- Email: [email protected]
A versatile chamber for the growth of duplicate monolayer cell cultures directly on microscope slides
Versatile growth other acid macromolecules at the histones. In Tetrahymena cells the caseseemsdifferent. There the amount of basic protein is higher at stationary than at exponential phase [IO]. The major amounts of RNA, DNA and basic proteins were soluble in 0.12 M NaCI, which indicates that aberrant nucleoprotein conditions prevail in the amoeba cell. A deviating nucleoprotein state is also implied by the nonstainability of the amoeba nucleus with ammoniacal silver and methyl green despite retained fast green and Feulgen stainability. The ratio basic proteins/nucleic acids was comparatively normal in the NaCl-soluble fraction and the distributional pattern of the soluble basic proteins is similar to that of ribosomal protein. Thus, the soluble proteins may be principally RNAassociated. The pattern of basic proteins in the insoluble fraction was different and the ratio basic proteins/nucleic acids was exceptionally high. It may be presumedthat the insoluble fraction contains the greater amount of those basic proteins which exist in macromolecular combinations other than nucleoproteins. Valuable technical aid was given by Miss Brita Nilsson. The investigation was facilitated by grants from the Royal Physiographical Society.
REFERENCES I. Agrell, I P S, Exptl cell res 42 (1966) 403. 2. -Ibid 43 (1967) 691. 3. - Ibid 50 (1968) 687. 4. Agrell, I P S & Christensson, E, Nature 191 (1961) 284. 5. Agrell, I P S & Karlsson, B, Exptl cell res 48 (1967) 634. 6. Alfert, M, & Geschwind, I, Proc natl acad sci US 39 (1953) 991. 7. Barnes, W &Jensen, T, J protozooll45 (1967), Suppl. 11. 8. Black, M M & Ansley, H R, J histochem cytochem 14 (1966) 177 9. Burton, K, Biochem j 62 (1955) 315. 10. Christensson, E, Arkiv zoo1 19 (1967) 297. I I. Johns, E W, Biochem j 104 (1967) 78. 12. Kjellstrand, P, Exptl cell res 53 (1968) 37. 13. Leuchtenberger, C, Gen cytochem meth 1 (1958) 220. 14. Lindh, N 0 & Brantmark, B L Anal biochem 10 (1965) 415. 15. Mundkur, B & Brauer, B, J histochem cytochem 14 (1966) 94. 16. Munro, H N & Fleck, A, Analyst 91 (1966) 78. 17. Neff, R J, Benton, W & Neff, R H, J cell bio123 (1964) 66 A. r’s
691801
chamber for cell cultures
433
18. Neff, R J, Ray, S A, Benton, W F & Wilborn, M, Meth cell physiol 1 (1964) 55. 19. Pearse, AC E,Histochemistry, p. 826. London (1960). Received March 20, 1969
A VERSATlLE CHAMBER FOR THE GROWTH OF DUPLICATE MONOLAYER CELL CULTURES DIRECTLY ON MICROSCOPE SLIDES G. E. LEVENSON,
Histology-Embryology Department, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pa 19104, USA’ and Strangeways Research Laboratory, Cambridge, UK
During studies on various cell types grown in monolayer cultures, the need arose for a culture vessel with an absolutely flat glass growth surface. Conventional dishesdo not have uniformly flat bottoms and cells tend to pool in the depressions. The inclusion of cover glassesin dishes to permit fixation and staining of cells has several disadvantages; cells become interposed between cover glass and the bottom of the dish thus interfering with inverted optics and visibility, and after histological processingit is very difficult to relocate microscopic fields that have been studied before fixation. The present system overcomes these problems and has additional advantages. (1) Both experimental and control cultures are on the sameslide and can be processedsimultaneously and identically. (2) The cells are cultivated on fresh surfaces. This prevents poor visibility due to scratching that frequently mars expensive dishes. (3) Cultures may be grown on membranes under the same conditions as on glass. (4) This uniform flat surface and the parallel glass-air-liquid interfaces provide excellent uniform optical conditions all across the chamber. (5) Entire cultures may be preserved for staining and subsequent study. (6) Large numbers of fixed cultures can be processedsimultaneously usingconventional slide carriers and staining dishes. 1 Author’s permanent address. Exptl
Cell Res 55
434
G. E. Levenson
1 cm
ti
Fig. I. Scale drawing of dual culture assembly as normally used showing a section through both chambers at their widest diameter. A, surface sealed with silicone stopcock grease, B, surface sealed with wax mixture or polyvinyl acetate. Shaded area indicates medium.
Methods The assembly consists of a 1 1/2” x 3” microscope slide, two cylindrical rings each of 22 mm inside diameter and 14 mm height. These rings have ground lips and lids (specially prepared by Bellco Glass Corp., Vineland, N.J.). The internal surfaces of the rings are thinly coated with silicone to prevent an exaggerated miniscus and to maintain an even fluid level. The sterile rings are positioned on sterile slides with the ground lip uppermost. Chambers are covered with lids and warmed to 70°C. A Vaseline-paraffin wax mixture (M.P. 50-55°C) is applied to the ring-slide junction, and flows rapidly round the junction by capillary attraction. After cooling, units are charged with 1 ml of cell suspension;the chambers are gassed,sealed with silicone grease, and incubated on plate glass strips which are kept level. Alternatively, components may be cemented with a non-toxic polyvinyl acetate (“Gelva” resin V-25, from Shawinigan Ltd., 118 Southwark St., London SWl.) obtained as a powder and dissolved in 95 % ethanol to form a thick clear fluid. Assemblies are permitted to dry overnight at 37°C to prevent air bubble formation in the cement and dry sterilized at 160°C. For fixation, medium is withdrawn, the cultures are washed with balanced salt solution, and fixative is applied by pipetting gently down the sides of the chambers. After fixation, waxcemented units are simply pulled apart; any remaining wax drops off in 70 76 alcohol or xylol during clearing. Preparations are made permanent with conventional media and round cover glasses.After fixing and washing, acetate-cementExptl
Cell Res 55
ed units are filled with 70% ethanol and left overnight in trays of 70 % ethanol after which the rings are easily removed; remaining cement is completely removed in the higher alcohols. The same chambers are easily and efficiently used for cell culture on Millipore membranes. The unground lips of the rings are ground on a flat plate with 320 mesh Carborundum powder. A coating of Millipore no. 1 cement (Millipore Corp., Bedford, Mass.) is applied and permitted to dry. A second layer is added and the rings are immediately pressedover the appropriate Millipore discs; rings and attached membranes are then quickly inverted to dry. Units are then assembledas usual, two per slide, and liquid Gelva cement is applied all round the ring-slidejunction by means of a hypodermic syringe and needle; the assembliesare dried as above and sterilized by dry heat at 100°C for 2 h. To prevent air from becoming trapped beneath the membrane, three droplets of “Gelva” may be appropriately placed on the slide before positioning the membrane and ring. After cells are plated and attached, two small slits are cut through the membrane at opposite sidesnear the glassrings. This permits complete replacements of medium beneath the membrane when cultures are refed. For fixation, membranes are cut out with a sharp scalpeland processed[ 11. To grow two cultures within the same chamber, a rectangle of coverslip glassmay be placed vertically into the chamber and sealed in with “Gelva” thus forming two hemi-chambers. This has proven very convenient for easy comparison of adjacent cultures in the samemicroscope field after partitions are removed and preparations fixed. Since the chamber base, a square slide, may be repositioned accurately in stage clamps, verniers or a field finder permit the relocation of a precise field for photography from day to day. The same field is easily relocated after fixation and staining. The author wishes to thank Mr T. R. Munro of the Strangeways Research Laboratory for certain technical suggestions, and Mr N. Applin for preparing the drawing. This work was done partly with support from USPHS DE 02047 and partly during the tenure of a USPHS National Cancer Institute Special Fellowship, no. 2-F3CA-31. 544-02.
TAA-induced REFERENCES Lillie, R D, Histopathologic technic and practical histochemistry, 3rd edn. McGraw-Hill, New York (1965).
MITOTIC ABNORMALITIES IN PROLIFERATING HEPATOCYTES INDUCED BY THIOACETAMIDE AT CERTAIN PERIODS AFTER PARTIAL HEPATECTOMY ST. MIRONESCU, Department of Morphology, Institute sf Endocrinology, Bucharest, Roumania Thioacetamide (TAA) elicits in rat liver striking changes in both interphasic and dividing hepatocytes. The nucleolar [IO-12, 161 and mitotic [14] alterations are two of the most prominent, and some of them were linked to the carcinogenic action of this drug [14, 161. Repeated applications are necessary to induce these morphological changes, since, as indicated by the studies in which 35S- and 3H-labeled TAA was used, during the first 24 h this drug is rapidly converted to acetate, via the acetamide pathway [20], and excreted in the urine [19,20]. Due to the rapid metabolism of TAA, the maximum and most uniform cytological effects were found 4 h after its subcutaneous administration [lo, 121. The relative rapid degradation of TAA within the liver tissue [20] and the possibility to obtain, after the subtotal liver resection, a sequence of macromolecular syntheses, mimming during the first 31 h, the ordered biochemical events occurring in the cell cycle [l-4], offers exploitable circumstances to test (a) whether single doses of TAA may induce changes in the regenerating liver, and (6) whether the biosynthetic steps, leading to a wave of synchronous DNA replication and mitoses [l, 41, exhibit variable degrees of sensitiveness toward the damaging action of this compound. The limits of interpretation of the biochemical changes developed in regenerating rat liver, concerning their resemblance to the processes operating during the cell life cycle of unstimulated hepatocytes, were outlined earlier [IS] and, more recently, they were extensively reviewed [2].
Materials
abnormalities
in hepatocytes
435
and methods
Partially hepatectomized [9] male Sprague-Dawley rats (89k3.4 g) divided in four groups of 8, 8, 6 and 12 animals, were inoculated intra-abdominally at 10, 20, 23 and respectively 26 h after operation with appropriate volumes of a 1 % solutionof TAA in 0.14 M NaCl, to achievein therecipientsthedrugconcentrationof 150mg/ g body weight.The two control rats of eachgroupreceivedappropriatevolumes of physiologicsaline.All the control and experimentalanimalswerekilled 31 h after surgery,between9-10a.m.,andthe remnantliver tissues werefixed in 10%neutralbufferedformaldehyde. Twenty p thick paraffin sections were prepared and stained by the Feulgenprocedure.At leastthree sectionsof each animal were checked for the presence of mitotic abnormalities (single or multiple chromosome bridges, acentric chromosome fragments and lagging chromosomes) in the anaphasic and telophasic stages of mitosis. At least 200 anaohases and telouhases were studied for each tested rat and’ the results were expressed as the mean number of abnormal anaphases and telophases/lOO anaphases and telophases. Thk data obtained-from the control rats were pooled and treated as for a single specimen. Statistical analyses were performed using Student’s t-test. The significance threshold was chosen at the level of P < 0.05.
Results Fig. 1 presents the variations of the different morphological types of abnormal anaphasesand telophases, occurring in regenerating rat liver cells after a single injection of 150 mg TAAjkg b.w. The data recorded in the controls showed that in saline-treated Sprague-Dawley rats, the occurrence of abnormal anaphases and telophases ranged between 7.5-8.5% of the total number of anaphasesand telophasesencountered 31 h after partial hepatectomy. Among these abnormalities 40-60 % were represented by the chromosomal bridges, about 30 % by the lagging chromosomes and lo-30 % by the acentric chromosome fragments. Compared with the control animals, the rats receiving 150 mg TAA/kg b.w., 10 h after they were partially hepatectomized, presented significantly increased proportions of both chromosome bridges and acentric chromosome fragments. Although increased, the frequency of the lagging chromosomes was found not statistically different from the values recorded in control rats, and this was also valid for all other intervals studied. The rats inoculated with TAA 20 h after surgery have presented extremely high proportions of chromosomal bridges, 50-75 % of anaphasic Exptl
Cell Res 5.5
REFERENCES I. Agrell, I P S, Exptl cell res 42 (1966) 403. 2. -Ibid 43 (1967) 691. 3. - Ibid 50 (1968) 687. 4. Agrell, I P S & Christensson, E, Nature 191 (1961) 284. 5. Agrell, I P S & Karlsson, B, Exptl cell res 48 (1967) 634. 6. Alfert, M, & Geschwind, I, Proc natl acad sci US 39 (1953) 991. 7. Barnes, W &Jensen, T, J protozooll45 (1967), Suppl. 11. 8. Black, M M & Ansley, H R, J histochem cytochem 14 (1966) 177 9. Burton, K, Biochem j 62 (1955) 315. 10. Christensson, E, Arkiv zoo1 19 (1967) 297. I I. Johns, E W, Biochem j 104 (1967) 78. 12. Kjellstrand, P, Exptl cell res 53 (1968) 37. 13. Leuchtenberger, C, Gen cytochem meth 1 (1958) 220. 14. Lindh, N 0 & Brantmark, B L Anal biochem 10 (1965) 415. 15. Mundkur, B & Brauer, B, J histochem cytochem 14 (1966) 94. 16. Munro, H N & Fleck, A, Analyst 91 (1966) 78. 17. Neff, R J, Benton, W & Neff, R H, J cell bio123 (1964) 66 A. r’s
691801
chamber for cell cultures
433
18. Neff, R J, Ray, S A, Benton, W F & Wilborn, M, Meth cell physiol 1 (1964) 55. 19. Pearse, AC E,Histochemistry, p. 826. London (1960). Received March 20, 1969
A VERSATlLE CHAMBER FOR THE GROWTH OF DUPLICATE MONOLAYER CELL CULTURES DIRECTLY ON MICROSCOPE SLIDES G. E. LEVENSON,
Histology-Embryology Department, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pa 19104, USA’ and Strangeways Research Laboratory, Cambridge, UK
During studies on various cell types grown in monolayer cultures, the need arose for a culture vessel with an absolutely flat glass growth surface. Conventional dishesdo not have uniformly flat bottoms and cells tend to pool in the depressions. The inclusion of cover glassesin dishes to permit fixation and staining of cells has several disadvantages; cells become interposed between cover glass and the bottom of the dish thus interfering with inverted optics and visibility, and after histological processingit is very difficult to relocate microscopic fields that have been studied before fixation. The present system overcomes these problems and has additional advantages. (1) Both experimental and control cultures are on the sameslide and can be processedsimultaneously and identically. (2) The cells are cultivated on fresh surfaces. This prevents poor visibility due to scratching that frequently mars expensive dishes. (3) Cultures may be grown on membranes under the same conditions as on glass. (4) This uniform flat surface and the parallel glass-air-liquid interfaces provide excellent uniform optical conditions all across the chamber. (5) Entire cultures may be preserved for staining and subsequent study. (6) Large numbers of fixed cultures can be processedsimultaneously usingconventional slide carriers and staining dishes. 1 Author’s permanent address. Exptl
Cell Res 55
434
G. E. Levenson
1 cm
ti
Fig. I. Scale drawing of dual culture assembly as normally used showing a section through both chambers at their widest diameter. A, surface sealed with silicone stopcock grease, B, surface sealed with wax mixture or polyvinyl acetate. Shaded area indicates medium.
Methods The assembly consists of a 1 1/2” x 3” microscope slide, two cylindrical rings each of 22 mm inside diameter and 14 mm height. These rings have ground lips and lids (specially prepared by Bellco Glass Corp., Vineland, N.J.). The internal surfaces of the rings are thinly coated with silicone to prevent an exaggerated miniscus and to maintain an even fluid level. The sterile rings are positioned on sterile slides with the ground lip uppermost. Chambers are covered with lids and warmed to 70°C. A Vaseline-paraffin wax mixture (M.P. 50-55°C) is applied to the ring-slide junction, and flows rapidly round the junction by capillary attraction. After cooling, units are charged with 1 ml of cell suspension;the chambers are gassed,sealed with silicone grease, and incubated on plate glass strips which are kept level. Alternatively, components may be cemented with a non-toxic polyvinyl acetate (“Gelva” resin V-25, from Shawinigan Ltd., 118 Southwark St., London SWl.) obtained as a powder and dissolved in 95 % ethanol to form a thick clear fluid. Assemblies are permitted to dry overnight at 37°C to prevent air bubble formation in the cement and dry sterilized at 160°C. For fixation, medium is withdrawn, the cultures are washed with balanced salt solution, and fixative is applied by pipetting gently down the sides of the chambers. After fixation, waxcemented units are simply pulled apart; any remaining wax drops off in 70 76 alcohol or xylol during clearing. Preparations are made permanent with conventional media and round cover glasses.After fixing and washing, acetate-cementExptl
Cell Res 55
ed units are filled with 70% ethanol and left overnight in trays of 70 % ethanol after which the rings are easily removed; remaining cement is completely removed in the higher alcohols. The same chambers are easily and efficiently used for cell culture on Millipore membranes. The unground lips of the rings are ground on a flat plate with 320 mesh Carborundum powder. A coating of Millipore no. 1 cement (Millipore Corp., Bedford, Mass.) is applied and permitted to dry. A second layer is added and the rings are immediately pressedover the appropriate Millipore discs; rings and attached membranes are then quickly inverted to dry. Units are then assembledas usual, two per slide, and liquid Gelva cement is applied all round the ring-slidejunction by means of a hypodermic syringe and needle; the assembliesare dried as above and sterilized by dry heat at 100°C for 2 h. To prevent air from becoming trapped beneath the membrane, three droplets of “Gelva” may be appropriately placed on the slide before positioning the membrane and ring. After cells are plated and attached, two small slits are cut through the membrane at opposite sidesnear the glassrings. This permits complete replacements of medium beneath the membrane when cultures are refed. For fixation, membranes are cut out with a sharp scalpeland processed[ 11. To grow two cultures within the same chamber, a rectangle of coverslip glassmay be placed vertically into the chamber and sealed in with “Gelva” thus forming two hemi-chambers. This has proven very convenient for easy comparison of adjacent cultures in the samemicroscope field after partitions are removed and preparations fixed. Since the chamber base, a square slide, may be repositioned accurately in stage clamps, verniers or a field finder permit the relocation of a precise field for photography from day to day. The same field is easily relocated after fixation and staining. The author wishes to thank Mr T. R. Munro of the Strangeways Research Laboratory for certain technical suggestions, and Mr N. Applin for preparing the drawing. This work was done partly with support from USPHS DE 02047 and partly during the tenure of a USPHS National Cancer Institute Special Fellowship, no. 2-F3CA-31. 544-02.
TAA-induced REFERENCES Lillie, R D, Histopathologic technic and practical histochemistry, 3rd edn. McGraw-Hill, New York (1965).
MITOTIC ABNORMALITIES IN PROLIFERATING HEPATOCYTES INDUCED BY THIOACETAMIDE AT CERTAIN PERIODS AFTER PARTIAL HEPATECTOMY ST. MIRONESCU, Department of Morphology, Institute sf Endocrinology, Bucharest, Roumania Thioacetamide (TAA) elicits in rat liver striking changes in both interphasic and dividing hepatocytes. The nucleolar [IO-12, 161 and mitotic [14] alterations are two of the most prominent, and some of them were linked to the carcinogenic action of this drug [14, 161. Repeated applications are necessary to induce these morphological changes, since, as indicated by the studies in which 35S- and 3H-labeled TAA was used, during the first 24 h this drug is rapidly converted to acetate, via the acetamide pathway [20], and excreted in the urine [19,20]. Due to the rapid metabolism of TAA, the maximum and most uniform cytological effects were found 4 h after its subcutaneous administration [lo, 121. The relative rapid degradation of TAA within the liver tissue [20] and the possibility to obtain, after the subtotal liver resection, a sequence of macromolecular syntheses, mimming during the first 31 h, the ordered biochemical events occurring in the cell cycle [l-4], offers exploitable circumstances to test (a) whether single doses of TAA may induce changes in the regenerating liver, and (6) whether the biosynthetic steps, leading to a wave of synchronous DNA replication and mitoses [l, 41, exhibit variable degrees of sensitiveness toward the damaging action of this compound. The limits of interpretation of the biochemical changes developed in regenerating rat liver, concerning their resemblance to the processes operating during the cell life cycle of unstimulated hepatocytes, were outlined earlier [IS] and, more recently, they were extensively reviewed [2].
Materials
abnormalities
in hepatocytes
435
and methods
Partially hepatectomized [9] male Sprague-Dawley rats (89k3.4 g) divided in four groups of 8, 8, 6 and 12 animals, were inoculated intra-abdominally at 10, 20, 23 and respectively 26 h after operation with appropriate volumes of a 1 % solutionof TAA in 0.14 M NaCl, to achievein therecipientsthedrugconcentrationof 150mg/ g body weight.The two control rats of eachgroupreceivedappropriatevolumes of physiologicsaline.All the control and experimentalanimalswerekilled 31 h after surgery,between9-10a.m.,andthe remnantliver tissues werefixed in 10%neutralbufferedformaldehyde. Twenty p thick paraffin sections were prepared and stained by the Feulgenprocedure.At leastthree sectionsof each animal were checked for the presence of mitotic abnormalities (single or multiple chromosome bridges, acentric chromosome fragments and lagging chromosomes) in the anaphasic and telophasic stages of mitosis. At least 200 anaohases and telouhases were studied for each tested rat and’ the results were expressed as the mean number of abnormal anaphases and telophases/lOO anaphases and telophases. Thk data obtained-from the control rats were pooled and treated as for a single specimen. Statistical analyses were performed using Student’s t-test. The significance threshold was chosen at the level of P < 0.05.
Results Fig. 1 presents the variations of the different morphological types of abnormal anaphasesand telophases, occurring in regenerating rat liver cells after a single injection of 150 mg TAAjkg b.w. The data recorded in the controls showed that in saline-treated Sprague-Dawley rats, the occurrence of abnormal anaphases and telophases ranged between 7.5-8.5% of the total number of anaphasesand telophasesencountered 31 h after partial hepatectomy. Among these abnormalities 40-60 % were represented by the chromosomal bridges, about 30 % by the lagging chromosomes and lo-30 % by the acentric chromosome fragments. Compared with the control animals, the rats receiving 150 mg TAA/kg b.w., 10 h after they were partially hepatectomized, presented significantly increased proportions of both chromosome bridges and acentric chromosome fragments. Although increased, the frequency of the lagging chromosomes was found not statistically different from the values recorded in control rats, and this was also valid for all other intervals studied. The rats inoculated with TAA 20 h after surgery have presented extremely high proportions of chromosomal bridges, 50-75 % of anaphasic Exptl
Cell Res 5.5