Senescence, the Last Half of Life

by G. Stanley Hall

The method of these remarkable achievements, now often repeated, is to put tiny bits of living tissue in a plasma of blood serum that will coagulate. The blood must be deprived of its cells by the centrifugal process and must generally be taken from the animal for which the tissue is to be cultivated or, at any rate, generally from the same species, although this is not without exceptions, for chicken tissue has been grown in the blood of human beings, dogs, and rabbits; morbid tissue, perhaps, like cancer, being most indifferent. The tissue is taken from an etherized subject, with every possible precaution against bacteria, chilling, or drying, and so liable is it to be killed by exposure to air that it is best dissected in serum. Both plasma and tissue are kept in cold storage and the time during which it can be thus kept varies very greatly with different animals. The bit of tissue must be very small because only the outer edge can get the nourishment when deprived of the normal blood circulation, for when the piece of tissue is large, all but the periphery dies. To see these changes of form, small bits of tissue are grown on the inside of a coverglass of a microscope slide that has been overlain with a prepared plasma, sealed with paraffin and put into an electric incubator provided with a microscope. The period before growth begins varies but when it occurs, the microscope shows the direct division of the nuclei and the growth taking the form either of layers or of radiating chains, depending on whether epithelial or connective tissue is being developed. Each tissue, whether normal or morbid, develops very precisely tissue of its own kind, and sometimes as, for example, with cancerous tissue, the growth is so rapid that it can be observed with the naked eye. This, of course, opens an immense field of observation and experiment, for example, immunity, protection against antibodies, redintegration, regulation of growth of the whole or parts, and perhaps especially rejuvenation and senility, to say nothing of the character and the influence of the secretions from all the glands. The trouble at first was that the artificial growth was so short-lived; but by changing the medium often and by frequent washing away of the waste products in a salt solution, it was found that the life and growth of these isolated bits of tissue could be very greatly prolonged. It seemed that the process of decay was due to the inability of tissues to eliminate waste products. So in 1912 Carrel’s problem was whether these effects could be overcome.

To solve this problem bits of the heart and blood vessels of a chick embryo were grown. These growths were immersed in salt solution for a few minutes and then placed in the new plasma and it was soon found that thus the tissue could be made to live on indefinitely. Growth is more rapid the earlier the stage of it and it soon declines; hence the advantage of using tissue from embryos. But by subjecting these artificial growths to washings it was found that they were many times greater at the end than at the commencement of the month, showing that they do not grow old at all. Thus C. Pozzi says:

The pulsations of a bit of heart which had diminished in number and intensity or ceased could be revived to a normal state by washing and passage through a new solution. In a secondary culture two fragments of heart, separated by a free space, beat strongly and regularly, the larger fragment 92, the smaller 120 times a minute. For three days the number and intensity of pulsations of the two parts varied slightly. On the fourth they diminished considerably in intensity, the large fragment beating 40, the smaller 90 times. When the culture was washed and placed in a new medium, the pulsations again became strong, the larger one 20, the smaller one 60 times a minute. At the same time, the fragments grew rapidly, and in eight hours they were united and formed a mass of which all the parts beat synchronously.

Pozzi again says:

On January 17 the fragment of a chicken heart embryo was placed in plasma. It grew readily on a thick crown of conjunctive cells. In three days the pulsations, which were regular and strong at the beginning, grew feeble and ceased completely, and this state continued for more than a month. On the 29th of February, the culture, which had been subjected to fourteen passages, was dissected and the central film placed in a new medium. After the fifteenth passage it contracted rhythmically, with pulsations as strong and frequent as on January 17, viz., from 120 to 130 per minute. During March and April this fragment of a heart continued to beat from 60 to 120 times per minute. As the growth of the conjunctive tissue became more active, it was necessary, before each passage, to extirpate the new connective tissue formed around the muscle. On April 17 the fragment beat 92 times, agitating all the mass of the tissue and the neighboring parts of the middle of the culture. On May 1 the pulsations were feeble and they were given their thirty-fifth passage. In the manipulation the muscular tissue was stretched and torn so that the contractions ceased.

Thus experiment seems to establish the fact that even connective tissue, composed of not the most highly developed but of vigorous though low-level cells, is immortal. Senility and death result because in normal conditions the blood does not succeed in removing waste products. Could science only wash them away in a living organism, life might be indefinitely prolonged. It is these connective tissues that give support to the textures that compose the body and that chiefly make up bone, cartilage, ligaments, and the lymph network, the cells of which are endowed with special properties of growth and play a great rôle in rejuvenating injured tissue. All this work, in a sense, started from Claude Bernard’s principle that the life of an organism is dependent on the interaction of its cells and the medium in which they grow. Thus, to understand the process by which the body develops and why it must yield to decay and death, we must inquire into the cause of the loss of character of these interactions; and this was impossible until tissue could be grown outside the body so that the processes might thus be brought within the range of the microscope and all its conditions under control. Carrel’s first effort, thus, was directed toward the way in which the medium affected the life of the cell and in constituting this medium of plasma from the blood of dogs and chickens he found that the older the animal from which the blood was taken, the less rapidly and extensively the tissues grew in it. In the blood of a relatively old animal the increase became so slight as to be practically nil. These comparative experiments were made, Grandcourt tells us, with the blood of animals from five months to five years of age, and there was enormously greater activity on the part of the blood of growing animals. Thus it would seem that when an animal attains its size and stops growing, its blood undergoes progressive changes till it lacks, more and more, the dynamic power of youth. So the problem was whether the plasma could be given the force of youth so far as its action on growing cells was concerned and this was accomplished by mixing it with juices extracted from the embryo. Experiments, too, were made with a strain of connective tissue cells that had been kept in artificial life for more than sixteen months. It was divided into two parts, one of which was grown on adult plasma and the other in a mixture of two parts, one of plasma and the other of embryonic juice. In two days the ring of tissue around the second part was three times as great as that around the first. Some of these tissues, passed through a salt solution 130 times, doubled their area in forty-eight hours. Another, washed 57 times, increased in volume fifteen times in ten days, etc. These rapid growths, however, could not be duplicated in normal plasma which was then further modified. Thus the different media have a pretty constant effect upon the rate of growth. Carrel says: “The special rapidity of the growth of the tissue depends so much on the composition of the medium that it may become possible to use as a reagent of the dynamic value of the humors of the organism a strain of cells adjusted to life in utero.” If human connective tissue could be preserved in the condition of permanent life as the connective tissue cells of a chicken are preserved, the value of the plasma of an individual might be approximated by the cultivation in it of a group of these cells and by the observation of the rate of their multiplication. Such observations do suggest some indication of certain values of the blood of an organism and may give us some clue to old age.

Thus in the course of development the activity of the tissue is apt to vary in the body as a whole and in its parts. It therefore became a question whether each particular condition was permanent or whether the dynamics of the cell changes through the action of the medium upon it. To determine this, several bits of tissue, each having its own dynamic power, were cultivated in media exactly alike and differences in the character of the growth were noted. Then the influence of the medium began to tell. Measurements of the changes undergone on the part, in turn of a fast- and slow-growing tissue, showed that the former had lowered its activity one-half in forty-eight hours, while the latter had multiplied its activity by six. This process continued until the level of uniformity was reached, when the conditions of growth remained equal in all cases. Thus it appears that though, in the beginning, certain substances that the tissues had accumulated had the effect of accelerating or retarding its activity in the medium, yet in time the latter overcame these conditions and growth was brought under the laws of its own special mechanism. Thus the sum of the investigations on the influence of the medium on cells is that it may not only change the dynamic possibilities of the tissue but the character of the change may be regulated by a carefully considered modification of the medium (Grandcourt).

All this work involves the theory that the cells make such demands upon the nutrition supplied by the medium that they deplete it and then become indirect means of introducing into the life process a chemically destructive activity (catabolism). The result is a gradual slowing down of cell growth, which is progressive aging and death. A very analogous course was that followed in the earlier artificial cultivations. The tissues lived a short span of days and then died. But the process of degeneration could be obviated by salt solutions and other processes so that tissues now grow in vitro for a year and a half and may continue to multiply faster than those of the embryo. Thus for such tissues senility does not exist and the question naturally arises whether we can ever hope to accomplish anything of this sort inside the body.

Carrel in 1914 reported a strain of connective tissue that had undergone 358 passages and had then reached the twenty-eighth month of its life in vitro. It was detached from the heart of a chick embryo seven days of age, which pulsated for 104 days and gave rise to a large number of connective tissue cells. These multiplied actively for the first two years, a great many cultures having been derived from this strain every week. The fragment of the tissue usually doubled in forty-eight hours, though rapidity of growth was subject to fluctuations. One striking result is seen by comparing the amount of tissue produced by a given culture in forty-eight hours this year with that produced in the same tissue by the same strain of cells a year before. This shows that the activity of the strain had increased, although this might, of course, be due to improvement of technic or possibly to a progressive adaptation to life in vitro. Carrel says: “Thus it is conclusively shown that the proliferating power of the strain has in no wise diminished. During the third year of independent life, the connective tissue shows greater activity than at the beginning of the period and is no longer subject to the influence of time. If we exclude accident, the connective tissue cells, like infusoria, may proliferate indefinitely.” In the latest report at hand one of these cultures had been kept alive and growing thus for seven and a half years.

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