The usefulness of human malignant tumor cellsgrown in tissue culture has been amply demonstrated by the numerous reports in the literatureon the HeLa strain of epithelial cell developed byGey and co-workers (.5). This report will describethe culture characteristics of three more humantissue culture cell strains derived from epidermoidcancers, one embryonal nhabdomyosareoma , andone sarcoma which, as yet, has not been completely adapted to continuous transfer. All thesestrains are derived from neoplasms which weregrown first in the irradiated and cortisone-treatedrat by one of the authors (8, 9).
MATERIALS AND METHODSTo begin a culture, the neoplastic tissue was finely minced
with scissors, suspended in culture media, and placed in eithera test tube, Carrel flask, or a 180-mi. milk dilution bottle, depending on the amount of tissue available. After a few daysof incubation at 87°C., an outcropping of sheets of cells couldusually be observed surrounding the minced pieces. When themedia became acid, the fluid was removed and the cells thereinspun out for implantation in another receptacle; fresh mediawas added to the original culture. When cells had grownsufficiently to form colonies of 1—2cm. or when they cornpletely covered the bottom of the container, they were removed by the addition of 0.5 per cent trypsin. This, afterincubation at 87°C. for 5—10minutes, was sufficient to floatoff the cells. After centrifugation the cells were resuspendedin either two Carrel flasks or two bottles. In some instanceswhen a very small amount of beginning material was available,cultures from tubes were transferred first to small Carrelflasks, then to large ones, and finally to bottles.
Once the cultures were established they were trypsinizedwhenever the growth had completely covered the bottom of thebottle. The rate of growth is a characteristic of the particular cell strain and will be described later. To prepare calture tubes for experiments, the “dry―tube method describedby Morann and Melnick (7) was used. The solid sedimentedcells with a few drops of human serum added were streakedwith a capillary pipette along the surface of a tube, previouslywarmed to 45°C., in much the same way that an agar slant is
S This work was supported in part by a research grant
(C-1355) from the National Cancer Institute of the NationalInstitutes of Health, Public Health Service; an Institutionalresearch grant from the American Cancer Society; and agrant from the Damon Runyon Memorial Fund.
Received for publication May 2, 1955.
streaked with bacteria. After 30 minutes at 4°C., 1.5 ml. ofculture media was added, and, following 2 days' incubation, asolid sheet of cells formed from the streaked site, and the tubescould be used for experiments.
Daily counts of the cells have been made in an effort todetermine the pattern of growth for each neoplasm. After implantation, 1 ml. of trypsin solution was added to each tube,and the cells were counted in a standard hemacytometer after30 minutes' incubation at 87°C. to provide an estimate of thenumber of cells implanted. Thereafter, daily counts were madein duplicate on tubes set up at the same time. Table 1 givesthe general pattern of growth for each neoplasm as exhibitedin this type of culture. Although the number of cells implantedvaried with different experiments, the general pattern wascharacteristic for each tumor. After 1 day of incubation therewas usually a decrease in the number of cells, probably owing
TABLE 1
DAILY CELL CouNTs5 ON CULTURETUBESOF HUMAN CANCER CELLS
to the fact that all those implanted were not viable. The mostrapid growth occurred within 2 days and in the instance ofH.Ep. #2, H.Ep. #3, and HeLa, represented the maximumattained, while the maximum for H.Ep. #1 was attained on the4th day and by H.Emb.Rh. #1 not until the 7th or 8th day.
When the neoplastic cells have been established in tissueculture, a definite routine is followed to determine if anychange occurs during their prolonged cultivation. Everymonth four bottles are set up with each tumor with two coverglasses in each bottle. The cells settling on the cover glassesare removed after 2 days' growth and stained with MayGrUnwald stain for comparison with previously and similarlyprepared slides. When the cells in the bottles have attainedtheir maximum growth (usually in 2-4 days dependingon the tumor) they are trypsinized, pooled, resuspended in asmall amount of media or balanced salt solution, and inoculated into irradiated, cortisone-treated weanling rats. When atumor has appeared (usually 10-14 days) the animal is sacrificed and the morphology of the neoplastic tissue is comparedwith that of previous tumors and with the original growth. As
598
Culture Characteristics of Four PermanentI. rTT ,@ I@' 11*Lines or i-iuman cancer @eiis
ALICE E. MOORE, LILLIAN SABACHEWSKY, AND HELENE WALLACE TOOLAN
(Virus Study Section, Division of Experimental Pathology, Sloan-Kettering Institutefor Cancer Research,Memorial Center, New York 9@?1,N.Y.)
MOORE ci al.—Culture Characteristh,s of Human Caiwer Cells 599
yet no histological change has been noted ; the characteristicsof each neoplasm appear to he preserved indefinitely.
RESULTSDzscRu.'rIoN OF TUMORS
H.Ep. #1 (Toolan).—This epidermoid carcinoma, the first to be established in serial passage inirradiated and cortisone-treated rodents and described previously by one of the authors (8), oniginated from a small biopsy specimen' obtainedfrom a carcinoma of the cervix. Some difficultywas experienced at first in establishing activegrowth in tissue culture, since it appeared to beharmed by trypsin; but cells from the nineteenthgeneration which had grown 13 days in the natwere more resistant to the treatment. Serial transfers in tissue culture have been made since Septemben, 1953. In the first passages the cells grewin colonies, and transfers could be made only at @-to 3-week intervals. Gradually the growth becamemore rapid and in single-cell form. Characteristically the cells do not form sheets as HeLa orH.Ep. #@do. Although the cells are often closelyapproximated, they appear to be more discretethan in either of the above tumors. The cells are oftwo types; the most numerous ones are large andangular, and the others are small and round. Atpresent the tumor grows rapidly and doubles itsbulk in@ days. Figure 1 shows the morphology ofthe tissue when removed from the patient; Figure@,an unstained tissue culture preparationof the
same neoplasm, demonstrates its rather angularcells; and Figure 8 illustrates the growth whicharose in an irradiated cortisone-treated rat afterne-implantation of trypsinized cells which hadbeen in culture for over 16 months.
H.Ep. #@ (Toolan).—After two generations inrats this tumor (also an epidermoid carcinoma),which had originally come from a man with a primary tumor of the larynx (Fig. 4), was implantedin tissue culture in September, 195@, by Dr. Audrey Fjelcte, using the plasma clot, roller drumtechnic. Its characteristics in this medium havebeen described (4). Since September, 1953, thesecells, which can easily be freed from the clot bytrypsinization,2 have been grown on the glass surface of bottles. They grow rapidly, doubling themselves in @—3days and forming sheets in the sameway as do the HeLa cells from which they aremorphologically almost indistinguishable (Fig. 5).When inoculated into an irradiated cortisonetreated nat, a typical epidermoid cancer forms(Fig. 6), which often contains vacuolated cells.
1 Obtained through the courtesy of Dr. Chester M.Southam.
S First done in our laboratory by Dr. Wilbur F. Noyes.
This vacuolation has not as yet been noted in theHeLa cells and may be a distinguishing characteristic of our cell line. Figure 4 shows the originalgrowth, in which the area of vacuolated cells isindicated.
H.Ep. #@(Tooh@n).—This neoplasm was derivedfrom a lymph node containing metastatic epidermoid cancer, grade III, primary in the buccalmucosa. It grows readily and rapidly in both ratsand eggs and was adapted to tissue culture 1 yearago. The cells can be started in culture at willfrom the animal implant and, even after prolongedcultivation, readily give rise to a characteristicneoplasm when implanted back into the animal.It grows in tissue culture at a somewhat slowerrate than do other lines, doubling itself in about Sdays. It does not form sheets but grows either assingle cells or a mosaic of flat clumps. The cellsthemselves are rather angular and may form amultinucleated mass. Occasionally, very large cellswith large nuclei are present in the clumps. Figure7 shows the original neoplasm; Figure 8, the cells
in tissue culture in the mosaic form; and Figure 9,the neoplasm which arose 10 days after inoculationof the cultured cells into the irradiated cortisonetreated rat.
H.Emb.Rh. #1 (Toolan).—This neoplasm, whichwas originally removed from the chest wall of a37-year-old male, is an embryonal rhabdomyosarcoma which was established in tissue culturefrom a 17-day-old growth (@0th generation in rats)received in June, 1954. It grew slowly at first, andthe first trypsinizations were carried out at approximately @-to 3-week intervals. It adapted itself well to tissue culture, however, and now istransferred at 3- to 4-day intervals. It grows inthick sheets which cover the bottom of the bottlecompletely with cells which are so transparentthat it would be difficult to detect the growthmacroscopically, were it not for the fact that theedges tend to curl. In this state it is impossible tosee the individual cells, but they are readily visiblein stained preparations where the characteristiccross stniations may be demonstrated by suitabletechnics. The tumor is characterized by large nuclei with many nucleoli and by its faintly staininglace-like cytoplasm (Fig. 11). Figure 10 shows theoriginal growth and Figure 1@that formed on thechonio-allantois of the egg after implantation oftissue culture cells imbedded in a plasma clot.
H.S. #1 (Toolan).—This neoplasm was a softpart sarcoma, primary origin unknown, removedfrom the calf of a 43-year-old male. It grows napidly and attains a huge size both in rats and in eggs(1), has grown very irregularly in tissue culture,and as yet is not in continuous serial passage. The
success or failure of the growth in culture seems todepend on the animal or egg implant selected fon aparticular experiment, some giving excellentgrowth and others none at all. A systematic investigation of the factors involved in these variationsof growth potential has not been made. Whengrowth occurs, however, the cells are charactenistic. They contain many vacuoles, a feature whichthey share with both the original neoplasm (Fig.13) and those grown in rat and on the chonioallantois of the egg. Figures 14 and 15 illustratetheir appearance in tissue culture.
GROWTH CHARACTERISTICSOF NEOPLASTICCELLS IN TISSUE CULTURE
Media.—The above-described tumors, plusHeLa and human fibroblasts derived from eitherembryonic or adult testicular tissue, were studied
TABLE 2
CoMPosITIoN OF MEDIA5 USED INGROWTH EXPERIMENTS
TYPES OF MEDIA#1 #5 #3 #4
70 80 85 9010 10 10 520 10 5
5
TABLE 3
SUMMARY OF GROWTH MEDIA EXPERIMENTS
GaowrsBest Good1@ All
land4 All1 2and5
landS 2and81 All1 All
H.Fibroblasts 5 1(Adult andembryonic)p The numbers indicate the type of media the compositions of which are
given in Table 5.
for their ability to grow in five different types ofmedia, the composition of which is shown inTable @2.Tubes were set up in duplicate and theirgrowth followed for 1 month without any changeof media. Two such experiments were done. Theresults, which are shown in Table 3, are arrangedaccording to the best growth, good growth, andlasting growth, to indicate the media in which thecells remained longest in good condition. Since theobservations were not quantitative, they must beregarded as a rough measurement of the impressions of two different observers.
In our laboratory all neoplasms and tissues such
as the human fibroblasts are routinely kept inmedia #1, designated BH (Bovine-human). Whenexperiments are to be done, the media is removedand replaced with media #5, designated Enders,since it was first described by that investigator(f). It has the advantage of containing no humanserum. The cells of all tumors, with the possibleexception of H.S. #1, keep very well for @2weeks atincubator temperature, at which time our expeniments are terminated.
The human serum is obtained from young, fasting adults and is collected by our blood bank3 inthe usual bottles, except that no anticoagulant isadded. After sterility tests the serum is placed intubes at 4°C. until used. We have never noticedany toxic effects from any sera.
Effect of pH.—BH medium (medium #1) wasadjusted in steps from pH 5.0 to pH 9.8 by theaddition of 0.1 N HC1 on NaOH. The adjustedmedium was then added to culture tubes in which
cells from each neoplasm had been streaked. Thetubes were observed for 3 weeks to determine:(a) the range of pH in which the cells could survivefor a 1-week period, (b) the range of pH at whichactive growth took place, and (c) whether any panticular pH range was best for growth. The resultsare given in Table 4.
In the cases of H.Ep. #3 and H.Emb.Rh. #1 itwas possible to compare the tissue culture strain ofcells with those isolated directly from the sameneoplasm grown in the rat. In each instance thecultured cells showed less tolerance to acidity, andbest growth seemed to take place in a more alkaline medium. To test the ability of H.Ep. #@ to
S The authors wish to thank Miss Mary Channon for her
MOORE et al.—Culiure Characteristics of Human Cancer CelLi 601
grow in media with an alkaline pH, three continuous transfers have been made into media atpH 9.0. Although the cells grew about 3 times asslowly as the controls kept at the routine pH(7.8), they formed sheets and retained their normalappearance. H.Ep. #3 failed to grow in the alkalinemedia.
All the neoplastic cells grew actively over afairly large range of pH, and it was often difficultto discover which was the best. The human fibroblasts grew in the most restricted range. Thesefindings roughly correspond to those reported byother authors (3).
In one experiment employing the growth ofH.Ep. @1,H.Ep. #@,HeLa, and HAF (human adultfibroblasts) in a pH range of 5.5 to 9.0, the fluidwas removed from the tubes after 1 week and thepH determined again. In each instance a changehad taken place in the direction of neutrality. Todetermine how quickly such an adjustment tookplace, the pH was determined at intervals aftertubes had been inoculated with H.Ep. #@cells inmedia of different pH's. The following changes hadtaken place in 1 hour: pH 5.7@to 6.61, pH 7.7 hadnot changed significantly, pH 8.9 to 8.5, and pH9.4 to 9.10. No further significant change in pHwas noted thereafter during the period of obsenvation; hence, one may assume that the growing cellshad exerted their buffering capacity very rapidly.It is possible that this is the determining factor inthe cells' ability to grow over such a wide range ofpH.
Effect of heat and ultraviolet on cells.—Experiments were done with all the different strains ofcells to determine how long they could be heatedat 56°C. in the water bath and still retain theirviability. Cells were trypsinized, centrifuged, andre-suspended in sufficient Gey's saline to make onemillion cells/ml. At the different time intervals0.1 ml. was removed and streaked on the warmedtube, and 0.9 ml. of BH media was added to thecultures which were placed in a stationary neceptacle for incubation at 37°C. The results of theseexperiments are reported in Table 5, showing thelast time interval at which good growth was obtamed and the time interval beyond which thecells failed to grow. Most of the cells grew wellafter being heated to 56°C. for 1 minute; two,HeLa and H.Ep. #1, could survive this temperature for@ minutes. The human fibroblasts,whether of adult or embryonic origin, appeared tobe the most heat-sensitive of all the cell linesstudied.
The effect of exposing similarly prepared cellsuspensions to ultraviolet light from a Hanovia
@,537 mercury vapor lamp, which delivered
@s0±15, 1O—@engs Km2/sec at a 10-cm. distance,was also studied. Three-tenths ml. of the cell suspension containing one million cells/ml was placedin a Maximow slide, and, after exposure at the diffenent time intervals, 0.1 ml. was placed in a tubeand 0.9 ml. of media added. The tubes were theninspected twice a week for growth. The last twocolumns in Table S give the results and show thatH.Emb.Rh. #1 and the human fibroblasts are verysensitive to ultraviolet radiation and that H.Ep. @1and H.Ep. #@are very resistant, whereas HeLa andH.Ep. #3 fall in a middle range.
DISCUSSION
The culture characteristics, morphology, andreaction toward heat and ultraviolet have beendescribed for four human neoplasms (three epidermoid carcinomas and one embryonal rhabdomyosancoma) which can be grown in lange quantities on glass surfaces in tissue culture. Wheneverpossible, the characteristics of another tumor,H.S. #1, have been described. It is not so corn
TABLE 5
EFFECTS OF HEAT AND ULTRAVIOLET LIGIrr ON THEGROWTH OF HUMAN C@us IN TISSUE CULTURE
HEATULTRAVIOLR?GoodSur
GoodSur‘I'UMORgrowthvivalgrowthvivalH.Ep.
#1H.Ep.#2H.Ep.#3H.Ernb.Rh.#11
mm.1 “1 “1 “ft
mm.1 “1 aI “5
ruin.15 “SOsec.
5 “10
mm.16SOsec.10aHeLa1
a24“1mm.3mm.Fibroblasts:Adult80
sec.1 mm.1 min.2mm,Embryonic30asec.10 sec.20 sec.
pletely established in culture, and we have beenunable to keep it in serial passage for longer than 6months. Since the other four neoplasms are readilycultivated, it is assumed that they will find thesame uses that have already been described for theHeLa cells. Each has been shown to be different inmorphology and in susceptibility to physicalagents, and it may be expected that their reactionsto viruses or chemicals may also vary.
The question is often raised whether theseneoplasms have changed during their prolonged
cultivation outside the human body. The followingfacts indicate that the original characteristics areretained: (a) When the long-cultivated cells areinoculated into an irradiated and cortisone-treatedanimal, growth takes place which is identical histologically with the tissue originally removed fromthe patient; (b) the human cells grown for longperiods in the rat, hamster, and egg have beenstudied immunologically and have. been shown to
maintain a steady human protein pattern (6) ; (c)studies of the chromosomes4 of the cultured cellsshow the persistence of characteristic human patterns; (d) finally, most of the cultivated cell typeshave been inoculated subcutaneously into humanvolunteers and have grown with the same histological characteristics as did the original tissue.'
The neoplastic cells have been shown to grow ina wide variety of media. Although we routinely use20 pen cent human serum, it may be possible toadapt the tumors to smaller amounts or to horseserum. Contrary to the experience of most workens, we have not found any batches of toxic serum.The rather wide range of pH over which the cellswill grow is surprising. The limited experiments reported here seem to indicate that such pH changesas take place occur very rapidly and that the pH isstabilized shortly after the cells begin to grow. Thereason for studying sensitivity to heat and ultraviolet was to determine similarities and differencesbetween the various cell lines. There were greatdifferences in regard to the ultraviolet sensitivityand lessen ones as far as heat was concerned, butthese appeared to be due to the individual charactenistics of the cell strain itself rather than to thetype of neoplasm. For example, one epidermoidcell strain (H.Ep. #@) was extremely resistant toultraviolet, whereas another (H.Ep. #3) was nelatively sensitive. These differences may also be useful in establishing any change which has takenplace during cultivation.
SUMMARY
The cultural and morphological characteristicsof three strains of human epidermoid cancer cells(H.Ep. #1, H.Ep. #@,and H.Ep. #3) and one human
4 Done by the courtesy of Dr. A@ R. T. Denues.
CThe reinoculation experiments will be published by Dr.
Chester M. Southam.
Fio. 1.—Epidermoid carcinoma from inguinal lymph nodebiopsy specimen (H.Ep. #1). H & E stain. X400.
FIG. 2.—H.Ep. #1 cells from a cellular colony grown intissue culture in BH media. Unstained. X400.
FIG. 3.—H.Ep. #1. Epidermoid cancer formed from theinoculation of cells grown continuously in tissue culture forl6months.H&Estain. X400.
Fia. 4.—H.Ep. #2. Epidermoid carcinoma of the larynx.Arrow indicates the vacuolated cells. X120.
Fio. 5.—H.Ep. #2 cells from a cover glass stained with MayGrunwald stain. X160.
Fio. 6.—H.Ep. #2. Epidermoid carcinoma produced in an
embryonal rhabdomyosancoma (H.Emb.Rh. #1)have been described. These tumors have beenpassed serially for periods of 8 months to@ yearsandhave maintained their original histological characteristics. One other tumor (H.S. #1), not yet permanently established in passage, has also beendescribed. The pH range and sensitivity of thetumor to heat and ultraviolet have been recorded.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical assistanceof Miss Margaret Moriarty.
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2. Ermxas, J. F. Bovine Amniotic Fluid as Tissue CultureMedium in Cultivation of Poliomyelitis and Other Viruses.Proc. Soc. Exper. Biol. & Med., 82:100—105,1953.
3. FISCHER, A. Growth of Fibroblasts and Hydrogen IonConcentration of the Medium. J. Exper. Med., 34:447-54,1921.
4. FJELDE,A. Human Tumor Cells in Tissue Culture. Cancer(in press).
5. Gsv, G. 0.; B@o, F. B.; and GEY, M. K. Responses of aVariety of Normal and Malignant Cells to ContinuousCultivation, and Some Practical Applications of TheseResponses to Problems in the Biology of Disease. Ann.N.Y. Acad. Sc., 58:976-99,1954.
6. KORNOOLD,L., and LIPAiu, R. Tissue Antigens of HumanTumors Grown in Rats, Hamsters, and Eggs. CancerResearch, 15:189—fl, 1955.
7. Moa.aNw, G. L., and MELNICK, J. L. Poliomyelitis Virus inTissue Culture. VI. Use of Kidney Epithelium Grown onGlass. Proc. Soc. Exper. Biol. & Med., 84:558—OS, 1953.
8. Tooi@a@i,H. W. Growth of Human Tumors in Cortisonetreated Laboratory Animals: The Possibility of ObtainingPermanently Transplantable Human Tumors. CancerResearch, 13:389—94,1958.
irradiated cortisone-treated rat 12 days after the subcutaneousinoculation of H.Ep. #2 cells which had been maintained forover 2 years in tissue culture. Arrow indicates vacuolated cells,also seen in the original tumor. X140.
Fio. 7.—H.Ep. #8. Microscopic appearance of the originalepidermoid carcinoma from a metastatic neck node, primaryin the buccal mucosa. X 160.
Fia. 8.—H.Ep.#8 after cultivation on cover glasses intissue culture. Stained with May-GrUnwald.X120.
Fio. 9.—The section of the neoplasm formed when cellsgrown in tissue culture for 6 months were inoculated intoirradiated and cortisone-treated rats. X 140.
1955;15:598-602. Cancer Res Alice E. Moore, Lillian Sabachewsky and Helene Wallace Toolan Cancer CellsCulture Characteristics of Four Permanent Lines of Human
