398
ENDOCRINE
AND REPRODUCTIVE
TISSUE
[34]
[34] P i n e a l G l a n d O r g a n C u l t u r e T e c h n i q u e s B y HARVEY M. SHEIN
Organ culture techniques which maintain intact mammalian or avian pineal glands for 24 hours or longer after explantation permit a wide range of biochemical studies to be carried out on these glands in vitro. 1-~ Organ Culture Methods Principles. Two different techniques of pineal organ culture have been employed. Method 1: The gland is clotted to the wall of a culture tube and incubated with nutrient medium on a slowly turning roller wheel in an air atmosphere at 37 ° with the culture tube sealed. Method 2: The gland is incubated at 37 ° in a stationary position on a platform at the interface between the nutrient medium and a continuous, watersaturated, gassing atmosphere of 95% 02 and 5% C02. The plasma clotroller wheel technique can be said to be more "physiological" in that it employs an air atmosphere; however, the continuously replenished enriched 02 atmosphere technique prevents the development of central necrosis in the cultured gland, which occurs reproducibly when the gland is cultured in an air atmosphere. The small volume of central necrosis which develops with the air atmosphere method is sufficiently reproducible among pineals prepared from inbred rats of the same strain, age, sex and weight so that the variation in synthesis of radiolabeled indoles and protein among a group of six to ten cultured pineal glands remains within a standard error of 15% of the means of the totals during 2 days of incubation. Nevertheless, the absence of necrosis with method 2 makes this technique preferable for most biochemical studies. Method 1
This technique has been used to maintain intact rat pineal glands in organ culture for periods up to 4 days. 6 1j. Axelrod, H. M. Shein, and R. J. Wurtman, Proc. Nat. Acad. Sci. U.S. 62, 544 (1969). 2H. M. Shein and R. J. Wurtman, Science 166, 519 (1969). D. C. Klein, G. R. Berg, and J. Weller, Science 168, 979 (1970). I. T. Lott, R. H. Quarles, and D. C. Klein, Biochim. Biophys. Acta 264, 144 (1972). s j. M. Rosner, G. Deelerq de Perez Bedes, and D. P. Cardinali, Li]e Sci. I0, 1065 (1971). 6H. M. Shein, R. J. Wurtman, and J. Axelrod, Nature (London) 213, 730 (1967).
[34]
PINEAL GLAND ORGAN CULTURE TECHNIQUES
399
TABLE I COMPOSITION OF N16 MEDIUMa Component
mg/liter
Component
mg/liter
NaCI KC1 Na2HPO4 • 7H20 MgSO4 • 7H20 CaCl_~. 2H20 KH~PO4 Glucose L-Arginine HCI I~-}tistidine HC1 L-Lysine HC1 L-Tryptophan B-Phenyl-L-alanine L-Methionine L-Threonine L-Leucine DL-Isoleucine DL-Valine L-Glutamic acid
7400.00 285.00 290.00 154.00 16.00 83.00 1100.00 37.50 37.50 80.00 20.00 25.00 25.00 37.50 25.00 25.00 50.00 75.00
L-Aspartic acid irProline Glycine L-Glutamine L-Tyrosine L-Cystine Hypoxanthine Thiamine • HCI Riboflavin Pyridoxine • ttCl Folic acid Biotin Choline Ca pantothenate Niacinamide i-Inositol NaHCO3
30.00 25.00 100.00 200.00 40.00 7.50 25.00 5.00 0.50 0.50 0. l0 0. l0 3.00 3.00 3.00 1.00 1200.00
T. T. Puck, S. J. Cieciura, and A. Robinson, J. Exp. Med. 108, 945 (1958).
Reagents Hanks' balanced salt solution Chicken plasma 7 C h i c k e m b r y o e x t r a c t (50% v / v in isotonic saline) 7 N16 c u l t u r e m e d i u m ( c o m p o s i t i o n in T a b l e I I ) N C T C - 1 3 5 c u l t u r e m e d i u m ( c o m p o s i t i o n in T a b l e I I ) L a c t a l b u m i n h y d r o l y z a t e ( 5 % ) in E a r l e ' s b a l a n c e d s a l t solution F e t a l calf serum, h e a t - i n a c t i v a t e d a t 56 ° for 30 m i n u t e s H o r s e serum, h e a t - i n a c t i v a t e d a t 56 ° for 30 m i n u t e s Penicillin G Streptomycin Amphotericin B Procedure. A l l steps a r e c a r r i e d o u t u n d e r a s e p t i c c o n d i t i o n s to m a i n t a i n s t e r i l i t y of t h e p i n e a l glands, t h e c u l t u r e vessels a n d t h e i r contents. R a t p i n e a l glands a r e r e m o v e d b y sterile dissection following d e c a p i t a t i o n a n d p l a c e d in a p e t r i dish c o n t a i n i n g H a n k s ' b a l a n c e d s a l t solution. E a c h i n t a c t p i n e a l g l a n d is t h e n c l o t t e d to t h e glass w a l l of a ~Prepared as described in R. C. Parker, "Methods of Tissue Culture," 3rd cd., Chapter 10. Harper (Hoeber), New York, 1961.
400
E N D O C R I N E AND REPRODUCTIVE TISSUE
[34]
TABLE II COMPOSITION OF NCTC-135 MEDIUM a
Component NaC1 KC1 CaCl~ (anhyd.) MgSO4 NaH2PO4 - H20 D-Glucose L-Alanine L-a-Amino-n-butyric acid L-Arginine HCI L-Asparagine • H~O L-Aspartic acid L-Cystine D-Glucosamine HC1 i-Glutamic acid L-Glutamine Glycine L-Histidine HC1 • H~O Hydroxy-L-proline L-Isoleucine L-Leucine L-Lycine HC1 L-Methionine L-Ornithine HCI ~Phenylalanine L-Proline L-Serine L-Taurine L-Threonine L-Tryptophan L-Tyrosine L-Valine Thiamine • HCI Riboflavin Pyridoxine - HCI Pyridoxal • HC1
mg/liter 6800.0000 400.0000 200. 0000 100. 0000 140. 0000 1000. 0000 31. 5000 5. 5000 31. 2000 9. 2000 9. 9000 10. 5000 3. 9000 8. 3000 135. 7000 13. 5000 26. 7000 4. 1000 18. 0000 20. 4000 38. 4000 4. 4000 9. 4000 16. 5000 6. 1000 10. 8000 4. 2000 18. 9000 17. 5000 16. 4000 25. 0000 0. 0250 0. 0250 0. 0625 0. 0625
Component Niacin Niacinamide D-Ca pantothenate Biotin Folic acid Choline CI Vitamin BI~ i-Inositol p-Aminobenzoic acid Vitamin A Calciferol Menadione Disodium ~-tocopherol phosphate Sodium glutathione Ascorbic acid Diphosphopyridine nucleotide Triphosphopyridine nucleotide (monosodium) Coenzyme A Cocarboxylase Flavin adenine dinucleotide Sodium uridine triphosphate Deoxyadenosine Deoxyguanosine Thymidine 5-Methylcytosine Tween 80 n-Glucuronolactone Sodium glucuronate • H~O Sodium acetate. 3H~0 Ethanol • for solubilizing lipid components Phenol red NaHCO3
mg/liter 0. 0625 0. 0625 0. 0250 0. 0250 0. 0250 1. 2500 10. 0000 0. 1250 0. 1250 0. 2500 0. 2500 0. 0250 0. 0250 10. 0000 50. 0000 7. 0000 1. 0000 2. 5000 1. 0000 1.0000 1. 0000 10. 0000 10. 0000 10. 0000 0. 1000 12. 5000 1. 8000 1. 8000 50. 0000 40. 0000 20. 0000 2200. 0000
V. J. Evans, J. C. Bryant, H. A. Kerr, and E. L. Schilling, Exp. Cell Res. 86, 439 (1964).
W a s s e r m a n tube, w h i c h has been c o a t e d p r e v i o u s l y w i t h a t h i n film of c h i c k e n p l a s m a (dispensed f r o m a P a s t e u r p i p e t t e ) by t h e a p p l i c a t i o n of a drop or t wo of chick e m b r y o e x t r a c t (dispensed f r o m a P a s t e u r
[34]
P I N E A L GLAND ORGAN CULTURE T E C H N I Q U E S
401
pipette)2 ,r Fifteen minutes later, when the clot is sufficiently firm, 0.5 ml of complete nutrient medium (see below) is added to the culture. The culture tube is then sealed with a rubber stopper and incubated in a standard tissue culture tube roller wheel at 37°C. ';'7 The complete nutrient medium ~ consists of 75.5% N16 medium, 4.0% NCTC-135 medium, 10.0% heat-inactivated fetal calf serum; 10.0% heat-inactivated horse serum, 0.5% of 5% lactalbumin hydrolyzate (Earle's), penicillin G (50 units per milliliter of medium), streptomycin sulfate (50 t*g per milliliter of medium), and amphotericin B (1 /,g per milliliter of medium). All the constituents of the complete nutrent medium, as well as the chick embryo extract, the chicken plasma, and the Hanks' BSS, can be obtained as standard preparations from Grand Island Biological Co., Grand Island, New York. For studies in which serotonin is to be assayed by spectrophotometric techniques, the media are prepared without phenol red, because this indicator dye interferes with the assay.
Method 2 Stationary Incubation at Interlace o] the Nutrient Medium and an Enriched 02 Atmosphere. This technique has been used to maintain intact rat pineal glands in culture without evidence of necrosis for periods up to 6 days? ,1° Reagents Modified B G J b culture medium (composition in Table III) Bovine serum albumin, Fraction V Procedure. All steps are carried out under aseptic conditions to mainrain sterility of the pineal glands, the culture vessels, and their contents. Pineal glands are obtained by sterile dissection following decapitation. One or two intact pineal glands are incubated on a stainless steel screen (see below) in a specially designed culture vessel (see below) at 37 ° in the presence of 0.5 ml of chemically defined, modified B G J b culture medium (Table I I I ) supplemented with fraction V bovine serum albumin °,~1,1-~ (Armour Pharmaceutical Co.) at a final concentration in the complete medium of 1 mg/ml, and are continually gassed with a mixture G. Yerganian, H. M. Shein, and J. F. Enders, Cytogenetics 1, 314 (1962). D. C. Klein and J. Weller, In Vitro 6, 197 (1970). This paper provides a sketch of the culture vessel with a pineal gland in place. ~*R. B. Berg and D. C. Klein, Endocrinology 89, 453 (1971). ~1L. G. Raisz, J. Clin. Invest. 44, 103 (1965). 12L. G. Raisz and I. Niemann, Endocrinology 85, 446 (1969).
402
ENDOCRINE AND REPRODUCTIVE TISSUE
[34]
TABLE III COMPOSITION OF MODIFIED BGJb MEDIUMa Component Calcium lactate Dihydrogen sodium orthophosphate Glucose Magnesium sulfate- 7H20 Potassium chloride Potassium dihydrogen phosphate Sodium bicarbonate Sodium chloride L-Lysine i-Histidine L-Arginine L-Threonine DL-Valine L-Leucine L-Isoleucine L-Methionine L-Phenylalanine i-Tryptophan L-Tyrosine L-Cysteine HCI L-Glutamine
mg/liter
Component
555.00 L-Alanine 90.00 L-Aspartic acid Glycine 10,000.00 L-Proline 200.00 L-Serine 400.00 Nicotinamide 160.00 Thiamine hydrochloride Calcium pantothenate 3,500.00 Riboflavin 5,300.00 Pyridoxal phosphate 240.00 Folic acid 150.00 Biotin 175.00 p-Aminobenzoic acid 75.00 a-Tocopherol phosphate 65.00 Choline chloride 50.00 Inositol 30.00 Vitamin B~2 50.00 Sodium acetate 50.00 Ascorbic acid 40.00 Phenol red 40.00 Streptomycin 90.00 Penicillin 200.00
mg/liter 250.00 150.00 800.00 400.00 200.00 20.00 4.00 0.20 0.20 0.20 0.20 0.20 2.00 1.00 50.00 0.20 0.04 50.00 50.00 20.00 50.00 100,000 units
a L. G. Raisz and I. Niemann, Endocrinology $5, 446 (1969).
of 95% oxygen and 5% carbon dioxide (v/v) in a water-saturated atmosphere. TM The stainless steel screen maintains the culture at the interface of the medium and the gassing atmosphere. The culture vessel is a U.S. Bureau of Plant Industry watch glass (The A. H. Thomas Comp a n y ) , fitted with a 1.5 mm mesh size stainless steel expanded screen (The Expanded Metal Company, Stranton Works, Hartlepool, England) platform fabricated in a machine shop to a 1.7 cm diameter with a curled edge 1 mm deep2 To maintain a water-saturated atmosphere during incubation, up to seven of the culture vessels are placed on a 9-cm disc of W h a t m a n No. 3M paper, previously soaked with 3 ml of incubation medium, in a 10-ml petri dish. Each petri dish is then incubated on one of five shelves (spaced 1 inch apart), in a small gas-tight Lucite chamber (with space for a total of 20 petri dishes), fabricated to these specifications in a machine shop, which is perfused continuously (0.3 liter per minute) with the gassing mixture2 ,1°
[35]
02 MICROTECHNIQUES WITH ISOLATED CELLS
403
Intact pineal glands from lO-month-old domestic ducks have also been maintained in organ culture for 24 hours without necrosis by the technique (Method 2) described above, with the modifications that each gland is cultured individually, and a different nutrient medium; i.e., medium 199 '3 is employed at a slightly higher temperature, 38°C. '~ '~J. F. Morgan, H. J. Morgan. and R. C. Parker, Proc. Soc. Exp. Biol. Med. 73, 1 (1950).
[35] U s e o f 0 2 M i c r o t e c h n i q u e s f o r A s s e s s m e n t o f Hormone Effects on Isolated Endocrine Cells B y LARS HAMBERGER, HANS HERLITZ~ and t~AGNAR HULTBORN
Methods for microgasometric measurements in biological material have been used and developed during the last four decades. In 1937 Linderstr0m-Lang introduced the Cartesian diver principle for this purpose. 1 The sensitivity of the original device was in the order of 10-~ to 10--" ~l per hour, which means a considerable increase in sensitivity compared to the Warburg technique. ~ In 1953 another Danish scientist 3 introduced the ampulla or microdiver respirometer with which cellular respiration could be determined in the order of 10-~ to l0 -~ ~l per hour2 An even more sensitive technique, the magnetic diver microgasometer, was introduced in 1964 * and recently further developed2 This technique gives reliable measurements in the magnitude of 10-6 to 10-; tL1 per hour, specifically interesting when taking into consideration that single tissue cells respire with intensities approximately 10-6 ~l per hour. One essential problem in this connection is concerned with procedures for alteration of the medium composition during an experiment. Numerous ingenious arrangements for addition of reactants and inhibitors to the incubation medium have been described for the Warburg technique. ~ However, in microscale this problem is even more intricate. Some of the techniques for addition of reactants and inhibitors in use for standarddivers and ampulla divers will be described in the following. I K. LinderstrCm-Lang, Nature (London) 140, 108 (1937). ~W. W. Umbreit, R. H. Burris, and J. F. Stauffer, "Manometric Techniques: A Manual Describing Methods Applicable to the Study of Tissue Metabolism." Burgess, Minneapolis, Minnesota, 1964. :~E. Zeuthen, J. Embryol. Exp. Morphol. 1, 239 (1953). ' M. Brzin and E. Zeuthen, C. R. Tray. Lab. Carlsberg 34, 427 (1964). '~S. Oman and M. Brzin, AT~d. Biochem. 45, 112 (1972).
ENDOCRINE
AND REPRODUCTIVE
TISSUE
[34]
[34] P i n e a l G l a n d O r g a n C u l t u r e T e c h n i q u e s B y HARVEY M. SHEIN
Organ culture techniques which maintain intact mammalian or avian pineal glands for 24 hours or longer after explantation permit a wide range of biochemical studies to be carried out on these glands in vitro. 1-~ Organ Culture Methods Principles. Two different techniques of pineal organ culture have been employed. Method 1: The gland is clotted to the wall of a culture tube and incubated with nutrient medium on a slowly turning roller wheel in an air atmosphere at 37 ° with the culture tube sealed. Method 2: The gland is incubated at 37 ° in a stationary position on a platform at the interface between the nutrient medium and a continuous, watersaturated, gassing atmosphere of 95% 02 and 5% C02. The plasma clotroller wheel technique can be said to be more "physiological" in that it employs an air atmosphere; however, the continuously replenished enriched 02 atmosphere technique prevents the development of central necrosis in the cultured gland, which occurs reproducibly when the gland is cultured in an air atmosphere. The small volume of central necrosis which develops with the air atmosphere method is sufficiently reproducible among pineals prepared from inbred rats of the same strain, age, sex and weight so that the variation in synthesis of radiolabeled indoles and protein among a group of six to ten cultured pineal glands remains within a standard error of 15% of the means of the totals during 2 days of incubation. Nevertheless, the absence of necrosis with method 2 makes this technique preferable for most biochemical studies. Method 1
This technique has been used to maintain intact rat pineal glands in organ culture for periods up to 4 days. 6 1j. Axelrod, H. M. Shein, and R. J. Wurtman, Proc. Nat. Acad. Sci. U.S. 62, 544 (1969). 2H. M. Shein and R. J. Wurtman, Science 166, 519 (1969). D. C. Klein, G. R. Berg, and J. Weller, Science 168, 979 (1970). I. T. Lott, R. H. Quarles, and D. C. Klein, Biochim. Biophys. Acta 264, 144 (1972). s j. M. Rosner, G. Deelerq de Perez Bedes, and D. P. Cardinali, Li]e Sci. I0, 1065 (1971). 6H. M. Shein, R. J. Wurtman, and J. Axelrod, Nature (London) 213, 730 (1967).
[34]
PINEAL GLAND ORGAN CULTURE TECHNIQUES
399
TABLE I COMPOSITION OF N16 MEDIUMa Component
mg/liter
Component
mg/liter
NaCI KC1 Na2HPO4 • 7H20 MgSO4 • 7H20 CaCl_~. 2H20 KH~PO4 Glucose L-Arginine HCI I~-}tistidine HC1 L-Lysine HC1 L-Tryptophan B-Phenyl-L-alanine L-Methionine L-Threonine L-Leucine DL-Isoleucine DL-Valine L-Glutamic acid
7400.00 285.00 290.00 154.00 16.00 83.00 1100.00 37.50 37.50 80.00 20.00 25.00 25.00 37.50 25.00 25.00 50.00 75.00
L-Aspartic acid irProline Glycine L-Glutamine L-Tyrosine L-Cystine Hypoxanthine Thiamine • HCI Riboflavin Pyridoxine • ttCl Folic acid Biotin Choline Ca pantothenate Niacinamide i-Inositol NaHCO3
30.00 25.00 100.00 200.00 40.00 7.50 25.00 5.00 0.50 0.50 0. l0 0. l0 3.00 3.00 3.00 1.00 1200.00
T. T. Puck, S. J. Cieciura, and A. Robinson, J. Exp. Med. 108, 945 (1958).
Reagents Hanks' balanced salt solution Chicken plasma 7 C h i c k e m b r y o e x t r a c t (50% v / v in isotonic saline) 7 N16 c u l t u r e m e d i u m ( c o m p o s i t i o n in T a b l e I I ) N C T C - 1 3 5 c u l t u r e m e d i u m ( c o m p o s i t i o n in T a b l e I I ) L a c t a l b u m i n h y d r o l y z a t e ( 5 % ) in E a r l e ' s b a l a n c e d s a l t solution F e t a l calf serum, h e a t - i n a c t i v a t e d a t 56 ° for 30 m i n u t e s H o r s e serum, h e a t - i n a c t i v a t e d a t 56 ° for 30 m i n u t e s Penicillin G Streptomycin Amphotericin B Procedure. A l l steps a r e c a r r i e d o u t u n d e r a s e p t i c c o n d i t i o n s to m a i n t a i n s t e r i l i t y of t h e p i n e a l glands, t h e c u l t u r e vessels a n d t h e i r contents. R a t p i n e a l glands a r e r e m o v e d b y sterile dissection following d e c a p i t a t i o n a n d p l a c e d in a p e t r i dish c o n t a i n i n g H a n k s ' b a l a n c e d s a l t solution. E a c h i n t a c t p i n e a l g l a n d is t h e n c l o t t e d to t h e glass w a l l of a ~Prepared as described in R. C. Parker, "Methods of Tissue Culture," 3rd cd., Chapter 10. Harper (Hoeber), New York, 1961.
400
E N D O C R I N E AND REPRODUCTIVE TISSUE
[34]
TABLE II COMPOSITION OF NCTC-135 MEDIUM a
Component NaC1 KC1 CaCl~ (anhyd.) MgSO4 NaH2PO4 - H20 D-Glucose L-Alanine L-a-Amino-n-butyric acid L-Arginine HCI L-Asparagine • H~O L-Aspartic acid L-Cystine D-Glucosamine HC1 i-Glutamic acid L-Glutamine Glycine L-Histidine HC1 • H~O Hydroxy-L-proline L-Isoleucine L-Leucine L-Lycine HC1 L-Methionine L-Ornithine HCI ~Phenylalanine L-Proline L-Serine L-Taurine L-Threonine L-Tryptophan L-Tyrosine L-Valine Thiamine • HCI Riboflavin Pyridoxine - HCI Pyridoxal • HC1
mg/liter 6800.0000 400.0000 200. 0000 100. 0000 140. 0000 1000. 0000 31. 5000 5. 5000 31. 2000 9. 2000 9. 9000 10. 5000 3. 9000 8. 3000 135. 7000 13. 5000 26. 7000 4. 1000 18. 0000 20. 4000 38. 4000 4. 4000 9. 4000 16. 5000 6. 1000 10. 8000 4. 2000 18. 9000 17. 5000 16. 4000 25. 0000 0. 0250 0. 0250 0. 0625 0. 0625
Component Niacin Niacinamide D-Ca pantothenate Biotin Folic acid Choline CI Vitamin BI~ i-Inositol p-Aminobenzoic acid Vitamin A Calciferol Menadione Disodium ~-tocopherol phosphate Sodium glutathione Ascorbic acid Diphosphopyridine nucleotide Triphosphopyridine nucleotide (monosodium) Coenzyme A Cocarboxylase Flavin adenine dinucleotide Sodium uridine triphosphate Deoxyadenosine Deoxyguanosine Thymidine 5-Methylcytosine Tween 80 n-Glucuronolactone Sodium glucuronate • H~O Sodium acetate. 3H~0 Ethanol • for solubilizing lipid components Phenol red NaHCO3
mg/liter 0. 0625 0. 0625 0. 0250 0. 0250 0. 0250 1. 2500 10. 0000 0. 1250 0. 1250 0. 2500 0. 2500 0. 0250 0. 0250 10. 0000 50. 0000 7. 0000 1. 0000 2. 5000 1. 0000 1.0000 1. 0000 10. 0000 10. 0000 10. 0000 0. 1000 12. 5000 1. 8000 1. 8000 50. 0000 40. 0000 20. 0000 2200. 0000
V. J. Evans, J. C. Bryant, H. A. Kerr, and E. L. Schilling, Exp. Cell Res. 86, 439 (1964).
W a s s e r m a n tube, w h i c h has been c o a t e d p r e v i o u s l y w i t h a t h i n film of c h i c k e n p l a s m a (dispensed f r o m a P a s t e u r p i p e t t e ) by t h e a p p l i c a t i o n of a drop or t wo of chick e m b r y o e x t r a c t (dispensed f r o m a P a s t e u r
[34]
P I N E A L GLAND ORGAN CULTURE T E C H N I Q U E S
401
pipette)2 ,r Fifteen minutes later, when the clot is sufficiently firm, 0.5 ml of complete nutrient medium (see below) is added to the culture. The culture tube is then sealed with a rubber stopper and incubated in a standard tissue culture tube roller wheel at 37°C. ';'7 The complete nutrient medium ~ consists of 75.5% N16 medium, 4.0% NCTC-135 medium, 10.0% heat-inactivated fetal calf serum; 10.0% heat-inactivated horse serum, 0.5% of 5% lactalbumin hydrolyzate (Earle's), penicillin G (50 units per milliliter of medium), streptomycin sulfate (50 t*g per milliliter of medium), and amphotericin B (1 /,g per milliliter of medium). All the constituents of the complete nutrent medium, as well as the chick embryo extract, the chicken plasma, and the Hanks' BSS, can be obtained as standard preparations from Grand Island Biological Co., Grand Island, New York. For studies in which serotonin is to be assayed by spectrophotometric techniques, the media are prepared without phenol red, because this indicator dye interferes with the assay.
Method 2 Stationary Incubation at Interlace o] the Nutrient Medium and an Enriched 02 Atmosphere. This technique has been used to maintain intact rat pineal glands in culture without evidence of necrosis for periods up to 6 days? ,1° Reagents Modified B G J b culture medium (composition in Table III) Bovine serum albumin, Fraction V Procedure. All steps are carried out under aseptic conditions to mainrain sterility of the pineal glands, the culture vessels, and their contents. Pineal glands are obtained by sterile dissection following decapitation. One or two intact pineal glands are incubated on a stainless steel screen (see below) in a specially designed culture vessel (see below) at 37 ° in the presence of 0.5 ml of chemically defined, modified B G J b culture medium (Table I I I ) supplemented with fraction V bovine serum albumin °,~1,1-~ (Armour Pharmaceutical Co.) at a final concentration in the complete medium of 1 mg/ml, and are continually gassed with a mixture G. Yerganian, H. M. Shein, and J. F. Enders, Cytogenetics 1, 314 (1962). D. C. Klein and J. Weller, In Vitro 6, 197 (1970). This paper provides a sketch of the culture vessel with a pineal gland in place. ~*R. B. Berg and D. C. Klein, Endocrinology 89, 453 (1971). ~1L. G. Raisz, J. Clin. Invest. 44, 103 (1965). 12L. G. Raisz and I. Niemann, Endocrinology 85, 446 (1969).
402
ENDOCRINE AND REPRODUCTIVE TISSUE
[34]
TABLE III COMPOSITION OF MODIFIED BGJb MEDIUMa Component Calcium lactate Dihydrogen sodium orthophosphate Glucose Magnesium sulfate- 7H20 Potassium chloride Potassium dihydrogen phosphate Sodium bicarbonate Sodium chloride L-Lysine i-Histidine L-Arginine L-Threonine DL-Valine L-Leucine L-Isoleucine L-Methionine L-Phenylalanine i-Tryptophan L-Tyrosine L-Cysteine HCI L-Glutamine
mg/liter
Component
555.00 L-Alanine 90.00 L-Aspartic acid Glycine 10,000.00 L-Proline 200.00 L-Serine 400.00 Nicotinamide 160.00 Thiamine hydrochloride Calcium pantothenate 3,500.00 Riboflavin 5,300.00 Pyridoxal phosphate 240.00 Folic acid 150.00 Biotin 175.00 p-Aminobenzoic acid 75.00 a-Tocopherol phosphate 65.00 Choline chloride 50.00 Inositol 30.00 Vitamin B~2 50.00 Sodium acetate 50.00 Ascorbic acid 40.00 Phenol red 40.00 Streptomycin 90.00 Penicillin 200.00
mg/liter 250.00 150.00 800.00 400.00 200.00 20.00 4.00 0.20 0.20 0.20 0.20 0.20 2.00 1.00 50.00 0.20 0.04 50.00 50.00 20.00 50.00 100,000 units
a L. G. Raisz and I. Niemann, Endocrinology $5, 446 (1969).
of 95% oxygen and 5% carbon dioxide (v/v) in a water-saturated atmosphere. TM The stainless steel screen maintains the culture at the interface of the medium and the gassing atmosphere. The culture vessel is a U.S. Bureau of Plant Industry watch glass (The A. H. Thomas Comp a n y ) , fitted with a 1.5 mm mesh size stainless steel expanded screen (The Expanded Metal Company, Stranton Works, Hartlepool, England) platform fabricated in a machine shop to a 1.7 cm diameter with a curled edge 1 mm deep2 To maintain a water-saturated atmosphere during incubation, up to seven of the culture vessels are placed on a 9-cm disc of W h a t m a n No. 3M paper, previously soaked with 3 ml of incubation medium, in a 10-ml petri dish. Each petri dish is then incubated on one of five shelves (spaced 1 inch apart), in a small gas-tight Lucite chamber (with space for a total of 20 petri dishes), fabricated to these specifications in a machine shop, which is perfused continuously (0.3 liter per minute) with the gassing mixture2 ,1°
[35]
02 MICROTECHNIQUES WITH ISOLATED CELLS
403
Intact pineal glands from lO-month-old domestic ducks have also been maintained in organ culture for 24 hours without necrosis by the technique (Method 2) described above, with the modifications that each gland is cultured individually, and a different nutrient medium; i.e., medium 199 '3 is employed at a slightly higher temperature, 38°C. '~ '~J. F. Morgan, H. J. Morgan. and R. C. Parker, Proc. Soc. Exp. Biol. Med. 73, 1 (1950).
[35] U s e o f 0 2 M i c r o t e c h n i q u e s f o r A s s e s s m e n t o f Hormone Effects on Isolated Endocrine Cells B y LARS HAMBERGER, HANS HERLITZ~ and t~AGNAR HULTBORN
Methods for microgasometric measurements in biological material have been used and developed during the last four decades. In 1937 Linderstr0m-Lang introduced the Cartesian diver principle for this purpose. 1 The sensitivity of the original device was in the order of 10-~ to 10--" ~l per hour, which means a considerable increase in sensitivity compared to the Warburg technique. ~ In 1953 another Danish scientist 3 introduced the ampulla or microdiver respirometer with which cellular respiration could be determined in the order of 10-~ to l0 -~ ~l per hour2 An even more sensitive technique, the magnetic diver microgasometer, was introduced in 1964 * and recently further developed2 This technique gives reliable measurements in the magnitude of 10-6 to 10-; tL1 per hour, specifically interesting when taking into consideration that single tissue cells respire with intensities approximately 10-6 ~l per hour. One essential problem in this connection is concerned with procedures for alteration of the medium composition during an experiment. Numerous ingenious arrangements for addition of reactants and inhibitors to the incubation medium have been described for the Warburg technique. ~ However, in microscale this problem is even more intricate. Some of the techniques for addition of reactants and inhibitors in use for standarddivers and ampulla divers will be described in the following. I K. LinderstrCm-Lang, Nature (London) 140, 108 (1937). ~W. W. Umbreit, R. H. Burris, and J. F. Stauffer, "Manometric Techniques: A Manual Describing Methods Applicable to the Study of Tissue Metabolism." Burgess, Minneapolis, Minnesota, 1964. :~E. Zeuthen, J. Embryol. Exp. Morphol. 1, 239 (1953). ' M. Brzin and E. Zeuthen, C. R. Tray. Lab. Carlsberg 34, 427 (1964). '~S. Oman and M. Brzin, AT~d. Biochem. 45, 112 (1972).
