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The development of patent blood vessels from adult human rib marrow in tissue culture.

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T H E DEVELOPAIENT O F PATENT BLOOD VESSELS
FROM ADULT HUMAN RIB MARROW
I N TISSUE CULTURE1
W. C. WOODARD AND C . M. POMERAT
Tissue Culture Laboratory, University of T e x a s , Medical Branch, Galveston
FIFTEEN FIGURES
INTRODUCTION
Following a preliminary report (Fulton, Parshley and
Simms, '49)' White and Parshley ('51) described the outgrowth of patent vessels in tissue culture from tibia1 marrow obtained from roosters and hens. These were maintained
in Carrel flasks in a healthy state for as long as three weeks
in a non-replenished medium consisting of a clot of homologous non-heparinized plasma and Simms ' physiological salt
solution in combination with a fluid phase made up of similar
components but with a higher dilution of the plasma.
Capillaries appeared on the 6th day and grew to a length
of almost 2mm in a week, attaining a width of from 7 to
100 p. These contained blood cells which moved due to the
streaming of fluid and the ameboid movement of the leucocytes. The mode of formation of vessels was found by White
and Parshley to agree closely with the accounts of capillary
formation in Z ~ T Ogiven by Lewis ('31) and in vivo by Clark
( '09, '18).
I n a valuable summary of the literature White and Parshley point out that several investigators failed to obtain growth
of vessels from mammalian bone marrow. Evidence is presented in support of the suggestion that a thick plasma clot
Aided by grants from the Ferris and Florence Smith Foundation f o r Research
in Plastic Surgery and the firm of Hoffmann-LaRoche, Inc.
663
'THE A N A T O Y I C A T > RECORD, VOI,. 117, NO. 4
UECEMBEK 1gs:j
664
U
'
. C. WOODARD A N D C. M. POMERAT
and an initial pH of 7.4 to 7.8 favor early capillary outgrowth
and subsequent maintenance.
The purpose of the present study mas (1) to find out
whether vessels could be grown from adult human bone marrow with the use of the cover slip-roller tube method which
would facilitate the introduction of experimental variables
within the fluid nutrient and ( 2 ) to make preparations ( a )
for still and cinematographic records in a phase contrast
optical system and (b) f o r fixed stained mounts.
MATERIALS ,4ND METHODS
The development of a method f o r the cultivation of blood
vessels from human bone marrow was based on the routine
procedure employed in this laboratory for roller tube cultures. This consists of embedding three to 5 explants in a
corresponding number of drops of heparinized rooster plasma
spread over the surface of 1 2 X 50mm cover glasses of no. 1
thinness. An equal number of drops of 7-day chick embryo
extract is mixed in by patting and stirring with the plasma.
Two such cover glasses are introduced back-to-back into
16 x 150mm test tubes. The clot is allowed to harden for
one to two hours before the addition of 2ml of a fluid nutrient. This consists of 50% ascitic fluid usually derived from
patients with carcinomatoses peritonei, 45% Gey 's balanced
salt solution and 5% embryonic extract. Penicillin at a final
concentration of 1000y/ml is included to reduce the chance
of contamination. Tubes are incubated a t 37°C. in a drum
rotating a t approximately 8 r.p.h.
Ribs obtained under aseptic conditions were brought to
the culture room within one-half to 8 hours. By means of
sterile instruments they were split open along their longitudinal axes and marrow was transferred to form cultures
according to the routine procedure. Some vascular sprouts
were observed, but in an effort to develop an ideal procedure
a series of experiments was conducted in order to evaluate
the importance of the critical variables which could be shown
to enhance vascular outgrowth. Material was obtained from
CULTURE O F VESSELS F R O M MARROW
665
1'7 patients. Eleven operations were for treatment of tuberculosis (thoracoplasties), 4 f o r bronchiectasis, one for chronic
niediastinitis, and one f o r diaphragmatic hernia. Marrow
from each subject provided from 20 to 150 cultures, a total
of some 1'700 having been used in obtaining the results herein
reported.
Advantage mas taken of Yarshley and White's ('51) findings, and marrow was embedded in thick clots, lout in subsequent observations this factor was not believed to be of prim a r y importance. The transfer of spicular fragments with
jc~veler'sforceps was not observed to yield the best results.
Curettage as well as the expression of marrow from bone
segments with the use of heavy forceps was soon abandoned
in favor of collecting bits, not exceeding l n i m in diameter,
of inarrow pulp on the tip of razor blade knives. Particularly
critical was the time of transfer of such marrow to the culture niedium after the removal of ribs a t surgery. The best
results obtained followed culture within 30 minutes. Tt is
suspected that anoxia in the rib marrow is one of the critical
factors in the survival of the eiidothelial elements.
In some series of experiments the clot was coniposed of
three parts rooster plasma, two parts homologous blood serum
a i d one p a r t embryonic extract. While inrufficient numbers
of explants mere embedded to reach definitive conclusions,
it is believed that this combination was superior to clots
coniposed entirely of avian materials. Additions to the clot
of nonmalignant or malignant ascitic fluid as well as liver
extract showed no clear improvement in the results. Clot
liquefaction, which always proves troublesome for long term
maintenance of patent vessels, appeared to be increased in
preparations containing rib marrow forcibly squeezed from
the ribs. S o attempt was made to reduce liquefaction by
means of the addition of crude soy bean trypsin-inhibitor
(cf., Morgan and Parker, '49). Increasing the concentration
of embryonic juice appeared to stimulate fibroblastic proliferation with a consequent crowding out of the vascular
ciido thelium.
666
W. C. WOODARD A N D C. &I. PO MERA T
It was found advisable erentually to allow three to 4 hours
rather than the usual period of one to two hours for clot
fixation before the addition of the fluid nutrient.
Variations in the composition of the fluid nutrient phase
included the addition of 5 y/ml of cortisone dissolved both
i i i acetone and in cellosolve, hut no noticeable difference was
observed under these conditions from the outgrowth obtained
from corresponding untreated controls following a 6-day
period of incubation. Since the marrow was derived in part
from tuberculous subjects, Rimifon and Ilarsilid were incorporated in the fluid medium. Some evidence of injury
was obtained in the presence of 50y/ml of Rimifon, while
no evideiice of stimulation could he found with the inclusion
of 2 5 y , although in this series of experiments the less favorable type of " squeeze " marrow was employed. Marsilid
proved toxic at 50 and 5 y/ml, but at 1y results resembled
those of untreated controls.
While the effect of pH v7as not systeniatically studied,
random samples of the nutrient fluid, using phenol red as
a n indicator, showed a p H of 7.8 to 8.0. This factor was
believed to be of no greater than secondary importance in
the development of vessels. Attention is called to the fact
that Trowel1 ('52) found that lymphocytes did not apppear
to be adversely affected by p H s ranging from 8.6 to 6.5 or
by CO, ranging from 0 to 15% in the gas phase.
Grateful ackno.i\rledgment is made to Dr. A. W. Harrison
for iiidispensahle aid in supplying ribs from surgical procedur c s.
RESCLTS
1. Stairzed preptrrutiov~ls. Cultures obtained by the rapid
transfer of bone marrow to clots composed of avian plasma
and a low concentration of embryonic extract but with some
human seiwm plus a fluid nutrient containing 50% ascitic
fluid from a malignant source and placed in roller tubes
exhibit rapid emigration of blood cells and elements believed
CULTERE O F VESSELS FROM MARROW
667
to be reticulum cells a few hours after incubation. Spindle
cells presumably of the fibroblastic series generally appear
after 48 hours and vascular sprouts can be identified on the
third day.
Under these conditions patent endothelial tubes are found
in approximately 50 to 60% of the explants. Beginning on
the 10th to the 12th day the explants undergo degenerative
changes. Outgrowth between the 5th and 7th days is ideal
f o r the study of the vessels and the activity of their cellular
content. This stage is represented in the accompanying illustrations. Stained preparations are shown in figures 1through
8. At low magnification (figs. 1 and 2) the large number of
rapidly outwandering elements of hematogenous origin can
be seen, while vascular sprouts are best demonstrated at the
higher magnifications used for figures 2 through 5. The occurrence of multiple tubeways along the margin of a mass
of fat rich marrow is shown in figures 2 and 3. These are
believed to arise from pre-existing capillaries and sinusoids
of the explanted marrow. Well developed vessels have a
broad base, a parallel sided median portion which may be
from 0.5 t o 1.5mm in length, and a cone shaped apical tip
(fig. 3 ) . A base with a diameter of from 50 to 80 p is common,
and the average width of the straight portion in many vessels
was approximately 25 p (figs. 4 and 9). The apical end was
closed (fig. 10) except in some vessels in which cells were
seen to escape but this may have been due to injury from
the microscope lamp.
TTsing oil immersion, culture preparations fixed with Helly
Zenker 's fluid and stained with hematoxylin-eosin-azure were
valuable for the study of the individual cell types. Figure
5 shows the nuclei of two endothelial cells. These were characteristically elongate, moderately basophilic and contained
two or more nucleoli of irregular size and shape. The cytoplasm of these cells was also quite blue. I n stained as well
as in living preparations studied with phase microscopy (fig.
lo), endothelial cells at the apical end characteristically
668
ITr.
C. WOODARD AND C . 78. POMERAT
showed iiiultiple fine branching processes extending into the
culture medium.
Luminal elements were predominately mature erythrocytes
(figs. 4 and 9) but many members of the leucocytic series,
particularly mature neutrophiles (fig. 5 ) , were characteristic.
Cells in this situation appear to resist degenerative changes
for longer periods than corresponding extravascular elements.
Extravascular cells included many erythrocytes but comprised a wide spectrum extending from elements believed to
be of the “blast” series (fig. 6 ) to members of the myelocytic
and lyinphocytic series (fig. 7) and macrophages filled with
debris including hemosiderin (fig. 8). Mitotic figures (fig.
7) were common. Negakaryocytes were occasionally found
in the zone of emigration.
Cultures incubated f o r 10 days were fixed with 10% formalin and treated with Rio-Hortega’s double silver impregnation modified for tissue cultures. A large amount of reticular
fibers radiates outward from the edge of the explant (figs.
14 and 15).
2. Phase .microscopy. Living cultures were photographed
with phase contrast microscopy to show cytological details
in the natural state (figs. 9-13). Figure 9 shows fat in adipose cells a t the bottom of the figure in an area where several
interlacing vessels arise. Endothelial cells appear to contain
large numbers of small granules of uniform size (figs. 9 and
10) resembling those seen in cells which are probably members
of the granulocytic series. Long, filamentous mitochondria
in the flattened ends of endothelial cells at the apical pole
are shown in figure 10, as well as slender branching processes,
sometimes of threadlike type. These features may be useful
in identifying outwandering spindle cells of endothelial origin.
The progressive changes in the position of intravascular
cells as seen in photomicrographs made a t various time intervals are shown in figures 11-13. Erythrocytic displacement
as a consequence of the locomotion of a granulocyte is demonstrated particularly well in figures 11 and 12.
C U L T U R E O F VESSELS FROXI MARROW
669
3. Phcrse c i m n m t o g r a p h y . Approximately 1000 feet of 16
mni time-lapse cinematographic records using phase contrast
microscopy were made of typical vessels in 5- to 7-day CUItures. F o r most sequences 8 frames per minute were taken,
while a few records were made a t lf.p.m. When projected
a t the standard rate of 16 f.p.m. these operations accelerated
the action by a factor of 120 and 960 times respectively. The
apparatus employed has been described by Pomerat (’51a).
The niost obvious and dramatic result observed in the film
1-ecord is the enormous activity of wandering cells. Of special
importaiice is the locomotion of elements presumably mostly
of the granulocytic type within the lumen of the vessels. These
are often seen to traverse the entire segment of the vessel
displacing other cells, particularly the erythrocytes, in the
course of their progress as already suggested by the analysis
of the still pictures (figs. 11 to 13). I n keeping with conclusions reached by other workers (14-hite and Parshley, ’51),
the jostling occasioned by this activity as well as by the
streaming of fluid is believed to account for the emigration
of passive erythrocytes to the extreme a p e s in the absence
of a circulatory a r c or the presence of muscular tissue
vithin these vascular systems. It is also thought that this
niechanism may be an important factor in the lurninization of
T W I V capillaries. Of special interest in the film sequences a r e
sereral records of rotatory motion by leucocytes against the
apical lurniiial margin of the terminal endothelial cell suggestive of what might be considered as a “boring operation.’’
The axes of spindles in dividing endothelial cells regularly
appeared a t right angles to the direction of the vessel.
DISCUSSION
The method described makes possible short term cultures
of patent blood vessels derived from adult human bone marrow. The source materials, particularly ribs which a r e ordinarily discarded, a r e available in useful amounts in large
hospital installations. The percentage of successful vascular
670
W. C. WOODAED A N D C. M. POMERAT
outgrowth makes experimental studies practical within the
limits of the requirements for its culture. The mode of cndothelial outgrowth, luminization, the development of acellular
fibrillar material, and the phenomenon of diapedesis are
among the areas of investigation which invite further study.
Using phase contrast time-lapse cinematographic technique,
especially in combination with a perfusion chamber (Pomerat,
'51b), the effect of blood serum obtained from patients suffering from acute diseases known to influence vascular
physiology upon patent vessels in culture offers attractive
possibilities. The action of drugs, particularly those influencing capillary permeability should prove interesting in
relation to the behavior of the intravascular elements and
the properties of the endothelial wall. Finally, it is suggested
that this use of rib marrow illustrates the type of challenge
which the tissue culture method offers f o r clinical-cytological
studies in which the activities of the tissues of particular
patients can be investigated directly under highly controllable
conditions recently stated by Pomerat ('52). By taking advantage of current tissue culture methods there may be profit
in special situations to perform studies directly upoii human
cells without endangering the patient and by-passing aiiimal
experimentation by utilizing readily available material ordinarily discarded in the course of surgical procedures.
SUMMARY
1. A method is described f o r the development of short
term cultures of patent blood vessels from human rib marrow.
The essential factors in the procedure include rapid transfer
of small bits of marrow free from spicules to plasma clots
coiisisting of heparinized rooster plasma, homologous serum
and a low concentration of chick embryo extract. Roller tube
cultures, utilizing a fluid nutrient phase consisting of 50%
ascitic fluid preferably of malignant origin, 45% Gey 's balanced salt solution, 5% embryonic extract, arid penicillin at
a final concentration of 1000 units/ml, appear optimal. Undei.
these conditions vascular tubes are found in 50 to 60% of
CULTURE OF VESSELS F R O M MARROW
671
the cultures which a r e considered ideal for experimental
studies between the 5th and 7th day of incubation but which
show signs of degeneration accompanying clot liquefaction
on the 10th to the 12th day.
2. S e w blood vessels as well a s extra- and intravascular
cells iii the outgrowth a r e described 1)oth from preparations
stained with hematoxylin-eosin-azure and in liviiig cultures
studied with still and time-lapse cinernatographic phase microscopy.
3. The mode o f capillary formation a s described by previous workers using in V i t i O and iiifra huniari in a i t r o preparations is confirmed.
4. Suggestions a r e made concerriiiig the opportunities afforded by short term cultures of vessels derived from adult
huiiian sources, which a r e readily available in surgical theaters, particularly with respect to the dynamics of vascular
pathology.
LITERATURE CITED
CLARK,ELIOTR. 1909 Observations on living lymphatics in the tail of the frog
larva. Am. J. Anat., 3: 183-198.
1918 Studies of the growth of blood-vessels in the tail of the frog
larva by ohservation a i d experiment on the living animal. Am. J. Anat.,
2‘3: 37-88.
FULTOP;,
J. B., M. S.PARSHLEY
AND H. S.SIXAZS1949 Growth of blood vessels
from adult tissue in zitro. Anat. Rec., 70.?: 37.
LEWIS,WARRENH. 1 9 3 1 The outgrowth of endotlielinin and capillaries in
tissue culture. Jolins Hopkins Hosp. Bull., A?: 248-253.
MORGAK,
J. P., AND R. C. P A R K E R 1949 The use of antitryptic agents i n tissue
culture. I. Crude soy bean trypsin-inhibitor. SOP.Exp. Biol. and Med.,
7 1 : 663-668.
I’OJIER.IT,
(”. M. 1931n Pictures for visualizing allergies. Rcsct1rc.h Rer., 5-8,
June.
___- 1931b Methods in Medical Research, Vol. IV. Year Book Publishers,
Chicago, Ill., 198-291.
1952 Dynainic cytology in experimental medicine. TT. R. Armed
Forces Med. J., 4 : 11-21.
TROWELL,0. A . 1952 The culture of lymph nodcs in &lo. J . Exp. Cell Res.,
3: 79-107.
1 9 5 1 Growth in witro of blood vessels from
WRITE, J. F., AND M. S.PARSHLEY
bone marrow of adult chickens. Am. J. Aiiat., 8 9 : 321-315.
PLATE 1
EXPLANITION OF’ FIGURES
1 and 2 Patent blood r e ~ s e l seniigiating from e q h i i t s of huiiiaii rib marrow
after 5 (1:rys ’ incubation. Helly Zeiiker fixation followed by Iieniatoxylin-eosiil
azure stain.
1 Entire euplant shown togetlier with snrlountling zone of rapidly emigrating
cells. A parallel series of eailotlrelial tubes are seen on tlie left side of tlie esplant. X 20.
2 Portion of tlie explaiited ~ n : i ~ r o with
w
the vessels shovn in figure 1 but a t a
higher inagnific.:ition. X 60.
G72
PLATE 1
CCLTCRE OK VESSELS FROX MARROW
W. C . WOODARD AND C . M. POMERAT
673
ESPI,IS.\TION
O F FIGURES
3 a n d 1 P a t e n t blood vcmels wiigratiiig f i oiii ex1il:rnts of human rib iimrro\v
a f t c r 5 days’ incubation. Helly Zrnker fixation follon.ed 1)- lieniatosyliii-ensillazure stain.
3
Series of newly fonned patcnt rcssels. X 180.
4 Tlic apical end of :I characteristic blood ressel showing its endotlie1i;il w i l l
and content of erythrocytes and leucoeytcs. Typical extravascular cell types are
rcl)wsented in the fieltl. X 600.
Gi4
675
PLATE 3
ESPL.\N.\TION
O F F lG U R E S
5-8 Same pre!)arntioii as sliowii in figures I to 4 but a t a ~iiagiiificatioiiof
npproxiiii:ttely x 1400.
5 Parallel arrangement of eiidotlielial wlls i n the miil-portion of n iiewly foriiied
wssrl slio\ving cli:ir:irtcristic iiuc.leolnr patterns aiid relntivc staining properties to
those of the blood seyics. Two iatr:iv:rswl:rr ~ieutropliilesare shown i n assorintioil
with groups of red lilood rells iii tire Juiiic~ii of the ca1)itl:iry.
G Cells sliwing (lee!) Iiasopliilic staining properties. The largest is bPJiewd to
rel)rvseiit a inyeloblast or even n 1ie1iiocytol)last.
T Evideiices of heiiiopoiesis including
ci~IIill mitosis.
:I
8 A iii:icroplinge Tyliirli w:is filled with
Iieiiiosideriii.
676
:I
yeJlowisti-green pigment rel~rcsenti~rg
PLATE 3
C U L T U R E O F VESSELS FROM MI\RROW
\V. C . WOODABD A N D 0. M. P O X E X A T
677
PLATE 4
ESk'L.ZSh'TIOS OF F I G U R E
9 O u t n m i d e r i n g vessels f r o m a i-(lay culture of human rib ii1ai~o\v. 1Slitlotlielial cc~lls allpear t o contain large numbem of small granules of uniforiii size.
T h e luiiien is packed v i t h l ~ l o o dcells. Fat i n the Ioiwr p a r t of the figure iiit1ic:ites
the 1oc:itioii of the exp1:int 1ii:irgiil f r o m \vliicIi the ~ c s s e l s1)roliferatetl. PIi:~se
contrast iiliwoscopy (rkirli 11, A 0 oil iiiiiiicisioii olijcctire). x 880.
6i8
CULTURE O F VESSELS FRO31 NIARKO\V
11‘. C. W O O D A R D A N D C. If. I’OJIERAT
PLATE 5
ESPLANATION OF FIGURES
1 0 Apical end of n rcssel showing the eliaracteristic fine braiicliiiig of tlie terminal endotlielial cell together with its rontent of filamentous niitocliondrin. Slagnifirntion al’proxiniately X 1200.
11-13 Capillary i n the zone of einigration from a 3-day cnltuie pliotograplicd
a t rarious intervals nitli the siinie terliniqne :IS that cinployed in figures 3-9 to
denionstr:~te the displacement of er) throcytes a s a I esult of tlie loconintor activity
of a gr:inulocytie cell. Magnification :ipproxiiiintely X 1230.
11 Initial pliotoniic~oglnpli.
12
Saine :is figure 11 h u t tlirec. minutes I:itci.
13
Saiiie a s figure 11 but 9 minutes Iatcr.
680
CUIJTT~REOF VESSELS FROM M.lRROW
PL.\TE
\V. C \ \ O O D 4 R l l A N D C . 31. POAlIEItAT
681
5
PLATE F
EIPI..\NATION
OF FIGUI<I?S
1 4 and 15 Ten-day-old culture of Iiuii~;inrib i i i a r r ~ ~fixed
r
in 1 0 % foriiinlin :ind
then st:iiiied with Rio-Hortega 's d o u l ~ l r silrer i~iiprcgnation modifietl fot. tissue
culture.
14 A portion of the es]-~l:intsliowiiig tlic rarlintiiig reticular fibe~.s. x 200.
15
S:iiiie
a s figure 1 4 but :it a Iiigher n ~ a g n i f i c a t i o ~x. 480.
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