and there was no tendency towards the formation of a cellular network. Refractile fat globules then became frequent in the cell cytoplasm.

When the cultures were fixed and stained with Giemsa stain the outline and structure of the cells became distinct. The cytoplasm appeared as a light blue, finely granular body with a more deeply stained, coarsely granular nucleus. The structures containing chromatin took on a deep purple stain. Figure 3 represents a photograph of stained individual cells highly magnified.

Although growth of human connective tissue derived from fetal heart tissue has been obtained which was as extensive as the growth of connective tissue derived from embryonic heart tissue of the chick, the growth of human tissue was usually less dense and less extensive. Fluctuations which occurred during the passages of human cultures were greater than those occurring in cultures of chick tissue. Liquefaction in human tissue cultures was more marked and eventually interfered with cell proliferation. In cultures of chick tissue the liquefaction that took place was slight and did not interfere with cell proliferation.

The fetal human tissue which was used in making cultures was generally obtained from fetal cadavers 4 to 6 months old; the embryonic chick tissue was obtained from 8 to 15 day old embryos. The latent period where human tissue was cultivated was usually from 16 to 18 hours. For chick tissue growth appears within 10 or 12 hours after the original fragment has been embedded.

SUMMARY.

A strain of human connective tissue was kept in a condition of active life in vitro for more than two months. When a medium has been devised the composition of which is more constant, it is reasonable to suppose that human connective tissue can be cultivated in vitro for an indefinite period.

THE CULTIVATION OF HUMAN SARCOMATOUS TISSUE IN

VITRO.

By JOSEPH R. LOSEE, M. D.
Pathologist to the Hospital

and

ALBERT H. EBELING.

(From the Laboratories of the New York Lying-In Hospital and of the Rockefeller Institute for Medical Research.)

THE first attempt to cultivate human malignant tumor in vitro was made in 1911 by Carrel and Burrows. Small fragments of tumor 1 Carrel, A., and Burrows, M. T., Jour. Exper. Med., 1911, xiii, 387.

were cultivated in normal human plasma and incubated. It was observed in some cases that after a few days the fragments were surrounded by many cells; but generally liquefaction of the medium occurred. The tissues were kept in a condition of survival for a few days, but no real cultures were obtained.

Lately it became possible to keep human fetal tissue, derived from fresh cadavers, in a condition of independent life for several generations2, and we therefore attempted to cultivate human sarcomatous tissue in the same manner.

TECHNIQUE.

The medium employed in these experiments was composed of equal parts of normal human plasma, Ringer's solution and varying quantities of extract.

The extract was prepared by cutting tissues obtained from fresh fetal cadavers into small pieces, and adding an equal quantity of Ringer's solution. After forty-eight hours in cold storage the substance was centrifuged and the supernatant fluid pipetted off. This fluid was used as extract in the making of cultures.

The tissues employed were obtained from recently excised sarcomatous growths3, and cultures were made about one and a half hours after excision. The primitive cultures were made by putting small, thin fragments of this tissue into the medium. After coagulation the cultures were immediately placed in the incubator and incubated at 38°C. for 24, 48, and 72 hours, the time of passage into fresh medium being governed by conditions which developed in the culture. Before the fragments in cultures were transferred into fresh medium they were washed in Ringer's solution for about one minute.

EXPERIMENTS.

Experiment 1, Series 1, Cultures 1, 2, 3 and 4.-Fragments from the periphery of an osteosarcoma1 were cultivated in equal parts of human plasma and Ringer's solution, to which one fourth part of extract was added. The cultures were made about one hour and a half after excision of the growth and incubated.

After 1, 6, and 18 hours' incubation there was no evidence of cell proliferation in any of the cultures. In twenty-four hours there was

2. Losee, J. R., and Ebeling, A. H., Jour. Exper. Med., 1914, xix, 593.

3. The sarcomatous growths were obtained by Dr. Carrel, through the courtesy of Dr. W. B. Coley, from some of his cases at the General Memorial Hospital, New York. Immediately after excision the tissues were carried to the Laboratories of the New York Lying-In Hospital.

Appears also in Jour. Exper. Med., Vol. xx, No. 2.

4. Pathological diagnosis: large round cell sarcoma.

still no growth to be observed and the medium had become liquefied around the fragments. The cultures were therefore washed and changed into fresh medium (first passage), to which one half part of extract was added. After twenty-four hours' incubation, growth was found to be present in culture 3, but no growth was observed in cultures 1, 2, and 4, and in forty-eight hours there was still no evidence of cell proliferation in these cultures; the medium had become liquefied and they were therefore discarded.

Culture 3 was cultivated in the same medium (second passage), and in forty-eight hours growth was apparent. This culture was transferred into fresh medium for twelve passages, during which time (twentyone days) growth was observed after each transfer into fresh medium. The culture was stained and photographed (figure 1).

Series 2, Cultures 1, 2, 3, and 4.-Fragments from the peripheral area of the same growth were cultivated in the same manner as series 1. After 1, 6, and 18 hours' incubation there was no evidence of cell proliferation. The medium was still in good condition. In twenty-four hours no growth was observed, and the medium about the pieces in cultures 1, 2, and 3 had become liquefied. Culture 4 was allowed to remain in the incubator. It was examined after forty eight and seventy-two hours, but there was no evidence of cell proliferation, although the medium was still in good condition. The culture was discarded. After twenty-four hours cultures 1, 2, and 3 were changed into fresh medium (first passage), to which one half part of extract was added.

Culture 1 after twenty-four hours showed no growth, and the medium was completely liquefied. It was changed into fresh medium (second passage), the same proportion of extract being added as in the previous passage. In twenty-four hours a few scattered cells were observed, but after forty-eight and seventy-two hours there was no further increase in the extent of cell proliferation. The culture was discarded.

Culture 2 (first passage) after twenty-four hours' incubation showed an area of cell proliferation, with no liquefaction of the medium. In seventy-two hours a good growth had developed. The medium was slightly liquefied. It was then changed into fresh medium (second passage) to which one fourth part of extract was added. After twenty-four hours growth had developed, and in forty-eight hours the area of cell proliferation was more extensive, but liquefaction of the medium had developed. The culture was changed into fresh medium (third passage) with the same proportion of extract added as in the previous passage. After twenty-four hours a few cells were observed in the medium surrounding the central fragment. In forty-eight hours there was no increase in the extent of growth and the medium had liquefied. The culture was again transferred into fresh medium (fourth passage), one part of extract being added. In twenty-four hours the medium had become liquefied and no growth was observed. The culture was changed

(fifth passage) into fresh medium, the same proportion of extract being added as in the previous passage. In twenty-four hours growth had developed, and in forty-eight hours it was more extensive, but the medium was almost completely liquefied. The culture was transferred (sixth passage) into fresh medium in the same manner as in the previous passage.

Fig. 1.

After twenty-four hours cell proliferation was observed, but small colonies of bacteria had also developed. The infection was general and the culture was discarded.

Culture 3 (first passage), after twenty-four hours' incubation, showed a few scattered cells which had grown out from the original fragment. In forty-eight hours the growth of new cells had increased, but

the medium was slightly liquefied. The culture was then treated (second passage) in the same manner as in the previous passage. After twentyfour hours good growth had developed and the medium was in good condition. After forty-eight hours the growth was more extensive, but after seventy-two hours there was no further increase and the medium was

slightly liquefied. The third passage into fresh medium was then made, the proportion of extract being increased to one part. After twenty-four hours' incubation the growth had developed and the medium was in good condition. In forty-eight hours the culture was growing actively and the medium had become slightly liquefied. The culture was subsequently changed into fresh medium for twenty-one more passages. The extent