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Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The purpose of this study was to identify culture conditions for maintenance of isolated mouse type II cells with intact surfactant protein (SP) and phospholipid production. Type II cells were isolated from 6-wk-old mice and cultured on Matrigel matrix-rat tail collagen (70:30 vol/vol) in bronchial epithelial cell growth medium minus hydrocortisone plus 5% charcoal-stripped FBS and 10 ng/ml keratinocyte growth factor. Under these conditions, type II cells actively produced surfactant phospholipids and proteins for at least 7 days. Synthesis and secretion of surfactant phospholipids and SP-A, -B, -C, and -D declined on day 1 of culture but recovered by day 3, reaching levels comparable to or exceeding freshly isolated cells by day 5. Abundant lamellar bodies were readily apparent in cells examined on days 5 and 7, and a surfactant pellet was recovered by centrifugation of media harvested on each day of culture. Secretion of SP-B, SP-C, and phosphatidylcholine was stimulated by phorbol 12-myristate 13-acetate and was inhibited by compound 48/80. When tested with a bubble surfactometer, surfactant secreted by type II cells on day 5 of culture lowered surface tension to 5.2 ± 2.3 mN/m. This is the first description of the synthesis and secretion of a functional surfactant complex by mouse type II cells after 7 days in primary culture.
surfactant; secretion; lung
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ISOLATED ALVEOLAR type II cells in primary culture have provided insight into the function of this important cell type in the lung. However, the rapid loss of the type II cell phenotype has limited the usefulness of this system. Manipulation of culture substratum and media has identified conditions that support the synthesis of surfactant proteins (SPs) and phospholipids in primary cultures of rat type II cells. Key substratum components include elements of extracellular matrix, such as the basement membrane extracted from Engelbreth-Holm-Swarm (EHS) tumor (commercially available as Matrigel), which contains laminin, type IV collagen, and heparin sulfate proteoglycan (24, 26). Interaction of type II cells with extracellular matrix is thought to promote a native cuboidal cell shape, which is important for type II cell function in vitro (25, 27). Keratinocyte growth factor (KGF; fibroblast growth factor-7) has been identified as a critical component of the culture medium, which likely reflects the importance of epithelial-mesenchymal interactions in vivo (28, 36). In addition to effects of media and substratum, the culture of type II cells at an air-liquid interface (by limiting the amount of apical medium and rocking the culture dish) has also been shown to enhance maintenance of the rat type II cell phenotype in vitro (7, 35).
Although considerable advances have been made in optimizing culture conditions for rat type II cells, comparable progress for mouse type II cell culture is lacking. The development of such a culture system is important, since it would allow the study of type II cells from a large number of transgenic mouse lines in which the SP-C promoter has been used to target transgene expression to the distal epithelium. An important step toward achieving this goal was the development of a reliable method for isolation of mouse type II cells in high yield and purity (4). These cells have been maintained in mixed culture for 7-14 days, but surfactant phospholipid and protein synthesis and secretion were not examined (32). In the present study, we describe culture conditions that preserve key aspects of the type II cell phenotype for up to 7 days in culture, including the synthesis and secretion of a functional surfactant complex.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials. Dispase was purchased from Fisher (Cincinnati, OH). CD45 and CD32 were purchased from BD PharMingen (San Diego, CA). Matrigel was obtained from BD Biosciences (Franklin Lakes, NJ). Human KGF was purchased from Peprotech (Rocky Hill, NJ). Bronchial epithelial cell basal medium (BEBM) and bronchial epithelial cell growth medium (BEGM) were obtained from Clonetics (Walkersville, MD). BEGM is BEBM that also contains bovine pituitary extract, triiodothyronine, retinoic acid, insulin, hydrocortisone, transferrin, epidermal growth factor, epinephrine, gentamicin, and amphotericin. FCS was purchased from HyClone (Logan, UT). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Rat tail collagen was prepared as previously described (19).
Isolation and culture of murine alveolar type II cells. Cells were prepared from 6-wk-old female C57B/6 mice by a modification of the method of Corti et al. (4). Mice were anesthetized with 0.2 ml Nembutal by intraperitoneal injection. The abdominal cavity was opened, and mice were exsanguinated by severing the inferior vena cava and the left renal artery. The trachea was isolated and cannulated with a 20-gauge luer stub adapter. The diaphragm was cut, and the chest plate and thymus were removed. With the use of a 21-gauge needle fitted on a 10-ml syringe, lungs were perfused with 10-20 ml 0.9% saline via the pulmonary artery. Dispase (3 ml) was rapidly instilled through the cannula in the trachea followed by 0.5 ml agarose (45°C). Lungs were immediately covered with ice for 2 min to gel the agarose. After this incubation, lungs were removed from the animals and incubated in 1 ml dispase for 45 min (25°C). Lungs were subsequently transferred to a 60-mm culture dish containing 7 ml of HEPES-buffered DMEM and 100 U/ml DNAse I, and lung tissue was gently teased from the bronchi. The cell suspension was filtered through progressively smaller cell strainers (100 and 40 µm) and nylon gauze (20 µm). Cells were collected by centrifugation at 130 g for 8 min (4°C) and placed on prewashed 100-mm tissue culture plates that had been coated for 24-48 h at 4°C with 42 µg CD45 and 16 µg CD 32 in PBS. After incubation for 1-2 h at 37°C, type II cells were gently panned from the plate and collected by centrifugation. Type II cells were resuspended in culture media and cultured under conditions detailed in RESULTS. The media were changed after the 1st day of culture and every 2 days thereafter.
For experiments requiring cell harvest, matrixes were solubilized by incubating cultures with dispase containing 1 mg/ml collagenase at 37°C for 60 min.SP synthesis and secretion. Type II cells were labeled with [35S]cysteine/methionine in MEM (cysteine/methionine deficient) containing 2% dialyzed FBS for 4 h and immunoprecipitated exactly as previously described (16). Total labeled protein was determined by trichloroacetic acid precipitation, and equal counts per minute of protein were precleared with normal rabbit serum. Lysates were sequentially immunoprecipitated by adding 30 µl protein G-Sepharose (Zymed, San Francisco, CA) and 5 µl of anti-rat SP-A antibody (8), pro-SP-C antibody (31), mature SP-B antibody (12, 34), or SP-D antibody (37). SDS-PAGE and autoradiography were performed as previously described (16). For Western blotting, gels were electrophoretically transferred to nitrocellulose and probed with the same antibodies used for immunoprecipitation or with antibody directed against recombinant, mature SP-C (21).
For secretion experiments, cells were used after 7 days of culture. The cells were washed three times with BEGM to remove extracellular surfactant, and secretagogues or inhibitors were added at time 0. Media were removed after 3 h, and cells were rinsed with 0.5 ml of fresh media. The media samples were combined, and cells were removed by centrifugation (130 g for 8 min). Surfactant pellets were then isolated by centrifugation of media (14,000 g for 30 min) and examined by Western blotting as noted above. Protein secretion was quantitated by scanning densitometry and expressed relative to control values as 100%.Surfactant phospholipid synthesis and secretion. For analysis of phospholipid synthesis, cells were incubated for 24 or 48 h with 1 µCi [14C]acetate/ml. After the cells were washed to remove free radiolabel, lipids were extracted with methanol, lipid, and aqueous phases generated with chloroform and 0.2 M KCl, and the lipid phase evaporated to dryness. After resuspension in chloroform, samples were spotted on preactivated silica gel plates for phospholipid determinations. Plates were run in the first dimension with chloroform-methanol-glacial acetic acid-water (195:75:24:12 vol/vol/vol/vol) and in the second dimension with tetrahydrofuran-methylal-methanol-2 M ammonium hydroxide (166:114: 31:17 vol/vol/vol/vol; see Ref. 2). Plates were dried, and phospholipids were visualized with iodine and compared with phospholipid standards. Phospholipids were harvested from the plates, and radioactivity was determined. Results for each phospholipid were expressed relative to total radioactivity.
[3H]phosphatidylcholine secretion was assessed as previously described for rat type II cells (20). Murine cells were labeled with 1 µCi/ml [3H]choline for 48 h before assay after 7 days in culture. Cells were washed to remove free label, and secretagogues or inhibitors were added at time 0. Media were removed after 3 h, and cells were rinsed with 0.5 ml of fresh media. The media samples were combined, and cells were removed by centrifugation (130 g for 8 min). Lipids were extracted with methanol from the cell and media samples, lipid and aqueous phases were generated with chloroform and 0.2 M KCl, and the lipid phases evaporated to dryness. Lipid disintegrations per minute (dpm) present in the media and cell samples were determined, and percent phospholipid secretion was calculated as dpm media/(dpm media + dpm cells) × 100%. Lactate dehydrogenase activity was determined in each sample as a measure of cytotoxicity. None of the agents tested had a significant effect on lactate dehydrogenase release by the cells.Quantity and surface activity of secreted surfactant. Media was collected between days 1 and 3, 3 and 5, and 5 and 7 of culture. The collected media were centrifuged at 150 g for 15 min to remove cells. The supernatant was then centrifuged at 48,400 g for 15 min. The surfactant pellet was resuspended in 0.9% saline, and the centrifugation was repeated to remove residual medium from the surfactant pellet. To assess phospholipid content, the surfactant pellet was suspended in 0.9% saline, and an aliquot was extracted with chloroform-methanol (2:1) for the phosphorus assay (9). The surface activity of secreted surfactant was measured with a captive bubble surfactometer at 37°C. The concentration of each sample was adjusted to 9 nmol phospholipid/µl and 3 µl of surfactant were applied to the air interface by microsyringe. Surface tension was measured every 10 s for 300 s to establish equilibrium surface tension before initiation of bubble pulsation. The minimum surface tension after 35% bubble volume reduction was measured at the fifth pulsation. The average bubble volume was 8.7 ± 0.5 µl.
Statistics. Differences among groups were determined by ANOVA and Newman-Keuls test using GB-Stat. All values are expressed as means ± SE, and significance was taken as P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Primary culture of murine type II epithelial cells.
Yields of type II cells from 6-wk-old C57B/6 mice varied from 4 to
6 × 106 cells/mouse. The purity of type II cell
preparations was typically >90%, as assessed by modified PAP stain
(5), electron microscopy, and immunostaining for SP-C.
Viability was >95%, as assessed by Trypan blue exclusion. Isolated
mouse type II cells were initially grown under conditions similar to
those optimized for the culture of rat type II cells (28).
Cells were seeded on Matrigel and cultured in DMEM containing 10 ng/ml
KGF. Under these conditions, SP-A, SP-B, SP-C, and SP-D were detected
by Western blot analysis of media samples or type II cell lysates after
7 days of culture, and DNA content of the cultures increased from
3.3 ± 0.1 µg/well on day 1 (n = 4)
to 9.1 ± 0.5 µg/well on day 7 (n = 4). The addition of hydrocortisone inhibited SP-C production by the
cells and significantly decreased the DNA content on day 7 (7.2 ± 0.7 µg/well, n = 4). Production of SP-A,
SP-B, SP-C, and SP-D was also maintained for 7 days when BEGM
containing KGF and without hydrocortisone (a normal component of BEGM)
was substituted for DMEM (Fig. 1). DNA content of the cultures under these conditions was 10.6 ± 1.0 µg/well on day 7 (n = 4), which was not
significantly different from the value obtained for cultures in DMEM
containing KGF. However, cells cultured for 7 days in BEGM contained
abundant lamellar bodies with typical concentric lamellae appearing
similar to freshly isolated cells and consistent with ongoing synthesis
of surfactant phospholipids and proteins (Fig.
2). Cells cultured in DMEM for 7 days
contained fewer lamellar bodies with disorganized and condensed lamellae. These cells also lost their cuboidal shape by 7 days. Therefore, BEGM was used for the remainder of the experiments. To
facilitate the formation of monolayers for secretion experiments, various concentrations of rat tail collagen were added to the Matrigel.
Consistent monolayers that maintained production of SP-C were observed
when type II cells were cultured on Matrigel-rat tail collagen (70:30
vol/vol) in BEGM (minus hydrocortisone) plus 5% charcoal-stripped FBS
and 10 ng/ml KGF (Fig. 3). Under these conditions, the cells would form monolayers suitable for secretion experiments and continued to produce SP-C (Fig.
4). Although cell aggregates comprised up
to 10% of the cultures (Fig. 3), aggregation did not preclude
secretion experiments, since the apical surface of the remainder of the
cells was exposed to the media. The following experiments were
performed with cells cultured under these conditions.
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SP synthesis.
Freshly isolated type II cells were cultured for 0, 1, 3, 5, or 7 days,
and SPs in cell lysates and media were analyzed by Western blotting.
After 1 day of culture, levels of mature SP-B [relative molecular mass
(Mr) = 16 kDa] and SP-C
(Mr = 4 kDa) peptides in cell lysates
declined relative to that in freshly isolated type II cells (Fig.
5). SP-B and SP-C production recovered by
day 3, reaching levels equal to or exceeding levels observed in freshly isolated cells by day 5 of culture. Consistent
with secretion of surfactant by type II cells, a white surfactant
pellet was easily detected after centrifugation of media isolated on alternate days of culture. Western analyses of the surfactant pellet
isolated from media detected both SP-B and SP-C mature peptides (Fig.
5). Similar results were obtained for SP-A and SP-D (Fig. 1).
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Surfactant phospholipid synthesis and lamellar body formation.
Incorporation of [14C]acetate into newly synthesized
phospholipids was used to determine surfactant phospholipid composition (Table 1). Consistent with the results of
Western analyses of SPs (Fig. 5), incorporation of labeled acetate into
phospholipid was lowest at early culture time points and increased on
days 5 and 7 of culture (Table 1). At each time
point examined, phosphatidylcholine was the most abundant phospholipid
species, constituting 74.5% (day 3) to 82.3% (day
7) of total surfactant phospholipid. The proportion of minor
phospholipid species (phosphatidylserine, phosphatidylethanolamine,
phosphosphatidylinositol, and phosphatidylglycerol) was remarkably
consistent, ranging from 12.4% (days 5 and 7) to 14.6% (day 3) of total surfactant phospholipid.
Sphingomyelin levels were lowest on day 7, resulting in a
lecithin-to-sphingomyelin ratio that was higher on day 7 than at any other time point. Unexpectedly, the levels of
phosphatidylglycerol declined significantly after day 1 and
remained relatively low for the remainder of the culture period.
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Surfactant phospholipid and protein secretion.
Because phosphatidylcholine synthesis was maximal on day 7 of culture, regulation of surfactant phospholipid and protein secretion was determined on this day. PMA stimulated secretion of surfactant phospholipid, SP-B, and SP-C, whereas compound 48/80 inhibited the
stimulated secretion (Table 2). The
-agonist terbutaline inhibited both basal secretion and
PMA-stimulated secretion of surfactant phospholipid, SP-B, and SP-C
when cells were cultured in BEGM (Table 2 and Fig.
8). Because terbutaline stimulates phospholipid secretion from rat type II cells cultured in DMEM, we
hypothesized the inhibitory effect of terbutaline, which we observed
was a result of epinephrine exposure for 7 days, since epinephrine is a
normal component of BEGM. We tested this hypothesis by culturing cells
in BEGM without epinephrine for 7 days. When cells were cultured in
BEGM without epinephrine for 7 days, terbutaline stimulated secretion
of SP-B, relative to control secretion, although addition of
terbutaline and PMA together resulted in greater secretion of SP-B than
either agonist added alone (Fig. 8).
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Surface properties of secreted surfactant. Media were collected between days 1 and 3, 3 and 5, or 5 and 7 of culture, and surfactant was isolated by centrifugation. The quantity of phospholipid in media was 36.2 ± 0.9, 22.1 ± 3.4, and 18.6 ± 0.9 nmol/ml for media collected on days 3, 5, and 7, respectively. To assess the surface properties of the secreted surfactant, the media were changed on day 3 and collected on day 5. A surfactant pellet was isolated by centrifugation and washed three times to remove serum proteins. The minimum surface tension detected with the captive bubble surfactometer was 5.2 ± 2.3 mN/m and ranged from 1.7 to 9.5 mN/m (n = 5).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Primary cultures of rat type II epithelial cells maintained on plastic dishes and in media containing FBS rapidly lose markers associated with the type II cell phenotype in vivo. Synthesis of both SPs (14, 24, 33) and phospholipids (17, 29) decreases with time in culture, with downregulation of SP expression occurring within hours of cell isolation. In addition to the loss of SP expression, the ability to sort transfected SP to the lamellar body is lost (15), suggesting that the regulated secretory pathway is also rapidly downregulated with time in culture. Consistent with this hypothesis, the number of lamellar bodies decreases during culture, and the cells become refractory to some secretagogues (29). These striking biochemical changes are accompanied by altered cell morphology, most notably a transition from the cuboidal epithelium seen in vivo to a flattened cell shape in vitro.
A variety of culture conditions have been shown to retard the loss of the type II cell phenotype in vitro. Culture of freshly isolated rat or guinea pig type II cells on components of extracellular matrix or amniotic membrane and under conditions that promote cuboidal cell shape preserve many characteristics of type II cells, including SP expression and the synthesis of surfactant phospholipids (22, 24-26). Accordingly, in the present study, we found Matrigel to be essential for sustained expression of SPs in cultured mouse type II cells. However, culture of type II cells on Matrigel resulted in the formation of multicellular aggregates unsuitable for secretion experiments in which cell apices were inwardly directed. Therefore, rat tail collagen was added to the Matrigel to produce a 30:70 mixture (collagen-Matrigel, vol/vol) that promoted formation of monolayers necessary for secretion experiments. As previously reported for rat type II cells, addition of human KGF to the culture medium enhanced cell proliferation and SP expression (28, 36). Both DMEM and BEGM supported cell proliferation in the presence of KGF to the same extent. However, fewer lamellar bodies were noted, and many cells lost their cuboidal shape when the cells were cultured in DMEM for 7 days and examined by electron microscopy. Therefore, BEGM was used for routine culture of mouse type II cells to maintain phenotype.
These optimized culture conditions resulted in ongoing synthesis and secretion of SP-A, SP-B, SP-C, and SP-D for up to 7 days. SP levels consistently declined after 1 day in culture but began to recover by day 3 of culture, reaching levels comparable to or exceeding those in freshly isolated cells by days 5 and 7. We hypothesize the decreases are the result of type II cell injury after exposure to protease during the isolation procedure. Synthesis of surfactant phospholipids was maintained during culture, with results comparable to those previously reported for rat type II cells cultured on EHS matrix (11). The level of newly synthesized phosphatidylcholine (75-82%; Table 1) detected over a 7-day period compared very favorably to the phosphatidylcholine content in mouse bronchoalveolar lavage fluid, which ranged from 75 to 85% of total surfactant phospholipid (1, 3, 10, 13). Consistent with the ongoing synthesis of surfactant phospholipids and proteins, typical lamellar bodies were readily detected in cultured type II cells; moreover, surfactant secretion was stimulated by secretagogues, reflecting an intact regulated secretory pathway. Importantly, surfactant secreted by cultured type II cells exhibited excellent surface tension-reducing properties in vitro. Taken together, these results indicate that key aspects of the type II cell phenotype, including surfactant synthesis, secretion, and function, are maintained for at least 7 days of primary culture. This is the first description of the synthesis and secretion of a functional surfactant complex by mouse type II cells in primary culture.
Surfactant recovered from the media of cultured murine type II cells was able to reduce surface tension to a relatively low value of 5.2 mN/m, quite similar to the value of 5 mN/m obtained for surfactant secreted from isolated rat type II cells cultured on plastic for 22 h (6). However, these values are somewhat higher than reported values for native and replacement surfactants (<1 mN/m; see Ref. 23). This result may be due in part to incomplete removal of serum proteins (an important component of the culture medium) from the isolated surfactant pellet. Serum proteins are known to markedly inhibit the surface tension-reducing properties of surfactant (30). It is also possible that changes in phosphatidylglycerol or other phospholipid components of surfactant contributed to the higher minimum surface tension upon bubble compression. In this regard, further optimization of the culture medium, such as addition of linoleic acid/albumin complex to increase phosphatidylglycerol levels (11), may improve the surface tension lowering properties of the surfactant secreted by cultured type II cells.
Regulation of surfactant phospholipid secretion has been extensively
studied in isolated rat type II cells, but these experiments were
performed in type II cells that had lost or were actively losing their
differentiated phenotype. Examination of the regulation of SP secretion
has not been possible with previously described culture systems. The
present system has allowed for the first time a direct comparison of
the regulation of surfactant phospholipid secretion with SP secretion
in cultured type II cells that are well differentiated. When cells were
cultured in BEGM, PMA stimulated secretion of surfactant phospholipid,
SP-B, and SP-C, whereas compound 48/80 inhibited the PMA-stimulated
secretion of phospholipid and proteins. The -agonist terbutaline
inhibited secretion of both surfactant phospholipid and SP-B and -C
when cells were cultured in BEGM. When cells were cultured in BEGM
without epinephrine (a normal component of BEGM), terbutaline
stimulated SP secretion. These latter results are consistent with
studies in rat type II cells and strongly suggest epinephrine in the
BEGM altered the effect of terbutaline on surfactant secretion. Why
chronic exposure to epinephrine alters the type II cell response to
-agonists is presently unclear. Although aggregation of the cells
was noted using the present culture system, the majority of cells
formed a monolayer. Overall, the results of secretion experiments in these cultured murine type II cells were similar to those previously reported for isolated rat type II cells and support the hypothesis that
SP-B and SP-C secretion are coregulated with phospholipid secretion.
This is in contrast to the constitutive secretion of SP-A and SP-D from
isolated rat type II cells (18). The amount of
phospholipid, SP-B, and SP-C secreted after stimulation is likely the
sum of component accumulation in lamellar bodies via both the
biosynthetic and endocytic (recycling) pathways. A careful analysis of
the rates of synthesis and recycling for individual surfactant
components will be required to assess the relative contributions of
these two pathways to lamellar body content.
In summary, culture of murine type II cells on Matrigel-rat tail collagen substratum and in media supplemented with KGF and carbon-stripped FCS resulted in maintenance of the hallmark features of the type II cell phenotype for at least 7 days. Surfactant phospholipids were synthesized and packaged into typical lamellar bodies. SP-B and SP-C were synthesized, appropriately processed, and secreted with phospholipids. Most importantly, secreted surfactant exhibited excellent surface tension-reducing properties. These culture conditions should facilitate analyses of type II cells isolated from transgenic and knockout mice.
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ACKNOWLEDGEMENTS |
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The expert technical assistance of Mary Falconieri, Emily Martin, and LeDong Ray is gratefully acknowledged.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Specialized Center of Research Grants HL-56387 (W. R. Rice), HL-57144 (J. M. Shannon), HL-61646 (M. Ikegami), and HL-56285 (T. E. Weaver).
Address for reprint requests and other correspondence: W. R. Rice, Div. of Pulmonary Biology, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: ricew0{at}chmcc.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 22, 2002;10.1152/ajplung.00302.2001
Received 2 August 2001; accepted in final form 15 March 2002.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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