Serum-free Medium for the in Vitro Growth of Normal and … · Human TCC cell lines, 647V (7), T-24 (5), and J82 (14) (all gifts from Dr. E. Bloom) growing in maintenance (serum-containing)

[CANCER RESEARCH 42. 2392-2397, June 1982]0008-5472/82/0042-OOOOS02.00

Serum-free Medium for the in Vitro Growth of Normal and MalignantUrinary Bladder Epithelial Cells1

Edward M. Messing,2 John L. Fahey, Jean B. deKernion, S. M. Bhuta, and J. Eric Bubbers

Division of Urology, Department of Surgery [E. M. M., J. B. D.], and the Departments of Microbiology and Immunology [J. L. F.], Radiation Oncology [J. E. B.Â¡,andPathology [S. M. B.], UCLA School of Medicine. Los Angeles, California 90024

ABSTRACT

A serum-free medium, DH-S1, is described which is valuable

for the establishment of primary cultures of normal and malignant transitional epithelium (transitional cell carcinoma of thebladder). Growth of epithelial cells in DH-S1 is facilitated but

that of fibroblasts is suppressed. Another established humantransitional cell carcinoma cell line, 647V, has grown continually in DH-S1 for over 36 passages. Morphological and anti-genie studies comparing 647V cells growing in serum-containing and serum-free medium reveal differences which are pro

nounced at low cell density but which almost disappear athigher densities. A new human transitional cell carcinoma cellline, LA-B1, is described whose initial growth was supportedby this serum-free medium.

INTRODUCTION

Tissue culture in vitro has proven invaluable in investigationsof cellular and subcellular mammalian systems. The survivaland growth of cultured cells in media free of serum has longbeen recognized as exceedingly valuable in the study of hormonal responses and metabolic functions because it eliminatesa "complex, undefined and variable" element from such investigations (4). In our studies of TCC3 of the bladder, we have

sought a serum-free medium to aid in investigations of growthfactors and cell surface antigens. A medium, DH-S1, has been

developed by modifications of previously described (4, 20)serum-free media, that has proven useful in supporting the

initial growth of both normal murine bladder epithelium andhuman TCC while suppressing the growth of fibroblasts. DH-

S1 has also supported the continued growth of an establishedhuman TCC cell line, thus aiding our efforts to detect a growth-

promoting factor in the supernatant from cultures of TCC cells.

MATERIALS AND METHODS

Serum-free Medium (DH-S1 )

The composition of DH-S1 is listed in Table 1. We have found that

attention to the following 2 technical considerations greatly facilitatesits production, (a) Hydrocortisone and prostaglandin E, should bedissolved together in 95% ethyl alcohol, divided into 0.05-ml aliquotscontaining 2.5 x 10~8 mol hydrocortisone and 12.5 jug prostaglandin

E,, and stored at -70Â°. (b) Triiodothyronine can be most easily

' This work was supported by USPHS Grants 16880, CA 09120, and CA

12880 awarded by the National Cancer Institute, Department of Health andHuman Services, and by the Concern Foundation of Los Angeles, Calif. 90024.

2 Present address, Division of Urology/Department of Surgery, University

of Wisconsin School of Medicine. 600 Highland Avenue, Madison.Wisconsin 53792.3 The abbrevations used are: TCC, transitional cell carcinoma: HBSS, Hanks'balanced salt solution; DMEM. Dulbecco's modification of Eagle's medium; NCS,

newborn calf serum; FCS. fetal calf serum.Received October 26, 1981 ; accepted March 12,1982.

dissolved by placing it in an alkaline solution (pH 10) and incubating at37" for 2 to 4 hr. We standardly dissolve 3.3 mg triiodothyronine in

100 ml HBSS (Flow Laboratories, Inc., McLean, Va.) to which NaOHhas been added to adjust the pH to 10.5 to 11. Appropriate dilutionsare then performed, and it is stored at -20Â° in 5-ml aliquots which

contain 2.5 mg insulin and 2.5 mg transferrin.

Preparation of Normal Murine Urothelium for in Vitro Growth

Bladders were removed aseptically from young adult female BALB/c mice (The Jackson Laboratory, Bar Harbor, Maine), and the epithelialsurface was excised and cut into pieces of 0.25 sq cm in area asdescribed by Bonar et al. (3). Serial enzymatic disaggregation wasthen performed with 0.3% collagenase type I (Sigma Chemical Co., St.Louis, Mo.)4 and 0.25% trypsin:EDTA solution (Flow Laboratories)(16). Two 1-hr digestions were performed at 37Â°, and supernatants

were pooled. Cells were counted with a hemocytometer, viability wasdetermined by trypan blue exclusion, and cells were plated in 25-sq

cm tissue culture flasks (Falcon Plastics, Cockeysville, Md.) at a densityof 4 to 6 X 104 viable nucleated cells/sq cm, in either DH-S1 orDMEM:Ham's F-12 plus 10% NCS (Grand Island Biological Co., Grand

Island, N. Y.). Cells were refed on the third day with the medium theywere initially plated in. Subsequent feedings (Day 7 and every 3 to 4days thereafter) for both groups of cultures were performed withDMEM:Ham's F-12 plus 10% NCS since those flasks initially containing

DH-S1 appeared to be free of fibroblasts by this time.

Establishment of New Human TCC Cell Lines

Specimens of human TCC were obtained at the time of bladderlavage, transported sterilely in cold 0.9% sodium chloride solution, andimmediately treated by mechanical disaggregation. After centrifuga-

tion, viability and counts were determined, and cells were plated at 1.5to 2 x 10" cells/sq cm in Falcon tissue culture flasks in either DH-S1or DMEMiHam's F-12 plus 20% FCS (Grand Island Biological Co.).

(Originally, NCS was tried, but this has been found to be far lesssuccessful in achieving growth than FCS). After 7 days, all flasks withadherent cells were refed DMEM:Ham's F-12 plus 20% FCS, and

subsequent refeedings continued at the same interval with this medium.Passages were performed by trypsinization and replating in FCS-con-

taining media. Cells for cytological study were grown to near confluence on sterile glass microscope slides in Petri dishes, air dried,stained with May-Grunwald-Giemsa, and examined by Dr. Dorothy

Rosenthal (Cytology Service of the Department of Pathology, UCLASchool of Medicine). Karyotypic analysis was performed by addingColcemid to subcultures when the cell growth was approximately 70%confluent. The subcultures were then returned to the incubator for 6 hrprior to harvesting. The cells were detached from the flask using trypsinand harvested following a modified technique described by Neu (13).The slides were put into a drying oven at 60Â°overnight in preparation

for the banding of chromosomes. The procedure used was a modification of the banding method of Seabright (19). Karyotypes wereprepared and examined by Dr. Richard L. Neu (Bio-Science Labora-

4 N. Hanna. Preparation of tumor cells for in vivo drug sensitivity testing.

UCLA, Department of Microbiology and Immunology Workshop No. 33. May 14to 15, 1981, Los Angeles, Calif.

2392 CANCER RESEARCH VOL. 42

Research. on September 29, 2020. © 1982 American Association for Cancercancerres.aacrjournals.org Downloaded from

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Serum-free Medium for Growth of TCC and Urothelium

Table 1DH-SÃDMEM:Ham's

F-12medium8HydrocortisoneProstaglandin

Ei6Putrescinedihydrochloride"Spermine

tetrahydrochlorideSpermidinetrihydrochloride6

Insulin(bovine)Transferrin(human)63,3'-5'-Triiodo-L-thyronineL-GlutaminecSodium

selenite50:50

(v/v)Sx10"'M25

ng/ml106M4

X 10""M4X10~*M5

/ng/ml5/ig/ml5X1Q-'2M0.292

mg/ml10-"M

Both purchased as liquids from Flow Laboratories, Inc., McLean, Va.' Sigma Chemical Co., St. Louis. Mo.' Grand Island Biological Co.. Grand Island, N. Y.

ALFA Inorganics, Danvers. Mass.

tories, Van Nuys, Calif.). Because of the large number of cells needed,cells for electron-microscopic examination were grown to confluence

and fixed in 2.5% glutaraldehyde and paraformaldehyde solutionbuffered by 0.1 M sodium carbodylate. Culture cells were then carefullyscraped off by means of a rubber policeman and collected. The cellswere then postfixed in 1% osmium tetroxide and pelleted. The specimen was dehydrated and embedded in epoxy resin. One-jum sections

were stained with toluidine blue. Ultrathin sections were stained withuranyl acetate and lead citrate. Electron micrographs were obtainedfrom a Zeiss EM-109 electron microscope.

Growth in soft agar was accomplished by plating a single-cell suspension of trypsinized cells on 35-mm-diameter Retri dishes (Costar,Cambridge, Mass.) containing an underlayer of 1 ml DMEM:Ham's F-

12 plus 15% heat-inactivated PCS + 0.55% agar(Difco Laboratories,Inc., Detroit, Mich.) and a plating layer of 1 ml DMEM:Ham's F-12 plus

15% heat-inactivated PCS + 0.35% agar. The cell concentrations usedwere 5 x 104, 10s, and 5 x 105 cells/plate. Plates were inspected

immediately after plating and then every 3 days thereafter.

Growth of Established TCC Cell Lines in Serum-free Medium

Initial Plating and Maintenance of Cell Lines in Serum-free Medium. Human TCC cell lines, 647V (7), T-24 (5), and J82 (14) (all giftsfrom Dr. E. Bloom) growing in maintenance (serum-containing) medium,were trypsinized, washed 3 times in HBSS (without CA2* and Mg2+),4

counted, and then plated in DH-S1 on Falcon tissue culture flasks at 2x 10" cells/sq cm. When cultures of 647V in DH-S1 reached conflu

ence, the medium was aspirated, the cells were trypsinized, and thereaction was stopped by the addition of an equivalent volume of 0.5%soybean trypsin inhibitor type IIS (Sigma) (4). If flasks were reused,they were washed twice with HBSS prior to replating in order to removeresidual trypsin. Freezing of cells was performed in Nunc freezing vials(Nunc-lnterMed, Roskilde, Denmark) using serum-free freezing medium consisting of 75% DH-S1, 15% anhydrous glycerol (J. T. Baker

Chemical Co., Phillipsburg, N. J.), and 10% dimethylsulfoxide (Sigma).

Frozen cells were stored in liquid nitrogen.Growth Curves. To determine the density and serum dependence

of each line, the following growth counts were performed. Cells wereharvested from serum-containing medium, washed as above, and thenplated in 35-mm-diameter Petri dishes (Costar). Media used were DH-S1 and DMEMiHam's F-12 plus 5 and 10% NCS (FCS for the newly

established line). Plating was performed at densities ranging from 3x 102 to 3 x 10" cells/sq cm at approximately 0.5-log increments on

Day 0. Cells were removed by trypsinization and were counted, andviability was determined on Days 1, 3, 5, 7, and 9. Media wereremoved, and cells were not refed during these experiments.

role of growth factors in the development and maintenance ofTCC, we felt it would be advantageous to have normal transitional epithelial cells. To determine whether normal urotheliumcould be grown in vitro without fibroblast overgrowth, we platedsterilely obtained mouse bladder epithelium after mechanicaland enzymatic digestion as a single-cell suspension in mediawith and without serum. Inspection of short-term (11 to 14

days) cultures of bladder cells has revealed that those initiallyplated in DH-S1 (and refed DMEMiHam's F-12 plus 10% NCS

at 1 week) were essentially free of fibroblasta. In contrast,those plated in serum-containing medium had epithelial cells

but were usually overgrown by fibroblasts. This occurred despite treatment with collagenase prior to plating (Fig. 1). Therefore, DH-S1 does not support fibroblast growth while support

ing epithelial cell growth. Growth beyond 3 weeks and passageof these cells have not been successfully accomplished.

Primary Human TCC Cell Lines. DH-S1 has proven valuablein the establishment of a permanent TCC cell line. Line LA-B1

was started from a bladder lavage specimen obtained from a57-year-old man with Grade III (9) Stage B, (1 2) nonpapillary

TCC. Its in vitro history is summarized in Table 2. After 30passages, a detailed study of the LA-B1 cells was done (Table

3). Karyotypic studies demonstrated that, while some of thecells were diploid and tetrapoid, G-banding revealed numerous

polyploid and aneuploid cells as well with considerable chromosome breakage (not shown). Marker chromosomes such asring, heterochromatic minute, or large subtelocentric, acrocen-tric, and submetacentric chromosomes as described by Sand-

berg (18) were not consistently found. Cytological examinationdemonstrated transitional epithelium with many malignant-ap

pearing cells using the criteria outline by Koss for exfoliatedcells (Fig. 2)5 (10). Transmission electron microscopy showed

an abundance of tonofibrils and junctional complexes (des-

mosomes) (Fig. 3). An asymmetrical unit membrane, as is seenin normal transitional epithelium,6 was not identified. These

cells demonstrated anchorage independence by forming multilayers in tissue culture and colonies in soft agar when platedat the 3 different densities tested. They have also grownsuccessfully after freezing [in Roswell Park Memorial InstituteTissue Culture Medium 1640 (MA Bioproducts, Walkersville,Md.) with 30% NCS and 10% dimethyl sulfoxide] and subsequent thawing.

Growth of TCC Cell Lines in Serum-free Medium (DH-S1).Four human TCC cell lines have been cultured in serum-free

medium (Chart 1). As demonstrated by cell counts at 24 hr, theplating efficiency of all these lines was always considerablyreduced in DH-S1. Only one cell line, 647V, demonstrated

vigorous growth in this medium (Chart 1, a and b). No 647Vcells grew, regardless of the concentration of serum at platingdensities of less than 3 x 103 cells/sq cm (not shown). Line

J82 survived without growth (Chart 1f) while line T24 grewpoorly (Chart 1cO. LA-B1, after over 30 passages in serum-

containing medium, had poor survival when reintroduced toDH-S1 (Chart 1e). T24, LA-B1, and J82 all grew at rates similar

to, or better than, those shown (Chart 1, d to f) in mediacontaining 5 and 10% serum at all plating densities tested (3

RESULTS

Normal Murine Transitional Epithelial Cultures. For ourstudies of both the immunobiology of TCC and the possible

5D. Rosenthal, personal communication.6 J. M. S. Caruthers and M. A. Bonneville. Biochemical and morphological

changes accompanying experimental rat urinary bladder cancer. National Bladder Cancer Project Investigators Workshop, June 7 to 10. 1981. Hershey, Pa.

JUNE 1982 2393



E. M. Messing et al.

Table 2History of TOC cell line. LA-B1

Day O Bladder lavage specimen plated Â¡nOH-S1Day 6 Refed DMEM:Ham's F-12 + 20% FCSaDay 34 First passage6Day 56 Second passage0Day 70 Third passage; subsequent passages every 7 days"Day 77 Cells were frozen in serum-containing freezing media"

Day 102 Subsequent passages every 3 to 7 daysDay 108 Cells frozen Day 77 were thawed and grown in

DMEM:Ham's F-12 + 20% PCS

Day 188 Cells plated in DH-S1 (see Chart 1e)Day 216 Cells plated in clonogenic assay

a The cells have subsequently remained in this medium.b 1:2 split; plated at 3.3 x 105 cells/25-sq cm flask.c 1:3 split; plated at 10e cells/75-sq cm flask." At 5 x 10s cells/75-sq cm flask."Two x 10' cells frozen; remainder of cells continued being passed. More

cells were frozen as the line was expanded.

Table 3Study of TCC cell line, LA-B1

Study Findings

Original tumor

Cell line (30 passages; Day 180)

In vitro growth characteristics

CytologyHistopathology

Cytology

Karyotype

Transmission electron microscopy

Form multilayersColony formation in

soft agar

Highly malignant (10)TCC, Grade III (9), Stage

B, (12)

Many malignant epithelialcells, consistent withtransitional cell origin(10)

Aneuploidy, polyploidy, nodiscrete markers (18)

Epithelial cells with desmo-somes and tonofibrils

YesYes

a See text and Fig. 2 for details.

x 102, 103, 3 x 103, 10", and 3x10" cells/sq cm). None of

these cells grew well Â¡nDH-S1 at any of the densities tested

(data not shown).Characteristics of 647V Cells Growing in DH-S1. Morpho

logical similarities and differences were observed in 647V cellsgrowing in media with and without serum. At low densitiesunder light microscopic examination in DH-S1, these cells

appear somewhat vacuolated with elongated projections (Fig.4a) which are not usually apparent in cells grown in serum-

containing medium at similar densities (not shown). However,as they approach confluence in DH-S1, cellular morphologyand ultrastructure become similar to that in serum-containing

medium (Figs. 4, b and c and 5, a and b).Antigenic comparisons were made of cells grown with and

without serum. Several murine monoclonal antibodies havebeen produced recently in our laboratory to human TCC celllines,7 and some of these react equally to 647V grown in

serum-containing and serum-free medium (at high cell densities) using a 2-layer 126l-labeled Staphylococcus protein A-

binding assay (22). At least 2 of these antibodies appear to beto tumor-associated antigens in that they react with all otherhuriian TCC cell lines tested (Table 4) but not with normal

human tissue obtained at surgery and autopsy (not shown).

' E. M. Messing, J. 6. Bubbers, and J. L. Fahey. Production and characteri

zation of murine hybridoma antibodies against human transitional cell carcinoma-associated antigens. A preliminary report, manuscript in preparation.

KT

,0

IO*

IO5

IO'

O--.

b".-â€¢oâ€”

O 2 IO I2 e sDAYS

IO I2468

DAYS

Chart 1. Growth of TCC cell lines in serum-containing and serum-free media.Cells were plated at indicated densities in DMEM:Ham's F-12 plus 10% NCS(FCSforLA-B1)(B), DMEM:Ham's F-12 plus 5% serum (A), and DH-S1 (O). Cell

counts were performed on indicated days. For details, see text. a. 647V (3 x104 cells/sq cm = 3 x 10s cells/plate); b. 647V (104 cells/sq cm); c. 647V (3x 103 cells/sq cm); d, T24 (3 x 10* cells/sq cm); e, LA-B1 (3 x 10" cells/sqcm); f, J-82 (3x10" cells/sq cm).

Table 4

Reactivity of monoclonal antibodies to TCC cell lines with 647V cells grown inserum-containing and serum-free medium

Reactivity8Hybridoma

supernatant2E1-F3C

9A7-F2647V

+bserum4.5

4.1647V

inDH-S13.74.0J822.3

3.1LA-B13.6 3.4T-243.0 4.4a Reactivity was determined by 2-layer '25l-labeled Staphylococcus protein A

radioimmunoassay (22). Values are the averages of 4 determinations, recordedas the ratio of absolute cpm '25I bound to background (hybridoma growth medium

instead of antibody-containing supernatant) cpm.b All TCC cell lines grown to subconfluence at the time of assay.c Antigens detected by hybridoma antibodies 2E1-F3 and 9A7-F2 are TCC

tumor-associated antigens.7

DISCUSSION

The medium which has been described is modified from thatreported by Taub ef al. (20) for canine kidney tubule cells andBottenstein ef al. (4) for rat neuroblastoma cell lines. Theseauthors emphasized the great utility of serum-free medium in

starting primary cultures (of kidney tubule cells) because italmost totally inhibited fibroblast growth. Polyamines, in theconcentrations suggested by Roszell ef al. (16), also wereadded by us for their antifibroblast effect (16) as well as fortheir contribution to ribosomal RNA and protein production (11,17). Use of DH-S1 has avoided the multiple medium changes

and washings recommended by Owens ef al. (15) for theestablishment of fibroblast-free primary cell lines from normal




and malignant human tissues. Our primary cultures, after becoming fibroblast free, have been fed a serum-containing me

dium to help establish permanent cell lines. Higgins et al. (8)suggested using horse serum (with polyamine-enriched media)

because horse serum contains low levels of amine oxidase (1 ),an enzyme which converts spermine to a toxic dialdehyde (2).We have been able to use PCS, however, without a horseserum intermediate step when changing from DH-S1 to serum-

containing media. Although both normal murine urothelium (3,15, 16) and human TCC (5, 7, 15, 21) have been grown inserum-containing media, our report of growth in DH-S1 represents the initial report of the growth of either in serum-free

medium. Application of this approach to normal human transitional epithelium is anticipated because of the success of thismedium with human TCC.

Cell lines of malignant transitional epithelium are more valuable for studies of tumor-associated antigens when sufficient

quantities of nonmalignant tissue from the same individual havebeen frozen and/or carried as a cell line (e.g., B-lymphoblas-toid cell line). We were able to do this in the case of our newlyderived TCC cell line, LA-B1 [e.g., have tissue and B-lympho-blastoid cell lines established by standard procedures (6)]. Thishas not been reported previously for TCC.

The initial (Chart 1a), as well as the continued, successfulgrowth of a human TCC cell line in serum-free medium has

demonstrated considerable dependence upon a high cell density (data not shown). This is presumably true not only becauseof technical factors, such as the reduced plating efficiency ofall TCC cell lines in this medium (Chart 1, a to f) and thetendency of 647V cells to clump after trypsinization (particularly when the reaction is not reversed by trypsin inhibitingactivity normally found in serum), but also because of thedensity-dependent nature of 647V in media which contain

serum as well as those which lack it (Chart 1, a to c). That thistendency is emphasized in serum-free medium is illustrated bymorphological aberrations of 647V grown at low density in DH-

S1 which largely disappear when a minimum density of approximately 3.5 x 104 cells/sq cm8 has been exceeded.

Enzymatic and mechanical trauma from the passage are notsufficient to cause the morphological change, since plating athigher densities (e.g., 3 to 4 x 10" cells/sq cm) in DH-S1

produces the morphological appearance of cells growing inserum-containing medium within 24 hr (data not shown). Moreover, the low-density appearance is very similar to that foundin those TCC cell lines (T24, J82, LA-B1 ) which grow poorly ornot at all in DH-S1 but which revert to normal morphology andgrowth characteristics within 12 hr8 after being refed serum-

containing media. We are presently investigating potential explanations for this density dependence including the possibleproduction of a growth-stimulating factor(s) which fosters and

maintains the return of 647V cells to normal morphology andgrowth.

' Unpublished observation.

Serum-free Medium for Growth of TCC and Urothelium

ACKNOWLEDGMENTS

We gratefully acknowledge the excellent technical assistance of DeborahClemens and the helpful advice and suggestions of Dr. Jane Bottenstein. Wealso wish to acknowledge Dr. Richard L. Neu of Bioscience Laboratories (VanNuys, Calif.) for his expertise and generosity in karyotyping LA-B1.

REFERENCES

1. Alarcon, R. A. Isolation of acrolein from incubated mixtures of spermine andcalf serum and its effects on mammalian cells. Arch. Biochem. Biophys..106: 240-242, 1964.

2. Bachrach, U., Abzug, S., and Bekierkunst, A. Cytotoxic effect of oxidizedspermine of Ehrlich ascites cells. Biochim Biophys. Acta, 134: 174-181.1967.

3. Bonar, R. A., Reich, C. F., Ill, and Sharief, Y. Canine urinary bladderepithelial cells: preparation for cell culture by enzyme dispersion. Urol. Res.,5. 87-94. 1977.

4. Bottenstein, J. E., Sato, G. H., and Mather, J. P. Growth of neurothelialderived cell lines in serum-free, hormone-supplemented media. Cold SpringHarbor Conf. Cell Proliferation, 6. 531 -544, 1979.

5. Bubenik, J., Baresova, M., Viklicky, V., et al. Established cell line of urinarybladder carcinoma (T-24) containing tumor-specific antigen. Int. J. Cancer,11: 765-773, 1973.

6. Chang. R. S., Hsieh, M. W., and Blankenship, W. Initiation and establishmentof lymphoid cell lines from the blood of healthy persons. J. Nati. Cane. Inst..47. 469-477, 1971.

7. Elliott, A. Y., Bronson, D. L., Stein, N., and Fraley, E. E. In vitro cultivationof epithelial cells derived from tumors of the human urinary tract. CancerRes., 36. 365-369, 1976.

8. Higgins, M. L., Tillman, M. C., Rupp, J. P., and Leach, F. R. The effect ofpolyamines on cell culture cells. J. Cell. Physiol., 74: 149-154, 1969.

9. Koss, L. G. Tumors of the urinary bladder. In: L. G. Koss (ed), Atlas of TumorPathology, Second Series, Fascicle II. Washington, D. C.: Armed ForcesInstitute of Pathology, 1975.

10. Koss, L. G. Tumors of the urinary tract and prostate. In: L. G. Koss (ed.),Diagnostic Cytology and its Histopathologic Bases, Ed. 3, pp. 749-81 7.Philadelphia: J. B. Lippincott Company, 1979.

11. Kuroda, Y., Mervick, W. C., and Shariner, R. K. Polyamines inhibit theprotein kinase 380-catalyzed phosphorylation of eukaryotic initiation Factor2a. Science (Wash. D. C.), 275. 415-416. 1982.

12. Marshall, V. F. The relation of the preoperative estimate to the pathologicdemonstration of the extent of vesical neoplasms. J. Urol.. 68. 714-723,1952.

13. Neu, R. L. Cytogenetic techniques and terminology. In: L. I. Gardner (ed.).Endocrine and Genetic Diseases of Childhood and Adolescence, p. 684.Philadelphia: W. B. Saunders Company, 1975.

14. O'Toole, C., Price, Z. H., Ohnuki, Y., and Unsgaard, B. Ultrastructure,

karyology and immunology of a cell line originated from a human transitionalcell carcinoma. Br. J. Cancer, 38. 64-76, 1978.

15. Owens, R. B., Smith, H. S., Nelson-Rees, W. A., and Springer, E. L. Epithelialcell cultures from normal and cancerous human tissues. J. Nati. CancerInst., 56. 843-849, 1976.

16. Roszell, J. A., Douglas, C. J., and Irving. C. C. Polyamine-stimulated growthof cultured rat urinary bladder epithelial cells. Cancer Res., 37. 239-243,1977.

17. Russell, D. H. The roles of the polyamines. putrescine, spermidlne, andspermine in normal and malignant tissues. Life Sci., 13: 1635-1647, 1973.

18. Sandberg, A. A. Tumors of the urinary tract. In: A. A. Sandberg (ed.). TheChromosomes in Human Cancer and Leukemia, pp. 503-511. New York:Elsevier/North-Holland Publishing Co.. 1980.

19. Seabright, M. A rapid banding technique for human chromosomes. Lancet,2:971, 1971.

20. Taub, M., Chuman, L., Saier, M. H., and Sato, G. Growth of Madin-Darbycanine kidney epithelial cell (MDCK) line in hormone-supplemented serum-free medium. Proc. Nati. Acad. Sei. U. S. A., 76: 3338-3342, 1979.

21. Williams, R. D. Human urologie cancer cell lines. Invest. Urol., / 7. 359-363,1980.

22. Zeltzer, P. M., and Seeger, R. C. Radioiodinated protein A from Staphy/o-coccus aureus microassay for antibodies bound to cell surface antigens ofadherent tumor cells. J. Immunol. Methods, 17: 163-175, 1977.

JUNE 1982 2395



E. M. Messing et al.

Fig. 1. a, 14-day primary culture of murine bladder epithelial cells initially plated in DH-S1 and refed serum-containing medium at 1 week. Note absence offibroblasts. x 200. b, 14-day mouse bladder cell culture treated identically to cells in a, except initial plating was in media with serum. Fibroblast overgrowth isobvious, x 200.

Fig. 2. Photomicrograph of LA-B1 cells, demonstrating characteristics which have been associated with malignancy (11 ). including increased nuclearcytoplasmicratio, many mitoses, and marked nuclear variability. Heterochromatin, prominence of nucleoli, and nuclear pleomorphism were accentuated on May-Grunwald-Giemsapreparations (not shown).5 x 200.

Fig. 3. Transmission electron-microscopic view of LA-B1 cells grown to high density revealing numerous junctional complexes (desmosomes) (arrow) typical ofepithelial cells including transitional epithelium.




5bFig. 4. a, 3-day culture of 647V cells plated at 1.2 x 10* cells/sq cm in DH-S1. Note cytoplasmic vacuoles and elongated projections. These are not found in

647V cultures of the same age which are plated in serum-containing media at the same or lower cell densities (not shown), x 200. b. 3-day culture of 647V cellsplated at 2.4 x 104 cells/sq cm in DH-S1. At higher cell densities, these cells are far more epithelial looking than those in Fig. 5a and are identical in appearance tothe same cells shown in serum (c). x 200. c, 3-day culture of 647V cells in DMEM:Ham's F-12 plus 10% NCS plated at 1.4 x 10" cells/sq cm. x 200.

Fig. 5. Transmission electron micrographs of 647V cells grown to high cell density in DMEM:Ham's F-12 plus 10% NCS (a) and DH-S1 (b). Few differences can

be seen between these cells grown in the 2 media. Tonofibrils and desmosomes (a/rows), typical of epithelial cells, are present on both groups of cells but appearto be slightly better developed on those cells grown in serum.



1982;42:2392-2397. Cancer Res Edward M. Messing, John L. Fahey, Jean B. deKernion, et al. Malignant Urinary Bladder Epithelial Cells

Growth of Normal andin VitroSerum-free Medium for the

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Serum-free Medium for the in Vitro Growth of Normal and … · Human TCC cell lines, 647V (7), T-24 (5), and J82 (14) (all gifts from Dr. E. Bloom) growing in maintenance (serum-containing)