Journal of Immunological Methods, 132 (1990) 145-146
145
Elsevier JIM 05676 Letter to the editors
A modified method for isolating viable alveolar type II cells from human lung tissue Frans J. Van Overveld, Wilfried A. D e Backer and Paul A. Vermeire Department of Pulmonary Medicine, Unioersity of Antwerp (UIA), B-2610.4ntwerpen- Wilrijk, Belgium
(Received 5 April 1990,accepted21 May 1990) Dear Editors, Both type I and type II alveolar cells are known targets for many toxic agents (Freeman et al., 1974; Crapo et al., 1980). In adult respiratory distress syndrome (ARDS) diffuse lung injury affects both type I and type II alveolar cells (Hasleton, 1983). An improvement in our knowledge of the pathogenesis of ARDS may thus depend on a better understanding of the acute toxicology of these cells. After lung injury, type II cells also retain the important ability tO replace type I cells in the repair of the epithelial surface of the alveolus (Adamson and Bowden, 1974). Although these type II pneumocytes only comprise 4% of the alveolar epithelial area, they represent 60% of alveolar epithelial cells by number (Crapo et al., 1983). Several methods have been employed to isolate and purify type II cells. Most of these methods deal with type II cells isolated from animal lung tissue. This prompted us to initiate in vitro studies of purified preparations of human type II cells. Normal human lung tissue was obtained during resections of bronchial tumours. Due to leaks on the cut surface cannulation and infusion of the airways with enzyme solutions could not be performed. The tissue was minced into 1-2 mm pieces. The fragments were washed extensively to remove blood cells as much as possible. Subsequently the
Correspondence to: F.J. Van Overveld, Department of Pulmonary Medicine, University of Antwerp (UIA), Universiteitsplein 1, B-2610Antwerpen-Wilrijk,Belgium.
lung fragments were digested during four consecutive exposures of 30 min at 37°C to 0.1% collagenase and 0.1% elastase. The enzymes were dissolved in Tyrodes' buffer supplemented with 1.2 m m ol / l MgC12, 1% gelatin and 0.006% deoxyribonuclease type I (TGMD). Dispersed cells were collected, filtered through sterile gauze, centrifuged and resuspended in TGMD. Erythrocytes were removed by an isotonic shock procedure (Roos and Loos, 1970). In our method removal of alveolar macrophages by differential adherence preceded gradient centrifugation in order to prevent obstruction of the gradient by macrophages. The cell suspension at 1.5-2 x 106 cells/ml was cultured for 90 min in 25 cm2 plastic flasks (7 ml/flask) during which time most of the alveolar macrophages adhered to the plastic. The nonadherent cells were removed and resuspended in 1-2 ml T G MD (max. 35 × 10 6 cells/ml). Percoll (Pharmacia, Uppsala, Sweden) densities of 1.040 and 1.090 g/m1 were prepared by diluting a stock solution of 1.100 g / m l with 10 × concentrated phosphate-buffered saline (PBS). The osmolality of the Percoll densities was 290 mosmol/kg. The less dense suspension was layered above the denser one. The cell suspension was then layered onto the gradient and tubes were centrifuged at 400 × g for 25 min. After centrifugation the cells that accumulated at the interface of both Percoll densities were removed, washed, resuspended in culture medium, and incubated in collagen-coated plates for 22 h at 37°C in a 5% CO2-95% air incubator. During this time type II cells adhered to the surface and contaminating cells were decanted. Cytocentrifuge smears of all cells fractions were prepared. Differential cell counts were performed
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V.(BiomedicalDivision)
146 by May-Grtinwald Giemsa staining. After fixation with glutaraldehyde, type II alveolar cells were identified by staining with tannic acid and a polychrome stain that showed both the granules and the cell outlines ( M a s o n et al., 1985). Cell viability was monitored b y 0.02% trypan blue dye exclusion. The viability of the nucleated lung cells exceeded 92% at each isolation step. Enzymatic digestion of h u m a n lung tissue yielded 13.4 + 1.9 × 106 (mean + SEM, n = 12) dispersed c e l l s / g wet lung tissue. This was considerably m o r e than previously reported yields of 1 - 4 x 106 c e l l s / g of tissue (Robinson et al., 1984; Edelson et al., 1989). The cell suspension obtained contained 32.0 + 7.9% type II cells. Using radioligand binding studies it was possible to show that the isolated cells did not express high-affinity fl-adrenergic receptor binding sites (Van Overveld, 1988). After incubation of the isolated cells in supplemented R P M I m e d i u m at 3 7 ° C a small n u m b e r of high affinity binding sites could be observed. This suggests that the total receptor population of the dispersed cells were not irreversibly impaired. After differential adherence of m a c r o p h a g e s type II cell purity was increased to 59.8 _+ 9.3% ( P = 0.05). The recovery of type II cells was then 88.0 +_ 10.4%. A further increase of their purity to 77.0 _+ 9.0% ( P = 0.05) was achieved with centrifugation across a discontinuous Percoll gradient. Percoll separation also effectively removed cellular debris (above the first Percoll suspension) and remaining red blood cells ( b o t t o m of the tube). After gradient centrifugation the recovery of type II cells was 81.7 _+ 6.3% and the total recovery was 55.2 _+ 2.0%. To date the total separation time required for isolation has been approximately 5.5 h (including adherence for 90 min). I n c u b a t i o n of the isolated cells for 22 h at 37 ° C thus resulted in type II cells which exceeded 90% purity with a seeding efficiency of 67.9 +_ 10.1%.
In conclusion, we report here a modified m e t h o d for isolating large n u m b e r s of viable alveolar type II cells f r o m h u m a n lung tissue. This system provides a useful model system for future studies of h u m a n alveolar cells and their role in lung injury.
References Adamson, I.Y.R. and Bowden, D.H. (1974) The type II cell as a progenitor of alveolar regeneration: A cytodynamic study in mice after exposure to oxygen. Lab. Invest. 30, 35. Crapo, J.D., Barry, B.E., Foscue, H.A. and Shelburne, J. (1980) Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen. Am. Rev. Respir. Dis. 122, 123. Crapo. J.D., Barry, B.E., Gehr, P., Bachofen, M. and Weibel, E.R. (1982) Cell number and cell characteristics of the normal human lung. Am. Rev. Respir. Dis. 125, 740. Edelson, J.D., Shannon, J.M. and Mason, R.J. (1989) Effects of two extracellular matrices on morphological and biochemical properties of human type II cells in vitro. Am. Rev. Respir. Dis. 140, 1398. Freeman, G., Juhas, L.T., Furiosi, N.J., Mussenden, R., Stephens, R.J. and Evan, M.J. (1974) Pathology of pulmonary disease from exposure to interdependent ambient gases (nitrogen dioxide and ozone). Arch. Environ. Health 29, 203. Hasleton, P.S. (1983) Adult respiratory distress syndrome-A review. Histopathology 7, 307. Mason, R.J., Walker, S.R., Shields, B.A., Henson, J.E. and Williams, M.C. (1985) Identification of rat alveolar type II epithelial cells with a tannic acid and polychrome stain. Am. Rev. Respir. Dis. 131, 786. Robinson, P.C., Voelker, D.R. and Mason, R.J. (1984) Isolation and culture of human alveolar type II epithelial cells. Am. Rev. Respir. Dis. 130, 1156. Roos, D. and Loos, J.A. (1970) Changes in the carbohydrate metabolism of mitogenically stimulated human peripheral lymphocytes. I. Stimulation by phytohaemagglutinin. Biochim. Biophys. Acta 222, 565. Van Overveld, F.J. (1988) Some aspects of mast cell subtypes from human lung tissue. Thesis, University of Utrecht, Utrecht, The Netherlands, p. 39.
145
Elsevier JIM 05676 Letter to the editors
A modified method for isolating viable alveolar type II cells from human lung tissue Frans J. Van Overveld, Wilfried A. D e Backer and Paul A. Vermeire Department of Pulmonary Medicine, Unioersity of Antwerp (UIA), B-2610.4ntwerpen- Wilrijk, Belgium
(Received 5 April 1990,accepted21 May 1990) Dear Editors, Both type I and type II alveolar cells are known targets for many toxic agents (Freeman et al., 1974; Crapo et al., 1980). In adult respiratory distress syndrome (ARDS) diffuse lung injury affects both type I and type II alveolar cells (Hasleton, 1983). An improvement in our knowledge of the pathogenesis of ARDS may thus depend on a better understanding of the acute toxicology of these cells. After lung injury, type II cells also retain the important ability tO replace type I cells in the repair of the epithelial surface of the alveolus (Adamson and Bowden, 1974). Although these type II pneumocytes only comprise 4% of the alveolar epithelial area, they represent 60% of alveolar epithelial cells by number (Crapo et al., 1983). Several methods have been employed to isolate and purify type II cells. Most of these methods deal with type II cells isolated from animal lung tissue. This prompted us to initiate in vitro studies of purified preparations of human type II cells. Normal human lung tissue was obtained during resections of bronchial tumours. Due to leaks on the cut surface cannulation and infusion of the airways with enzyme solutions could not be performed. The tissue was minced into 1-2 mm pieces. The fragments were washed extensively to remove blood cells as much as possible. Subsequently the
Correspondence to: F.J. Van Overveld, Department of Pulmonary Medicine, University of Antwerp (UIA), Universiteitsplein 1, B-2610Antwerpen-Wilrijk,Belgium.
lung fragments were digested during four consecutive exposures of 30 min at 37°C to 0.1% collagenase and 0.1% elastase. The enzymes were dissolved in Tyrodes' buffer supplemented with 1.2 m m ol / l MgC12, 1% gelatin and 0.006% deoxyribonuclease type I (TGMD). Dispersed cells were collected, filtered through sterile gauze, centrifuged and resuspended in TGMD. Erythrocytes were removed by an isotonic shock procedure (Roos and Loos, 1970). In our method removal of alveolar macrophages by differential adherence preceded gradient centrifugation in order to prevent obstruction of the gradient by macrophages. The cell suspension at 1.5-2 x 106 cells/ml was cultured for 90 min in 25 cm2 plastic flasks (7 ml/flask) during which time most of the alveolar macrophages adhered to the plastic. The nonadherent cells were removed and resuspended in 1-2 ml T G MD (max. 35 × 10 6 cells/ml). Percoll (Pharmacia, Uppsala, Sweden) densities of 1.040 and 1.090 g/m1 were prepared by diluting a stock solution of 1.100 g / m l with 10 × concentrated phosphate-buffered saline (PBS). The osmolality of the Percoll densities was 290 mosmol/kg. The less dense suspension was layered above the denser one. The cell suspension was then layered onto the gradient and tubes were centrifuged at 400 × g for 25 min. After centrifugation the cells that accumulated at the interface of both Percoll densities were removed, washed, resuspended in culture medium, and incubated in collagen-coated plates for 22 h at 37°C in a 5% CO2-95% air incubator. During this time type II cells adhered to the surface and contaminating cells were decanted. Cytocentrifuge smears of all cells fractions were prepared. Differential cell counts were performed
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V.(BiomedicalDivision)
146 by May-Grtinwald Giemsa staining. After fixation with glutaraldehyde, type II alveolar cells were identified by staining with tannic acid and a polychrome stain that showed both the granules and the cell outlines ( M a s o n et al., 1985). Cell viability was monitored b y 0.02% trypan blue dye exclusion. The viability of the nucleated lung cells exceeded 92% at each isolation step. Enzymatic digestion of h u m a n lung tissue yielded 13.4 + 1.9 × 106 (mean + SEM, n = 12) dispersed c e l l s / g wet lung tissue. This was considerably m o r e than previously reported yields of 1 - 4 x 106 c e l l s / g of tissue (Robinson et al., 1984; Edelson et al., 1989). The cell suspension obtained contained 32.0 + 7.9% type II cells. Using radioligand binding studies it was possible to show that the isolated cells did not express high-affinity fl-adrenergic receptor binding sites (Van Overveld, 1988). After incubation of the isolated cells in supplemented R P M I m e d i u m at 3 7 ° C a small n u m b e r of high affinity binding sites could be observed. This suggests that the total receptor population of the dispersed cells were not irreversibly impaired. After differential adherence of m a c r o p h a g e s type II cell purity was increased to 59.8 _+ 9.3% ( P = 0.05). The recovery of type II cells was then 88.0 +_ 10.4%. A further increase of their purity to 77.0 _+ 9.0% ( P = 0.05) was achieved with centrifugation across a discontinuous Percoll gradient. Percoll separation also effectively removed cellular debris (above the first Percoll suspension) and remaining red blood cells ( b o t t o m of the tube). After gradient centrifugation the recovery of type II cells was 81.7 _+ 6.3% and the total recovery was 55.2 _+ 2.0%. To date the total separation time required for isolation has been approximately 5.5 h (including adherence for 90 min). I n c u b a t i o n of the isolated cells for 22 h at 37 ° C thus resulted in type II cells which exceeded 90% purity with a seeding efficiency of 67.9 +_ 10.1%.
In conclusion, we report here a modified m e t h o d for isolating large n u m b e r s of viable alveolar type II cells f r o m h u m a n lung tissue. This system provides a useful model system for future studies of h u m a n alveolar cells and their role in lung injury.
References Adamson, I.Y.R. and Bowden, D.H. (1974) The type II cell as a progenitor of alveolar regeneration: A cytodynamic study in mice after exposure to oxygen. Lab. Invest. 30, 35. Crapo, J.D., Barry, B.E., Foscue, H.A. and Shelburne, J. (1980) Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen. Am. Rev. Respir. Dis. 122, 123. Crapo. J.D., Barry, B.E., Gehr, P., Bachofen, M. and Weibel, E.R. (1982) Cell number and cell characteristics of the normal human lung. Am. Rev. Respir. Dis. 125, 740. Edelson, J.D., Shannon, J.M. and Mason, R.J. (1989) Effects of two extracellular matrices on morphological and biochemical properties of human type II cells in vitro. Am. Rev. Respir. Dis. 140, 1398. Freeman, G., Juhas, L.T., Furiosi, N.J., Mussenden, R., Stephens, R.J. and Evan, M.J. (1974) Pathology of pulmonary disease from exposure to interdependent ambient gases (nitrogen dioxide and ozone). Arch. Environ. Health 29, 203. Hasleton, P.S. (1983) Adult respiratory distress syndrome-A review. Histopathology 7, 307. Mason, R.J., Walker, S.R., Shields, B.A., Henson, J.E. and Williams, M.C. (1985) Identification of rat alveolar type II epithelial cells with a tannic acid and polychrome stain. Am. Rev. Respir. Dis. 131, 786. Robinson, P.C., Voelker, D.R. and Mason, R.J. (1984) Isolation and culture of human alveolar type II epithelial cells. Am. Rev. Respir. Dis. 130, 1156. Roos, D. and Loos, J.A. (1970) Changes in the carbohydrate metabolism of mitogenically stimulated human peripheral lymphocytes. I. Stimulation by phytohaemagglutinin. Biochim. Biophys. Acta 222, 565. Van Overveld, F.J. (1988) Some aspects of mast cell subtypes from human lung tissue. Thesis, University of Utrecht, Utrecht, The Netherlands, p. 39.
