STUDIES ON THE GROWTH OF TISSUES IN VITRO

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1 6o STUDIES ON THE GROWTH OF TISSUES IN VITRO VI. THE EFFECTS OF SOME TISSUE EXTRACTS ON THE GROWTH OF PERIOSTEAL FIBROBLASTS BY O. A. TROWELL, M.B., B.CHIR. AND E. N. WILLMER, M.A. From the Department of Physiology, Cambridge (Received 25th July 1938) (With Six Text-figures) INTRODUCTION IT is well known that mesenchyme tissues (fibroblasts) can be maintained for long periods in an active state of growth and cell proliferation when cultured in media containing plasma and saline extracts of embryonic tissues. It is recognized that this prolonged growth is due rather to the embryo extract than to the plasma, although the nutrient substances in the plasma may play an important role. Previous workers (Carrel, 1913, 1922; Ebeling, 1924; Semura, 1931; Walton, 1914), from measurements of the areas attained by cultures when treated with extracts of adult tissues, have stated that with one or two exceptions extracts of adult tissues have very much less power of promoting growth than extracts from embryonic tissues. Only spleen, thyroid, leucocyte and Rous sarcoma extracts have been regarded as at all important. Several questions therefore suggest themselves. In the first place, do the organs of the body lose their growth-promoting properties at equal rates, and if not, which organs maintain them for the longest time? Secondly, if the stimulating action is localized in special organs or tissues, can it be correlated with any known biological or chemical properties characteristic of those tissues? Fowler (1937) has recently made extracts from different parts of the chick embryo with a view to ascertaining if certain regions are more potent than others, but apart from the fact that the anterior region was slightly more growth promoting than the rest, no very significant results were obtained and the above questions remained unanswered. Now that quantitative methods for the measurement of growth are available by the photographic technique (Willmer & Jacoby, 1936) it seemed profitable to make a comparative study of the growth-promoting powers of the different organs of older birds with a view to solving some of these problems. Another problem also presented itself. It is possible that growth in the adult does not cease because growth-stimulating substances fail, but rather because their action is abolished by the antagonism of inhibitors. It has been claimed (Walton, 1915; Baker & Carrel, 1927) that in blood there exist both growth-promoting and

2 Studies on the Growth of Tissues in vitro 61 growth-inhibiting substances, and that the latter (associated with the lipins) increases while the former (associated with the proteins) decreases with increasing age of the animal. Extracts of adult organs might therefore show actual inhibition of growing cells as has indeed been claimed for certain liver extracts (Walton, 1914). METHODS The active substance in embryo tissues is immediately soluble in saline solutions, and consequently the extracts from the tissues of older animals were made in a manner closely similar to that used for the preparation of embryo extract, so that the preparations might be expected to contain the same active substances if such were present in the tissues. The animal was killed by administration of ether, and the required organ or tissue was extirpated immediately after death. The tissue was then weighed in a tared sterile tube, minced with scissors, and 3 c.c. of Tyrode solution added for each gram of tissue. It was then allowed to extract in the Tyrode solution for 20 min., centrifuged, and the supernatant fluid decanted off and preserved on ice until required. Since embryo extract loses its properties quite rapidly even when kept at o C, the tissue extracts were never kept for more than a few days before use. In certain cases where the organs were small the extracts were made rather more dilute, but when this was necessary the fact has been noted in the tabulation of the results. Organs have been taken mostly from the 3-monthold fowl, but comparisons have been made with extracts made from organs of birds respectively 1 month, 9 months, and more than 20 months old. For estimating the activity of the extracts the method was as follows. Strains of periosteal fibroblasts derived from the os frontale of the 13-day-old chick were cultured on cover-slips for at least a fortnight before being used. Selected cultures were then transferred to flasks containing a dilute plasma coagulum, and kept for 48 hr. with a supernatant fluid phase of Tyrode solution alone. By the end of that time the residual growth energy was practically exhausted, and the activity of the extract could be tested by replacing the Tyrode solution by 1 c.c. of the extract and recording the subsequent growth and behaviour of the culture photographically. The details of this technique have already been described (Willmer & Jacoby, 1936). The effects of the extracts on the mitotic index (the percentage of cells dividing per hour) were noted, and in some cases the effects on the general activity of the cells were estimated by measurements of the migration rate of the cells. RESULTS In Fig. 1 the growth-promoting activity of three representative organ extracts are compared with the activity of 15 % embryo juice whose effects on growth are well established. It will be seen that all the extracts from organs of older animals are far less potent than is embryo juice at the same concentration. It should, however, be stated that embryo extract when made in the manner employed for the preparation of the extracts used in these experiments corresponds to somewhat less than 15%, more nearly to 12^%.

3 62 O. A. TROWELL and E. N. WILLMER The growth-stimulating capacity of various organ extracts tested have been plotted in Figs. 2 and 3 in descending order of magnitude of their effects. It will be seen that the brain extracts are the most active of all. This in itself is somewhat surprising, since the brain of ah" organs is composed of cells in which division is believed to cease at a very early stage in life. The thyroid, second in order on fig. 2, behaved somewhat irregularly, but it can undoubtedly give rise to extracts which are very active. Owing to the small Time in hours after adding extract Brain (14 exps.) (1-5 % per hr.) Thymus (11 exp«.) (0-9 % per hr.) Spleen (20 exps.) (0-4% per hr.) Fig. 1. Graph showing the effects of three representative organ extracts on the growth of chick periosteal fibroblasts in contrast to the effect of 15 % embryo juice. The organ extracts were prepared from fowls which were 3 months old or more. Mean mitotic index during period hr. are indicated in brackets together with number of experiments performed. 15 % embryo juice (3-6 % per hr.). quantity of available tissue the extracts used were somewhat below normal strength, i.e. between o-i and 0-3 g./c.c. The irregular behaviour of thyroid extracts may in part be explicable on the ground that the glands may have been in different states of activity. The strength of the extract seemed to bear little relationship to its activity as a growth-stimulating agent. Nevertheless, it is unlikely, though not impossible, that the activity is due to thyroxin itself since, if it were due to that, the extract would probably be active in very low concentration as it appears to be in the body, whereas the extracts were completely inactive when diluted ten times or more.

4 Studies on the Growth of Tissues in vitro 63 Similar difficulties in obtaining sufficient quantities of fowl pituitary have made it impossible to include the anterior lobe in this analysis, but in the experiments tried no extracts from the fowl pituitary, however small the dilution (two anterior Brain (7) Aver«g mitotic index from 10th o24th br. 1-3 Thyroid (7) i.1 B J3 8 Thymus (6) TcstU (5) Ovary (5) '.s u a 8 Bone marrow (6) Liver (4) Kidney (3) 0-3 Upper curve adu Lower curve 3 mo plccn (11) 0-2 Muscle (4) Time in hours after the addition of the extract Fig. 2. Chart showing the effects of various tissue extracts from the 3-month-old fowl on the growth of perio8teal fibroblasts. The numbers in brackets indicate the number of experiments.

5 6 4 O. A. TROWELL and E. N. WILLMER lobes minced in i c.c. of Tyrode solution) gave any indication of growth promotion. A few experiments have been done using commercial extracts of the anterior lobe (Parke Davis Antuitrin), and in contrast to the results of Semura (1931) there was no indication of any growth-stimulating action. Antuitrin was added to cultures in the same way as the other extracts have been applied, and it was used in concentrations varying from 1 : 10 to 1 : 1000 in Tyrode solution. Semura found that in a concentration of 1 : 100 it stimulated the growth of hanging drop cultures as indicated by the area reached by the treated cultures in comparison with that reached by controls. There is the possibility that in the commercial extract there is sufficient preservative to interfere with the normal growth of the cells, but I c 3 o h fi *4-2-0' I "g v CQ ~5 -o J a "a, a Xi i -i r J be a. 6 1 s v f I ā eg 3 a o I c o g en I nn Fig. 3. Chart showing the effects on the growth rate of chick periosteal fibroblasts following the addition of extracts of organs from the 3-month-old fowl. The effects of certain other organ extracts are also inserted for comparison. The numbers in brackets indicate the number of experiments. Semura himself states that in concentrations below 1 : 10 the effects of this are negligible. In none of the present experiments was there any significant increase in the number of cells undergoing division in whatever concentration the extract was applied, nor was there any sign of damage to the cells. A few other experiments were performed with pituitary extracts made from fresh ox pituitary glands (anterior lobe). These glands were extracted in the same way as the other organs have been extracted for these experiments, but since the original gland tissue used was not obtained under aseptic conditions the final product was filtered through a Berkefeld filter. This filtration may well reduce the potency of substances akin to those present in embryo tissue juice but should not have any effect on reducing the activity of the growth hormone. Nevertheless, such extracts had no power of increasing the number of cell divisions in cultures to which they were applied.

6 Studies on the Growth of Tissues in vitro 65 Admittedly these experiments are only of a somewhat preliminary character, but they indicate that the growth hormone has no marked effect on the frequency of cell division when applied directly to cells in vitro. On the basis of these findings it is very unlikely that the potency of embryo and other active tissue extracts depends on the endocrine substances ordinarily associated with growth, since these substances are active in the body in extremely minute concentrations which must have been equalled or exceeded in the experiments here described. It is perhaps worth emphasizing that in the preparation of the brain extracts the pituitary body was carefully excluded. In a discussion on the activity or absence of activity of extracts of the endocrine glands, the results of one or two experiments with extracts of the adrenal glands are not without interest. The extracts were made in the usual manner though again somewhat dilute. They produced no very marked effect on the frequency of cell division in cultures to which they were applied, but some difficulty was experienced in photography since the whole medium turned black owing to the formation of melanin from the adrenalin originally present. This serves to illustrate a rather important point in connexion with all experiments of this character on the growth of tissues in vitro. In the body, hormones are constantly circulating in minute amounts, and if they are destroyed they can be readily replaced, thus maintaining, if need be, a constant concentration. In vitro, however, if the substance is destroyed it probably cannot be replaced, and consequently it either disappears entirely from the environment of th«cells or else it has originally to be added in doses so large as to be quite unphysiological. Such constant perfusion apparatus as has recently been developed by Carrel & Lindberg may prove to be the key to the solutions of such problems. Returning now to the other tissue extracts whose effects are summarized in Figs. 2 and 3, the thymus is found to occupy the third place on the list. Extracts of this organ gave very constant results and showed considerable growth-promoting activity, but considering that it is an organ which many workers associate especially with the growth of the animal the growth rates obtained were not so high as might have been expected. The relation of the age of the thymus to its growth-promoting power was found to be significant and is discussed later in this paper. Less active than the thymus, yet still effective growth stimulants, were found to be the testis, ovary and bone marrow, all giving extracts of about the same potency. The domestic fowl has no definable lymph glands, but extracts made from the lymph glands of rabbits of comparable age showed activity of the same order as that of the bone marrow of the bird. That is to say they are slightly less potent than extracts from the thymus. Extracts of thymus from the rabbit are comparable in activity with those from the bird. The active substances do not appear therefore to be species specific and are similar in this respect to the active substance or substances in embryo extract. All the other organ extracts tried have with one exception proved to have only insignificant activity or no activity at all. These include liver, kidney, muscle, and spleen, and spleen is the exception. The behaviour of the spleen extracts was unexpected. Closely similar in JEB-XVli 5

7 66 O. A. TROWELL and E. N. WILLMER structure to lymph glands, and containing considerable numbers of leucocytes, the spleen should give, and in the hands of other workers has given, extracts of considerable growth-promoting power. Nevertheless, all the experiments which were performed with extracts of the spleens of 3-month-old birds produced negative results, or results which could not, for instance, be compared with the results from the thymus. It was thought at first that perhaps the concentration was too high, since a gelatinous precipitate often formed in the supernatant fluid and since the cells nearly always became granular and rather fatty. Spleen extracts were therefore diluted io, 100 and 1000 times, and then applied to cultures but still with the same negative results. Now the spleen is, of course, an organ which generally contains a considerable quantity of blood, and it was possible that this might have some inhibitory action on growth. Experiments using spleens emptied of blood showed Brain Age of bird in months Fig. 4. The effect of the age of the bird on the activity of the extracts from its organs. The numbers in brackets indicate the number of experiments. that this was not a significant factor. However, it was found in the course of these experiments that the age of the animal might be significant, and therefore the effects of spleen extracts from animals of different ages were systematically investigated. The results are summarized in Fig. 2. It is clear from this figure that, contrary to the usual belief that young tissues provide growth-stimulating substances, which are not to be found in adult tissues, the extracts from adult spleens were active, whereas similar extracts from the spleens of birds 1 and 3 months old were without any stimulating effect. The spleen of the adult therefore is quite actively growth-promoting, and the experiments here described on such extracts confirm the findings of other workers. A curious effect of several of the extracts from adult spleens was to cause mitoses in the latent period. This may have exaggerated the general effect but it cannot wholly account for it. Reference to Fig. 4, in which are summarized the results of a similar comparative study of the effects of age on the growth-promoting properties of the different

8 Studies on the Growth of Tissues in vitro 67 organs, shows that there is no regular relation between age and growth-promoting effect in the various tissues studied. Both spleen and brain seem to show definite increase in growth promotion with age. Several other tissues, e.g. muscle and liver, decrease, while the thymus remains almost constant (see Fig. 5). It must be remembered that the thymus in birds suffers no marked involution as it does in most mammals. It was possible that growth inhibitory factors might be present in the young spleen, and so this was tested by adding spleen extracts to cultures actively growing 2- Thymus 1 month 4 expe. Average mi to tic Index from 10th to 24th hr. 1-2 Thymus 3 months 6 exp«. 0-9! o ' B S Thymus 9 months 2 expe Thymus 2 yeare 3 exps. 1-0 o~t Time in hours Fig- 5- Graphs showing the effects on the growth of periosteal fibroblasts of extracts of thymus gland, taken from birds of different ages. in the presence of embryo tissue extract (Fig. 6). The effects were not significantly different from those obtained by adding an equal quantity of Tyrode to cultures under the same conditions. Interesting results were also obtained by adding spleen extracts from 3-month-old birds to hanging drop cultures in plasma, for it was found that although large areas of growth were obtained, the numbers of mitoses present at the end of a 36 hr. growth period were even less than in the controls in Tyrode solution. The average area of eighteen cultures in spleen extract and plasma was 6-05 sq. mm. The mitoses averaged 0-3 per culture. In fourteen cultures grown in Tyrode and plasma the average area was 2-68 sq. mm. and the mitoses were 1-2 per

9 68 O. A. TROWELL and E. N. WILLMER culture. In these cultures therefore the migratory activity of the cells is increased, but the mitoses are unaffected or slightly decreased in the spleen extract. This seems to be an instance of an independence of the migratory activity of cells and their mitotic activity. Usually these two processes are very closely associated Fig. 6. Graphs showing the effects of replacing embryo extract on cultures or periostea! fibroblasts by (A) spleen extract from 3-month-old bird, and (B) Tyrode solution. DISCUSSION The results described in this paper are of interest from two particular points of view. In the first place the loss of growth-promoting power with age is not as universal and regular as had previously been supposed, and secondly, the persistence of this power in the different organs does not follow the distribution that might have been expected. It might have been justifiably assumed that growth-promoting substances could most likely be extracted from those organs which were themselves growing most actively; but this is found to be only partially true. Active extracts can be obtained from the lymph glands and testis, which are certainly organs in which active cell division is going on. The ovary, however, has as much growthpromoting capacity as the testis, yet the rate of cell division in it is probably not nearly so great. Moreover, the thymus gland is considerably more active than either, and this gland does not normally contain so many dividing cells. The same is true of the thyroid and still more so of the brain, for that organ of all those present in the adult is the one in which cell division is at a minimum and yet it provides the most potent extracts. Obviously then growth-promoting power cannot with certainty be attributed only to the organs which are themselves actively growing.

10 Studies on the Growth of Tissues in vitro 69 Another justifiable assumption might have been that extracts of organs containing a high proportion of nuclei would provide active growth-promoting extracts, but this again is not altogether borne out by the results of experiments described in this paper, where, for example, young spleen is seen to behave differently from adult spleen without any major change in histological structure, and where ovary is conspicuously more active than kidney. It is necessary therefore to fall back on some other hypothesis in order to account for the apparently anomalous distribution of growth-promoting substances among the organs of the animal body. To formulate this it is necessary to find some common factor among the organs which are most active. So far there is little that can be said. There seems to be no direct correlation between the general metabolic activity of the tissue and its content of growth substances. The rate of respiration of a tissue is no indication of its power to stimulate growth. On the other hand, ability to obtain energy by anaerobic glycolysis does seem to show some correlation with growth-promoting power. This capacity is generally well developed in rapidly growing tissues. For example, embryonic tissues are particularly able to glycolyse. Among the organs of the adult with pronounced glycolytic powers may be mentioned the lymph glands, bone marrow, thymus, all of which are active growth promoters, and finally brain which is very active in both directions. This hypothesis fails in connexion with the thyroid gland which has no pronounced glycolytic powers but does seem able to stimulate growth. It is, of course, possible that the activity of the thyroid is dependent not on its content of embryonic extract-like substances but on its content of hormone, although this is not altogether consistent with the results obtained when cultures were treated with very dilute extracts in which there would probably be as much hormone as is normally circulating in the blood stream. Semura (1931) and Ebeling(i924) both find this organ to promote the growth of tissues, and in Ebeling's experiments the thyroid was active when growing in the same medium as the experimental culture. Possibly, therefore, especially in the light of the variable results obtained, it may be best to treat the thyroid as a special case, at any rate until more is known about its effects. Apart from this the correlation between glycolysis and growth-promoting power is sufficiently close to suggest that it might be worth while to investigate the relationship further. There is now also reason to believe that growth may be connected with the particular form of glycolysis which is best described as non-phosphorylating glucolysis, indicating the direct breakdown of glucose without the intervention of phosphates as in the muscle cycle. Experiments which give support to this hypothesis will be described in a forthcoming paper. Finally, emphasis should be laid on the fact that the presence in a tissue of the substances which promote growth does not necessarily mean that they have any functional activity in that direction in the body. It is doubtful, for example, whether the brain or thymus are secreting growth-promoting substances into the blood or lymph streams in the body.

11 70 0. A. TROWELL and E. N. WILLMER SUMMARY 1. Tissue extracts have been made from several organs of growing and adult fowls, and the growth-promoting properties of these extracts have been tested on periosteal fibroblasts growing in vitro. 2. The growth-promoting properties are most pronounced in brain extract and diminish in the sequence: thyroid, thymus, testis, ovary, bone marrow, liver, kidney and muscle extracts. Extracts made from the spleens of adult cocks were found to be far more efficient growth-promoting agents than extracts made from the spleens of young birds. 3. The growth-promoting power of a tissue extract cannot always be correlated with the age of the tissue from which the extract was made, with the rate of growth of that tissue, nor with its nuclear content. The authors wish to express their appreciation of the invaluable technical assistance rendered by Mrs Simon-Reuss. They also wish to record their indebtedness to the British Empire Cancer Campaign for the grant which has made this work possible. REFERENCES BAKER, L. E. & CARREL, A. (19*7). J. exp. Med. 45, 205. CARREL, A. (1913). J. exp. Med. 17, 14. (192a). J. exp. Med. 36, 385. EBELINO, A. H. (1924). CJi. Soc. Biot., Paris, 90, FOWLER, O. M. (1937). J. exp. Zool. 76, 235. SEMURA, S. (1931). Arch. Zeltforsch. 10, 349. WALTON, A. J. (1914). J. exp. Med. 20, 554. (1915)- Pr c- roy. Soc. B, 87, 45a. WILLMER, E. N. & JACOBY, F. (1936). J. exp. Bid. 13, 337.