Aust. J. exp. Biol. med. Sci. (1966), 44. pp.

287-300

THE GROWTH OF MOUSE BONE MARROW CELLS IN VITRO

by T. R. BRADLEY' AND D . M E T C A L F -

(Frnni the Department of Physiology, University of Melbourne; and the Cancer Rescarcli Unit, Walter and EHza Hall In.stitute, Parkville, N.2, Vietoria). {Accepted j or pidiliciilion lOth March, 196S.)
Summary. A simple in vitro technique is de.'iCTihc'd for the ^niwth nf colonies trorii single cell suspensioiiM of iiioii.se lione marrow. The system involves the plating of marrow cells in ayar on feeder layers of other cells, those from 8-day-ol<l mouse kidney and 17th day mouse embryo bcinp: shown to he the most efficient types of feeder layers. Approximately 400 folonies per 1X10'' nucleated marrow colls were jjrown, using kidney cell feeder layers. A linear relatioTiship between the nnmher of cells plated and tlie number of colonies developing was demonstrated. In comparison with the marrow cells, lymph node or thyinns eells did not form colonies, but a .small number of colonies wa.s formed nsing spleen cells. Early in the development oi the colonies the dominant cell type was a large monoimclear cell with cytoplasm filled witli j^anuk-s .staining metaeliromatically with tohiidine blue. With growth of the colony, cells with ring or hoiseslioe sliapcd nnelei appeared, and a progression with further eolony jjrowth to smaller eells with segmented nuclei similar to polymorphonuclear l)Iood c<'lls was observed.

INTRODUCTION. A variety of teehni(iue-s has been used differentiation of bone ma]ro\\ cells in recently by Woodliff (1964). However, been described wliich allow tjuantitative Ul vitro using large numbers of eclIs. in attempts to study the growth and vitro. These have lieen reviewed as yet, simple teehnitiues have not work on bone marrow cell growth

The method described here depends on tlie nse of feeder layers and allows large numbers of colonies to be grown from bone marrow single cell suspensions, the i.solation of individual colonie.s at variou.s times of incubation and snbsetinent study of staining reactions and functional activity of tlie colony lorrninii; eells and their progeny. 1 Supported by grants from the Anti-Cancer Council of Victoria and the Anna Fuller Fund. New Haven. Connecticut. - Snpportetl by the Carden Fellowship Fiiitd of the Anti-Cancer Council of Victoria.

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T. R. BRADLEY AND D . METCALF

MATERIALS AND METHODS.

Mvdniin. Eagle's minimal essential medium (Eagle, 1959) siippleniented with sudium pyruvate 1 mM, 1-serine 0-2 niM, foetal calf scniin (10%) and trypticase soy broth (10% Baltimore Biological Laboratory) was used for all e.xperinients (ElOlO). The medium was made np as tloiible strrngth (E1010x2) for dilution witii equal voliimos of agar. Mice. Inhred C57BI mice were used for all experunents. Prcpartilion of cell smpenshnfi. All tissues were removed ascptically and treated .is follows: Bone marrow. Femora were dissected free of mascle. removed by dislocation at the knee-joiut and cut with seissors at the projciinal end. Using a sterile syringe and 21 C. needle. 2 ml. of ElOlO was foreed through each femur and the extrnded marrow pipetted up and down in plastic tiii)es to give a su.spen.siou of single cells. The feiiiora of 8-day-old lnke were cleaned, minced with scissors, pipetted up and down, clnmps allowed to settle aud the supernatant single cell su.spensions taken of with a Pasteur pipette. Thymus. The glands were luinccd with scissors, iiipetted, clumps allowed to settle and the supernatant single cell suspensions taken ofE with a Pasteur pipette. Splcf^n. As for thymus. Kidney. Tlie required uumber of kidneys were minced with scissors, tiypsinized (0-2% trypsin (Difco 1:250) in Hanks' balanced salt solution) (B.S.S.) in a suspension culture flask, centrifuged at 800 r.p.m. for 10 min., washed twice with ElOlO and resuspended in ElOlO. Liver. Portions of liver were minced with scis.sors and pipetted in Hanks' B.S.S. without Ca++ or Mg+ + , clumps were allowed to settle, the supernatant pipetted off, centrifuged and the cells resuspended in ElOlO. Embryo. 17th clay embrj'os were decapitated, luinccd with scissnrs and trypsfiu'zeil in the same way as the kicbiey. Peritonrol cavity celh. 10 ml, of Hanks' B.S.S. were injected intraperitoneally iuto eacli mouse, immediately withdrawn, centrifuged at 800 r.p.m. for 10 min. and the cells resuspended iu a small volume of ElOlO. Blood. Blood was centrifuged in narrow tulies, the bully coat layer removed aud resuspended in ElOlO. Cell counting. All counts were made using a standard haemocytometer. obvious red cells being excluded from the eouuts. Plating procedure. For all experiments 60 nun. pyrex glass petii dishes were used. Basal layers of nutrient agar or nutrient agar containing various cell suspensions as "feeder layers" were plated by mixing E1010x2 with an equal volume of 1% agar (previously melted and cooled to 40) and 5 ml. idiquots poured per dish. Bone marrow single cell suspensions were then plated on top of the basal layer by suspending the cells iu E1010x2 mixing with an equal volume of 0-6'i agar (previously melted and cooled to 40*^) and 2 ml. aliquot.s poured per dish. The dishes were incubated at 37" in a humid incubator continuously gassed with 5? CO., in air. Colony counting. Colonies developing in the top agar layer were counted, without staining, using a binocular dissecting microscope (X16) with ligliting offset to the side of the dish. Examination of colonies. Colonies to be stained were sucked up with a small amount of surrouudiug agar in fine Pastenr pipettes and deposited on microscope slides. For examination of nuclear morphology a drop of 0-5% aceto-oreein in 45!S atjueous acetic acid

MARROW COLONIES IN VITRO

289

was added to each colony for 30 min. The aceto-orcein was then drained off\ a drop of glycerol added and a covcrslip placed over the colonies. For Wright's aud toluidine blue staiuing the colonies were fi.xed with methyl alcohol and then stained in the usual manner. To detect nucleated red cell precursors the method of Lo Bue, Dornfest, Gordon, Hurst and Qiiastler (1963) was used. Estimates of the viability of cells in mature colonies were made using nigrosin (Kaltenbach. Kaltrnbach and Lyons, 1958). One ml. of a 0-5% solution nf uigrosiii in ElOlO was added to the dishes and allowed to penetrate the agar layer containing the colonies. The colonies were then jiiekcd np and examined microscopically.

RESULTS. When bone marrow, tliymns, blo(Kl and spleen cells from 2-month-old C57B1 mice were plated as single eell suspensions in thin Q-3% nutrient agar layers on top of ()-5% nutrient agar layers, only tlu; bone marrow cells showed a tendency to develop into colonies. The growth was limited, the number of cells per colony lieing usually lietwecn 20 and 50 after 14 days' incubation. These very small unstiuuilated colonies aie referrt-d to us micro-colonies. The effect of cell suspensions in the bottom agar layer on the growth of tlie bone marrow cells in the upper layer was inve.stigatcd, since feeder layers
TABLK 1,
(irou-th nf boiif emrrov> cnhnie.i from, i' monlh olif mice on various feeiler Itii/prs.

Ago of donof animals for feeder layers

Feeder layer Suurc-f) of c-ella

Nil

No. of cellH
(X 10")

No. of (.'oionies' (per 1 K 10'' cells) 0^

Tia3Us from 2 m,->nth old mice

Thymus
10

Spleen Kiduoy Liver Peritoneal Cavity cells Thymua Spleen Kidney Liver

r>
iO 5 10 10 10 .1 JO
iy

7 45 2 11 79 98 0 0 0 0 4 21 0 5 472 547 2 8

Tissues from 8 day old mice

10 .5 10 10

\lth day embryo

Whole embryo
10

Colonies couiiLod at tl) days of int^ubatiou. Each value is average of 2 replicates from each of 6 mice = 12 obBervationa.

290

T. R, BRADLEY AND D . METCALF

have been iLSt-d successfully in Iiciuid stationary culture to facilitate tlic giowtli of some cell types {Puck, Marcu,s and Ciecura, 1956). Cell suspensions from some tissue.s were found to be capable of stimulating the growth of colonies from the marrow cells. These colonies contained up to 2,000 cells and are referred to as macro-colonies. All colony counts are counts of macro-colonies only. Table 1 shows the effects of various feeder layers on the growth of the bone marrow cells. Cells from 17th day embryo,s increased the growth of the bone marrow cells, both the number of colonies and the size of individtial colonies. Using cells from tissues of 8-day-old mice as feeder layer cells, only those from the kidney were successful in indueing bone marrow cell growth. Although the S-day-old kidney cells regularly stimulated growth of more colonies than did the embryo cells, colony sizes were approximately the same on both types of feeder layer. A fairly eonsistcnt difference between the kidney and embryo feeder layers was that colonies growji on kidney cells were at maximum size at 10 days, whereas the eolonies grown on embryo cells were slightly slower to develop and usually reached a maximum size at 12 days. Thymus cells horn 8-day-oId mice showed a slight feeder layer activity at a level of 10 X 10" feeder cells per dish, but the colony size and numbers were not comparable with those obtained using feeder layers of embryo or kidney cells. Spleen eells showed only a very slight aetivity and liver cells no activit\' in stimulating marrow eell growth.

Fig. 1. Colonies of 2-nionth C57B! nioiise bonr nuirrow cells grown in vitro on 8-clay C57Bi kiduey feeder layer cells; incubation time 10 days (XIO).

MARROW COLONIES IN VITRO

291

Cells from 2-nK)nth-()Icl tissues were not so siiecessfiil as feeder layers, altlioiigli kidney cells showed some activity. Thymus cells from 2-inonth-old mice showetl slightly more activity than those from 8-da>-okl mice, but again the colonies were not as well developed as those grown on embryo or 8-daykidncy feeder cells. Fig. I shows the macro-eolonies on a portion of a dish in which kidney feeder cells from S-day-oId mice were used for the growth of bone manow eells from 2-m(jnth-old mice. The pattern of bone marrow response was not appreciably changed when bone marrow eells from S-day-oId miee were plated on varions feeder layer cells (Table 2). However, the cokmy sizes and nnmbers were not as large as those found using bone marrow cells from 2-month-old mice.

TABLE -2. of hone mnrrow colonies from 8 dni^ old mice on vanous feeder hn/pr-^. Feeder layer Agt! of donor animals for feeder layers Source of cells Nil Tissues from 8 day old mice Thymus Spleen Kidney Liver nth day I'mhrijo Whole embryo 10 T', 10
i)

No. of foedt^r cells (x 10)

No. of colonies^ (per 1 X U) cells) 0^ 0 0 0 0 156 185 8 21 (i8 HI

10 5 10 5 10

(.'olfjiiLoa countfil iit 10 days of iiieubation. Eafh vahu! is tlio averago of 2 replicattis from eath of 0 mice ^ 12 observations.

The abilit)- of embryo and 8-day-kidncy feeder cells to promote growth of cells from other tissues was investigated by plating bkwd, spleen, thymus and lymph nwle cells as the target cells in the upper agar layer compared with those of bone marrow from the same animals. No colonies were fonnd with thymus or lymph node cells on either kidney or embiyo feeder layers and only an occasional colony with blood cells on kidney feeder layers. With spleen cells a small number of colonies regularly developed on the kidney feeder layers, but these were always mueh smaller than those found with bone marrow cells (Table 3).

T. R. BRADLEY AND D. METCALF

TABLE 3.
grou-tli of col on ie.') from rniriou.'! fV.swf/c.v on kiihiey ami enihri/ofepi/er li'iiprs.

Tar {jet cells Blood

Feeder layer cells (;") X 10 o'i'lls) NIL Kidney (8 day) Einbryo NIL Kidney (8 day) Einbryo NIL Kidney {8 day) Embryo NIL Kidney (8 day) Embryo NIL
Kidney (8 day) Embryo

No. of colonies' {per 1 x 1 0 ^ cells) 1 0 0 17 I

ooo

Spleen

Thymus

Lymph Node

0 0
0 384 I'.'-. 6

Bone Marrow

s rountfd at 10 days' incubation. * Kafih vftlue ia tbe average of ! rojjlicates from each of 4'niict! = 8 obsprvfitions.

In order to demonstrate tluit the colonies counted were solely in the upper agar layer, the colonies in a number of dishes were counted, then the top layer was removed by tilting the dish, allowing the npper agar layer to slide off, and the dish rc-inspected. No colonies were left after this procedure. Further, when nutrient agar withotit cells was plated on kidney or embryo feeder layers, no colonies were found in tho upj^x'r agar layer, indicating that the feeder layer cells were not capable of migiating into the tipper layer. It was also noted that no colonies formed in the 0-.5% agar basal layer from kidney or embryo cells.
TABLE 4.
The effect of increasing ugar concentration oti the growth of bone marrow roiovie-s.

Upper agar layer concentration 0-3% 0-4%

O-r.%

No. of colonies On 8 day old mouse kidney feeder cells. * 393^

286 42

On 17th day mouse embryo feeder cells.' 312

206

'2'1

5 X 10" eells/flml. feeder layer, 0-5^;, ajiar. Eaeh observation is the average of i replioato observations.

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293

The growth of bone marrow cells from 2-inoiith-old mice on 8-day-kidney and embryo feeder layer eells was tested not only in 0 3% bnt also in 0-4% and 0-5% agar. Growth was sliglitly less in 0-4% agar and severely restricted in 0-5% agar layers (Table 4). Using S-day-oId mouse kidney feeder layers, an experiment was made plating different numbers of marrow eells in the upper layer and the maerocolonies were coimtod after 14 days' incnbation. Fig. 2 shows the plot of

3OO-

y = - 2 9-1-0 OOO42

2OO-

lOO-

3 4 5 6 7 a NUMBER OF CELLS PLATED X 10

15 62 X

10

CELLS PLATED NUMBER OF COLONIES EXPT 1 EXPT. 2

I2S <,05 2 5x10^ 5 X 10^ I . I O ^

7 .4 3 .3

8 ,8 10 J O

2 4 . 22 23

46 , 4 8 42 .49

98 .132 IB6,223 4,B.3,2 IOB,9O 202,206 398.48O

Fig. 2. The relatioiis)iip between the iiiiiiiher of 2-iiionth C57B1 lunie nuirrow cells plated and the nnmher of (olonies developed on 8-day C57B1 kidney feeclfr layers (5 x 10" cells per dish); incubation time 14 days,

the nnmber of ailonie.s obtained against the number of eells plated and the data from the experiments. The dose response relationship is a linear one with the line very nearly passing through the origin on extrapolation.
Examination of colonies. 1. In situ. Tliree types of macro-colonies formed on either embryo or kidney feeder layers (a) eompaet clusters of eells occurring at any level through the agar layer, (b) diffuse colonies which very often extended from the bottom to the top of the agar layer, and (c) monolayer type colonies usually on the

T. R. BRADLEY AND D. METCALF bottom of the agar layer. No consistent differenees in macroscopic colony morphology were found which eould be ascribed to a difference in feeder layer cells. 2. Microscopy. Examination of micro-colonies (20-50 cells per colony) grown for 14 days on nutrient agar without feeder cells showed, with acetoorcein .staining, that the dominant eell type was that of a large (20-25 jx diameter) mononuclear cell with the nticleus either central or eccentric and occupying approximately one-third of the cell (Fig. 3). With Wright's stain

Fig. 3. A micro-colony of 2-month C57B1 mouse bone marrow cells grown on nubient agar and stained with aceto-orcein; infuiiation time 14 days. All of the cells are of ihe mononuclear type {X400).

the nuclei often stained light green and the cytoplasm was filled with purple red granules (0-1-0-5 /x diameter). Tliese granules stained metachromatically with toluidine blue. Macro-eolonies were examined at various stages during their development. One hundred and twenty colonies were picked up after 4-6 (la\s of growth on kidney feeder layers and were .stained with aceto-orcein. OF these 110 were composed predominantly of large mononuclear eells similar to those previously described in the control micro-colonies. llowe\er, the colonie.s often contained large eells with ring or horsc-shoe-shaped nuclei and very little cytoplasm (Fig. 4). The other 10 colonies were found to be \ery nein'ly pnre cultures of the large cells with ring or horse-shoe-shaped nnelei. Mitotic figures were numerous, an average of 4% of cells being eonnted at various stages of mitosi.s.

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295

Fig. 4. Portion of a macro-colony of 2-nionth C57B1 mouse bone murrow cells grown on 8-day C57BI kidney feeder layer cells and stained with aceto-oreeiii; inci!))iition time 6 days. Note mitotic figure and a number of eells with ring or liorse-slioe-shaped nucld. (X400.)

Fig. 5. Portion of a macro-colony of 2-m)nlh C57B1 mouse bone marrow eells; grown on 8-diiy C57BI kidney feeder layer cells and stained with aceto-orccin; incubation time 8 lays. Note the presence of a mitotie fij;ure, large mononndear cells, eells with ring-shaped imdei and cells witli partially segmented nuclei. (X400.)

296

T. R. BRADLEY ANO D. METCALF

Colonies examined after 6-8 days of growth showed greater variation in size and types of cells, the principal change being the appcariuice of stages of segmentation of nuclei in more of the cells uiul the iippearanee of smaller cells (approximately 10 ju, diameter) with nuclei similar to polymorphonuclear marrow cells (Fig. 5). Colonies examined at later stages showed an increase in the numbers of small polymorphonuclcar-like cells. M^hen stained with Wright's stain or toluidine blue, the cytoplasm of the large cells of the macro-colonies w^as filled witli granules staining metachromatically and indistinguishable from those described in control micro-colonies (Fig. 6). As the macro-colonies matured, the smaller cells developing were often

Fig, 6. Portion of a macro-colony of 2-iiiontli C^TIil iiumse hone marrow t-t'lls grown on 8-day C.57BI kitlney feeder layer cells and stained with toliiidine blue; iiicnhation time -1 days. Note the large numbers nf granules present in the cytophism of nciirly ;ill eells. (.\ 1000.)

poor in, or devoid of, the.se cytoplasmic granules, but the large cells in such colonies were rich in granules. None of the cells in mature colonies showed evidence of cytoplasmic haemoglobin when stained with tlic method of Lo Bue et al. {1963). When colt)nies were tested with nigrosin at 8-10 days' incubation, very few cells in each of tle colonies tested took up the d\'e, indicating that the majority of the cells could be classified as viable.

MARROW COLONIES IN VITRO DISCUSSION.

297

The technique described is a simple reprcKluciblc method for the growth of large colonies from bone marrow cell ix)pulation.s, thus allowing quantitative experiments to be conducted with these cells. The regression data obtained is compatible witli the assumption that the colonies arise from single cells, but further studies are necessary to establish this. It is interesting to note that the results obtained with this in vitro technique are comparable with those of Till and McCull(]ch (1961) who measured the number of colonies developing in the spleens of irradiated C57B1 mice after intravenous injection of bone marrow cell suspensions. In their experiments Till and McCulloch obtained an average of 11 colonies x^er 1 x 10^ cells injected, whereas the present technique yields an average of 40 colonies for the same number of cells plated. In this in intro techni(iue, however, there is as yet no evidence of development of erythroid cell colonies, as Till and McCulloch found with their in vivo technique. The metachromatic staining reaction of the cytoplasmic granules of the large mononuclear cells with toluidine blue appears to be evidence for their classification as mast cells. However, the possibility must be considered that the granules are composed of agar taken up by the cells during growth. Agar (an acidic polysaccharide) also stains metachromatically with toluidine blue and Dougherty (1961) has shown that cells in vivo and in vitro exhibit cytoplasmic granules following treatment with a variety of sulphated polysaccharides. If the large mononuclear cells are mast cells, then it would appear that the control micro-colonies (the majority of cells in the early stages of growth of the stimulated macro-colonies) and variable numbers of cells in the more mature macro-colonies are mast cells. Consequently, the large numbers of cells with nuclei typical of non-segmented cells of the myeloid series and, later in the colony development, of smaller cells with nuclei similar to polymor^jhs would be considered as progeny of these mast cells. However, if the metachromatic granules are "urtefact" agar granules, then the large mononuclear cells could be considered as the stem cells some of whose progeny undergo growth and maturation in vitro which is fairly typieal of cells of the myeloid series. In tlie light of the results obtained with acetoorcein staining this view seems the most tenable one at present, although such interpretations are necessarily tempered by consideration of the possibility that colonies do not arise solely from single cells. For instance, it is possible that initial g)<nvth of one cell type may occur but that on expansion of the colony to a level where other cells are contacted these latter eells may then be stimulated to divide and contribute progeny to what appears macroscopically to be one colony. Assuming a 100^ cloning efficiency of the cell types alile to grow in this system, the colony forming cells would be present at a level of about 0-05% in

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T. R. BRADLEY AND D . METCALF

these bone marrow cell suspen.sions. Since this level is low, it is obvious that development into a more efficient system will dcpi'iid on siicccssfnl fractionation of bone marrow LCII suspensit)ns similar to lliat n!:id by Cudkowitz, Bennct and Shearer (196-1). ind at the same time MKII a technique may provide evidence of the cell tyjjes capable ot giving rise to colonies. The lack of grow th of blood, thymus and I\ niph node cells even on kidney feeder layers suggests that there are \'iry few or no cells in these tissues with growth potential equivalent to those of the bone marrow cells. However, there may be \'ery small numbers of sticli cells in these tissues which dillCrcnt conditions of incubation might disclose. There is uiidoiibtcclly a lairK' constiuit small number of cells with growth potential present in adult spleens. With the staining techniques used IKTC no major differences between spleen and bone marrow colonies ha\e been fouTid, but further tests arc necessary to ascertain the identity of the colony fonning cells in these tissues. The growth of colonies from the marrow cell suspensions depends, not only on the use of the appropriate feeder la\er cells, but also on the use of soft agar as the supporting medium. In contrast to statioiiar\ h({uid cultures, neither stem cells nor their progeny are required to adhere to a substrate sucli as glass or feeder cells and are not lost in refeeding procedures. The superiority of kidney cells as feeder ia\ers tends to suggest that these cells contribute something s^x-cific to stimulate the gro\\tli of the bone marrow cells. Kidney has been suggested as a source of leukopoietin G (Bierman, 1964) and of er\ thropoictin (Jacobson, Coldwasser, Fried and I'lzak. 1957). However, in the present expc^rimcnts there is no evidence of development of cells of the erythroid series. Also, Penington (1963) has produced evidence that eiythrt)poietic acti\it\' can be extracted from li\(r tissue as well as kidney and, in our experiments, liver cells were cmnplclcly inactive in stiuuilating the growth of the marrow cells. Thus it would seem reasonable to examine the possibilit)- tliat the kidnc\' cells are acti\c b\' \irtue of secretion or release of substances similar to leukopoietin G, or that they act merely by some less specifie conditioning of the culture medium. The slight but definite activity of thymus cells as feeder layers, whilst not impressive compared with those of embryo and kidney, is enough to suggest that tlie role of thymus cells as feeder layers for bone marrow cells should be investigated further. The method appears to ofFer jTOssibilities for more quantitative' examination of problems connected with proliferation antl differentiation of bone marrow cell types. The meditim used in the present sttidy was a simple one, and changes in tliis, the semi-solid support medium and the addition of possible humoral factors to the culture uitdium may provide more precise techniques suitable tor the examination or tlirection of diilerentiative trends in bone marrow cells.

MARROW COLONIES IN VITRO

1 1 1 icrograpiis.

299

We are indebted to Mr. j . Smith for preparation of tlie plioto-

REFERENCES. wtAN, II. R. (1964): 'Characteristics Lo BUE, J., DORNFEST, B . S.. CORDON, of k'likopoietin G in animals ;ind man.' A. S., HuBST, j . . and QUASTLEH, H . Ann. N.Y. Acad. Set, 113, 753. (1963): 'Marrow distribution in rat femurs determined by cell enmneration Cvimowrr/., 0.. BENNET. M., and SIIEAHEH, and Fe"'-' labeling.' I'loc. Soc. E.xp. Biol. G. M. (1964): 'Pluripotent stem cell and Med., 112, 1058. function of mouse marrow "lympliocytc".' Science, 144, 866. PENINCTON, D . C . (1963): 'Erytluopoiotin in tissues; features of the renal erythroDouGiiEHTV, T. II. (1961): In ConfLTcnte poictiu-produciiig system'- in Memoir.s of on the Bioloffy of Connective Tissue Soc. for Endocrinol. No. 13. 201. Gflls. Arthritis and Rheumatism Conference Series No. 7, 219. PUCK, T . T.. MARCUS, P. L, and C^rECUiiA, EACLE, H . (1959): 'Amino acid metabolism in mammalian cell cultures,' Science, 130, 432.
JACOBSON, L. O.. GOLUWASSER, E., FRIED,

S. J. (1956): "Clonal growth of mammalian cells in vitro.' J. Exp. Med., 103, 273.
TILL, J. K.. and McCuuLocn, E. A.

VV.. and Pi.zAK, L. (1957): 'Role of the kidney in erythropoiesis.' Nature, 179, 633.
KALTKNDACH, J. P.., KALTENBAGH, N . . and

(1961): 'A direct meas\irement of the radiation sensitivity of normal mouse bone marrow cells." Rttdlutioii lies., 14, 213. VVooDLiFF, II. J. (1964): 'Blood and bone marrow cell cultm-e.' Eyre and Spottswoode, London.

LYONS, W . E . {1958): 'Nigrosin as a dyt' for differentiating live and dead ascites cells.' Exp. Cell Res., 15, 112.