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«MR. PRESIDENT AND GENTLEMEN,These researches on the Cultivation of Human and other Tissues were carried on in the Marcus Beck Laboratory, in this ...»

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Ebe iRopal ZocietV of Hebticuite.

MIarcus Beck Laboratory Reports.-No. 1.

The Cultivation of Human Tumour Tissue in vitro.

By DAVID THOMSON, M.B., Ch.B.Edin., D.P.H.Cantab.,' and

JOHN GORDON THOMSON, M.A., M.B., Ch.B.Edin.2

Read before a Meeting of the Society on May 11, 1914.

The PRESIDENT, Sir FRANCIS H. CHAMPNEYS, Bt., M.D., in the Chair.

MR. PRESIDENT AND GENTLEMEN,These researches on the Cultivation of Human and other

Tissues were carried on in the Marcus Beck Laboratory, in this building, by Dr. J. G. Thomson, as Beit Memorial Research Fellow, and by myself, as Grocers' Research Scholar, under the directorship of Sir Ronald Ross. The work represents the first-fruits of the Laboratory, and it is only;right that on this account we should make a brief reference to the memory of Marcus Beck. It would be indeed ungracious not to take special notice of the fact that the facilities we have enjoyed are due to the respect which he inspired, and which led to the generous donation to which the Laboratory owes its existenee. A portrait of Marcus Beck hangs in the Laboratory, and underneath are his favourite verses, one of which reads as follows "I need not be missed if another succeed me, To reap down those fields which in Spring I have sown, He who ploughed and who sowed is not missed by the reaper, He is only remembered by what he has done."

Grocers' Research Scholar.

I Beit Memorial Research Fellow.

8 D. Thomson & J. G. Thomson: Cultivation of Tissue in vitro Those among us who did not know Marcus Beck do not miss him, but we are now reaping the advantage from the Laboratory which he has inspired, and on account of which he will always be remembered. Before proceeding with my lecture I wish also to thank the President, the Council, and Mr. MacAlister for the facilities that they have given us in connexion with our work in this building.

(I) INTRODUCTION.

THE discovery of the method of cultivating tissues in vitro by Harrison (1910) has opened up a wide field for research. It is unwise to prophesy how far this new power will develop, but undoubtedly it will lead to discoveries of great importance. In June, 1913, we attempted to cultivate human tumour tissue in the medium employed for cultivating the malarial parasite in vitro-viz., in fresh defribrinated human blood containing 0 5 per cent. of glucose. Small nieces of breast carcinoma were placed in test-tubes containing this meium and incubated at blood heat. Smears of the surface of these portions of tumour before inoculation, and again after one to two weeks' incubation, showed that new elongated cells had developed in the medium on the surface of the tissue. These were probably new connective tissue cells.

This medium and method were, however, unsatisfactory. In September, 1913, one of us (D. Thomson) visited the Rockefeller Institute in New York and through the kindness of Dr. Carrel, whose excellent work on this subject is well known, was able to acquire the technique necessary for this research. Carrel has worked chiefly with the tissues of the chick embryo, and we have attempted the cultivation of human tissue on similar lines. The human tissue necessary was obtained at the Middlesex Hospital from operations performed by Sir John BlandSutton, to whom we are much indebted. As soon as the tumour or other human tissue was removed from the patient it was placed in a sterile bowl. A small piece was immediately cut out with aseptic precautions and transferred to a test-tube containing 5 to 10 c.c. of sterile Ringer's solution. This was placed in a Thermos flask to which some water about blood heat was added. The tissue was in this way transferred to the laboratory as quickly as possible and then planted into the necessary medium and incubated at 37.50 C. The space of time which elapsed between the removal of the tissue from the patient and its transference in the culture medium to the incubator was as a rule about two hours.

The Royal Society of Medicine (II) THE CULTURE MEDIUM.

It seemns to be generally believed that the tissue of a given animal can only be cultivated in the plasma of that species of animal, so that to grow chick tissue the plasma of a fowl is employed. The tissues of the frog, rabbit and guinea-pig have been grown always in the plasma of the frog, rabbit and guinea-pig respectively. With this idea it seemed necessary to employ human plasma for the cultivation of human tissues. We commenced our work, therefore, by using human blood plasma for the medium, but found this inconvenient and troublesome, chiefly on account of the rapidity with which human plasma clots.

It was found later by one of us (D. Thomson) that hunman tissues could be grown satisfactorily in fowl plasma, and as this plasma can be kept fluid in paraffined tubes in the ice-chest for several months, it formed a much more convenient medium for our work. In this research the total number of inoculations of pieces of human tissue into various modifications of plasmatic media was fifty. Nine different tissues were employed-viz,, liver, uterine fibroma, papillomatous tumour of the ovary, ovary, skin epithelium, scirrhous cancer of the breast, cancerous lymphatic gland, voluntary muscle, and scrapings of epithelial cells from the inner surface of the Fallopian tube. Of these nine species of tissue, only three showed distinct and definite growth in the medium-viz., the papilloma of the ovary, the ovarian tissue itself and the cancerous lymphatic gland. Slight growth took place in the case of liver and fibroma. It seemed to us that extract of the tissue itself should be added to the fowl plasma medium in order to get good growth. No extract was added in the case of the liver, and this possibly explains the slight growth in this case. Also in the case of the fibroid scirrhous cancer of the breast and the fibroma of the uterus no extract was added, because the tissue was so hard that we were unable to get an extract from it. Hard fibrous tissues did not grow well, but whether this was due entirely to the lack of extract.we are unable to state.





Soft tissues, such as could be easily mashed up for the purpose of getting an extract grew best, but whether this was due to the addition of the extract to the medium we are not prepared to state. Later we found that the best growth of human tissue which -we have so far obtained occurred in a medium composed of fowl plasma one part plus chick embryo extract one part. The growth was more successful in this medium than in the medium composed of fowl plasma one part plus 10 D. Thomson & J. G. Thomson: Cultivation of Tissue in vitro extract of the given tissue one part, so that it would seem that extract of chick embryo can take the place of the human tissue extract.

It would appear to us, therefore, that for tissue growth one should employ plasma plus extract, and that the plasma and extract do not require to be specific. There would seem to be no reason, therefore, why certain human tissues and tumours should not be successfully transplanted into a fowl, since the tissues of certain mammals, including man, grow successfully in fowl plasma plus extract of chick embryo.

(III) TECHNIQUE.

The medium is transferred by means of sterile Wright's pipettes to a sterile cover-slip. A tiny piece of the tissue is then placed in this medium and the cover-slip inverted over a hollow in a thick glass slide.

The cover-slip is hermetically sealed to the slide by painting melted paraffin around the margin of the former. The preparation is then placed in the incubator at 37.50 C. A culture of this kind can be examined at any time by the low powers of the microscope, and sometimes even the oil-immersion lens can be focused on the growing cells.

(N.B.-The plasma medium clots after sonme time so as to form a transparent jelly. This clotting occurs much more quickly when extract of chick embryo is added.) (IV) THE GROWTH OF TIlE TISSUES.

We have attempted to illustrate this by means of successive photomicrographs of the live tissue.

(a) Papillomna of the Ovary.-This was a very soft tumour about the size of a child's head. It was described by the pathologist as an intracystic papilloma not truly malignant. The tissue of this tumour was apparently composed entirely of round cells, there being apparently no fibrous stroma present. A tiny portion, about 1 mm. in square area, was placed in a medium composed of fowl plasma one part, Ringer's solution (containing 0 5 per cent. of glucose) one part, and extract of the tumour in Ringer's solution one part. On the third day of incubation definite buds of growing tissue appeared. On the fifth day these were quite distinct, as shown in fig. 1, Plate I; and on the eighth day the amount of growth had considerably increased (fig. 2). This growth consisted of a solid extension of epithelial cells. As the growth extended it caused some liquefaction of the medium, which was of a gelatinous consistence, The Royal Society of Medicine 11 and in the more liquefied parts the new growing cells were more scattered (fig. 3), but as a rule they remained in contact with each other by means of long, fine protoplasmic connexions (figs. 7, 8 and 9, Plate II). The new actively proliferating cells varied markedly from the cells of the original tissue planted in the medium. The appearance of the original cells is seen in fig. 4; while fig. 6 shows the original cells on the left and the new growing cells on the right. The latter are also seen in figs. 7, 8, 9 and 10. It is distinctly noticeable, therefore, that the new actively growing cells are large and amceboid, with long processes which communicate with each other, and they also contain large highly refractilq granules. The original cells, on the other

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Papilloma of ovary. Growth in vitro. Diagrams A, B and C represent the remarkable amoeboid shapes of the growing cells. ( x 600.) hanad, are. much smaller. They show no amoeboid processes and do not exhibit amceboid movement, and they contai'n few or no' large refractile granules. The amoeboid movement of the larger growing cells was very remarkable, as illustrated in the diagrams A, B and C. On the fourteenth day of incubation the new cells began to show signs of degeneration (fig. 11), and on the fifteenth day almost complete autolysis took place (fig. 12). There was no bacterial infection to account for this autolysis. It is possible that the cells, after having lost their vitality, were dissolved by their own ferments. We were unable to determine the exact nature of the highly refractile granules; most probably they were fat droplets. The smaller granules were seen to run 12 D. Thomson & J. G. Thomson: CultivatioiL of Tissue in vitro

EXPLANATION OF PLATE I.

FIG. 1.-Tissue: Intracystic papilloma of human ovary (bordering on malignancy). Medium : Fowl plasma one part, Ringer's solution plus glucose one part, extract of the tissue one part. Photomicrograph of live tissue taken on the fifth day of incubation shows commencing growth. (x 40.) FIG. 2.-The same specimen, eighth day of incubation, showing further growth. (x 40.) FIG. 3.-Another portion of the same specimen, tenth day of incubation, showing outgrowth of large epithelial cells. (x 75.) FIG. 4.-Photomicrograph of unstained wet film of original tumour cells.

400.) (x FIG. 5.-Photomicrograph of unstained wet film of the tumour cells after incubating for thirty days in ascitic fluid. Note the star-shaped masses of crystals. (x 400.) FIG. 6.--Photomicrograph of growing cells (same specimen as figs. 1, 2 and 3), tenth day of incubation. Original cells on left, growing cells on the right. Note the increase in size of the growing cells and also the appearance of large granules. (x 400.)

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FIG. 7.-Same specimen of live tissue, ninth day of incubation. Shows ameeboid processes in the growing tumour cells. Fine granules are seen at the terminal ends of these processes. (x 400.) FIG. 8.-Same specimen of live tissue, ninth day of incubation. Shows amceboid processes of the larger tumour cells. (x 720.) FIG. 9.-Same specimen of live tissue, ninth day of incubation. Shows similar processes; one process ends in a mass of granules. (x 720.) FIG. 10.-Same specimen of live tissue, ninth day of incubation. Shows the large size of the growing cells which always contain granules. (x 720.) FIG. 11.-Same specimen of live tissue, fourteenth day of incubation.

Shows commencing autolysis of the growing cells. (x 400.) FIG. 12.-Same specimen of live tissue, fifteenth day of inc.ubation. Shows complete autolysis of the growing cells. (x 400.) PLATE II.

–  –  –

(DE Thomson, photo.) 16 D. Thomson & J. G. Thomson: Cultivation of Tissue in vitro along the pseudopodia of the cells at times, and were frequently extruded from the ends of those processes into the surrounding medium. Fig. 7 shows the granules in the fine pseudopodia very clearly. Another point of interest in connexion with this tumour tissue was the development of stars of fine crystals (fig. 5, Plate I) after the culture preparation had been kept for about four weeks. These stars also developed in the tibes of ascitic fluid to which pieces of the original tumour had been added, and incubated for a month or more. They appeared to resemble tyrosine crystals in some respects, and evidently they indicate the presence of some chemical substance in the tumour.

(b) Carcinomatous gland from the neck, secondary to carcinoma of the floor of the mouth. Small pieces of this tissue were inoculated

into six variations of fowl plasma medium as follows:

(1) Fowl plasma one part plus extract of the cancer gland one part.

(2) Fowl plasma one part plus extract of the cancer gland one part and of chick embryo one part.

(3) Fowl plasma one part plus extract of chick embryo one part.

(4) Fowl plasma one part plus Ringer's solution two parts plus cancer gland extract two parts.

(5) Fowl plasma one part plus glucose Ringer's solution one part and cancer extract one part.

(6) Fowl plasma one part plus ascitic fluid one part plus cancer gland extract one part.



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