The culture of mature organs in a synthetic medium

Experimental

118

THE CULTURE

Cell Research 16, 118-147 (1959)

OF MATURE ORGANS MEDIUM

IN A SYNTHETIC

0. A. TROWELL Medical

Research Council Radiobiologicul

Unit, Harwell,

Berkshire,

England

Received April 11, 1958

ORGAN culture originated in the Strangeways Laboratory about thirty years ago [46, 91. But, so far, the method has been chiefly used for the culture of embryonic organ rudiments. Such cultures not only maintain their histological organization but grow in size and differentiate in a more or less normal manner until they become too large to survive in vitro. The reasons why the method has been largely restricted to embryonic organs appear to be (1) whole organs from embryos are so small that they can be cultured entire, (2) embryonic tissues, in contrast to mature tissues, are remarkably resistant to the rather anoxic conditions prevaijing in this type of culture, and (3) interest was chiefly centred on the study of morphogenesis in vitro. If, by some adaptation of this method, it were possible to keep fully differentiated organs (or parts thereof) alive in vitro without either growth or dedifferentiation, the way would be opened for many experimental studies on organ physiology, metabolism and pathology. The purpose of this paper is to describe such a method and to show that by its use many mature organs can be maintained in vitro in an entirely synthetic medium and retain their normal histological appearance for about a week. The basic method and the synthetic medium were developed originally for the culture of lymph nodes [50, 511. Since then, the apparatus has been improved and the medium simplified. The current methods and the results obtained in the culture of almost all the organs of the rat will now be described. Recent work by others on parallel lines will be referred to in the Discussion. METHODS Apparatus.-The standard culture chamber is shown in Fig. I. It houses up to 20 cultures, each about 2 mm diameter, in 5-6 ml of fluid culture medium. This

chamber, which is made mostly of aluminium and has a built-in gas reservoir, will be called the Type II chamber to distinguish it from earlier patterns [48, 501 which were made of perspex. The culture medium is contained in a shallow dish (D) made of hard glass or of fuzed silica (Vitreosil). These dishes are made from ordinary capsules (crystallizing Experimental

Cell Research 16

Culture of mature organs dishes) 48 mm outside diameter, by grinding them down to an outside height of 10 mm. In the dish stands a square metal grid (G) which is made by bending over the two ends of a flat piece of perforated metal to make short legs. The top of the grid is 25 ): 25 mm and the legs are 4 mm high. The material used is stainless steel “expanded metal”, 1.5 mm mesh, 0.005 inch thick (The Expanded Metal Co., Burwood House, Caxton St., London S.W.1). In earlier work [5Oj the grids were made from

Fig. l.-Scale drawing of the Type II culture chamber. On the left in vertical section, on the right as seen from above and horizontal section through gas outlet B. The solid black part is aluminium. L, plate-glass iid; R, slicione rubber sealing ring; D, culture dish; G, perforated metal grid. A, B, rubber tube connexions for gas inlet and outlet respectively. Fourteen cultures are shown in position.

tantalum wire gauze; these grids, though satisfactory in use, were costly and rather difficult to fabricate owing to the soft nature of the material (the top of the grid must be absolutely flat). The expanded metal grids are very easy to make and altogether more robust. On top of the grid is placed a 27 x 27 mm piece of lens paper (Green’s Cl%). Sufficient medium is present just to reach and wet the lens paper and the cultures are planted on the wet paper. During the course of cultivation certain organs may adhere to the paper and entangled fibres of paper may cause difficulty in the subsequent histological section cutting. We have not experienced much trouble on this score, but, if necessary, the difficulty can be avoided by using a sheet of 2 per cent agar, 1 mm thick, in place of the lens paper. This is made by pouring a predetermined volume of molten 2 per cent agar (Davis) in 0.7 per cent NaCl into a stainless steel tray of known area. When set, squares of agar are cut out with a knife. In experiments in which cultures are to be exposed to accurate doses of X-radiation, it is advantageous to use agar sheets 3 mm thick, in order to minimize “back scattering” of r&i&ion from the metal grid. The gas chamber (shown black in Fig. 1) is machined out of a length of 3 inch diameter aluminium bar. The plate glass lid (L) and the silicone-rubber sealing ring (R) are held on by an external screw clamp (not shown in Fig. 1). Fig. 2 Shows a Experimenial

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120

convenient arrangement in which the clamps for 6 chambers are permanently mounted on a common baseboard. The chambers can be used in an incubator, but it is much more convenient to have them on a table in a small dust-free room maintained at 37°C which also contains the gas cylinder and gas line. A removable perspex diaphragm (P) divides the chamber into an upper compartment (35 ml) which is the culture chamber proper and a lower compartment (150 ml) which is the gas reservoir.

10 CM --.....I

j F W T----I

Fig. 2.-Arrangement of lid clamps and gas line for a set of six Type II chambers. The left-hand part of the drawing is in plan, the right-hand in elevation, The lid of each chamber is held on by a perspex (lucite) bar, E, which is screwed down by wing nuts on long bolts fastened to the baseboard. P, glass plug for closing gas outlet; F, bobbin type flowmeter; W, gas wash bottle with sintered-glass plate; T, tube for adjusting gas pressure and keeping it constant. The 3-way taps are shown set for passing gas through number 4 chamber. Four I mm diameter holes in the diaphragm are quite sufficient to maintain gaseous equilibrium between the two compartments under all conditions of culture. The gas mixture, which is normally 5 per cent CO, in oxygen, enters by a rubber tube attached at A and leaves by a similar tube attached at E. These tubes and all other rubber tubing used in the gas line and also the sealing ring (R) are made of “translucent silicone rubber” (Esco Rubber Ltd., London, N.16), which is non-toxic and can be sterilised by dry heat. If ordinary rubber is used it absorbs oxygen at a surprising rate and a considerable negative pressure develops. The standard rate of gas flow is 75 ml/min, measured by a Rotameter flowmeter (F) and adjusted by raising or lowering tube T (Fig. 2). Sometime before the experiment, the empty chamber (i.e. without culture dish) is sealed up and gas is passed through it at this rate for 30 minutes, after which the outlet is closed with a glass plug (P). This is sufficient to displace all the air. When the time arrives to insert the culture dish (with cultures already planted) the gas flow is restarted before the chamber is opened. Under these conditions gas is passing upwards through the holes all the time and there is no possibility of air getting into the lower compartment however long the lid is open. After the lid is closed the gas flow is continued for a further 4 minutes, which IS sufficient to displace all the air from the upper compartment. In the same way the gas flow is restarted before opening the chamber for manipulations such as changing the medium or removing cultures. Experimenfal

Cell Research 16

Culture of mature organs The total gas space, with dish and medium in place, is 170 ml. Twenty organ eultures produce about 2 ml of CO, per day, by respiration and glycolysis, and consume about 1.5 ml of oxygen, so, if the chamber is left for 3 days, the CO, should rise from 5 to 8.5 per cent and the oxygen fall from 95 to 92.4 per cent. In practice, the CO, does not rise so much as this, probably because some escapes by diffusion through the silicone rubber. The chambers are normally opened every 3 days for the purpose of changing or reconditioning the medium. This involves 5-10 minutes re-gassing every third day, which is usually all that is required. The perspex chambers originally used [48, 501 had to be regassed twice a day. The apparatus is cleaned and sterilised as follows The aluminium chambers, lids, sealing rings and gassing attachment tubes are sterilised at 110°C for 1 hour. The perspex diaphragms are rinsed in ether and put in a sterile box. The culture dishes and all other glassware used for culture work or medium-making are scrubbed out with a mildly abrasive cleaning compound (“Gumption”), rinsed in hot water, immersed in pure nitric acid overnight, then well rinsed in glass distilled water, dried and sterilised. The stainless steel grids are briefly boiled in 10 per cent sodium carbonate, rinsed, immersed in pure nitric acid overnight, then rinsed, dried and sterilised at 110°C for i hour. The lens paper is washed in Ether AR., 4 changes, followed by glass distilled water 12 changes and overnight, after which it is dried, cut to size and sterilised at 110%. During and after cleaning, the grids and paper are handled only with forceps. It may be mentioned that the gas chambers, here made of aluminium, can also be made of stainless steel or perspex, but not of brass. We have bad uniformly bad results with brass chambers and have traced this to the “creeping” of traces of metal into the culture medium where it can be demonstrated by chemical tests. Synthefic Medium T8.-In a previous paper [51] a medium called TACPI was described which consisted of inorganic salts, glucose, 19 amino acids, cocarboxylase, p-aminobenzoic acid and insulin, together with chloramphenicol as antibiotic and phenol red as indicator, This medium has now been modified as follows (1) seven amino acids have been omitted, the remaining 12 being those which Eagle and coworkers [7] found essential for the maintenance of a variety of human cell lines in vitro; (2) the concentrations of the 12 amino acids have been reduced to approximate those found optimal by Eagle et al., except that 0.3 mM cysteine has been used in place of 0.05 rnfil cystine; (3) the concentration of phosphate has been increased from I mM to 3 mM in the light of the work of Waymouth [53]; (4) thiamine is used instead of cocarboxylase, because it is obtainable in purer form; (5) in TACPI the tryptophane must have been destroyed during heat sterilization of tbe stock solution; this is now avoided. The new medium is called T8 and its composition is given in Table I. The medium is made up, just before use, from the following five stock solbltions each of which contains the constituents mentioned at 20 times the concentration given in Table I. AA contains all the amino acids except cysteine. The tyrosine should be dissolved in the final volume of glass distilled water first, heated to about 90°C to facilitate solution. Then add the remaining amino acids, introducing the tryptophane last when the solution is cool. Solution S. NaCl, KCl, CaCl,, MgSO,.

Solution

Experimenfal

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Solution BP. NaHCO,, phenol red. Solution P IC. p-Aminobenzoic acid, insulin, chloramphenicol crystalline). In addition, 0.3 ml N. HCl per 100 ml of solution the insulin. Solution GP TC. Glucose, NaH,PO,, thiamine, cysteine. TABLE

NaCl KC1 CaCl, MgSO, * 7H,O NaH,PO, * 2H,O NaHCO, Glucose L-Arginine HCl L-Cysteine HCl L-Histidine HCI L-Isoleucine L-Leucine a Concentration

I. Composition

mM/l

mg/lOO ml

104

610

6

45

2

22

1

25

3

45

33.5

282

22

400

0.1

2.1

0.3

4.7

0.05

1.0

0.2

2.6

0.2

2.6

of medium

(“Chloromycetin”, is added to dissolve

T8.

L-Lysine HCl DL-Methionine DL-Phenyl-Alanine DL-Threonine L-Tryptophane L-Tyrosine L-Valine Thiamine HCl P-Aminobenzoic acid Insulin Chloramphenicol Phenol red

mM/l

mg/lOO ml

0.2

3.6

0.05a

1.5

0.1=

3.3

0.2a

4.8

0.02

0.4

0.1

1.8

0.2

2.3

0.05

1:7

0.25

3.5

0.001 0.1

5.0

0.03

1.0

3.0

of L-form.

Solution BP is put up in 3 ml amounts in small ampoules made from 8 mm bore hard-glass tubing. The ampoules are sealed off and then sterilised at 105°C for 1 hour. The other four solutions are sterilised by suction through No. “5 on 3” porosity sintered-glass filters and stored in hard glass test tubes with silicone rubber stoppers. Solution GPTC is made up freshly on each occasion; solution PIC should not be more than a month old, but the other three solutions can be stored for at least 6 months at room temperature without deterioration. To make up IO ml of medium, 0.5 ml volumes of each of the five stock solutions are added to 7.5 ml of fresh glass distilled water. Equilibrated with 5 per cent CO, at 37°C the pH is 7.6. Animals.-We have found by experience that, for cultures approximately spherical, the diameter must not exceed about 2 mm, otherwise necrosis occurs in the centre from lack of oxygen. So it is desirable, in theory, to select animals in which the organ in question is about 2 mm in size and can be cultured whole. In practice however, this is not always feasible, for organs such as lung, liver and kidney will always be too large to use whole. Furthermore we have found in the case of organs with fairly uniform histological pattern, such as thyroid,‘prostate, ovary and lymph node, that no better results are to be obtained by culturing the whoIe organ of a small animal than by using a one half or quarter piece cut from the organ of a larger animal. In our experience the 4 week-old rat, weighing about 60 g, is the most generally useful animal, and most of the work described here was done with it. In this animal Experimental

CtW Research

16

Culture of mature organs

12.3

the adrenal, pituitary, pineal, parathyroid, ovary and lymph nodes can be used whole, and cultures of narrow tubular organs, such as ureter, ductus deferens, and uterine horn, can be of unlimited length. The adult mouse appears to be equally suitable but our experience with it is more limited. Richter [32-341 has used adult bats, which have conveniently small organs. For certain organs we have used rats of 16, 20 or 200 g as will be described later. Apart from convenience of size, we have usually found no difference in the cultural behaviour of organs taken from rats of different age, ranging from 1 week to 3 months. Procedure.-The empty culture chambers are set up in tbe 37°C room and filled with 5 per cent CO, in oxygen as already described. The culture dishes, each containing a grid, are set out in the culture room, each one in a petri dish. The required volume of medium is made up, allowing 6 ml for each chamber and an extra 5 ml for dissection, etc. Medium is now pipetted into each dish, at one side of the grid. After about 5 ml has been added it rises far enough to wet the underside of the grid at one corner. Addition is now continued, drop by drop, until, spreading across the underside of the grid, tbe medium just reaches the opposite corner. This is the correct volume of medium to use and no further adjustment is required. With it, the cultures will be quite wet enough for nutritional purposes and will not wash off if the dish is tilted. A piece of lens paper is -then carefully lowered onto each grid. If, in any chamber, it is planned to remove some of the cultures before others, it is best to have the lens paper in several strips so that one strip with its attached cultures can be removed without disturbing the others. It is necessary to make sure that the paper is thoroughly wet and that no air remains entrapped beneath it. The prepared culture dishes can now be left in the petri disbes for up to 2 hours if necessary. The animal (rat or mouse) is anaesthetised, outside the culture room, with a 50 per cent CO,-50 per cent 0, mixture. It is then killed and bled out, by decapitation if no organs from the head and neck are needed, or by opening the chest and cutting the heart if the bead and neck is required. By means of rubber bands attached to the feet, the animal is firmly extended on and fastened to a metal plate (15 x 10 cm). The whole animal is then liberally swabbed with 80 per cent alcohol and passed into the culture room through a service hatch. The skin is widely reflected, with instruments kept in a beaker of 80 per cent alcohol. Then, with fresh sterile instruments, the organ or organs are freely exposed but not touched. The organ is now flooded with a few drops of medium, dissected free with fresh instruments and transferred to a pool of medium in a cavity slide. When the organs required for one chamber have been collected they are cleaned free of fat and connective tissue with two cataract knives. Small organs such as adrenal and pituitary are usually cultured whole, but large ones such as liver and lung must be cut into suitably-sized pieces with the cataract knives. The cultures are then transferred to the lens paper and arranged in some sort of order which is recorded, SO that each one is individually identifiable. It is wise to cut off the top left-hand corner of the lens paper, to preserve orientation. The best instrument for lifting the cultures is a “spud” made by mounting a platinum wire in a glass rod and hammering out the end to make a flat spoon about 3 mm diameter. The gas flow tbrough the chamber is now restarted, the dish is inserted, and the flow continued for a further 4 minutes. The chamber can now be left for three days, after which the medium must be either “changed” or “‘reconditioned”. Experimenial

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To “change” the medium, 4 ml are removed and 4 ml of fresh medium added. It appears however that deterioration of the medium after three days is due to two factors only-exhaustion of glucose and increased acidity from accumulation of lactic acid. This can be rectified by adding isotonic glucose and isotonic NaHCO, to the medium,

a procedure

which

we call “reconditioning”

the medium.

We have found

in fact that, over a 9 day culture period, rather better results are obtained by reconditioning the medium every 3 days than by changing it. A 5.5 ml volume of medium is normally reconditioned by adding 0.2 ml of 5.83 per cent glucose and 0.5 ml of 1.36

per cent NaHCO, and then, after mixing, removing 0.7 ml of medium. This increases the glucose concentration by 175 mg/lOO ml and the NaHCO, by 100 mg/lOO ml. When the cultures are to be studied histologically, the lens paper is lifted off with

cultures attached and placed in formol-sublimate fixative (9 ~01s. of 6 per cent HgCl,, 1 vol. of precipitate-free 40 per cent formaldehyde) for 2 hours. After washing, the cultures are gently detached from the paper, dehydrated solve) and embedded and cut in ester wax [3]. For cultures

priate to use 8 changes of 2-ethoxyethanol

in 2-ethoxyethanol (celloof average size it is appro-

(total time 3 hours) and 2 changes of

ester wax, 20 mins. each. The cultures are usually sectioned in the vertical plane; if the top of each is lightly touched with indian ink just before fixation, the ink is

visible in the final section and identifies the top side of the culture. RESULTS

The results are judged from the histological appearances of cultures maintained for 2, 3, 6 and 9 days in uifro. In the histological descriptions the following terms will be used. “Unaltered” means that neither the appearance of the cells nor the general architecture of the organ differs appreciably from in vivo controls. “Healthy” means that not more than 2 per cent of the cells in any tissue of the organ are degenerate or dead. “Necrosis” means death of cells, and is usually recognised by pyknosis of the nuclei. Necrosis is “central” when confined to the central part of the culture, “focal” when confined to a small area elsewhere, or “scattered” when randomly distributed cells in a particular tissue or layer are dead. “Regularly” means that the same result was obtained in each of a number of experiments, and that it may therefore be anticipated with some confidence. “Usually” means that the result was obtained much more often than not, but that failures do occur. “Sometimes” means that the result was obtained in more than one, but not in every, experiment. For convenience, the various organs and structures cultivated will be grouped as follows-(l) tubes, (2) exocrine glands, (3) kidney, (4) lung, (5) endocrine glands, (6) gonads, (7) skin, (8) adipose tissue, (9) haemopoietic organs, (10) nervous system, (11) eye, (12) organ associations, (13) humoral influence of one organ on another. Experimental

Cell Research 16

Culture of mature organs In the following descriptions, the figure in brackets organ is the total number of cultures studied, including ascertain optimal size of animal and culture.

125 after the title of eat early experiments to

Tubes Ureter (54).-The ureter of a 60 g rat or an adult mouse is only 0.4 mm in diameter, so the cultures can be any length and 5-7 mm is convenient. Regularly healthy for 4 days, usually for 6. After 6 days progressive necrosis of the muscle often occurs, though the epithelium survives for at least 9 days (Fig. 3). The epithelium always migrates out from the cut ends (Figs. 4, 5) and, after covering the ends, progresses for some distance over the outside of the ureter. As mitoses are rarely seen, this process is assumed to be migration rather than growth. The migrated epithelium is only one or two cells thick. The lumen epithelium is apparently not thinned by this migratory loss of cells, probably because the whole culture shortens. The epitheliai nucleoli usually enlarge. Ductus deferens (la).-The ductus of a 20 g or 60 g rat or of an adult mouse is narrow enough to culture at any length and 5-7 mm is convenient. Regularly healthy for 9 days. The epithelium migrates over the cut ends and then back over the outside, but not so extensively as in the ureter. The migrated epitbelium is cubical and non-ciliated; the lumen epithelium usually remains columnar and ciliated (Fig. 7) but it sometimes becomes stratified (Fig and closely resembles the normal transitional epithelium of the ureter. have not discovered why this occurs in some experiments and not in others. Numerous mitoses are usually seen in the epithelium during the first few days (Fig. 7) and occasionally even at 9 days. Tbe epithelial nucleoli usually enlarge a little. In older cultures the muscle always survives longer than the e&helium and the distinction between inner circular and outer ~o~g~t~di~a~ layer is maintained to the end. In all other organs studied the muscle died before the epithelium. Uterus (56).-The uterine horns of 20 g rats are cultured at any len 5-7 mm being convenient. In 60 g rats the uterine horn is rather maria in size. Usually it is about 1 mm diameter and can be cultured in 5-7 mm lengths, but if it is wider (up to 1.5 mm) only 3 mm lengths should be used. Usually healthy for 6 days, sometimes for 9. Focal patches of necrosis are apt to occur in the inner muscle layer for unknown reasons. The epitbeli~m always migrates out as a cubical layer over the cut ends and then back over ingrowths. the outside (Fig. 9), where it sometimes gives rise t gland-like attens from columnar As culture Iproceeds, the lumen epithelium gradually Eqerimenial

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0. A. Trowel1

126

to low cubical, probably because scattered cells are dying and not being replaced. A little pyknotic debris is often found in the lumen. Mitoses are rarely seen. The epithelial nucleoli enlarge and after 9 days are remarkably prominent (Fig. 10). The muscle cells become shorter and fatter and come to resemble tibroblasts, while the distinction between circular and longitudinal muscle layers is mostly lost. This apparent dedifferentiation of plain muscle has not been seen in any of the other organs studied. Uterine cultures usually show active movement during the first day or so; they crawl about on the grids, disturb other cultures, and sometimes fall off the edge of the grid and drown. Trachea (16).-Transverse rings, 1 mm wide, are cut from the trachea of a 60 g rat and planted with axis vertical to the grid. Regularly healthy and unaltered for 4 days, after which a little scattered necrosis appears in the subepithelial connective tissue and the epithelium progressively flattens owing to loss of cells (Fig. 28). The epithelium and cartilage however look fairly healthy for 9 days. Mitoses are sometimes seen in the epithelium. The right and left main bronchi of 60 g rats may be cultured as short tubes, with axis parallel to the grid, and behave in the same way as the trachea. Arteries (6).-Long lengths of the internal spermatic artery or branches of the mesenteric arteries are very suitable for culture. Regularly unaltered for 9 days. It is noteworthy that the elastic libres and laminae are perfectly preserved.

Exocsine Glands Salivary

dibular,

glands

sublingual

(72).-Pieces and parotid

Figs. 3$34.-Sections of organ cultures. haemalum and eosin. Fig. 3.-Ureter

of 60 g rat, cultured

about 2 X 2 X 1 mm, cut from the submanglands of the 60 g rat, usually remain healthy Fixed in formol-sublimate,

for 9 days.

Fig. 4.-Ureter of adult mouse, cultured tissue on outside of ureter.

cut in ester wax, stained with

x 250.

for 4 days. x 250. Epithelium

migrating

over adipose

Fig. 5.-Ureter of 60 g rat with lymph node of adult mouse, cultured migration seen in upper right quadrant. Fig. 6.-Ductus thelium.

deferens

of 20 g rat,

cultured

Fig. 7.-Ductus

deferens of 20 g rat, cultured

for 2 days. x 19. Epithelial > for 10 days. x 250. Squamous metaplasia of epi-

for 2 days. x 250. Mitoses in epithelium.

Fig. 8.-Ductus deferens with lymph node, both from adult Lymphocytes in lymphatics of ductus. Fig. 9.-Uterine Fig. lO.-Uterine (In this culture Experimenfal

horn of 56 g rat, cultured

mouse, cultured

for 3 days. x 250. Uterine

epithelium

horn of IO g rat, cultured for 9 days. x 250. Enlargement the uterine lumen was greatly distended.) Cell Research 16

for 6 days. x 55. on the outside.

of epithehat

nucleoli.

Culture of mature organs

127

0. A. Trowel1

128

for 6 days (Fig. ll), and sometimes for 9. They are unaltered except for some reduction in the zymogen and mucin content of the cells. In two out of eight experiments, in most of the cultures, the epithelium of the ducts underwent a remarkable squamous metaplasia (Fig. 12) and there was a good deal of mitosis in this metaplastic epithelium even after 6 days. Pancreas (14).-Pieces of pancreas from 60 g rats do not survive. After 24 hours all the cells, including connective tissue and blood vessels, have completely disappeared and only a ghostly framework of reticulin is left. Presumably the trypsin becomes activated and digests the cells. Other organs (e.g. lymph nodes) cultured in the same dish are, however, not affected. Exorbital lachrymal gland (18).-Pieces, 2 X 2 X 1 mm, from the 60 g rat are usually healthy for 6 days. Mammary gland (20).-Small lobules, about 2 mm diameter, are taken from rats in late pregnancy. Except for a little scattered necrosis in the connective tissue, the cultures are regularly healthy for 6 days (Fig. 13). There is a progressive reduction in the secretion content of the cells. Prostate (28).-The bilobed ventral prostate is used. In 20 g rats a whole lobe is planted, while in 60 g rats the lobe is cut into several flat pieces, each about 2 mm diameter. Regularly healthy and unaltered for 6 days, usually for 9 (Fig. 14). Seminal uesicZe (12).-The seminal vesicle of the 60 g rat is cut into 4 pieces. Regularly healthy and unaltered for 6 days. By 9 days the epithelium has often undergone some squamous metaplasia. Liver (39).-The results have been unsatisfactory. Using the 60 g rat, we have cultured small 0.5 mm thick slices (as used in Warburg manometry) and also pieces (1.5 x 1.5 mm) cut from the thin edge of the small lobe which lies in the lesser curvature of the stomach. Within 2 days there is, in the first place, a large central necrosis. The outer surviving zone is broadest in the upper part of the culture where it is, rather constantly, 0.2 mm wide. Secondly, Fig. il.-Submandibular

gland of 60 g rat, cultured

Fig. 12.-Submandibular ducts.

gland

Fig. 13.-Mammary Fig. 14.-Prostate

for 6 days. x 250. Usual appearance.

of 60 g rat, cultured

gland of pregnant

rat, cultured

gland of 60 g rat, cultured

for 6 days. x 250. Squamous for 6 days. x 250.

for 9 days. x 250.

Fig. 15.-Lung

of 60 g rat, cultured

for 6 days. x 250. Artery

Fig. 16.-Lung

of 60 g rat, cultured

for 6 days. x 250.

Fig. 17.-Kidney Fig. 18.-Lung Experimental

cortex

of 10 g rat, cultured

of 60 g rat, cultured Cell Research

16

and bronchiole.

for 9 days. x 250.

for 10 days. x 250.

metaplasia

of

Culture of mature organs

9 - 593701

Experimental

Cell Reseurch 16

130

0. A. Trowel1

even in this surviving zone there is scattered necrosis, the nuclei disappearing mostly by karyollysis, and after 4 days all the cells show vacuolar degeneration which is followed by cytoplasmic shrinkage. The nucleoli in these degenerating cells are however larger than normal. Westfall et al. [54] cultured a pure strain of liver cells in synthetic media and showed that it had a specific requirement for glutamine, but we have added glutamine and also many other possible nutrients to medium T8 without improving the results. Foetal rat liver gave better results; most of the hepatic cells remained healthy for 4 days, but the haemopoietic cells in: the sinusoids were all dead by then and obscured the picture. Kidney

(24)

Tangential shces or 1.5 mm cubes cut from the kidney cortex of 10 g or 60 g rats are used. As with the liver, there is some central necrosis. But the outer surviving zone usually remains healthy and unaltered for 9 days, except for some changes in the renal corpuscles. The renal corpuscles regularly undergo a curious change which begins after 3 days”, is complete in 6 days and still the same at 9 days. The capillary and connective tissue elements of thc,iglomerulus become completely necrotic and are reduced to a hyaline mass. The inner layer of Bowman’s capsule, on the other hand, survives and becomes transformed into a columnar epithelium often two cells deep; the outer layer of Bowman’s capsule remains unaltered. The renal corpuscles thus came to look rather like ovarian follicles (Fig. 17). Although, at first sight, this looks like an active epithehal metaplasia it is probably purely passive and results from a great contraction of the superficial area of the epithelium, so that cells originally expanded and flattened become bunched up and columnar. Normally, the inner layer of Bowman’s capsule is closely applied to the tortuous capillary loops of the glomerulus and is in fact highly convoluted. But in. the cultures, with necrosis of the glomerulus, these infoldings disappear and it is evident that the surface area of the epithelium is greatly reduced” The cut surfaces of the cultures become covered with a cubical epithehum of uncertain origin and the renal tubules remain mostiy unaltered.

Lung (46) Rats weighing 20 g or 60 g are used and 1.5 x 1.5 mm pieces a.re cut from the thin edge o*I ibe upper lobe of the right lung. Usually healthy for 9 da.ys, though sorr~c~lmes the alveolar epithelium shows a little scatthered necrosis. The alvcoi::L i’ c~>~heliai cells always enlarge and become roughly cubic& or pc:Yygo-raa’L( ?‘~f;, ?B), which is probably due to shrinkage of the aireok:;. BY

Culture of mature orpms the 9th day the lumina of the alveoli have nearly or completely and the tissue looks curiously like liver (Fig. 18). The bronchioles are healthy and unaltered for 9 days (Fig. 15). Endocrine

131 disappear-e and arteries

Glands

Thyroid (43).-The lateral lobe in 60 g rats is the right size for cuhure but difficult to remove without damage. It is better to use half the lateral lobe of adult (200 g) rats. Regularly healthy and unaltered for 6 days (Fig. 19). Later the interfollicular connective tissue starts to die, but the follicular epithelium is usually healthy for 10 days. There is usually some reduction in colloid; the follicles shrink a little and the epithelial cells become somewhat taller in consequence. Ultimately, the outer follicles degen.erate before the central ones. Parathyroid (IO).-These glands lie embedded in the thyroid. They are cultured with the thyroid, not dissected free. Regularly healthy and unaltered for PO days (Fig. 20). Pituitary (48).-The whole pituitary of the 60 g rat is used. It is almost rectangular in shape and measures 3 X 2 X 1 mm. This is rather larger than the general run of cultures but it survives without central necrosis (Fig. 2%). The anterior lobe, which makes up the greater part of the organ, has no arterial blood supply [5j; it is supplied by venous blood from the hppophpseal portal vessels and must therefore be accustomed to a rather poor oxygen supply. In the cultures, the pars anterior is regularly healthy for 9 days (Fig. 22) except that some vacuolar degeneration occurs at a short distance inside the extreme outer rim-as seen in Fig. 2 1 I This vacuolation develops regularly in this position between the 6th and 9th day, and nowhere else, and it is difficult to account for the location. The pars intermedia is regularly healthy and unaltered for 9 days (Fig. 23). Sometimes the pars intermedia contains a considerable number of mitoses even after 6 days, though none are found ~II ~iaa at this age. The pars nervosa is regularly healthy for 9 days. It apparently becomes more and more cellular until eventually it looks remarkably like the adrenal cortex (Fig. 23). Probably the cells simply crowd together as the interv enjng nerve fibres degenerate and disappear.

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the outer cortex and the medulla are usually healthy for 9 days (Fig. 25). The same happens when half or quarter pieces of larger adrenals (60 g rat) are used, so the result is not related to any metabolic or nutritional gradient within the culture. The dead zone appears to correspond approximately to the zona fasciculata. In normal rats of this age the boundary between the glomerular and fascicular zones is not clear, and there is no reticular zone. Thinking that this degeneration of the cortex might be due to absence of ACTH we cultured several pituitary glands along with, and even in contact with, the adrenals, but the fascicular zone degenerated just the same (see also Section 12e). Addition of ascorbic acid to the medium had no effect. In some cultures, after 6 or 9 days, clusters of newly-formed young cells had appeared in the outermost part of the cortex (Fig. 26). It is probable that these were new glomerulosa cells developed from the capsule, but we could not be certain. Schaberg [35] cultured small pieces of adrenal from 5 day-old rats on a plasma clot and found the same necrosis of the zona fasciculata and regeneration of zona glomerulosa from the capsule as reported here. Gonads Ovary (36).-The mature ovary (60 g rat) is not very satisfactory because large mature or maturing follicles always degenerate within a few days. But it is only fair to record that in the ovaries of “normal” 60 g Wistar rats, both in our own colony and in colonies maintained elsewhere, we have found a surprising amount of scattered necrosis in the follicular epithelium of ripening follicles. Good results have been’obtained with the ovaries of 10 g (1 week old) rats which contain only immature follicles. These are cultured whole and are usually healthy and unaltered for 9 days (Fig. 24).

Fig. lS.-Thyroid

of 200 g rat, cultured

Fig. 20.-Parathyroid

for 7 days. x 250.

of 200 g rat, cultured

for 7 days. x 250.

Fig. 21.-Pituitary of 60 g rat, cultured for 7 days. x 17. The intraglandular below it, the pars intermedia and the pars nervosa. Fig. 22.-Pituitary

of 60 g rat, cultured

cleft is seen and,

for 9 days. x 250. The pars anterior.

Fig. 23.-Pituitary of 60 g rats, cultured for 6 days. x 250. From top left to bottom anterior, intraglandular cleft, pars intermedia, pars nervosa. Fig. 24.-Ovary

of 10 g rat, cultured

Fig. 25.-Adrenal zona fasciculata,

of 10 g rat, medulla.

Fig. 26.-Adrenal from the capsule. Experimental

for 9 days. x 250.

cultured

of 10 g rat, cultured

Cell Research 16

right-pars

for 3 days. x 250. From left to right-zona for 6 days. x 250. New glomerulosa

glomerulosa,

tissue differentiating

Culture of mature oryans

133

Ezperimenfal

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Testis (28).-Attempts to culture isolated testis tubules were quite unsuccessful., It ‘tias found better to cut out a small bunch of tubules from the testis of a 200 g-rat and plant this directly on the grid without any cleaning up or unravelhng. Usually a few tubules survived well for 3 days, but most degenerated and’idisintegrated. In the surviving tubules the cells appeared normal with the-usual number of mitotic and meiotic figures. Cultures maintained in air instead’ of 95 per cent 0, survived slightly better for the first 3 days, but were all dead by 6 days. / SFcin (40) Skin from the neck of the full-term rat foetus is used. This is more or less mature in that it is already keratinized, though the hair follicles are only just developing. This skin may be cultured, raw surface down, in quite large sheets. It is an advantage of the lens paper method that thin sheets such as skin, mesentery, retina, remain fully extended: on a plasma clot or on agar they roll up into balls. Regularly healthy and unaltered for 3 days (Fig. 27). By 6 days, though usually still healthy, the boundary between epidermis and dermis is lost (Fig. 29). Presumably the basement membrane disappears so that epidermal cells and tibroblasts intermingle. By 6 days a good deal of extra keratin has been formed and the thickness of the cellular layer is reduced. No mitoses are seen. Ultimately the dermis dies (scattered necrosis) before the epidermis. These cultures do just as well in air as in oxygen. Adipose

Tissue

White adipose tissue (27).-Flattish pieces, about 2 mm in diameter, are obtained by cutting “leaves” from the cord of fat which accompanies the internal spermatic vessels in the 60 g rat. Regularly healthy for 9 days, with some reduction in size of the fat cells. The nucleoli usually enlarge. Brown adipose tissue (12).-This is taken from the posterior abdominal wall in the region of the renal hilum or the bifurcation of the aorta. In surprising contrast to white fat this tissue survives rather badly, many of the Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.

27.--Skin of foetal rat, cultured for 3 days. x 250. 28.-Trachea of 60 g rat, cultured for 6 days. x 250. 29.-Skin of foetal rat, cu!tured for 6 days. x 250. 30.---Spinal ganglion of 200 g rat, cultured for 6 days. x 250. 31.-Lymph node of 66g rat, cultured in air for 4 days. x 17. Only the outer rim survives. 32:~--Sympathetic ganglion (juxta adrenal) of 60 g rat, cultured for 4 days. x 250. 33.--Pituitary of 60 g rat, cultured in air for 2 days. x 17. Only the outer rim survives. 34.-Adrenal of IO g rat, cult.ured in air for 2 days. x 17. Only the outer rim survives.

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cells are dead within 2 days though some live for 6 days. The cells of brown fat are histologically very similar to those of the adrenal cortex and there is evidence that they actually secrete similar hormones and participate in the alarm reaction [41]. As mentioned already (p. 132) the adrenal cortex also survives badly under these conditions of culture. Haemopoietic

Organs

Lymph node (1310).-In our experience, which is considerable, the most reliable nodes are the lumbar and sacral of 60-80 g rats. These are used whole or cut transversely into 2 or 3 pieces, depending on size. Regularly healthy for 6 days, sometimes for 9. Although the number of dead lymphocytes present does not usually exceed 2 per cent at any one time, there is nevertheless a steady loss of these cells, partly by death and partly by migration out of the culture. After 6 days the depletion is quite noticeable. Sometimes the upper part of the culture is much more depleted than the lower, but just as often vice versa. Many of the lymphocytes move from the interstices of the reticulum and collect in large numbers in the sinuses. The cytological appearances suggest that after about 4 days many of the reticulum cells are differentiating into large lymphocytes, and some of the small lymphocytes into monocytes or macrophages. These findings have been described elsewhere [51, 52] and will not be recapitulated here. In several experiments, cervical and mesenteric nodes from 60 g rats gave equally good results; but in other experiments the results were bad, though lumbar and sacral nodes from the same individual rats were quite satisfactory. This is not related to individual animals or litters and there is some indication of a seasonal effect; thus, cervical nodes seem to be satisfactory from October to March but not from April to September. Thymus (53).-Pieces, 1.5 X 1.5 mm, are cut from a thin edge of the organ, using rats of any age and also adult mice. The results have been inconsistent and in most cases unsatisfactory. Usually the lymphocytes died quite quickly and few were left after 4 days, but sometimes, and for no apparent reason, they survived quite well for as long as 6 days. Spleen (44).-Pieces cut from the spleen of new born, 20 g and 60 g rats have been tried but all were regularly unsatisfactory. All the haemopoietic cells, including lymphocytes, were dead within 3 days, though the reticulum cells survived much longer. Culture in air gave similar results. Bone marrow (14).--S mall pieces of rat femoral bone marrow were used and found uniformly unsatisfactory. Nearly all the haemopoietic cells were dead within 3 days, but the reticulum cells survived. Experimental

Cell Research 16

Culture of mature organs Nervous

System

Brain (33).-Pieces about 1.5 mm cube are cut from the superficial part of the cerebral cortex along the edge of the longitudinal fissure. These must be cut out in situ with very thin sharp knives (made from razor blades) and transferred directly to the grid. The nerve cells in the peripheral part of the culture die, almost certainly from trauma, but in the central part about twothirds of them survive for 6 days. These cells, though surviving, are much shrunken both in nucleus and cytoplasm, and Nissl’s granules have disappeared. Addition of glutamine to the medium did not improve the results. Cultures in air did no better, usually rather worse. Cultures similarly prepared from the cerebellar cortex survived badly; nearly all Ihe cells were dead wit 3 days. It should be mentioned that if fragments are cut from an excised piece of brain with cataract knives in the ordinary way, the initial trauma is so great that all the cells in the explant die and become pyknotic within an hour. Spinal gangZion (23).-The first and second lumbar roots of a fully grown rat are exposed by posterior laminectomy. Each posterior root is cut at either end, removed with its associated ganglion and cultured whole. e’sually about 60 per cent of the ganglion cells remain healthy for 6 days and contain normal Nissl granules (Fig. 30); the others slowly degenerate. In degeneration, Nissl substance disappears first, then the cytoplasm becomes increasin eosinopbilic and the nucleus slowly fades away by karyolysis. More ganglion cells survive in the lower part of the culture than in the upper. This suggested the possibility of oxygen poisoning, but when ganglia were cultured in air all tbe cells died within 6 days. The satellite (Schwann) cells surrounding each ganglion cell are regularly healthy and unaltered for 6 days, usually for 9. Most of the axons degenerate in the first few days. Sympathetic ganglion (5).Small ganglia closely associated with adrenal glands, sacral lymph nodes or seminal vesicles were sometimes cultured (unknowingly) with these organs. Usually healthy and unaltered for 9 days (Fig. 32), and therefore surviving much better than spinal ganglia. The rat adrenal medulla always contains some ganglion cells [29] and in adrenal cultures these survived well for 9 days. Eye (132) In a medium of 15 per cent rat serum in TACPI, whole eyes of 8-l 1 day old CBA mice survived fairly well for 8 days. In the same medium, isolated retinae of mice and rats aged 9-14 days survived well for 40 days, but only Esperimenfal

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if they were cultured in air. When retinae were,cultured in 95 per cent oxygen, or even 60 per cent, the cells were rapidly killed by oxygen poisoning. The isolated rat retina survived well in TACPI alone, but the mouse eye and retina seemed to require some serum. These results, obtained in joint work with Dr. D. R. Lucas, have been fully described elsewhere [22] and are only briefly mentioned here. Organ Associations It would be interesting to culture two different organs with their cut surfaces in direct apposition, to see if their tissues fused, interpenetrated or otherwise interacted. Gaillard [lo] was the first to;study “Confronted explants” as he called them. He cultured small fragments of anterior pituitary and posterior pituitary in direct apposition in a single drop of medium and found that tissue resembling pars intermedia differentiated at the contact zone. More recently Wolff and his coworkers have studied the intergrowth of a large number of embryonic organ rudiments in this way [56]. The behaviour of mature organs in this respect has hardly been explored and there would seem to be a wide field in which the organ culture method could be used to study organ intergrowth and in vitro grafts, free from the complications of immunity reactions. So far we have studied only a few cases. It seems best to call these organ “associations” [56] rather than chimeras or parabioses, which have slightly different connotations. Organ associations are homologous or heterologous according to whether the organs are anatomically the same or different, and homoplastic or heteroplastic according to whether the organs come from the same or different species. Lymph node-lymph node.-When half a rat lymph node is planted with its cut surface in apposition to half a mouse lymph node the tissues fuse completely and nothing further happens. The two cultures become a single “organ” and it is only possible to tell which half is which by a difference in the thickness of the capsule. This is an example of a homologous heteroplastic association. Lymph node-Adipose tissue.-When half a rat lymph node is cultured with its cut surface in contact with adipose tissue, white or brown, from rat or mouse, the tissues fuse but there is no migration of lymphocytes into the fat or vice versa. Lymph node-Ureter.-A convenient heterologous association, which we have studied rather fully, is made by wrapping a 3 mm length of ureter, horseshoewise, round about two thirds of the perimeter of a small (1.5 mm) lymph node culture. Either 60 g rats or adult mice can be used: The epithelium Experimental

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migrates from the cut ends of the ureter and covers the free surface of the lymph node, whether this be capsule or cut surface of lymphoid tissue. phocytes from the cut surface of a node do not invade the outer wall of the ureter or ureteric epithelium which may have grown over them. There is just a fusion of tissues with mutual respect for each other’s territory. Fig. 5 shows such an association, but it is not quite typical because the ureter was not completely freed from adipose tissue. Heteroplastic associations-rat node and mouse ureter, or mouse node and rat ureter-behave no differe from homoplastic ones, over a period of 6 days. We bad anticipated that lymph node might produce antibodies against the ureter. In pursuance of this idea we tried to produce an active state of immunity in the lymph nodes before the experiment began. Dr. P. L. T. Ilbery kindly immunised adult mice for us, by administering a primary injection of rat bone marrow, fatlowed 4 weeks later by a secondary injection of minced rat ureter given subcutaneously into both hind legs. One week later we cultured the lumbar lymph nodes from these mice in association with normal rat ureter, but the ureters survived healthily for 4 days, as did the lymph nodes. Similar results were obtained when mice were immunised with rat lymph node material and their lymph nodes subsequently cultured with -rat lymph nodes. It is generally believed that, in the whole animal, “graft immunity” is carried in the lymphocytes and not in the plasma. So these negative results in vitr might be ascribe nodes penetrated to the fact that no lymphocytes from the immunised lymp tbe ureters. In many (but not all) of the ureters, rat or mouse, cultured in association with mouse lymph nodes there were many mitoses in the epithelium. This was not found when rat lymph nodes were used It seems therefore that mouse lymph nodes liberate some substance which stimulates mitosis in ureteric epithelium. Lymph node-Ductus deferem--In general this behaved like the lymph nodeureter association, but the epithelial migration was not so extensive. ‘very often, lymphocytes found their way into the lymphatics of the ductus wall, wherein they travelled for long distances in considerable numbers (Fig. 8). is never happened in the ureter. Mouse lymph nodes did .not stimulate mitosis in the ductus epithelium. B)ituitarZI-Adrenal.-The capsules fused and the two organs remained distinct. Some necrosis occurred in .the pituitary because its oxygen supply was blocked on one side. There, was some evidence of a hormonal effect of the anterior pituitary on the adrenal cortex. The zona fasciculata died, quickly in the usual way; but after 6 days most of the glomerulosa cells had enlarged, Experimenfal

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become vacuolated and apparently differentiated into new fasciculata cells. An exactly similar effect was found by Schaberg [36] who cultured small pieces of adrenal with small pieces of anterior pituitary, from 5 day-old rats, on a plasma clot. Humoral

Injluence

of one Organ on Another

If different organs are cultured in the same dish, but not in contact, there is the possibility that they may influence each other by humoral means. In this way, both the secretion and the action of hormones in vitro could be investigated and, with 20 organs in one dish, the number of permutations and combinations could provide a lifetime study. Preliminary experiments on these lines have so far been disappointing. For example, when 4 pituitaries were cultured with 6 thyroids or with 6 adrenals there was no effect on the thyroids or adrenals in 6 days. Probably more preliminary work is necessary to discover the optimum conditions for demonstrating these effects. For instance how many pituitaries should be put with how many thyroids and in what volume of medium?-and is serum necessary? One positive result has been obtained. When 8 adrenals were cultured with 8 lymph nodes, the adrenals released into the medium sufficient cortical hormone to kill about 50 per cent of the lymphocytes in 2 days. From previous work with pure cortisone [49] it was deduced that the hormone concentration in the medium must have been equivalent to about 0.03 mMcortisone (lO,~g/ml). Schaberg and de Groot [37] cultured small pieces of rat adrenal cortex on a plasma clot and measured the corticosteroid content of the medium by a spectrophotometric method. Their culture/medium ratio was roughly the same as ours and they found a corticosteroid concentration of 3-4 ,ug/ml after 3 days, which is in good agreement. Lymph-node cultures are not.affected by the presence of pituitary, ovary salivary gland, pancreas, prostate, uterus, spleen, brain, liver or thymus cultures in the same dish. DISCUSSION

In the original method devised at the Strangeways Laboratory [9], embryonic organs are cultured in air on a clot made from cock plasma and chick embryo extract. This medium is favourable to growth and differentiation, but for mature organs it has the disadvantage of procuring an outgrowth of fibroblasts and of liquefying under the digestive action of the culture. Furthermore the medium can be changed only by transplanting the cultures. But despite these difficulties certain post-embryonic organs can be cultured by Experimental

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Culture of mature organs

141

tbis method provided the cultures are very small and embryo extract is kept to a minimum. The following organs have been successfully maintaine this way: uterus of rabbit [4] and mouse [12], ovary of new born rat [25, 261, seminal vesicle of guinea pig [4] and mouse [la], prostate of mouse 6 weeks old [IS] and 6 months old [19], human parathyroid [I I], and thyroid, pituitary and adrenal of the young rat [27]. Parker [30] was the first to draw attention to the importance of oxygen and glucose in the culture of adult tissues, though the advantage of culturing in oxygen was in fact disc.overed by Loeb 1211 who explanted adult organs in test tubes as long ago as 1897 [20]. Parker cultured 0.75 mg pieces of adult spleen in a serum-saline medium reinforced with glucose, contained in a flask filled with oxygen. Pieces of adult rat thyroid [ 151, human endometrium [31] and rabbit skin [28] have been successfully maintained for a few days by essentially similar methods. In 1952 I devised a method for culturing adult organs on the surface of cotton wool soaked with a serum-saline medium containing 0.4 per cent of glucose, in oxygen [48]. This was an attempt to combine the desirable features of the Strangeways and the Parker methods. Later, in order to use a much larger volume of medium and more cultures per dish, 1 introduced the metkod of culturing on lens paper supported by a grid of tantalum gauze [SO]. This method had the advantage that the medium could be sampled, changed or added to, without disturbing the cultures. Biochemical studies were particularly in mind. At the same time 3 per cent of CO, was added to the oxygen gas phase in order to stabilise the pH. When pure oxygen or air is used the medium becomes very alkaline from loss of CO,. The method was use tially for the culture of lymph nodes [51]. Other workers used it for c of prostate [39] and thyroid [40] of adult mice, and for adult rabbit spleen which was shown to synthesize antibody in vitro [23, 451. The method was modified by Chen [2] who discovered that certain brands of lens paper would float of their own accord, so he dispensed witk the metal grid. Using this floating lens paper method he successfully cultured a variety of embryonic organ rudiments in air. Chen’s method was successfully used by Richt and his co-workers for the culture of the ovary and uterus [33], thyroi parathyroid [32] and adrenal [34] of the adult bat (Tadasida mezicnnaji-a88 in air. It is interesting that these workers found the same early degeneration of the inner part of the adrenal cortex and of the maturing ovarian follicles that we noted in our rat cultures. The floating lens paper method was used by Sidman [42] for the culture of brown adipose tissue from the rat. The same early necrosis was found as we report in this paper, though it was later disEzperimentai Ceil Research16

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coveredb that survival was improved by addition of insulin and culturing at 33°C [43]. It may be noted that floating lens paper will support about 4 cultures but it will not support 20 cultures on a square inch, for which reason we still prefer to use metal grids. Schaffer [38] found that, although lens paper was satisfactory for most mammalian organs, it was a great nuisance with chick organs. These grew into the paper and the incarcerated paper tibres caused trouble in the subsequent section-cutting. So he used instead an openweave rayon (cellulose acetate) fabric, coated with silicone to make it float. The cultures were fixed and dehydrated on the fabric and the rayon was then dissolved away in acetone. We have used rayon fabrics in place of lens paper on the metal grids and found them satisfactory except that the rayon threads cut into,the bottom of the cultures and make grooves. As explained in the Methods section, if adherence to lens paper proves troublesome, it is best to use a thin sheet of agar instead. In the method described here, the chief improvement is the new chamber which keeps the gas phase much more constant. The results reported here are definitely better than those obtained in the original perspex chambers, and this is attributed to stabilisation of the CO, and pH. In the original chambers, which had to be regassed twice a day, there was a 12-hourly cycle of COi’from 3 to 8 per cent of pH from 7.8 to 7.2. The m.edium T8 is thought to give slightly better results than the original TACPI; but itschief virtue is simplicity. It might well,be supposed that addition of further nutrients would prolong the survival of the cultures but we have,.not been encouraged in this belief. A variety of substances, including ascorbic acid 0.3’mM, glutamine m2M, m-inositol 0.1 mM, have been added to T8 without. any noticeable effect on histological appearances or survival times. It .has been shown by Eagle, and co-workers that both glutamine [7] and :m-inositol [8] are essential for, the long term survival of various cell lines in tissue culture. Furthermore, addition to T8 of egg yolk, yeast extract, chick embryo extract, rat brain extract or rat thymus extract has not, so far, appreciably improved the results, .except in the case of liver. Further work on the culture of liver is still in progress. The Importance

of Oxygen

Except in a few instances, the oxygen gas phase is indispensable to our method. The solubility of oxygen in biological media is very low; had it been higher the problems of evolution and of organ culture would have been greatly simplified. Twenty organ cultures consume, per hour, a volume of Experimental

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oxygen equal to that dissolved in 2.5 ml of medium. It is now welt known that adult (in contrast to embryonic) tissues will not survive under anoxic conditions, so it is clear that diffusion of oxygen from the gas phase willi be a critical factor in the survival of ad&t organ cultures. As oxygen diffuses through the culture, some of it is consumed by the cells and the concentration will be minimal at the centre. If we consider a spherical culture surrounded by pure oxygen (gas) and take the limiting case in which the concentration of oxygen at the centre just reaches zero, then the Iimiting radins is given by the folloiving formula [ 141 r = I/S

CD/A,

where d is the oxygen consumption in ml/ml tissue/nun, C is the external oxygen concentration in atmospheres, D is the diffusion constant of oxygen, and r is in cm. A will vary with the organ, but, as an example, the figure of 0.023 for lymph node cultures [48] may be used. The best available figure for 11 is 1.98 x lop5 [l].,With these figures, r becomes 0.7 mm. So, in theory, central necrosis should occur if the diameter of a spherical culture exceeds 1.4 mm. In practice we find that the limiting size is rather larger-about 2 mm. This is probably because towards the centre of the culture the actual oxygen consumption becomes submaximal, so the oxygen concentration falls off less steeply than is assumed in the theory. As expected, it has been found that the limiting size depends on the normal oxygen ~~~~s~l~~tio~ [I7 ] of the organ, being smaller in organs with high consumption (kidney, liver) and larger in organs with low consumption (uterus, ovary, skin, lung, fat). If it be furtber assumed that the spherical culture is surrounded by a stationary layer of medium 1 mm thick, it can e calculated that the critical volume is reduced by a factor of 3 [48], which explains why cultures must be supported on the grid so that they project well into the gas phase; if submerged a large central necrosis occurs. If air is used instead of oxygen, r becomes 0.32 mm and the critical volume is reduced by a facior of 10. We have cultured organs in air and found that this prediction is largely confirmed. Standard-sized cultures of thyroid, pituitary, pineal, adrenal, ovary, uterus and lymph node cultured in air show, after 2 days, only a peripheral rim surviving; the larger central part is necrotic (Figs. 31, 33, 34, and compare Fig. 33‘ with 21). Some organs, however, survive as well in air as in oxygen, but this is probably because they are either very thin or narrow (ureter, artery, bronchus, skin) or have a very How oxygen consumption (adipose tissue). It may be asked: why bother to construct the special chambers required EzperimeniaE

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for culture in oxygen, why not use much smaller explants in air? The answer is that the trauma incidental to dissecting out or cutting out explants is, relative to the size of the explant, much greater in the case of smaller fragments. Also, smaller cultures are less able to “condition” the immediately adjacent medium to their liking. Whatever the explanation, it is our experience that small cultures in air do less well than larger ones in oxygen. It may be enquired if the use of oxygen involves a risk of oxygen poisoning. Biochemists working with tissue slices have shown that pure oxygen at atmospheric pressure does poison brain [24] but not other tissues investigated [6]. Lucas and Trowel1 [22] found that isolated retinae of mice and rats cultured by this method were poisoned when 95 per cent or 60 per cent oxygen was used, but they survived in air. With the possible exception of the testis, none of the other organs dealt with here has shown any sign of oxygen poisoning. Organs whose survival in oxygen was unsatisfactory (liver, spleen, brain, spinal ganglion) did no better in air, usually worse. But this only shows that cultures failed for other reasons, it does not prove that they are unharmed by oxygen. It is known that among the various mammalian species, the QO, of tissue slices in vitro is inversely proportional to body weight [ 161. Mice and rats, the smallest species, have the highest QO, and are, from this point of view, the least suitable for organ culture. In the horse for example, the QO, of most organs is only half as great as in the rat [16], so it should be feasible to use cultures three times larger in volume. Presumably the whale would be better still. Work on these lines will be published later. GENERAL

CONCLUSIONS

It is possible to draw some general conclusions about the behaviour of mature organs cultured under the conditions and in the medium described here. 1. In general, connective tissue stroma survived worst, plain muscle better and epithelium best. This may be a defect of the medium, though addition of various vitamins, including ascorbic acid, did not alter the results. Richter and co-workers [32-341 found that in cultures of adult bat organs the capillaries were the first elements to die. We have been unable to decide whether the pyknosis seen in the stroma represents death of fibrocytes or of capillaries. This is in marked contrast to the extensive dedifferentiation, proliferation and outgrowth of connective tissue which occurs when organ fragments are cultured in a plasma clot by. traditional tissue culture methods. Experimental

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2. Epithelial cells closely massed with a minimum of stroma, as in parathyroid, pineal, pars intermedia of pituitary, and stratified epithelium, did better than epithelial cells in single layers or scattered groups (acini). 3. Mitosis occurred only in epithelium. It was frequently, but not constantly, seen in the ductus deferens, trachea and pars intermedia of %he pituitary, occasionally in ureter and lymph nodes, very rarely elsewhere. 6. In many organs the nucleoli of the epithelial cells enlarged progressively during the period of culture. The same occurred in the reticulum cells of lymph nodes. Nucleolar enlargement has been supposed to indicate an increased rate of protein synthesis [47], but it is doubtful if such is the case here. The nucleoli of rat liver cells enlarge (in uiuo) in certain amino acid deficiencies [44] and this would seem to be more relevant to our findings. .5* Explants naturally thin and flat or which flatten out on culture, such as prostate, mammary gland, pituitary, lymph nodes and adipose tissue, generally do better than those which remain spherical or cubical, such as liver, kidney, ovary and adrenal. This is probably because they are in better equilibrium with both the gas phase and the medium. In %beory, from the standpoint of diffusion of oxygen and metabolites, thin organ slices should be better than cubes or spheres of the same volume. But the trauma of cutting them is very much greater and for this reason they are useless for organ culture ecprate for 2-hour biochemical experiments. 6. The attempts to culture brain, liver, thymus, spleen, bone marrow and testis must be counted unsuccessful. The cause was not discovered, but oxygen poisoning and any simple deficiency of the medium seemed to be excluded. It is likely that trauma was a major factor. 7. The best results were obtained with the ductus deferens, pituitary, pineal, parathyroid and prostate, all of which regularly survived for at least 9 days in a remarkably normal state of histological preservation. The aim of this work was to maintain mature organs in as near normal a state as possible, judged histologically. There was, in most cases, no growth, differentiation or dedifferentiation; indeed any such would be “abnormal”and foreign to the physiological and biochemical purposes ultimately in mind. is the right term to use for this work, It may be questioned if organ “culture” for culture implies some connotation of growth and multiplication. “‘Maintenance” or “conservation” would be more correct, but perhaps less informative. The method should be suitable for various short-term experimen the action of hormones, drugs, radiations, etc., and also for metabolic st It is hoped that this general account will define more clearly its possibilities and its limitations. 10 - 593701

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0. A. Trowel1

146

.A culture method is described by which mature organs, or parts thereof, from rats and mice may be maintai:iied in vitro in a simple synthetic medium for the purpose of short term exg,eriments. The following organs could regularly be kept in a satisfactory sts?e of histological preservation for 6-9 days: ureter, ductus deferens, uterus, trachea3 arteries, salivary glands, mammary gland, prostate, seminal vesicle, J.r:~ng,thyroid, parathyroid, pituitary, pineal, ovary, skin, white adipose tissuer lymph nodes, symphathetic ganglia. Partial survival was obtained in the case af kidney, adrenal and spinal ganglion. The survival of brain, liver, thynmusj spleen, bone marrow, testis and pancreas was unsatisfactory. I am indebted to my chief technician, Mr. W. R. Lush, for several suggestions which have been incorporated in the technique. Most of the culture work was done by Mr. W. R. Lush and Miss E, Feakman, working in tandem. They also made all the histological preparations. Miss Mu Tebbutt was responsible for ‘the conscientious preparatory work which the method demands. The aluminium chambers were made in the workshop of this Unit, under direction of Mr. C. F. Wright. REFERENCES 1. CARLSON, T., J. Am. Chem. Sot. 33: 1324 (1911). 2. CHEN, J. M., Exptl. Cell Research 7, 5~1.8(1954). 3. CHESTERMAN, W. and LEACH, E. H., Quart. J. Microscop. Sci. 97, 593 (1956). 4. COWARD, R.. Bull. biol. France et Beta. 77. 120 (1943). 5. DANIEL, %. $f. and PRICHARD, M. M. L., J. Physiol. i33, 4P (1956). 6. DICKENS, F., Biochem. J. 40, 145 (1946). 7. EAGLE, H., OYAMA, V. I. and IZVY, Fkf., Arch. Biochem. Biophys. 67, 432 (1957). 8: EAGLE, H., OYAMA, V. I., LEFY; M. and FREEMAN, A. E., J. Biol. Chem. 226, 191 (1957). 9. FELL, H. B. and ROBISON, 10.~ GAJLLARD, P. J., Actualitb (1942).

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1

8,

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Experimenfal

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