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Experimental Cell Research 104 (1977) 95-99

THE EFFECT

OF VARIOUS

COLONY-STIMULATING HUMAN

MARROW

SOURCES

ACTIVITY IN AGAR

OF

ON NORMAL CULTURE

I. D. C. DOUGLAS and B. M. J. PICKERING Royal Marsden Hospital and Institute of Cancer Research, Downs Road, Sutton, Surrey, UK

SUMMARY A series of normal human bone marrows was induced to form colonies in agar in vitro by means of peripheral blood white cell feeder layers or medium conditioned by various cell cultures as sources of colony-stimulating activity (CSA). The number of colonies grown from any marrow was found to vary, depending on the CSA used but no one source produced a consistently higher colony yield from ah marrows. This indicates that caution is required in the interpretation of results when the agar colony assay is used for comparative studies of precursor cells in human bone marrow until a consistent source of CSA can be used for all such experiments.

In vitro colony growth from bone marrow cells was achieved initially with mouse [ 1, 21 and later with human marrow [3]. There are two basic requirements of the method: immobilization of the dividing cells and the provision of colony-stimulating activity (CSA), without which cell growth is limited. For comparative studies of marrow function using this technique, it is important to maintain constant conditions within the experimental system. Many methods have been described, all basically similar, with perhaps the most notable variation being the source of CSA. This has resulted in a wide range of “normal” values and can clearly lead to confusion when comparisons are attempted. This survey reports the effect of exposing marrow cells simultaneously to a number of different GAS. 7-761812

MATERIALS

AND METHODS

One hundred and eightytwo marrow samples were chosen as being cytologically within normal limits. In all, 375 experiments were performed on these specimens.

Preparation of marrow samples Bone marrow was aspirated, usually from the anterior iliac crest, using a 2-syringe technique. A small sample was withdrawn into a dry syringe and smears were made. A further aspiration of 2-5 cm3 was made into a second syringe, previously rinsed with heparin (1 GO0U/cm3, without preservative). This was allowed to sediment at room temoetature for 30-60 min. when the cell-rich plasma was removed. The cells were washed 3 times in modified McCoy’s 5A medium [3], resuspended and counted using a haemocytometer.

Preparation offeeder layers Ten to 20 cm3 of venous blood was taken into hepatin from each patient at the time of marrow aspiration and treated in the same way as the marrow. The appropriate volume of the washed cell suspension was added to a mixture of molten agar and modified McCoy’s SA medium plus 15% foetai bovine serum (of which a single batch was used throughout), at 38°C. The final Exp

Cell

RPS 104 (1977)

96

Douglas and Pickering

Table 1. Overall results from marrows studied No. of marrows tested Source of CSA

Total

Colonies

White cell feeders Colon CM HEK CM Skin CM Foetal lung CM Macrophage CM Dialysed colon CM HeLa cell CM Endotoxin mouse serum

E 78

47 43 45 9 0 1 0 0 0

31 3 4 11 3 11

cell count was 10D/l and agar concentration, 0.5%. Aliquots of 1 cm3 of the mixture were pipetted into 35 mm plastic Petri dishes, resulting in lo6 cells/plate, and allowed to gel at room temperature.

Preparation of conditioned media (CM)

Clusters

Contaminated

No growth

; 3 3

10 4 2 1

:: 28 18

1

In ah cases, the Petri dishes were sealed in humiditied plastic boxes, gassed with 10% CO* in air and incubated at 37°C for 10 days. Colonies were counted using an inverted microscope at x40 magnification. Any aggregate of more than 50 cells was scored as a colony and, if less than 50 cells, as a cluster.

RESULTS

Skin and colon. Cells were obtained from biopsies of

normal skin and of colon at laparotomy. The cells were established as monolayer cultures and had been growing for 3 months when used to condition medium. Modified McCoy’s 5A medium plus 10% foetal bovine serum was harvested from the cultures twice per week. Human embryonic kidney (HEK). Kidneys were obtained from foetuses aged from 16-19 weeks of gestation. Each pair was trypsinized, the cell suspension washed in Fischer’s medium and 1.2x lo8 cells seeded into medical flats with 10cm3 of Fischer’s medium plus 10% foetal bovine serum. The bottles were gassed with 5% CO, in air and incubated at 37°C. Medium was harvested each week. CM from all cultures was centrifuged, filtered and stored in aliquots of 2 cmS, either at 4°C or frozen, the latter being thawed at 37°C before use.

Setting up of cultures Using feeder layers. Bone marrow cells were added to

agarlmedium at 38°C to a final cell count of 2x108/1 and agar concentration of 0.3% 1 cm3 was pipetted over the previously prepared feeder layer and allowed to gel. The feeder layer was controlled by setting up a plate with agar/medium overlay without bone marrow cells. Unstimulated controls of the bone marrow cells were layered over blank agar/medium. Using CM. 0.2 cm3 of CM was placed in each Petri dish. Bone marrow cells were added to agar/medium to a concentration of 2.5~ lo”/l. 0.8 cm3 of this suspension was pipetted into each Petri dish, so that the resulting volume of 1 cm3 contained 2x lo5 cells in 0.3 % agar. Unstimulated controls were set up using 0.2 cm3 of ordinary McCoy’s 5A medium in place of CM. Exp Cell Res 104 (1977)

:. 11 3 11

The incidence of spontaneous colony formation in control plates was low and colonies were few in number. Where applicable, these have been subtracted from colony numbers in the stimulated cultures. Table 2. Variation in colony-forming according

ability

to source of CSA

Results expressed as means of 34 plates ColoniesRx 1oJbone marrow cells Patient

Feeder layer

Colon CM

Ki McL Ch Pe Tu Em MO Re Ma EY An Ja Ri

173 113

192 Clusters 24 19 63 No growth 16 43

:; No growth 24 Clusters 27 5 14

f No growth

HEK CM

16 18 19 19 14

Effect of CSA on normal marrow

Table 3. Variation in colony-formation different types of CM

with

Table 5. Loss of activity storage of CM

resulting

97 from

Coloniesdx 1oJbone marrow cells

Colonies/2 x 10” bone marrow cells

Patient

Colon CM

HEK CM

Colon CM (a)

St

18 :(!I

4 13 38 Ill 19 25 48

Ha Gi Ca co Bu Tu

37 15 19 49

Late

Colon CM(b)

Me Ba

Clusters Clusters

193 173

k

Clusters 1

425

Patient

Early

Wa Ba Te

51 55 18

The relative efficacy of several sources of CSA is compared in table 1. Some sources were tested against a few marrows but there were other problems with this only but were uniformly unsuccessful in method. For example, of eight specimens of stimulating, although these marrows pro- HEK set up, only six produced active CM: duced colonies in response to other CSAs activity was most marked after about 10 tested simultaneously. Of all sources, three days of culture and was maintained for might be considered successful-peripheral some 2 weeks thereafter but fell off once the blood feeders, colon CM and HEK CM. ceil iayer thickened. Trypsinization and The simultaneous testing of several reseeding at 2 weeks did not result ‘in a sources of CSA against a single marrow al- renewal of activity. Considerable variation ‘in colony yield lowed a comparison of the relative potency of each source. Peripheral blood white cell was also observed using CM as the source feeders were effective but were neither con- of CSA (table 3). Colon CM was apparently sistently better nor worse than CM (table 2). better for some marrows than HEK (Ha) In some cases, more colonies were grown in but HEK was superior for others (Ca). response to feeders (McL) but the reverse The activity of CM from a single cell culture was also found to be inconsistent from could also be true (Tu, MO). From a practical point of view, CM was batch to batch (table 4). Storage, whether at regarded as more convenient than feeders 4°C or frozen had the effect of reducing the CSA in CM (table 5). Although a batch of colon CM stimulated colony formation Table 4. Variation of CSA in different when newly harvested, a loss of activity batches of CM from the same cell culture was apparent when it was compared with a Colonies/2x lo5 bone marrow cells fresh, active batch one month later. Patient HEK (a) HEK (b) HEK (c) This storage effect might account for some of the batch variation in table 4 but Na 50 38 21 the superiority of the earliest medium in Gr 36 36 31 Ga 27 21 21 some cases (Na, Sh) suggests that this is not 49 19 co the complete explanation and that there is 27 21 t: Ho 35 15 21 GI probably some inherent variability in CSA 35 65 Sh production by the HEK cells. Exp Cc// RPS 104 (1977)

98

Douglas and Pickering

Thus, no correlation was observed between the number of colonies obtained under the influence of different sources of CSA, nor was there any one source consistently better for all marrows.

there is therefore no reason to suppose that cells even from a single individual must necessarily produce the same level of CSA on every occasion. It has also been shown [7] that the placing of possibly incompatible cells in close proximity in this way may, in some cases, have an adverse effect on coloDISCUSSION ny formation. Evidence on the relative roles of cells in Measurement of the colony-forming ability of human bone marrow has potential clini- the production of CSA is conflicting. It was cal application since it provides a quantita- assumed by the early workers that granulotive measure of early progenitor cells as op- cytes were the primary cell type and some posed to a morphological evaluation of more recent work [8,9] appears to confirm more mature cells. It has, however, a num- this. Conversely, other investigators [ 10, ber of disadvantages, principally that there 11, 121 stress the importance of the monois no “reference” cell line nor any source cyte; and there are reports that granuloof CSA which supplies a constant level of cytes may actually be inhibitory [7, 13, 141. stimulation. This leads inevitably to a lack The properties of these subpopulations of cells almost certainly contribute to the of standardization in culture conditions. There appear to be no contributions to variability of results obtained using leukocyte feeder layers. the literature in which there is a definitive The use of CM offers an escape from discussion of the incidence of positive colosome of the disadvantages of feeder layers. ny growth. The figures in table 1 indicate that there may be a failure rate of 30-40%, To bring this comparison in line with many regardless of the source of CSA used and other series, a concentration of 20% CM which cannot be accounted for by inade- was chosen, as this seems to be the most quacies in the culture system, e.g. affecting widely used [15, 16, 171. Inspection of the foetal bovine serum or culture medium. If figures in table 2 might suggest that HEK this is a general finding, it is a primary indi- CM was superior to feeders but all the recation that caution should be used in the sults from this group of marrows are low. interpretation of results. The same is not true of colon, yet figures in Perhaps the most important variable in table 3 show that, in some cases, this is the literature is the source of CSA. In their better than HEK. The results for these maroriginal description of the method, Robin- rows in table 2 are, therefore, probably arteson & Pike [3] used a feeder layer of human factual. CM is more convenient for setting peripheral blood white cells and these have up and reading plates but variation in CSA remained perhaps the most popular feeder (table 4) and instability on storage (table 5) cells for investigation of both normal [4] suggest it is no more satisfactory than and abnormal [5] marrows. This practice is feeder layers in terms of obtaining a conopen to question, since cells from different stant level of colony yield from different individuals will almost certainly produce marrows. different levels of CSA. Furthermore, seIn order to carry out proper comparative rum CSA levels can be shown to vary, for studies on human marrow using the agar example, in response to infection [6] and colony assay, it is necessary that each Exp Cell Res I@# (1977)

Effect of CSA on normal marrow

specimen be stimulated to produce the maximum number of colonies of which it is capable; or, at least, that each marrow be exposed to the same level of stimulation as every other. Failure to comply with one of these conditions must inevitably produce numbers of colonies which will vary with the source of CSA and make precise comparisons impossible. We are grateful for the help of Dr S. D. Lawler, Director of the Fetal Tissue Bank, Royal Marsden Hospital (MRC supported) who provided the foetal kidneys.

REFERENCES 1. T$nik,

D H & Sachs, L, J cell physio166 (1%5)

2. Bradley, T R & Metcalf, D, Aust j exp biol med sci 44 (1966) 287. 3. Robinson, W A & Pike, B L, Symposium on hematopoietic cellular differentiation (ed F Stohlman) p. 249. Henry M Stratton, New York (1%9).

99

4. Morris, T C M, McNeill, T A &Bridges, J M, J clin path01 27 (1974) 776. 5. Moore, M A S, Spitzer, G, Williams, N, Metcalf, D & Buckely , J, Blood 44 (1974) 1. 6. Foster, R, Metcalf, D, Robinson, W A t Bradley, T R, Brit j haematol 15 (1968) 147. 7. Haskill, J S, McKnight, R D & Galbraith, P R, Blood 38 (1971) 788. 8. Greenbera, P L, Nichols. W C & Schrier.I S L.I New EngTmed 284 (1971) 1225. 9. Heit, W, Kern, P. Kubanek. B & Heimnel. . , H. Blood 44 (1974).511. 10. Chervenick, P A & LoBuglio, A F, Science 164 (1972) 166. 11. Golde, D W & Cline, M J, J clin invest 51 (1972) 2981. 12. Senn, J S, Messner, H A & Stanley, E R, Blood 44 (1974) 33. 13. Paran, M, Ichikawa, Y & Sachs, L, Proc natl acad sci US 62 (1%8) 81. 14. Broxmeyer, H E, Baker, F L & Galbraith, P R, Blood 47 (1976) 389. 15. Iscove, N N, Senn, J S, Till, J E & McCulloch, E A, Blood 37 (1971) 1. 16. Cowan, D H, Clarysse, A, Abu-Zahra, H, Senn, J S & McCulloch, E A, Ser hematol V (1972) 179. 17. Ogawa, M, Bergsagel, D E & McCulloch, E A, Blood 42 (1973) 851. Received May 6, 1976 Accepted July 16, 1976

Exp Cell Res 104 (1977)