Erythropoietin in Vitro
A Culture Plate Method for Marrow
E U G E N E T. B A R T L E Y , J R . , B.S., A N D E. R. P O W S N E R ,
M.D.
Nuclear Medicine and Research Services, Veterans Administration
Hospital, Allen Park, Michigan and Department of Pathology,
Wayne State University School of Medicine, Detroit, Michigan
ABSTRACT
Bartley, Eugene T., Jr., and Powsner, E. R.: Erythropoietin in vitro: A culture
plate method for marrow. Am. J. Clin. Pathol. 61: 69-73, 1974. A lowvolume, culture-plate method for the study of the effects of erythropoietin
(ESF) on bone marrow cultures has been developed. This method is relatively
rapid and conserves ESF, bone marrow, and culture materials. It is suitable for
the dissection of the mode of action of ESF and other human serum factors in
vitro. T h e culture plate, fabricated from Teflon, contains 104 wells. A culture
volume of 260 /ul. in each well contains the following: rat bone marrow
(2.6 X 106 cells) suspended in NCTC 109 medium and fetal bovine serum
mixed with test ESF and 55 Fe in rat serum. In diese cultures hemin radioactivity
in the presence of ESF is approximately two to six times the control value at 24
hours. Utilization of carrier-free iron facilitates measurement of die smaller
amounts of heme synthesized. (Key words: Erythropoietin; Tissue culture;
Bone marrow culture; Heme synthesis; Culture plate; Radioiron.)
COMPARED WITH in vivo and immunochemical methods, in vitro technics for the
measurement and detection of erythropoietin have been advantageous for the
study and dissection of the mode of action
of this hormone on the bone marrow normoblast. 2 8 1 7 2 2 2 3 Technics for both in vivo
and in vitro study and assay have been
reviewed. 18 Methods available for bone
marrow have r e q u i r e d relatively large
volumes. Lower-volume m e t h o d s for
peripheral blood using volumes in the
range described here have been available
for more than a decade. 1 1 2 , 2 0 More recently
a method employing commercial plastic
plates has beer, applied successfully to
low-volume cultures of ESF-stimulated
fetal mouse liver hematopoietic cells. 4
N C T C - 1 0 9 (MiAssociates,
Inc., Bethesda,
crobiological
M a r y l a n d ) w a s s t o r e d a t 4 C . Immediately
p r i o r t o c u l t u r e > 0 . 1 ml. of 1.0 mM.
Received June 13, 1973; accepted for publication
June 28, 1973.
L-glutamine (Nutritional Biochemical
Corp., Cleveland, Ohio) was added to 10
69
T h e s e technics have not been directly
applicable to bone marrow cultures. T h e
present p a p e r describes a low-volume,
culture-plate method for bone marrow
which requires only 0.26 ml. per well. It is
an adaptation of the culture tube technics
previously described by us a n d by
others. 1819,21,26 - 27 A comparison of the new
and old technics is presented here. T h e
culture-plate technic has resulted in a
significant conservation of time a n d
material,
Methods
Tissue cukure medium
70
BARTLEY AND POWSNER
A.J.C.P.—Vol. 61
ml. of NCTC-109; a mixture of one part (0.077 mCi., 0.28 ml.) were added to 5.0 ml.
CO2 to 19 parts air was passed through a rat serum and treated as above.
0.2-fim. filter (Millipore Corp., Bedford,
A Teflon culture plate was fabricated by
Massachusetts) and bubbled into the me- milling 104 flat-bottomed holes into a block
dium for 5 minutes. This gassed mixture of Teflon. Press-fitted glass pins were used
was used to flush bone marrow aseptically to suport a glass cover 0.1 cm. above the
from the femurs and tibias of 150-200-Gm. block (Fig. 1). Prior to each use, the Teflon
rats (Sprague-Dawley Strain, Holtzman block and its coverplate were rinsed succesCo., Madison, Wisconsin). The cells were sively in 1 N HCl, multiple changes of tap
gently dispersed by aspirating three times water, 7x detergent (Linbro Chemical Co.,
into a syringe without a needle and counted New Haven, Connecticut), distilled H2O,
electronically as previously described. 9
a n d double-distilled H2O, a i r - d r i e d ,
wrapped
in aluminum foil, and autoclaved.
Fetal bovine and rat serum (MicrobiologExpressed as fractions of the final culture
ical Associates, Inc.) p r e p a r e d without
hemolysis were inactivated, 13 divided into mixture, the components of the initial culaliquants sufficient for a single experiment, ture mixture were:
and stored at - 2 0 C.
Bone marrow cell suspension in
E r y t h r o p o i e t i n was p r e p a r e d from
N C T C - 1 0 9 c o n t a i n i n g 3.6
phenylhydrazine anemic sheep plasma. 28
x 107 cells/ml.
0.28
In brief, plasma was adjusted to pH 5.5 with
Fetal bovine serum
0.40
0.1 N HCl, heated at 70 C. for 10 minutes,
Test preparation (ESF) in NCTCdialyzed at 4 C. for 16 hours against 10
109
0.19
volumes of 0.00375 M NaCl + 0.0025 M
NaH2P04, pH 5.5, centrifuged, and stored
Subtotal 0.87
at - 2 0 C. This anemic sheep plasma fraction was passed through a 0.2-/nm. filter The final culture mixture is:
(Nalge Co., Rochester, New York) prior to
Initial culture mixture
0.87
use.
0.13
Rat serum widi 55 Fe
The activity of all preparations of ESF
was determined in the polycythemic mouse
Total
1.00
assay.5 Preparations were standardized by
8
comparison with ESF standards A and B.
For each well, 225 fil. of the initial culture
In all experiments the ESF concentration is mixture were dispensed with a repeatingexpressed as units per milliliter of the final type sterile syringe and incubated at 37 C.
volume of the culture. During the initial in an atmosphere of one part CO2 to 19
incubation (0-22 hours) prior to addition of parts air. T h e gas was passed through a
radioactive iron solution, the ESF concen- fritted bubbler at approximately 1 liter per
tration is a p p r o x i m a t e l y 15 per cent min. At 22 hours, 35 fi\. of 55 Fe in rat serum
greater. For the culture plates, 55FeCl3 in were added to each well with a syringe mi0.5 N HCl, carrier-free (2,400 Ci. per Gm.) croburet (Micro-Metric Instrument Co.,
( I n t e r n a t i o n a l Chemical a n d N u c l e a r Cleveland, Ohio) and mixed widi a sterile
Corp., Irvine, California) was mixed with pasteur pipet. Incubation was continued an
rat serum. For these cultures 55 Fe, 3.2 /u.g. additional 4 hours.
iron (7.7 mCi., 0.75 ml.) was added to each
Cultures were quantitatively transferred
5-ml. vial of serum, incubated at 37 C. for 1 from the well by repeated rinsing with cold,
hour, and stored at - 2 0 C. prior to use. 4-C. saline solution (0.9 Gm./l.). T h e comThe tube method utilized "FeCta in dilute bined rinsings were placed in 12 X 7 mm.
HCl, specific activity 15 Ci. per Gm. plastic test tubes (Falcon Plastics, Los
(Union C a r b i d e C o r p . , T u x e d o , New Angeles, California). T h e cells were cenYork). For these cultures 5.2 fj-g. of iron trifuged at 180 X g for 10 minutes. The
January 1974
71
ERYTHROPOIETIN IN VITRO
12 em
1.2
Cm
i
1.2 cm
I
—+ + +
+ + +
+ t
Mill 104 holes
0.625cm dia—^
+
i
T
4-
+
+ + +
+
+
4-
+
+
4-4-
+ + + + + + +
+ +
4- +
+
+
+
+
+
+
+
4 - 4 - +
4-
+
\ „© + +
•-«§© +
+
+
+
+
+
4-4-
4-
+
+
4-4-
4-
4-
4-
4-
4-4-
+ + + •+ 4- + +
+ +
-m
©
4-+
- 0 . 3 x 7x11cm
glass plate
+
+
1
1.2 cm
+
+
(
8cm
*5Z
-*-jo.8[-«-
J_
+
+ + +
+ + +
L.l
0.8cm
+ + +
4 glass air space pins
0.1cm air s p a c e /
/»m
1
fril'iim; '!
5 g|a88 locating
m
^
i! i l l ii •!
.LJ.JI<.JUJ
pins
press f i t into teflon
1.8cm
FIG. 1. Teflon culture block with a glass cover plate resting on glass pins.
pellet was resuspended in cold saline solution and recentrifuged three times.
A butanone-water partition method 2 4
for extracting heme was modified as follows: After the final wash, 200 /xl. of cold
distilled water and 100 /xl. of cold Drabkin's
cyanmethemoglobin reagent 7 were added
to the pellet and mixed vigorously. The
cold suspension was centrifuged at 700 x g
for one hour and 200 /xl. of the supernatant
pipetted into a small glass test tube; 20 /xl. of
1 N HC1 and 400 /xl. of 2-butanone were
added. The tubes were stoppered, mixed
forcefully for 15 seconds, and then kept at 4
C. for o n e h o u r . F r o m the u p p e r
(butanone) layer, 200 /xl. were transferred
into a small glass test tube, stoppered
loosely with cotton, kept at - 2 0 C. for 15
minutes, then vacuum-dried. Butanone
was r e m o v e d completely to p r e v e n t
butanone quenching in the liquid scintillation counting. Using liquid scintillation
fluid,18 the dried contents were quantitatively washed into vials and counted.
Bacterial cultures, both aerobic a n d
anaerobic, were used to monitor possible
contamination at each stage of the bone
marrow and culture preparation. A culture
without 55 Fe was used for total and viable
cell counts, the latter by the trypan blue
exclusion method. 3 2 5 Whenever necessary,
new medium, containers, and other materials were used in cultures prepared in tandem with the old materials to insure
continuity.
Technics for the larger-volume test tube
culture method were used as described
previously 2 except that the marrow and incubation components were modified proportionately to match those described here
for the culture plates.
Results
Hemin radioactivity increased with increasing erythropoietin concentration (Fig.
2). For the culture plate method, the lowest
concentration of ESF causing a detectable
increase is approximately 0.03 units/ml. As
the ESF concentration is increased from
0.03 to 0.22 units/ml., the response increases from 2.4 to 5.8 times the control.
T h e slope of the log dose versus response
line is 3.8, expressed as change in relative
hemin radioactivity divided by the change
in the logarithm of the ESF concentration
in units per ml. T h e standard error of this
slope is 0.3. T h e corresponding slope and
72
BARTLEY AND POWSNER
1
—I
0.03
1—i—i i i i i i
0.06 0.08 0.11
ESF units/ml
rr~
0.22
FIG. 2. Log dose-response curve: hemin synthesis vs
erythropoietin concentration. Each point represents
55 cultures for the tube method and 25 cultures for the
plate method. T h e mean count rates for the control
cultures were 859 and 178 c.p.m., respectively. T h e
grey area r e p r e s e n t s the s t a n d a r d e r r o r of the
slope ± 0.3.
standard deviation for the previous test
tube method using the same ESF preparation are similar, 3.3 ± 0.3.
In preliminary experiments a commercially available plastic plate proved unsatisfactory. Iron was incorporated into heme,
but ESF caused no increase in the incorporation. This was confirmed on two separate
lots of this plate, but was not further investigated. Sterile plastic test tubes were also
tested, but the effect of ESF was less consistent than with the glass tubes regularly used
in these and previous experiments.
Discussion
T h e method described was developed to
simplify the investigation of erythropoietin
effects in vitro. T h e use of marrow permits
study of the effects on the cells themselves.
T h e reduction in volumes shortens the time
required for marrow collections, e.g., one
rat required for the plate method vs. nine
rats for a typical test tube assay, and conserves test material and supplies. Preparation of glass culture tubes extends over a
day and a half, while a Teflon plate can be
A.J.C.P.—Vol. 61
readied within two hours. T h e plates permit rapid and repetitive pipetting and can
be filled in about a fifth the time required
for tubes.
The cells were morphologically intact in
smears p r e p a r e d a n d stained with
Leishman's stain. Approximately 90 per
cent appeared to be viable using the trypan
blue exclusion method. In our laboratory
the culture tube technic has been shown
previously to r e s p o n d to S t a n d a r d A,
Standard B, and to Step III material. 11 Although our anemic sheep plasma fraction
was of relatively low specific activity compared with these other materials, the sensitivities of the older tube and newer plate
methods were sufficient to permit the use of
this more available fraction in the present
experiments.
T h e reduction of culture volume and cell
number in the culture plate method resulted in a corresponding reduction in total
heme synthesis. In early plate cultures the
low-specific-activity 55 Fe was used, and the
net count rates of processed samples from
control and maximally ESF-stimulated cultures were about 33 and 127 c.p.m., respectively. In the experiments reported here,
carrier-free 55 Fe was used to compensate
for the smaller amount of heme synthesized. Using the carrier-free 55 Fe, the net
count rate of the isolated hemin was 178
c.p.m. in the control culture and 563 c.p.m.
in the maximally-stimulated culture. These
represent a four- to five-fold increase in
hemin activity. This may be compared with
the approximately hundredfold increase in
the amount of iron radioactivity added. Assuming that the concentration of endogenous iron is approximately the same in all
plasma samples, this increase in radioactivity added should have produced a hundredfold or greater increase in iron specific
activity in the culture medium. The reason
for the much smaller increase in hemin
specific activity is not apparent.
Iron may be assumed to be bound to rat
transferrin during the one-hour incubation
before its addition to the cultures. T h e total
concentration of the iron added was less
than 1 fig. per ml., or less than a third the
January 1974
ERYTHROPOIETIN IN VITRO
total iron-binding capacity of rat transferrin. This makes it p r o b a b l e that the
radioiron is presented to the cells primarily
on rat transferrin, not on fetal bovine
s e r u m . 1 0 1 4 T h e r e l a t i o n s h i p between
hemin radioactivity and heme synthesis in
vitro has been considered previously. 23
Cantor has described the use of commercially available plates for the culture of fetal
mouse liver hematopoietic cells.4 Volumes
used were similar to those described here.
He found that 5 per cent CCh was detrimental to the growth of these cultures, and
substituted N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid in sealed cultures
to maintain culture p H . O u r cultures
grew well in Teflon culture plates when
exposed to 5 per cent CO2. T h e possibility that plastic is toxic to bone marrow
cells only in the presence of CO2 was not
investigated.
Antibiotics were not used. Preservatives
in commercially prepared solutions such as
55
Fe, heparin, and saline solution were
avoided.
Acknowledgments. Dr. Isadore A. Bernstein, University of Michigan Medical School, Ann Arbor, prepared
the erythropoietin fractions, Mildred L. Werle provided valuable assistance, John Brnetich provided the
illustrations, and the Medical Illustration Service of
the Allen Park Veterans Administration Hospital took
the photographs.
References
1. Arakaki DT, Sparkes RS: Microtechnique for culturing leukocytes from whole blood. Cytogenetics 2:57-60, 1963
2. Berman L, Powsner ER: Review of methods for
studying maturation of human erythroblasts in
vitro: Evaluation of a new method of culture of
cell suspensions in a clot-free medium. Blood
14:1194-1212, 1959
3. Black L, Berenbaum MC: Factors affecting the
dye exclusion test for cell viability. Exp Cell Res
35:9-13, 1964
4. C a n t o r LN, Morris AJ, Marks PA, et al:
Purification of erythropoietin-responsive cells
by immune hemolysis. Proc Nad Acad Sci USA
69:1337-1341, 1972
5. Cotes PM, Bangham DR: Bio-assay of erythropoietin in mice made polycythaemic by exposure to air at a reduced pressure. Nature 191:
1065-1067, 1961
6. Cotes PM, Bangham DR: T h e international reference preparation of erythropoietin. Bull W H O
35:751-760, 1966
7. Drabkin DL: Unaltered globin, and crystalline,
synthetic (reconstituted) myoglobin. J Biol
Chem 158:721-722, 1945
73
8. Erslev AJ, Silver RK: In vitro studies of erythropoiesis. Semin Hematol 4:315-326, 1967
9. Fly MN, Powsner ER: Electronic counting of cells
from human bone marrow. Am J Clin Pathol
36:224-226, 1961
10. Gallien-Lartigue O, Goldwasser E: Hemoglobin
synthesis in marrow cell culture: T h e effect of
rat plasma on rat cells. Science 145:277-279,
1964
11. Goldwasser E, Kung C K H : Progress in the
purification of erythropoietin. Ann NY Acad
Sci 149:49-53, 1968
12. Junge U, Hoekstra J, Wolfe L, et al: Microtechnique for quantitative evaluation of in vitro
lymphocyte transformation. Clin Exp Immunol
7:431-437, 1970
13. Kabat EA, Mayer MM: E x p e r i m e n t a l Immunochemistry. Springfield, 111., Charles C
Thomas, 1958, p 123
14. Katz J H , Jandl J H : T h e role of transferrin in the
transport of iron into the developing red cell,
Iron Metabolism. Berlin, Springer-Verlag,
1964, p 118
15. Kinard FE: Liquid scintillator for the analysis of
tritium in water. Rev Sci Instrum 28:293-294,
1957
16. Krantz SB, Gallien-Lartigue O, Goldwasser E:
T h e effect of e r y t h r o p o i e t i n upon heme
synthesis by marrow cells in vitro. J Biol Chem
238:4085-4090, 1963
17. Krantz SB, Jacobson LO: Erythropoietin and the
Regulation of Erythropoiesis. Chicago, T h e
University of Chicago Press, 1970, p 118
18. Krantz SB, Jacobson LO: Erythropoietin and the
Regulation of Erythropoiesis. Chicago, T h e
University of Chicago Press, 1970, p 6
19. Morton HJ, Isaacs RJ: Cultivation of rat bone
marrow. I. Preliminary studies and some effects
of hemolyzed blood as nutrient. J Nat Cancer
Inst 39:795-803, 1967
20. Parker JW, Lukes RJ: A microculture method for
lymphocyte transformation studies in the clinical laboratory. Am J Clin Pathol 56:174-180,
1971
21. Powsner ER, Berman L: Correlation of radioactive hemin formation with morphologic alterations in cultures of human bone marrow. Blood
14:1213-1222, 1959
22. Powsner ER, Berman L: Erythropoietin-induced
DNA synthesis in normoblasts in vitro: Effect of
actinomycin-D. Life Sci 6:1713-1722, 1967
23. Powsner ER, Berman L: Effect of erythropoietin
on DNA synthesis by erythroblasts in vitro.
Blood 30:189-197, 1967
24. Teale FW: Cleavage of the haem-protein link by
acid methylethylketone. Biochim Biophys Acta
35:543, 1959
25. Tennant JR: Evaluation of the trypan blue technique for determination of cell viability. Transplantation 2:685-694, 1964
26. Thomas ED, Lochte HL Jr, Stohlman F Jr: Attempts to develop an in vitro system for the assay
of erythropoietin. J Lab Clin Med 55:311-318,
1960
27. Ward HP: An in vitro assay of erythropoietin. Proc
Soc Exp Biol Med 125:370-374, 1967
28. White WF, Gurney CW, Goldwasser E, et al:
Studies on erythropoietin, Recent Progress in
Hormone Research. Edited by G Pincus. New
York, Academic Press, 1960, pp 219-262