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Culture of the mature hamster adrenal
Experimental
274
CULTURE
OFTHEMATURE
P. A. KENDALL, Departments
HAMSTER
G. R. ZIMMERMAN
of Physiology
Cell Research 21, 274-278 (1960)
ADRENAL1
and G. E. FOLK,
and Pathology, College of Medicine, Iowa City, Iowa, U.S.A.
State University
JR. of Iowa,
Received September 10, 1959
MAINTIIKANCE of an isolated
endocrine gland by the technique of organ culture offers a unique opportunity to study secretory processes and allied biochemical and cellular changes, under controlled environmental conditions. Attempts have been made to culture intact adrenals of the rat [3, X] and bat [4], but these have been attended by early degeneration of the zona fasciculata. This note reports maintenance of the adrenal of the adult hamster, and results to date indicate that preservation of all parts of the structure appears to be considerably more feasible in this species than in those hitherto investigated. The histological effects of adding ACTH to the nutrient were also investigated, and our findings differ once again from those previously recorded for the rat. Preliminary experiments have already been described in detail, together with the development of the procedure now in use (21. MATERIALS
AND
METHODS
The culture technique as finally developed is a modification of Trowell’s [7] original lens paper method, substituting for the metal grid a piece of sponge, through which a 0.5 cm hole has been bored vertically. Each piece was approximately 0.1 cm x 2 cm x 0.4 cm thick (fine pore, cosmetic, cellulose sponge, Du Pont Co.) The sponge is completely covered by nutrient in the culture vessel (in our apparatus, 10 ml of nutrient are required) before placing a strip of lens paper to cover the hole. Excess nutrient can then be drawn off from beside the sponge, the lens paper acting to seal the hole and maintain a continuous column of liquid in it. An organ placed on the paper over the hole thus has optimum contact with both gaseous and liquid phases, and cannot sink. Moreover, the level of nutrient outside the sponge is clearly not critical. Three ml are arbitrarily removed, leaving a final volume of 7 ml. Culturing is in 12 in. circular dishes, equipped with side arm closed by a rubber sleeve. The cover is sealed on with vacuum grease, leaving a volume of air of about 5 ml around the organ. Our nutrient, designated RLY +lO per cent HS, consists of 10 per cent horse serum and 90 per cent of a solution of the following composition: 0.74 per cent NaCl; 0.06 per cent KCl; 0.02 per cent CaCl,; 0.02 per cent KH,PO,; 0.02 per cent MgSO, * 7H,O; 0.268 per cent Na,HPO, * 7H,O; 0.4 per cent glucose; 0.5 per cent 1 Supported by a grant from the U.S. National Professor J. T. Bradbury for his careful criticism Experimental
Ceil Research 21
Science Foundation. of this manuscript.
The authors are grateful
to
Culture of hamster adrenal
275
lactalbuminhydrolysate; 0.1 per cent yeast extract; 200 units/ml penicillin; 200 pg/ml streptomycin. (The disodium phosphate is introduced in the form of a 2.68 per cent solution containing hydrochloric acid to bring the pH to 7.4). Control experiments showed that the phosphate buffer maintained the system at a pH of 7.1-7.2. Adrenals were taken from male hamsters (90-120 g) which had been killed by decapitation. Where no ACTH was to be added to the medium, the complete culturing assembly, with the final 7 ml of nutrient, was prepared beforehand, leaving only the lid to be sealed once the organ was in place. Each organ was cultured in a separate dish. In many experiments organs were weighed before culturing, in which case they were first allowed to equilibrate with the nutrient for ten minutes in the unsealed vessel. Where ACTH was to be included, a slightly different procedure was adopted in order to minimize premature inactivation of ACTH, by principles possibly present in the horse serum (cf. findings of Pincus, Hopkins and Hechter [3]). This insured that the organ was subjected to the full nominal dose of ACTH at least initially. To this end, ACTH (Park, Davis and Co.) was dissolved in Tyrode’s solution in such a quantity that 0.2 ml of this solution, when diluted to IO ml with nutrient, would give the required concentration of ACTH. The culture vessel was prepared containing only the sponge block and 0.2 ml ACTH-Tyrode solution. The cleaned organ was placed aside in fresh Tyrode or Ringer’s solution, while 10 ml of nutrient were added to the ACTH, after which the organ was transferred at once to the culture dish and placed in the nutrient beside the sponge. The lens paper was then set in place, and the organ was lifted to its usual position, before extracting the excess nutrient and proceeding as previously. Nutrient was replenished once daily, where culturing was to be continued, by exchanging about half of it for fresh medium. This was accomplished by tilting the dish and extracting by syringe as much liquid as possible (3-3.5 ml) through the sidearm without disturbing the sponge block; at the same time a needle was inserted attached to a long, sterile, cotton wool-filled gassing-tube, to avoid a partial vacuum and to introduce fresh air. When replenishing medium containing ACTH, ACTHTyrode was added to give the same concentration of fresh ACTH, with respect to in the particular culture. the amount of fresh nutrient, as was used initially Adrenals were cultured at 37°C for periods of 1-5 days, and the effect of ACTH was investigated at concentrations of zero, 20, 100, 500 and 2000 I.U. per litre of nutrient. Many organs were weighed at the beginning and end of the culture period, but none, in these recent experiments, were removed during culture for weighing as in the earlier series. Organs were fixed in Bouin’s solution, and stained with hematoxylineosin. Freshly excised adrenals were used as controls to check fixation and staining procedure. RESULTS
In absence of ACTH, cellular integrity was usually good after three days’ maintenance, although fasciculata cells stained less intensely than in sections of fresh tissue and there was enlargement of intercellular spaces (compare Figs. 1 and %). \Vhere areas of necrosis occurred, there vvas no emphasis on any one zone, and healthy and degenerate regions would he seen side by Experimenfal
Cell Research 21
276
P. A. Kendall, G. K. Zimmerman and G. E. Folk, Jr.
Figs. I-6.--Sections of hamster adrenals, fixed in Bouin’s solution, stained with hcmaloxylin eosin. Fig. I.-Freshly excised (control). Fig. 2.- Cultured 3 days without added ACTH. Good preservation, enlargement of intercellular spaces. Fig. 3. --Cultured 5 days without added ACTH. Pretlominantly healthy. Fig. 4:-Culturccl 1 day, with ACTH in nutrient, concentration 500 I.U./liLre. Note subcapsular vacuolization. Fig. 5.-A Fig. 1, I,ow power. Note extent of subcapsular vacuolization, and accentuation of fasciculata cords. Fig. fi.PCulturetl 3 days, with ACTH in nutrient, concentration 500 I.U./litre. Only a trace of subcapsular vacuolization (cf. Fig. 5).
side. After four or five days in rdtro, the appearance of the tissue was qualitatively similar, and although there were large patches of extensive necrosis, many areas reaching from periphery to centre of the cortex appeared to he healthy (Fig. 3). The influence of AC’I’H was studied hvith nine adrenals. Three adrenals were maintained in nutrient containing ACTH for one day, three t\vo days, anti three three days; there \vere three dose levels of AC’I’H (‘LO, 100, 500 I.U. per litrc of nutrient). In this experiment there were three controls, \vhich were given the ‘l’yrode hath before transferring to nutrient containing no AC’I’H, where they were maintained one, two, anti three days respectively. The most striking efyect of AC’I’H was in the suhcapsular area of the zona glomcrulosa \vhere there was widespread nuclear pyknosis and marked atrophy Experimenfal
Cell Hesearch
21
Culture of hamster adrenal
278
P. A. Kendall, G. K. Zimmerman anp G. E. Folk, Jr.
of some cells and cytoplasmic vacuolization of others (Figs. 4 and 5). There was also some shrinkage in the fasciculata region and accentuation of the cellcords, but no cytoplasmic vacuolization of the type described by Schaberg [6]. These changes, which were more outstanding after one day (Fig. 5) than after two days and only just visible in organs fixed at three days (Fig. 6), were noticeable at concentrations of ACTH of ‘20 I.lT./litre, hut were most pronounced at 500 I.V./litre, the highest concentration used in this group. =\CTH had no observable histological ell‘ect after more than three days of culture. In no case where ACTH was absent was any subcapsular vacuolization observed. There \vas little obvious correlation betlveen changes of Tveight and histological integrity, most organs tending to remain at 80 per cent of their initial \veight after more than three days in vitro. DISCUSSION
Since healthy areas were present in all zones of the cortex for at least five days, suitable modification of the technique should, in theory, enable the entire cortex of an adrenal gland to be maintained. Since histological appearance alone is an insufficient criterion for the assessment of viability of the tissue, experiments are in progress in an attempt to analyze for ketosteroids in nutrient and gland after culturing. In comparing our observations with those of Schaberg [C;! concerning the action of AC’I’H, it should be noted that the volume of the medium was larger than that used in his technique, and in the present experiments the pH was maintained more consistently (7.1 to 7.2) than in his studies. Also it should be noted that the hamster adrenal differs somewhat from that of the rat in its reaction to ACTH in uivo, due to absence of deposited cholesterol in the hamster organ [ 11. ‘I’herc is relatively little cytological dil‘ference between glomerulosa and fasciculata cells in the latter species (Fig. 1). In summary, perhaps the success in maintaining hamster adrenals for 3-3 days is attributable to species difference, which might show itself to advantage in attempts to culture other hamster organs. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8.
ALPERT, M., Endocrinology 46, 166 (1950). KENDALL, P. A., MELTZER, M. R. and ZIMMERMAN, G. Ii., Proc. Iowa Acad. Sci. 66,414 (1959). PINCUS, G. H., HOPKINS, T. F. and HECHTER, O., Arch. Biochem. Biophys. 37, 408 (1952). RICHTER, I<. M., RITCHESOX, B. R. and CLOUD, S., Anat. Xecord 128, 607 (1957). SCHABERG, A., Anat. Record 122, 205 (1955). __ hoc. Koninkl. Ned. Akad. Wetenschup. 60, 463 (1957). TROWELL, 0. A., Ezptl. Cell Research. 6, 246 (1954). __ ibid. 16, 118 (1959).
Experimental
Cell Research 21
274
CULTURE
OFTHEMATURE
P. A. KENDALL, Departments
HAMSTER
G. R. ZIMMERMAN
of Physiology
Cell Research 21, 274-278 (1960)
ADRENAL1
and G. E. FOLK,
and Pathology, College of Medicine, Iowa City, Iowa, U.S.A.
State University
JR. of Iowa,
Received September 10, 1959
MAINTIIKANCE of an isolated
endocrine gland by the technique of organ culture offers a unique opportunity to study secretory processes and allied biochemical and cellular changes, under controlled environmental conditions. Attempts have been made to culture intact adrenals of the rat [3, X] and bat [4], but these have been attended by early degeneration of the zona fasciculata. This note reports maintenance of the adrenal of the adult hamster, and results to date indicate that preservation of all parts of the structure appears to be considerably more feasible in this species than in those hitherto investigated. The histological effects of adding ACTH to the nutrient were also investigated, and our findings differ once again from those previously recorded for the rat. Preliminary experiments have already been described in detail, together with the development of the procedure now in use (21. MATERIALS
AND
METHODS
The culture technique as finally developed is a modification of Trowell’s [7] original lens paper method, substituting for the metal grid a piece of sponge, through which a 0.5 cm hole has been bored vertically. Each piece was approximately 0.1 cm x 2 cm x 0.4 cm thick (fine pore, cosmetic, cellulose sponge, Du Pont Co.) The sponge is completely covered by nutrient in the culture vessel (in our apparatus, 10 ml of nutrient are required) before placing a strip of lens paper to cover the hole. Excess nutrient can then be drawn off from beside the sponge, the lens paper acting to seal the hole and maintain a continuous column of liquid in it. An organ placed on the paper over the hole thus has optimum contact with both gaseous and liquid phases, and cannot sink. Moreover, the level of nutrient outside the sponge is clearly not critical. Three ml are arbitrarily removed, leaving a final volume of 7 ml. Culturing is in 12 in. circular dishes, equipped with side arm closed by a rubber sleeve. The cover is sealed on with vacuum grease, leaving a volume of air of about 5 ml around the organ. Our nutrient, designated RLY +lO per cent HS, consists of 10 per cent horse serum and 90 per cent of a solution of the following composition: 0.74 per cent NaCl; 0.06 per cent KCl; 0.02 per cent CaCl,; 0.02 per cent KH,PO,; 0.02 per cent MgSO, * 7H,O; 0.268 per cent Na,HPO, * 7H,O; 0.4 per cent glucose; 0.5 per cent 1 Supported by a grant from the U.S. National Professor J. T. Bradbury for his careful criticism Experimental
Ceil Research 21
Science Foundation. of this manuscript.
The authors are grateful
to
Culture of hamster adrenal
275
lactalbuminhydrolysate; 0.1 per cent yeast extract; 200 units/ml penicillin; 200 pg/ml streptomycin. (The disodium phosphate is introduced in the form of a 2.68 per cent solution containing hydrochloric acid to bring the pH to 7.4). Control experiments showed that the phosphate buffer maintained the system at a pH of 7.1-7.2. Adrenals were taken from male hamsters (90-120 g) which had been killed by decapitation. Where no ACTH was to be added to the medium, the complete culturing assembly, with the final 7 ml of nutrient, was prepared beforehand, leaving only the lid to be sealed once the organ was in place. Each organ was cultured in a separate dish. In many experiments organs were weighed before culturing, in which case they were first allowed to equilibrate with the nutrient for ten minutes in the unsealed vessel. Where ACTH was to be included, a slightly different procedure was adopted in order to minimize premature inactivation of ACTH, by principles possibly present in the horse serum (cf. findings of Pincus, Hopkins and Hechter [3]). This insured that the organ was subjected to the full nominal dose of ACTH at least initially. To this end, ACTH (Park, Davis and Co.) was dissolved in Tyrode’s solution in such a quantity that 0.2 ml of this solution, when diluted to IO ml with nutrient, would give the required concentration of ACTH. The culture vessel was prepared containing only the sponge block and 0.2 ml ACTH-Tyrode solution. The cleaned organ was placed aside in fresh Tyrode or Ringer’s solution, while 10 ml of nutrient were added to the ACTH, after which the organ was transferred at once to the culture dish and placed in the nutrient beside the sponge. The lens paper was then set in place, and the organ was lifted to its usual position, before extracting the excess nutrient and proceeding as previously. Nutrient was replenished once daily, where culturing was to be continued, by exchanging about half of it for fresh medium. This was accomplished by tilting the dish and extracting by syringe as much liquid as possible (3-3.5 ml) through the sidearm without disturbing the sponge block; at the same time a needle was inserted attached to a long, sterile, cotton wool-filled gassing-tube, to avoid a partial vacuum and to introduce fresh air. When replenishing medium containing ACTH, ACTHTyrode was added to give the same concentration of fresh ACTH, with respect to in the particular culture. the amount of fresh nutrient, as was used initially Adrenals were cultured at 37°C for periods of 1-5 days, and the effect of ACTH was investigated at concentrations of zero, 20, 100, 500 and 2000 I.U. per litre of nutrient. Many organs were weighed at the beginning and end of the culture period, but none, in these recent experiments, were removed during culture for weighing as in the earlier series. Organs were fixed in Bouin’s solution, and stained with hematoxylineosin. Freshly excised adrenals were used as controls to check fixation and staining procedure. RESULTS
In absence of ACTH, cellular integrity was usually good after three days’ maintenance, although fasciculata cells stained less intensely than in sections of fresh tissue and there was enlargement of intercellular spaces (compare Figs. 1 and %). \Vhere areas of necrosis occurred, there vvas no emphasis on any one zone, and healthy and degenerate regions would he seen side by Experimenfal
Cell Research 21
276
P. A. Kendall, G. K. Zimmerman and G. E. Folk, Jr.
Figs. I-6.--Sections of hamster adrenals, fixed in Bouin’s solution, stained with hcmaloxylin eosin. Fig. I.-Freshly excised (control). Fig. 2.- Cultured 3 days without added ACTH. Good preservation, enlargement of intercellular spaces. Fig. 3. --Cultured 5 days without added ACTH. Pretlominantly healthy. Fig. 4:-Culturccl 1 day, with ACTH in nutrient, concentration 500 I.U./liLre. Note subcapsular vacuolization. Fig. 5.-A Fig. 1, I,ow power. Note extent of subcapsular vacuolization, and accentuation of fasciculata cords. Fig. fi.PCulturetl 3 days, with ACTH in nutrient, concentration 500 I.U./litre. Only a trace of subcapsular vacuolization (cf. Fig. 5).
side. After four or five days in rdtro, the appearance of the tissue was qualitatively similar, and although there were large patches of extensive necrosis, many areas reaching from periphery to centre of the cortex appeared to he healthy (Fig. 3). The influence of AC’I’H was studied hvith nine adrenals. Three adrenals were maintained in nutrient containing ACTH for one day, three t\vo days, anti three three days; there \vere three dose levels of AC’I’H (‘LO, 100, 500 I.U. per litrc of nutrient). In this experiment there were three controls, \vhich were given the ‘l’yrode hath before transferring to nutrient containing no AC’I’H, where they were maintained one, two, anti three days respectively. The most striking efyect of AC’I’H was in the suhcapsular area of the zona glomcrulosa \vhere there was widespread nuclear pyknosis and marked atrophy Experimenfal
Cell Hesearch
21
Culture of hamster adrenal
278
P. A. Kendall, G. K. Zimmerman anp G. E. Folk, Jr.
of some cells and cytoplasmic vacuolization of others (Figs. 4 and 5). There was also some shrinkage in the fasciculata region and accentuation of the cellcords, but no cytoplasmic vacuolization of the type described by Schaberg [6]. These changes, which were more outstanding after one day (Fig. 5) than after two days and only just visible in organs fixed at three days (Fig. 6), were noticeable at concentrations of ACTH of ‘20 I.lT./litre, hut were most pronounced at 500 I.V./litre, the highest concentration used in this group. =\CTH had no observable histological ell‘ect after more than three days of culture. In no case where ACTH was absent was any subcapsular vacuolization observed. There \vas little obvious correlation betlveen changes of Tveight and histological integrity, most organs tending to remain at 80 per cent of their initial \veight after more than three days in vitro. DISCUSSION
Since healthy areas were present in all zones of the cortex for at least five days, suitable modification of the technique should, in theory, enable the entire cortex of an adrenal gland to be maintained. Since histological appearance alone is an insufficient criterion for the assessment of viability of the tissue, experiments are in progress in an attempt to analyze for ketosteroids in nutrient and gland after culturing. In comparing our observations with those of Schaberg [C;! concerning the action of AC’I’H, it should be noted that the volume of the medium was larger than that used in his technique, and in the present experiments the pH was maintained more consistently (7.1 to 7.2) than in his studies. Also it should be noted that the hamster adrenal differs somewhat from that of the rat in its reaction to ACTH in uivo, due to absence of deposited cholesterol in the hamster organ [ 11. ‘I’herc is relatively little cytological dil‘ference between glomerulosa and fasciculata cells in the latter species (Fig. 1). In summary, perhaps the success in maintaining hamster adrenals for 3-3 days is attributable to species difference, which might show itself to advantage in attempts to culture other hamster organs. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8.
ALPERT, M., Endocrinology 46, 166 (1950). KENDALL, P. A., MELTZER, M. R. and ZIMMERMAN, G. Ii., Proc. Iowa Acad. Sci. 66,414 (1959). PINCUS, G. H., HOPKINS, T. F. and HECHTER, O., Arch. Biochem. Biophys. 37, 408 (1952). RICHTER, I<. M., RITCHESOX, B. R. and CLOUD, S., Anat. Xecord 128, 607 (1957). SCHABERG, A., Anat. Record 122, 205 (1955). __ hoc. Koninkl. Ned. Akad. Wetenschup. 60, 463 (1957). TROWELL, 0. A., Ezptl. Cell Research. 6, 246 (1954). __ ibid. 16, 118 (1959).
Experimental
Cell Research 21