Quantifying Proliferation of Cultured Human and Rabbit Airway Smooth Muscle Cells in Response to Serum and Platelet-derived Growth Factor Stuart J. Hirst, Peter J. Barnes, and Charles H. C. Twort Respiratory Research Laboratories, Smooth Muscle Research Group, Division of Medicine UMDS, St. Thomas' Hospital, and Department of Thoracic Medicine, National Heart and Lung Institute, London, United Kingdom

Development of suitable methods for the quantification of the proliferative response of airway smooth muscle (ASM) cells in culture will assist the investigation of the cellular mechanisms underlying the hyperplasia and hypertrophy of ASM as seen in asthmatic airways. In this study, two rapid and simple colorimetric assays have been modified to enable the growth of human bronchial and rabbit tracheal smooth muscle in culture to be assessed. One method depends upon the reduction by living cells of the tetrazolium salt MTT to form a blue formazan product, whereas the other relies on rapid binding of the dye Coomassie brilliant blue to protein at acidic pH. Experiments demonstrated the validity of both assays in quantifying the proliferative response of cultured human and rabbit ASM cells. The increase in optical density observed for each assay correlated directly, throughout the duration of culture, with the increase in cell number determined by hemocytometry in human and rabbit ASM cells proliferating in response to fetal calf serum (1.25 to 10 %). This relationship held also for rabbit tracheal ASM cells proliferating in response to the heterodimer of platelet-derived growth factor (1 to 50 ng/ml). Application of these methods to adherent proliferating cultures of human and rabbit ASM cells demonstrated their adaptability to the generation of growth curves in response to serum and to a defined growth factor. These methods allow both total cellular protein and proliferation to be estimated in human and rabbit ASM cells in culture, using assays that are rapid, reproducible, inexpensive, and easy to perform while negating the use of radioisotopes. It is intended that these additional methods should be useful in delineating some of the mechanisms that might contribute to the proliferative response of these cells - particularly since there has been a resurgence in interest in culturing smooth muscle cells derived from the airways.

One of the most striking and characteristic pathologic features of chronic severe asthma is an increase in the smooth muscle mass of the airways (1-4). Both hyperplasia (increase in cell number) and hypertrophy (increase in cell size) may contribute to this increase. The maintenance of airway smooth muscle (ASM) cells in culture, which retain their physiologic responsiveness to agonists implicated in inflammatory airway disease, has only recently been described (5-7), but may provide a useful model of ASM cells in vivo. The development of methods that allow accurate and repro-

(Received for publication June 15, 1992) Address correspondence to: Stuart 1. Hirst, Ph.D., Respiratory Research Laboratories, Smooth Muscle Research Group, Division of Medicine UMDS, S1. Thomas' Hospital, Lambeth Palace Rd., London SEI 7EH, United Kingdom. Abbreviations: airway smooth muscle, ASM; Dulbecco's modified Eagle's medium, DMEM; fetal calf serum, FCS; 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide, MTT; Dulbecco's Ca2+/Mg2+-free phosphate-buffered saline, PBS; platelet-derived growth factor, PDGF; human recombinant platelet-derived growth factor heterodimer, PDGF-AB; sodium dodecyl sulfate, SDS. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp. 574-581, 1992

ducible quantification of the proliferative response of ASM cells to mitogenic stimuli could greatly enhance our understanding of the cellular and molecular basis for the regulation of growth in these cells. Here we describe two rapid and easily performed colorimetric assays for the quantification of ASM growth: cleavage of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the mitochondrial enzyme succinyl dehydrogenase (8) and determination of total cellular protein using the Coomassie brilliant blue (G-250) technique (9). The cleavage of MTT into a blue-colored formazan product by the mitochondrial enzyme succinyl dehydrogenase (10), occurring only in living cells, has long been used to assess cell survival and has more recently been applied to assaying proliferation (8). Augmentation of protein synthesis and expression (i.e., hypertrophy) is estimated using the capacity of the dye Coomassie brilliant blue (G-250) to give a color reaction when bound to protein at acidic pH (11) without the need {or prior lysis of cells. We have applied these methods to human and rabbit bronchial smooth muscle cells in culture to assess their validity

Hirst, Barnes, and Twort: Quantifying Airway Smooth Muscle Proliferation

in quantifying growth in response to both serum and to a defined smooth muscle mitogen - platelet-derived growth factor (PDGF). The potential mitogenic effect of PDGF on ASM cells was investigated as it exists in large amounts in inflammatory cells recruited into the asthmatic airway, such as the alveolar macrophage and platelet (see reference 12). These methods have not previously been applied to human or any other species of smooth muscle in culture, including airway smooth muscle, nor have the mitogenic properties of PDGF been demonstrated in these cells. A preliminary account of this study has been communicated to the American Thoracic Society (13).

Materials and Methods Cell Culture Rabbit tracheal smooth muscle cells were obtained according to the methods described by Chopra and colleagues (14), while human bronchial smooth muscle cells were obtained according to those described in Twort and van Breemen (6, 15). Briefly, adult male New Zealand white rabbits (2 to 3 kg) were administered a lethal intravenous dose (50 to 60 mg/kg) of pentobarbitone. The trachea was removed immediately and under aseptic conditions cleaned of adhering connecting tissue. The epithelium was removed by firm scraping across the luminal surface with a rounded scalpel blade. The trachealis muscle was then cut away from the cartilage prior to enzymatic dispersion. Similarly, human bronchial smooth muscle was obtained during lung resections from patients (50 to 65 yr of age) of either sex undergoing surgery for carcinoma of the bronchus. After removal of the epithelium, portions of smooth muscle not invaded by the carcinoma were dissected free of adherent connective and parenchymal tissue. Both human and rabbit smooth muscle strips were subjected to the same enzymatic dispersion protocol, described in detail elsewhere (14, 15), prior to seeding on to 35-mm dishes (Falcon Labware; Becton-Dickinson and Co., Oxford, UK) at a density of 2 to 5 X l()5 viable cells/ml in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (DMEM-lO% FCS) supplemented with sodium pyruvate (1 mM), L-glutamine (2 mM), non-essential amino acids, gentamicin (50 ttg/ml), and amphotericin B (1.5 ttg/ ml). Cell viability assessed by trypan blue exclusion was > 95 %. Cells were cultured in a humidified atmosphere containing 5 % CO2 in air. Fresh culture medium was replaced in both cell types every 72 h. After several days in culture (14 to 21 days for human and 6 to 10 days for rabbit), the cells grew to confluence. Cells were subcultured by removal from the plastic base of each dish with a plastic scraper, followed by mechanical dispersion by repeated pipetting in DMEM-lO% FCS for 1 to 2 min prior to transferring to a 25-cm2 flask (Falcon). At confluence, the cells were then subcultured as above into a larger flask (75-cm 2 growth area). Confluence in these flasks was usually achieved in 10 to 12 days for human cells and 5 to 7 days for rabbit cells, after which further subculturing allowed half of all the cells to be maintained for experimental use and the other half for further subculturing in

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75-cm2 flasks to continue each cell line. Using this approach, cells could be maintained in culture over many passages (usually 10 to 12). Preliminary experiments reported in Chopra and colleagues (14) and Twort and van Breeman (15) have shown only minimal loss of surface Ca2 + mobilizing agonist receptors over these passages. Using indirect immunofluorescence techniques, each cell type stained for both smooth muscle actin and myosin. When examined under the light (6) and electron (16) microscope, both cell types displayed all the characteristics of smooth muscle cells in culture (17). Confluent cells (passages 5 through 11) were harvested for experimental use by scraping the base of several 75-cm2 flasks containing 6 ml Dulbecco's Ca2 + /Mg2+-free phosphate-buffered saline (PBS). Cells were washed twice by centrifugation (200 X g for 5 min) in PBS, and the viable yield was determined by hemocytometry (trypan exclusion > 97 %) prior to resuspension at an appropriate density in either PBS or DMEM. Determination of the Linearity Range for Each Assay MIT assay. Harvested washed cells were resuspended in DMEM-lO% FCS and aliquoted (500 ttl) into 24-well cluster plates (Falcon Labware) prior to serial dilution (1:2) in duplicates. To each well, 100 ttl of an appropriate MTT concentration (dissolved in PBS and filtered through a 0.2 ttm filter before use to remove any blue formazan product) was added immediately after diluting the cells, which were then incubated for 3.5 h at 37°C. The resulting blue formazan product (seen as blue crystals within the cell cytosol under the light microscope) was solubilized overnight (16 h) at 37°C by the addition of 500 ttl of 10% sodium dodecyl sulfate (SDS) in 0.01 M HCl to each well. A sample (150 ttl) from each duplicate well was transferred to a 96-well microplate (Falcon Labware), and the optical density determined by automated spectrophotometry (Dynatech Multiscan MR 700; Billingshurst, UK) against a reagent blank (no cells) at a test wavelength of 570 nm and a reference of 630 nm. Coomassie blue protein assay. Harvested and washed cells were resuspended in PBS, aliquoted, and then diluted in duplicate across 24-wellcluster plates according to the design used for the MTT assay (see above). The plates were immediately placed in an oven at 65°C for approximately 3 h; this effectivelydried the samples to leave a protein film. The Coomassie blue reagent (200 ttl; BioRad, Hemel Hempstead, UK) was added to each well for 5 min, prior to addition of 800 ttl distilled water. The color reaction was allowed to develop for at least 5 min before determining the optical density against a reagent blank (test, 630 nm; reference, 490 nm). By reference to a standard curve, generated with bovine serum albumin, the total amount of protein in each well could be determined. Determining the Optimum Incubation Time of MTT with the ASM Cells Experiments to determine the kinetics of MTT cleavagewere carried out by incubating adherent human and rabbit cells with MTT (0.8mg/ml) over an 8-h period. This was repeated over several days of culture until confluence was reached.

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Quantifying the Proliferative Response Harvested and washed cells were resuspended in DMEM and seeded at a density of 2 x 104 cells/well (l x 104 cells/ern') in 250-1L1 aliquots into 24-well cluster plates containing 250 ILl DMEM at varying FCS concentrations. On each day of culture, proliferating cells from three plates were removed from the incubator-one plate for each assay and the third for direct comparison of cell numbers by hemocytometry. MIT assay. The growth medium was removed by gentle aspiration, exposing the cell monolayers which were washed twice with 500 ILl DMEM-lO% FCS. The medium was then replaced with DMEM-lO% FCS (500 ILl) to which 100 ILl MTT (5 mg/rnl in PBS; final concentration, 0.8 mg/rnl) was added to each well, and the cells were incubated at 37°C for an appropriate period prior to overnight solubilization with SDS and the change in optical density was determined. Protein assay. A similar washing procedure to the MTT assay was adopted using PBS instead of DMEM-lO% FCS. Preliminary experiments (not shown) indicated that washing the cell monolayers twice with PBS was sufficient to remove any soluble protein present in the media. The plates were then air-dried and the protein content of each well was determined as previously described. Hemocytometry. In the third plate, the medium was removed and the cell monolayers were resuspended in 250 ILl PBS containing 0.02% (wt/vol) trypsin and EDTA at room temperature for 2 min before counting in a Neubauer hemocytometer. At least 120 cells from each well were counted in order to minimize counting error. Materials All tissue culture reagents (DMEM, PBS, FCS, sodium pyruvate, r.-glutamine, non-essential amino acids, amphotericin B, and gentamicin) were obtained from Gibco BRL (Paisley, UK). Human recombinant platelet-derived growth factor heterodimer (PDGF-AB) was also purchased from Gibco BRL. MTT, SDS, bovine serum albumin (fraction 5, essentially fatty acid-free), elastase (type 1), collagenase (type 1), trypsin (type III), insulin, ascorbic acid, transferrin, and trypan blue were all purchased from Sigma

(Poole, UK). Coomassie blue (G-250) protein reagent concentrate (supplied in 55 % phosphoric acid and 15% methanol) was purchased from BioRad (Hemel Hempstead, UK). EDTA (disodium salt) was obtained from BDH (Poole, UK). Statistical Analysis All values for cell number determined by hemocytometer counts and optical density changes were calculated as mean ± SEM. In some instances, the SEM was very small and fell within the symbols shown in the figures. Correlations between optical density and adherent cell number were assessed using the correlation coefficient (r). These values were tested for statistical significance using Geigy's Scientific Tables (28). A P value < 0.05 was considered significant.

Results Relationship between Absorbance and Cell Number in Suspension In both species of ASM cell studied, the absorbance determined in the two assays was directly proportional to the number of cells in suspension (Figure 1). In these experiments, the standard deviations were mostly less than 5 % of the values shown and in many cases fell within the symbols for each line. Varying concentrations of MTT (0.4, 0.8, and 1.6 mg/rnl), incubated with cells for 3.5 h, showed an increasing sensitivity of the assay up to 0.8 mg/rnl MTT (results not shown). Taking into account the cost of the reagent and the lack of any further increase in sensitivity at 1.6mg/rnl, a concentration of 0.8 mg/rnl MTT was chosen for all subsequent experiments. Increasing the concentration of SDS for solubilization from 10% to 15% (wt/vol) did not enhance the sensitivity of the assay (results not shown). Optimal Incubation Time for MTT in Adherent Cultures The kinetics of MTT (0.8 mg/rnl) cleavage (i.e., formazan production) was studied over an 8-h period on each day of culture until confluence was reached in DMEM-lO% FCS (Figure 2). On each day, the cell number in each well was monitored by hemocytometry. There was a rapid production, which could be measured as early as 15 min after incu-

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Discussion Recently, there has been renewed interest into the processes that lead to thickening of the airway wall of asthmatic patients (7, 18, 19) and its possible relationship to airway hyperreactivity in vivo (4, 20, 21). The thickening occurs primarily by hyperplastic and hypertrophic changes of bronchial smooth muscle (2, 4). Colorimetric assays for cell growth and survival in vitro, which can be carried out in convenient cluster plates in combination with repeating pipettes and an automated scanning spectrophotometer, offer significant advantages in speed,

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992

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simplicity, and cost over more conventional assay systems employing the uptake of radiolabeled compounds (22, 23). Here we have chosen two such rapid and simple techniques, the MTT and Coomassie blue protein assays and tested their validity in quantifying proliferation of cultured ASM cells. The main alternative method for assaying the growth rate of cells in culture relies. upon uptake of the radiolabel ['Hlrhymidine. To date, this has been the method used in most of the reports investigating the proliferative response of cultured vascular (e.g., reference 24) and airway smooth muscle cells (e.g., references 18 and 19). This method has a number of disadvantages both from a practical (expense, safety, and capital expenditure on equipment) and conceptual perspective. These problems are discussed in more depth by Oliver and associates (23). Briefly, [3H]thymidine uptake is dependent not only on the proportion of cells entering S-phase, but also on the total number of cells present. Secondly, it is inherent in the interpretation of [3H]thymi-

dine uptake values that the radioligand has been incorporated into newly synthesized DNA and not merely taken up by the cell or indeed degraded by the action of secreted enzymes (25). In other words, uptake of pH]thymidine may occur in the absence of actual cell division. Not surprisingly, therefore, other more reliable quantitative methods that are less laborious and time-consuming have been sought. It was not, however, the intention of this study to compare the MTT and Coomassie blue assays with uptake of ['Hlthymidine, but with a more definitive marker of hyperplasia, actual cell counts. MTT Assay The cleavage of MTT to a blue formazan derivative by living cells is clearly an effective principle on which to base a growth (and survival) assay (8, 26). Methodologic problems encountered during the initial development of this assay included difficulty in solubilizing the formazan product with-

Hirst, Barnes, and Twort: Quantifying Airway Smooth Muscle Proliferation

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Quantifying proliferation of cultured human and rabbit airway smooth muscle cells in response to serum and platelet-derived growth factor.

Development of suitable methods for the quantification of the proliferative response of airway smooth muscle (ASM) cells in culture will assist the in...
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