A CLIPPING OF BLACK WING COVERTS.

In clipping, only the first and second rows of wing coverts are taken.

1. FEATHERS OR DOWN TAKEN FROM THE NECK AND HEAD OF AN ADULT BIRD. The two larger, one at each side of the group, are from the white ring toward the base of the neck of the cock, the long hair-like middle feather is from the head, and the others from the neck. The "hair" is seen to be a greatly elongated barb.

MANIFESTED LIFE OF TISSUES OUTSIDE OF THE

ORGANISM.

By ALEXIS CARREL and MONTROSE T. BURROWS.

(From the Laboratories of the Rockefeller Institute for Medical Research, New York.)

I. INTRODUCTION.

Fragments of tissues and organs of mammals and other animals can be kept outside of the organism in a condition of manifested life, when they are placed under certain conditions in a proper culture medium. Their life is essentially characterized by an active growth of the cells from the original fragment into the medium where they undergo direct or indirect division. These cells cover a wide area of the medium, and are often very densely packed. They grow during a period of time which varies from 5 or 6 days to more than 20 days, without any evidence of necrobiosis. The cells which have wandered into or have been born in the plasmatic medium can be transplanted into a new medium and produce a very luxuriant generation of cells. A culture of tumor transplanted into the body of an animal can take and grow rapidly.

The idea of cultivating tissues as previously defined is far from being new. Many experimenters have already thought of the possibility of growing tissues outside of the body, and several have attempted to develop adequate method for it. In 1897 Leo Loeb thought that the culture of tissues in an artificial medium outside of the body was possible, and stated that he had found a method for accomplishing it. But the technique and the results of his experiments have not been published. In 1907 Harrison demonstrated in a series of splendid experiments, made in the anatomical department of Johns Hopkins University, that embryonic tissue of the frog, transplanted into coagulable lymph, will develop normally. The central nervous system of a frog embryo, covered with fluid from the lymph sac of an adult frog, produced long nerve fibers. These experiments demonstrated that the nerve fibers are really an outgrowth from a central neurone. But they demonstrated also a very much more important fact, the possibility of growing tissue outside

the body. At this time Carrel was engaged in the study of the laws of cicatrization of tissues and cutaneous wounds of mammals, and resolved to use for that purpose the method of Harrison. Then Burrows, under the guidance of Prof. Harrison himself, adapted the method to the cultivation of tissues of the chick embryo; that is, of a warm-blooded animal. Then, in September, 1910, at the Rockefeller Institute we succeeded in cultivating in vitro, adult tissues of mammals.

We used at first the culture method of Harrison, that is, of small pieces of tissue suspended in a hanging drop of plasma. Afterwards we developed a method of culture on a plate, which permitted us to grow large quantities of tissues. It became therefore possible to observe many new facts.

It was found at first that almost all the adult and embryonic tissues of dog, cat, chicken, rat, and guinea pigs could be easily cultivated in vitro. According to their nature these tissues generate connective or epithelial cells, which grow into the plasmatic medium in continuous layers, or in radiating chains. The tissue fragments may surround themselves completely with dense new tissue, or, on the contrary, the new cells may spread over the surface of the medium. We observed the direct division of the nuclei during the life of the cells, and many karyokinetic figures in the fixed and stained cultures. Other experiments showed that the life in vitro of the tissues, which varies from about 5 days to about 20 days, can be prolonged by secondary and tertiary cultures, and that a new generation of thyroid, splenic and sarcomatous cells can be obtained from cells which have developed outside the body. We succeeded quickly also in cultivating malignant tissues such as the Rous chicken sarcoma, the Ehrlich and Jensen sarcoma of the rat, a primary carcinoma of the breast (dog) and two human tumors, a sarcoma of the fibula, and a carcinoma of the breast. A culture in vitro of the Rous sarcoma transplanted into a chicken caused the development of a sarcoma. Meanwhile the method has been applied successfully in the laboratory of Prof. MacCallum by Drs. Lambert and Hanes, who cultivated the Ehrlich sarcoma of the rat. We applied also the method of cultivation of tissues in vitro to several problems of the redintegration of normal tissues and of the biology of malignant tumor.

The results obtained in this and other laboratories are already too numerous to be described in this article. We will indicate only the technique, the general characters of the cultures, and some of the applications of this new method.

II. TECHNIQUE.

The new technique consists essentially in depositing small fragments of living tissues in fluid plasma or in an artificial medium. The cultures belong to three types-the small cultures in a hanging