Improvement Of Human Nasal Epithelial Cells Culture And Ions Transport Evaluation

The nasal epithelial cells have two important functions of a protective barrier and the transport of ions and water. Epithelial ion and water transport is the important foundation of mucociliary transport. Further researches of electrophysiological facilitate our understanding of physiology and pathology relating to nasal and sinus mucous. Not only is the CF a well known typical channel disease, but also chronic sinusitis and nasal polyps may be pathophysiologically relevant in elevated rates of ion transport. Traditional methods for electrophysiological research such as patch clamp techniques mainly require single cell model. It is unavoidable that channels function will be disturbed by mechanical and enzymatic dissociation. In contrast, Ussing chamber technique has attracted more and more attentions for its no enzymatic and mechanical damage, and a variable control of the solution in two sides of chamber. Cell culture technique is the basis of Ussing chamber studying, and for this reason it becomes very important to us. Developing a primary culture model of differentiated human nasal epithelium which grown on semipermeable membranes at an air-liquid interface formed confluent polarized monolayer epithelium with high transepithelial resistances that suit for Ussing chamber electrophysiological studies is a challenge to us. To our knowledge, such research has not been reported domaesticly.In vitro models of airway epithelial cells are established for years. However, there are too many kinds of methods, medium and supplements being used, all of which make researchers confusion and the result is very variable. Deep understanding the difference between different culture mediums, and developing a primary culture model with wide application especially suiting for ussing chamber is a meaningful work. This paper presents our research about nasal epithelial culture. To establish a novel primary culture model of differentiated human nasal epithelium, meanwile investigate its characteristics in the respect of cell morphology, ciliary beat frequency, histochemistry, electrophysiological function, and demonstrate its important features of native human nasal epithelium. We have made the preliminary observations about electrophysiological features of nasal polyps by the end.OBJECTIVE:To develop a wide applicability primary culture model of differentiated human nasal epithelium.METHODS:Epithelial cells were removed from 27 patients'polyp or normal nasal mucosa by 0.1% Protease XIV and 0.001% DNase disaggregation and counted using a hemocytometer. Different cell culture mediums (Wu's medium, BEGM, modified Gray's medium:the modification including EGF was reduced to 0.01ng/ml, the concentration of BPE was the same as BEGM's) and medium supplements, culutre interface model were tested, and the outcome were evaluated by cell proliferation, differentiation, morphology, composition, ciliogenesis, ciliary beat frequency and adherence rate.①.Nasal epithelial cells Purity:cells (2 specimens, n= 8) were plated in serum-containing medium on plastic dishes for three days. The fibroblasts were stained with mouse monoclonal anti-human vimentin, and cell number, proportion was calculated by Image-Pro Plus 6.②.The effect of serum and Wu's medium on the cell adherences to the matrix (ONE-way AN OVA):cells disaggregated from 6 specimens, were plated in three 96-well microtiter plates (the cells of one 96-well microtiter plate were came from 2 specimens), and each plates contain 32 wells which have cells. Those wells were divided into 4 groups: Wu's medium +collagen pretreatment,10%FBS+Wu's medium+collagen pretreatment, serum containing medium(10%FBS)+ collagen pretreatment, serum containing medium(10%FBS), and an extra control group. Cells were calculated by MTT method.③. Effects of different serum-free mediums on cells morphology:cells (6 specimens,3 groups, n=6) were divided into 3 groups: Wu's medium, BEGM and serum-containing group, and the morphology were record.④.The quantity and distribution of basal cells:cells were plated in ALI culture medium on pore culture inserts establishing an air-liquid interface culture mode and plastic dishes in immersion mode respectively (2 groups,3 specimens, n=6). Basal cells were stained with mouse monoclonal anti-human CK14 after 7 days'culture. The number of CK14 positive cell in serum containing medium group were calculated by Image-Pro Plus 6 and the ALI medium group were injuried artificially to make a deletion of part cells a day before immunohistochemical study.⑤.The result of ciliogenesis in air-liquid interface ALI culture medium:mouse monoclonal anti-human p-Tubulin were used to stain the ciliated cells after 21 days culture(3 specimens, n=12),and Image-Pro Plus 6 were used to calculate the cells number.⑥. Evaluation two medium (Wu's and serum-containing medium) with ciliary beat frequency:cells (3 specimens, n=15) were cultured with Wu's and serum-containing medium respectively, and the CBF were record from the first day to the ninth day. The data was analyzed by two-way ANOVA.⑦. Evaluation the CBF in air-liquid interface ALI culture medium:the CBF was record in the 14th and 30th day respectively (1 specimens, n=15). And after 60th culture the morphology of the cells were record.⑧.Scanning electron microscope:cells were cultured on porous supports in ALI medium, and scanning electron microscope was performed on day 14.RESULTS:①.Purity of nasal epithelial cells. The enzymatic dissociation of the specimen could result in a typical yield of 4.2±1.8×106 cells/specimen (Mean±SEM, n=8) with high viability.②. The effect of serum and Wu's medium on the cell adherences to the matrix(ONE-way ANOVA). On the 1st day, there were significant differences between the Wu's treatment+collagen pretreatment and serum-containing medium whithout collagen pretreatment, serum-containing medium+collagen pretreatment and serum-containing medium whithout collagen pretreatment respectively (P<0.05). On the 3rd day, the cell proliferation of the Wu's was higher than other three groups significantly (P<0.05), while no significant difference was shown between the rest groups (P>0.05). On day 5 the comparison between groups is the same as the 3rd day.③. Effects of different serum-free mediums on cells morphology:In Wu's group and serum-containing medium group the cells appeared irregular and to be form stratified squamous epithelium. Cultured more than 2 weeks, most original ciliary epithelial cells disappeared, some of which died. Cells cultured in the BEGM medium had a regular shape, but no differentiation of ciliary epithelial cells after 2 weeks cultured.④. Amount of CK14 positive basal cells in serum-containing medium group was 23±5%(Mean±SEM, n=6),but on CK14 expression in the luminal surface in the ALI culture system. Artificial damage to the cells resulted in high CK14 expression.⑤. Ciliary beat frequency (CBF) was correlated with the categories of culture medium and the incubation time. Serum-containing medium have a higher CBF than Wu's with statistically significant difference (P<0.01, two-way ANOVA analysis)⑥.The morphology of the cultures in an air-liquid interface (ALI) culture system was characterized by inverted microscope. The cells remained polarization,orderly, without no squamous epithelization or vacuolization, and revealed ciliary epithelial cells differentiation.up to day 14.On day 21 the coverage rate (Mean±SEM, n=12) of the cilia was 20.4±3.2%, while stratified epithelium occurred partly on day 60. The CBF (Mean±SE, n=15) amounted to 8.8±1.7Hz,8.5±1.3 Hz respectively on day 7, there was no statistically significant difference between the two groups by the mean of T test.⑦. Scanning electron microscope observation:A monolayer columnar epithelium cells were densely covered with microvilli on the 14th day of culture, some of which were induced differentiation to a ciliated epithelial cell.CONCLUSIONS:①.The enzymatic dissociation of the specimen could get high quantity,quality,purity human nasal epithelial cells;②. serum and collagen have a synergism on cell attachment;③.serum decreases the proliferation and induces squamous differentiation;④.High [Ca](1.05mM) and EGF(25ng/ml) inhibites ciliogenesis even in the presence of High RA(50nM);⑤.ALI media on semipermeable membranes at an air interface supports a good cilia differentiation and a long time culture;⑥.there is no basal cell on lamina in well differentiation nasal epithelial,and basal cell may be play a important role in restoration process of epithelial cell after impairment.Objective:To evaluate the model of human nasal epithelial by bioelectric properties and make comparison between different culture periods by transepithelial bioelectric properties.To make a preliminary research of human nasal polyps epithelial channels.Methods:The methods of cells culture were the same as part one.4 nasal polyps epithelial were dissociated and plated in 16 wells of Trans Well porous supports.Those wells were divided into two groups randomly(14d gruop,45d gruop) formed confluent within 4 days, and were mounted in Ussing chambers when culture time exceed 14 days or 45 days. PD (Transepithelial potential difference),Isc (short circuit current) were measured and Rt(transepithelial resistance) were calculated by Ohm's law.After that,lamina was exposed to a series of channel blocker or agonist (Amiloride, Forskolin, DIDS in sequence) and Basolateral side was exposed to bumetanide finally.RESULTS:After 14 days or 45 days of primary culture, the human nasal epithelial cells formed a tight junction and no significant morphological differences were observed. The basal Isc and resistence(R) in Group 14 days were 44.7±2.8μA/cm2 and 347.7±28Ω/cm2 respectively, while in Group 45 days were 18.1±1.9μA/cm2,625.7±88Ω/cm2 respectively, there was significant difference between the two groups(n=8, P<0.05). The share of the amiloride-sensitive Isc in the basal Isc were 92±2%,85±3% respectively, no significant difference (n=8, P>0.05) showed in comparison of the two groups. There was a statistically significant group difference (P< 0.05) in the activity of each drug-sensitive channel. Forskolin activated Cystic fibrosis transmembrane conductance regulator(CFTR), can also activate Ca2+-activated Cl" channels (CaCC) and Na+/K+/2Cl- cotransport channel(NKCC),while Cl- secretion via apical CFTR was sharply decreased following blocking NKCC.Conclusion:①. In primary culture of human nasal epithelial cells formed intercellular tight junction and high transepithelial resistance afer 14 days and 45 days, which corresponded with the requests of electrophysiological studies.②. The amiloride sensitive epithelial Na+ channel(ENaC), Cystic fibrosis transmembrane conductance regulator(CFTR) and Ca2+-activated Cl channels are located in the apical membrane of nasal epithelial cells.Na+/K+/2Cl-cotransport channel (NKCC) are located in the basolateral membrane.③.Experiments proved that the amiloride-sensitive Isc in the basal Isc was prevailing,which indicates abnormality of sodium ion transport might be involved into the development of nasal polyposis.④. Cl- secretion via apical CFTR was positively correlated with the activity of the basolateral NKCC⑤. There must be close communication between CFTR, CaCC, NKCC, ENaC, and those channels may be activated or suppressed by each other. By this way ASL (apical surface lequid) and electrophysiology could be maintained in a normal state.