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. Author manuscript; available in PMC: 2022 Apr 28.
Published in final edited form as: Cells Tissues Organs. 2021 Apr 28;210(1):10–23. doi: 10.1159/000514200

Protein Substrate Alters Cell Physiology in Primary Culture of Vocal Fold Epithelial Cells

Emily E Kimball a,c, Lea Sayce b,c, Xiaochuan C Xu b, Chase M Kruszka d, Bernard Rousseau b,c
PMCID: PMC8222167  NIHMSID: NIHMS1664490  PMID: 33910192

Abstract

The basement membrane interacts directly with the vocal fold epithelium. Signaling between the basement membrane and the epithelium modulates gene regulation, differentiation, and proliferation. The purpose of this study was to identify an appropriate simple single-protein substrate for growth of rabbit vocal fold epithelial cells. Vocal folds from three New Zealand white rabbits (Oryctolagus cuniculus) were treated to isolate epithelial cells, and cells were seeded onto cell culture inserts coated with collagen I, collagen IV, laminin, or fibronectin. Transepithelial electrical resistance (TEER) was measured and phase contrast microscopy, PanCK, CK14, and E-cadherin immunofluorescence were utilized to assess for epithelial cell-type characteristics. Further investigation via immunofluorescence labeling was conducted to assess proliferation (Ki67) and differentiation (Vimentin). There was a significant main effect of substrate on TEER, with collagen IV eliciting the highest, and laminin the lowest resistance. Assessment of relative TEER across cell lines identified a larger range of TEER in collagen I and laminin. Phase contrast imaging identified altered morphology in the laminin condition, but cell layer depth did not appear to be related to TEER, differentiation, or morphology. Ki67 staining additionally showed no significant difference in proliferation. All conditions had confluent epithelial cells and dispersed mesenchymal cells, with increased mesenchymal cell numbers over time; however, a higher proportion of mesenchymal cells was observed in the laminin condition. The results suggest collagen IV is a preferable basement membrane substrate for in vitro vocal fold epithelial primary cell culture, providing consistent TEER and characteristic cell morphology, and that laminin is an unsuitable substrate for vocal fold epithelial cells and may promote mesenchymal cell proliferation.

Keywords: Vocal Fold, Epithelium, Cell Culture, Basement Membrane, Culture Substrate

Introduction

In vitro cell culture models are used ubiquitously across many areas of research and provide an isolated, well-controlled environment in which to test biological hypotheses. In most cases, the in vitro model provides a more cost-effective and efficient means to study cellular phenomena, with the expectation that results will generalize to the in vivo environment. Cell culture literature has shown that the substrate on which cells are grown can significantly affect the growth and physiology of the cells, and it is important to consider this when developing a new cell culture model system [Charrier et al., 2018; Liberio et al., 2014].

In vocal fold biology, the development of a model in which to study the vocal fold epithelium is critical to identifying the unique features of this tissue system. As the most superficial layer of the vocal fold, the epithelium presents a physical barrier to the underlying lamina propria, is often considered the first line of defense against the forces of vibration, and shows structural damage following vibratory trauma [Gray, 2000; Levendoski et al., 2014; Leydon et al., 2014; Mizuta et al., 2017a; Rousseau et al., 2017; Kimball et al., 2019]. As such, maintenance and repair of the epithelial barrier is critical to withstanding repeated vibratory stresses in the healthy vocal fold [Rousseau et al., 2017; Kojima et al., 2014; Novaleski et al., 2016; Rousseau et al., 2011]. Several different species have been utilized as in vivo and in vitro models of human vocal fold biology and physiology, and these include pig, dog, sheep, rabbit, and rat [Sivasankar et al., 2010; Rousseau et al., 2003; Hanson et al., 2010; Nakagawa et al., 1998; Alipour et al., 2011; Akhtar et al., 1999; Ling et al., 2010]. Each of these models has its benefits and drawbacks, as is the case in any model system. However, each can be optimized by carefully selecting the conditions under which we conduct our experiments, such that generalizability to human vocal fold epithelia is maximized.

To characterize the effects of phonotraumatic damage to the vocal folds, the New Zealand white rabbit (Oryctolagus cuniculus) provides a well-established in vivo phonation model [Ge et al., 2009]. The similarities between the rabbit and human vocal folds with respect to vocal fold extracellular matrix components make it an ideal, small-animal model for investigation of vocal fold biology, physiology, and pathophysiology [Branski et al., 2005; Ge et al., 2009; Rousseau et al., 2004; Thibeault et al., 2003]. Further, presence and localization of critical ion channels is preserved between rabbit and human vocal fold tissue, through the epithelium and lamina propria layers [Gartling et al., 2020]. The rabbit vocal fold epithelium is slightly less stratified (3–4 cells thick) than the human epithelium (6–7 layers), but the overall structure is biomechanically comparable with a robust basement membrane, serving as the capsule that encloses the viscous lamina propria [Maytag et al., 2013; Pitman et al.; Hertegård et al., 2003]. Because of the in vivo utility of the rabbit model of phonation and vocal fold injury, it is important to establish a cell culture protocol that allows for parallel, controlled investigation at the cellular level in rabbit vocal fold tissue. Mizuta et al. described a primary vocal fold epithelial cell culture model using cells harvested from rabbit larynges, which was used to examine trans-epithelial electrical resistance (TEER) as a proxy for epithelial barrier integrity [Mizuta et al., 2017b]. In this technique, cells are seeded onto porous cell culture inserts coated with collagen I and allowed to grow to confluence, at which point the electrical resistance through the cell layer can be measured [Srinivasan et al., 2015]. In the model described, optimization was centered around culture media, seeding density, and the presence or absence of feeder cells. Collagen I was selected as a growth substrate due to previous studies describing its use in the culture of corneal epithelial cells, another non-keratinized, stratified squamous epithelium [Spurr-Michaud, and Gipson, 2013]. However, the typical makeup of the vocal fold epithelial basement membrane does not include collagen I as one of its main components [Dikkers et al., 1993].

Other studies have sought to better replicate the complex in vivo environment in an in vitro experimental paradigm using a combination of gel substrates and native vocal fold fibroblast co-culture. Fibroblasts of the vocal fold, like many other mesenchymal cell types, do not require a substrate and can grow directly on tissue culture treated plastic [Chen, and Thibeault, 2008; Graupp et al., 2018; Lou et al., 2019]. Unfortunately, vocal fold epithelial cells do not fare well in these substrate-free conditions, and instead require additional support to promote adhesion and proliferation in culture. Attempts have been made to create a suitable environment for epithelial cells through use of co-culture with vocal fold fibroblasts, both in simple culture and through use of a gel-matrix paradigm [Imaizumi et al., 2013; Leydon et al., 2013; Walimbe et al., 2017]. In each of these conditions, epithelia achieved morphology similar to that of the in vivo vocal fold setting, and these systems allow for investigation of the interplay between the two primary cell types within the vocal fold tissue. However, these cell culture models do not allow for the independent investigation of the vocal fold epithelium in isolation, which is of equal importance in elucidating the vocal fold tissue response to acute and chronic injury. A lone study by Erickson-DiRenzo et al. demonstrated effective culture of porcine vocal fold epithelium using Collagen IV, although the choice to use Collagen IV was not discussed [Erickson-DiRenzo et al.]. The challenge of the current study lies in creating an environment suitable for rabbit vocal fold epithelial growth in the absence of fibroblasts, and to further quantify the relative contributions of each basement membrane component in the establishment and maintenance of a healthy and intact epithelial barrier.

The basement membrane of the epithelium consists primarily of 5 components: laminin, heparin sulfate proteoglycan, entactin, collagen type IV, and fibronectin [Leblond, and Inoue, 1989]. The anchoring of the basement membrane to the basal epithelial cells is modulated through integrins, which are responsible for converting mechanical signals from the extracellular matrix into chemical signals within the cell [Berrier, and Yamada, 2007; Harburger, and Calderwood, 2009; Sun et al., 2016]. This interaction ultimately elicits downstream changes that may affect gene regulation, cell polarity, migration, differentiation, and proliferation [Berrier, and Yamada, 2007]. Proteins of the basement membrane and superficial lamina propria interact directly with integrins in the cell membrane and can use this interaction to modify cell physiology and signaling [Lodish et al., 2000]. This communicative pathway between the extracellular matrix and the epithelial layer is critical to homeostasis and may be impaired in structural vocal fold pathologies such as nodules or scar, where the fibrous makeup of the basement membrane and superficial lamina propria is altered [Benninger et al., 1996; Bless, and Welham, 2010].

In the development of a cell culture protocol to investigate normal and pathological mechanisms, it is important to consider the individual components of the basement membrane, and identify which of these components (e.g. collagen I, laminin, collagen IV, or fibronectin) will provide the best translation from in vitro to in vivo. Erickson-DiRenzo et al. sought to establish an isolated primary epithelial vocal fold culture method derived from porcine larynges, and demonstrated successful cultures with normative epithelial morphology and protein markers and minimal fibroblast contamination using a collagen IV protein-substrate [Erickson-DiRenzo et al.], supporting further investigation into in vitro substrates for the support of vocal fold epithelial cell proliferation. No other studies to date have investigated the interaction between substrate and epithelium, although many have acknowledged the likelihood of this interaction playing a critical role in tissue integrity and homeostasis in other tissue types [Frisch, and Francis, 1994; Coraux et al., 2008; Suzuki et al., 2003].

The objective of the current study was to investigate the role of protein substrate coating on epithelial cell physiology in cells harvested from rabbit larynges, such that protein substrate interactions and the subsequent pathways influencing epithelial changes in vitro might be elucidated in future studies. Characteristics used to determine optimal culture conditions were: (a) reproducible and characteristic progression of transepithelial electrical resistance as described previously [Mizuta et al., 2017b] and (b) presence of characteristic epithelial cell markers and morphology. To achieve this, rabbit vocal fold epithelial cells were cultured on inserts coated with either collagen I (a commonly used protein substrate, on which the rabbit vocal fold epithelial cell culture was first established) or one of the constituent proteins known to be present in the vocal fold basement membrane (collagen IV, laminin, or fibronectin). The differential cellular response was evaluated for each condition. Due to a lack of information regarding relative abundance of each of the constituent proteins within the vocal fold basement membrane, and to reduce confounding variables in this study, all substrates were applied to cell culture membranes at the same protein concentration, 0.5ug/cm2, which was the collagen I concentration used in the seminal paper by Mizuta et al [Mizuta et al., 2017b] and demonstrated to be successful in other primary cell lines derived from stratified squamous epithelia [Spurr-Michaud, and Gipson, 2013]. TEER data was collected across protein substrates, and further analysis of cell morphology, cell proliferation and stratification, and cell differentiation was conducted.

Materials and methods

All animal procedures were approved by the Vanderbilt University Institutional Animal Care and Use Committee (Approval #: M16100162).

Cell culture plate coating (P0 and P1):

All plates were coated with collagen I as follows; 2mL of 37.5 μg/mL collagen solution (Advanced Biomatrix PureCol) diluted in PBS was added to each well. The plates were incubated at 37°C for 2.5 hours, and the remaining liquid was aspirated with a sterile pipette. The plates were rinsed with PBS, re-sterilized under UV light for 30 minutes, and stored at 4°C for up to 3 weeks prior to use.

Experimental cell culture insert coating (P2):

For each protein substrate (collagen I, collagen IV, laminin, or fibronectin), 0.6mL of 37.5 μg/mL protein solution was added to a cell culture insert (Millipore, PET 1μm), for a final concentration of 0.5ug/cm2. Solutions were prepared per manufacturer specifications: collagen I (Advanced Biomatrix PureCol) was diluted in PBS, collagen IV (Corning Collagen IV, mouse) was diluted in 0.05M HCl, laminin (Corning Laminin Ultrapure, mouse) was diluted in PBS, and fibronectin (Millipore Fibronectin, bovine plasma) was diluted in 37°C PBS. Inserts were left to dry overnight in a dark biosafety cabinet with the fan on. In the morning, each insert was rinsed with PBS, wrapped in parafilm, and stored at 4°C for up to 3 weeks prior to use.

Tissue harvest:

Normal larynges were harvested from three male New Zealand white breeder rabbits (3.0 –3.3kg) following sedation and euthanasia. Three rabbit primary cell lines were established (one per animal) for this study.

Epithelial cell preparation and seeding:

Epithelial cells were cultured according to procedures previously described [Mizuta et al., 2017b]. Culture medium was composed of DMEM/F12 (1:1 with 1-glutamine, 15 mM HEPES, 1 mM CaCl2, GIBCO), 10% fetal bovine serum (FBS, HyClone), penicillin (100 U/mL), streptomycin (100 μg/mL, HyClone), epidermal growth factor (10 ng/mL, Peprotech), insulin (5 μg/mL, Sigma-Aldrich), adenine (24 μg/mL, Sigma-Aldrich), hydrocortisone (0.4 μg/mL, Sigma-Aldrich), cholera toxin (0.1 nM, Sigma-Aldrich), and triiodo-L-thyronine (2 nM, Sigma-Aldrich).

In brief, excised vocal fold tissue was treated with 66 U/mL Dispase II (Roche Life Science) at 37°C for 4 hours and the epithelial layer was removed and treated with 0.05% trypsin/0.02% EDTA solution (Sigma-Aldrich) at 37°C for 20 minutes. The cells from each vocal fold were seeded into wells on a 6-well plate and co-cultured with mitomycin C-treated feeder cells at a density of 2.0 × 104 cells/cm2.

Epithelial cell passaging:

When the cells were locally confluent (at approximately passage 0, day 7), cells were dissociated and resuspended in two stages: a 2 minute treatment with 0.05% trypsin/0.02% EDTA at 37°C was used to remove feeder cells and any contaminant cells (e.g. fibroblasts) which more readily dissociate, and then cells were washed with PBS and re-treated with 0.05% trypsin/0.02% EDTA for 15 minutes at 37°C to dissociate and suspend epithelial cells. Cells were counted and subcultured into passage (P) 1 at a seeding density of 2.2 × 104 cells/cm2, co-cultured with mitomycin-C treated feeder cells at a seeding density of 2 × 104 cells/cm2 on collagen I-coated 6-well plates. When P1 cells reached 70–80% confluence (~P1 day 4), cells were counted and seeded at P2 onto cell culture inserts (Millipore, PET 1μm) coated with each of the experimental proteins (5μg/cm2 of collagen I, collagen IV, laminin, or fibronectin), at a density of 2.2 × 104 cells/cm2 without feeder cells. P2 cells were used for experimental manipulation in this study, as previous reports found that P2 for rabbit vocal fold epithelial cells maintained cell culture stability while maximizing output through expansion from P0 [Mizuta et al., 2017b].

Feeder cell preparation and seeding:

For P0 and P1 of culture, cells were co-cultured with feeder cells (3T3-Swiss Albino, ATCC CCL®−92™, ATCC) onto collagen I coated plates. For P0 and P1, feeder cells were treated with 10 μg/mL mitomycin-C (Sigma-Aldrich) at 37°C for 3 hours prior to seeding to halt proliferation and seeded concurrently with the epithelial cells on a collagen I-coated 6-well plate at a density of 2.0 × 104 cells/cm2.

Transepithelial electrical resistance:

Unlike other measures of epithelial barrier integrity that require fixation of cells, TEER can be measured repeatedly in live cells and has a well-documented correlation with other more direct measures of barrier permeability [Duff et al., 2002; Muendoerfer et al., 2010; Chen et al., 2015]. The TEER values measured correlate to ion permeability, and while they do not necessarily represent epithelial barrier permeability to other macromolecules or cytokines, TEER is still a valuable proxy for overall barrier integrity [Ferruzza, 2013]. To evaluate the functional integrity of the epithelial cell layer, P2 TEER was measured using an epithelial voltohmmeter (EVOM2, World Precision Instruments) equipped with a measurement chamber (EndOhm). TEER was measured in each well daily from days 4–18 of P2 in all three cell lines. To convert the digital readout to area-normalized units, the following formula was applied: resistance of a unit area (Ω*cm2) = Resistance (Ω) x Effective Membrane Area (cm2).

Phase contrast imaging:

Phase contrast imaging of cell morphology provided an indication of cellular confluence and cell shape, with normal growth parameters that are well characterized [Weinstein et al., 1975]. Phase contrast images were collected on days 4 and 16 of passage 2. Images were captured using a Nikon Eclipse Ts2 microscope with DS-Fi2 color camera using a 10x objective. Characterization was undertaken by a blinded reviewer with 25 years of cell culture experience.

Cross-sectional H&E imaging:

To evaluate stratification between protein substrates cell culture inserts from cell line 2 were fixed in 10% formalin on days 7, 10, 13, and 16, paraffin processed, and sectioned at a thickness of 10μm. Cross sectional slices of each cell layer were stained with Hematoxylin and Eosin, and images were captured using a Nikon Eclipse 90i microscope with Nikon Ds-FI2 color camera and a 20x objective. Images were randomized and analyzed in ImageJ by a blinded rater with 25 years of cell culture experience. The grid plugin was applied to images and 10 epithelial depth measurements were taken using the measure tool at equal distances along the membrane.

Immunofluorescence labeling:

Cell culture inserts were fixed with methanol on days 4, 7, 10, 13, 16, and 19. Fixed cells were permeabilized with 0.1% Triton-X100 before undergoing immunofluorescence labeling. Inserts were immunolabeled with primary antibodies (described below), tagged with a corresponding fluorescent secondary antibody (Goat α Mouse 594, Invitrogen 35510, 1:300; Goat α Rabbit 594, Invitrogen A-11012, 1:200), and counter stained with 4’,6-diamidino-2-phenylindole (DAPI) and mounted using Vectashield (Vector Laboratories). All antibodies were validated using primary(−) and secondary(−) control conditions, and blocking was achieved using 10% goat serum (Sigma-Aldrich G9023) in PBS.

Inserts from each cell culture line were labeled for E-cadherin (monoclonal Mouse α E-cadherin, BD Transduction Laboratories 610181, 1:100), PanCK (monoclonal Mouse α PanCK, Abcam ab80826, 1:50) and CK14 (monoclonal Mouse α CK14, Abcam Ab9220, 1:50) to confirm presence of cells of epithelial origin.

Use of the Ki67 antibody is the gold standard for quantifying proliferation in cells due to its presence in cells in all active phases of the cell cycle, and is used in this study to calculate growth fraction [Scholzen, and Gerdes, 2000]. Cell proliferation was assessed via fluorescence immunolabeling with a monoclonal antibody to Ki67 (polyclonal Rabbit α Ki67, Abcam ab15580, 1:100). Three non-overlapping images of each labeled insert were captured using a Zeiss upright axioplan widefield microscope with a Hamamatsu ORCA-ER camera and 20x objective. For each protein condition at each time point, the percent of cells exhibiting Ki67 staining was calculated using ImageJ [Schneider et al., 2012].

A more detailed assessment of cell differentiation was achieved using antibodies targeted against proteins specific to different cell types. In this case, E-cadherin, Pan CK, CK14 (epithelial cell markers) and vimentin (a marker of mesenchymal cells) were used. Cell differentiation was assessed using immunofluorescence co-labeling of vimentin (a mesenchymal marker; rabbit α vimentin, Abcam ab45939, 1:500) and E-cadherin (an epithelial cell marker; mouse α e-cadherin, BD Transduction Laboratories 610181, 1:100). Three non-overlapping images were captured of each labeled insert. Images were captured using a Nikon spinning disk confocal microscope with an Andor DU-897 EMCCD camera. Z-stacks were obtained throughout the visual plane using 5μm optical sectioning with a 20x objective and 2×2 image stitching. A maximum intensity projection of each z-stack was used for quantitative analysis. Percent positive vimentin staining area was calculated for each image. Images were blinded and randomized, and an intensity threshold was manually selected using ImageJ. Percent positive area was calculated based on this manual threshold. For intra-rater reliability, 10% of samples were redundantly analyzed (R2=0.81).

Statistical analysis:

All data was analyzed using Prism V8.21 (GraphPad, San Diego, CA). For each set of quantitative data collected (TEER, percent proliferation, and percent vimentin) a Levene’s test was applied to assess parametricity and a two-way analysis of variance (ANOVA) was computed. A post-hoc Tukey test was performed at each time point for pairwise comparison and multiplicity adjusted P values were calculated. A mixed-effects model with Geisser-Greenhouse correction and post-hoc Tukey test was performed to assess relative changes in TEER from day 4 across cell lines. Statistical significance was determined using an alpha level of 0.05.

Results

Confirmation of epithelial cell phenotype:

Cultures were determined to be primarily epithelial, with positive E-Cadherin, Pan-CK, and CK14 observed by immunohistochemistry of cell culture inserts at P2 day 4 (Fig 1a, b, & c), with minimal Vimentin labeling (Fig 1b), and cobble-stone morphology observed by phase contrast imaging at P2 Day 4 (Fig 1d).

Fig 1. Representative immunolabeling of P2, Day 4 cell cultures to demonstrate epithelial cell phenotype.

Fig 1

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(a) Immunofluorescence labeling against Pan-CK (green) and DAPI nuclear counterstain (blue) at x20 magnification, (b) Immunofluorescence labeling of E-cadherin (red), Vimentin (green) and DAPI (blue) at x20 magnification, (c) Immunofluorescence labeling of CK14 (green) and DAPI (blue) at x20 magnification, (d) Phase-contrast imaging at x10 magnification. Scale bar = 50μm.

Transepithelial electrical resistance:

The range of TEER measured in this study (approximately 200–4000 ohms·cm2) is consistent with that of other airway epithelial cultures reported in the literature, where healthy airway epithelia range from 300–3000 ohms·cm2 depending on origin and other factors such as temperature and media contents [Srinivasan et al., 2015]. Srinivasan et al. also reported TEER values near 250 ohms·cm2 in diseased bronchial epithelial cells, providing a relative functional threshold for barrier integrity in airway epithelia. In all three cell lines assessed, two-way ANOVA revealed significant main effects of both protein substrate and time point on TEER (p<0.0001; Fig 2, Supplemental Data S1–S3). Trends were reproduced across cell lines with peak TEER observed across all cell lines between days 9 and 12. A main effect of substrate protein was significant (p<0.0001). Post-hoc Tukey testing revealed that the collagen IV condition produced significantly higher TEER values compared to laminin across all cell lines (p<0.0001; Fig 2a, b, &c), and significantly higher TEER than collagen I in cell lines 1 and 2 (p<0.0001; Fig 2a, &b) and was approaching significance (p=0.084; Fig 2c) in cell line 3. TEER values for cells in the laminin condition were consistently lower than all other conditions for the duration of the experiment (p<0.0001; Fig 2a, b, &c). The fibronectin condition produced variable results, with peak TEER values comparable to collagen IV in cell line 1 and 3 (p=0.2209, and p=0.9921; Fig 2a, &c), while significantly lower TEER was observed in cell line 2 (p<0.0001; Fig 2b). Relative TEER change from day 4 demonstrated significant differences between all conditions (laminin vs. all p<0.0001, collagen I vs collagen IV, p=0.0179, collagen I vs fibronectin, p=0.0351), excepting fibronectin vs. collagen IV (p=0.9923). Assessment of the min/max and median % TEER change displayed large variations from the median in both collagen I and fibronectin across all timepoints, while laminin and collagen IV demonstrated more predictable change (Fig 2d).

Fig 2. TEER measurements on days 4–18 of passage 2.

Fig 2

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(a,b,c) Epithelial Cell Lines 1, 2 and 3 respectively. Collagen I is depicted in dark blue with a circular marker, laminin is depicted in red with a square marker, fibronectin is depicted in fuchsia with a downward pointing triangular marker, and collagen IV is depicted in green with an upward pointing triangular marker. Error bars represent standard deviation. Peak TEER windows are marked with brackets. (d) Relative change from baseline (%) across all cell lines. Bars display minimum and maximum values with median marked by the black line within each bar. Collagen I is depicted in dark blue and is the bottom bar for each time point, laminin is depicted in red and is the second from bottom bar for each time point, fibronectin is depicted in fuchsia and is the second bar from the top of each time point, while collagen IV is depicted in green and is the top bar in each for each timepoint displayed.

Cell morphology:

Cells cultured on laminin demonstrate altered cell morphology. On day 4, cells in all conditions except the laminin condition were confluent. On day 16, cells in the collagen I, collagen IV, and fibronectin conditions all shared regular, round, cobblestone-like cell morphology. However, cells in the laminin condition presented with altered morphology characterized by long, spindle-like cell shapes at day 16 (Fig 3).

Fig 3. Representative phase contrast images of passage 2 cells from Rabbit Epithelial Cell Line 1.

Fig 3

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Images captured at 10X magnification. Panels (a)-(d) are representative of cell morphology in the collagen I, laminin, collagen IV, and fibronectin conditions respectively, on day 4 of passage 2. Panels (e)-(h) are representative of cell morphology in the same conditions on day 16 of passage 2. Scale bar: 200μm.

Vimentin expression:

Vimentin-expressing cells were more abundant in the laminin protein substrate condition. A subset of cells was fixed on days 4, 7, 10, 13, 16, and 19 and immunolabeled for vimentin. Two-way ANOVA revealed a main effect of time and protein substrate on vimentin expression (p<0.001; p<0.0001; Fig 4a, Supplemental Data S4). Post-hoc Tukey testing revealed that on days 4, 13, 16, and 19, the laminin condition had significantly higher vimentin expression than any other condition (Fig 4e, p<0.05).

Fig 4. Immunofluorescence to detect Vimentin and Ki67.

Fig 4

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(a) Percent area positive for vimentin expression during passage 2, marking mesenchymal cell presence, (b) percentage of cells in passage 2 positive for Ki67, a marker of cell proliferation. Collagen I is depicted in dark blue and is the right-most bar for each time point, laminin is depicted in red and is the second from right bar for each time point, fibronectin is depicted in fuchsia and is the second bar from the left of each time point, while collagen IV is depicted in green and is the left bar in each for each time point displayed. Error bars represent standard deviation. (c-f) Representative images of Vimentin immunolabelling (red) and DAPI counterstaining (blue) in cell culture inserts at P2 day 19, x20 magnification; (c) collagen I, (d) fibronectin, (e) laminin, (f) collagen IV. (g-j) Representative images of Ki67 immunolabeling (red) and DAPI counterstaining (blue) in cell culture inserts at P2 day 4, x20 magnification; (g) collagen I, (h) fibronectin, (i) laminin, (j) collagen IV. Scale bar: 100μm.

Proliferation:

Epithelial cell proliferation is not affected by protein substrate. A subset of cells was fixed on days 4, 7, 10, and 13 and immunolabeled for Ki67 (Fig 4b, I, Supplemental Data S5). Two-way ANOVA revealed a main effect of time on proliferation (p<0.0001), but no main effect of protein substrate (p=0.38). By day 10, percent proliferating cells in all conditions was less than 3%.

Cell layer depth:

Depth of epithelial cell layer does not appear to impact TEER, and all substrate types yielded a stratified epithelial layer (Fig 5a–d). Epithelial cell layer depth was evaluated in cells that had reached confluence: days 7, 10, 13, and 16 to evaluate whether lower TEER values for laminin and fibronectin in cell line 2 could be attributed to epithelial stratification (Fig 5e). Significant main effects of protein substrate (p<0.0001), P2 cell culture day (p<0.0001), and interaction of protein substrate and P2 cell culture day (p<0.0001) were observed; however, these findings were not aligned with TEER values observed in each substrate. Multiple comparisons revealed no significant difference in epithelial depth between laminin and collagen I (p= 0.2083), laminin and collagen IV (p= 0.0504) or fibronectin and collagen IV (p=0.4382). A significant difference was determined between laminin and fibronectin (−4.31±1.173μm, p= 0.0024); however, this finding was contrary to TEER values for the substrates. Likewise, collagen I and collagen IV demonstrated significant differences (5.253±1.129μm, p<0.0001), but while collagen I demonstrated a thicker epithelial layer, collagen IV demonstrated higher TEER values at all time points excepting day 16. Significant differences were also observed between collagen I and fibronectin (6.8±1.243, p<0.0001).

Fig 5. Hematoxylin and Eosin staining of epithelial cross sections and measurement of epithelial depth.

Fig 5

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Representative images of epithelial cross sections on P2 day 13; (a) collagen I, (b) collagen IV, (c) fibronectin, and (d) laminin. Scale bars: 50μm. (e) depicts measures of epithelial depth across P2 day 7, 10, 13, and 16. Individual measurements are marked by blue circles for collagen I, green squares for collagen IV, purple triangles for fibronectin, and red carrots for laminin. Averages for each are indicated by a horizontal black line.

Discussion

In the current study, several different proteins found in the basement membrane of vocal fold tissue in humans and rabbits were tested as a growth substrate for vocal fold epithelial cells in vitro. Collagen I is a standard protein used in cell culture for plate/insert coating and was therefore used as the standard condition for this study [Yang, and Nandi, 1983]. Three additional proteins –collagen IV, fibronectin, and laminin –were chosen due to their endogenous presence within the basement membrane of epithelia [Leblond, and Inoue, 1989], and coated the membranes at an equal concentration to our standard collagen I condition. The data presented demonstrate vocal fold epithelial cell responsiveness to protein substrate, suggesting that the protein composition of the basement membrane and superficial lamina propria affects the physiology of the vocal fold epithelial barrier in vivo. Based on the findings from this study, the use of collagen IV as a growth substrate supports stable growth of rabbit vocal fold epithelial cells in vitro, promoting reproducible epithelial barrier function and normal cellular physiology and morphology, as determined by TEER, phase contrast imaging, measure of epithelial depth, and the presence and absence of epithelial and mesenchymal cell-type markers respectively.

Fibronectin protein substrate demonstrated variable outcomes in the three cell lines studied, likely due to fibronectin’s inherent multi-formational properties. Studies into the dynamic nature of fibronectin’s interaction with the extracellular matrix and integrins has shown that the polymerization of fibronectin is of critical importance[Patel et al., 2006]. Denaturation or re-folding of fibronectin’s 3-dimensional structure may have contributed to changes in the polymerization of fibronectin in our system, yielding inconsistent TEER values [Sottile, and Hocking, 2002]. With previously described sensitivity and tendency to change formation during routine handling, it was determined that fibronectin was not the best candidate for consistent epithelial cell culture in this context. Further replication of the experiments presented in this study are needed to confirm the inconsistencies observed, and to elucidate the mechanisms at play.

Of note, while culture with collagen I revealed the expected changes in TEER trend over time [Mizuta et al., 2017b], TEER was significantly lower than collagen IV in two of the three cell lines characterized, and relative change from baseline between cell lines was demonstrated to be inconsistent for vocal fold epithelial cells, which may impair reproducibility of results in future experiments. Cell morphology, cell proliferation, epithelial depth, and cell differentiation were assessed, but on their own these factors do not wholly explain the observed differences in TEER between the collagen I and collagen IV conditions.

The absence of significant differences in cell proliferation or epithelial depth between collagen I and collagen IV, and the inverse relationship between epithelial depth and TEER between these two substrates suggest that TEER differences are not related to confluence or stratification; a finding which is supported by the cell morphology observed at day 16, where both conditions promoted the growth of a confluent, cobblestone-like morphology (Fig 3), and aligns with previous studies suggesting that collagen I and collagen IV both exert biological activity through binding of a common triple-helical domain [Baer, and Bereiter-Hahn, 2012].

A lack of clear relationship between epithelial depth and any of the other parameters investigated further confirms the complexity of the in vitro system and the substrate-cell interactions at play in the conditions assessed. Although statistical significance was reached for some comparisons above, few meaningful differences were observed, and all substrates facilitated the formation of a stratified epithelium. Of note, there was a relative increase in thickness of the epithelial layer in the collagen I condition at day 10 only (Fig 5e), which may be the result of a similar mechanism to the hyperstratification characteristic of re-epithelialization following vocal fold injury [Branski et al., 2005]. The increase in thickness at this time point delayed the stabilization of depth in collagen I, further supporting an advantage to the use of collagen IV, for which epithelial depth achieved relative stability at day 10 while maintaining high TEER.

There is also no clear relationship between TEER and cell differentiation (Fig 4a). No significant differences in vimentin-positive staining between the collagen I and collagen IV conditions were identified at any time point investigated (Fig 4b). Therefore, there are likely other mechanisms by which collagen I and collagen IV differentially interact with the epithelial layer which were not investigated in this study. Emechukwu et. al. [Enemchukwu et al., 2016] investigated the contributions of extracellular matrix (ECM) properties on culture of Madin-Dark Canine Kidney cells and determined that ECM elasticity, adhesive ligand density, and ECM protease degradability were all key factors in lumenogenesis and apicobasal polarity. In an A6 kidney-derived epithelial cell line, the presence of a collagen I and III mixed-substrate was associated with increased TEER due to higher conductance (and not higher density/length) of tight junctions, likely due to increased phosphorylation of ZO-1 [Jaeger et al., 1997]. While it is plausible that collagen I and collagen IV may elicit different mechanical, tight junctional, or protein expression/modifications, these parameters were not assessed during this study and warrant further investigation.

TEER values remained low in the laminin condition across all cell lines, even at day 16 when the cells reached confluence and a stratified cell layer was present. Further analysis of cells in this condition over time showed a lack of cell confluence early, however percent proliferation was not decreased relative to other protein substrates. These data together suggest that fewer of the seeded cells initially adhere to the laminin-coated insert. This finding is consistent with data suggesting that laminin is required for cellular polarization, but in order to fulfill this function and form a basement membrane, cell anchoring mediators such as β−1 integrins and dystroglycans are recruited [Li et al., 2003]. Additionally, the authors speculate that mechanical property changes modulated by interaction of laminin scaffold with the covalently bonded crosswork of collagen IV polymer [Morrissey, and Sherwood, 2015] may be important in determining the behavior of cells at the basement membrane [Li et al., 2003]. It seems plausible that absence of any of one of these interactions may have reduced epithelial cell adherence, but we did not seek to quantify recruitment of these components during our study. Despite there being fewer adherent cells early in P2 in the laminin condition, proliferation is still intact, and as such the cells do eventually reach confluence in the cell culture insert by day 16 (Fig 3f) with comparable epithelial layer depth to collagen I and collagen IV. However, even at cellular confluence with stratification, cell morphology is different and corresponding TEER values are significantly lower than in any other protein substrate condition.

Upon investigation of cellular differentiation in the laminin condition, a potential explanation of decreased TEER values begins to emerge. In the laminin condition, vimentin expression is significantly greater than in any other protein condition, suggesting that many of the confluent cells are not epithelial cells. Instead, these cells are a distinct population of mesenchymal cells growing alongside the epithelial cells. In the other conditions, these cells are few compared to the epithelial cells, but in the laminin condition they make up a large proportion of the cells cultured on the insert (Supplemental Fig S6). The origin of the mesenchymal cells observed at P2 remains unclear from the current study. They may be de-differentiated cells originating from cells of an epithelial origin [Baer, and Bereiter-Hahn, 2012], or they may be lingering vocal fold fibroblasts (carried over from the initial tissue dissection) that have been encouraged to grow more in the laminin condition than in the other conditions. A similar phenomenon was observed in studies by Walimbe et al. and DiRenzo-Erickson et al. where a primary bronchial/tracheal or laryngeal epithelial cell culture yielded some vimentin-positive cells among the confluent epithelial layer [Walimbe et al., 2017; Erickson-DiRenzo et al.]. Walimbe et al. posited that epithelial-mesenchymal transition may have been at play, but noted that a confluent epithelial barrier was still conserved in the presence of these mesenchymal cells. They noted that in the presence of vimentin-positive cells, the epithelium simply grows over and around these cells, maintaining an intact epithelial barrier superficial to any present mesenchymal cells. In the present study, although the relative localization of vocal fold epithelial cells in and mesenchymal cells was not investigated explicitly, retrospective review of the z-stacks collected prior to compression into the maximum intensity projections used for analysis offered some additional insight. The E-cadherin and vimentin labeled cells indeed largely appear to exist in separate planes, consistent with the observations of Walimbe et al. and measured TEER values that suggest the formation of an intact and contiguous superficial epithelial barrier. It appears that only in the laminin condition was the presence of mesenchymal cells sufficient to inhibit the formation of a complete and intact epithelial barrier.

Cell morphology confirms this differential cell physiology across substrate conditions, as many cells in the laminin condition appear spindle-shaped with elongated cell bodies instead of exhibiting characteristic epithelial cobblestone appearance in phase-contrast imaging. Further post-hoc review of fluorescence imaging confirms vimentin positivity of these spindle-shaped cells, and also shows that E-cadherin positivity tends to be less intense where these spindle-shaped cells congregate (see Supplemental Fig S6). This suggests that an intact epithelial barrier did not form on top of the mesenchymal cell population in some places in the laminin condition. These vimentin-only patches appeared very rarely or not at all in the other substrate conditions. This cell morphology is consistent with mesenchymal cells such as fibroblasts, and these cells do not produce epithelial barrier proteins. If these cells do not produce the proteins required to create an intact barrier, paracellular spaces will be larger, and TEER values will be decreased, as was observed in this study. While the origin of these cells is unclear, laminin has been described to both suppress epithelial-mesenchymal transition [Chen et al., 2013] and promote epithelial-mesenchymal transition in cooperation with TGF-β1 [Giannelli et al., 2005] and through multiple laminin-5 receptors [Li et al., 2017; Scott et al., 2019] in cancer progression. As such, further investigation is warranted to characterize the mesenchymal cells observed in this condition.

A major limitation of this study is the inherent variability in TEER measured between cell lines, and the inherent difficulty in understanding what differences in TEER mean, especially when values are all within what would be considered a functional range in other types of epithelia [Srinivasan et al., 2015]. Vocal fold epithelial cells are extremely sensitive to experimental manipulation and require careful handling to establish successful cultures [Baer, and Bereiter-Hahn, 2012]. In the recent study by Erickson-DiRenzo, 70% of cultures established were successful using porcine vocal fold tissue, while success rates in human glottic epithelial cells were around 40% [Erickson-DiRenzo et al.]. While all lines established for this experiment displayed the morphological characteristics expected and achieved confluence, it’s possible that factors such as length of time from harvest, and physical shock during dissection may have influenced the culture [Baer, and Bereiter-Hahn, 2012]. While this study sought to optimize cell culture through the use of known components of the vocal fold epithelial basement membrane, the use of a single protein in isolation is not reflective of the basement membrane in vivo. As such, a substrate which provides a combination of protein components (e.g. Matrigel) may facilitate improved cell line stability and reproducibility [Hughes et al., 2010; Kleinman, and Martin, 2005]. Further, the use of a single coating concentration across substrates may have similarly limited optimization of cell culture conditions. It is possible and likely that altering concentrations of each protein substrate independently may further influence cell stability and culture reproducibility. The current study confirmed that protein substrate does affect epithelial cell culture outcomes, and although we hypothesize that substrate concentration also influences the parameters discussed, this was not evaluated in this study and should be pursued in future experiments.

The in vivo makeup of the vocal fold epithelial basement membrane is of great interest given the findings of the current study, and better characterization of the basement membrane in healthy and pathologic vocal fold tissue is of critical importance to the optimization of primary cell culture. This line of work also has the potential to lend great insight into the mechanisms involved in vocal fold wound healing in normal tissue as well as in the presence of scar and other lesions that affect the fibrous makeup of the basement membrane in vivo.

Conclusions

The interaction of vocal fold epithelial cells with the protein components of the basement membrane in vivo is complex. The basement membrane regulates a wide range of functions and behaviors including cell adhesion, cell morphology, cell differentiation, and epithelial barrier integrity [Morrissey, and Sherwood, 2015]. As such, these interactions should be considered in the development of an in vitro cell culture system. The goal of any cell culture model is to closely replicate the environment and physiologic cell responses seen in vivo; as such it is imperative that basement membrane interactions are considered in the development of an in vitro cell culture system.

While not all of the possible mechanisms of interaction were expressly demonstrated or investigated in the current study, the data presented support a major role of the basement membrane protein matrix in the integrity of the isolated epithelial cell layer in vitro. The interaction of basal epithelial cells with collagen IV promotes a consistently strong epithelial barrier. The exact mechanism of the stabilization in TEER values achieved in the collagen IV condition compared to the other protein substrates remains unclear, as there is no clear relationship with the other measures investigated (cell proliferation, epithelial depth, cell differentiation, cell morphology). On the contrary, interaction of cells with laminin instead promotes the growth of mesenchymal-type cells in rabbit epithelial cell lines. Further investigation of the mechanisms by which laminin promotes mesenchymal cell growth in vitro and in vivo is still needed; however, the relationship identified in this study suggests that the abundance of laminin in the basement membrane in vivo may play a role both in vocal fold wound healing and also in the development of fibrous lesions.

Understanding the interaction of the vocal fold epithelium with each component of the basement membrane and underlying fibrous lamina propria will guide future in vivo studies of vocal fold injury and repair. Further, we have demonstrated that choice of protein substrate in vitro has significant consequences to cell morphology and physiology. Care should be taken in making this choice in any tissue type such that the in vitro model can accurately reflect the in vivo environment in a species-specific manner.

Collagen IV appears to be the optimal protein substrate for primary rabbit vocal fold epithelial cells, with stabilization of culture achieved by P2 day 10, offering an optimal starting point for further experimental manipulation. However, further optimization of protein substrate concentration and media composition will inevitably affect the timeline for cell culture in this system. These parameters were chosen based on the observed study parameters across the cell lines used here, and each cell culture model system should be similarly independently assessed based on relevant in vivo environmental factors, such as basement membrane components and extracellular interactions.

Supplementary Material

1

S1 Table. Epithelial Cell Line 1 TEER Data.

S2 Table. Epithelial Cell Line 2 TEER Data.

S3 Table. Epithelial Cell Line 3 TEER Data.

S4 Table. Epithelial Cell Line 1 Vimentin Expression Data.

S5 Table. Rabbit Epithelial Cell Line 1 Proliferation Data

S6 Figure. Immunofluorescence of E-cadherin and Vimentin. E-cadherin (in green) and Vimentin (in red) on day 13 of P2 in the (a) collagen IV substrate condition and (b) laminin substrate condition. (a) shows contiguous labeling of E-cadherin across the field of view even in the presence of Vimentin-positive cells, consistent with observations in the collagen I and fibronectin conditions. (b) shows a distinct group of vimentin-positive cells with distinct spindle-shaped morphology (top left quadrant), with a visible decrease in E-cadherin labeling in the same region, suggesting discontinuity of the epithelial barrier in the laminin condition.

Acknowledgement

This study was supported in full by a research grant from the National Institute on Deafness and Other Communication Disorders of the National Institutes of Health (R01DC015405, PI: Rousseau). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

The authors would like to acknowledge the contributions of Dr. Maria Powell and Gary Gartling toward the final preparation of this manuscript.

Funding Sources

This study was supported in full by a research grant from the National Institute on Deafness and Other Communication Disorders of the National Institutes of Health (R01DC015405, PI Rousseau).

Footnotes

Statement of Ethics

The procedures used in this study were performed in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, National Institutes of Health Guide for the Care and Use of Laboratory Animals, and Animal Welfare Act (7 U.S.C. et seq.). The animal use protocol was approved by the Institutional Animal Care and Use Committee of Vanderbilt University Medical Center (protocol # M16100162).

Disclosure Statement

Rousseau receives research funding from the National Institute on Deafness and Other Communication Disorders of the National Institutes of Health (grant numbers: R01DC015405, PI: Rousseau, R01DC016236, PI: Rousseau, and R01DC017397, PI: Branski). Kimball, Sayce, Xu, and Kruszka have no conflicts of interest to declare.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

S1 Table. Epithelial Cell Line 1 TEER Data.

S2 Table. Epithelial Cell Line 2 TEER Data.

S3 Table. Epithelial Cell Line 3 TEER Data.

S4 Table. Epithelial Cell Line 1 Vimentin Expression Data.

S5 Table. Rabbit Epithelial Cell Line 1 Proliferation Data

S6 Figure. Immunofluorescence of E-cadherin and Vimentin. E-cadherin (in green) and Vimentin (in red) on day 13 of P2 in the (a) collagen IV substrate condition and (b) laminin substrate condition. (a) shows contiguous labeling of E-cadherin across the field of view even in the presence of Vimentin-positive cells, consistent with observations in the collagen I and fibronectin conditions. (b) shows a distinct group of vimentin-positive cells with distinct spindle-shaped morphology (top left quadrant), with a visible decrease in E-cadherin labeling in the same region, suggesting discontinuity of the epithelial barrier in the laminin condition.

NIHMS1664490-supplement-1.pdf (1.4MB, pdf)

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