Isolation and culture of alveolar type II cells.

Isolation

and culture of alveolar type II cells

LELAND

G. DOBBS

Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, California 94143-0130

DOBBS, LELAND, G. Isolation and culture of alveolar type II celk. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L134-L147, 1990.-The alveolar type II cell performs many important functions within the lung, including regulation of surfactant metabolism, ion transport, and alveolar repair. Because type II cells comprise only 15% of all lung cells, it is difficult to attribute specific functions to type II cells from studies of whole lungs or mixed cell cultures. At the present time, there is no passaged line that exhibits the full range of known type II cell functions. For these reasons, investigators have used isolated type II cells to study alveolar cell biology, biochemistry, and molecular biology. This review addresses many of the issues involved in isolating and culturing type II cells, including the choice of a method of isolating cells, the importance of using specific markers of the differentiated type II cell phenotype, and the problems of maintaining these differentiated phenotypic characteristics in tissue culture. lung function; trypsin; epithelia; elastase

surfactant;

ALVEOLAR TYPE 11 CELL (dvedar type 2 Cell, granular pneumocyte, granular pneumonocyte, type II pneumonocyte, giant corner cell) performs a variety of important functions within the lung, including regulation of surfactant metabolism, ion transport, and alveolar repair in response to injury. Because type II cells comprise only 15% of all lung cells (27,28,54), it is difficult to attribute specific functions to type II cells from studies of whole lungs or mixed cell cultures. At the present time, there is no passaged cell line that exhibits the full range of known type II cell functions. For these reasons, investigators have used isolated type II cells to study alveolar cell biology, biochemistry, and molecular biology. Since the first method of isolating type II cells was published in 1974 (66), there have been many reports of methods to isolate type II cells. This review addresses some of the issues involved in isolating and culturing type II cells. Although references will be made to type II cells obtained from fetal or abnormal adult lungs, the major focus of the review concerns type II cells isolated from adult experimental animals.

THE

ISOLATION

OF TYPE

II CELLS

Anatomic, Species, Strain, and Age Considerations

The alveolar epithelium is comprised of two morphologically distinct cell types, called type I and type II cells, which rest on a basement membrane. Alveolar cells have tight intercellular junctions (115). Type I cells, which constitute 8-10% of all lung cells, are very large cells, the thin cytoplasmic extensions of which cover >95% of the alveolar surface (28). Type II cells comprise 15% of all lung cells, but cover ~5% of the alveolar surface. Type L134

1040-0605/90

$1.50 Copyright

lung; differentiation;

lamellar

bodies;

II cells are cuboidal cells of a size intermediate between the smaller endothelial and interstitial cells and the larger macrophages and type I cells (27, 28,54). The size of type II cells varies both among species and within species. Type II cells in humans and baboons have been reported to be larger than those in rats (27). Within one standard deviation of the mean, the average volume of rat type II cells varies by -50% [calculated from data of Crapo et al. (28) and Haies et al. (54)]. Type II cell size may also vary with the state of lung growth and repair. Because cell size changes during the cell cycle (57), it is not surprising that type II cells are larger in lungs undergoing early postpneumonectomy lung growth, (129), treatment with 85% 0, (28), or treatment with silica (95). Size variation in type II cells becomes a matter of some importance when techniques of differential sedimentation are used to isolate cells (see Differential sedimentation).

Methods have been described to isolate type II cells from a variety of different species (See Table 1). In studies with rats, some workers have found the yield of type II cells to be higher from specific-pathogen-free (52, 91) animals or younger animals weighing ~300 g (107). In our laboratory, we obtain better cell yields and viabilities when we minimize the risk of infection by using specific pathogen-free rats within a few days of receiving the animals. Dissociation of Type II Cells From Lung

To isolate cells from organs, the cells of choice must be dissociated from their attachments to other cells and to the extracellular matrix. Several different strategies have been used to accomplish this. In some organs, such

0 1990 the American

Physiological

Society

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INVITED

1. Yield and purity

TABLE

of type II cells isolated by different methods Crude

Ref.

Kikkawa

Species

1974

(66)

Mason

1977

(89)

Fisher

1977

(44)

Greenleaf

n

(52)

Rat

1) 2)

Cryst. trypsin, 3 mg/ml DNAase, 30 pg/ml

13

Cryst.

31 5

Cryst. trypsin, 3 mg/ml DNAase, 30 pg/ml

1980

(45)

Rat

9

Dobbs

1980

(32)

Rat

1) 26 2) 86 3)102

Skillrud

1984

Goodman

(123)

1982

(50)

5-12”

55

Fisher

trypsin,

Trpysin, DNAase,

10 mg/ml

2.5 mg/ml 10 pug/ml

Cryst. trypsin, 0.1 mg/ml Cryst. trypsin, 3 mg/ml Porcine elastase, 40 U/ml”

Rat

Porcine DNAase,

Rat

DNAase, 1 mg/ml + I) Elastase 4 U/ml or 2) Trypsin, 2.5 mg/ml

elastase, 35 U/ml 0.2 pg/ml

1984

(20)

Rat

25

Elastase,

7 U/ml

1986

(121)

Rat

10

Elastase, Trypsin, ml DNAase

12.6 U/ml (type III) 25 mg/

Weller

1986

(135)

Rat

Dobbs

1986

(33)

Rat

Richards

(142)

1987

50

Massey

1987

1987

Kikkawa

Rat

112

Finkelstein

Leary

1982 (72)

Robinson

(71)

1989

1977 1986

(98) (120)

1984

8"

Rabbit Rabbit

32

Rabbit

1983

Augustin-Voss (7) Pfleger

(43)

(108)

20

Cow

126

Hamster

Human

30 U/ml

Density

gradient

(Ficoll)

Density

gradient

(albumin)

38

Density

gradient

(Ficoll)

46

I) Density 2) Density elutriation

28-34

68 31

Density

40 30

Adherence

4.9

-48 48

Protease

70 pig

(albumin) +

%Type

0.4

95

8-20b

60-67

4

73

7-lib

67

gradient gradient gradient

Albumin or 1 Metrizamide

gradient

(Percoll)

Density

gradient

Removal lectin

of macrophages agglutination

+ unit

Density

gradient

5 11 25

60 63 80

7

92

3-6

j

gravity by

9

90

12

77

(Percoll) + Adherence to plates coated with leukocyte-common antigen

10-20

90

400-500

trypsin,

-40 -40 -90 -50f

3 mg/ml

54

Adherence to plates with IgG

11

1) Laser flow cytometry Acridine Orange 2) Elutriation

34

-50

I, 10 mg/ml

Density

gradient

coated

35

89

with

2 x 105/h

85 95

(Percoll)

Laser flow cytometry monoclonal antibody type II cells

with against

Elutriation

-14 -20 -32 -20f

82

3 x lo”/h

96

0.6

65

Density

gradient

(Ficoll)

20-30

95

Cryst. trypsin, 0.25 mg/mlh Elastase, 0.3 mg/ml DNAase, 10 pg/ml

Density

gradient

(Ficoll)

10

80-90

6 U/ml porcine type mg/ml 10 pg/ml

180

Trypsin,

21-29’

Elastase,

3 mg/ml trypsin,

3 mg/ml

elastase,

30 U/ml

Laser flow cytometry phosphine 3R

300 IX,

Collagenase CLS II, 0.5 pg (=75 U)/ml Elastase, 0.55 pg/ml (lOU)/ ml

Cryst.

II

j

Trypsin

Porcine

13

gradient gradient

Cells

Adherence

81

81

DNAase, 250 fig/ml I ) Trypsin, 0.15 mg/ml 2) Trypsin, 0.25 mg/ml 3) Trypsin, 0.50 mg/ml 4) Elastase, 42 U/ml

Elastase, Trypsin, 0.025 DNAase,

Rabbit

Guinea

32

II

No. cells X 106/animal

I, 60 pg/ml

Elastase,

Cryst.

Mouse

(67)

1982

Lafranconi

Sikpi

Rat

(93)

1975

%Type

Purification Method

82

72

Collagenase

6 Funkhouser (47)

Purified

Elastase, 80 pg/ml Collagenase (CLS), 2 mg/ ml Trypsin, 0.5 mg/ml DNAase I, 50 pg/ml

Rat

(107)

Mixture

Density Density Density

1) 51 2) 26

Brown

1986

(45jd

32-59

Simon

Wilson

Cell

No. cells X 106/animal

Trypsin”

Rat

1979

Enzymes

Rat Rat

L135

REVIEW

40 U/ml

50 400

1.3-4.8'

21

34-39

with

Lavage + removal of macrophages by magnetism

5 X lo5 cells/h

98

36

88

Density

gradient

(Percoll)

2-3g

96

Density

gradient

(Ficoll)

4

70

Density gradient + Elutriation

(Metrizamide)

Density

(Metrizamide)

gradient

a The time of enzymatic digestion varied from 5 to 60 min. After 20 min digestion, the yield was 10 X mixture. The yield of purified cells was calculated for a 20-min digestion. b Greater yields from pathogen-free and 3 different lots compared. d Calculated from original report, 1 g lung tissue z 1 lung. e In orcein/elastin nitroanilide units, divide by 7. f This study investigated the effect of body weight (age) on cell yield. Yields are and are interpolated to the same weight animals for elastase from data in original publication. g 8 pools of 7-12 the protease combinations and concentrations on cell yields was examined. i Per gram of lung tissue. j Purity more days.

j

lo6 cells/animal in the crude cell animals. ’ Trypsin from 2 sources units; to calculate Suc-(L-ala)3-pgiven for 200-g animals for trypsin mice/pool. h The effects of varying assessed only after culture for 1 or


L136

INVITED

as the spleen, where the cellular composition is more homogeneous and cellular attachments are fewer or less tight than in the lung, gentle mechanical forces are sufficient to liberate relatively homogeneous populations of cells. In most other organs, cell dissociation requires more rigorous techniques. Amsterdam and co-workers (6) found that different chemical treatments were required to disrupt intracellular junctions or cell-matrix adhesion points of pancreatic cells. These workers reported that desmosomes separate and intercellular tight junctions undergo rearrangement when exposed to EDTA, but intercellular tight junctions are resistant to enzymatic treatment. In contrast, the pancreatic basement membrane can be digested with collagenase but is unaffected by calcium chelation. Detailed studies comparing the effects of various chemical agents on alveolar ultrastructure have not been published. Calcium chelation, enzymatic treatment, or both, have nevertheless been empirically used in all methods of isolating type II cells. Most methods of isolating type II cells begin with vascular perfusion of the lung to remove blood and endobronchial lavage to remove alveolar leukocytes. Although perfusion effectively removes erythrocytes from capillaries, substantial populations of leukocytes remain within capillaries and airspaces after perfusion and lavage (66,and L. G. Dobbs and M. C. Williams, unpublished observations). Because the numbers of these inflammatory cells increase when animals are infected, type II cell isolations are more successful when lungs are free from bacterial or viral infections. After perfusion and lavage, cells are dissociated by treatment of the lungs with proteolytic enzymes. The most common enzymes used to free type II cells from the lung have been trypsin, pancreatic elastase, and collagenase; DNAase is often added to minimize cell clumping. Because there can be considerable variation in commercially available preparations of enzymes, it is useful to have a detailed description about the sources, lots, and characteristics of the enzymes used to isolate cells in each published method. Several common problems may complicate the choice and use of enzymes. 1) Enzymes obtained from different vendors may vary in quality. 2) Commercial enzyme preparations may contain other enzymes in addition to those on the bottle label. For example, preparations of crude typsin usually contain elastase and chymotrypsin in addition to trypsin. Collagenase often contains trypsin and chymotrypsin. 3) The adjective crystalline does not ensure that an enzyme preparation is pure and homogeneous. 4) In published methods of cell isolation, it is sometimes difficult to determine the actual enzymatic activity that was used to dissociate cells, because the enzyme concentration was expressed as weight per volume rather than in units of enzymatic activity. 5) Various vendors may express enzymatic activity in different units. This may cause confusion about how much enzyme was actually used to isolate cells. For example, one unit of elastastolytic activity as measured by the succinyl-(L-alanine)y-p-nitroanilide method (41) (used by Worthington Biochemical, Freehold, NJ) is equivalent to seven units as meas-

REVIEW

ured by the orcein/elastase method (110) (used by Elastin Products, St. Louis, MO). Many different enzyme cocktails have proven effective in isolating type II cells. The manner in which the enzyme is delivered to the alveolar surface may influence cell yield. Both in rabbits (43) and rats (Dobbs, unpublished observations), instillation of enzyme via the trachea is more effective in liberating cells than is incubation of minced lung tissue with enzymes. There are few studies that compare cell numbers and purities obtained with defined amounts of different enzymes, solely and in combination, and no studies of optimal enzymatic conditions for dissociating type II cells in different species. Finkelstein et al. (43) showed that the addition of small amounts (2.5-25 pg/ml) of trypsin to elastase (0.3 mg/ml purified elastase) improved cell yield. We have found similar results when 50 pg/ml crystalline trypsin is added to elastase (30 orcein/ elastin U or 4.3 Suc(ala)3-p-nitroanalide U/ml). Elastase appears to be more selective than trypsin or collagenase in dissociating alveolar cells. When elastase is instilled into the lung, the alveolar epithelium is selectively loosened or removed from the underlying basal lamina (32). Although elastic fibers in the interstitial and perivascular spaces are disrupted or degraded, the interstitium remains remarkably intact. In contrast, treatment with trypsin (66) or collagenase (74) liberates interstitial and endothelial cells as well as alveolar cells. Because there is no easy method of separating type II cells from interstitial and endothelial cells (see below), the use of elastase can provide a significant advantage over trypsin or collagenase because it reduces the number and types of contaminating cells. When proteolytic enzymes are used to isolate cells, it is always appropriate to ask whether enzymatic treatment alters certain cellular functions (and, if so, reversibly or irreversibly) damages cells. Examples of enzymatic treatment altering cellular functions are common. For example, trypsin concentrations greater or equal to 100 pg/ml have been reported to alter functions of rat parotid acinar cells (86) and macrophage Fc receptors (14). Type II cell functions can also be affected by protease treatment. Finkelstein and Mavis (42) showed that the specific activities of NADPH cytochrome c reductase and CDP choline: 1,2 diacylglycerol cholinephosphotransferase are reduced in subcellular fractions prepared from lung tissues and type II cells treated with proteases. Goodman and Crandall (50) observed a dramatic reduction in dome formation (and, by inference, in ion transport) in cultured type II cells when trypsin rather than elastase had been used to isolate the cells. The differences in dome function were dramatic even after 8 days in culture, demonstrating that the method used to isolate cells can influence cell functions for prolonged periods. More detailed information about how methods of type II cell isolation modulate specific cellular functions would be helpful to investigators in choosing a method to isolate type II cells for a particular purpose. Separation

of Type II Cells from Other Lung Cells

Differential sedimentation. Most methods of separating different types of cells rely on techniques of differentiai


INVITED

sedimentation. According to the basic differential sedimentation equation, the sedimentation velocity of a cell varies in direct proportion to cell density (wt/vol) and in proportion to the square of cell diameter (15). Type II cells and other lung cells overlap in both size (27, 28, 54) and density (66), making it difficult to separate type II cells from a mixture of lung cells by techniques of differential sedimentation alone. In the first published description of a method of isolating type II cells, Kikkawa and Yoneda (66) described a procedure to alter the density of some nontype II cells. Cells were incubated with a colloidal suspension of barium sulfate. Macrophages that phagocytosed barium became denser than the type II cells that did not phagocytose barium. The heavier macrophages could then be separated from the type II cells on a density gradient. Although other investigators have successfully used similar strategies, employing particulate material of varying compositions, there are several problems inherent to these procedures. Not all macrophages ingest particles, making the separation by density of macrophages and type II cells incomplete. Some phagocytic particles cosediment with type II cells; the final preparation of cells may contain these particles in addition to type II cells. The material used to construct the density gradient may also alter cellular functions (33). Other methods of isolating type II cells, such as centrifugal elutriation (52, 142), also rely on properties of differential sedimentation. The underlying difficulty in isolating type II cells by sedimentation properties is that crude mixtures of lung cells contain non-type II cells which, because they overlap in size and density with some type II cells, cosediment with type II cells. To obtain relatively pure populations of type II cells, it is necessary to discard those type II cells which sediment with other cell types. This process reduces the cell yield. Many methods employing differential sedimentation to isolate type II cells recover ~10% of the total number of type II cells in the lungs, raising questions about how representative the isolated cells are of the total type II cell population. Adherence in tissue culture. Because macrophages adhere more rapidly to tissue culture surfaces than do type II cells and because, in the presence of serum, type II cells are more adherent than most lymphocytes, it is possible to purify type II cells by differential adherence in culture. Type II cells adhere slowly (over 3-48 h) to tissue culture surfaces. It is easy to obtain type II cells in >90% purity by culturing cells for l-2 days (32, 45, 89). However, type II cells undergo extensive morphological and biochemical changes in this time (see BEHAVIOR OFTYPEIICELLSCULTUREDONTISSUECULTUREPLASTIC WITH SERUM); functional studies of cells cultured for

this time must therefore be interpreted with caution. Methods not employing differential sedimentation or prolonged culture. Several other methods avoid some of the difficulties inherent in differential centrifugation and prolonged tissue culture. Simon and co-workers (121) used lectin agglutination to remove macrophages from a mixture of lung cells, ultimately obtaining a population of type II cells that was -7580% pure. Several groups have used flow cytometry to isolate type II cells. Rochat and co-workers (109) have recently shown that a com-

REVIEW

L137

bination of natural fluorescence and orthogonal light scatter can be used to distinguish type II cells from macrophages and monocytes. Although it is theoretically possible to isolate unstained cells by differences in these properties, methods using flow cytometry to purify type II cells have to date relied on staining cells with fluorescent markers (47, 72, 109, 142). When type II cells (47, 72, 142) rather than non-type II cells (109) have been tagged with fluorescent markers, the effects of the markers on cellular functions need to be assessed.Because of the length of time involved in cell sorting, laser flow cytometry yields relatively low numbers of type II cells. As cell sorting becomes faster, these techniques may become more attractive. Two newer methods have used techniques of differential adherence to specific molecules to purify type II cells. The conditions of enzymatic digestion of the lung can be adjusted so that the initial mixture of dissociated cells (“crude cell mixture”) contains mostly type II cells, macrophages, and leukocytes. The macrophages and leukocytes can be removed by “panning” the crude cell suspension on plates coated with either antibody specific for leukocyte common antigen (135) or with nonspecific immunoglobulin G (IgG) (33). By virtue of binding, respectively, either to leukocyte common antigen or to Fc receptors, the majority of non-type II cells in the crude cell mixture can be removed. Both of these methods provide some practical and theoretical advantages over earlier methods. Practical advantages include higher yields, ease of procedure, and cheaper costs in comparison with methods of density gradient centrifugation. Both practical and theoretical advantages are that a much larger proportion of type II cells (20-35% of type II cells in the lung) is recovered, that cells are not selected by criteria of size or density, and that the plating efficiency of the cells is improved. Because a higher proportion of type II cells are recovered and because cells are not cultivated by size or density, the likelihood of isolating a subpopulation of type II cells with distinct properties is reduced. There are theoretical disadvantages to these methods, which to date await experimental testing. One potential problem is that exposure to immunoglobulin may alter type II cell functions. Although there is no direct evidence that exposure to IgG may directly affect type II cells, which lack Fc receptors, an interaction among immunoglobulins, immune effector cells, and type II cells is possible. Leukocytes bound to IgG-coated surfaces may release effector molecules that could act secondarily on type II cells. Type II cells both express class II (Ia) molecules of the major histocompatibility complex (56, 109) and synthesize complement components (128), suggesting that type II cells may have the capacity to interact with the immune system. These issues need further investigation. Isolation of Type II Cells From Fetal Lungs Several methods have been described for isolating type II cells from fetal rat lung. Early studies of cultures of fetal rat lung explants demonstrated that the explants have the capacity to differentiate into alveolar-like structures. Sorokin (125) made the interesting observation that explants obtained from fetal lungs of different ges-


L138

INVTTED

tational ages differentiate at similar rates when placed in tissue culture. Lung explants from early, mid, or late gestational fetuses all differentiate during a few days in culture. Differentiation correlates with the time spent in culture rather than with gestational age plus time in culture. Ballard et al. (8), working with explants of human fetal lung tissue, showed that differentiation in vitro can be accelerated by treatment with dexamethasone and triiodothyronine. Several groups (8, 43, 53) have developed methods for isolating type II cells from explants that have been allowed to differentiate in vitro. Similar to the lung explant models are the “organotypic” cultures of lung tissue (36, 100, 101). In these cultures, cells are first liberated from fetal lung tissue by enzymatic treatment and are then cultured on collagen sponges. The mixed cell cultures form alveolar-like structures from which type II cells can subsequently be isolated. Although studies of these fetal type II cell model systems have provided much useful information about

REVIEW

alveolar cell development, there are certain disadvantages to these methods. One is that cells are cultured for several days before they are used. Another is that the gestational age of the cells in culture cannot be easily related to that of the animals from which the cells were isolated because differentiation occurs in culture in the absence of exogenous factors such as hormones and growth factors. There are a few methods of isolating fetal type II cells without prolonged tissue culture. Methods used to isolate adult type II cells are generally not directly applicable to fetal systems. Fetal type II cells early in gestation are difficult to recognize and, because they contain few lamellar bodies and abundant glycogen, probably have different sedimentation characteristics from adult type II cells. Batenburg and co-workers (11) have described a method for isolating type II cells from Is-day-old fetal rat lungs by differential adherence, and Scott and coworkers (116) have described a method for isolating type

A 1)

Spin cells

(density

2)

Let slides

dry 15 hrs

3)

Incubate Imparts

4)

Rmse

5)

Incubate

6)

Rinse

7)

Incubate

m 50%

ethanol

for 1 5 mr

8)

Incubate

I” 80%

ethanol

for 15 set

9)

Incubate

in 95%

ethanol

for 15 set

10)

Incubate

in 100%

11)

Incubate

in xylene

ethanol

12)

Incubate

I” xylene

for 1 ml”

13)

Mount

14)

Inspect

* Harris’ gravity

of 2 x 105 cells/ml)

in cytocentrlfuge

at 5-1Oxg

slides in hematoxylin solution’ for 3-4 m m (longer a darker blue color to the granules) by dIppIng

slides

m lIthum in water

in dIstIlled

carbonate

two to three

**for

stalnlng

time

t!mes

2 mm

once

ethanol

for 30 set 1 :t for 30 set

cells at 630x-1000x

hematoxylin,

water

solution

for 5 men

commercially

** Lithium carbonate solution: Make lithium carbonate to 158 mls distilled

magnlflcation, available,

count filtered

fresh by adding water

at least 400 cells through

Whatman

2ml of a saturated

#l

filter by

solution

of

FIG. 1. A: protocol for modified Papanicolaou stain for staining inclusions in type II cells. [Modified from Kikkawa and Yoneda (66).] B and C: light microscopic views of stained cells (~1,400). Type II cells can be identified by presence of dark blue inclusions: some of the inclusions are refractile. B, cells isolated by elastase digestion before purification; C, cells after purification by adherence on plates coated with IgG (35).


INVITED

II cells from fetal rabbits by density gradient centrifugation. Because the type II cell phenotype changes during development, careful characterization of the cell populations obtained from fetal animals is particularly important. Isolation

of Type II Cells from Abnormal Lungs

Type II cells have been successfully isolated from the lungs of animals treated with 85% oxygen (28) or silica (64, 95). Uhal (131) reported isolation of type II cells from a lung after contralateral pneumonectomy had been performed. In general, type II cells obtained from these lungs are hypertrophic when compared with type II cells obtained from control animals. Because type II cells in abnormal lungs may have different characteristics from type II cells in normal lungs, some changes in isolation methods may be necessary. Choice of Methods of Isolating Type II Cells Most of the published methods of isolating type II cells provide adequate populations of cells for further study. Although it has not been common practice to do so, the selection of a method to isolate cells might well be based on specific needs, such as avoidance of specific enzymes or a need to achieve certain cell yields. Whatever the method, it is wise to monitor the yield and purity of each cell preparation, since these may vary considerably from day to day. Common problems that may adversely affect the yields and purities of type II cells include subclinical infections in laboratory animals, which may cause large increases in the numbers of pulmonary leukocytes and macrophages, and the occasional lots of proteolytic enzymes, which liberate a significant number of fibroblasts. Routine examination of crude cell mixtures, final purified cell preparations, and cells maintained in culture are important to monitor the yields, purities and viabilities of the isolated cell population. The modified Papanicolaou stain (66) is an easy method by which differential cell counts can be determined. A brief protocol describing the stain and a photograph of stained cells from crude and purified cell mixtures are shown in Fig. 1. MAINTENANCE

QF TYPE

II CELLS

IN

TISSUE

CULTURE

Type II cells cultured on plastic surfaces gradually lose their differentiated morphologic and biochemical characteristics (31, 34, 88, 89), as do many other epithelial cell types. Assessment of the differentiated state involves determining whether a cell possessesthe full complement of differentiated functions of a particular cell type in vivo. Some characteristics, such as morphological phenotype, can easily be compared with cells in situ. It is more difficult to decide about the normal range of other cellular characteristics, such as cellular content of or biosynthetic rates of marker molecules. Therefore, assessment of the differentiated state in culture is somewhat arbitrary, depending on the markers selected, the techniques used to identify or quantitate the markers, and the assumptions about what constitutes the “normal state.” As we learn more about normal type II cell functions, we revise our concepts about how we should assess the state of differentiation of cells in culture. Table 2

L139

REVIEW TABLE

2. Identifying

Morphological Typical lamellar

characteristics of type II cells

bodies

B iochemical Surfactant components Lipids (saturated PC, PG) Proteins (SP-A, SP-B, SP-C) Surface markers Lectin binding: Maclura pomifera, MPA-gp -200 Monoclonal antibodies to cell surface antigens Heymann nephritis antigen(s) Cal Enzymes cu-Glucosidase Alkaline phosphatase Cytokeratins -

contains a list of some markers that have been used to characterize differentiated type II cells. Most of these markers are not unique to type II cells, a fact that should influence both the design and the interpretation of experiments. Markers of Differentiated

Type II Cell Phenotype

Morphological characteristics. The morphological hallmark of the type II cells is its striking intracellular organelle, the lamellar body, which contains surfactant. Although lamellar bodies can be seen in cells by phase contrast or interference contrast microscopy, they cannot easily be differentiated at the light microscopic level from other intracellular inclusions, including triglyceride droplets. Even at the electron-microscopic level, it is difficult to distinguish lamellar bodies from lamellar inclusions seen in a wide variety of different cell types. These problems have been discussed in detail by Mason and Williams (90). Figure 2, in which electron micrographs of type II cells from rat and human species are compared with an electron micrograph of serum-starved fibroblasts, illustrates some of the difficulties involved in using lamellar inclusions alone to identify type II cells. The lamellar bodies in human and rat type II cells have different morphological appearances; some of the lamellar inclusions in the serum-stained fibroblast appear similar to the lamellar bodies, particularly those in the human type II cell. Biochemical markers. Cellular content (the balance of synthesis and catabolism), localization, and metabolism of specific “marker” molecules are commonly used to assessthe differentiated state of cells. Some of these specific molecules have known functions; the function of other biochemical markers is not known. PULMONARY

SURFACTANT

LIPIDS

AND

PROTEINS.

1)

Surfactant lipids. Because type II cells are the main cellular source of pulmonary surfactant, the lipid and protein components of surfactant have been used as biochemical markers for type II cells. To date, no single surfactant component has been found to be unique to type II cells. Phosphatidylglycerol and saturated phosphatidylcholine, which are found in high relative abundance in surfactant and in type II cells, are also components of non-surfactant-related cell membranes and organelles. Nonetheless, because type II cells contain unusually high proportions of phosphatidylglycerol and


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saturated phosphatidylcholine, the relative abundance of these phospholipids has been used as a marker for type II cell differentiation. Phospholipids isolated from highly purified (>90%) freshly isolated type II cells contain -10% phosphatidylglycerol and 70% phosphatidylcholine (32, 52); 45-50% of the phosphatidylcholine is fully saturated (i.e., contains saturated fatty acids at both the Cl and the C2 positions) (32). In comparison, phospholipids isolated from several lines of cultured fibroblasts contain Cl% phosphatidylglycerol and 45-50% phosphatidylcholine; only 20-25% of the phosphatidylcholine is fully saturated (90). When the phospholipid profile of cultured cells falls between these two points, the interpretation of the findings becomes difficult. The incorporation of radioactive precursors into various phospholipid classes has been used to assess whether these lipids are being actively synthesized. In early studies, the incorporation of radioactive choline into phosphatidylcholine was often used as a measure of surfactant synthesis. By itself, choline incorporation into phosphatidylcholine is not a good marker for differentiation of type II cells because it may reflect general membrane synthesis in addition to surfactant synthesis. Both radioactive acetate and glycerol have been commonly used to label surfactant lipids. In freshly isolated cells, the pattern of incorporation of these radioactive precursors into various phospholipid classes is quite similar to the relative proportion of phospholipids by content. Methods used to separate phospholipid classes must be chosen carefully. One-dimensional thin layer chromatography, which is adequate to separate phospholipids obtained from some sources, gives falsely high values for phosphatidylglycerol in experiments with type II cells when radioactive acetate is used as a precursor (65). Therefore, in this type of experiment, it is preferable to use twodimensional thin layer chromatographic techniques to resolve phospholipid classes (65). When comparing data in these types of experiments, it is also important to choose the denominator carefully for assessing the relative distribution of lipid classes. For example, type II cells cultured for 24 h with radioactive acetate incorporate ed lectins, it appears that the terminal glycosylation of the type II cell antigen differs from the Hep 2 cell antigen recognized by Cal. 2) Antibodies against surface proteins of type II cells. Funkhouser and associates (47) have produced a monoclonal antibody against a 146-kDa protein present on the apical surface of rat type II cells. Within the alveolar epithelium, the antibody is specific to type II cells. This antibody is not organ specific, also binding to epithelial cells of the renal proximal tubule and the small intestine. Observations from two other groups have demonstrated cross-reactivity between other renal, intestinal, and type II cell antigens. Glycoprotein GP 300, a pathogenic autoantigen causing Heymann nephritis, can be localized to the apical plasma membrane of type II cells, in addition to the apical plasma membranes of renal, intestinal, epididymal and yolk sac epithelia (21). Raychowdhury et al. (106) recently reported that GP 300 shares partial homology with the low-density lipoprotein (LDL) receptor. Other investigators (85, 122) have implicated a renal glycoprotein of molecular mass 600 kDa in the pathogenesis of Heymann nephritis. A polyclonal antibody to this protein immunoprecipitates antigens of molecular mass 270-290 kDa from membranes prepared from type II cell preparations (84). At this time, it is uncertain whether the antibodies against the two renal glycoproteins (of 300- and 600-kDa mass) recognize the same or different antigens. Brody and co-workers (19) have shown that human type II cells bind Cal, a monoclonal antibody raised against an extract of Hep 2 cells; a cell line derived from a human laryngeal carcinoma. Although Cal recognizes a glycoprotein common to many malignant cells, it binds only to type II cells in the normal lung. In areas of

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Ll41

hyperplastic lung, Cal also binds to nonciliated bronchiolar cells (Clara cells) in addition to type II cells. ENZYMES. Two enzymes have been proposed as markers for type II cells. One is cu-glucosidase.Although this enzyme is not unique to type II cells, there is a report that an a-glucosidase active at neutral pH may be unique to surfactant and type II cells (30). A second enzyme proposed to be a marker for the differential phenotype of type II cells is alkaline phosphatase. Although cells from many organs have alkaline phosphatase activity, alveolar macrophages lack phosphatase activity (24). For this reason, histochemical demonstration of alkaline phosphatase has been used to distinguish type II cells from alveolar macrophages (37). It is not known whether the particular phosphatase found in type II cells is a gene product unique to type II cells or whether it is similar to alkaline phosphatase from other cellular sources (59, 68, 134) C~TOKERATINS. Cytokeratins are intermediate filament proteins found in all epithelial cells. There are more than 20 different proteins, which can be separated by immunologic and electrophoretic methods (96). Paine and co-workers (97) characterized the patterns of cytokeratin synthesis in freshly isolated type II cells. Freshly isolated rat type II cells synthesize four cytokeratins, which, by their electrophoretic mobilities and Western blot analysis, correspond to human cytokeratins No. 7, 8, 18, and 19. GENERALCONSIDERATIONSABOUTCULTURE OFTYPEIICELLS

The ideal set of culture conditions would allow type II cells to express their full range of functions and thus permit investigators to modulate these functions by altering conditions of culture. Evaluation of the differentiated state of type II cells should therefore involve as many markers of type II cell functions as possible. This is particularly important because most markers of type II cell functions are not unique for type II cells and because our understanding of the factors that control expression of the type II cell phenotype is incomplete. BEHAVIOROFTYPEIICELLSCULTUREDONTISSUE CULTUREPLASTIC WITH SERUM

The behavior of type II cells cultured on tissue culture plastic in the presence of 510% fetal bovine serum has been characterized in some detail. Over l-7 days in culture, type II cells undergo extensive morphological changes. The cells spread over the tissue culture dish, lose their cuboidal appearance, and become flattened. Lamellar bodies decrease in size and in number; most cells eventually lose their lamellar inclusions (31, 34). Biochemical changes occur even more rapidly. Both the content and biosynthesis of phosphatidylcholine and phosphatidylglycerol decrease over 24-48 h of culture (34, 89). The content of SP-A (80, 138) and mRNA for SP-A (80) decrease dramatically within 24 h of culture. During this time, the relative biosynthesis of neutral lipids increases (34, 65). The pattern of surface markers changes as type II cells are cultured. Type II cells bind less MPA and more RCA-


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I (34) and other lectins (63), although the expression of some MPA-binding proteins may not decrease (18). Expression of a surface antigen specific for type I cells in situ appears and then increases as type II cells are maintained in culture (35). Enzyme and cytokeratin patterns also change with tissue culture. Expression of alkaline phosphatase (37) decreases as type II cells are cultured and the pattern of cytokeratin synthesis changes (97, 143). During 7 days in tissue culture, synthesis of cytokeratin No. 19 dramatically decreases and synthesis of cytokeratin No. 18 increases (97). From studies of cytokeratin synthesis in fetal lung cells and in cells cultured on substrata other than plastic, it appears that cytokeratins will be useful in helping to define the differentiated state of type II cells. These observations suggest that type II cells on plastic lose those morphological and biochemical characteristics associated with the differential type II cell phenotype at different rates over several days in culture. In addition, cultured type II cells acquire some characteristics associated with the type I cell phenotype. Whether type II cells undergo transdifferentiation in culture or whether they are merely dedifferentiating is unclear at this time. The behavior of type II cells cultured on plastic in the presence of serum has been used for comparison to the behavior of type II cells cultured under other conditions. STRAGETIES FOR OF DIFFERENTIATED

PROMOTING EXPRESSION PHENOTYPE IN CULTURE

Investigators studying various other types of epithelial cells have been successful in discovering conditions that preserve the differentiated state of the cells, albeit with varying degrees of success. Several general observations have emerged from many studies with other cell types. First, it is important to use more than one marker to assess the state of differentiation of a cell (73). Second, many factors can affect expression of the differentiated state. Among these are cell shape (46), substrata (38, 39, 51, 73, 139), serum (9, 39, 55), hormones (5, 55, 105), growth factors (9), various cofactors (58, 130), lectins (lo), and cell-to-cell interactions (78, 114, 127). Third, which factors are important and how these factors interact may vary considerably among different cell types. The omission or inclusion of one factor may alter the expression of cell function, promoting expression of either the differentiated or the dedifferentiated state; cells may require a complex combination of factors in order to express differentiated functions (39, 62, 79). Substrata and Cell Shape

In vivo, there are cytochemical differences in the basement membrane-associated microdomains of type I and type II cells (112). Type II cells deposit a basal lamina as they are maintained in culture (92), secreting wellcharacterized components of the extracellular matrix such as fibronectin and type IV procollagen (29, 111) as well as less well-characterized collagen-binding proteins (29). Fetal type II cells synthesize proteoglycans (124). By inference, the basal lamina underlying type II cells in situ is at least partially derived from the type II cells themselves.

REVIEW

In culture, substrata of preformed matrix compon .ents can profoundly alter cellular functions (see above) (22 9 48, 82). Type II cells adhere to various preformed extracellular matrices and purified matrix components. Clark and co-workers (23) showed that type II cells adhere more avidly to fibronectin-coated surfaces than to surfaces coated with laminin or types I, II, or IV collagen; the fibroblastic cell-attachment domain of fibronectin mediates the adherence to fibron .ectin. The ways in which extracellular matrix can modulate the expression of type II cell-differentiated characteristics has been recently reviewed (104). Several different biological matrices have been used in attempts to promote type II cells to express differentiated characteristics in culture. Type II cells cultured either on floating type I collagen gels (49) or on matrices prepared from bovine cornea1 endothelial cells (92) have relatively well-preserved ultrastructural characteristics, but exhibit a pattern of phospholipid biosynthesis similar to that of cells cultured on tissue culture plastic (34,49). When collagen gels are used as a substratum, morphological characteristics are better preserved when the gel is detached and allowed to float in the culture medium; this permits cells to assume a more cuboidal shape (49, 119). Type II cells cultured on human amn iotic membrane (83) have an improved m.orphological appearance and show a small increase in relative phosphatidylcholine synthesis; Cott et al. (26) also reported an increase in the neutral lipid fraction in type II cells cultured for 48 h on human amnion. Substrata can affect ion transport. Cells cultured on amniotic basement membrane have higher resistance values and lower short-circuit currents than cells cultured on collagen-coated filters (25). When type II cells are cultured on the gels made from the Engelbreth-Holm-Swarm tumor (EHS), they form spherical clusters surrounding a central lumen (13, 118). Although the effect of EHS on lipid synthetic patterns of type II cells are modest (small increases in PC and saturated PC, no differences in PG when compared with cells cultured on plastic), there are striking increases in the content of surfactant proteins SP-A and in the mRNAs for all three surfactant proteins. As cells are maintained in culture on EHS matrix for 8 days, there is a progressive increase in the expression of mRNAs for surfactant proteins SP-A and SP-B; expression of SP-C mRNA does not increase (117, 118). Attempts to determine which of the components of the EHS matrix (including laminin, type IV collagen, heparin sulfate proteoglycans, and nidogen/entactin) are responsible for these striking effects have so far met with limited success. Laminin comprises >90% of the EHS tumor matrix. Laminin may promote retention of the cuboidal shape of type II cells (103). Although culture of type II cells on laminin-coated surfaces retards the appearance of 46kDa acidic cytokeratins associated with dedifferentiation (l43), laminin by itself does not promote the expression of mRNA for surfactant proteins (102, 117). The form in which laminin is presented to cells may determine whether it promotes expression of differentiated characteristics (70). In contrast to the effects of EHS matrices on type II cells, fibronectin-coated surfaces appear to promote the


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loss of some differentiated type II cell characteristics (l43), while stimulating incorporation of [3H]thymidine (102) by the cells. How matrix components affect expression of the differentiated phenotype of type II cells is uncertain. In other cell systems, matrix components can modulate gene expression by both receptor-mediated mechanisms (136) and cytoskeletal and cell shape changes (46, 60, 61). Which of these and other possible mechanisms will prove relevant to type II cells is a matter of current investigation. Cell-to-Cell

Interactions

Cell-to-cell interactions are important in the development and maintenance of the differentiated state in a variety of different organ and cell systems (78,114, 127). In the lung, the classical studies of Alescio (Z-4), Spooner (126), and Wessells (137) clearly show that bidirectional mesenchymal-epithelial interactions may dictate the course of subsequent lung differentiation. In primary cultures of hepatocytes, epithelial-epithelial cell interactions have also been shown to be important in regulating gene expression (12). The influence of cell-to-cell interactions on the expression of the differentiated phenotype in type II cell cultures has been studied by Shannon and co-workers (119), who cultured type II cells on flattened or floating collagen gels seeded with feeder layers of various cell types: fibroblasts from various sources, bovine aortic endothelium, and rat mammary tumor epithelial cells. Type II cells cultured on attached (nonfloating membranes) membranes with feeder layers are similar to cells cultured on plastic, whereas cells cultured on floating collagen gels containing feeder cell layers have to some extent improved expression of differentiated characteristics (65) (see below). Coculture of mesenchymal cells and type II cells may be in part responsible for the in vitro differentiation of fetal lung explant and “organotypic” cultures (see above). Because the effects of cell shape, substrata, cell-to-cell interactions and soluble factors interact, these systems involving more than one cell type are difficult to construct and the results are difficult to interpret. Nevertheless, such studies should provide important insights about how individual gene expression is controlled in type II cells. Soluble Factors Type II cells cultured on many substrata, including tissue culture plastic and floating collagen gels, are poorly responsive to soluble factors, such as hormones (49,119). Cott and co-workers (26) demonstrated that sera from different animal sources had different effects on the relative synthesis of phospholipid classes. Cells cultured with rat serum incorporated higher percentages of radioact,ive acetate into phosphatidylcholine and saturated phosphatidylcholine than cells cultured with fetal bovine serum. These authors postulated that the varying concentrations of linoleic acid in various sera might be responsible for some of their observations. They found that supplementing media with linoleic acid stimulated acetate incorporation into phosphatidylcholine but in-

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hibited the incorporation of radioactive precursors into phosphatidylglycerol. Culturing type II cells on floating collagen gels containing feeder layers partially restores cellular responsiveness to soluble factors. Kawada and co-workers (65) found that hydrocortisone and CAMP decreased the incorporation of radioactive acetate into neutral lipid; the relative proportion of neutral lipids, although less than without hormones, remained high. Type II cells cultured on EHS gels in conditions of low serum concentration (1%) plus hydrocortisone and CAMP showed an increase in relative synthesis of saturated phosphatidylcholine and phosphatidylglycerol and a decrease in relative synthesis of neutral lipids. No responses to soluble factors were seen when higher concentrations of serum (5%) were used, once again illustrating that precise combinations of soluble factors and matrix components may be needed in order to detect changes in expression of differentiated characteristics. Under certain conditions of cell isolation, ascorbic acid and glutathione improve subsequent plating efficiency and incorporation of radiolabeled choline and leucine (94). PROLIFERATION

OF TYPE

II CELLS

In the adult lung, type II cells have the capability of proliferating (1, 17, 40). Type II cells isolated from adult animals exhibit little proliferation in tissue culture. Cells cultured on plastic with fetal bovine serum have a nuclear labeling index of ~0.5% (77). Leslie and co-workers (77) compared autoradiography, 3Hthymidine incorporation, and cell numbers in cultures of type II cells. Although extracellular matrix prepared from bovine cornea1 endothelial cells and various soluble factors (rat serum, EGF, insulin, cholera toxin) stimulated [3H] thymidine incorporation, cell labeling, and DNA content, these agents did not cause cell proliferation; the number of cells in culture remained the same. These observations showing that type II cells can be stimulated to synthesize DNA without undergoing mitosis are important because they demonstrate that [“HI thymidine incorporation by itself cannot be used as an index of cell proliferation in type II cell cultures. Because primary cultures of type II cells also contain some non-type II cells, autoradiographic techniques are important to identify the types of cells that are being stimulated. Leslie and co-workers have shown that soluble factors from macrophages (75) and a heat- and trypsin-sensitive factor in bronchoalveolar lavage fluid from normal rats (76) stimulate DNA synthesis in type II cells. Rannels and co-workers (103) have examined in more detail the effect of extracellular matrix and matrix components on [3H]thymidine incorporation in type II cells. Both fibronectin and a fibronectin-rich matrix produced by type II cells stimulate [“HI thymidine incorporation. In contrast, both the EHS matrix and soluble components released from this matrix inhibit [“HIthymidine incorporation (102). Unlike type II cells isolated from adult animals, cultures of the dissociated alveolar-like structures obtained from organotypic cultures of fetal lung cells have the capacity to proliferate (100). Which cells actually divide and whether the presence of non-type II cells in the


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cultures is critical to cell division is unclear. Manipulation of type II cells obtained from younger animals or from injured lungs may prove important in further studies of type II cell proliferation. C ONCLU

REVIEW

12.

SIONS

Many different methods have been developed to isolate type II cells in a high degree of purity from the lung. Although most of these methods provide adequate numbers of cells for further study, the effects of various isolation techniques on specific cellular functions has only been partially elucidated. Most of the studies referred to in this review have utilized type II cells obtained from adult rodents. Studies of fetal and neonatal cells, cells obtained from various species, and cells obtained from lungs undergoing injury and repair, may change our thinking about how type II cells function. By varying conditions of tissue culture, we have gained some insight into the factors that may be important in regulating expression of the differentiated state of alveolar cells. This area of research is exciting and the experimental variables are large. Such studies have the potential for expanding our knowledge about lung development, differentiation, and repair. The author is grateful for the expert technical assistance of Robert Gonzalez and Lennell Allen and the expert manuscript preparation of Pattie Weinmann Schwartz. Dr. Mary Williams, Dr. Jo Rae Wright, and Robert Gonzalez made helpful suggestions about the manuscript. The author is supported in part by National Heart, Lung, and Blood Institute Grants HL-24075-11 and HL-4195802. Address for reprint requests: L. G. Dobbs, Cardiovascular Research Institute and Dept. of Medicine, University of California, San Francisco, CA 94143-0130.

13.

14.

15.

16. 17.

18. 19.

20.

21.

22.

23.

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INVITED 34. DOBBS, L. G., M. C. WILLIAMS, AND A. E. BRANDT. Changes in biochemical characteristics and pattern of lectin binding of alveolar type II cells with time in culture. Biochim. Biophys. Acta 846: 155-166,1985. 35. DOBBS, L. G., M. C. WILLIAMS, AND R. GONZALEZ. Monoclonal antibodies specific to apical surfaces of rat alveolar type I cells bind to surfaces of cultured, but not freshly isolated, type II cells. Biochim. Biophys. Acta 970: 146-156, 1988. 36. DOUGLAS, W. H., AND P. J. CHAPPLE. Characterization of monolayer cultures of type II alveolar pneumocytes that produce pulmonary surfactant in vitro. Dev. Biol. Stand. 37: 71-76, 1976. 37. EDELSON, J. D., J. M. SHANNON, AND R. J. MASON. Alkaline phosphatase: a marker of alveolar type II cell differentiation. Am. Rev. Respir. Dis. 138: 1268-1275, 1988. 38. EMERMAN, J. T., J. ENAMI, R. PITELKA, AND S. NANDI. Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes. Proc. Natl. Acad. Sci. USA 74: 4466-4470, 1977. 39. ENAT, R., D. M. JEFFERSON, N. RUIZ-OPAZO, Z. GATMAITAN, L. A. LEINWAND, AND L. M. REID. Hepatocyte proliferation in vitro: its dependence on the use of serum-free hormonally defined medium and substrata of extracellular matrix. Proc. NatZ. Acad. Sci. USA 81: 1411-1415, 1984. 40. EVANS, M. J., R. J. STEPHENS, AND G. FREEMAN. Transformation of alveolar type 2 cells to type 1 cells following exposure to NO,. Exp. Mol. Pathol. 22: 142-150, 1975. 41. FEINSTEIN, G., A. KUPFER, AND M. SOKOLOVDKY. N-acetyl-(LALA)3-p-Nitroanilide as a new chromogenic substrate for elastase. Biochem. Biophys. Res. Commun. 50: 1020-1026, 1973. 42. FINKELSTEIN, J. N., AND R. D. MAVIS. Biochemical evidence for internal proteolytic damage during isolation of type II alveolar epithelial cells. Lung 156: 243-254, 1979. 43. FINKELSTEIN, J. N., AND D. L. SHAPIRO. Isolation of type II alveolar epithelial cells using low protease concentrations. Lung 160: 85-98, 1982. 44. FISHER, A. B., AND L. FURIA. Isolation and metabolism of granular pneumocytes from rat lungs. Lung 154: 155-165, 1977. 45. FISHER, A. B., L. FURIA, AND H. BERMAN. Metabolism of rat granular pneumocytes isolated in primary culture. J. Appl. PhysioZ. 49: 743-750, 1980. 46. FOLKMAN, J., AND A. MOSCONA. Role of cell shape in growth control. Nature Lond. 273: 345-349, 1978. 47. FUNKHOUSER, J. D., L. B. CHESHIRE, T. B. FERRARA, AND R. D. PETERSON. Monoclonal antibody identification of a type II alveolar epithelial cell antigen and expression of the antigen during lung development. Dev. Biol. 119: 190-198, 1987. 48. GEHLSEN, K. R., L. DILLNER, E. ENGVALL, AND E. RUOSLAHTI. The human laminin receptor is a member of the integrin family of cell adhesion receptors. Science Wash. DC 241: 1228-1229, 1988. 49. GEPPERT, E. F., M. C. WILLIAMS, AND R. J. MASON. Primary culture of rat alveolar type II cells on floating collagen membranes. Exp. Cell. Res. 128: 363-374, 1980. 50. GOODMAN, B. E., AND E. D. CRANDALL. Dome formation in primary cultured monolayers of alveolar epithelial cells. Am. 9. Physiol. 243( Cell Physiol. 12): C96-ClOO, 1982. 51. GOSPODAROWICZ, D., AND C. ILL. Extracellular matrix and control of proliferation of vascular endothelial cells. Clin. Invest. 65: 1351-1364, 1980. 52. GREENLEAF, R. D., R. J. MASON, AND M. C. WILLIAMS. Isolation of alveolar type II cells by centrifugal elutriation. In Vitro 15: 673-684, 1979. 53. GROSS, I., R. M. FREEDMAN, C. M. WILSON, AND S. LINDSEY. Organotypic culture of fetal rat lung: evaluation and comparison with organ culture. Am. Rev. Respir. Dis. 123: 313-319, 1981. 54. HAIES, D. M., J. GIL, AND E. R. WEIBEL. Morphometric study of rat lung cells. I. Numerical and dimensional characteristics of parenchymal cell population. Am. Rev. Respir. Dis. 123: 533-541, 1981. 55. HAMMOND, S. L., R. G. HAM, AND M. R. STAMPFER. Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract. Proc. Natl. Acad. Sci. USA 81: 5435-5439, 1984. 56. HARBECK, R. J., N. W. GEGEN, D. STRUHAR, AND R. MASON. Class II molecules on rat alveolar type II epithelial cells. Cell Immunol. 111: 139-147, 1988.

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Isolation and properties of type II alveolar cells from rat lung.

Isolation and identification of type 2 alveolar epithelial cells.

Isolation and characterization of lung resident mesenchymal stem cells capable of differentiating into alveolar epithelial type II cells.

Physiologic and toxicologic responses of alveolar type II cells.

Inhibitors of choline transport in alveolar type II epithelial cells.

LysoTracker is a marker of differentiated alveolar type II cells.

The freeze-fracture study of alveolar type II cells and alveolar content in fetal rabbit lung.

Sodium-dependent phosphate and alanine transports but sodium-independent hexose transport in type II alveolar epithelial cells in primary culture.

Knockdown of Drosha in human alveolar type II cells alters expression of SP-A in culture: a pilot study.

Isolation of cells that retain differentiated functions in vitro: properties of clonally isolated type II alveolar pneymonocytes.

Silver nanowire interactions with primary human alveolar type-II epithelial cell secretions: contrasting bioreactivity with human alveolar type-I and type-II epithelial cells.

Fetal mouse alveolar type II cells in culture express several type II cell characteristics found in vivo, together with major histocompatibility antigens.

Oxidants and antioxidants in alveolar epithelial type II cells: in situ, freshly isolated, and cultured cells.

Activation of type II cells into regenerative stem cell antigen-1(+) cells during alveolar repair.

Hypertrophy and hyperplasia of alveolar type II cells in response to silica and other pulmonary toxicants.

Amphibian cells in culture. II. Isolation of drug-resistant variants and an asparagine-independent variant.

Reactivity of alveolar epithelial cells in primary culture with type I cell monoclonal antibodies.

Effect of iron chelators on paraquat toxicity in rats and alveolar type II cells.

Augmentation of superoxide dismutase and catalase activity in alveolar type II cells.

Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium.

Expression and function of aquaporin-1 in hyperoxia-exposed alveolar epithelial type II cells.

Competition between paraquat and putrescine for uptake by suspensions of rat alveolar type II cells.

High density lipoprotein promoting proliferation and migration of type II alveolar epithelial cells during inflammation state.

A morphometric examination of type II alveolar epithelial cells in normal and isolated-perfused dog lungs.