The Type 1 Alveolar Lining Cells of the Mammalian Lung I. Isolation and Enrichment From Dissociated Adult Rabbit Lung Paul Picciano, PhD, and Robert M. Rosenbaum, PhD

With removal of large numbers of macrophages by airway lavage, Type 1 cells were isolated in heterogeneous cell populations following the stepwise dissociation of lung tissue. Using a carefully timed collagenase-trypsin digestive sequence at 37 C, unwanted cellular and noncellular lung components were minimized prior to selective release of Type 1 cells. Resulting heterogeneous cell suspensions containing wellpreserved Type 1 cells, as determined by electron microscopy, were layered onto a shallow gradient (3 to 6% Ficoll in minimal essential medium [MEM I) and separated at unit gravity into enriched subpopulations of various cell types. These included various fractions enriched with respect to Type 1 cells (70%), Type 2 cells (82%), and macrophages (81%). Identification of Type 1 cells following their isolation and gradient enrichment was established by light microscopic staining techniques and by specific cell surface characteristics in vitro as visualized by electron microscopy. (Am J Pathol 90:99-122, 1978)

OF THOSE PAPERS dealing with the cellular dispersal of adult lung,1-4 all have described, primarilv, the isolation of Type 2 cells of the terminal airway. Tvpe 1 pneumocvtes, which in the rat are known to account for approximately 15% of alveolar epithelial cells,5 have not been systematically isolated. Consequently, little is known about the Type 1 cell. In addition to serving as part of the air-blood barrier in conjunction with Type 2 cells, the Type 1 cell is recognized as possibly functioning in the transport of fluids between alveoli and blood."9"Moreover, these cells are noted to be highly susceptible to injury following treatment with levels of edematogenic agents (eg, 02, 03, NO2) known to cause damage in the terminal airwav.1013 So little is known about this important cell type that any information regarding its structure and function would be useful. A better understanding of Type 1 cells could be achieved with the use of isolated cells: cell culturing and biochemical and metabolic studies would be possible with the availabilitv of purified fractions of this cell tvpe. In this paper, thereFrom the Department of Pathology. Albert Einstein College of Medicine. Bronx. New York Supported by Program Project HL-16137 and Contract HR5-2952 from the National Heart. Lung and Blood Institute Dr Picciano is the recipient of Young Investigator Grant HL 21396 from the National Heart. Lung and Blood Institute Accepted for publication September 8. 1977. Address reprint requests to Dr Robert M. Rosenbaum. Department of Pathology. Albert Einstein College of Medicine. 1.30 Morris Park Avenue. Bronx. NY 10461

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fore, we describe a procedure by which pulmonary Tvpe 1 cells were isolated from rabbits. This involved the dissociation of lung into dispersed heterogeneous cell populations and the subsequent enrichment of Type 1 cells bv sedimentation at unit gravity. Specific details describing the ultrastructure and surface characteristics of the isolated Tvpe 1 cell are considered in a separate paper.14 Materals and Methods Young adult male New Zealand white rabbits (0.3 to 1.3 kg) were lightly heparinized (100 units/kg) and killed with pentobarbital sodium (Nembutal) (100 mg kg). The trachea was transected and cannulated; the pulmonanr trunk was then catheterized with a No. 15 polyethylene catheter via the right ventricle. Following en bloc evisceration of the lungs, heart, and trachea, the heart itself was dissected awav at the level of the auricles. Blood was removed from the surface of the preparation by rinsing in 300 ml of 0.13 NI saline at room temperature. The trachea and the lungs were placed in a beaker of saline and evacuated at a pressure of 13 mm Hg for 10 minutes to render them gas-free. The trachea was immediately clamped to minimize reentry of air, and the lungs and trachea \vere weighed. Examination by both light and electron microscopy of this material confirmed minimal damage by these procedures.

Dissociation of Lung Tissue The clamp was removed and the lungs lavaged as described by Brain and Frank,5 with some modification. The lungs were slowly filled for 1 minute with 3.3 ml of buffered saline (0.13 N\) per gram of lungs via the tracheal cannula. This medium was allowed to remain for an additional minute, and the lungs were emptied during the third minute. Five additional cycles were required to complete the lavage. Suspension of the entire preparation in saline during this procedure prevented drying of the tissue and allowed for even filling of the lungs by eliminating hydrostatic gradients.15 W7e have found that a pH of 7.2 is optimal for effective lavage. In addition, 100 units ml of penicillin and 100 gg/ml of streptomycin were effective in minimizing bacterial contamination in further handling of this tissue. The lavage step was necessary for removal of a large number of macrophages from subsequent cellular isolates. Their elimination had a significant effect on the success of isolating Type 1 cells as enriched populations. \1acrophages obtained as such were processed for staining (see Light Microscopy). Examination by both light and electron microscopy of this material confirmed minimal damage by these procedures. The tracheal-lung preparation was suspended in a sealed lucite chamber with the trachea open to room air and perfused via the pulmonary trunk catheter with 100 ml of calcium-magnesium-free Mfoscona's saline (C\MF, 32 C).'6 pH 7.2. and antibiotics (100 9g ml kanamycin [Kantrex]. 7.5 jg ml amphotericine B [Fungizone]. 100 units ml penicillin G, and 100 gg /ml streptomycin sulfate) at a rate of 11 ml min at 28 C using a Har-ard syringe pump. Temperature control was maintained by a constant temperature water jacket surrounding the perfusion chamber. This was followed by 130 ml of 0.10%5 tr-psin (32 C, Difco, 1: 250) in CNIF. pH 7.2. During the course of perfusion. a minimal vacuum (equivalent to 20 cm H20 pressure) was used to rhythmically expand and contract the lungs at 3 times per minute during CM1F perfusion only. Following completion of the perfusion procedure, the lungs were removed from the chamber and the well-perfused regions of lung were cut into 2.0-sq-cm pieces. Six grams of this tissue was put into the barrel of a 10-cc syringe and passed twice through a No. 16 WN7introb needle. Fragments resulting from this step w-ere washed by equally distributing the tissue to three siliconized (Siliclad. Clay Adams. Parsippanv, NJ) 125-mi Erlenmeyer flasks, each containing 20 ml

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of minimal essential medium (MEM)/0.1% bovine serum albumen (BSA, Fraction V, Armour Pharmaceutical Co., Phoenix, Ariz) with antibiotics and shaken at 37 C in a Dubnoff Metabolic shaker for 10 minutes at 120 cpm. The tissue was removed from suspension by aspiration using a No. 16 Wintrob needle and again was equally distributed to three siliconized flasks, each containing 20 ml of 0.1% collagenase (Type 2, Worthington Biochemical Corp., Freehold, NJ)/0.1% BSA and antibiotics in MEM. These suspensions were incubated for 15 minutes and the tissue was removed from the enzvme solution as described above. The collagenase treatment was repeated and the tissue was removed from suspension by centrifugation at 1000 X g for 10 seconds in siliconized Corex tubes. At this stage of dissociation, the tissue consisted primarily of large clumps or strands of cells devoid of most interstitial framework, as demonstrated by electron microscopy. (See Figures 3 and 4.) Subsequent treatment with a trypsin-ollagenase cocktail was required to effect complete dissociation. Cells flushed from pulmonary vasculature by CMF and trypsin perfusion and cells released by lung fragments during MEM and collagenase washes were recovered by centrifugation and processed for staining. (See Light Microscopy.) Fragments of lung following MEM wash and each collagenase wash were prepared for transmission electron microscopy as described below. (See Electron Microscopy.) Collagenase-treated fragments were next incubated in the Dubnoff bath (37 C, 130 cpm) for 8 minutes in two siliconized flasks, each containing 20 ml of 0.05% trypsin/0.02% EDTA/0.1% collagenase/0.1% BSA in CMF. At this time, to reduce viscosity, 0.2 ml of 5 mg/ml solution of DNase (Type 2, Sigma) in CMF was added to each suspension. Following an additional 2 minutes of shaking, the lung cells were cleared of nondissociated tissue by centrifugation at 1000 x g for 30 seconds. The supematants were diluted with an equal volume of cold MEM/10% fetal calf serum (FCS) and centrifuged at 500 X g for 10 minutes at 4 C. The resultant pellets of cells were dispersed in cold MEM/0. 1% BSA. An aliquot was removed for staining and the remaining cells were placed on ice. Nondissociated tissue was again trypsinized for 8 minutes followed by treatment with DNase for an additional 2 minutes. Undigested tissue was removed by aspiration. Cell suspensions were diluted with 3 parts cold MEM/10% FCS, poured through a fine mesh nylon screen (147.2 openings/inch, 112 u/opening), and centrifuged at 500 X g for 10 minutes. The cells were resuspended in cold MEM/0.1% BSA, and an aliquot was removed for staining. The remaining cells were combined with those already on ice. A complete summary-outline of these procedures is found in Text-figure 1. Enridhment of Lung Ces at Unit Gravity The equipment and procedures used for enrichment of Type 1 cells have been described in detail for enrichment of cell types from rat adenohypophysis," which were separated into enriched cell populations by sedimentation at unit gravity through a gradient of 0.3 to 3.0% BSA in medium 199. A modification made for lung cell separation has been the substitution of Ficoll for BSA as a separation medium. To charge the separating chamber, a heterogeneous population of 9 to 10 million lung cells in 5 ml of MEM/0.1 % BSA/0.01 % EDTA was used. The enrichment of cell types from lung was accomplished by allowing pneumocytes to sediment at unit gravity through a shallow gradient (3% to 6% Ficoll in MEM/0.1% BSA/0.01% EDTA with antibiotics, pH 7.2) at room temperature. After layering cells thinly onto the gradient as described by Hymer et al,17 lung cells were then removed through the top of the chamber as 10-ml fractions by displacement with 10% sucrose in 0.15 M NaCl. Cd Prevato

The integrity or preservation of cells isolated and enriched by the above procedures were evaluated initially by the dye exclusion test and, ultimately, by electron microscopy.

Isolated tracheal - lung preparation

1. Lavage: 6 washes with 0.15 M NaCl, pH 7.2 2. Process for staining.

Lavaged Lung 1. Perfuse with CMF, pH 7.2, followed by CMF, 0.1% trypsin, pH 7.2 (28 C). I

9

Cells in perfusate: process for staining

a

Perfused lung 1. Pass 6 g well-perfused lung through 1 6-gauage Wintrob needle 2 times. 2. Wash fragmnts in MEM-0.1% BSA in Dubnoff shaker bath (120 cycles/nrin, 37 C) for 10 min. 3. Remove tissue from suspension by aspiration.

1-Singie cells in suspension: proess for staining

Washed fragments 1. Resuspend f ragrents in MEM, 0.1 % collagenase, 0.1% BSA. 2. Shake 15 min. 3. Remove fragments by aspiration.

I-

Single cells in suspension: process for staining

Collagenase-treated fragments 1. Repeat 1 5-min collagenase treatment. 2. Remove fragments from suspension by aspiration or by centrifugation at 1000 x g for 10 sec.

I

-0

Single cells in suspension: process for staining

Collagenase-treated fragments 1. Resuspend fragments in 0. 1 % col lagenase, 0.05% trypsin, 0.02% EDTA, 0.1% BSA in CMF. 2. Shake (130 cycles/min, 37 C) for 8 min.; add 0.2 ml of DNase solution (5mg/mi CMF) per flask and shake for an additional 2 min. 3. Remove tissue from suspension by centrifugation at 1000 x g for 30 sec. I

Single cells in suspension 1. Dilute cells with equal volume of cold MEM, 10% FCS. 2. Centrifuge at 500 x g for 10 min (4 C). 3. Resuspend cells in MEM, 0.1 % BSA; remove aliquot for staining and place remainder of cells on ice.

Trypsin-treated lung fragments 1. Repeat trypsinization step for 8 min. 2. Add DNase as above.

Single cells in suspension 1. Dilute with 3 parts cold MEM, 10%

FCS. 2. Pour through a fine mesh nylon screen. 3. Centrifuge and resuspend cells as in preceeding step; remove cell aliquot for staining. 4. Combine remaining cells with those on ice from the first trypsin digestion.

Enrichment of cell types by separation at unit gravity 1. Load 9 x 106 to 10 x 106of combined cell suspensions resulting from trypsin digestion of lung tissue. 2. Allow to separate into discrete bands (1.25 hr) at room temperature. 3. H-arvest 10-rm fractions. 4. Process cells for staining or electron microscopy.

TEXT-FIGURE I-Outline of method used for dissociation of rabbit lung.

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The latter was determined to be more reliable in assessing cell damage, since we have found frequently that populations of cells which exclude dye to a high degree (98%) sometimes contain numerous cells in the process of dying when studied in ultrathin sections. However, for this study dye exclusion was used routinely for determining the number of cells that were "intact," ie, those cells possessing membrane integritv sufficient for the exclusion of solutes such as trypan blue, according to the procedure of Phillips.1' Electron microscopy was used as a means of evaluating membrane integrity, cytoplasmic edema, and mitochondrial swelling, which were deemed critical in estimating the degree of injury incurred by cells during all stages of tissue dissociation. Liht Mr

Dispersed populations of fixed lung cells and paraffin sections of intact tissue were stained by the Herlant's tetrachrome procedure."' This method offers a high degree of differentiation among the different cell types comprising lung and has permitted us to use light microscopy for identification of isolated Type 1 cells with reference to similarly stained intact tissue. Cells (1 X 10') in MEM/0.1% BSA (0.2 ml/slide) were concentrated onto glass slides by a Shandon cytocentrifuge and immediately placed into BouinHollande fixative for 24 hours." Duplicate slides were prepared for each of the cell suspensions resulting from dissociation and gradient cell enrichment. For comparison with cells in situ, intact tissue was removed from air-inflated lung and fixed in Bouin-Hollande fixative for 1 week. Fixed lung was then rinsed in saturated aqueous LiCO3 and embedded and sectioned according to conventional techniques. On embedding lung samples, sections were taken through a descending series of toluene and alcohol (to 70%). The sections were placed in 1% iodine/70% alcohol for 5 minutes before proceeding with the remainder of the procedure used for staining of dispersed cells. Differential counts of stained cells were obtained by counting 1000 cells on duplicate slides from each experimental series. Electro M'cocp

Whole lung tissue from norrnal animals and fragments obtained during dissociation of lung were placed in a drop of cold fixative on a paraffin sheet and minced into 0.5-cu-mm fragments using a degreased razor blade. Fixation with cold (4 C) 3.0% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) was allowed to proceed for 2 to 3 hours, after which the fragments were rinsed in additional buffer. Fragments were postfixed with 2.5% osmium tetroxide in 0.2 M sodium cacodylate, pH 7.2, for no longer than 3 hours. The tissue was dehydrated and cleared in graded alcohol followed by propylene oxide and embedded in Araldite-Epon. Blocks were trimmed on an LKB Pvramatome and sectioned with an LKB Ultratome. Sections were examined with a Siemens 102 electron microscope. Cells obtained from gradient fractions following sedimentation at unit gravitv were centrifuged into BEEM capsules, fixed, and processed according to procedures described by Rosenbaum and Picciano.4 In addition to cutting ultrathin sections by the above method, 0.2-Mu sections of identical material were prepared to provide "thick" sections for light microscopic visualization. In this way, preservation of isolated cells and monitoring of the dissociation procedure was achieved. Thick sections were stained with toluidine blue 0 and examined with phase microscopy.

Results Dissocation of LuM rTm.

The dissociation of lung tissue can be conveniently divided into three phases: initial treatment with trypsin by perfusion, digestion of fragment

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interstitium by collagenase, and dispersal of tissue with trypsin to form a heterogeneous suspension of single cells. The effectiveness of perfusion with trvpsin as a primary step toward the complete dissociation of lung may be seen in Figures 1 and 2, which depict cross sections through rabbit lung septums. The action of this enzyme was presumed to "loosen" alveolar cells from their respective basement membranes without disturbing intercellular junctions. There were, however, early signs of separation of cytoplasm at many points between adjacent Type 1 cells. Virtually all cells of alveoli appeared intact, although the apical surface of some Type 1 cells showed a slight folding (corregation). Many mitochondria of Type 1 cells stained deeply with osmium tetroxide, indicating an increase in osmiophilia. This effect was due, possibly, to matrix changes, which in turn may be caused by changes associated with altered metabolism, such as seen with loss of phosphorvlative control.2'21 The cytoplasm of Type 1 cells characteristically exhibited frequent packets of free ribosomes interspersed among numerous pinocytic vesicles and fragments of rough endoplasmic reticulum. Moreover, interstitial changes appeared to be minimal since, as a whole, the interstitium was intact and collagen bundles were comparable to those seen in sections of in situ lung. Further breakdown of lung tissue into component cells was achieved by treatment of tissue fragments with collagenase. Immediately following perfusion with trypsin, well-perfused areas (which were less resilient than untreated or poorly perfused tissue) were easily fragmented mechanically by passing through a Wintrob needle. This step served to increase the accessibility of collagenase to interstitial collagen. Following 15 minutes of collagenase digestion, the basic effects brought about by trypsin perfusion were exaggerated (Figure 3). The most significant effect on lung induced bv collagenase treatment was the breakdown of the interstitium. Some collagen framework remained, but masses or bundles of collagen could not be detected. While Type 1 cells still retained respective intercellular junctions, basement membranes were further removed. This latter effect was most probably a secondary response to the mechanical action of shaking during incubation and the interstitial breakdown due to collagenase, as well as the tryptic activity in preparations of "Type 2" collagenase. At this stage, it was possible to find dead or dying cells as marked bv the loss of internal organization (Figure 3). Following incubation with collagenase for 30 minutes, a marked dissolution of the interstitium was evident. Cells were free of their basement membrane and existed mainly as clumps, many of which consisted of Tvpe 1 cells only or of Type 1 and Type 2 cells (Figure 4). These cells maintained their intercellular junctions. At this stage, mitochondria of

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Types 1 and 2 epithelia appeared to have recovered, no longer demonstrating increased osmiophilia. Typical of Type 1 cells following removal from lung by collagenase, however, was a surface blebbing phenomenon (zeiosis) by which they were easily recognized. To our knowledge, no other cell in lung behaves in this wvay following enzymatic isolation. Details of Type 1 cell blebbing are considered in a subsequent paper.14 Cell clumps thus achieved were dissociated into single cells by exposure to trxpsin-EDTA. Intercellular junctions wvere broken to produce a heterogeneous population of lung cells, wshich was further separated into enriched subpopulations by sedimentation of cells through 3 to 6% Ficoll in MEM at unit gravitv. Cell Preservation

In our hands, viability of suspensions rich in Ty-pe 1 pneumocytes derived from final trvpsinization was routinely 855%, as indicated bv trvpan blue dve exclusion methods. Ultrastructural examination of this material before and after Type 1 cell enrichment at unit gravity demonstrated cells which appeared normal. Signs of reversible injury, however, could be detected in some Type 1 cells, including cytoplasmic vacuolization, hydropic swvelling of endoplasmic reticulum, and mitochondrial swelling (Figure 3). Identfication of Isolated Cells

Followving isolation of cells from lung, Ty-pe 1 pneumocvtes w-ere recognized by light microscopy based on cell surface phenomena and specific staining characteristics. As mentioned above, Type 1 cells wvere particularlv subject to zeiosis, resulting in an irregular cell outline and frequentlyan irregular cell shape (Figure 4).14 Type 1 cells, wvhen stained according to Herlant's tetrachrome procedure,19 w,ere recognized by red-purple. round nuclei offset by blue-green cytoplasm. Such staining patterns wvere confirmed by similarly stained Type 1 cells in situ in paraffin sections. Other cell tvpes from lung, such as white blood cells and lymphocytes, were easily recognized by similar morphology and staining criteria as seen in conventional blood-smear preparations. Red blood cells stained yellowvorange. Macrophages were observed as possessing pink nuclei -ith lightgreen cytoplasm in situ as well as in cx-tospin preparations of macrophages obtained by pulmonary lavage. Macrophage nuclei wvere eccentrically positioned and either oval or concave. Numerous vacuoles were randomly distributed throughout the cytoplasm, and the overall shape of these cells was round. Type 2 pneumocy-tes were observed as possessing light-blue, round

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nuclei eccentrically positioned in deep-blue cytoplasm. Inclusions (cytosomes) were frequently seen in regular array within the basophilic cytoplasm. The Type 2 cell was round to "clam-shaped." Reco of Cls From Lung

The removal of macrophages from lung by pulmonary lavage prior to dissociation of lung into single cells was essential for eventual enrichment of Type 1 cells by gradient sedimentation. The number of cells which may be layered onto a gradient for 1 X g separation cannot exceed a critical limit;22 therefore, the starting cell population must contain as many Type 1 cells as possible. Employing the procedure described in Materials and Methods, 4 X 106 to 5 X 10" cells/g (wet weight) of lung were routinely recovered from lavage fluids. Greater than 90% of this yield was macrophages, whereas approximately 2 to 3% was blood cells. The remainder of cells could be identified as airway epithelium (eg, ciliated epithelium). Numerical data pertaining to the release of Type 1 cells during the dissociation of lung tissue are found in Table 1. Duplicate cytospin pellets of cells recovered from each of the stages of tissue digestion (Text-figure 1) were prepared and stained as described in Materials and Methods. One thousand cells per slide were counted and categorized as RBC, WBC, lymphocytes (collectively referred to as blood cells), macrophages, Type 1 cells, or Type 2 cells. For the purpose of this study all other cells were categorized as "other," pending further clarification of the tinctorial response to the dye baths by this heterogeneous group of cells. The data are expressed as percent cell type based on the number of total cells counted or on the number of total nonblood cells counted (since the number of blood cells recovered from MEM, collagenase, and trypsin washes varies greatly from one rabbit to another). The percent distribution of nonblood cell types recovered based on nonblood cells counted at each stage of dissociation is depicted in Text-figure 2. These results were essentially duplicated with four experiments. It can be seen from these data that Type 1 cells were released in greatest numbers, as were other cells, by trypsin digestion. Conversely, blood cells, macrophages, and Type 2 cells were primarily recovered from the pretrypsin MEM and collagenase washes. When expressed as percent cumulative recovery of specific cell types at various stages of digestion (postperfusion) (Table 2), this trend was confirmed. Seventy-eight percent of the blood cells, 85% of the macrophages, and 73% of the Type 2 cells released from lung fragments were recovered from MEM and collagenase washes. However, only 23 % and 41 % of Type 1 and "other" cells, respectively, were released prior to final treatment with trypsin. Conversely,

Vol. 90, No. 1 January 1978

ALVEOLAR LINING CELL ISOLATION

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Figure 3-Electron micrograph montage of alveolar cells from a trypsin-perfused rabbit, with lung fragments exposed to collagenase (15 min). Many Type 1 cells (arrows) have become free of the interstitium (int), but intercellular junctions remain intact at this stage of the digestion procedure. At several sites, these intercellular connections have started to rupture, a process completed with final trypsinization. Two dying cells are marked by asterisks and show swelling, disruption of their sparse mitochondrial population, and loss of intranuclear organization. as = alveolar space. (X

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Figure 4-A cluster of attached Type 1 and 2 epithelial cells following trypsin perfusion and in vitro collagenase treatment (30 min) but not final trypsin treatment. Released from their basement membrane, such clusters still retain intercellular junctional complexes (arrows) that will be broken with a subsequent exposure to trypsin. The margins of the Type 1 cell have begun to develop surface blebs, a characteristic following release from the interstitium by collagenase. T2 = Type 2 epithelial cell. (x 9000)

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Figure 5-A Type-i-cell-enriched unit gravity gradient preparation from a heterogeneous rabbit lung cell suspension. Of the approximately 16 cells or parts of cells shown, all but 4 can be identified as Type 1. Ready identification can be made by means of the cytoplasmic surface shown either with blebs or more irregular vesicular shapes. m = macrophages, x = unidentified cells, d1 = dying Type 1 cell with typical electron-dense nucleus and fine membrane fragments. (x 5000)

The type 1 alveolar lining cells of the mammalian lung. I. Isolation and enrichment from dissociated adult rabbit lung.

The Type 1 Alveolar Lining Cells of the Mammalian Lung I. Isolation and Enrichment From Dissociated Adult Rabbit Lung Paul Picciano, PhD, and Robert M...
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