(CANCER RESEARCH 27, 726-730, April 1967]

Tissue Culture Studies on Murine Virus-induced Leukemia Cells:Isolation of Single Cells in Agar-Liquid Medium1

MARIA C. PATULEIA2 AND CHARLOTTE FRIEND2

Division of Microbiology, Sloan-Ketlering Institute for Cancer Research, New York, \ew York WOei

SUMMARY

Maturation along the erythrocytic line is observed in long-termcell cultures of murine virus-induced leukemic cells. In attemptsto determine the relationship between the different morphologictypes in these cultures, an adaptation of the Sanders technic ofplating and cloning cells utilizing an agar-liquid medium was devised. This method permitted the cloning of single cells. The progeny of each clone consisted of primitive cells, proerythroblasts,and erythroblasts. From these data the possibility is consideredthat the primitive cell characteristic of the murine leukemia understudy is an erythrocyte precursor.

INTRODUCTION

The cells of a murine virus-induced leukemia propagated invitro exhibit the capacity to mature along the erythrocytic line(5). The fact that these long-term cultures are composed ofprimitive cells and erythroblasts in various stages of developmentprompted the attempt to clone single cells to determine whetherthe progeny of each would possess similar properties. The recentdetailed report of Sanders et al. (6), describing the bacteria-likegrowth of mammalian cells in semisolid agar, suggested that suchmethods could be adapted to our studies. With the use of a modification of the Sanders technic which includes the addition ofnutrient fluid to cover an agar droplet, it has not only been possible to plate the leukemic cells in semisolid agar but has alsoallowed the precise observation of the development of clones fromsingle cell isolates.

MATERIALS AND METHODS

Cells. The cells for plating were obtained either directly fromtransplantable reticulum cell sarcomas originating from subcutaneous implants of spleen or liver from leukemic DBA/2 mice(4) or from tissue culture lines established from a number ofthese tumors.

The cells of Passage 13 to 40 of the tissue culture line, designated Sq 3A were used throughout this study. They grow insuspension, double in number in approximately 14 hours, and are

1This work was supported in part by NCI Grant CA 08748 andHealth Research Council of the City of New York Contract U-1096IIC2 Ext.

2 Present address: Department of Experimental Cell Biology,Mt. Sinai School of Medicine, New York, N. Y.

Received August 3, 1966; accepted December 1, 1966.

maintained in Basal Medium Eagle with Earle Balanced SaltSolution (powder media [G-ll], Grand Island Biological Co.,Grand Island, N.Y. [BME/EBSS]) plus 10% calf serum.

Sterile, disposable 60- x 15-mm Falcon tissue culture plasticPetri plates were used. The cultures were routinely kept at 37°Cin a water-jacketed incubator saturated with 5% COj.

When tumor was the source of the cells, it was finely minced inmedium and allowed to stand at room temperature for 10 minutes to enable the larger fragments to settle. The number of livingcells in the supernatant, determined by counting in a standardhemocytometer with 10% trypan blue, was adjusted to containIO6cells per ml.

Media. Semisolid media, designated ULS15, which containeda final concentration of 0.45% agar was prepared as follows: 45ml of 2x concentrated Basal Medium Eagle with Hanks BalancedSalt Solution (powder media [G-12], Grand Island BiologicalCo., Grand Island, N. Y. [BME/HBSS]) without NaHCO3) 24ml of distilled H20, 15 ml of calf serum, 1 ml of 7.5% NaHC03,15 ml of 3% Difco Bacto-agar thoroughly washed according tothe method of Dulbecco and Vogt (l).

The liquid media, designated OLS15 was prepared as follows:84 ml of BME/HBSS without NaHC03, 15 ml of calf serum, 1ml of 7.5% NaHCOs-

Plating. The procedure is described in detail since the varioussteps proved critical for obtaining optimal growth of the cells insemisolid agar.

The first three components of ULS15 contained in an 8-ozprescription bottle were placed in a 45°Cwater bath while thestandard IO6cells were further diluted in tenfold steps in OLS15

media. Duplicate tubes of each dilution were made.To the first items of ULS15, which generally reached 45°Cby

this time, the agar and the NaHCOs solution were added andthe mixture maintained at this temperature, to avoid undesirablejelling.

To each tube containing 0.2 ml of a given cell suspension, 0.8 mlof warmed ULS15 was added. After gentle mixing three times bypipetting, 0.5 ml was slowly placed on the bottom of the Petridish. The droplet formed a semisolid pearl approximately 20 mmin diameter within 5 to 10 minutes. When this occurred, 7 ml ofOLS15 were slowly added to each dish and the plates were thenplaced on a tray in the COs incubator.

The liquid medium was changed once a week. In withdrawingthe medium, the pipet was placed at the bottom of the dish at anangle with the wall. Fresh medium was added at a slow rate ofdelivery in order not to disturb or lacerate the pearl.

Passages were made by aspirating individual colonies from the

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Murine Virus-induced Leukemia Cells

TABLE 1Plating Efficiency of Murine Leukemia Cells in Semisolid Agar Liquid Medium

CellsourceDirectly

fromtumor"Cell

cultureline6Replating

from colonies in agar6Numbers

of cellsplatedIO»ConfluentConfluentConfluent10«ConfluentConfluentConfluent10»Isolated

and confluentcoloniesIsolated

and confluentcoloniesIsolated

and confluent colonies10*20

colonies16.5

colonies44

colonies10'1.5

colonies4

colonies5.3

colonies

" Pooled results of 3 experiments.0Pooled results of 5 experiments.

agar pearl with a Pasteur pipet and resuspending the cells in 1ml of OLS15. There were generally about 3 X IO6cells per large

colony. For replating, serial dilutions were made and 0.2 nil ofeach dilution placed in duplicate serologie tubes. Into each tube,0.8 ml of ULS15 was added and agar pearls prepared as previouslydescribed.

Cloning. Single cells in microdrops were obtained utilizing amethod comparable to that described by Sanford (7) and Wildyand Stocker (8). With a finely drawn out Pasteur pipet, several1- x 1-mm drops of the diluted cell suspension were placed at thebottom of a plastic Petri dish. The drops were immediately observed under the inverted microscope and the number of cellsper drop was counted. A drop containing a single cell, preferablyin the middle of the plate, was selected. It was examined carefully to exclude the presence of any other cell hidden close to theperiphery or in a different optical plane.

Each drop with a single cell was aspirated carefully into amicropipet containing an equal volume of ULS15 media whichhad been maintained at 45°Cand immediately placed inside a

marked area of the Petri plate. This area, 5 mm in diameter, wasetched in the plastic plate with the proximal end of a heatedhypodermic needle. It served not only as a reference for locatingthe small drop of agar but also as a trap for confining it. Fig. 1shows a single cell resting at the periphery of the agar pearl.Once the drop was set in the trap, it was again examined underthe inverted microscope to confirm that only a single cell existedin the agar and to mark its position clockwise for future reference.After approximately 2 minutes, the drop of agar solidified and 7ml of OLS15 were added. The [liâteswere then placed in the CO2incubator. Observations were made daily and in some instances,time-lapse photography was utilized to follow the progression of

the growth of the colony.Bioassay. DBA/2 mice, approximately 6-8 weeks of age, ob

tained from Jackson Memorial Laboratories, Bar Harbor, Maine,were used to assay the tumor-producing potential of the tissue-

cultured cell clones. Swiss ICR mice of the same age obtainedfrom Millerton Farms, Millerton, N. Y., were used to assay forvirus titrations since the DBA/2-derived cells are not expected

to grow in these randomly bred adult mice. Each inoculum of0.2 ml containing approximately 2 X IO5cells was administered

intraperitoneally or subcutaneously to the DBA/2 mice andintravenously to the Swiss mice.

RESULTS

Plating Efficiency. Plates to which IO6 cells were addedshowed microscopic groups of cells scattered in the agar pearls bythe 7th day (Fig. 2). Macroscopically, the pearl appeared slightlyopaque. On about the llth day, minute colonies were also visibleon the agar seeded with higher dilutions of cells. By the 3rd week,all colonies were visible macroscopically (Fig. 3). Since cellssloughed off into the supernatant fluid at about this time,colonies were routinely counted on the 21st day after seeding.

Table 1 contains the results of experiments in which the platingefficiency of cells of three different sources were compared after21 days of incubation. Tenfold dilutions were made, three platesbeing used for each cell dilution. Cells were obtained from: themouse tumors from which the tissue culture lines originated;the Sq 3A tissue culture line of murine virus-induced leukemiccells; and from colonies which resulted from replating cells ofeither of the other two.

Confluent growth was observed at the highest cell concentrations making the isolation of individual colonies difficult (Fig. 4).When 10l or IO2cells were plated, the colonies were discreet and

could be readily counted. As was observed by Sanders et al.(6), the efficiency of plating was greatest when the cells werereplated from the agar colonies and lowest when the cells weretaken directly from the tumors.

Cloning. Each single cell isolated was observed daily andphotographs taken to document the growth. After the number ofcells exceeded 26, photographs were taken at longer intervals of

time. It was frequently observed, however, that some clonedcells underwent a number of divisions but the cultures died before there were sufficient progeny cells to assay.

Clone Morphology. At least three types of colonies werenoted when the culture lines were cloned. In the first, growth wasrapid and the cells doubled in a matter of hours. The whole pearlof agar was covered with cells within 21 days. Usually, two kindsof cells were observed in such colonies, one rather flat and irregular in shape and the other réfringentand round. The flat cellsalways occupied the front line of the spreading growth (Fig. 5).

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Maria C. Patuleia and Charlotte Friend

The second type of colony was characterized by the confinedarea of its growth. The cells formed a closely packed colony whichremained in this state for about 2 weeks before starting a rapidspread (Fig. 6).

In the third colony type, groups of cells appeared to growrapidly for about 7 days and then slowed down almost to astandstill for about 2 weeks. During this time, the cells seemedto be degenerating and multiplying at an equal rate so that theviable cell number seemed to remain stationary. The cells appeared to be more pleomorphic than those in Type 1 or 2 coloniesand giant cells were present (Fig. 7). At approximately the 3rdweek flat cells, irregular in outline, appeared on the periphery ofthe colony and from then on the spreading type of growth commenced.

Cell Morphology. Individual colonies were picked from theagar and the cells resuspended in OLS15 medium. Cell lines derived from each clone were thus established and maintained influid medium for further observation.

Invariably, the donai population consisted of the primitivestem cells and the proerythroblasts at various stages of maturation.These cells were undistinguishable from those observed in ourcontinuous lines initiated directly from leukemic tissue (5). Thecells from a clone developed from a single cell are shown in Fig. 8.

Bioassays. The bioassays of the agar-grown colonies gaveuniform results. Twenty-six of the fifty-two cloned cultures werebioassayed. Within 10-12 days after the subcutaneous inoculationof 0.2 ml of a cell suspension, containing 2 X IO5cells, all of the

DBA/2 mice developed tumors at the site of inoculation. Thesewere identical with the reticulum cell sarcomas from which theculture lines originated as confirmed by histologie examination.When the DBA/2 mice were inoculated intraperitoneally, intra-peritoneal tumor with ascites developed within 20 days afterinoculation.

Adult Swiss ICR mice inoculated intravenously with 0.2 ml ofthe same cell suspension generally remained free of leukemiaduring a 3-month observation period. However, Swiss miceinoculated with suspension of spleen from the DBA/2 micebearing the tumor resulting from the tissue culture cell bioassay,developed leukemia, indicating that the virus was still associatedwith the cells. The amount of virus synthesized by the tissue-cultured cells is generally insufficient to infect adult Swiss micebut is a most effective antigen for immunizing them (3).

DISCUSSION

Attempts to plate leukemic cell lines in agar under the specialconditions of humidity recommended by Sanders et al. (6) metwith no success since the cells appeared to disintegrate after thethird day. However, it was noted that when plates of semisolidagar had some areas of condensed water, the cells not only survived longer in those areas but also multiplied as long as fluidpersisted. These findings suggested that the agar surrounded byliquid medium might provide a more favorable environment for

the desired colonial type of growth. This proved to be the casefor the cells adapted readily to this combined medium and themethod permitted precise examination of both cell and colonymorphology.

In our studies, the number of colonies actually in each agarpearl was lower than expected from the calculated number of cellsdistributed in each plate. One possible explanation for this discrepancy, discounting any errors due to inaccurate counting ordiluting, is that every cell that passes the trypan blue test doesnot necessarily have the capacity to multiply. It may be thatonly stem cells possess the ability to proliferate indefinitely.Other cells in more advanced stages of maturity may have alimited growth potential. This may account for the fact that somesingle cell isolates had been observed to undergo a number ofdivisions but did not progress to form clones. It was, therefore,impossible to bioassay them.

The cloning technic described, permitted the analysis of theprogeny of single cell isolates. Each clone contained a mixedpopulation of primitive cells and proerythroblasts. This may bedue to the fact that either the primitive cell, presumably anerythrocytic precursor, is the only cell capable of propagatingto the extent necessary to form a colony, or (under still as yetundetermined conditions) proerythroblasts may give rise toprogeny which possess the ability to dedifferentiate to stemcells. The first of these possibilities appears more likely. Theevidence suggests that the primitive cell characteristic of themurine leukemia under study (2) is probably an eo'throcytic

precursor.

REFERENCES

1. Dulbecco, R., and Vogt, M. Plaque Formation and Isolation ofPure Lines with Poliomyelitis Viruses. J. Exptl. Med., 99: 167-182, 1954.

2. Friend, C. Cell-free Transmission in Adult Swiss Mice of aDisease Having the Character of a Leukemia. J. Exptl. Med.,106: 307-318, 1957.

3. Friend, C. Immunologie Relationships Among Some of theMurine Leukemia Viruses. In: W. J. Burdette (ed.), VirusesInducing Cancer, pp. 51-60. Salt Lake City: University ofUtah Press, 1966.

4. Friend, C., and Haddad, J. R. Tumor Formation with Transplants of Spleen or Liver from Mice with Virus-Induced Leukemia. J. Nati. Cancer Inst., SS: 1279-1289,1960.

5. Friend, C., Patuleia, M. C., and deHarven, E. ErythrocyticMaturation in vitro of Murine (Friend) Virus-Induced Leukemic Cells. NCI Monograph 22, 505-522, 1966.

6. Sanders, F. K., Burford, B. O., and Borenfreund, E. Bacteria-like Growth of Mammalian Cells. J. Cell Sci., in press.

7. Sanford, K. K., Earle, W. R., and Likely, G. D. The Growthin vitro of Single Cell Isolated Tissue Cells. J. Nati. CancerInst., 9: 229, 1948.

8. Wildy, P., and Stocker, M. Multiplication of Solitary HeLaCells. Nature, 181: 1407-1408,1958.

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FIG. 1. A single cell in an agar droplet. A. Edge of the agar; T, Trap formed by the etched area on the plastic Petri dish which con

fines the agar droplet. X 1060.FIG. 2. A colony in the agar pearl on the 7th day of cultivation. X 840FIG. 3. An agar pearl containing 4 macroscopic colonies on the 21st day after plating 10 cells.FIG. 4. An agar pearl showing confluent growth on the 21st day after plating 1 X IO4cells.

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FIG. 5. A clone of Tj^pe I morphology, growing on a droplet of agar. Arrow notes flat cells moving away from the loosely packed

cells of the clone. X 840.FIG. 6. A clone Type II morphology the cells of which are densely packed in a confined area of growth. A, Edge of agar. X. 540.FIG. 7. A clone of Type III morphology containing many dead cells intermixed with viable cells. Giant cells are present. X 840.FIG. 8. Preparation of cells derived from a single cloned cell. Primitive cells, proery throblasts, and basophilic normoblasts are present.

Wright stain, X 1000.

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1967;27:726-730. Cancer Res   Maria C. Patuleia and Charlotte Friend  Cells: Isolation of Single Cells in Agar-Liquid MediumTissue Culture Studies on Murine Virus-induced Leukemia

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