The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome – associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 Ω×cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.
1. Culturing Calu-3 Cells for Use in Transwell Cultures
Safety Measures: Perform all procedures in a biosafety cabinet using sterile culture technique.
2. Growing Polarized Calu-3 Liquid-covered Cultures (LCC)
Safety Measures: Perform all procedures in a biosafety cabinet using sterile culture technique.
3. Evaluating Resistance Development of Calu-3 LCC
Safety Measures: Perform all procedures in a biosafety cabinet using sterile culture technique.
4. Infecting Polarized Calu-3 LCC with Respiratory Virus
Safety Measures: Perform all procedures in a biosafety cabinet using sterile culture technique, at a biosafety level appropriate for the virus being used.
When grown as liquid-covered cultures (LCC) in Transwell culture systems, as illustrated in Figure 1, Calu-3 cells polarize, developing distinct apical and basolateral surfaces. Following the method described here, the trans-epithelial electrical resistance (TEER) of Calu-3 LCC reaches a plateau at or above 1,000 Ω×cm2 within 3 weeks after seeding, an example of which is shown in Figure 2. The tight junctions formed between polarized cells prevent passive equilibration of small molecules between the apical and basolateral compartments. Thus, a modified sodium fluorescein equilibration assay is used to confirm polarization of Calu-3 LCC 15,17,18 . As the TEER of Calu-3 cell monolayers in LCC increases, the amount of fluorescein that passively equilibrates into the basolateral compartment decreases. Once the TEER is 1,000 Ω×cm2, the amount of fluorescein that equilibrates into the basolateral compartment is ≤ 1%, as shown in Figure 3; therefore, Calu-3 LCC are considered to be fully polarized when the TEER is ≥ 1,000 Ω×cm2. The peak TEER measurement of Calu-3 LCC may vary from experiment to experiment. However, once TEER values plateau for any given experiment, a fully polarized, uninfected Calu-3 LCC may be stable for 5 through 12 weeks post-seeding. Absence of resistance development in Transwell-cultured Calu-3 may be caused by several factors as outlined in Table 1. Once the TEER of Calu-3 LCC plateaus at or above 1,000 Ω×cm2, the model is ready to be used to examine airway epithelial cell responses to respiratory pathogens, including respiratory syncytial virus (RSV). Exposure to RSV results in a more rapid decline in polarized culture integrity compared to a mock-infection of cells (Figure 4).
Figure 1. Cross-section representation of Transwell-cultured cells. Cells are grown on the apical surface of the insert membrane.
Figure 2. Development of trans-epithelial electrical resistance (TEER) and polarization of Calu-3 cells after seeding in Transwell inserts. At each time point, TEER is presented as median Ω x cm2 ± SEM of 32 independent wells from one representative experiment.
Figure 3. Passive equilibration of sodium fluorescein into the basolateral compartment of Calu-3 cell monolayers is inhibited as cells become polarized. Cumulative data from four independent experiments is presented. Each data point represents an individual measurement.
Figure 4. RSV infection of polarized Calu-3 LCC accelerates a decline in monolayer integrity. Polarized Calu-3 cells were infected with RSV-A2 at an MOI=1 on day 0, and polarity was monitored for 8 weeks post-infection. One representative experiment is shown. Data are presented as median resistance of 8 independent wells per infection ± SEM per time point. *p<0.05 between mock- and RSV-A2-infected cultures, as determined by Student’s t-test.
Issue | Resolution(s) |
Resistance is not measurable |
|
Resistance develops, but full polarization (≥1,000 Ω×cm2) is not attained |
|
Cells fully polarize, but resistance is not consistent from one reading to the next |
|
Table 1. Trouble-shooting problems that may arise when culturing Calu-3 cells in Transwells. Multiple factors contribute to full polarization of Calu-3 cells in liquid-covered cultures, the most likely of which are highlighted in this table.
When establishing Calu-3 LCC in Transwell inserts, cells may not polarize at all, or may not fully polarize, as defined by a TEER ≥ 1,000 Ω×cm2 and ≤ 1% sodium fluorescein dye equilibration between the apical and basolateral compartments. In addition, Calu-3 cells in LCC may fully polarize, but TEER may be inconsistent between measurements. Although fluctuations in TEER measurements of Calu-3 LCC are normal from day to day, once fully polarized, dramatic swings in TEER are not expected until the culture naturally declines with age, which may be as little as 5 weeks or as long as 12 weeks after seeding.
The ability of Calu-3 LCC to polarize depends in part on how cells are maintained and subcultured before use in the Transwell system. Cells that have grown beyond 90% confluence as a monolayer during subculturing, that have been subcultured more than 10 times from frozen stock, or that have not been supplied with fresh medium on a regular schedule are less likely to fully polarize, and any polarization is likely to decline rapidly. Incomplete or total lack of polarization may also be attributed to variation in the material and pore size of Transwells used for Calu-3 LCC, and lot-to-lot variation in Transwells of similar composition and pore size may also affect polarization. Larger pore sizes can allow Calu-3 to grow through the Transwell membrane into the basolateral compartment, preventing the culture from polarizing. An absence of polarization may also be due to bacterial growth, indicated by clouded culture medium, which leads to subsequent breakdown of the tight junctions between Calu-3 cells.
Variable TEER measurements of Calu-3 LCC may be caused by mechanical disruptions of the Calu-3 LCC cell monolayers, the inserts, or the plates themselves. Medium changes and TEER measurements should be performed without pipette tips or electrode leads touching the cells. While performing these operations, care should be taken to avoid introducing air bubbles into the apical and basolateral compartments, which will disrupt the ability of the voltohmmeter to detect resistance. The ability of the voltohmmeter to detect resistance in a culture that is actually polarized may also limited by protein buildup on the electrode leads. This build-up may be removed with gentle sanding, or may be corrected by replacing the electrode.
Once Calu-3 LCC completely polarize and the TEER is no longer increasing, Calu-3 LCC are ready for use as an in vitro model for characterizing host lung epithelial cell responses to respiratory infection. This system permits better characterization of directional responses to pathogens compared to monolayer-cultured lung cell lines traditionally used to study respiratory pathogens, such as A549 and HEp-2 cells, with the additional advantages of Calu-3 LCC being more rapid to develop, more easily obtained, and less expensive to generate than primary, polarized, differentiated NHBE. Similar to NHBE, polarized Calu-3 demonstrate tight junction formation, and produce mucins. However, unlike NHBE, polarized Calu-3 cells do not differentiate into layers of basal cells and ciliated columnar epithelial cells, and few polarized Calu-3 cells develop cilia-like projections19. Thus, although useful for examining polarized responses of airway epithelial cells to respiratory insult, polarized Calu-3 LCC are not an ideal model to examine airway development or remodeling in response to respiratory insult or injury. Mucus production by cultured cells in vitro may impact cellular infectivity, as well as release of infectious virus and virus spread in a polarized model, and a direct comparison of the mucus production between polarized Calu-3 LCC and polarized, differentiated NHBE has not been reported. A549 and HEp-2 cells are easier to culture than Calu-3 cells, however, unlike Calu-3 LCC, they do not form polarized cultures when grown on Transwell inserts, and are thus not ideal models for examining in vitro the responses of polarized epithelial cells to respiratory virus infection.
The authors have nothing to disclose.
The authors wish to thank Elisabeth Blanchard for her technical assistance. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
REAGENTS | |||
Calu-3 | ATCC | HTB-55 | |
0.05% Trypsin – 0.02% EDTA | Gibco/Invitrogen | 25300 | |
Eagle’s Minimum Essential Medium (EMEM) | Gibco/Invitrogen | 07-00100DK | |
Fetal bovine serum (FBS) | HyClone | SH30070.03 | Heat-inactivate, 56 °C, 30 mins |
Non-essential amino acids 100X (10 mM) | Gibco/Invitrogen | 11140 | Store at 4 °C in the dark |
L-glutamine | Gibco/Invitrogen | 25030 | |
HEPES | Gibco/Invitrogen | 15630 | |
EMEM-10% FBS (EMEM-10%) | Supplement EMEM with heat-inactivated FBS to 10% serum, sterile-filter | ||
EMEM-20% FBS + supplements (EMEM-20%+S) | Supplement EMEM to final concentrations: heat-inactivated FBS, 20%; 1X amino acids; 2 mM L-glutamine; 10 mM HEPES; sterile-filter | ||
24-well Transwell plates | Corning Costar | 3472 | 3 μm pore size, polyester |
Trypan blue | Gibco/Invitrogen | 15250 | |
Ethanol | Sigma | E7023 | Prepare to 70% using sterile dH2O |
Dulbecco’s PBS (D-PBS) | Invitrogen | 14040 | |
Non-fluorescent buffer | 118 mM NaCl; 4.75 mM KCl; 2.53 mM CaCl2×H2O; 2.44 mM MgSO4; 1.19 mM KH2PO4; 25 mM NaHCO3 in sterile water; sterile-filter | ||
Sodium fluorescein | Sigma | 6377 | 1 mg/ml in sterile non-fluorescent buffer; sterile-filter, protect from light; store at 4 °C up to 6 months |
EQUIPMENT | |||
Voltohmmeter | World Precision Instruments | ||
STX2 electrode | World Precision Instruments | ||
ELISA plate reader | Capable of measuring A486 or A490 |