clonal growth of epithelial cells from normal adult human … · rested swiss 3t3 mouse feeder...

[CANCER RESEARCH 41, 2294-2304, June 1981]0008-5472/81 /0041-OOOOS02.00

Clonal Growth of Epithelial Cells from Normal Adult Human Bronchus

John F. Lechner,1 Aage Haugen, Herman Autrup, Irene A. McClendon, Benjamin F. Trump, and Curtis C.

Harris

Human Tissue Studies Section, Laboratory of Experimental Pathology, National Cancer Institute, Bethesda 20205 Â¡J.F. L., A. H., H. A., I. A. M., C. C. HJ, andDepartment oÃPathology, University of Maryland, Baltimore, Maryland 21201 [B. F. J.]

ABSTRACT

Normal primary epithelial cell cultures devoid of fibroblasticcells have been developed from tissue expiants of adult humanbronchi. Conditions for clonal growth of secondary cultures ofbronchial epithelial cells were optimized by coculturing thehuman cells with mitomycin C growth-arrested Swiss 3T3

mouse feeder cells, lowering the calcium concentration ofmedium M199, and supplementing it with hydrocortisone, insulin, cholera toxin, epidermal growth factor, and 1.25% fetalbovine serum. The epithelial cells grew for an average of 35population doublings and had the normal human karyotype,expressed keratin and blood group antigen epithelial cellmarkers, metabolized benzo(a)pyrene, and were capable ofdifferentiating into both ciliated and squamous cells. This culture system makes it potentially possible to investigate variousaspects of differentiation and carcinogenesis in human bronchial epithelial cells.

INTRODUCTION

Most human malignant neoplasms are of epithelial origin.Thus, the need to develop techniques for culturing normalhuman epithelium to study carcinogenesis has been emphasized repeatedly (4, 7). Our laboratory has been using humantissues for carcinogen metabolism (8) and tissue-mediated

mammalian cell mutagenesis (12) studies. In addition, bronchialepithelial cells in primary culture have been characterizedextensively (15, 27). This study was undertaken to developreproducible methods for obtaining fibroblastic cell-free repli

cative epithelial cell cultures capable of being subculturedseveral times. Here we report these methods. In addition, thispaper describes optimized conditions for their clonal growth.The monolayer cultures exhibited many of the characteristicsnormally ascribed to human bronchial epithelial cells includingthe ability to metabolize B(a)P2 and to differentiate into ciliated

and squamous cells.

MATERIALS AND METHODS

Cultures and Growth Media. Human bronchial tissue wasobtained from either immediate autopsy or surgery sources

' To whom requests for reprints should be addressed.2 The abbreviations used are: B(a)P, benzo(a)pyrene; CT, cholera toxin; EGF,

epidermal growth factor; HC, hydrocortisone; ISN, insulin; FBS, fetal bovineserum; PET, 0.1% trypsin:0.02% ethylene glycol bisQS-aminoethyl ether>-W.W.A/'.A/'-tetraacetic acid:1% polyvinyl-pyrrolidine prepared in 4-(2-hydroxy-ethylH-piperazine-ethanesulfonic acid-buffered 0.9% NaCI solution; FN, (human) fibronectin; DMEM, Dulbecco's modified minimum essential medium; ECGS,

endothelial cell growth supplement; MSA, multiplication-stimulating activity.

Received November 19, 1980; accepted March 10, 1981.

(30). Normal-appearing specimens were immersed in L-15

culture medium and transported on ice to the laboratory. Lungtissue was trimmed away, and the bronchus was cut into large(2- x 3-cm) fragments. To facilitate removal of mucus and to

promote reversal of ischemie damage (29), these pieces werenext placed in culture, epithelial side up, according to procedures detailed previously (8, 26, 27). These cultures wereincubated at 36.5Â°; the medium was changed every other day.

After 3 to 5 days in culture, the bronchial tissue was cut witha scalpel into smaller (0.5-sq cm) pieces, placed epitheliumside up as expiant cultures (4 to 5 explants/60-mm dish), and

incubated in growth medium prepared without CT. Growthmedium consisted of modified M199 medium [modified bylowering the calcium concentration to 0.6 ITIMand adding 20m.M4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 20 ngEGF per ml, 10 ng CT per ml, 5 X 10~7 M HC, 0.2 fig ISN per

ml, trace elements (16), 50 fig gentamycin per ml, and 1.25%Sephadex G-10-filtered FBS (13)]. The final concentration ofeach trace element was: 3 X 10~5 mM H2SeO2; 1 x 10~6 mMMnCI2 4H2O; 5 x 10~4 mM NaSiO3 9H2O; 1 x 10~6 mM(NH4)6M07O24 4H2O; 5 x IO'6 mM NH4VO3; 5 x 1Q-7 mMNiSO4 6H2O; and 5 X 10~7 mM SnCI2 2H2O. This medium was

replaced with fresh medium every 3 to 4 days. After 8 to 11days of incubation, epithelial cells radiated outward on thesurface of the culture dish from the tissue more than 1.5 cm.The tissue pieces were then transferred to new culture dishesto reestablish primary cultures, and mitomycin C growth-arrested Swiss 3T3 mouse feeder cells (3 x 105/60-mm dish)

(23) were added to the outgrowth cultures. These cocultureswere incubated in growth medium (containing CT) for an additional 3 to 5 days and then subcultured. The feeder cells werefirst removed by vigorously pipeting the cultures after they hadbeen incubated for 1 min with 0.05% crystalline trypsin:0.01 %ethylene glycol bis(/?-aminoethyl ether)/V,A/,W,A/'-tetraaceticacid: 0.5% polyvinylpyrrolidine prepared in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered 0.9% NaCI solution(16). The bronchial epithelial cells were subsequently dissociated into single cells by incubation in PET for 5 to 10 min atroom temperature. Dissociated bronchus cells were washed,pelleted by 5 min of centrifugaron (125 x g), resuspended ingrowth medium, and replated in FN-coated dishes at variouscell densities. One to 2 hr later, 3 x 105 feeder cells were

added to the cultures. Growth medium was replaced after 1day of incubation and, subsequently, biweekly. FN coating wasaccomplished by dissolving FN in L-15 medium and adding 50jug/60-mm dish. After 2 to 5 hr of incubation at 36.5Â° for

absorption, the supernatant was removed.Peptide growth factors and FN were obtained from Collabo

rative Research, Inc., Waltham, Mass.; CT was purchased fromSigma Chemical Co., St. Louis, Mo.; ISN was a gift of the Eli

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Human Bronchial Epithelial Cells in Culture

Lilly and Co., Indianapolis, Ind.; and HC was obtained fromSteraloids Inc., Wilton, N. H. Media were prepared by the NIHMedia Unit.

Several cell types other than human bronchial epithelial cellswere used in this study. Human bronchial fibroblastic cells,HBF 310, were-developed from expiants grown in PFMR-4

medium (16) containing 10% FBS. Swiss 3T3 mouse andhuman squamous carcinoma (SCC-15) cells, gifts of Dr. J.

Rheinwald, Sidney Farber Cancer Institute, Boston, Mass.,were grown in DMEM supplemented with 10% bovine serumand M199 containing 7% FBS, respectively. Human prostaticcarcinoma cells, PC-3 (14), were obtained from Dr. M. E.

Kaighn, Pasadena Foundation for Medioal Research, PasadenaCalif., and grown in PFMR-4 medium containing 1% FBS.Human bladder carcinoma, HT-1376, human embryonic lungfibroblasts, IMR-90, human lung carcinoma, A-549, and mouse

BALB/c 3T3 cells were obtained from the American TypeCulture Collection, Rockville, Md., and grown according totheir prescribed protocols (11 ).

Mitomycin C growth-arrested feeder cells were prepared by

first treating a semiconfluent culture for 2 hr with mitomycin C(2 /ig/ml) followed by 2 hr of incubation in DMEM. The cellswere dissociated with PET and washed with growth medium.These cells could be maintained at 4Â°for up to 48 hr prior to

their use as feeder cells without discernible loss of growth-

promoting activity.Clonal Growth Assay. Clonal growth rates (R) were used to

measure the mitogenic potency of putative mitogens. Thesevalues were obtained using methods described previously (17).Colonies were fixed after 8 to 10 days of incubation with 10%formalin and stained with rhodanile blue (23) to differentiatethe bronchial epithelial cells from the 3T3 feeder cells. Themean number of cells per clone in 18 randomly selectedcolonies (6/dish) was determined. To derive R, the Iog2 of theaverage number of cells per clone was divided by the numberof days of incubation.

Enzyme kinetic parameters were used to quantify the influence of mitogen concentration on clonal growth rates. Datawere analyzed using the Lineweaver-Burke transformation ofthe Michaelis-Menten equation using statistically weighed linear regressions of dose-response data (17-19). The theoretical

maximal growth rate (RMAX),the reciprocal of the Y intercept,was defined as the growth rate at infinite mitogen concentration, whereas the mitogen concentration at which half-maximalgrowth rate occurred (KSit09en)was the negative reciprocalvalue of the X intercept. Student's t test (5) was used toevaluate significant differences between the RMAXor KS'l09en

values derived from different experimental groups.Characterization. Measurement of B(a)P metabolic products

was accomplished, using previously published methods (2). Thefeeder cells were thoroughly removed from the epithelial cultures prior to adding fresh growth medium containing B(a)P(1.5 fiM\ 66 Ci/mmol). The cultures were incubated for 24 hr.Media were then harvested for analysis of B(a)P metabolites,and the cells were dissociated with PET, pelleted, and counted.

Epithelial cell cultures were fixed and processed in situ fortransmission and scanning electron microscopy using procedures published previously (21, 27). Mucin-containing cellswere identified using alcian blue-periodic acid-Schiff histolÃ³g

ica! staining (15). Immunoperoxidase staining (15) was used todetect epithelial cell markers, e.g., keratin and blood group

antigens. Chromosomal analysis was performed using standardprocedures (14).

RESULTS

Epithelial Outgrowths from Expiant Cultures. Previouswork (15, 26, 27) had shown that ISN (0.2 mg/ml), HC (5 x10~7 M), and serum (10%) supplements were growth stimula

tory for primary expiant outgrowth cultures of human bronchialepithelial cells. However, fibroblastic cells were commonlyobserved in these cultures, though to a lesser extent whenputrescine was also incorporated into the medium (26). In anattempt to eliminate fibroblast contamination, different serumconcentrations were tested. It was found that reduction in theserum level from 10 to 2% inhibited fibroblastic cell growthwithout significantly affecting the size of the epithelial celloutgrowths. Although mitotic epithelial cells were seen in serum-free media (M199 with trace elements), reduction in the

serum concentration to less than 1% markedly reduced thesize of the outgrowths. Therefore, 1.25% (0.4 mg serum proteinper ml) serum supplementation was chosen as an optimalconcentration, since cultures which developed in this mediumwere free of fibroblastic cell contamination, as monitored byboth phase microscopy and rhodanile blue staining.

Growth factor supplementation experiments were undertaken to increase the size of the epithelial cell outgrowthswhich developed after 10 days of incubation in low-serum-

containing medium. Platelet growth factor and fibroblast growthfactor were ineffective at concentrations varying from 0 to 1unit/ml and 0 to 100 ng/ml, respectively, whereas EGGS,MSA, and EGF were stimulatory. Addition of ECGS (100 fig/ml) increased the diameter of the outgrowths approximately2.5-fold. These outgrowths were comprised predominantly ofsmall epithelial cells; large squamoid-appearing cells were rare,

and fibroblastic cells were not noted. Similar results wereobtained with MSA (10 ng/ml). On the other hand, EGF (5 to20 ng/ml) not only increased the size of the outgrowths 2.5-

fold but also increased cell multilayering. Blood group antigensand keratin proteins were detected by immunoperoxidasestaining, and patches of cells with beating cilia were oftennoted. On the other hand, mucus-containing cells were rare

(less than 1%) and were usually localized to the multilayeredregions of the colonies.

Ultrastructural studies (Fig. 1) showed that the predominantcells in the outgrowth populations contained numerous tonofil-

aments, tight junctions, and desmosomes. EGF significantlyreduced the number of desmosomal junctions; the other growthfactors were ineffective in this regard.

The outgrowth cultures continued to divide for more than 3weeks so long as the expiant tissue remained contiguous withthe monolayer portion of the culture. Removal of the expianttissue resulted in rapid damping of mitotic activity followed byan apparent increase in cell area. Four to 5 days later, the cellsappeared to be desquamating, 2 to 3 days later eventuallysloughing into the medium. Similar results were obtained if theexpiant tissue was only moved to another area of the culturedish. Although a new outgrowth of dividing epithelial cellsdeveloped around the expiant tissue, the original culture underwent desquamation.

Repeated transfer of expiant tissue to a new dish was foundto be a good method to initiate additional primary cultures of

JUNE 1981 2295



J. F. Lechner et al.

human bronchial epithelial cells. For example, over a period of10 months, 20 successive cultures, each estimated to havemore than 200,000 epithelial cells at the time of tissue transfer,were established from a single piece of bronchus. Except forcontaminated specimens (15% of the donors), sequential outgrowth cultures from more than 20 donors have been initiatedusing this technique. To date, donor age has not had any effecton the number of times the tissue could be transferred. Tissuesfrom 2 immediate autopsy donors aged 17 and 81 years haveboth been transferred sequentially more than 20 times. Usingkeratin, blood group, and mucin staining and ciliated cells ascriteria, no difference between the primary and 20th epithelialoutgrowth cells were noted.

Subculture and Clonal Growth of Bronchial EpithelialCells. Initially, it was necessary to develop conditions whichmaintained mitotic activity in post-explant transfer cell cultures.Since several media formulations (CMRL 1066, DMEM, L-15,M199, PFMR-4) were found to be unsuccessful, feeder cellcultures were tested. Mitomycin C growth-arrested Swiss 3T3

mouse cells were coincubated with the epithelial cells after theexpiants were transferred. The bronchial epithelial cells continued to divide under these coculture conditions.

Previous attempts to subculture adult human bronchial epithelial cells had been only marginally successful (27). However,these experiments were done without feeder cells. Initially,when feeder cell-cocultured bronchial epithelial cells were

dissociated with PET into single cells and replated (5000 cells/60-mm dish) in M199 medium containing 10% FBS, 18 to 23%

of the cells were observed to reattach as single cells; smallclumps of cells failed to reattach and remained as floatingaggregates. However, the reattached single cells failed to grow.On the other hand, cells plated by the same procedure butcocultured with feeder cells developed into colonies of rapidlydividing epithelial cells. These secondary clonal cultures wereagain subcultured when the colonies contained between 50 to100 cells (9 days; approximately 6 population doublings).Subculturing was repeated 5 times before the cells failed togrow into colonies. Subsequently, every expiant culture frommore than 20 immediate autopsy and surgery specimens wassubcultured sequentially 4 to 5 times at clonal densities. In allcases, feeder cells were necessary for clonal growth. Fibro-

blastic colonies were not seen in any subcultures.Effect of Calcium on Clonal Growth. Recent investigations

have shown that reduction in the calcium ion concentrationincreased the plating efficiency of human epidermal cells (10,20). Therefore, the calcium ion concentration was titrated usingCa2+-free M199 medium supplemented with 2% FBS, trace

elements, ISN, and HC. Although colonies developed in mediaranging from 60 to 1800 fiM Ca2+, highest plating efficiency

and most rapid growth occurred between 200 and 700 /Â¿M(Chart 1). Therefore, the Ca2+ concentration was routinely

adjusted to 600 JUM.The calcium concentration also influencedclonal morphology and cell shape (Fig. 2). In high Ca2+ con

centrations, the cells were small and packed tightly into colonies with discrete borders which demarcated them from the3T3 feeder cells. Progressive reduction of the Ca2+ concentra

tion caused the border between the epithelial and feeder cellsto become less distinct. Furthermore, the epithelial cells wereflatter and less tightly apposed. A decreased number of des-mosomal junctions was observed when the Ca2+ was reduced

to less than 400 /Â¿M(Fig. 3).

Effect of Serum on Clonal Growth. Cell multiplication rateswere dependent upon the serum concentrations at levels lessthan 5%, and more than 10% serum supplement was inhibitory(Chart 2). Lineweaver-Burke analysis of these data showedthat the half-maximal growth rate (KmBSPvalue) was attained

using 0.45 mg serum protein per ml (1.4%).Mitogenic Action of Putative Growth-promoting Agents.

Each growth factor normally incorporated into growth mediumwas assessed. HC or EGF improved both plating efficiency andgrowth rate whereas ISN and FN predominantly increased theplating efficiency (Table 1). Thus, HC, ISN, and EGF wereroutinely added to growth medium, and plates were precoatedwith FN. Other peptide growth factors, e.g. fibroblast growthfactor, MSA, platelet growth factor, and EGGS were tested atconcentrations shown to be mitogenic for other cells (3, 13,16) both in lieu of and in combination with EGF. None wasfound to influence plating efficiency or growth of the bronchialepithelial cells. CT, reported to promote growth of severalhuman epithelial cell types (6), was also tested. Dose-responseexperiments showed that CT was markedly stimulatory (Table1).

The influence of EGF and CT on the serum requirement forclonal growth is shown in Chart 2. Kinetic analysis of these

Â£

0.51.000 2,000

Chart 1. Influence of calcium on plating efficiency and clonal growth rate ofhuman bronchial epithelial cells. Plating efficiencies based upon mean number ofcolonies per triplicate plating; growth rates based upon number of cells per 18randomly selected colonies.

> 1.0

Ã irÃ¯0.9co

Â§0.7

I5 0.6o.OÂ£-0.5

0.4

â€¢â€”â€¢,Serum alone

X-X, Senim plus EGF

0â€”0, Serum plus EGF and CT

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 1.5 3.0

FETAL BOVINE SERUM PROTEIN, mo/ml

I I I,..

1.0 2.0

FETAL BOVINE SERUM, %

Chart 2. Influence of EGF and CT on clonal growth rate of human bronchialepithelial cells as a function of serum concentration: serum alone; serum plus 20ng EGF per ml; serum plus 20 ng EGF and 10 ng CT per ml. Growth rates basedupon number of cells per 18 randomly selected colonies for each point.





data showed that these factors together spared the serumrequirement more than 20-fold (Table 2).

Effect of Feeder Cell Type and Density on Clonal Growth.Clonal plating efficiency and growth rates of human bronchialepithelial cells were compared directly using different culturesof mitomycin C growth-arrested feeder cells (Table 3). The

Swiss 3T3 mouse feeder cells were superior althoughBALB/c 3T3 mouse, isogenic human bronchial epithelial (HBE310), and human prostatic carcinoma (PC-3) feeder cells allsupported clonal growth but were less efficient. All the other

Table 1

Effect of growth factors on plating efficiency and clonal growth rate of humanbronchial epithelial cells

GrowthmediumWithout

CTWithoutCTonlyWithout

FN"Without

ISNWithoutHCWithout

EGFWith

CT0.1ng/ml1ng/ml10ng/ml100

ng/mlPE"4.01.91.30.41.86.010.015.014.0R0.740.650.58e0.38e0.45e0.790.890.970.94ea PE, plating efficiency (number of colonies + number of cells inoculated x

100); R, growth rate as number of population doublings per day." FN, 50 (ig/60-mm dish; ISN. 0.2 fig/ml; HC, 5 X 10~7 M; EGF, 20 ng/ml.

Using Student's f test (p < 0.05), comparing value with result using growth

medium without CT.

Table 2Effect of EGF and CT on Kâ„¢SPand RuifSP values with human bronchial

epithelial cells

AdditionsNone

EGF0EGF + CT8Kâ„¢spa

(mg serum

protein/ml)0.45(0.12)"

0.31 (0.07)"0.02(0.01)"RiÃx8"

(population

doublings/day)0.67

(0.08)0.96(0.13)"1.02(0.10)"

K"SP, half-maximal growth rate; RT'FBSP,theoretical maximal growth rate;

values derived by extrapolation using statistically weighted least-squares regressions.

" Numbers in parentheses, S.E.cdAt 20 ng/ml.

Significance (p < 0.05), comparing value with result with no additives usingStudent's t test.

8 At 10 ng/ml.

Table 3

Effect of feeder cell type on plating efficiency and clonal growth rate of humanbronchial epithelial cells

Each 60-mm plate was inoculated with 5000 human bronchial epithelial cells,HBE 310, and 3 x 105 feeder cells.

FeedercellSwiss

3T3BALB/c3T3HBE310HBF

310ePC-3A-549SCC-

15HT1376IMR-90PE"18.53.25.9<0.021.8<0.02<0.02<0.02<0.02R1.080.7060.846NC0.73"NCNCNCNC

PE, plating efficiency (number of colonies â€¢*number of cells inoculated x

100; mean of triplicate platings); R, growth rate (number of population doublingsper day, based upon mean number of cells per 18 colonies); NC, no colonies.

Significance (p < 0.05), comparing value with result using Swiss 3T3 mousefeeder cells using Student's f test.

c Human bronchial fibroblastic cells, isogenic to human bronchial epithelial

cell.

feeder cell cultures failed to support clonal growth of thebronchial epithelial cells.

Dose-response experiments were used to compare thefeeder cell activities of Swiss 3T3 and HBE 310 cells (Chart 3).Kinetic analysis of these data showed that the half-maximalcolony-forming efficiency was 5-fold greater using Swiss 3T3feeder cells. Further, both the theoretical maximal colony-form

ing efficiencies and theoretical maximal growth rates weresignificantly less using HBE 310 feeder cells (Table 4).

Conditioned Media. Conditioned media (6, 31) were testedas a means to eliminate the feeder cell requirement for clonalgrowth of the bronchial epithelial cells. Semiconfluent Swiss3T3 and PC-3 cultures, as well as human bronchial, HBE 310,

expiant cultures, were incubated for 2 days in growth medium.These media were harvested subsequently, filtered, and testedimmediately in FN-coated 60-mm dishes inoculated with 5000

epithelial cells. These conditioned media were used both at fullstrength and diluted 1:3 with fresh growth medium and supplemented with CT (10 ng/ml), EGF (20 ng/ml), and serum (2 to20%). None of these conditioned media supported clonalgrowth of the bronchial epithelial cells.

Inasmuch as the putative soluble feeder cell growth factor(s)might be unstable, epithelial cells were inoculated at low density (250 cells/sq cm) into the central portion of FN-coated 60-mm dishes and surrounded by a ring of 3 x 106 Swiss 3T3

mouse feeder cells. Although these plates were incubated in arocking chamber to improve diffusion of any putative factors,no epithelial colonies appeared.

Characterization of the Replicative Epithelial Cells. Numerous criteria were used to characterize the replicative humanadult bronchial epithelial cultures using the standardized conditions. Scanning electron microscopic observations showedcolonies comprised of cells covered with various numbers ofshort microvilli; the cell borders were apposed (Fig. 4). Ultra-

structural studies showed that the cells had features identicalto those described for the expiant outgrowth cultures. The cellsfailed to grow in soft agar (16) at a density of 100 cells/cumm, and chromosomal analysis showed that the cells retainedthe normal human karyotype (2n = 46) throughout the replicative phase. Epithelial cell markers, i.e., keratin and donor-

specific blood group antigen (15), were detectable in bothearly- and late-passage clonal cultures.

Mucin-stained cells were detected rarely and only in oldermultilayered colonies. /3-Retinol acetate in doses varying from10~9 to 10~6 M failed to affect either clonal growth rate or the

number of mucin-stained cells. Patches of ciliated cells were

seen commonly in cultures maintained for more than 50 days.To demonstrate this, 3 x 103 secondary-passage epithelialcells were initially inoculated with 3 x 105 feeder cells and

incubated in growth medium. Feeder cells were changed atweekly intervals. Three to 4 weeks after inoculation, mitoticactivity ceased. After an additional 3 weeks, ciliated cells werenoted; their numbers increased with continued incubation (Fig.5). This phenomena was noted in cell cultures developed fromearly as well as 17th-explant transfer outgrowth cultures.

B(a)P metabolites were quantified for both bronchial epithelial and isogenic bronchial fibroblastic cells. The rate of metabolism per epithelial cell (0.77 pmol/106 cells/24 hr) was morethan 2.5-fold greater than per fibroblastic cell (0.28 pmol/106

cells/24 hr). The spectrum of water-soluble metabolites (sulfate esters, glucuronides, and glutathione conjugates) re-

JUNE 1981 2297




covered from the epithelial, fibroblastic, and tissue cultures didnot differ significantly. However, the epithelial cells produceda greater percentage of organosoluble metabolites (Table 5).

0.610.000 30,000 100,000 300,000

FEEDER-CELLS PER 60mm DISH

Chart 3. Comparison of mitomycin C growth-arrested feeder cell type as afunction of number of cells per 60-mm dish on colony-forming efficiency andclonal growth rate of human bronchial epithelial cells. Top. human bronchialepithelial feeder cells; bottom, Swiss 3T3 mouse feeder cells. Colony-formingefficiencies based upon mean number of colonies per triplicate platings; growthrates based upon number of cells per 18 randomly selected colonies for eachpoint.

Table 4Differential effect of feeder cell type on colony-forming efficiency and growth

rate of human bronchial epithelial cells, HBE 310

HBE 310 celts (5000) were initially inoculated. Two hr later, one half of theplates received various concentrations of mitomycin C growth-arrested Swiss3T3 mouse feeder cells, whereas the other half received various concentrationsof mitomycin C growth-arrested HBE 310 feeder cells. Thus, attachment of cellsto the dish was not a differential factor in the experiment.

cell3T3

HBE 310Colony-forming

efficiencycppl-tMder

cella18.5(1.2)"5.9

(0.5)KS-"-51.8(6.1)130.8(20.2)Growth

rateCFEB5Â»-1.09(0.19)

0.84 (0.27)K!T"

c"'8.3(1.1)

9.2 (3.5)

CFEÃ¼ÃAx*1"'"". theoretical colony-forming efficiency; values derived by extrapolation using statistically weighted least-squares regressions; KIT*** c<-l,number of feeder cells ( x 103) per 60-mm dish at which half-maximal effect occurred;R MAT"" ce". theoretical maximal growth rate; values derived by extrapolation using

statistically weighted least-squares regressions.6 Numbers in parentheses, S.E.

Table 5

Organosoluble metabolites of B(a)P formed by cultured bronchial cells andtissue

MetabolitesOrganosoluble

(%)7,10/8,9-Tetrol7,9/8,10-Tetrol7,9/1

0/8-Tetrol7,9,10/8-Tetrol7/8,9-Triol

andfrans-9,1O-Diolfrans-4,5-Diolfrans-7,8-Diol3-Hydroxy9-HydroxyQuiÃ±onesUnidentifiedEpithelial

cells826.22.8"16.22.60.844.90.4S.I0.91.5NO24.6Fibroblasticcells18.13.8d27.21.70.750.60.14.8NO"1.1ND9.2Tissue027.29.1d6.45.73.530.30.33.50.33.61.233.2

' HBE 310 bronchial epithelial cells.b HBF 310 fibroblastic cells, isogenic to HBE 310.c Cultured HBE 310 tissue.d Percentage of organosoluble metabolites.8 Not detectable.

DISCUSSION

This study shows that replicative epithelial cultures free offibroblastic cell contamination can be routinely establishedfrom normal adult human bronchial tissue. In addition, clonalgrowth of these epithelial cells was accomplished. These cultures exhibited significantly enhanced growth and longevitycompared with previously published reports (27). These cultures were comprised of normal epithelial cells as defined bytheir exhibition of many of the characteristics commonly ascribed to normal bronchial epithelial cells. The application ofseveral techniques went into the development of these culturemethods, including: initiating expiant cultures in low-serum-

supplemented medium; cocultivating epithelial outgrowth cultures with mitomycin C growth-arrested Swiss 3T3 mousefeeder cells; repeatedly initiating replicative cultures from asingle piece of bronchial tissue by sequential transfer; utilizingfeeder cell cocultivation to induce clonal growth; and usingdose-response experiments quantified by enzymatic kinetic

analysis procedures to optimize growth conditions.Procedures for growing replicative cultures of a few types of

normal human epithelial cells have been published (6, 10, 16,20, 22-25, 28, 31, 33). However, reports describing theirclonal growth are rare. Growth-arrested feeder cells are not

required for clonal growth of all normal human epithelial celltypes (16, 20). However, this technique has been successfullyapplied previously for clonal growth of keratinocytes (6, 23)and breast (28) and cervical epithelial cells (25). Under theconditions tested, bronchial epithelial cells showed a feedercell requirement for growth. The composition of nutrient medium markedly influences serum and growth factor requirements and vice versa (3, 20). Thus, the growth responses (andfeeder cell requirement) observed when bronchial epithelialcells were cultured in modified M199 medium might havediffered markedly if other nutrient media had been used. Forexample, human skin keratinocytes can be grown at clonaldensities without feeder cells using an optimized-nutrient medium (20). However, the data suggest that contact between thebronchial epithelial cells and feeder cells might be necessaryfor growth-promoting activity. Neither conditioned media nordouble cell plating without cell-cell contacts supported clonal

growth. The pseudopodial overlapping of the epithelial cells bythe feeder cells seen in scanning electron microscopic (Fig. 4,insert) photographs further corroborates this suggestion.

Whereas several types of cells could serve as feeder cellsfor breast (28) and cervical (25) epithelial cells, the feeder cellrequirement of the bronchial cells was highly specific. Swiss3T3 mouse feeder cells were 5-fold more efficient than the

epithelial feeder cells for colony formation but equally efficientin supporting clonal growth. This suggests that there may be 2aspects to the feeder cell phenomenon: (a) that contact withthe 3T3 feeder cell may stimulate single epithelial cells to divideenough times until (b) the colony contains sufficient numbersof cells to "cross-feed" itself.

Improved growth conditions with low-serum supplement andfeeder cells were developed using clonal-growth dose-response experiments. Based upon these experiments, the Ca2+

concentration was reduced to a concentration similar to thatfound optimal for human skin keratinocytes (10, 20). FurtherISN, HC, EGF, and CT supplementation of reduced-Ca2+ M199

medium were all found to be stimulatory. When this supple-





mented medium was used in conjunction with FN-coated

dishes, the serum requirement for clonal growth was markedlyreduced to a concentration similar to that reported for otherhuman epithelial cell types using optimized conditions (16, 20).

Sequential expiant transfer has been used to reestablishoutgrowth cultures from both bronchial (27) and prostatic (32)tissue. However, successful outgrowths from more than 20sequential transfers have not been reported previously. Sincedonor age does not appear to influence the number of expiantoutgrowths, the 3-dimensional configuration of the expiant, in

which epithelial and stromal contacts and relationships areretained, may be important in maintaining a pool of replicativeepithelial cells necessary for repeatedly initiating a large number of outgrowth cultures. Nonetheless, over a period of 1year, a 0.5-sq cm expiant may provide 20 cultures, eachconsisting of a population of 200,000 epithelial cells which inturn can be stimulated to undergo 20 to 30 population doublings before division ceases.

The epithelial cells exhibited several features ascribed tonormal bronchial epithelium. Initiating these cultures in low-serum-containing media supplemented with specific growth

factors may have contributed to expressing their differentiationpotential (1). Culture conditions influenced their differentiation.Calcium and EGF both modulated the number of desmosomaljunctions. Further, the absence of feeder cells stimulated mul-

tilayering and desquamation. Subcultured epithelial cells wereable to differentiate into ciliated cells. This was noted only inquiescent cultures and took 4 to 6 weeks to be expressed. Thisphenomenon is being investigated further.

Cultures of human bronchial tissue enzymatically activatevarious chemical carcinogens, e.g., B(a)P, into their proximateand ultimate carcinogenic metabolites (2, 8). Cell culturesinitiated from bronchus expiant cultures maintained this abilityat least through the first 3 passages without any loss of totalactivity. Metabolism 2.5-fold higher was found in the epithelial

cells compared to the fibroblast cells from the same donor.Similar results were noted using autoradiography; 4-fold more

grains were located in the epithelium than in the stroma (9).The profile of B(a)P organosoluble metabolites was qualitativelycomparable in all cultures. However, although the percentageof proximate carcinogen (7, 8-diol) was similar, more B(a)Pdiol-epoxide II, as measured by formation of 7,9/8,10- and7,9,10/8-tetrols and 9,10-diols, was formed by the cell cul

tures than by the tissue; phenols and quiÃ±ones were onlyformed in minor amounts.

The features of the in vitro system now developed make itpotentially possible to investigate numerous problems in bronchial cell differentiation and pathobiology, including chemicalcarcinogenesis. Large quantities of human adult bronchial epithelial cells can be obtained readily. Thus, carcinogenesisexperiments designed to use large numbers of cells at risk canbe repeated numerous times with several different agents usingcells derived from the same donor. The cells express thefeatures of normal bronchial epithelium. Finally, they metabolize carcinogens considered to be relevant in the etiology ofhuman lung cancer.

ACKNOWLEDGMENTS

The authors wish to thank Dr. J. Rheinwald, Dr. M. E. Kaighn, Dr. R. G. Ham,Dr. G. D. Stoner. Dr. S. P. Banks-Schlegel, Dr. A. Fornace, and Dr. U. Saffiotti

for valuable suggestions. The technical assistance of W. Pettis, H. Tate, J.Quintero, M. Brugh. F. Jackson, N. Woodside, and P. MeÃ³la and the secretarialassistance of S. Dorfman were appreciated greatly.

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.

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Fig. 1. Electron micrograph of the epithelial cell outgrowth showing the effect of EGF on desmosomes and tonofilaments. A, no EOF; B, 20 ng EOF per ml.X 6500.

2301




%Jâ€¢â€¢Â«â€¢

- *~TÂ¡ f v "

2CFig. 2. Phase-contrast micrograph showing foci of epithelial cells growing with different concentrations of Ca2*; A, 60 Â»IMCa2*; B, 200 JIMCa2*. C, 700 piMCa2*

D, 1800|uMCa2*. x 165.




pl^h /^^H//â€¢t

â€¢>.,â€¢V:,'->"V \. "*â€¢â€¢'V

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Fig. 3. Electron micrograph showing Ca2'-mediated effect on desmosomes and tonofilaments; A, numerous tonofilament bundles and desmosomes are observedat 1800 /IM Ca2*; B, decreased number of tonofilament bundles and desmosomes at 200 Â¡IMÃ‡a2*,x 2000.

2303




Fig. 4. Scanning electron micrograph showing part of an epithelial cell colony (Â£)surrounded by Swiss 3T3 mouse feeder cells (FI. x 2600. InserÃ¬,pseudopodialoverlapping (arrow) of an epithelial cell by a feeder cell, x 3000.

Fig. 5. Scanning electron micrograph of ciliated subcultured epithelial cells observed after 6 weeks incubation, x. 3600.




1981;41:2294-2304. Cancer Res John F. Lechner, Aage Haugen, Herman Autrup, et al. BronchusClonal Growth of Epithelial Cells from Normal Adult Human

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