Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro

Abstract

The purpose of this study was to compare cell growth characteristics, ciliated cell differentiation, and function of human nasal epithelial cells established as explant outgrowth cultures or dissociated tissue cultures. Human nasal mucosa of the uncinate process was obtained by endoscopy and epithelial cell cultures were established by explant outgrowth or dissociated tissue culture methods. Epithelial cell growth characteristics were observed by inverted phase contrast microscopy. Ciliated cell differentiation was detected by β-tubulin IV and ZO-1 immunocytochemistry. Basal and ATP-stimulated ciliary beat frequency (CBF) was measured using a high-speed digital microscopic imaging system. Both the explant and dissociated tissue cultures established as monolayers with tight junctions and differentiated cell composition, with both types of cultures comprising ciliated and non-ciliated epithelial cells. Fibroblasts were also frequently found in explant cultures but rarely seen in dissociated tissue cultures. In both culture systems, the highest ciliated cell density appeared at 7th–10th culture day and declined with time, with the lifespan of ciliated cells ranging from 14 to 21 days. Overall, 10% of the cells in explant cultures and 20% of the cells in the dissociated tissue cultures were ciliated. These two cultures demonstrated similar ciliary beat frequency values at baseline (7.78±1.99 Hz and 7.91±2.52 Hz, respectively) and reacted equivalently following stimulation with 100 μM ATP. The results of this study indicate that both the explant outgrowth and dissociated tissue culture techniques are suitable for growing well-differentiated nasal ciliated and non-ciliated cells, which have growth characteristics and ciliary activity similar to those of nasal epithelial cells in vivo.

Keywords

ciliated cells / ciliary beat frequency / dissociated tissue culture / explant culture / nasal epithelial cells

Introduction

There is a plethora of evidence in the literature that the airway epithelium plays an important physicochemical role in the etiology of airway disease [1-3]. The ciliated pseudostratified columnar epithelial cells are the predominant cell type of the respiratory epithelium, with each cell having between 200 to 300 cilia, which beat in tandem at a particular frequency to clear the inhaled particulate matter trapped in mucus released by interspersed goblet cells. Evidence from several studies suggests that cilia clearly play a major role in maintaining the functional integrity and defense of the respiratory tract and that efficient mucociliary clearance is dependent on coordinated patterns of ciliary activity [4]. Indeed, there is evidence that mucociliary clearance dysfunction is involved in the pathogenesis of several respiratory diseases such as chronic rhinosinusitis, cystic fibrosis, chronic bronchitis, and asthma [5], although the mechanisms underlying this dysfunction in the various diseases are not entirely clear [4,6].

Primary cell cultures of human nasal epithelial cells provide an ideal in vitro model to study the role of these cells in the pathogenesis of respiratory mucosal disease [7], as well as the mechanisms involved in ciliogenesis, ciliary movement, mucus secretion, nasal mucosal infection, and the effect of novel therapeutic agents affecting these processes [8]. Although different techniques have been employed to culture human nasal epithelial cells, the primary goal of in vitro cell culture is to achieve differentiated morphology and biochemical features, resembling original tissue as closely as possible [9]. The aim of this study was thus to culture and compare the epithelial cell characteristics, ciliated cell differentiation and function of human nasal epithelial cells, established as either explant outgrowth cultures or dissociated tissue cultures.

Materials and methods

Reagents

Dulbecco’s Modified Eagle’s Medium (DMEM), Dulbecco’s Modified Eagle’s Medium/Ham’s F12 (DMEM/F12) and antibiotics were purchased from GIBCOBRL (Grand Island, NY, USA). Bronchial Epithelial Growth Medium (BEGM) was from Lonza (Walkersville, MD, USA). Anti-zona occludens-1 (ZO-1) antibody was obtained from Invitrogen Corporation (Carlsbad, CA, USA). Goat anti-mouse IgG, H & L chain specific fluorescein conjugate and goat anti-rabbit IgG, H & L chain specific rhodamine conjugate were obtained from Merck Chemicals Ltd. (Darmstadt, Germany). 4′, 6-diamidino-2-phenylindole (DAPI) was from Beyotime Biotech (Nantong, China). Adenosine triphosphate (ATP), human placental collagen, protease, anti β-tubulin IV antibody and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Explant outgrowth culture of human nasal epithelial cells

Human nasal epithelial cells were established as explant outgrowth cultures as described previously [10]. In detail, mucosa of the uncinate process was obtained by endoscopy from patients with chronic rhinosinusitus. The samples were immediately placed into isotonic sodium chloride solution (normal saline) and transported to the laboratory, where the tissue was prepared for cell cultures. The dissected mucosa was cut into approximately 0.5-mm squares, plated on human placental collagen, type IV-coated glass coverslips, and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. The explants were incubated at 37°C in a 5% CO₂ in air atmosphere, and observed for cell outgrowth after 2-3 days. The culture medium was changed 48 hourly until the cells had grown to confluency.

The study protocol was reviewed and approved by the local ethics board of Beijing TongRen Hospital, Capital Medical University. Informed consent was obtained from all the participants.

Culture of human nasal epithelial cells from dissociated nasal mucosal tissue

Human nasal epithelial cells were established from dissociated nasal mucosal tissue using an adaptation of the methods published by Nlend et al [11]. The dissected mucosa was incubated in 0.05% protease (type XIV) in DMEM overnight at 4°C. At the end of incubation, 5.0%FBS was added to the medium to stop the enzymatic digestion, and the epithelial cells were released from the tissue matrix by vigorous shaking. The cell suspension was centrifuged at 50 g for 10 min, and the cells were resuspended in fresh DMEM/F12: BEGM 1:1 medium, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were incubated at 37°C for 1 h in plastic culture dishes to remove the fibroblasts and the epithelial cells were plated on human placental collagen, type IV-coated glass coverslips at a density of 5 × 10⁵/ml in a submerged fashion in DMEM/F12: BEGM 1:1 medium. The cells were incubated at 37°C in a 5% CO₂ in air atmosphere. The culture medium was changed 48 hourly and cultured as above for the explant cultures.

Dual-label fluorescent cytochemistry

Cultured human nasal epithelial cells were fixed in a 50∶50 mixture of methanol-acetone and after rinsing in PBS, treated with 0.3% triton X-100, blocked with 5% skimmed milk and incubated overnight at 4°C with β-tubulin IV antibody (1:500 dilution) and ZO-1 antibody (1∶100 dilution). The cells were then incubated with FITC-conjugated goat anti-mouse IgG, 1:800, and rhodamine-conjugated goat anti-rabbit IgG, 1:500 for 40 min at 37°C, and following counterstaining with DAPI nuclear stain, and the cells were examined by microscopy using an Olympus IX81 confocol microscope (Tokyo, Japan).

Measurement of ciliary beat frequency (CBF)

Measurements of CBF were made according to a previously published method [12]. Briefly, cell cultures were equilibrated at 25°C for 10 min and following treatment with sterile Hank’s balanced salt solution (HBSS), the ciliated epithelial cells were observed by phase contrast microscopy using an Olympus IX71 inverted microscope (Tokyo, Japan) equipped with a × 40, 1.3 NA, Ph 4, oil immersion objective. Images ciliary movement were captured on a high speed CCD camera (TM-6710cL, Pulnix America, Sunnyvale, USA), equipped with a frame grabber (Meteor, Matrox Co., USA) and recording software (StreamPix 3.16.5, Norpix Corporation, USA). The phase-contrast images of ciliary movement were recorded at 240 frames per second and evaluated using a specialist image analysis program (IPLab v3.65a, Scanalytics, Inc., USA). The CBF was measured using a 3-s waveform (~720 frames) that was generated by the variation in gray level intensity of the phase-contrast images resulting from the repetitive motion of cilia. The frequency of each ciliary beat cycle was determined from the period of each cycle of the gray intensity waveform, bydigitally extracting the average gray value from the image data set in a region of interest near the ciliary tip when the cilia were lying in the rest phase position and then plotting this value with respect to time (i.e., frame number).

Basal CBF of the cultured cells was assessed in five randomly selected 7-10 days old cultures established from either explants or dissociated tissue, by recording ciliary activity in at least 10 random locations in each culture. Similarly, the effect of an exogenous stimulus (100 μM ATP) on the CBF of these cells was also assessed in randomly selected cultures for three seconds every 1 min for 5 min.

Statistics

All data were expressed as means±SEM. Differences in basal CBF observed in the cultures established from explants or from dissociated tissue were assessed using the t-test, whereas differences in ATP-stimulated cells were assessed using a Repeated Measures ANOVA followed by Bonferroni’s multiple comparison test. Values of P<0.05 were considered to be statistically significant.

Results

Epithelial cell growth characteristics in explant outgrowth and dissociated tissue cultures

Assessment of cultures, demonstrated that epithelial cells were adhered to the growth surface in both explant and dissociated tissue cultures by 24 h of culture and ciliated cell and ciliary activity were evident by 48 h. The number of ciliated cells increased with time in both culture types, with the highest ciliated cell density being evident at 7th-10th day of culture and overall, lifespan of these cells being 14 to 21 days.

Assessment of the cell types in the cultures demonstrated both the explant and dissociated tissue cultures comprise ciliated and non-ciliated epithelial cells. In contrast, explant cultures also comprised fibroblasts, whereas this cell was rarely seen in cultures established from dissociated nasal tissue.

Ciliated cell differentiation in explant outgrowth and dissociated tissue cultures

Immunocytochemistry for β-tubulin IV, a ciliary cell marker, and ZO-1, a tight junction marker, was performed to determine the differentiation and confluence of cultured epithelial cells. Fig.1 shows confocal image of epithelial cells at day 7 in explant (A) and dissociated tissue (B) cultures, and indicates that both type of cell cultures established as monolayers with tight junctions and differentiated cell composition with cilia.

Estimation of ciliated cell density indicated that 10% of the cells in explant cultures were ciliated, compared to 20% of the cells in the dissociated tissue cultures at confluence.

CBF in explant outgrowth and dissociated tissue cultures

Assessment of basal CBF demonstrated that these cultures were not significantly different in this regard, with the cells from explants demonstrating a mean CBF value of 7.78±1.99 Hz and cells from dissociated tissue demonstrating a mean CBF value of 7.91±2.52 Hz (Fig. 2).

The changes in CBF in response to 100 μM ATP are shown in Fig. 3. ATP induced a rapid and marked increase in CBF in both cultures, with the CBF in explant cultures increasing to 132.45%±7.86%, 129.03%±12.63%, 131.59%±11.72%, 137.68%±9.94% and 129.9%±15.20% of baseline values at 1, 2, 3, 4, 5 min, respectively, after ATP treatment. Similarly, in dissociated tissue cultures, the CBF increased to 136.94%±22.34%, 143.47%±14.44%, 143.74%±13.4%, 144.11%±13.71% and 139.25%±12.13% of the baseline CBF at 1, 2, 3, 4 and 5 min, respectively, after ATP treatment.

Discussion

Explant outgrowth and dissociated tissue cultures are two basic approaches to establishing cultures of differentiated respiratory cells, with the cultures from dissociated tissue being established as adherent (which can be further divided as submersion and air-liquid interface [ALI] cultures), suspension and sequential cultures. In the present study, we compared the epithelial cell growth characteristics, ciliated cell differentiation and function of human nasal epithelial cells grown as explant or dissociated tissue cultures, and found that the cell growth characteristics and ciliary function were similar for both types of cultures. However, the density of ciliated cell in dissociated tissue cultures was higher than in explant cultures. Additionally, the cell types in the two culture types were different, with the explant cultures being composed of non-ciliated and ciliated epithelial cells and fibroblasts, while the dissociated tissue cultures comprised mostly non-ciliated and ciliated epithelial cells.

The explant outgrowth cell culture technique is one of the first system developed to grow epithelial cells in vitro, whereby the tissue (biopsy) specimens are cut into small explants less than 1mm³ in size and immersed in culture medium on uncoated or coated plastic supports. Our study showed that in explant cultures, the epithelial cells differentiated well and ciliated cell density approached around 10%. Furthermore, the basal CBF values of 7-10 Hz noted for nasal epithelial cells in the present study were comparable to those noted by others in vitro [13-15] and in vivo [16], and were additionally sensitive to the application of an exogenous stimulus such as ATP. While these findings suggest that the explant cell culture technique is a reliable cell culture system for respiratory cells, and has been shown to be suitable, especially for investigations on the immune response to pathogens and allergens [17] and the epithelial barrier function in allergic rhinitis. One limitation of this culture method is the presence of non-epithelial cell types in explants makes the experimental results difficult to predict and/or to interpret. Another disadvantage is the longer time needed to establish confluent cell cultures, compared with the cultures starting from dissociated cells [9].

Dissociated tissue cell culture is one of the most widely used techniques for culturing respiratory epithelial cells. This technique entails gentle digestion of the nasal mucosa specimens with a protease (pronase) at a low temperature to release single cells or small cell aggregates, which are seeded in culture medium. In contrast to the explant culture technique, the major advantage of the dissociated tissue culture technique is that it allows easier isolation of the epithelial cells in higher quantity and with apparently greater purity. Furthermore, contaminating cells such as fibroblasts can be reduced dramatically by culturing the isolated cells in serum-free medium or adherence to plastic supports over a course of specified time, prior to culturing the epithelial cells in appropriate growth medium. The present study has indicated that number of ciliated cells were also greater in these cultures than in explant outgrowth cultures, and that the lifespan of the ciliated cells was about 14-21 days. Furthermore, the ciliated cells maintained a constant basal and ATP-stimulated ciliary reactivity over the culture period. Submerged dissociated tissue culture is an ideal culture model suitable for lots of epithelial research without special requirements, such as pharmacological and toxicological studies.

Air-liquid interface (ALI) culture is another widely used culture system for respiratory epithelial cells. In this method, the dissociated cells are grown on a permeable insert and exposed basolaterally to the feeding culture medium while their apical surface is exposed to air, thus simulating the conditions for the airway respiratory epithelium in vivo. The ALI culture system effectively allows the establishment of a much higher level differentiation of cells in a pseudostratified type epithelium culture containing ciliated, secretory, and basal cells compared with the culture methods employing submerged cells [18]. Although cells grown by the ALI technique show better morphology, differentiation, polarity and ion transport properties, the major limitation of this technique is that it is very time-consuming and rather costly, with poor epithelial cell proliferation and lengthy cell growth. Another major drawback is that the layer of the differentiated cells is viable for comparatively short periods, and in some cases the cells start to detach from the culture vessels within 14 days of culture [19]. Despite, ALI culture represents the most promising culture system for pharmacological, transport and mucociliary studies; it is especially useful for some special experiments such as measuring the transport and metabolism of drugs across the cell layer and within the cells due to the presence of a permeable insert supporting adherent cells.

In summary, the present study compared the epithelial cell growth characteristics, ciliated cell differentiation and function between explant outgrowth and dissociated tissue cultures established from human nasal mucosa tissue. We showed that the epithelial cells differentiated well in both types of cultures and with cilia beating and reacting sensitively toward exogenous stimuli. Neither of these techniques appears to be superior to the other, and each one could be employed effectively, depending on the specific scientific outcomes to be investigated.

References

[1] Kojima T, Go M, Takano K, Kurose M, Ohkuni T, Koizumi J, Kamekura R, Ogasawara N, Masaki T, Fuchimoto J, Obata Ki, Hirakawa S, Nomura K, Keira T, Miyata R, Fujii N, Tsutsumi H, Himi T, Sawada N. Regulation of tight junctions in upper airway epithelium. Biomed Res Int 2013(2013): 947072

[2] Kato A, Schleimer RP. Beyond inflammation: airway epithelial cells are at the interface of innate and adaptive immunity. Curr Opin Immunol2007; 19(6): 711-720

[3] Devalia JL, Davies RJ. Airway epithelial cells and mediators of inflammation. Respir Med1993; 87(6): 405-408

[4] Gudis D, Zhao KQ, Cohen NA. Acquired cilia dysfunction in chronic rhinosinusitis. Am J Rhinol Allergy2012; 26(1): 1-6

[5] Antunes MB, Gudis DA, Cohen NA. Epithelium, cilia, and mucus: their importance in chronic rhinosinusitis. Immunol Allergy Clin North Am2009; 29(4): 631-643

[6] Gudis DA, Cohen NA. Cilia dysfunction. Otolaryngol Clin North Am2010; 43(3): 461-472, vii

[7] Comer DM, Elborn JS, Ennis M. Comparison of nasal and bronchial epithelial cells obtained from patients with COPD. PLoS ONE2012; 7(3): e32924

[8] Bukowy Z, Ziętkiewicz E, Witt M. In vitro culturing of ciliary respiratory cells—a model for studies of genetic diseases. J Appl Genet2011; 52(1): 39-51

[9] Dimova S, Brewster ME, Noppe M, Jorissen M, Augustijns P. The use of human nasal in vitro cell systems during drug discovery and development. Toxicol In Vitro2005; 19(1): 107-122

[10] Zhang L, Han D, Song X, Wang K, Wang H. Effect of oxymetazoline on healthy human nasal ciliary beat frequency measured with high-speed digital microscopy and mucociliary transport time. Ann Otol Rhinol Laryngol2008; 117(2): 127-133

[11] Nlend MC, Bookman RJ, Conner GE, Salathe M. Regulator of G-protein signaling protein 2 modulates purinergic calcium and ciliary beat frequency responses in airway epithelia. Am J Respir Cell Mol Biol2002; 27(4): 436-445

[12] Jiao J, Wang H, Lou W, Jin S, Fan E, Li Y, Han D, Zhang L. Regulation of ciliary beat frequency by the nitric oxide signaling pathway in mouse nasal and tracheal epithelial cells. Exp Cell Res2011; 317(17): 2548-2553

[13] Devalia JL, Sapsford RJ, Wells CW, Richman P, Davies RJ. Culture and comparison of human bronchial and nasal epithelial cells in vitro. Respir Med1990; 84(4): 303-312

[14] Rhee CS, Min YG, Lee CH, Kwon TY, Lee CH, Yi WJ, Park KS. Ciliary beat frequency in cultured human nasal epithelial cells. Ann Otol Rhinol Laryngol2001; 110(11): 1011-1016

[15] Dimova S, Vlaeminck V, Brewster ME, Noppe M, Jorissen M, Augustijns P. Stable ciliary activity in human nasal epithelial cells grown in a perfusion system. Int J Pharm2005; 292(1-2): 157-168

[16] Robertson A, Stannard W, Passant C, O’Callaghan C, Banerjee A. What effect does isoflurane have upon ciliary beat pattern: an in vivo study. Clin Otolaryngol Allied Sci2004; 29(2): 157-160

[17] Liu Z, Lu X, Wang H, You XJ, Gao QX, Cui YH. Group II subfamily secretory phospholipase A2 enzymes: expression in chronic rhinosinusitis with and without nasal polyps. Allergy2007; 62(9): 999-1006

[18] Agu RU, Jorissen M, Willems T, Augustijns P, Kinget R, Verbeke N. In-vitro nasal drug delivery studies: comparison of derivatised, fibrillar and polymerised collagen matrix-based human nasal primary culture systems for nasal drug delivery studies. J Pharm Pharmacol2001; 53(11): 1447-1456

[19] Werner U, Kissel T. Development of a human nasal epithelial cell culture model and its suitability for transport and metabolism studies under in vitro conditions. Pharm Res1995; 12(4): 565-571

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg