TissUE CULTURE STLEDEES 0N AVMJ-J
LYMFHOID TUMGR

Thesis fer the Degree: ca: 5‘“. S;
MECHIGAN STATE LC‘LJZW

w 1 ' . I!
121‘ .1e~. ..3 i 5.: . r 5‘"
meager? i. uizififiu

r"; 'E‘Vti
Tar-3.1.11

{finals

This is to certify that the

thesis entitled

Tissue Culture Studies on.Avian Lymphoid Tumor.

presented by

Robert H. Bussell

has been accepted towards fulfillment
of the requirements for

M.S. degree in Bacteriology

Major professor

Date January 30; 1952

0-169

 

 

TISSUE CULTURE STUDIES ON AVIAN

LYMPHOID TUMOR

By

ROBERT H. QUSSELL

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Bacteriology and Public Health

1952

 

'THESH‘}

 

4/17/5’ 1-
'.” ,
(,3:

ACKNOWLEDGMENTS

The author wishes to express his sincere thanks to Dr.
Walter N. Mack for his supervision, interest and many valuable
suggestions which have made this investigation possible.

Grateful acknowledgment is also due to Dr. H. J. Staf-

seth for his assistance during the writing of this thesis.

TABLE OF CONTEN TS

Page

INTRODUCTION . . . . . . . . . . . . . . 1
HISTORICAL . . . . . . . . . . . . . . . 8
MATERIALS AND METHODS . . . . . . . . . l4
EXPERIMENTAL . . . . . . . . . . . . . l 7
Experiment I . . . . . . . . . . . . . . 1?
Experiment 11 . . . . . . . . . . . . . 22

Plates I-XIV . . . . . . . . . . . . . 27-40
Experiment III .. . . . . . . . . . . . . 41
DISCUSSION . . . . . . . . . . . . . . . 43
SUMMARY . . . . . . . . . . . . . . . . 49

BIBLIOGRAPHY..............50

IN TRODUC TION

There have been many experimental approaches to the
cancer problem. Three of the main ones are those concerned
with (1) genetics (2) cellular physiology and biochemistry and
(3) attempts to demonstrate viruses and virus-like agents as
the cause of cancer. During the past 50 years an ever-widening
number of tumors and tumor-like processes of animals have
been discovered. Many of these have been shown to have eti-
ological relationships with viruses and virus-like agents. Pos-
sibly the only reason why such agents have not been demon-
strated in some of the human tumors is the high degree of
specificity shown by these tumors and their agents. In general
their specificity is such that only homologous hosts can be used
in experimental work with these tumors. Thus, the same situ-
ation has risen that has always plagued microbiologists working
in the realm of human diseases, namely, that experimental
progress is the lack of a satisfactory experimental host. In
the absence of such a host, similar diseases in animals must
receive the attention of the laboratory investigator. This is the

situation that prevails in a great deal of the cancer research

done today. The fowl is among the animals which have con-
tributed greatly to the study of neoplastic diseases.

The first tumor-like process shown to be caused by a
filterable virus was fowlpox (Marx and Sticker, 1902). Since
that time many viruses have been shown to cause proliferative
lesions of many different types, both in animals and in man
(Kidd, 1948, 1950).

The first neoplastic disease transmitted by a filterable
agent was demonstrated by Ellerman and Bang (1908). They
were able to transmit both lymphomas and leukemia of chickens
by inoculating blood and cell-free filtrates of organs from the
diseased birds into healthy birds. A few years later Rous (1911)
succeeded in transmitting a sarcoma of fowl, which had been
previously identified as a neoplasm, by means of a filterable
agent. A year later Rous (1912) suggested a more widespread
use of this and other transplantable tumors of the fowl as ma-
terial for cancer research.

The description of many transmissible tumors of the
fowl have followed the work of Rous. Practically all these
tumors have been transmitted by the use of agents other than

living tumor cells. This property has been accepted as

characteristic of this group of tumors by Claude and Murphy
(1933). However, there are several characteristics of this group
of cell-free and filterable agents which cause many to hesitate
calling them viruses. First of all, when filtrates are inoculated
into animals there is a latent period before tumors and death
are produced. This not the case when live tumor cells are
transmitted directly, tumors and death following in a relatively
short period of time. As Claude and Murphy (1933) pointed

out, bacteria and viruses show the same degree of susceptibil-
ity to ultraviolet light whereas the filterable tumor agents are
far more resistant.

They also pointed to the fact that these agents have a
definite selective action with regard to the type of tissues which
they will affect. This, of course, is a property they have in
common with true viruses. However, some of the tumor agents
are bound or inactivated by the tissues to which they show their
specific affinity. Claude and Murphy (1933) stated that this was
not true of the viruses. However, since the discoveries of
Hirst (1941, 1943) and the development of the cell receptor
theory, this view becomes more questionable. Hirst (1941)

first discovered that influenza virus would agglutinate red blood

4
cells and later (1943) showed that the cells of the respiratory
tract would absorb the influenza virus. These are the cells
which influenza virus selectively attack.

The sporadic occurrence of tumors in nature has also
been advanced as an argument against any theory proposing
viruses as their etiological agents. Andrews (1934) proposed
that a latent virus exists in apparently normal tissues, which
in turn requires some sort of stimulation before malignancy
is produced. This would provide an excellent explanation if
the theory could be proved by experimental evidence. As yet,
the presence of such an agent in normal tissues has not been
adequately demonstrated. If some method, possibly a serologi-
cal test, for demonstrating infection with such a latent agent
could be developed, this problem might be solved. An analogy
may be drawn here between a well-known virus and these tu-
mor agents to show the significance of their detection if they
exist in supposedly normal tissues. Before adequate means of
detecting the presence of poliomyelitis virus were available,
investigators were unable to explain the sporadic occurrence
and the seemingly noncontagious nature of the disease. The

discovery of a suitable experimental animal (Landsteiner and

Popper, 1909) definitely established poliomyelitis as an infec-
tious disease and provided a means for the detection and identi-
fication of the etiological agent. A year later Netter and Le-
vaditi (1910) developed the neutralization test. This provided
an indirect method for determining the distribution of the virus
in nature, which was shown to be widespread in spite of the
sporadic occurrence of paralytic cases.

Shrigley (1951) stated that probably the greatest handi-
cap to the study of the agent of Rous' sarcoma is the lack of
a means to determine it quantitatively. This is also true of
the other agents producing tumors in fowls and other animals.
It is also a situation which must be solved if these agents are
to be considered viruses in the sense that we think of them
today.

It may be said that these tumor-producing agents do
have properties in common with the well-known viruses. They
are both ultramicroscopic, filterable and their multiplication is
intimately associated with living susceptible tissues and cells.

It was not long after the development of techniques for

the cultivation of living cells _i_n_ vitro that Carrel (1925, 1926)

 

applied these techniques to the study of the agent of Rous'

sarcoma. He was able to propagate this agent in the presence
of chick embryo tissue, chicken monocytes and chicken spleen.
Since that time relatively few investigations have appeared in

the literature applying these techniques to the study of tumor-
producing agents. However the literature dealing with the cul-

tivation of the tumors in vitro is voluminous.

 

The lymphoid tumor used in the following studies is a
transplantable tumor originally described by Olson (1941). Later,

in the hands of Burmester _e_t_ a_l_. (1946, 1947), this tumor was

shown to contain a filterable agent. The filtered plasma from
tumor—bearing birds was also shown to contain this agent. It
failed to produce tumors at the site of inoculation and required
incubation periods of the order of four to six months before
tumors developed. This tumor may be considered to be a
form of visceral lymphomatosis or a lymphocytoma which may
be defined as a malignant neoplastic disease, the undifferenti-
ated lymphocyte being the type cell of the tumor (Olson, 1940).
Chrétien (1951) was able to show that this tumor would
maintain its malignancy when cultivated in 1112 by reinoculation
of tumor tissue into chickens and the resultant production of

tumors at the site of inoculation. Working with the filterable

agent described by Burmester it. 11: (1946, 1947), she was un-
able to produce tumors by the inoculation of normal spleen
material, growing .13. vitro, to which this agent had been added.
This thesis is essentially a continuation of the work by
Chrétien (1951) on attempts to detect the presence of this agent
using tissue culture techniques. An effort was also made to
determine if a magnetic field would affect the growth of this

tumor in vitro .

 

HIS TORICAL

There are many excellent reviews concerning viruses
and filterable agents and their relationships to tumors. One
of the more recent ones (Shrigley, 1951) discusses a wide va-
riety of tumors of animals. Kidd (1948, 1950) discussed vari-
ous proliferative lesions caused by viruses and the reasons for
placing some of these viruses along with sunlight, tar and many
other substances in the category of carcinogenic agents. Claude
and Murphy (1933) gave a summary of the work done up to that
time on the various transmissible tumors of the fowl and pre-
sented several good reasons for not placing many of the filter-
able agents isolated from these tumors in the category of vi-
ruses.

For discussions concerning the tumor with which this
thesis deals and other closely-related conditions in the fowl,
the reader is referred to the works of Olson (1940), Jungherr
(1948) and Chrétien (1951),

For a historical presentation and descriptions of the
methods of tissue culture the reader is referred to the mono-

graphs of Parker (1950) and Cameron (1950). An excellent

summary of tissue culture techniques, as applied to the study
of viruses, may be found in the paper by Robbins and Enders
(1950).

The present literature review will deal only with the
cultivation of tumor-producing agents in £1152.

Carrel (1925), while working with the agent of Rous'
sarcoma, was the first to attempt to change normal cells into
malignant ones 51.. 3.3.35.9. by means of a tumor-producing agent.
By isolating pure cultures of cells from the Rous sarcoma
tumor growing -1_n_ M’ he was able to show that it was the
macrophages and not the fibroblasts which are responsible for
the malignancy of this tumor. He then proceeded to add the
Rous sarcoma agent to pure cultures of monocytes. They rap-
idly acquired the characteristics of malignancy, as shown by
inoculation into chickens. Some of these cultures appeared as
normal growing tissue although they produced tumors when
inoculated; however, more often specific changes took place in
the infected cultures. The cultures which showed the specific
changes produced tumors more rapidly than the ones which
showed no such changes. Carrel and Ebeling (1926) were able

to show that, although monocytes under ordinary cultural conditions

10
never were transformed into fibroblasts, the Rous sarcoma agent
produced this change in such cultures. They described other
conditions which will produce this change in the absence of the
Rous sarcoma agent. They’ attempted to explain this phenome-
non on the basis of an adaptive change, the sensitive cell trans-
forming itself into a type of a cell which is not sensitive to the
action of the Rous agent. Carrel (1926) proved conclusively

that this agent multiplies i_n_ vitro and that this multiplication

 

depends upon the presence and the nature of the cells contained
in the cultures. Tumors were produced after inoculation of cul-
tures having incubation periods ranging from 4 to 30 days after
addition of the agent. Successful results were obtained with
leucocytes, spleen fragments and embryo pulp. As he had
shown before, experiments utilizing pure cultures of fibroblasts
were all negative.

Ludford (1937) was the next to study the infection of
cells in tissue cultures with the Rous sarcoma agent.. He also
performed similar experiments with the agent of the Fujinami
sarcoma, which is a similar tumor of the fowl. His object of
reopening these studies was to clarify the confusion which ex-

‘9

isted at that time as to whether the monocyte or the fibroblast

11
was the malignant element of these sarcomas. Berkefeld fil-
trates of both the Rous and the Fujinami sarcomas were added
to cultures of fibroblasts and cultures of the buffy coat obtained
from fowl blood. Inoculation of the fibroblasts treated with
these agents produced tumors, but in only one case did cultures
of the buffy coat produce a tumor. After varying periods of
time the cultures were treated with immune serum to inacti-
vate any free virus in the cultures. When the cultures were
treated 'in this manner only the inoculation of the fibroblast
cultures resulted in tumor formation. This suggested to Lud-
ford that in Carrel's previous experiments the production of
the tumors by the monocyte cultures was due to the presence
of the agent in the medium. His conclusion was that the fibro-
blast and not the monocyte was the cell which was sensitive to
infection with these agents.

Furth _e_t_ _a_l_. (1934, 1937) described experiments dealing
with the cultivation of agents producing various types of leuko—
sis in fowls. The majority of their experiments deal with the
cultivation of the tumor-producing cells. Their observations
concerning the cultivation of the various agents in the presence

of normal cells are as follows: Working with their virus 13

12
(a complex sarcoma leukosis agent), they failed to demonstrate
survival of the agent in tissue cultures of normal fibroblastic
cells from chicken embryonal leg muscle and in adult chicken
spleen cultures. They concluded that this agent was destroyed
in the presence of these normal cells. Their agent was pre-
pared by freezing the tumor tissue at -310 C. After thawing,
the material was centrifuged and the supernatant fluid used as
a source of the agent. The same results were obtained by cul-

tivation of their virus 1 (which produces a form of erythroleu-

kosis) in the presence of normal cells. In another series of
experiments they attempted to cultivate a virus which produces
leukosis only (virus 1), in the presence of sarcoma tissue. The
results of their inoculations showed that only sarcomas were
produced and no leukosis, thus proving that the leukotic agent
did not survive in the presence of common sarcoma cells.
Doljanski and Pikovski (1942) were able to show that the

agent of hemocytoblastosis (strain T of Engelbreth-Holm) would

I
survive in the presence of normal bone marrow and normal
fibroblasts for as long as 178 days. In the absence of living

cells the agent lost its activity within 24 hours. Because of

the large number of serial transfers made, the authors concluded

13
that there was a real increase of the leukotic agent in 11.13.13:
Both the cell cultures and the cell-free supernatant fluid re-
mained infective throughout these experiments. However, they
failed to observe any changes in the appearance of the cultures
or the individual cells when compared with similar cultures
without the addition of the agent.

Chrétien (1951), working with the agent of the avian lym-
phoid tumor, with which this thesis deals, was unable to pro-
duce tumors by the inoculation of normal spleen fragments which
had been cultivated in the presence of this agent in 111233. How-
ever, she was able to show that the addition of the agent pro-
duced morphological changes in the growth pattern of the nor-

mal spleen fragments cultivated in vitro.

MATERIALS AND ME THODS

Avian Lymphoid tumor. This tumor, designated as strain RPL

 

12 by the U. S. Regional Poultry Research Laboratory, East
Lansing, Michigan, was maintained throughout these studies by
serial passage in the pectoral muscle of chickens. Alternate
passages of this tumor material from chickens to tissue cul-
tures were maintained throughout the majority of these experi-
ments. This technique was first used by Chrétien (1951), whereby
she proved that this tumor would maintain its malignancy when

cultivated in vitro.

 

Tissue culture methods. The following techniques were employed

 

during this investigation: Carrel flask, 25-ml. Erlenmeyer
flasks, and the double cover slip method described by Parker

(1950).

Physiolgical solution. Hanks' solution was used throughout

 

these experiments. It was prepared according to a modifica-
tion of the formula given by Hanks and Wallace (1949). The
solution was prepared from stock solutions as needed and stored

in 25- or 50-ml. Erlenmeyer flasks. Hanks' solution was

15
prepared from the stock solutions at least once a week. Peni-
cillin and streptomycin were added to Hanks' solution to make
a final concentration of 25 units of penicillin per ml. and 125

micrograms of streptomycin per ml.

Plasma and serum. The same group of chickens was used

 

throughout these experiments. They were bled by cardiac punc-
ture as the plasma or serum was needed. Heparin was used
as the anticoagulant in obtaining the plasma. After removing
the cells from the blood, the plasma and serum were stored

in the refrigerator until used.

Embryo extract. The embryo extract was prepared from 9- to

 

ll-day-old chicken embryos. They were removed from the
shells aseptically and then rinsed in Hanks' solution to remove
any excess red blood cells. The eyes and feet were removed
and the embryos placed in a mortar. After cutting up the em-
bryos with a pair of curved scissors and grinding them lightly
with a pestle, two m1. of Hanks' solution was added for each
embryo used. This made approximately a 1:2 dilution of the
embryo pulp as recommended by Cameron (1950). The mixture

was then allowed to stand at room temperature for 30 minutes

16

before centrifuging at approximately 2,000 r.p.m. for 10 min-
utes. The supernatant fluid was drawn off, sealed in small
test tubes and stored in the frozen state until used. Just be-
fore use, the extract was thawed at room temperature and

clarified by light centrifugation.

”Agent." All samples of the “agent" used in these experi-
ments were obtained from the U. S. Regional Poultry Research
Laboratory, East Lansing, Michigan. Burmester and Cottral
(1947) described the methods of preparation of the “agent" in
that laboratory. In general, either filtration or centrifugation
or both methods have been used to render the tumor suspen-
sions free of intact cells. Plasma from chickens with active
growing tumors was also used as a source of the agent in one

experiment.

Sterility checks. All nutrient fluids added to the tissue cul-

 

tures were checked for sterility. Plain nutrient broth (Difco)
was the medium employed. Only occasionally was contamina-
tion encountered, this usually being caused by a mold. Occa-
sionally contamination of the Hanks' stock solutions occurred,

making it necessary to prepare new solutions.

EXPERIMENTAL

Experiment I

The first group of experiments was concerned with at-
tempts to detect the presence of the agent when it was added
to normal lymphoid tissue growing i_n \_r_i_t_r_<_)_. After the addition
of the agent to the cultures, which consisted of normal spleen
fragments from 17-day-old chicken embryos, daily observations
were made. Controls of normal spleen without added agent
were used to make comparisons. The cultures were inoculated
into chickens at the end of observation periods which ranged
from 7 to 12 days. These inoculations were made to deter-
mine if the normal cells had become malignant, or if by some
mechanism the incubation period of this agent could be short-
ened. Burmester and Cottral (1947) found that this agent re-
quires an incubation period of four to six.months before tumors
may be demonstrated in the inoculated chickens.

The Carrel flask technique was used throughout this
group of experiments. Briefly it consisted of embedding small

fragments of spleen (approximately one cubic mm.) in a plasma

18
clot consisting of: 0.3 Inl. plasma, 0.6 ml. Hanks' solution and
0.1 ml. of embryo extract. After complete coagulation of the
plasma clot a liquid nutrient (Cameron, 1950) consisting of 40
per cent serum, 40 per cent Hanks' solution and 20 per cent
embryo extract was added to the cultures. The nutrient fluid
was changed every two or three days. This procedure consists
of removing the old nutrient, bathing the culture in fresh Hanks'
solution for 15 to 30 minutes and then adding fresh nutrient.
The agent, in varying quantities, was added to the cultures af-
ter they had been allowed to proliferate for different intervals
of time. In each case the nutrient fluid was changed just prior
to the addition of the agent so that the cultures could be left
undisturbed for at least two days before it was necessary to
change the nutrient fluid.

In one series of cultures in this experiment the agent
was diluted 1:2 with the plasma used in the formation of the
clot. A similar method was used by Doljanski and Pikovski
(1942) in their studies of the agent of fowl leukosis in tissue
cultures.

At the end of the observation periods the tissue frag-

ments were removed from the cultures and separated from the

19
clot. After the addition of a small amount of Hanks' solution,
these fragments were then ground in a mortar to make a cell
suspension. Each such cell suspension was inoculated into the
pectoral muscle of two chickens (approximately 1-2 months old),
each chicken receiving half of the inoculum which was usually
between 0.5 and 1.0 ml.

A synopsis of the cultures in this experiment is given

below.

 

 

 

Amount of Age Of Age of
A cut Added Cultures Cultures
g When Added When Inocu-
(ml.)
(days) lated (days)
d' . : ‘
11 l 2 With 0 7
plasma
0.4 2 9
0.4 5 11
0.25 2 10
0.25 5 12

Results. Active migration of the round type of cells could be
seen within a few hours after the planting of the spleen frag-
ments. After 24 hours an extensive proliferation of the frag-

ments had taken place. During the third day of cultivation the

20
fibroblast-like cells began to make an appearance and they in-
creased rapidly thereafter.

Difficulty was encountered in making observations of the
cultures which were made after diluting the agent with the plasma.
This was due to the cloudy nature of the solution which contained
the agent, leaving the plasma clot slightly opaque. The cultures
which received 0.4 ml. of the agent were slightly cloudy but ob-
servations were possible. This difficulty required alteration of
the methods in later experiments.

As far as could be determined, no specific morphological
changes were observed in the cultures to which the agent was
added. On the third day of incubation, one of the cultures (0.4
ml. agent added on the second day) seemed to show a greater
proportion of the fibroblast-like cells than the controls. The
digestion of the plasma clot seemed to be retarded in the cul-
tures which contained the agent. This might have been due to
an interference with the multiplication of the cells, although no
gross differences were observed in the extent of proliferation
shown by these fragments.

Chickens inoculated with these cultures were observed

for 3 months and all failed to develop tumors within this period

of time. Several of the birds died after two months but all
failed to show signs of visceral lymphomatosis or tumors at

the site of inoculation.

21

22

Experiment II

A second series of tissue cultures was set up to elimi-
nate some of the difficulties encountered in the first experi-
ment.

The controls in the previous experiment consisted of
normal spleen fragments to which no agent had been added.

To eliminate personal error in the observation of the normal
spleen fragments, and spleen fragments to which the agent had
been added, ”unknowns” were prepared. Samples of chicken
plasma as well as tissue extracts, some containing the agent
and some not, were prepared at the U. 5. Regional Poultry
Research Laboratory. These preparations appeared the same
when observed and were designated by numbers only. The
identity of these ”unknowns,” was not revealed to the investi-
gator until the experiments were completed. Attempts were
then made to determine which of these ”unknowns” contained
the agent. The cultures were observed daily to determine if
there were any differences in their gross or microscopic char-
acteristics. Tissue cultures of spleen fragments, one contain-
ing the agent and a second culture not containing the agent, were

prepared at the same time to serve as controls.

23

In addition to the Carrel flask type of cultures as de-
scribed in Experiment I, the double cover-slip method, as
described by Parker (1950), was also used. This technique
consists of attaching a small cover-slip to a larger one by
means of a drop of Hanks' solution. A small fragment of the
tissue is then placed into the plasma clot which is prepared
by mixing one drop of plasma with one drop of embryo extract
on the small cover-slip. A drop of the "unknown" specimen

was also mixed with the two substances making up the clot.

A few drops of the nutrient fluid described in Experiment I
were added to the cultures and a large cover-slip was placed
over a depression slide and sealed with paraffin.

It was not possible to test all tissue cultures for in vivo

 

production of tumors because of the limited space available for
housing the chickens. In the first series of cultures, the indi-
vidual cultures were carried in duplicate. After 9 days of cul-
tivation (0.4 ml. “unknown" added on second day), the cells
from the duplicate cultures were removed from the [plasma
clot and placed in a mortar. The nutrient fluid from both
cultures and enough physiological saline were added to make

approximately two ml. of fluid. The cells were ground with a

24
pestle and the resulting cell suspension inoculated into the pec-
toral muscle of two chickens (approximately 2-4 months old),
each receiving one-half of the cell suspension or approximately
a one-ml. inoculum.

Table I gives a summary of the details and results of

the chicken inoculations in this experiment.

Results. It was not possible to determine which of the ”un-
known” samples contained the agent and which did not. As

far as could be determined, no specific morphological differ-
ences were observed in the cultures which received the unknown
samples of the agent.

The results of the animal inoculations were also nega-
tive. The chickens inoculated with these cultures were observed
for a period of three months. Several of the chickens died from
other causes during the observation period; all failed to show
signs of visceral lymphomatosis or tumor formation at the site
of inoculation.

During the course of later experiments, two of the above
inoculated chickens (series No. l) were inoculated with one ml.
of a 20 per cent fresh tumor cell suspension for serial propa-

gation of the tumor. These two chickens failed to develop

25

 

 

 

 

 

 

 

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26
tumors. All other chickens used for serial propagation of this
tumor received similar inoculations. They developed tumors at
the site of inoculation within a period of 5 to 11 days. When
the identity of the ”unknown“ samples was revealed, it was
found that one of the chickens had been originally inoculated
with material from cultures containing normal spleen fragments
to which no agent was added. The other chicken had been
originally inoculated with material from cultures containing

normal spleen fragments to which the agent was added.

Plate I - Tissue Culture - 4—hour Growth. Normal Spleen
Fragment. Magnification - 120x.

Plate II - Tissue Culture - 24-hour Growth. Normal Spleen
Fragment. Magnification - 36X.

 

28

 

Plate I

 

Plate II

Plate III - Tissue Culture - 72-hour Growth.
Fragment. Magnification 36X.

Plate IV - Tissue Culture - 72-hour Growth.

No rmal Sple en

Normal Spleen

Fragment, Agent Added 24 hours Previously. Mag-

nification 36X.

 

30

 

Plate III

"dvz ._
a“)

 

Plate IV

Plate V - Tissue Culture - 72-hour Growth. Normal Spleen
Fragment. Magnification Approximately 72X.

Plate VI - Tissue Culture - 72-hour Growth. Normal Spleen
Fragment, Agent Added 24 hours Previously. Mag-
nification Approximately 72X.

 

32

 

   

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vw

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’94 .‘I‘? {Y’T' . .
"' 72.! :n' .' b"? “(1.1.
A, 11. “A: "A! I l 3" -

Plate VI

Plate VII - Tissue Culture - 5-day Growth. Periphery of Nor-
mal Spleen Fragment. Magnification 36X.

Plate VIII - Tissue Culture - 5-day Growth. Periphery of Nor—
mal Spleen Fragment. Magnification Approximately
72X.

 

         
  
 

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‘gs‘. at: ,
kfl- a _ «4.1
-. 1‘.“ is... ..
: . 1' .73).. h. ,
. ".1”,
I’ ‘...~

 

Plate VIII

     
   

34

Plate IX - Tissue Culture - 5-day Growth. Normal Spleen
Fragment, Showing Outgrowth of New Cells. Mag-
nification 516X.

Plate X - Tissue Culture - 5-day Growth. Normal Spleen
Fragment, Showing Outgrowth of New Cells, Agent
Added 3 days Previously. Magnification 516X.

 

36

 

Plate IX

 

Plate X

Plate

Plate

XI-

XII -

Tissue Culture - 7-day Growth. Periphery of
Normal Spleen Fragment, Agent Added 5 days
Previously. Magnification 36X.

Tissue Culture - 7-day Growth. Periphery of
Normal Spleen Fragment, Agent Added 5 days
Previously. Magnification Approximately 72X.

38

 

Plate XI

 

Plate XII

Plate XIII - Tissue Culture - 7-day Growth. Normal Spleen
Fragment, Showing Outgrowth of New Cells. Mag-

nification 516X.

Plate XIV - Tissue Culture — 7-day Growth. Normal Spleen
Fragment, Showing Outgrowth of New Cells, Agent
Added 5 days Previously. Magnification 516X.

40

 

Plate XIII

 

Plate XIV

41

Experiment III

Fardon (1940) and Katzberg (1951) observed that when

two small fragments of tissue are cultivated in vitro there oc-

 

casionally is observed a field of attraction between the two frag-
ments. The pattern formed resembled the lines of force in a
magnetic field.

With this in mind an experiment was set up to deter-
mine if a magnetic field would affect the i2 m growth of the
avian lymphoid tumor. Normal spleen and heart fibroblasts
growing in litre were also placed in a magnetic field.

The only reference in the literature found, dealing with
a similar experiment, was that of Ingvar (1920). He applied
weak galvanic currents to tissue cultures consisting of central
nervous system tissue of the chick. He was able to show that
the current had a directing influence upon the cells and fiber
outgrowth. This occurred almost entirely along the lines of
force in the galvanic field. The cell processes growing toward
the cathode differed morphologically from those growing toward
the anode. He concluded that electrical forces play a role in

the formative processes in morphogenesis.

42

Two horseshoe magnets with a field strength of approx-
imately 125-150 gauss were used in this experiment. The Car-
rel flask type of culture as described in previous experiments,
was used to cultivate the tissues.

The first trial consisted of placing one culture contain-
ing a single fragment of tumor in the magnetic field. A con-
trol culture, which was not placed in the magnetic field, con-
sisting of a single fragment of tumor, was used to make com-

parisons. The culture placed in the magnetic field grew rapidly,

but the control culture failed to grow.

The experiment was repeated, but this time two control
cultures were used. This time the results of the first trial
were reversed. The fragment in the culture placed in the mag-
netic field failed to grow and the fragment in each control cul-
ture grew. Similar experiments were set up using cultures of
normal spleen and heart fibroblasts from a l7-day-old chick

embryo.

Results. These experiments failed to indicate that the mag-

netic field influenced the growth of tissues _1_I_1_ vitro. It should

 

be stated that the experiments were of a preliminary nature

and should receive more investigation.

DISCUSSION

As Robbins and Enders (1950) pointed out, there are two
general methods of demonstrating the presence of a virus in
tissue cultures. The first method is to show that the material
removed from the culture exhibits the characteristic activity of
the virus in question. This method of demonstrating the pres-
ence of the agent of the avian lymphoid tumor was impractical
because of the unusually long incubation periods (up to 300 days)
required. Experiments (of this nature should be performed with
this agent to determine if the agent will remain active in the
presence of living cells.

It was hoped that the incubation period could be short-
ened by the addition of this agent to normal lymphoid tissue
growing i_n_ v_it_12. If this agent was able to convert the normal
cells into malignant ones, the incubation period would be ex-
pected to be somewhat like that following the inoculation of live
tumor cell suspensions (approximately 7-15 days). If this had
happened, a more rapid method of detecting the agent would be

available .

44
Inoculation with the cultures containing normal spleen
fragments, to which the agent was added, and subsequent fail-
ure of tumor formation in the chickens could be explained in
several ways.
Direct inoculation of this agent into chickens requires
a long incubation period before tumors develop. Thus, contin-
ued stimulation over a long period of time seems to be neces-
sary for the conversion of the normal into the malignant cell

in vivo. If this continued stimulation is necessary for the con-

 

version to take place in vitro, the failure could be explained on
the basis of the characteristics of spleen cultures growing in

vitro. Maximow and Bloom (1948) showed that lymphocytes cul-

 

tured i_n m2 rapidly develop into macrophages and then turn
into fibroblasts. Since the lymphocyte is the type-cell of the
tumor, its apparent absence from cultures of spleen tissue
within a few days would explain the failure of these cultures
to produce tumors. However, this fails to explain why the
tumor may be cultivated in li_t;_r_<_)_ (Chrétien, 1951) without loss
of malignancy. It may be possible that the malignant lympho-
cytes do not undergo the changes which have been observed

of the normal ones. Intimate physiological mechanisms, lacking

45
in tissue cultures, may be necessary for the conversion of the
normal into the malignant cell in this case. The conditions
used for the cultivation of tissues 39. 1122 are a great deal

different from those which involve the growth of tissues in vivo.

 

Such factors as the presence or absence of inhibiting factors,
the growth-promoting principles involved and the influence of
the animal as a biological unit must be considered. Influenza
virus, in the presence of chick embryo tissue, multiplies .12
zit—1'2 at 37° C. At a temperature of 410 C. (approximately the
normal body temperature of the chicken) multiplication does not
take place (Enders and Pearson, 1941). Enders (1948) used
these facts to explain the resistance of the chicken to infection
with influenza virus. A similar explanation might possibly ac-
count for the failure here, since the cultures were incubated
at 370 C. instead of 410 C.

The number of cells contained in the inoculum may not
have been adequate to elicit tumor formation had they been
malignant. An attempt was made in the second experiment to
overcome this difficulty. Some of the cultures to be inoculated
were carried in duplicate. This resulted in the chickens re-

ceiving an inoculum containing approximately twice the number

46
of cells inoculated in Experiment I. The results of these tests
were also negative.

A second method of demonstrating the presence of a
virus in tissue cultures is by detecting some abnormal change
in the tissue or cells which is caused by the virus in question
(Robbins and Enders, 1950).

Chrétien (1951) was able to detect morphological changes
in tissue cultures of spleen fragments to which the agent of
this tumor was added. Similar experiments were performed
during this investigation but all failed to indicate that morpho-
logical changes took place in cultures to which the agent was
added.

Carrel and Ebeling (1926) were able to convert pure
cultures of monocytes into fibroblasts by the addition of the
Rous sarcoma agent, but they also described other conditions
during which this conversion may take place. When the mono-
cytes were observed to congregate in masses of dead tissue
the conversion took place in the absence of the agent.

Doljanski and Pikovski (1942) were unable to demonstrate
an abnormal change in cultures to which the agent of fowl leu-

kosis (hemocytoblastosis, strain T1) was added.

47

The fact that two of the chickens, used for inoculation
of cultures containing normal spleen incubated with the agent,
later resisted an inoculation of live tumor cells must be ex-
plained. Burmester and Prickett (1944), working with the same
lymphoid tumor, were able to show that chickens surviving ap-
propriate dosages of live tumor cells were later resistant to
subsequent challenge inoculations with these cells. Active growth
and later regression of the tumors had taken place in all cases.
At first it was believed that the chickens surviving the challeng-
ing inoculations in this experiment may have developed a sim-
ilar type of resistance. When the condition of the unknown
samples was revealed, it was discovered that one of the chick-
ens had received a control preparation which did not contain
the agent. It is believed that these chickens survived the chal-
lenge inoculations due to their age (approximately 4-1/2 months)
or to their state of health. The chickens were kept under very
crowded conditions and when they were inoculated with the tu-
mor cell suspension it was noted that they were in poor phys-
ical condition. It is known that young chickens and those in
good physical condition are more susceptible to the transplan-

tation of tumors than older and unhealthy ones (Claude and

48
Murphy, 1933). It is possible that the inoculum may have been
improperly administered, however, 30 other such inoculations
during these experiments, proved to be 100 per cent success-
ful.
The experiments dealing with the effects of a magnetic

field on the growth of the tumor in vitro were of a preliminary

 

nature only. During the first trial it was noted that a tumor
fragment grew better when the culture was placed in a magnetic
field. The control culture failed to show growth. The experi-
ment was repeated with proper controls and no stimulation in
growth could be detected.

Similar experiments were performed to see if a mag-
netic field would affect the growth of fragments of spleen and
heart fibroblasts from a chick embryo. These experiments
failed to show that the magnetic field had any consistent effect

on the growth of these tissues in vitro.

 

It is suggested that this possibility be investigated fur-
ther. The magnets employed had a field strength of 125-150
gauss. It may be that a stronger magnet could influence the
growth of these tissues. An experiment using growth measure-
ments similar to those described by Parker (1950) would also

be valuable .

 

SUMMARY

Tissue cultures of normal spleen tissue to which the cell-
free agent of an avian lymphoid tumor was added failed

to produce tumor formation upon inoculation into chickens.

Attempts to detect morphological changes in cultures re-

ceiving ,the agent were entirely negative.

Placing tissue cultures containing avian lymphoid tumor
tissue, normal chick embryo spleen and heart fibroblasts
in a magnetic field (125-150 gauss) failed to reveal any
differences in the rate of growth in these cultures when

compared with controls.

10.

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