Isolation and Long-Term Culture of Diploid Mammalian …cancerres.aacrjournals.org/content/canres/22/5_Part_1/556.full.pdf · and definition of the factors affecting cell morphol

Isolation and Long-Term Culture of Diploid

Mammalian Cell Lines

JEAN FERGUSONANDANN WANSBROUGH

(Commonwealth Serum Laboratories, Parkville, Victoria, Australia)

SUMMARY

Cells have been established in continuous culture from human and animal tissues,both normal and carcinomatous. From 197 specimens, 105 could be maintained in vitrofor varying lengths of time, 27 for more than 6 months. In all cases the cells were foundto be diploid. Cell lines with both epithelial and fibroblastic morphology have beenisolated in medium 199 with added fetal calf serum, but all lines maintained over longperiods have eventually become fibroblast-like. Following the additional use of othersynthetic media, cells with epithelial morphology have retained their characteristicshape for up to 3 months.

Cells derived from mammalian tissue have beenused for biochemical and cytological studies, particularly with respect to cultural characteristicsof the rapidly multiplying "continuous" cell

lines (2,4,5). These heterogeneous and heteroploidcell populations are a valid analytical tool withincertain limitations. Cloned and characterized linesare being widely used but give little basis for comparison with the normal cell in vivo (J8, 20).

In recent work, lines of fibroblast-like cellsfrom normal tissues have been isolated and propagated in vitro without apparent alteration of theirmorphology or cultural behavior (15, 22), and inaddition these cells have retained the diploidchromosome number of the original tissue (16).

Technics for the cultivation of normal cellspermit studies on the morphology and behavior ofdifferent cell types. Recent improvements in mediaand definition of the factors affecting cell morphology in vitro will extend the application of suchtechnics. In this paper the methods for establishing fibroblast-like cell lines with morphologicaland biochemical characteristics of primary tissueoutgrowth are described. Preliminary investigations into the propagation of diploid epithelial-like cells are also reported.

MATERIALS AND METHODSSolutions.â€”

Phosphate-buffered saline (P.B.S.) (3)Trypsin, 0.25 per cent (Nutritional

Received for publication September 5, 1961.

Biochemicals Corporation (1:300)in P.B.S.)

L Y medium (11)199 medium (14)858 medium (13)N.C.T.C. 109 ( 6)N. 16 (16)

Additives.â€”Sera:

Human pooled, individual and cordRabbitCalf, fetal calfHorse, pooledMonkey, rhesus and cynomolgus.

After collection all sera are passed throughCorning ultra-fine sintered glass filters. Calf serumand individual human sera are prepared weeklyand stored at 4Â°C. for periods not exceeding 1

month; all other sera and extracts are stored atâ€”30Â°C. until required.

Before use individual human sera and fetal calfserum batches are tested on sensitive cell lines forcapacity to support growth and for toxicity.

Extracts.â€”Chick embryo : modification of method

of Bryant el al. (1)Beef embryoFowl plasma: freeze-dried and stored

at 4Â°C.

Fetuin: modification of method ofFisher, Puck, and Sato (10)

556

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FERGUSONANDWANSBROUGHâ€”Cultureof Diploid Mammalian Cell Lines 557

Tissue samples.â€”

Human :Skinâ€”Pinch biopsies, adult and child

(from inside of forearm).Pieces taken at operation (normal skin

taken from border of wound).Foreskin taken at circumcision.

Amnionsâ€”from normal deliveries.Cervical samplesâ€”biopsies taken at clin

ical examination for carcinoma.Normal and tumor tissue removed at

operation.Animal:

Normal tissues taken aseptically from laboratory animals.

Culture vessels.â€”Pyrex screw-capped bottles

(160 ml.), tubes (160 X 25 mm.), and Petridishes (60 mm.) were used.

Initiation of cell cultures.â€”Various procedures

for isolating cultures have been used.Three methods were compared for small tissue

specimens (less than 2 gm.).A. Plasma clot method : Minced tissue fragments

moistened with chick embryo extract were allowedto clot on the plasma-coated surface of tubes orPetri dishes, and culture-medium was added.

B. Trypsinization : The tissue was minced asfinely as possible in a 60-mm. Petri dish and incubated for 10-15 minutes at 37Â°C. with an aliquot

of trypsin determined by the size of the sample.The suspension from a small biopsy (<2 mg.)had 0.8 ml. of serum added. The suspension fromlarger samples was divided among several dishes,each moistened with 0.8 ml. serum. The finalvolume of the trypsin solution should be less than1:10 of the volume in the dish. When the fragments had settled, 3.2 ml. of basal medium wasadded gently without disturbing the tissue, andthe dish was incubated for 72 hours before themedium was changed.

C. Trypsinization and plasma: A combinationof methods A and B was attempted. The tissuesuspension in trypsin was dropped on the plasma-coated surface of the culture vessel and allowed tosettle before the culture fluid was added.

Large tissue specimens were treated in thefollowing ways to produce single cell suspensions.

D. Amnion trypsinization : Following the method of Ferguson and Tobin (9), the membrane wasgiven one 3- to 4-hour extraction with trypsin, and

the cell yield was inoculated to give a final concentration of 0.3-0.4 X IO6 cells per ml. of culture

medium.E. Mass trypsinization: A cell suspension was

obtained from tissues of more than 2 gm. by the

method of Youngner (23), with trypsin used forrepeated extraction of the chopped tissue (10- to20-mg. pieces). Cells were inoculated to give aconcentration of 0.2 X IO6 cells per ml. of culture

medium.F. Liver extraction: After laparotomy the he

patic artery was ligated. The inferior vena cavawas pierced, and the liver was perfused liberallywith normal saline through a cannula inserted intothe portal vein. The inferior vena cava was thenclamped off and medium 199 injected forciblythrough the cannula with the aid of a 50-ml. syringe. The volume of medium used was variedbetween 50 and 150 ml. per experimental animal.The well inflated liver was removed, chopped, andextracted by agitation in medium 199 or trypsindiluted to 0.125 per cent with medium 199, thusproducing a single cell suspension.

^Maintenance and propagation of cultures.â€”

Petri dish cultures: A humidified incubator withan atmosphere of 5 per cent CO2 in air was used tomaintain a pH of 7.1 in the medium. After thefirst 3 days the medium was replaced 3 timesweekly. Cells which had become attached to theglass might stretch and begin multiplying up to afortnight after trypsinization. The time taken fora continuous cell sheet to form depended on theoriginal amount of tissue and varied from a fewdays to a month. Subculture was carried out whenthe surface of the vessel was about 50 per centcovered, the medium being withdrawn and 0.2-

0.5 ml. trypsin added. After 1 or 2 minutes thecells were gently rubbed free from the surface withthe tip of a pipette and suspended in the trypsinsolution. The cell suspension from the first subculture might be left in the original dish and freshmedium added. Rapid multiplication followed;the culture became confluent and could be dividedbetween two dishes every few days by the methoddescribed above. After three passages in Petridishes the culture was well enough established tobe transferred to bottles or stored at â€”65Â°C. (7).

Bottle cultures : Cells obtained from large specimens by methods D, E, and F can be establishedin bottles directly. The medium was replaced onthe 4th day. After primary inoculation at theoptimum concentration the cell sheet should beconfluent within a week. Subculture was carriedout by removing the medium, washing the cellsheet once with P.B.S., and incubating the bottleat 37Â°C. with trypsin for 15-30 minutes (1-2 ml/

160-ml bottle). The cell suspension obtained wasdivided between two bottles containing fresh culture medium.

Growth media.â€”All components used are listedabove. Fibroblast lines were initiated and propa-



558 Cancer Research Vol. 22, June 1962

gated in medium 199 with fetal calf serum. Someestablished lines could be maintained with calfserum.

Liver and kidney cultures have been grown inmedium 199 or 858 with fetal calf serum (5-15 per

cent) and with or without N.C.T.C. 109 (5 percent) added. Amnion cultures were grown in fetalcalf or human serum (cord or adult pooled).

Storage of tissue samples.â€”Although specimens

were generally cultured as soon as possible, theycould be stored in medium 199 before use. Am-nions were washed and left overnight while sterility was confirmed (9). Operation specimens received in the afternoon were washed, chopped into1- to 2-gm. pieces and left overnight in freshmedium at 4Â°C. Chloramphenicol (20-50 mg/ml

medium) was added if infection was suspected.Skin biopsies have been stored in a few ml. ofmedium at 4Â°C. or room temperature for up to

1 week.Storage of cells.â€”Cells were stored in ampoules

at â€”65Â°C., each ampoule containing 2X IO6

cells in 1 ml. of growth medium, with 10 per centglycerol added (7). After thawing, the cell suspension was not washed but added directly tofresh growth medium in a fresh Petri dish or bottle.

Chromosome enumeration.â€”After three pas

sages mitoses were frequent, and the chromosomenumber of a culture could be determined (8).

RESULTSCell lines established.â€”A variety of normal and

carcinomatous tissues from human sources andnormal animal tissues were investigated. Excluding those contaminated with microorganisms, of197 specimens cultured 170 showed a primarygrowth of viable cells, judged by their stretchedappearance on the glass. In total, 105 cultureswhich were still multiplying after the third transfer in vitro were regarded as successful isolations.Of these, 27 lines were propagated in vitro for morethan 6 months and were considered establishedlines; 35 others were stored before 6 months hadelapsed. The remainder were lost, due to inadequate media in the case of epithelial lines, toxicityof serum or contamination. These results are summarized in Table 1.

Following the system of Foley et al. (12), a newcell line was not considered to be 'isolated' until

three serial passages had been made, and this linewas not considered to be 'established' until it had

been carried in culture in the laboratory for 6months.

Culture methods.â€”The cells obtained from tryp-

sinization of a large tissue specimen rarely failed toform a culture which would multiply for at leasta few weeks in vitro. Before the system of Petri

dish cultures in a CO2 incubator was used, cellsfrom small tissue samples could not be successfullypropagated. Later use of methods A, B, and C allgave satisfactory results, but because of its convenience method B was mostly emploved (seeTable 2).

Serial passage of cells derived from normal animals was attempted before the use of the Petridish system of culture. As shown in Table 1 in nocase was a line established.

Nutritional requirements.â€”In early attempts to

propagate cells in primary culture biological additives and various media and sera were used.Embryo and yeast extracts gave variable results.Horse, rabbit, and monkey sera were often toxic.Amnion cultures did not grow significantly betterin LY medium than in 199. Puck's medium N.16

and lower concentration of trypsin gave similarresults to 0.25 per cent trypsin and 199.

However, the introduction of fetal calf serumwas found to be an important factor for the continued multiplication of a culture after transfer.Apart from amnion cultures, which grew similarlyin human, human cord, or fetal calf serum, allprimary cultures grew consistently better in fetalcalf serum.

Morphology and chromosome number.â€”Although

a mixture of cell types often appeared in primarycultures, all the lines we established were fibro-blast-like in appearance, but morphology differedslightly according to the tissue of origin. Thechromosome number of several of these stocklines was checked at intervals during their establishment (Figs. 1, 2). The normal diploid numberwas found in all cases investigated, each line having been in continuous culture for at least 6months. In no case was a cell line found with otherthan the diploid number (Table 3). In addition, noalteration of chromosome morphology was notedduring cultivation of these lines. Cells tested afterstorage at â€”65Â°C. were also found to retain their

diploid configuration.Storage of cells.â€”Stock fibroblast lines have

been stored at â€”65Â°C. and later tested for via

bility. After a period ranging from 1 to 9 monthsof storage twenty cell lines were tested for capacityto grow. All except three cultures yielded viablecells which were propagated and re-stored. In somecases the number of viable cells recovered fromthe ampoule has been counted and recorded as apercentage of the original (Table 4). Culturalcharacteristics were found to be unaltered, andthe condition of the cells at the time of storagewould appear to be the most important factoraffecting the recovery rate.

Growth from tissue samples stored for up to 7days at various temperatures was compared with



TABLE 1

ORIGINOFISOLATEDANDESTABLISHEDCELLLINES

Tissue oforiginNormal

humanSkin:Biopsy, normalforinvestigationOperation

specimen,adultForeskinThyroidKidneyCervixSpleenBladderAumionMalignant

humanCervicalbiopsyCarcimona,

vaginalungbreastbladdercolonrectumkidneyNormal

animalMonkey,kidneylungspleenthyroidliverHorse,

kidneyRabbit,kidneyliverRat,

kidneyskindiaphragmlungBeef,

embryoskinTotal:No.specimenscultured925417615111114212425537621624521214197No.specimensshowingprimarygrowth820314514110391221547021413221214170No.

celllinesisolated7142135411017133741113214105No.celllinesestablished62131102227

TABLE 2

METHODSOFCCLTURINGSMALLTISSUESAMPLES

METHODA.

ExpiantB. TrypsinizationC. Trypsinization+plasmaNo.CULTURESATTEMPTED28

10114NO.

SHOWINGPRIMARY

GROWTHUn-Â»tretchedcells2

234Stretched

cells2678

10No.

SHOWINGSUBSEQUENT

GROWTHMultiplicationaftertransfer

115

605Serial

propaga

tionaftertransfer

31

555




controls of the same samples cultured when received. The samples tested were a carcinoma ofthe rectum, two pieces of adult skin taken atoperation, and nine foreskin specimens. Each wasdivided in four to nine parts; two parts were cultured immediately as controls and the rest storedand cultured on subsequent days. Apart from theexaggeration of previous microbial contamination,the difference in successful isolation and multiplication of cultures from control and stored sampleswas not significant for samples stored for 7 dayseither at room temperature or at 4Â°C. Of twosamples each stored at 37Â°C. for 3 and 5 days,

one was successfully cultured on the 3d day only.A skin biopsy taken at autopsy 24 hours afterdeath was successfully cultured after a further 2days at room temperature.

Comparison of growth from normal and malignant sites.â€”Cells grown from tissues from malig

nant sites have shown a similar morphology tothose grown from normal tissues. Isolations frommalignant tissues were more difficult to achieve,but subsequent establishment of cell lines was

TABLE 3

CHROMOSOMENo. OFCELLLINESISOLATEDFROMHUMANTISSUES

ORIGINOFCELLNormal

skinCa.cervixNo.

LINES

ESTAB

LISHED910CHROMOSOME

NO.AFTER25PASSAGESNo.lineatested97No.lineswith

chromosomeno.

of4697No.

lineswithchro

mosomeno.otherthan

4600

found to occur with greater frequency (see Table5). Although epithelial-like cells were present inthe initial stages of a large number of cultures(Fig. 3), they did not survive propagation, sinceapparently the method and medium we had chosentended to favor fibroblast-like cells (Fig. 4). Attempted propagation in the same medium of epithelial-like colonies isolated from earlier skin andcarcinoma cultures resulted in initial multiplication of the cells followed by elongation and declinein the rate of metabolism over one or two transfers. Our experience with trypsinized cells frompredominantly epithelial tissues such as kidneyand amnion was the sameâ€”the epithelial-like cells

multiplied during a few passages, then degeneratedor became overgrown by fibroblasts. The epithelial-like cell type grown from trypsinized thyroidappeared to elongate during its initial passagesand then behaved as a fibroblast-like cell. Four ofseven rectal or colon carcinomas received over a6-month period produced cultures of short bipolar cells which, in later passages, constantly appeared as a mixture of distinct epithelioid andfibroblast-like cells.

Epithelial-like cell lines.â€”The last part of our

investigation was concerned with propagation inthe diploid state of epithelial-like cells from nor-

TABLE 5

ESTABLISHMENTOFCELLLINESFROMTISSUEFROMNORMALANDMALIGNANTSITES

TissueoforiginNormal

humanMalignanthumanNo.

specimenscultured9064No.isolations5724No.linesestablished1814

TABLE 4SURVIVALOFDIPLOID CELLLINES AFTERSTORAGEAT â€”65Â°C.

TlBSCEOFORIGINOF

LISERectal

carcinomaColoncarcinomaCervicalbiopsyCervicalbiopsyCervical

biopsyThyroidSpleenSkinPER

CENT CELLS SURVIVINGAFTERSTORAGE

TIME (iNMONTHS)1402515t47381958V4V56V7VVVVV8VV0VVVREMARKSOf

9 other lines tested 6 were stillviableat9months7

lines tested

V = viable cells present but not counted.



FERGUSONANDWANSBROUGHâ€”Cultureof Diploid Mammalian Cell Lines 561

mal tissues. Rabbit liver and human kidney werechosen for preliminary experiments.

Cultures from human kidney consisted of uniform, small epithelial cells. With 199 as the basalmedium the concentration of 5 per cent fetal calfserum proved to be optimal, giving the highestmultiplication rate over the first few passages. Afterthis period of rapid growth the cultures entered aphase where cell enlargement continued without celldivision, the cells becoming granular and vacuo-lated and eventually degenerated. Eleven cultureswere studied in this medium, and apart from onewhich gave rise to a fibroblast line requiring ahigher serum concentration the latest transferfollowed by multiplication occurred 50 days afterprimary trypsinization. Following the recommendation of Puck et al. (16), 5 per cent N.C.T.C. 109was added to a mixture of 199 and 5 per cent fetalcalf serum. Under these conditions the initialgrowth rate improved, and the onset of cell degeneration was delayed. Comparisons were madebetween 858 and 199 as basal media for each oftwo kidney cultures. A marked difference wasnoted after the first two transfers. Control bottlesin 199 and 5 per cent fetal calf serum showeddegeneration of the cells by 40 days, whereas thosein 858 with the serum concentration increased to15 per cent were still small, multiplying epithelialcells after 85 days and nine transfers in the firstcase, 74 days and five transfers in the second.

Rabbit liver cultures showed a similar reactionto more complex basal media. Four cultures wereinitiated by the inflation trypsinization technic.The cell suspension appeared to contain four celltypesâ€”large irregular cells with a granular cytoplasm, large ovoid cells with a displaced distinctnucleus, and granular cytoplasm and small regularcells of varying diameters with and without granules. In primary culture occasional large ovoid orirregular cells could be identified attached to theglass, but these did not multiply. Thus, the celltypes which formed a cell sheet appeared to fallinto four groups ranging from epithelial-like tofibroblast-like cells, the ratio depending on themedium used. Further study of these cell types,their origin and behavior in cultured media, is inprogress.

DISCUSSIONCultures grown from a variety of tissues have

shown that cells may be propagated which areinitially epithelial in appearance. These cells will,in most cases, stretch out to give a fibroblast-likeappearance with variation of the culture mediumnot affecting the result.

Fibroblast lines have been reported to declinein growth rate after a period in vitro (16, 22). Three

of our lines have been kept growing continuouslyin vitro for longer than 12 months, the remainderbeing stored satisfactorily. One of these lines hasshown no decline in growth rate; the others havebeen poor cultures since their accidental exposureto toxic serum.

We have found that diploid lines can be initiated and propagated under conditions of pH andtemperature similar to, but less stringent than,those recommended by Puck et al. (16). However,conditions of culture do have to be very carefullycontrolled if only a few cells are available. Sanfordet al. (19) have shown that there is a critical ratiobetween the volume of growth medium and thesize of the cell population. When their numberfalls below a certain level in the growth medium,cells do not appear to be able to adjust the fluidsufficiently for their survival and proliferation.

Carcinomatous tissues have also yielded fibroblast-like cells in culture which are sensitive tocultural conditions and are more difficult to propagate than cells derived from normal tissue. Therelationship between these cultured euploid cellsand the carcinoma cell is difficult to ascertain,especially since epithelial-like cells are also frequently present.

The experience of Puck et al. (16) is that euploidcells from any source adopt an elongated configuration in culture. Our studies with other tissueshave underlined the importance of the variablemedium requirements of different cell types. Itwould appear that factors in fetal calf serum andembryo extract do aid fibroblast-like growth. Celldensity is also an important factor. When the concentration of cells is high the medium requirements are less stringent.

Several workers have noted the appearance intheir cultures after a few weeks in vitro of a veryrapidly multiplying cell of compact appearance.This cell rapidly overgrows all the other cells andcontinues to maintain this high rate of divisionindefinitely. These 'transformed' cell lines are

found to be heteroploid, with chromosome numbers almost double the diploid number of the original tissue. Probably the most widely known ofsuch lines is the HeLa cell, which was isolated byScherer, Syverton, and Gey in 1953 (21).

During the serial passage of their amnion cultures Zitcer and Dunnebacke (24) noted the regular appearance of a rapidly multiplying epithelial-like cell which could be maintained in continuousculture over long periods. Pulvertaft et al. (17)cultured thyroids showing various pathologicalconditions and were able to demonstrate the appearance of a fast growing cell type morphologically similar to HeLa cells.




However, sudden transformations have notbeen noted in our cell systems, and it is thoughtthat this is due to the stringent cultural conditions.It is possible that in a system which is lackingsome cultural requirement only a particular cellwill be able to survive prolonged cultivation. Thiscell may be able to adapt to this particular environment better than any of the other cells, or,alternatively, such a cell could have arisen from achance mutation.

ACKNOWLEDGMENTSWe would like to thank Dr. M. Sherlock for arranging the

collection of the human tissue samples. Several Melbourne hospitals kindly provided us with these specimens, and, in particular, we are indebted to Dr. J. D. Hicks and Mr. E. S. R.Hughes of the Royal Melbourne Hospital and to Dr. J. C.Laver and the late Mr. G. G. Godfrey of the Royal Women's

Hospital. We are also grateful to Dr. P. L. Bazeley and Mr.John \Vhite for their interest in the project.

REFERENCES1. BRYANT,J. C.; EARLE,W. R.; and PEPPERS,E. V. Effect

of Ultracentrifugation and Hyaluronidase on Filtrabilityof Chick Embryo Extract for Tissue Culture. J. Nat'l. Cancer Inst., 14:189-225, 1953.

2. " . .1.11i.i. L. L. ; McAiAESTER,R. M.; and WAGNER,B. M.Criteria for Determining Malignancy in Tissue CultureCell Lines in the Albino Rat. Special Publications, N.Y.Acad. Sci., 6:343-55, 1957.

3. DULBECCO,R., and VOGT,M. Plaque Formation and Isolation of Pure Lines with Poliomyelitis Viruses. J. Exp.Med., 99:167-82, 1954.

4. EAGLE,H.; OYAMA,V. J.; LEVY,M.; and FREEMAN,A. E.Myo-inositol as an Essential Growth Factor for Normaland Malignant Human Cells in Tissue Culture. J. Biol.Chem., 226:191-206, 1957.

f. EARLE,W. R. Production of Malignancy in vitro: MouseFibroblast Cultures and Changes Seen in Living Cultures.J. Nat'l. Cancer Inst., 4:165-212, 1943.

6. EVANS,V. J.; BRYANT,J. C.; MCQUILLAN,W. T.; FIORA-MONTI,M. C.; SANFORD,K. K.; and EARLE,W. R. Studiesof Nutrient Media for Tissue Cells in Vitro; Protein-freeChemically Defined Medium for Cultivation of Strain L.Cells. Cancer Research, 16:77-86, 1956.

7. FERGUSON,J. Long-Term Storage of Tissue Culture Cells.Aust. J. Exp. Biol. Med. Sci., 38:389-94, 1960.

8. . Chromosome Studies of Human Cells in TissueCulture. Med. J. Aust., 1:40-43, 1962.

9. FERGUSON,J., and TOBIN,J. O'H. Preparation of Cell Cultures from Human Amnions. Brit. M. J., 1:144-46,1958.

10. FISHER, H. W.; PUCK,T. T.; and SATO, G. Molecular

Growth Requirements of Single Mammalian Cells: TheAction of Fetuin in Promoting Cell Attachment to theGlass. Proc. Nat'l. Acad. Sci., 44:4-10, 1958.

11. FOGH,J., and LUND,R. O. Continuous Cultivation of Epithelial Cell Strain (F.L.) from Human Auiniotic Membrane. Proc. Soc. Exp. Biol. & Med., 94:532-37, 1957.

12. FOLEY,G. E.; DROLET,B. P.; MCCARTHY,R. E.; GOULET,K. A.; Doxos, J. M.; and FILLER, D. A. Isolation andSerial Propagation of Malignant and Normal Cells inSemi-defined Media: Origin of C.C.R.F. Cell Lines. CancerResearch, 20:930-41, 1960.

13. HEALY,G. M.; FISHER,D. C.; and PARKER,R. C. Nutrition of Animal Cells in Tissue Culture. X. Synthetic Medium No. 858. Proc. Soc. Exp. Biol. & Med., 89:71-77,1955.

14. MORGAN,J. F.; MORTON,H. J.; and PARKER,R. C. Nutrition of Animal Cells in Tissue Culture. I. Initial Studies onSynthetic Medium. Proc. Soc. Exp. Biol. & Med., 73:1-8,1950.

15. PUCK,T. T.; CIECIURA,S. J.; and FISHER, H. W. ClonalGrowth in Vitro of Human Cells with Fibroblastic Morphology. J. Exp. Med., 106:145-58, 1957.

16. PUCK,T. T.; CIECIURA,S. J.; and ROBINSON,A. Geneticsof Somatic Mammalian Cells. III. Long Term Cultivationof Euploid Cells from Human and Animal Cells. J. Exp.Med., 108:945-59, 1958.

17. PTLVEHTAFT,R. J. V.; DAVIES,J. R.; WEISS, L.; andWILKINSON,J. H. Studies in Tissue Cultures of HumanPathological Thyroids. J. Path. Bact., 77:19-32, 1959.

18. ROTHFELS,K. H.; AXELRAD,A. A.; SIMINOVITCH,L.;McCuLLOCH,E. A.; and PARKER, R. C. The Origin ofAltered Cell Lines from Mouse, Monkey and Man, as Indicated by Chromosome and Transplantation Studies. CaÃ±ad.Cancer Conf., 3:189-214, 1959.

19. SANFORD,K. K.; EARLE,W. R.; and LIKELY,G. D. TheGrowth in vitro of Single Isolated Tissue Cells. J. Nat'l.Cancer Inst., 9:229-46, 1948.

20. SATO, G.; FISHER, H. W.; and PUCK, T. T. MolecularGrowth Requirements of Single Mamalian Cells. Science,126:961-64, 1957.

21. SCHERER,W. F.; SYVERTON,J. T.; and GEY, G. O. Studiesin the Propagation in vitro of Poliomyelitis Viruses. IV.Viral Multiplication in a Stable Strain of Human Malignant Epithelial Cells (Strain HeLa) Derived from an Epi-dermoid Carcinoma of the Cervix. J. Exp. Med., 97:695-710, 1953.

22. SWIM,H. E., and PARKER,R. F. Culture Characteristics ofHuman Fibroblasts Propagated Serially. Am. J. Hyg., 66:235-43, 1957.

23. YOUNGNER,J. S. Monolayer Tissue Culture 1. Preparationand Standardisation of Suspension of Trypsin DispersedMonkey Kidney Cells. Proc. Soc. Exp. Biol. & Med., 85:202-5, 1954.

24. ZITCER,E. M., and DUNNEBACKE,T. H. Transformationof Cells from the Normal Human Amnion into Established Strains. Cancer Research, 17:1047-53, 1957.

FIG. 1.â€”Chromosome preparation from culture derivedfrom normal skin, 3d passage. Mag. X 2,000.

FIG. 2.â€”Chromosome preparation from culture derivedfrom same skin sample as above but at 26th passage. Mag.X 2,500.





Fio. 3.â€”Epithelial-like cells derived from human kidney,3d passage. Mag. X400.

FIG. 4.â€”Fibroblast-like cells derived from human embryoskin, 18th passage. Mag. X400.



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1962;22:556-562. Cancer Res Jean Ferguson and Ann Wansbrough LinesIsolation and Long-Term Culture of Diploid Mammalian Cell

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Isolation and Long-Term Culture of Diploid Mammalian …cancerres.aacrjournals.org/content/canres/22/5_Part_1/556.full.pdf · and definition of the factors affecting cell morphol