It was early demonstrated by Carrel and Burrows that the life of the tissues in vitro which varied in different experiments from 5 to 20 days could be prolonged by a process of successive transfers of the culture to an indefinite period. Cells which were nearing the end of their life and growth in one culture need only be transferred to a new culture medium to keep on growing and multiplying. Dr. and Mrs. Warren H. Lewis made the important discovery that tissues of the chick embryo could be cultivated outside the body in purely inorganic solutions, such as sodium chloride, Ringer's solution, Locke's solution, etc. No growth in these inorganic cultures took place without sodium chloride. Growth was prolonged and increased by adding calcium and potassium. If maltose or dextrose, or protein decomposition products were added proliferation of the cells increased.

By the method of transfer to fresh nutrient media Carrel has been able to keep cultures of tissue from the heart of the chick embryo alive for a long period of years. In a letter recently received he says: "The strain of connective tissue obtained from a piece of chick heart

PENNARIA. Restitution mass six days old, completely metamorphosed, with developed
Op. perisarc of original mass; x, perisarc of outgrowth adherent to glass.

(From

FIG. 6. CULTURE OF OLD STRAIN OF CONNECTIVE TISSUE. 8 months old, lacking 2 days. 48 hours' growth. x20. (Ebeling.)

1614 passage. 8 years and

is still alive and will be nine years old the seventeenth of January, 1921." Figure 6 is a photograph showing the present condition of this culture.

This is indeed a remarkable result. It completes the demonstration of the potential immortality of somatic cells, when removed from the body to conditions which permit of their continued existence. Somatic cells have lived and are still living outside the body for a far longer time than the normal duration of life of the species from which they came. I think the present extent of Carrel's cultures in time fully disposes of Harrison's criticism to the effect that we are "not justified in referring to the cells as potentially immortal or even in speaking of the prolongation of life by artificial means, at least not until we are able to keep the cellular elements alive in cultures for a period exceeding the duration of life of the organism from which they are taken. There is at present no reason to suppose this cannot be done, but it simply has not been done as yet." I have had many years' experience with the domestic fowl, and have particularly studied its normal duration of life, and discussed the matter with competent observers of poultry. I am quite sure that for most breeds of domestic poultry

the normal average expectation of life at birth is not substantially more than two years. For the longest lived races we know this normal average expectation of life cannot be over four years. I have never been able to keep a Barred Plymouth Rock alive more than seven years. There are on record instances of fowls living to as many as 20 years of age. But these are wholly exceptional instances, unquestionably far rarer than the occurrence of centenarians among human beings. There can be no question that the nine years of life of Carrel's culture has removed whatever validity may have originally inhered in Harrison's point. And further the culture is just as vigorous in its growth today as it ever was, and gives every indication of being able to go on indefinitely, for 20 or 40, or any desired number of years.

The potential immortality of somatic cells has been logically just as fully demonstrated in another way as it has by these tissue cultures. I fully agree with Leo Loeb when he says that the proof of potential immortality "can just as well be supplied by serial transplantation of tissues in the living body; but we can, I believe, go further, and state that as far as such potential immortality of tissues can be proved, the proof has already been given through the long-continued, apparently endless serial transplantation of tumors. Now tumor cells are merely ordinary somatic cells living under special conditions; and we may, therefore, conclude that, in the same sense as protozoa and germ cells, also, certain ordinary mammalian somatic cells possess a potential immortality." Loeb first announced this important conclusion nineteen years ago. To him unquestionably belongs the credit for first perceiving that death was not a necessary inherent consequence of life in the somatic cell, and demonstrating by actual experiments that somatic cells could, under certain conditions, go on living indefinitely.

Before turning to the next phase of our discussion let us summarize the ground we have covered up to this point. We have seen that by appropriate control of conditions it is possible to prolong the life of cells and tissues far beyond the limits of longevity to which they would attain if they remained in the multicellular body from which they came. This is true of a wide variety of cells and tissues differentiated in various ways. Indeed, the range of facts which have been ascertained by experimental work in this field probably warrants the conclusion that this potential longevity inheres in most of the different kinds of cells of the metazoan body, except those which are extremely differentiated for particular functions. To bring this potential immortality to actuality requires, of course, special conditions in each particular case. Many of these special conditions have already been discovered for particular tissues and particular animals. Doubtless, in the future many more will be worked out. We have furthermore seen that in certain cases the physico-chemical nature of the conditions necessary to insure the continuance of life has been definitely worked

out and is well understood. Again this warrants the expectation that, with more extended and penetrating investigations in a field of research which is really just at its beginning, we shall understand the physics and chemistry of prolongation of life of cells and tissues in a great many cases where now we know nothing about it,

One further point and we shall have done with this phase of our discussion. The experimental culture of cells and tissues in vitro has now covered practically all the essential tissue elements of the me tazoan body, even including the most highly differentiated of those tissues. Nerve cells, muscle cells, heart muscle cells, spleen cells, connective tissue cells, epithelial cells from various locations in the body, kidney cells, and others have all been successfully cultivated in vitro. We may fairly say, I believe, that the potential immortality of all the essential cellular elements of the body either has been fully demonstrated, or else has been carried far enough to make the probability very great that properly conducted experiments would demonstrate the continuance of the life of these cells in culture to any definite extent. It is not to be expected, of course, that such tissues as hair, or nails, would be capable of independent life, but these are essentially unimportant tissues in the animal economy as compared with those of the heart, the nervous system, the kidneys, etc. What I am leading to is the broad generalization, perhaps not completely demonstrated yet, but having regard to Leo Loeb's work, so near it as to make little risk inhere in predicting the final outcome, that all the essential tissues of the metazoan body are potentially immortal. The reason that they are not actually immortal, and that multicellular animals do not live. forever, is that in the differentiation and specialization of function of cells and tissues in the body as a whole, any individual part does not find the conditions necessary for its continued existence. In the body any part is dependent for the necessities of its existence, as for example nutritive material, upon other parts, or put in another way, upon the organization of the body as a whole. It is the differentiation and specialization of function of the mutually dependent aggregate of cells and tissues which constitutes the metazoan body which brings about death, and not any inherent or inevitable mortal process in the indidividual cells themselves.

3. SENESCENCE

A careful and unprejudiced examination will suffice to convince anyone of open mind, I think, that much of the literature on senescence is really of no fundamental importance, because it has unwittingly reversed the true sequential order of the causal nexus. If cells of nearly every sort are capable, under appropriate conditions of living indefinitely in undiminished vigor, and cytological normality, there is little

ground for postulating that the observed senescent changes in these cells while in the body, such as those described by Minot and others, are expressive of specific and inherent mortal processes going on in the cells, or that these cellular processes are the cause of senescence, as Minot has concluded. It would rather appear that these visible cytological changes are expressive of effects not causes, and that they are the effects of the organization of the body as a whole as a system of mutually dependent parts, and not of specific, inherent and inevitable cellular processes.

Cells in culture in vitro, as we have seen, do not grow old. We see none of the characteristic senescent changes in them. From these facts it is a logically cogent induction to infer that when cells show the characteristic senescent changes, which were discussed in the preceding paper, it is because they are reflecting in their morphology and physiology a consequence of their mutually dependent association in the body as a whole, and not any necessary progressive process inherent in themselves. In other words we may justifiably, in the light of our present knowledge as I believe, regard senescence as an attribute of the multicellular body as a whole, consequent upon its scheme of morphologic and dynamic organization. This attribute is reflected morphologically in the component cells. But it does not originate in the cells, nor does it ever occur in the cells when they are removed from the mutually dependent relationship of the organized body as a whole. In short senescence is not a primary attribute of the physiological economy of cells as such.

If this conception of the phenomenon of senescence is correct in its main features, as I believe it is, it shows the essential futility of attempting to investigate its causes by purely cytological methods. On the other hand, by clearing away the unessential elements, it indicates where research into the problem of causation of senescence may be profitable.