THE COMPATIBILITY O F RAT AND MOUSE CELLS I N MIXED TISSUE CULTURES'

MORGAN HARRIS 2

Department of Pathology, University of Pennsylvania, and The Cl-istar Zns t i t~ ' fe Of Anatomy and Biology, Philadelphia

INTRODUCTION

Transplantation of tissues and organs between different species of higher animals may succeed in embryonic hosts (Murphy, '26)' but similar combinations are not compatible in adult stages (Loeb, '30). Thus while both normal and tumor tissues from rats and mice may be cultivated on the chorioal- lantoic membrane of young chick embryos (Murphy, '12, '13, '14a), and even human skin will proliferate in this environ- ment (Goodpasture, Douglas, and Anderson, '38)' such grafts are only temporarily successful and regress if maintained in hosts beyond the eighteenth day of incubation (Murphy, '14b). Interspecific transplants in adult birds and particularly in mammals are rapidly destroyed (Loeb, '30). Regression of lieteroplastic grafts in adult mammals is due to the develop- ment of antagonistic responses to the engrafted tissues, as shown by the studies of Loeb and Addison ( '09, '11)' Loeb ('20, '21), Loeb and Harter ('26)' Fleisher ('21, '22), and others. These reactions involve humoral factors as well as the connective tissue, lymphocytes, and polymorphonuclear leuco- cytes of the host. Much of the work to date on antagonism in heteroplastic grafting is concerned with describing the de- tailed pattern of the host response under conditions where the species used as host or the type of tissue transplanted varied.

This investigation has been aided by a grant to Dr. Warren H. Lewis from the

George Leib Harrison Research Fellow, University of Pennsylvania, 1941-1942. International Cancer Research Foundation.

1 O i

THE AXATOIICAL RECORD, VOL. 87, NO. 2 OCTOBER, 1943

108 MORGAN HARRIS

The causative mechanisms of tissue reactions to alien grafts are less well known. This may be traced, in part, to the natural disadvantages of transplantation as a method of studying tis- sue compatibility. In the living animal, it is difficult or impos- sible to separate individual aspects of tissue reactions from the total host response. I n particular, tyansplantation does not reveal whether the responses by connective tissue, lympho- cytes, and leucocytes are due to an intrinsic incompatibility be- tween the cells of host and graft, or whether cellular activity of this type is conditioned by more general reactions occurring in the body of the host as a whole. On the one hand, cellular an- tagonism may be due to a direct local reaction between alien cells or substances given off by these cells, and the surrounding tissues of the host. Alternatively, tissue reactions might con- ceivably arise not as local phenomena, but as the result of focal sensitization to the graft, spreading secondarily over the whole body. I n this connection, it is of interest to know whether a direct incompatibility exists between isolated cells of two different mammalian species, or whether cells of the two forms, if removed from complicating factors present in the body as a whole, may grow freely in association with one another.

The present investigation was undertaken with the fore- going question in mind. Studies were carried out on mixed tissue cultures containing normal rat and mouse tissues ex- planted together. Particular attention was given to the pattern of outgrowth, cytological appearance, and cellular behavior in such cultures. These criteria were used as a measure of compatibility between isolated cells of the two species.

MATERIALS 'AND METHODS

Tissues to be explanted were removed aseptically from new- born rats and mice. Heart, spleen, and kidney were selected as representative tissue types. For culturing, the roller tube technique as developed by Gey ('33) and W. H. Lewis ('35) was followed throughout. As a routine, cultures were planted in chicken plasma. and a supernatant, fluid added. I n some

COMPATIBILITY O F RAT Ah’D MOUSE CELLS 109

series of experiments, this fluid consisted of rat serum, with or without the addition of chick embryonic extract. In other experiments a completely heterologous medium was employed, the details of which will be described subsequently. At inter- vals of 2 or 3 days the cultures were washed in Locke solution and fresh supernatant fluid added. The large size of the cul- ture vessels obviated necessity for subculturing during culture periods ranging up to 36 days.

EXPERIMENTAL

Growth ifi a heterologous medium To rule out any possible influence of specific factors in the

serum on tissue antagonism, a number of cultures were made in a medium entirely foreign to both rat and mouse cells. The fluid phase included chick embryo extract, Loclre solution, and human cord serum (from placental blood obtained after birth), in a 1 : 3 : 3 ratio.

In an initial group of twenty-three experiments, fragments of rat and mouse heart were explanted together. The paired explants were placed in contact, closely adjacent, or at some distance from one another in the plasma clot, several pairs of explants being placed in each roller tube. Control cultures contained only mouse or rat heart, also in paired arrange- ment. During the first few days of culture in both experi- mental and control tubes, numbers of cells migrated outward f rom the explants, forming well-developed growth zones around the central fragments. Peripherally, these zones con- sisted primarily of mesothelium, with macrophages and fibro- blasts forming the bulk of the remainder. In experimental cultures where mouse and rat explants developed adjacent to each other, the inner margins of the two growth zones came into contact. Among this line, rat and mouse fibroblasts, macrophages, and mesothelial cells intermingled freely. On subsequent days, outward migration continued from the two explants, resulting in an intimate interweaving of mouse and rat cells. I n this way, the growth zones around adjacent rat and mouse explants fused smoothly without any clear line

110 MORGAN HARRIS

of demarcation between the two types of cells. There was 110

evidence of abnormal cellular accumulation or reaction along the line of junction between the two explants. Fibroblasts, as well as the other elements present, appeared completely indif- ferent to the foreign cells, even in close proximity. Except for progressive growth, the composite structure formed by the fused explants was maintained without change during culture periods of 21-36 days. The central portions of the two heart fragments continued to pulsate for 2 or 3 weeks, although independently of one another in all cultures. Such composite cultures did riot differ in degree of growth or cytological normality from control cultures containing paired fragments of rat or mouse heart only.

In another series of experiments, pieces of mouse spleen were explanted in association with rat spleen. Both were also cultured in separate tubes as a control procedure. In twenty- eigli t cultures of the experimental type, polymorphonncleizr leucocytes, lymphocytes, and macrophages moved out in grea It numbers from the central explants during the first 24 hours of culture. Between adjacent explants, the amoeboid activity of these elements resulted in a mixed zone of outwandering cells from the two species. No sign of antagonism was appar- ent in such random aggregations of ra t and mouse cells. Out- ward migration was approximately equal on all sides of the central explants, and the subsequent movements of the cells were apparently uninfluenced by the nearby alien tissue. I n particular, these elements showed no tendency in vitro to surround or react to cells of the opposite species, o r to move specifically in the direction of the foreign explant. The sur- vival period of lymphocytes and polymorphonuclear leuco- cytes in both experimental and control cultures was limited to 48 or 72 hours. After this time, the growth zones around splenic explants came to consist of fibroblasts, macrophages, and mesothelium. Between nearby rat and mouse fragments, these peripheral regions united in a structure similar to that described for heart explants. Both rat and mouse cells re-

COMPATIBILITY O F RAT AND MOUSE CELLS 111

mained normal in appearance and growth continued until the experiments were terminated at 21 days.

Rat spleen and mouse kidney, or mouse spleen and rat kid- ney, were cultured together in a final group of twenty-two experiments. Similar results were obtained with either com- bination. Appropriate control cultures were set up containing kidney and splenic explants, both from the same species. The growth of kidney explants was variable, but large sheets of renal epithelium appeared around a number of fragments within 2448 hours of culture. Where rat and mouse fragments had been placed close together, lymphocytes, polymorphonu- clear leucocytes, and macrophages were frequently present near the kidney cells in great number. These foreign cells, however, did not appear incompatible in any way with the sheets of renal epithelium, and their presence near kidney explants resulted from normal outward migration and the proximity of the two explants, rather than specific accumu- lation in the vicinity of the alien tissue.

Kidney epithelium in both experimental and control cul- tures was overgrown and replaced after several days with fibroblasts and macrophages originating in the same explant. A connection was subsequently established between the growth zones surrounding kidney and splenic fragments. No further significant changes occurred over a 21-day period.

Growth i m rat serum

I n a parallel group of experiments, rat serum was substi- tuted as a supernatant fluid in cultures, in place of a com- pletely heterologous medium. Thus the rat cells were growing in a homologous serum, while mouse explants were exposed to an alien environment as in thc foregoing experiments. This provided a means of testing whether tissue incompatibility depends on interaction of cells with a foreign serum, that is, whether an antagonism would develop against mouse cells in these mixed cultures if rat serum were present.

The specific combinations of rat and .mouse tissues in these cultures were identical with those employed in the first group

112 MORGAN HARRIS

of experiments, namely, paired explants of rat and mouse heart or spleen, together with a third series containing rat spleen and mouse kidney. The results of these various experi- ments may be treated together in a general description. In nearly all cultures both rat and mouse explants showed slightly better growth than in human cord serum and Locke solution; the effect was somewhat more marked on rat cells. This harmonizes with the finding of Lambert and Hanes ('lla) that nearly all mammalian sera exercise a slight in- hibiting effect on alien cells in tissue culture.

Aside from minor effects on growth, the sequence of events in these cultures followed closely the pattern described for corresponding cultures in the completely heterologous medium. Outgrowths from rat and mouse fragments eventu- ally united whenever the explants were sufficiently close for this to occur. Mouse cells in such cultures were invariably in good condition and showed no difference in cytological appear- ance from controls. During a culture interval of 21 days, there was no evidence of overgrowth or cellular antagonism by rat cells in the cultures.

DISCUSSION

These experiments demonstrate a complete indifference between rat and mouse cells maintained in close association in tissue cultures over a period of several weeks. Isolated cells from the two species are clearly compatible, therefore, as evidenced by the normal migration, growth, and appear- ance of individual cells in these cultures. It is of interest to compare these results with the descriptions of cellular phe- nomena in or around heterotransplanted mammalian tissue in vivo, as summarized by Loeb ( '30). A marked lymphocytic reaction against heterografts does not develop for a week or more ; this harmonizes well with the present experiments, where lymphocytes during their survival period of 48-72 hours in cultures, showed no cytotaxis for foreign cells. Poly- morphoneuclear leucocytes, on the other hand, often accumu- late around heterotransplants soon after operation : there was

COMPATIBILITY OF RAT AND MOUSE CELLS 113

no evidence of similar activity in tissue cultures. The behavior of fibroblasts in mixed cultures also contrasts strongly with their reported activity in the vicinity of foreign cells in vivo. In the body, this activity includes abnormal accumulation of fibroblasts near the foreign graft, as well as processes of encapsulation and infiltration. Such activity in the neighbor- hood of alien cells did not occur in any cultures in the present experiments. Even when directly in contact in culture, rat and mouse fibroblasts appeared entirely independent of, and un- affected by the foreign cells present. It is especially important that toxic phenomena, which play a major role in the destruc- tion of heterotransplants in the body, were completely lacking in vitro. During culture periods up to 36 days, rat and mouse cells in mixed cultures were uniformly healthy and could not be distinguished from similar cells in control cultures con- taining tissue from one species only.

These findings are significant in their bearing on Loeb's concept of tissue antagonism, as set forth in his theory of organismal differentials ( '30). Loeb assumes that cells in an alien environment give off factors, which either immedi- ately or after direct interaction with the foreign body fluids, become toxic. In heterotransplantation these injurious factors, or heterotoxins, are themselyes responsible for much damage to the graft, and further, serve to attract the lymphocytes, polymorphonuclear leucocytes and connective tissue of the host. These toxins initiate the antagonistic reactions of the host, whether in homoplastic or hetroplastic transplantation, and thus assume a central importance in the theory. The formation of toxins is conceived to be a local affair occurring directly between the transplant and body fluids surrounding it; to immune reactions Loeb attributes a secondary or com- plemen t ary influence.

If tissue antagonism originates from a direct toxic reaction by cells in a foreign medium, it is of considerable interest that tissues may be successfully cultivated in alien sera, as shown by Lambert and Hanes ( ' l l a ) and many other workers. In discussing these results Loeb has pointed out that in ordinary

114 MORGAN HARRIS

tissue culture technique the explant is surrounded by a relatively small amount of non-circulating fluid. Under these conditions the supply of toxins acting on the explant is assumed to be locally exhausted. The absence of host tissues in such cultures is mentioned as a further complicating factor. These objections, however, do not apply to the present ex- periments. The roller tube technique as used here has the dual advantage of using a relatively large volume of fluid medium which is kept in constant circulation by rotation of the tubes. I n this way a fresh supply of serum is continually acting on the explant, and any toxic factors produced are cir- culated throughout the medium. Furthermore, these experi- ments have demonstrated that mouse cells grow normally in roller tube cultures containing both rat serum and actively metabolizing rat cells, the latter corresponding to “host” tissue. Under these circumstances there was no sign of toxic factors, either acting directly on mouse cells, or serving to attract rat fibroblasts.

It is difficult to harmonize these results with the concept that sensitizing toxins are produced by a local interaction between graft and foreign body fluids. It is easier to suggest tentatively that sensitization may be set up by central agencies in the body of the host against foreign protsins, or organismal differen- tials from the graft. Sensitizing factors could then spread through the body and while not acting directly on the graft, could modify the behavior of host tissues toward the implant. That an animal previously inoculated with an alien graft does exhibit an altered reaction to repeated transplants of the same tissue is evident, if the tissue used has a high growth energy, as for example tumor or embryonic tissue. It is a familiar fact that a number of tumors will grow for a time in alien hosts but later regress, leaving the animal immune from the growth of subsequent transplants of the tumor. M. R. Lewis ( ’40) has shown further that the development of such immunity, in the case of certain mouse tumors, occurs well before regression of the primary transplant sets in. Likewise the experiments of Rous ( ’10) indicate that mouse embryonic tissue, inoculated

COMPATIBILITY O F RAT AND MOUSE CELLS 11.5

into adult mice as a primary graft, will grow vigorously for a short time, although retrogression eventually occurs. A second injection of embryonic tissue in the same mouse does not grow at all. On the other hand, Loeb ('18) found that repeated transplantation of thyroid in rats failed to accelerate tissue responses to the grafts, and on this basis concluded that previous inoculation does not sensitize the host. Thyroid, however, is characterized by a low growth energy, especially in an unfavorable environment, and this disadvantage might obscure any differences between primary and secondary im- plants. Moreover, in determining whether or not the host has been sensitized by a previous inoculation, the growth or failure to grow by transplanted tissues possessing a strong growth potential would seem to be a more reliable criterion than the time relationships of host responses.

Further experiments are necessary to evaluate the fore- going hypothesis more completely, as related to the cellu- lar phenomena in tissue reactions. Direct toxic action on heterografts may be ascribed at least in part to other, typical immune reactions. Lambert and Hanes ( ' l lb ) , Lumsden ( '28), the writer 3, and others have demonstrated that sera from animals bearing heterotransplanted tumors are directly toxic to the corresponding cells in cultures. Since similar cytotoxins are not developed in mixed cultures of rat and mouse cells, it is probable that they do not form locally, but in a manner analogous to other typical antibodies.

SUMMARY

1. Paired rat and mouse tissue fragments have been cultured together in roller tubes. Heart, spleen and kidney from the two species were explanted in varying combinations.

2. I n all cultures, adjacent rat and mouse fragments through cellular outgrowth eventually fused together to form a single, composite structure, stable over culture periods ranging up to 36 days.

Unpublished data.

116 MORQAN HARRIS

3. Fibroblasts, lymphocytes, and polymorphonuclear leuco- cytes of both species migrated out normally from explants, without any attraction toward the foreign cells present. No evidence of encapsulation or specific accumulation in the vicinity of alien elements was observed. 4. Rat and mouse cells apparently are physiologically com-

patible in vitro. Toxic phenomena were completely absent in mixed cultures and both rat and mouse cells remained normal in cytological appearance.

5 . The bearing of these results on current concepts of an- tagonism to transplanted tissues is discussed.

I am indebted to Dr. E. B. Krumbhaar for helpful criticism, and to Dr. and Mrs. Warren H. Lewis fo r many constructive suggestions during the present work, as well as for the gen- ei*ous provision of laboratory facilities.

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-___ 1922 Iiiiiiiuiiity in relation to transplanted tissue. J. Med. Res.,

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GOODPASTVRE, E. W., E. DOUGLAS AND K. ANDERSON A study of human skin grafted on the chorioallantois of chick embryos. J. Exp. Med.,

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