THE EFFECT OF PROLONGED CULTIVATION IN VITRO UPON THE PATHOGENICITY OF YELLOW FEVER VIRUS

1. Experimental evidence is presented to show that prolonged cultivation of yellow fever virus in vitro results in a change in its pathogenicity, and that this change varies with the type of tissues used for the cultivation. 2. In the tissue cultures used for the propagation of the virus, three different types of tissues were used. They included whole mouse embryo, chick embryo from which the head and spinal cord had been removed, and testicular tissues of mice and guinea pigs. 3. The changes in the pathogenicity of the virus cultivated for a period of over 3 years in a medium containing the tissues of whole mouse embryo were not striking. The viscerotropic virulence of the virus appeared somewhat diminished, in that when injected subcutaneously into rhesus monkeys or hedgehogs it failed to produce a fatal infection, although there is evidence to indicate that a generalized infection takes place as demonstrated by the appearance of virus in the circulating blood in relatively high concentration during infection. The neurotropic virulence of the virus remained unaltered during the cultivation in this medium. 4. The changes in the pathogenicity of the virus cultivated in medium containing tissues of chick embryo from which the head and spinal cord had been removed were very pronounced. The viscerotropic virulence of the virus was lost to a large extent. When injected subcutaneously into monkeys there was as a rule a very mild generalized infection, as demonstrated by the minimal quantities of virus found in the circulating blood. Its neurotropism was also much diminished. When injected into monkeys intracerebrally, it no longer produced a fatal encephalitis but only a moderate febrile reaction, followed by recovery and solid immunity to reinoculation with a highly virulent strain of virus. When injected intracerebrally into mice, the mortality ratio was not diminished but the incubation period was markedly prolonged. 5. The changes in the pathogenicity of the virus cultivated in medium containing testicular tissues were somewhat similar to those observed after cultivation in chick embryo medium which contained only a minimal amount of nervous tissue. Its viscerotropic affinity had been largely lost and only very small amounts of virus were found in the circulating blood of monkeys inoculated subcutaneously. Given intracerebrally, it produced death from encephalitis in monkeys. The incubation period in mice inoculated intracerebrally with this virus was also prolonged but somewhat less so than with the virus grown in chick embryo tissues without the central nervous system.     

THE USE OF YELLOW FEVER VIRUS MODIFIED BY IN VITRO CULTIVATION FOR HUMAN IMMUNIZATION

The response of rhesus monkeys to a subcutaneous inoculation with varying amounts of virus modified by prolonged cultivation in vitro has been studied. The tissue components of the medium consisted of chick embryo tissue containing minimal amounts of nervous tissue. The immunity produced in monkeys, as measured by the antibody titer developed, has no relation to the amount of virus inoculated. Monkeys inoculated subcutaneously with the tissue culture virus are rendered immune to a subsequent injection of a highly virulent yellow fever virus. This resistance is already present 7 days after vaccination. The subcutaneous inoculation of the culture virus into immune persons leads to a substantial increase of the serum antibody titer. The results of vaccinating eight normal persons with culture virus are presented. The reactions were minimal. The highest temperature recorded following vaccination was 37.4°C. The sera taken from the eight vaccinated persons 2 to 4 weeks after inoculation with the tissue culture virus showed the presence of yellow fever antibodies.     

STUDIES ON THE PROPAGATION IN VITRO OF POLIOMYELITIS VIRUSES : I. VIRAL MULTIPLICATION IN TISSUE CULTURES EMPLOYING MONKEY AND HUMAN TESTICULAR CELLS

Poliomyelitis virus was propagated in vitro successfully in extraneural tissues. Suspended tissue fragment cultures and combined plasma clot-suspended tissue fragment cultures of monkey or human testicular tissues were employed. Five strains representative of poliomyelitis virus were maintained for from 36 to 263 days in the suspended tissue fragment type of culture. The dilution factors calculated by tissue replacements for the eight serial passages ranged from 10^7.8 to 10^44.5 and when assessed by fluid replacements, from 10¹⁵ to 10^95.3. The LD₅₀ for each strain of Type 2 virus was determined for selected transfers. The identify of each strain of virus was established by neutralization tests and histopathological findings in monkeys dead from the injection of tissue culture virus. Control experiments and other tests made known that propagation of poliomyelitis virus did not occur in the absence of viable testicular cells and that an extraneous virus was not inadvertently acquired during the course of these studies.     

EXPERIMENTAL STUDIES OF THE NASOPHARYNGEAL SECRETIONS FROM INFLUENZA PATIENTS : VII. FURTHER OBSERVATIONS ON THE CULTURAL AND MORPHOLOGICAL CHARACTERS OF BACTERIUM PNEUMOSINTES.

After artificial cultivation for a period of over 3 years Bacterium pneumosintes has maintained its original morphological and cultural characteristics, when grown in the original medium. Adaptation to a saprophytic existence has been accompanied by a loss of pathogenicity. Our strains now grow readily under strictly anaerobic conditions in a variety of media with peptone broth as a base, enriched with fresh tissue, blood, or by the growth of other bacteria. Surface colonies have been obtained on blood agar plates in. an anaerobic jar. These various methods of cultivation are adapted to special purposes. In broth cultures Bacterium pneumosintes grows in larger forms than in the ascitic fluid-tissue medium, but the identity of the microorganisms is proved by their serological reactions and by reversion to the minute forms on transfer to the original medium.     

STUDIES ON YELLOW FEVER IN SOUTH AMERICA : V. TRANSMISSION EXPERIMENTS WITH CERTAIN SPECIES OF CULEX AND A?DES

1. Yellow fever virus has been transmitted from monkey to monkey both by the bites of Aëdes (Ochlerotatus) scapularis which had fed upon monkeys infected with yellow fever and by the injection of the ground up bodies of such mosquitoes. 2. A fatal infection has been obtained by the injection of the ground up bodies of Aëdes (Ochlerotatus) serratus, which had previously fed on an infected monkey, and a mild infection has been secured by the similar injection of Aëdes (Taeniorhynchus) taeniorhynchus. 3. No definite infection has been secured either by the bites or by the injection of Culex quinquefasciatus (C. fatigans). However, some of the experimental animals bitten by this species have been relatively immune following inoculations of blood or tissues containing virus.     

THE SUSCEPTIBILITY OF MARMOSETS TO YELLOW FEVER VIRUS

1. It has been possible to introduce yellow fever virus into the small Brazilian monkeys, Callithrix albicollis and Leontocebus ursulus, by the bites of infected mosquitoes and to carry the virus through a series of four passages in each species and back to rhesus monkeys by the bites of Stegomyia mosquitoes fed on the last marmoset of each series. 2. Five specimens of L. ursulus were used. Four developed fever, and all died during the experiments. At least two showed liver necroses comparable to those found in human beings and rhesus monkeys that died of yellow fever. 3. Twenty specimens of C. albicollis were used. Very few showed a temperature reaction following the introduction of virus. Of those that died, none had lesions typical of yellow fever as seen in certain other species of monkeys and in humans. 4. The convalescent serum from each of five C. albicollis protected a rhesus monkey against yellow fever virus, but the serum from a normal marmoset of the same species was found to be non-protective.     

INFECTIVITY OF BLOOD DURING THE COURSE OF EXPERIMENTAL YELLOW FEVER

Five M. rhesus fatally infected with yellow fever virus ran varied and typical courses, death occurring from 82 hours to 10 days after infecting. Batches of A. aegypti were fed daily on each monkey and specimens of blood injected into other animals. By mosquito transfer, the virus was found to be circulating in the peripheral blood 1 or 2 days after the infecting and the same interval before the onset of fever; in one instance, mosquitoes became infectious by feeding on a monkey 12 hours after its inoculation. Mosquitoes continued to acquire infectivity during the febrile period and for 1 day thereafter, except in one instance when death occurred during fever which prevented post-febrile testing. By subinoculation of blood, the disease was transferred before and after, as well as during the same interval as in mosquito transmission. In one of two attempts, the virus was carried by this means as early as 12 hours after the donor animal was infected. Following the first day of the post-febrile period, blood transmissions were irregularly fatal beyond the period infective for mosquitoes. These results point to a remarkably rapid multiplication of the virus in the animal host, in one case a blood subinoculation (0.5 cc.) being successful at the first test 24 hours after the donor monkey was bitten by only 2 A. aegypti. The regular acquisition of infectivity by mosquitoes fed during the incubation period is of especial interest in indicating the infectivity of human cases for mosquitoes before the appearance of clinical symptoms. This offers one explanation for the insidious propagation of epidemics of yellow fever and should be useful in the institution of control activities during an outbreak of this disease.     

THE FILTRABILITY OF YELLOW FEVER VIRUS AS EXISTING IN THE MOSQUITO

The virus of yellow fever as it exists in Aëdes aegypti mosquitoes, both in their so-called infective stage and in the intermediate condition termed the "incubation period", is capable of passing through Berkefeld N filters when suspended in normal monkey serum, although earlier investigators have shown that the virus from infective mosquitoes will not do so when suspended in physiological salt solution. The virus of yellow fever as it exists in mosquitoes behaves with regard to filtration through Berkefeld N filters as does the virus in the blood of infective monkeys.     

STUDIES OF A MURINE STRAIN OF POLIOMYELITIS VIRUS IN COTTON RATS AND WHITE MICE

1. A neurotropic murine virus was isolated by passing poliomyelitis virus (SK strain) from the monkey to cotton rats and white mice. 2. The murine virus has been grown in tissue culture consisting of embryonic mouse brain in ox serum ultrafiltrate. 3. The symptoms and lesions produced by the murine infection compare in all respects with those of poliomyelitis in monkey and man. 4. The murine virus, while highly pathogenic for mice and cotton rats, is non-pathogenic for albino rats, guinea pigs, and rabbits. It possesses limited pathogenicity for rhesus monkeys. 5. Although producing no paralysis in the above mentioned refractory animals, the murine virus may be recovered in active form from neural and extraneural sites of infected albino rats, guinea pigs, and monkeys, but not from rabbits. 6. The identity of the murine and monkey virus is further suggested by cross-neutralization between the murine virus and homologous (SK) and related (Aycock) antipoliomyelitis sera, as well as between homologous and related monkey poliomyelitis virus and antimurine virus sera. 7. Immunization of monkeys with live murine virus, in the form of mouse brain or tissue culture, seems to confer some degree of resistance against subsequent infection with the homologous poliomyelitis monkey virus. 8. The presence of the murine virus in the central nervous system of infected monkeys appears to interfere with the propagation of SK and Aycock poliomyelitis monkey virus in the same animal.     

THE TRANSMISSION OF YELLOW FEVER : EXPERIMENTS WITH THE "WOOLLY MONKEY" (LAGOTHRIX LAGO-TRICHA HUMBOLDT), THE "SPIDER MONKEY" (ATELEUS ATER F. CUVIER), AND THE "SQUIRREL MONKEY" (SAIMIRI SCIREUS LINNAEUS)

1. Saimiri sciureus has been infected with yellow fever virus, both by the inoculation of infectious blood and by the bites of infective mosquitoes. Some of the monkeys have died, showing lesions, including hepatic necrosis, suggesting yellow fever as seen in human beings and in rhesus monkeys. Virus has been transferred back to M. rhesus from infected Saimiri both by blood inoculation and by mosquito bites. The virus undoubtedly has been maintained through four direct passages in Saimiri. Reinoculations of infectious material into recovered monkeys have not given rise to invasion of the blood stream by virus. Sera from recovered animals have protected M. rhesus against the inoculation of virus. 2. It has been possible to pass the virus to and from Ateleus ater by the injection of blood or liver and by the bites of mosquitoes. The livers from two infected animals have shown no necrosis. The serum from one recovered monkey proved to be protective for M. rhesus. 3. Only three out of twelve Lagothrix lagotricha have reacted to yellow fever virus by a rise in temperature. Probably none have died as a result of the infection. In only one instance has the virus been transferred back to M. rhesus. The sera of recovered animals have had a protective action against yellow fever virus.     

THE RELATIONSHIP OF YELLOW FEVER OF THE WESTERN HEMISPHERE TO THAT OF AFRICA AND TO LEPTOSPIRAL JAUNDICE

1. The yellow fever now in South America, the present yellow fever of Africa and the historic yellow fever of Panama and other American countries are the same disease. This conclusion is based on cross immunity tests in monkeys with strains of yellow fever virus from Africa and Brazil and on tests of sera from 25 persons, who had recovered from yellow fever in various places and at various times, for the power to protect monkeys against African or Brazilian virus strains. 2. Cases of leptospiral jaundice (Weil''s disease) were present among those diagnosed as yellow fever in the recent epidemic in Rio de Janeiro. This is shown by the isolation of cultures of leptospirae from the blood of two patients by H. R. Muller and E. B. Tilden of The Rockefeller Institute, and by the demonstration by us of protective power against leptospirae and absence of protective power against yellow fever virus in the sera from two persons after recovery. The isolation of leptospirae by Noguchi and other investigators from the blood of occasional patients in past epidemics of yellow fever in a number of American countries indicates that leptospiral jaundice was present then as well and was diagnosed clinically as yellow fever. 3. The absence of protective power against leptospirae shown by the Brazilian sera which protected against yellow fever virus and the absence of protective power against yellow fever virus in the sera that protected against leptospirae point to the probability that American yellow fever is not the combined effect of leptospirae and yellow fever virus. The position of L. icteroides, isolated by Noguchi during yellow fever epidemics, now appears to be not that of a secondary invading microorganism in cases of virus yellow fever, but that of the incitant of a form of infectious jaundice, sometimes fatal, often coincident in its appearance with typical yellow fever and apparently indistinguishable from it clinically. This leptospiral disease has not hitherto been separated from true yellow fever. Noguchi''s discoveries become; therefore, of the greatest significance in respect to the epidemiology and causation of yellow fever and of infectious jaundice, previously confused one with the other. In all outbreaks of supposed yellow fever hereafter the existence of the two kinds of jaundice, one due to yellow fever virus and the other to leptospirae will have to be taken into account. Only the former probably is spread by mosquitoes and requires anti-mosquito measures for its control. 4. The only difference observed by us between the American and African strains of yellow fever virus was a pronounced difference in virulence for monkeys. The virulence of the two African strains studied was very high while that of the one American strain was highly variable and usually low.     

CONTRIBUTIONS TO THE STUDY OF THE MECHANISM OF THE GROWTH OF CONNECTIVE TISSUE

When connective tissue cells have been cultivated for a certain length of time in a medium which has been repeatedly changed, a definite relation arises between the rate of growth of the cells and the composition of the medium. It is possible, by adding to the culture medium a given quantity of certain substances, such as embryonic juices, to foresee the extent to which a fragment of tissue composed of a given strain of cells will increase in a given time. The rate of growth of a strain of cells can be accelerated or retarded by the addition to the medium of activating or retarding substances. The dynamic condition of a strain of connective tissue cells, which have been living in a given medium for some time, is not a definitely acquired characteristic, but a temporary state, and is the product or function of the medium in which the cells are living, and is readily modified merely by altering the composition of the medium. A knowledge of the characteristics of the growth of connective tissue described has led to a new result,—the indefinite proliferation of a strain of connective tissue cells outside of the organism. The strain of connective tissue originally obtained from a fragment of chick embryo heart, which had been pulsating in vitro for 104 days, was still actively alive after sixteen months of independent life and more than 190 passages. The rate of proliferation of the connective tissue sixteen months old equalled and even exceeded that of fresh connective tissue taken from an eight day old embryo. It appears, therefore, that time has no effect on the tissues isolated from the organism and preserved by means of the technique described above. During the sixteenth month of life in vitro the cells increased rapidly in number and were able in a short time to produce a large quantity of new tissue. This fact, therefore, definitely demonstrates that the tissues were not in a state of survival, as was the case in certain earlier experiments, but in a condition of real life, since the cells of which they were composed, like microorganisms, multiplied indefinitely in the culture medium.     

Latent viral infection of cells in tissue culture. I. Studies on latent infection of chick embryo tissues with psittacosis virus

Chick embryo tissues maintained for from 11 to 28 days in Hank's balanced salt solution lost their capacity to support the multiplication of psittacosis virus. The virus however infected such cells, as active multiplication of the virus occurred on the addition of beef embryo extract to this nutritionally poor medium at any period up to 28 days of cultivation in balanced salt solution. The virus remained in a state of latency for as long as 15 days in these starved cells in a non-infectious phase. These results obtained in this in vitro model system for the investigation of latent infections of cells with psittacosis virus suggest that cell nutrition as well as an alteration in the immunological defenses of the host may prove to be an important factor in the activation of latent viral infections.     

STUDIES ON SOUTH AMERICAN YELLOW FEVER : III. TRANSMISSION OF THE VIRUS TO BRAZILIAN MONKEYS PRELIMINARY OBSERVATIONS

Yellow fever virus from M. rhesus has been inoculated into a South American monkey (Cebus macrocephalus) by blood injection and by bites of infected mosquitoes. The Cebus does not develop the clinical or pathological signs of yellow fever. Nevertheless, the virus persists in the Cebus for a time as shown by the typical symptoms and lesions which develop when the susceptible M. rhesus is inoculated from a Cebus by direct transfer of blood or by mosquito (A. aegypti) transmission.     

SOME CONDITIONS OF THE REPRODUCTION IN VITRO OF THE ROUS VIRUS

A method has been developed by which the susceptibility of chickens to Rous virus can be tested, and the virulence of eight or ten different fluids compared in a single animal. The results of five series of experiments made with this technique can be summarized as follows: When a medium composed chiefly of chicken serum and Tyrode solution and containing no fresh tissues is inoculated with filtered extract of Rous and other sarcomas and incubated for 48 hours, it never produces a tumor after being injected into chickens. The virus has apparently been destroyed or at least has lost activity. But, in a solid medium, composed chiefly of serum and Tyrode solution and containing fragments of fresh tissues, the virus is found to increase readily, as shown in the first series of experiments. In the course of 15 months, the experiments have been repeated many times, with identical results. Flasks containing embryo pulp or leucocytes inoculated with filtered extract of sarcoma are used to keep on hand a constant supply of the Rous virus. The cultures of monocytes inoculated with the filtered extract often assume the appearance of the cultures of Rous sarcoma. They may also remain normal to all appearance despite the circumstance that the virus is multiplying within the medium. It is not certain that the activity of Rous virus is always accompanied by cell lesions, but there is no doubt that its increase depends on the presence of fresh tissues within the medium. The disappearance of the Rous virus from a medium that does not contain any fresh tissue may be interpreted as follows: the agent has been destroyed; or it is still present in a concentration lower than 1 in 50,000, which is the concentration required to produce a tumor even in the more susceptible chickens; or, according to the hypothesis of Gye, it is present in an inactive form. In the above experiments, the fresh tissues added to the medium might conceivably have enabled the virus to keep its full activity, through supplying the conditions requisite therefor, or they might merely have furnished an activating substance. The value of Gye''s hypothesis was tested in a series of experiments. The results indicate that the tumor-producing virus present in the cultures was not composed of two parts, an inactive part multiplying in the medium, and an activating part supplied by the tissues. In another series of experiments, the relations between the reproduction of the virus and the quantity of the tissues contained in the medium were studied. The presence of a small fragment of leucocytic film or spleen tissue was sufficient to prevent the virus from disappearing. Approximately 1 c.mm. of spleen tissue in 3,000 c.mm. of medium may on occasion maintain a concentration of Rous virus in this fluid sufficient to produce a tumor upon inoculation into chickens. But this rarely happens. Generally when the medium contained only one fragment of spleen or leucocytic film, or 1 drop of embryonic pulp, the virus disappeared rapidly. When the quantity of tissue was from five to nine times larger, an abundant production of virus was practically always found. It became obvious that the quantity of active virus present in a medium containing multiplying cells depends upon the amount of tissue in the medium. In the fourth series of experiments, the kind of cells needed for the multiplication of the virus was ascertained. Rous virus was found to disappear rapidly from the fluid of cultures of fibroblasts, while it multiplied readily in cultures of leucocytes, the total volume of both tissues being approximately the same. It should be remembered that strains of fibroblasts obtained from Rous and other sarcomas very rarely produce tumors upon inoculation into chickens, while the inoculation of cultures of macrophages from the same tumors practically always determines their appearance. The fifth series of experiments showed that the cell metabolism is an important factor in the reproduction of the virus. When the activity of tissues had been suppressed or very much decreased by freezing, no virus was produced, while it multiplied readily in the control. The lack of oxygen for a period of 24 or 48 hours stopped cell proliferation, and at the same time the production of Rous virus ceased. However, the fluid of some of these dead or inactive cultures, after 6 days incubation, was still able to give rise to a small tumor upon inoculation into a chicken. There is an evident relation between the proliferating activity of the tissues and the production of the virus. But the agent may persist for several days in association with dead tissues. It may be concluded that the reproduction in vitro of the active virus depends on the presence of fresh tissues in the culture and upon the quantity, the activity, and the nature of the cells contained in the medium.     

CHARACTERISTICS OF A STRAIN OF LYMPHOCYTIC CHORIOMENINGITIS VIRUS ENCOUNTERED AS A CONTAMINANT IN TISSUE CULTURES OF RABIES VIRUS

A strain of lymphocytic choriomeningitis virus has been encountered, which grows readily in mouse embryo, serum, Tyrode culture media. Its origin is not definitely known but appears to be either the mouse brain tissue or, more probably, the monkey serum. This strain gives clear cut results on filtration tests through Elford membranes, establishing the size of the virus, according to formula, as 33 to 50 mµ. The strain shows a high and uniform virulence in W-Swiss mice. This appears to be due in part, at least, to the age and strain of mice employed for passage and titration. The strain has been found to be more virulent in young than in old mice, especially following intraperitoneal inoculation. Finally, the strain, when given as a vaccine intraperitoneally in amounts as small as 160 intracerebral lethal doses, induces an immunity against subsequent intracerebral inoculations of as much as 10,000 lethal doses.     

ON THE NUTRITIONAL REQUIREMENTS IN VITRO OF NORMAL AND MALIGNANT MOUSE EPITHELIUMS

The cultivation in vitro of mouse tissues derived from normal organs from 86 spontaneous epithelial tumors of the mammary gland, and from 27 Ehrlich carcinomas, has been undertaken, together with a study of the properties of the various cell types. 1. The tissues liquefied fibrin from mouse and rat plasma more readily than fibrin from chicken plasma. Clots made of chicken plasma alone, if thoroughly washed, did not inhibit the migration of the cells. Normal and tumor tissues liquefied fibrin from the mouse, rat, and chicken more actively than Ehrlich carcinoma did. 2. Mouse epitheliums, both normal and malignant, showed greater activity than connective tissue cells from the same origin and were not overgrown by the latter. 3. Mouse epithelium was more active in rat serum than in mouse or chicken serum and in embryonic juice from chickens, mice, and rats. None of these fluids, however, supported cell proliferation indefinitely except in the case of Ehrlich carcinoma. 4. These results indicate that mouse tissues possess nutritional requirements which are different from those of fibroblasts and epithelial cells of other animals. Nutritive media that suffice for prolonged cultivation of the normal and malignant tissues of the rat and the fowl, and also of Ehrlich carcinoma, are not suitable for the cultivation of adult mouse epithelium derived from normal organs or from spontaneous mammary gland tumors. 5. Rat serum supported the life of spontaneous tumors for a limited period of time only, whereas it enabled the Ehrlich carcinoma to proliferate indefinitely. Normal organs and spontaneous tumors were not capable of invading normal tissues as Ehrlich carcinoma did.     

JENNERIAN PROPHYLAXIS BY MEANS OF INTRADERMAL INJECTIONS OF CULTURE VACCINE VIRUS

The second revived strain of culture vaccine virus has been propagated through 130 culture passages during a period of 3 years. It seems to be adapted to in vitro cultivation and still has an intradermal titer (rabbits) of 1 to 100,000 or 1 to 1,000,000. Intradermal inoculations in human beings of 0.1 cc. amounts of culture virus diluted from 2.5 to 10 times result in primary takes in unvaccinated people and immune reactions or accelerated takes in individuals previously successfully vaccinated. Primary takes produce an immunity to standard strains of calf lymph. Culture virus mixed with purified gum acacia (2.5 per cent), frozen, desiccated, and sealed in vacuo retains its activity for a month at 37°C., and when the dried virus is resuspended in saline solution it is suitable for intradermal vaccination of human beings.     

CULTURES OF ORGANIZED TISSUES

An artificial organism, if one may so term it, composed of a complex of tissues, was cultivated for a long period of time. Small fragments of intestine from chick embryos 20 to 21 days old were placed in a suitable medium. The epithelium proliferated and completely covered the fragment of intestine after 4 to 6 days. A small body was thus formed, round or oblong in shape, surrounded by cylindrical epithelium and containing epithelial, connective, and muscle tissues, endothelium, and ameboid cells. After a month''s cultivation in vitro, no necrosis had occurred. Therefore, it may be assumed that, through the intestinal epithelium, the medium supplied the intestinal tissue with sufficient nourishment. No uncontrolled proliferation took place after the epithelium bad surrounded the entire fragment. The cultivation of complex tissues will facilitate the study of the interactions of the different cells under various conditions. In some experiments, pure cultures of epithelial cells were grafted into such an "organism" without difficulty. The growth of malignant cells could be studied in the same way. When the "organism" was placed in a fluid medium, the epithelium remained normal but the stroma disappeared. It seems that plasma played an important rôle in the maintenance of the tissues in their normal condition.