells during the culture period. The cell number greatly increased after 48 h culture with or without Y-27632 treatment; however, a six-fold increase in the cell number was observed following treatment with Y-27632 from 48 to 72 h. On the contrary, only a three-fold increase in cell number was reported in the absence of Y-27632 at the same time point (Fig. 1c).
Fig. 4. Enhanced expression of salivary gland cell markers by ROCK inhibition. (a) Immunostaining of α-amylase and C-Met in rat salivary tissue. (b) Immunostaining of α-amylase and C-Met in isolated salivary gland cells with or without Y-27632 treatment. (c) Western blot analysis of C-Met expression in isolated salivary gland cells with or without Y-27632 treatment. R(+): cultured cells with Y-27632, R(−): cultured cells without Y-27632 treatment. Scale bar = 50 μm.Consistent with these observations, a significant increase in the number of proliferating cells was observed in the Y-27632 treatment group than in cells without inhibitor treatment (Fig. 2a and b). Furthermore, Edu- positive proliferating cells exponentially increased in Y-27632 treat- ment group after 72 h and the numbers of Edu + Protectant medium proliferating cells were about three-fold increased in Y-27632 treated cells compared to control culture after 96 h (Fig. 2a and b). These data indicate that ROCK inhibition in the salivary gland cells may facilitate cell growth, as evi- dent from the increase in cell proliferation.
3.3. ROCK inhibition increases the motility of salivary gland cells
The effect of ROCK inhibition on the migration of salivary gland cells was assessed with the in vitro scratch assay. Images were obtained with JuLi™ Br live cell analyzer from 5 min to 24 hfollowing treatment of cells with Y-27632 (Fig. 3). The time-lapse images at five time points (0, 4, 8, 10, and 23 h) after scratching showed that Y-27632 treatment increased the migration of salivary gland cells and the cells reached over 90% confluency after 10 h (Fig. 3a). In the absence of Y-27632, the scratched area was slowly closed and the cells reached confluency at23 h (Fig. 3a). To confirm this morphological observation, we per- formed a comparative analysis of the percentage confluency at different time points with an automated program. Cells treated with Y-27632 reached confluency within averagely 13.5 h of treatment, whereas those without Y-27632 treatment took 23.5 h to reach confluency (Fig. 3b). Furhermore, we accessed the cell migration rate in the cul- ture, then, Y-27632 treatment significantly increased cell migration rate of salivery gland cells (Fig. 3c). Thus, ROCK inhibition not only enhance the survival, growth, and proliferation of salivary gland cells but also increase their motility.
3.4. ROCK inhibition induces the expression of salivary gland cell-specific markers
As hyposalivation causes reduction in the salivary flow rate, it is important to maintain the secretory functions of the in vitro cultured salivary gland cells. Thus, we assessed the expression of markers for acinar and ductal cells that play pivotal roles in the secretory functions of the salivary gland. The activities of antibodies against acinar cells (α- amylase) and ductal cells (C-Met) in the mouse salivary gland tissue were evaluated (Fig. 4a). Staining results showed that α-amylase was well stained in the cluster-structured acinar cells in the mouse tissue, while C-Met was stained well in the ductal structure of the mouse tissue (Fig. 4a).To determine whether ROCK inhibition may induce the secretory function of the salivary gland under in vitro condition, we cultured salivary gland cells and analyzed the expression of markers at 72 and 96 h (Fig. 4band Gentamicin molecular weight c). In the absence of Y-27632 treatment, the cultured cells showed the expression of α-amylase at 72 h but lost α-amylase expression at 96 h (Fig. 4band c) and no C-Met expression was reported (Fig. 4b and c). On the contrary, Y-27632 treatment maintained the expression of α-amylase even at 96 h (Fig. 4b) and induced the ex- pression of C-Met in salivary gland cells (Fig. 4b). The increase in the expression of ductal cell marker, C-Met, following ROCK inhibition was also demonstrated by western blot analysis (Fig. 4c). Therefore, ROCK inhibition in the salivary gland cells may result in the maintenance of the secretory function of the cells.
4. Discussion
Hyposalivation as a consequence of curative radiotherapy in pa- tients with head and neck cancer may reduce the function of salivary glands. As there is no effective therapy for hyposalivation, transplan- tation of primary cultured salivary gland cells has been considered as a therapeutic approach (Nelson et al., 2013). However, acinar cells of the salivary gland easily lose their functions in in vitro cultures (Fujita- Yoshigaki et al., 2009). Furthermore, these cells take a long time to reach confluency for their use in transplantation with the current methods of primary cell culture.The enzyme ROCK mediates various cellular functions related to cell morphology, motility, and survival. In our experiments, we observed that the treatment of salivary gland cells with ROCK inhibitor resulted in an increase in the adhesion of cells onto the cell culture plate surface and enhanced their survival, growth, and proliferation. From these observations, we suggest that the treatment of primary mouse sub- mandibular gland cells with ROCK inhibitor may protect them from cell death during the initial attachment stage and in prolonged cultures. In addition, ROCK inhibition may offer advantages related to cell pro- liferation and growth in cultures. We demonstrated that ROCK inhibi- tion in salivary gland cells may increase the migration of cells. Cell motility is an important factor for cell therapy. In the clinical applica- tion of mesenchymal stem cells (MSCs), homing of the injected cells into the target tissue is a significant issue (De Becker and Riet, 2016).
Therefore, we hypothesize that injection of cultured salivary gland cells in the presence of a ROCK inhibitor may provide better therapeutic applications through increased homing capacity of the cells.In the presence of Y-27632, the cultured salivary gland cells ex- hibited prolonged expression of markers of acinar and ductal cells of the salivary gland. Primary salivary gland cells rapidly lose their secretory functions in vitro (Wigley and Franks, 1976). Furthermore, parotid acinar cells may change to duct-like cells during in vitro epithelial- mesenchymal transition (Fujita-Yoshigaki et al., 2009). However, we found that the expression of α-amylase was retained in cells cultured in the presence of Y-27632. Furthermore, the inhibitor treatment also increased the expression of C-Met in salivary gland cells. Thus, ROCK inhibition in salivary gland cells may suppress the transition of acinar cells to ductal cells and amplify the number of ductaland acinar cells in the salivary cell culture.
5. Conclusion
In summary, our study demonstrates that ROCK inhibition may offer several advantages during the culture of primary salivary gland cells, such as increased cell survival, adhesion, growth and proliferation, migration, and cell type-specific marker expression. Therefore, we conclude that ROCK inhibition in primary salivary gland cells may be used as a effective strategyfor their easy and convenient application in clinical settings.