Exp. Clin. Endocrinol. 99 (1992) 34-38
Experimental and Clinical Endocrinology © 1992 Johann Ambrosius Barth
The Effect of Serum Concentrations and Sources on DNA Synthesis and Insulin Secretion of Cultured Islets Institute of Diabetes "Gerhardt Katsch" (Director: Prof. Dr. U. Fischer), Karlsburg/Germany
Key words: Serum - Islet DNA synthesis - Insulin secretion - Islet culture
significantly inhibited the islet DNA synthesis as well as the glucose stimulated insulin secretion after 2, 4 and 8 days of culture, HS at different concentrations failed to inhibit both, the
Summary: We investigated the effect of different sera, as newborn calf serum, fetal calf serum, rat serum and human serum at various concentrations on biofunctions of cultured pancreatic islets. Islets isolated from newborn LEW. i W rats were maintained at 37°C in TCM 199 containing 10 mmol/1 glucose and either 1%. 10% or 50% newborn calf serum (NCS), fetal calf serum (FCS), serum obtained from adult syngeneic rats (RS) or different batches of human serum (HS). While exposure of islets to increasing concentrations of NCS
Introduction The maintenance of pancreatic islets in tissue culture represents a suitable method for preservation of viable islets before transplantation into diabetic recipients. In comparison to freshly isolated islets the pool of cultured islets is characterized by a higher percentage of viable and functionally intact islets, since substantially affected tissue (probably as result of ischemia or of the preparation procedure) is lost during the cultivation period. However, to simulate an in vivo like environment for tissue maintenance or even for multiplication of insulin producing cells before their implantation, appropriate culture conditions have to be established. A lot of investigations were done to find a suitable medium for islet culture (Andersson, 1987; Nielsen, 1981) or to test the effects of various nutrients on islet cell function and proliferation (Andersson et al., 1976; Andersson, 1974; Buitrago et al., 1975; Gylfe, 1978; Swenne et al., 1980; Chick, 1973; King and
Chick, 1976; King et al., 1978; Swenne, 1982). Only
islet DNA synthesis and the secretory response to glucose at each time point investigated. The action of FCS, RS and 2 further batches of HS on islet DNA synthesis was analysed by short-term culture. The supplementation of the medium with 50 % FCS and RS resulted in a marked decrease of H-thymidine incorporation into islet DNA similar to that observed with 50 % NCS. In contrast, exposure of islets to different batches of HS never resulted in an inhibition of DNA synthesis. Moreover, the 50% concentration of HS 465 caused a significant stimulation of thymidine incorporation.
few informations (Andersson et al., 1976; King et al., 1977; Ono et al., 1979) are available concerning the serum component of the medium in supporting growth and function of endocrine pancreatic tissue in vitro although serum is known to be essential for maintenance of islets in culture. Up to now no satisfactory conditions for islet culture are described in the literature. Therefore, we comparatively investigated the effect of
different concentrations of NCS, together with FCS, which are very often used sera in islet culture, and of HS on DNA synthesis, insulin content and insulin release of cultured rat islets. In addition, we determined the DNA synthesis of pancreatic islets in response to sera of other
animal sources as well as different batches of human serum.
Material and Methods Collagenase (160 U/mg, Worthington Biochemical Corpora-
tion, Freehold New Jersey) isolated islets from Lewis rats
Downloaded by: Universite Laval. Copyrighted material.
Annemarie Dunger, I. Klöting, W. Besch and H. J. Hahn
35
A. Dunger et al., Pancreatic Islet Cell Culture
(8-12 days of age) were cultured free-floating in TCM 199
Statistical analysis
(Difco Laboratories) containing 10 mmolJl glucose and either
All results are given as mean ± S.E.M. of n different experiments. Statistical significance was checked by Student's t-test.
Laboratories) or fetal calf serum (FCS, Difco Laboratories). Furthermore, pooled serum from adult syngeneic rats (RS) or 3 different batches of human serum (HS, Institut für Impfstoffe
Dessau, Germany) indicated by the number were used. The
Results
cultures were maintained at 37°C for a period of 2, 4 and 8 days in a humidified atmosphere (97 % air, 3% CO2). The medium
In contrast to HS 532, an increase of NCS concentration in the culture medium resulted in a significant decrease of islet DNA synthesis at day 2, 4 and 8 (Fig. 1). A marked
was changed every 2-3 days. For estimation of DNA content and synthesis groups of 40 islets incubated with 74 KBq/ml (methyl-3H)-thymidine
reduction of DNA synthesis during the first 4 days in
(UVVVR-Prag, tSFR, Spec. act. 560 OBq/mmol) for 16h were twice washed and ultrasonically disrupted. Duplicate ahquots of the homogenate were taken for fluorometric assay of DNA content (Kissane and Robins, 1958) and for measurement of radioactivity (Ziegler et al., 1982). The glucose-induced islet insulin release was assayed radioimmunologically in a postcultural incubation (Besch et al., 1987). For this purpose the cultivated islets were firstly incubated in Krebs-Ringer bicarbonate buffer (KRBB, Hahn, 1978) with 5 mmol/l glucose for 30 min at 37°C. After preincubation the islets were transferred into KRBB containing 1.5, 5, lO or
20mM glucose and incubated for 2 h at 37°C. The secretion rates are given in pmoh/islet per 120 min. The insulin content of
pancreatic islets was determined radioimmunologically after their ultrasonication in an acidified solution (Hahn et al., 1974).
[DPMIpg DNA]
culture was evident in islets of both, NCS and HS supplemented culture. A further decrease of islet DNA synthesis was observed when islets were cultured in the presence of NCS, for more than 4 days. In the presence of HS (1 % or 10%) the further decrease in DNA synthesis was completely prevented (Fig. 1). As depicted in Table I the islet insulin content was neither affected by the NCS concentrations used, nor by the cultivation time. However, at the 50% concentration, HS significantly affected the insulin content of the cultured islets. Increased NCS concentrations generated a distinct inhibition of glucose-stimulated insulin secretion investi-
HS 532
NCS
io
20
is 12
Fig. 1 DNA synthesis of rat pancreatic islets cultured for 8 days in the presence of
8-
newborn calf serum (NCS) or human serum (HS 532). Serum concentrations:
4
l%(I/O), l0%(A/A),50%(/D). **
o
p < 0.01 in comparison to the values at day 2 of culture, ++ p p < 0.01 compared to 1% serum ± p < 0.05 compared to 10% serum, ' p < 0.01 compared to l0% FCS and RS, compared to 50% NCS, FCS and RS. The number of experiments is indicated at the bottom of the bars
Downloaded by: Universite Laval. Copyrighted material.
O
A. Dunger et al., Pancreatic Islet Cell Culture
37
parable incorporation of labelled thymidine into islet DNA (as already found with HS532), the exposure of
however, noteworthy that the culture time dependent decrease in glucose-stimulated insulin release was pre-
islets to 50% of HS 465 resulted in a significant stimulation of replication.
vented when NCS was substituted by HS. In short-term experiments it was found that FCS and NCS, belonging to the standard sera being used in islet cell and tissue culture (Federlin and Bretzel, 1981), and RS have an inhibitory effect on DNA synthesis compar-
Serum provides hormonal and/or nutritional factors
able to that of NCS. Interestingly, in contrast to the
which are necessary for growth and survival of cultured cells (Pace and Clements, 1981). Medium for islet culture was originally supplemented with 10% newborn calf serum (Hales and Mimes, 1968). The presence of 10 % serum was thought to be sufficient to sustain the secretory potential of pancreatic B-cells (Ziegler et al., 1983). Our investigations succeeded in demonstrating that the functionality of cultured islets is dependent on the serum concentration if NCS is used as serum source. Increasing the NCS concentration from 1 % via 10% to 50% resulted in a significant inhibition of islet DNA synthesis as well as B-cell function documented by glucose stimulated insulin release after 2, 4 and 8 days of islet culture. In respect of stimulated insulin secretion are the results of special significance since they were obtained in a postcultural incubation period in the absence of serum.
results with NCS, FCS or RS, the experiments with ilS have shown that in no case any inhibitory effect on DNA synthesis was present, indicating that certain factors from
When the same experiments were carried out with medium supplemented with human serum instead of NCS
no marked concentration-dependent serum effect was found. That is, the DNA synthesis of islets cultured in the presence of 1 %, 10 % or 50 % HS was comparable. In addition, islets cultured in the presence of 50% HS for 2
to 8 days exhibited in contrast to NCS cultured islets a significantly higher insulin release when stimulated with glucose. The results clearly demonstrate that growth and functionality of pancreatic islets in tissue culture to a high degree depend on the serum source used.
In contrast to the DNA synthesis and the stimulated insulin release, the islet insulin content was not affected by different amounts of NCS, which may be the result of an unaffected insulin storage though islet secretory responsiveness was influenced by a high NCS concentra-
tion. There are pieces of evidence indicating that a diminished hormone release is often related with a better maintenance of insulin biosynthesis and islet insulin content (Hales and Milner, 1968; Gylfe, 1978; Ziegler et al., 1983). An opposite behaviour may exist in islets cultured in the presence of 50% HS, where an increased insulin secretion is connected with a reduction in islet insulin content.
Independent whether pancreatic islets were maintained in tissue culture medium supplemented with NCS or HS, in both cases a decrease in DNA synthesis with duration of culture time could not be prevented. The fall in DNA synthesis as function of cultivation time is currently a still
unsolved problem, which was also observed by other authors using neonatal and adult islet cells (King et al., 1978; Ono et aI., 1979; De Clercq et al., 1980). It is
HS might have a generally beneficial influence on growth and function of endocrine pancreatic tissue under in vitro
conditions. Therefore, in our opinion HS seems to be a more suitable supplement for the cultivation of pancreatic islets before their use for transplantation. Acknowledgements. The authors wish to thank Mrs. G. Strauch,
Mrs. M. Henkel and Ms. K, Gumm for skillful technical assistance.
References Andersson, A.; Hellerström, C.: Metabolic characteristics of isolated pancreatic islets in tissue culture. Diabetes 21 (1972) 546-554. Andersson, A.; Gunnarsson, R.; Hellerström, C.: Long-
term effects of glucose on insulin release and glucose oxidation by mouse pancreatic islets maintained in tissue culture. Biochem. J. 140 (1974) 377-382. Andersson, A.; Gunnarsson, R.; Hellerström, C.: Longterm effects of a low extracellular glucose concentration on glucose metabolism and insulin biosynthesis and release of mouse pancreatic islets maintained in tissue culture. Acta Endocrinol. (KBH) 82 (1976) 3 18-329. Andersson, A.: Isolated mouse pancreatic islets in culture: Effects of serum and different culture media on the insulin production of the islets. Diabetologia 14 (1987) 397-404. Barnes, G.; Sato, G.: Methods for growth of cultured cells in serum-free medium. Anal. Biochim. 102 (1980)
255-280. Besch, W.; Woltanski, K. P.; Keilacker, H.; Diaz-Alon-
so, J. M.; Schulz, B.; Amendt, P.; Kohnert, K. D.; Ziegler, M.: Measurement of insulin in human sera using a new RIA kit. 1. Insulin determination in the absence of insulin antibodies - conventional assay and micro modifi-
cation. Exp. Clin. Endocrinol. 90 (3), (1987) 264-270.
[71 Buitrago, A.; Gylfe, E.; Hellman, B.; Idahl, L. A.; Johansson, M.: Function of microdissected pancreatic islets cultured in a chemically defined medium. I. Insulin content and release. Diahetologia 11(1975) 535-540.
[81 Chick, W. L.: Beta cell replication in rat pancreatic monolayer cultures. Effects of glucose, tolbutamide, glucocorticoid, growth hormone and glucagon. Diabetes (1973) 687-693. [91 De Clercq, L.; Schmidt, G.; De Laere, P.; Remade, C.: Nuclear events in B-cells of young and senescent rat islets in organ culture. Cell Biol. Int. Rep. 4 (1980) 817-827. [10] Federlin, K.; Bretzel, R. G.: Islet isolation, culture and cryopreservation. In: Intern. Workshop, Giessen 1980. Eds. Federlin, K., Bretzel, R. G., Stuttgart u. a.; Thieme
u.a., 1981.
Downloaded by: Universite Laval. Copyrighted material.
Discussion
Exp. Clin. Endocrino!. 99 (1992) 1
38
E.: Protection of the pancreatic beta-ce]! glucoreceptor mechanism for insulin secretion during cul-
Federlin, K.; Bretzel, R. G., Stuttgart: Thieme 1981, pp.
tuie in chemically defined medium. Biochem. J. (1978) 959-964.
Ono, J.; Takaki, R.; Okano, H.; Fukuma, M.: Long-term culture of pancreatic islet with special reference to the Bcell function. In Vitro 15 (1979) 95-102.
174
Hahn, H. J.; Hellman, B.; Lernmark, B.; Täljedahl, I. B.: Insulinsekretion isolierter Veränderung der Langerhans'scher Inseln durch Behandlung mit Neuraminidase. Acta Bio].
Med. Germ. 32 (1974)
375-383. Hahn, H. J.: Die isolierte Langerhans'sche Insel - ein Modell zur Untersuchung der insulinsekretion in vitro. Endokrinologie 71(1978) 308-324. Hales, C. N.; Milner, D. G.: Cations and the secretion of insulin from rabbit pancreas in vitro. J. Physiol. 199 (1968) 177-187. King, D. L.; Chick, W. L.: Pancreatic beta cell replication
- effects of hexose sugars. Endocrino]. 99 (1976) 1003-1009. King, D. L.; Chick, W. L.; Kitchen, K. C.: Pancreatic
69-80.
Pace, C. S.; Clements, R. S.: Myo-inositol and the maintenance of 3-cell function in cultured rat pancreatic islets. Diabetes 30 (1981) 621-625. Swenne, 1.; Bone, A. J.; Howell, S. L.; Hellerström, C.: Effects of glucose and aminoacids on the biosynthesis of DNA and insulin in fetal rat islets maintained in tissue culture. Diabetes 29 (1980) 686-692. Swenne, I.: The role of glucose in the in vitro regulation of cell cycle kinetics and proliferation of fetal pancreatic 13cells. Diabetes 31(1982) 754-760.
Ziegler, B.; Kohnert, K. D.; Noack, S.; Hahn, H. J.: Effects of 3-isobutyl-1-methyl-xanthine on secretory response, cAMP accumulation and DNA synthesis of islets from postnatal and adult Wistar rats. Acta Bio!. Med.
von Wasieiewski, E.; Chick, E. W., Excepta Medica,
Germ. 41(1982)1171-1177. Ziegler, B.; Wilke, B.; Woltanski, K. P.; Knospe, S.;
Amsterdam 1977, p. 109.
Hahn, H. J.: Protection of insulin secretion by magnesium
King, D. L.; Kitchen, K. C.; Chick, W. L.: Pancreatic
during islet culture: Independence of cAMP or insulin
beta cell replication - relation to insulin secretion. Endocrino]. 103 (1978) 1321-1327. Kissane, J. M.; Robins, E.: The fluorometric measure-
accumulation.
beta cell replication. In: Pancreatic Beta Cell Culture. Eds.
Exp.
Clin.
Endocrino].
82
(1983)
199-207.
ment of deoxyribonucleic acid in animal tissues with
Accepted: 2 October 1990
special reference to the central nervous system. J. Biol. Chem. 233 (1958) 184-188. Nielsen, J. H.: Human pancreatic islets in tissue culture. In: Islet isolation, culture and cryopreservation. Eds.
Correspondence address: Dr. Annemarie Dunger, Institut für Diabetes "Gerhardt Katsch", Greifswalder Str. 11 a, O-2201 Karlsburg, BRD
Downloaded by: Universite Laval. Copyrighted material.
[li] Gylfe,
Experimental and Clinical Endocrinology © 1992 Johann Ambrosius Barth
The Effect of Serum Concentrations and Sources on DNA Synthesis and Insulin Secretion of Cultured Islets Institute of Diabetes "Gerhardt Katsch" (Director: Prof. Dr. U. Fischer), Karlsburg/Germany
Key words: Serum - Islet DNA synthesis - Insulin secretion - Islet culture
significantly inhibited the islet DNA synthesis as well as the glucose stimulated insulin secretion after 2, 4 and 8 days of culture, HS at different concentrations failed to inhibit both, the
Summary: We investigated the effect of different sera, as newborn calf serum, fetal calf serum, rat serum and human serum at various concentrations on biofunctions of cultured pancreatic islets. Islets isolated from newborn LEW. i W rats were maintained at 37°C in TCM 199 containing 10 mmol/1 glucose and either 1%. 10% or 50% newborn calf serum (NCS), fetal calf serum (FCS), serum obtained from adult syngeneic rats (RS) or different batches of human serum (HS). While exposure of islets to increasing concentrations of NCS
Introduction The maintenance of pancreatic islets in tissue culture represents a suitable method for preservation of viable islets before transplantation into diabetic recipients. In comparison to freshly isolated islets the pool of cultured islets is characterized by a higher percentage of viable and functionally intact islets, since substantially affected tissue (probably as result of ischemia or of the preparation procedure) is lost during the cultivation period. However, to simulate an in vivo like environment for tissue maintenance or even for multiplication of insulin producing cells before their implantation, appropriate culture conditions have to be established. A lot of investigations were done to find a suitable medium for islet culture (Andersson, 1987; Nielsen, 1981) or to test the effects of various nutrients on islet cell function and proliferation (Andersson et al., 1976; Andersson, 1974; Buitrago et al., 1975; Gylfe, 1978; Swenne et al., 1980; Chick, 1973; King and
Chick, 1976; King et al., 1978; Swenne, 1982). Only
islet DNA synthesis and the secretory response to glucose at each time point investigated. The action of FCS, RS and 2 further batches of HS on islet DNA synthesis was analysed by short-term culture. The supplementation of the medium with 50 % FCS and RS resulted in a marked decrease of H-thymidine incorporation into islet DNA similar to that observed with 50 % NCS. In contrast, exposure of islets to different batches of HS never resulted in an inhibition of DNA synthesis. Moreover, the 50% concentration of HS 465 caused a significant stimulation of thymidine incorporation.
few informations (Andersson et al., 1976; King et al., 1977; Ono et al., 1979) are available concerning the serum component of the medium in supporting growth and function of endocrine pancreatic tissue in vitro although serum is known to be essential for maintenance of islets in culture. Up to now no satisfactory conditions for islet culture are described in the literature. Therefore, we comparatively investigated the effect of
different concentrations of NCS, together with FCS, which are very often used sera in islet culture, and of HS on DNA synthesis, insulin content and insulin release of cultured rat islets. In addition, we determined the DNA synthesis of pancreatic islets in response to sera of other
animal sources as well as different batches of human serum.
Material and Methods Collagenase (160 U/mg, Worthington Biochemical Corpora-
tion, Freehold New Jersey) isolated islets from Lewis rats
Downloaded by: Universite Laval. Copyrighted material.
Annemarie Dunger, I. Klöting, W. Besch and H. J. Hahn
35
A. Dunger et al., Pancreatic Islet Cell Culture
(8-12 days of age) were cultured free-floating in TCM 199
Statistical analysis
(Difco Laboratories) containing 10 mmolJl glucose and either
All results are given as mean ± S.E.M. of n different experiments. Statistical significance was checked by Student's t-test.
Laboratories) or fetal calf serum (FCS, Difco Laboratories). Furthermore, pooled serum from adult syngeneic rats (RS) or 3 different batches of human serum (HS, Institut für Impfstoffe
Dessau, Germany) indicated by the number were used. The
Results
cultures were maintained at 37°C for a period of 2, 4 and 8 days in a humidified atmosphere (97 % air, 3% CO2). The medium
In contrast to HS 532, an increase of NCS concentration in the culture medium resulted in a significant decrease of islet DNA synthesis at day 2, 4 and 8 (Fig. 1). A marked
was changed every 2-3 days. For estimation of DNA content and synthesis groups of 40 islets incubated with 74 KBq/ml (methyl-3H)-thymidine
reduction of DNA synthesis during the first 4 days in
(UVVVR-Prag, tSFR, Spec. act. 560 OBq/mmol) for 16h were twice washed and ultrasonically disrupted. Duplicate ahquots of the homogenate were taken for fluorometric assay of DNA content (Kissane and Robins, 1958) and for measurement of radioactivity (Ziegler et al., 1982). The glucose-induced islet insulin release was assayed radioimmunologically in a postcultural incubation (Besch et al., 1987). For this purpose the cultivated islets were firstly incubated in Krebs-Ringer bicarbonate buffer (KRBB, Hahn, 1978) with 5 mmol/l glucose for 30 min at 37°C. After preincubation the islets were transferred into KRBB containing 1.5, 5, lO or
20mM glucose and incubated for 2 h at 37°C. The secretion rates are given in pmoh/islet per 120 min. The insulin content of
pancreatic islets was determined radioimmunologically after their ultrasonication in an acidified solution (Hahn et al., 1974).
[DPMIpg DNA]
culture was evident in islets of both, NCS and HS supplemented culture. A further decrease of islet DNA synthesis was observed when islets were cultured in the presence of NCS, for more than 4 days. In the presence of HS (1 % or 10%) the further decrease in DNA synthesis was completely prevented (Fig. 1). As depicted in Table I the islet insulin content was neither affected by the NCS concentrations used, nor by the cultivation time. However, at the 50% concentration, HS significantly affected the insulin content of the cultured islets. Increased NCS concentrations generated a distinct inhibition of glucose-stimulated insulin secretion investi-
HS 532
NCS
io
20
is 12
Fig. 1 DNA synthesis of rat pancreatic islets cultured for 8 days in the presence of
8-
newborn calf serum (NCS) or human serum (HS 532). Serum concentrations:
4
l%(I/O), l0%(A/A),50%(/D). **
o
p < 0.01 in comparison to the values at day 2 of culture, ++ p p < 0.01 compared to 1% serum ± p < 0.05 compared to 10% serum, ' p < 0.01 compared to l0% FCS and RS, compared to 50% NCS, FCS and RS. The number of experiments is indicated at the bottom of the bars
Downloaded by: Universite Laval. Copyrighted material.
O
A. Dunger et al., Pancreatic Islet Cell Culture
37
parable incorporation of labelled thymidine into islet DNA (as already found with HS532), the exposure of
however, noteworthy that the culture time dependent decrease in glucose-stimulated insulin release was pre-
islets to 50% of HS 465 resulted in a significant stimulation of replication.
vented when NCS was substituted by HS. In short-term experiments it was found that FCS and NCS, belonging to the standard sera being used in islet cell and tissue culture (Federlin and Bretzel, 1981), and RS have an inhibitory effect on DNA synthesis compar-
Serum provides hormonal and/or nutritional factors
able to that of NCS. Interestingly, in contrast to the
which are necessary for growth and survival of cultured cells (Pace and Clements, 1981). Medium for islet culture was originally supplemented with 10% newborn calf serum (Hales and Mimes, 1968). The presence of 10 % serum was thought to be sufficient to sustain the secretory potential of pancreatic B-cells (Ziegler et al., 1983). Our investigations succeeded in demonstrating that the functionality of cultured islets is dependent on the serum concentration if NCS is used as serum source. Increasing the NCS concentration from 1 % via 10% to 50% resulted in a significant inhibition of islet DNA synthesis as well as B-cell function documented by glucose stimulated insulin release after 2, 4 and 8 days of islet culture. In respect of stimulated insulin secretion are the results of special significance since they were obtained in a postcultural incubation period in the absence of serum.
results with NCS, FCS or RS, the experiments with ilS have shown that in no case any inhibitory effect on DNA synthesis was present, indicating that certain factors from
When the same experiments were carried out with medium supplemented with human serum instead of NCS
no marked concentration-dependent serum effect was found. That is, the DNA synthesis of islets cultured in the presence of 1 %, 10 % or 50 % HS was comparable. In addition, islets cultured in the presence of 50% HS for 2
to 8 days exhibited in contrast to NCS cultured islets a significantly higher insulin release when stimulated with glucose. The results clearly demonstrate that growth and functionality of pancreatic islets in tissue culture to a high degree depend on the serum source used.
In contrast to the DNA synthesis and the stimulated insulin release, the islet insulin content was not affected by different amounts of NCS, which may be the result of an unaffected insulin storage though islet secretory responsiveness was influenced by a high NCS concentra-
tion. There are pieces of evidence indicating that a diminished hormone release is often related with a better maintenance of insulin biosynthesis and islet insulin content (Hales and Milner, 1968; Gylfe, 1978; Ziegler et al., 1983). An opposite behaviour may exist in islets cultured in the presence of 50% HS, where an increased insulin secretion is connected with a reduction in islet insulin content.
Independent whether pancreatic islets were maintained in tissue culture medium supplemented with NCS or HS, in both cases a decrease in DNA synthesis with duration of culture time could not be prevented. The fall in DNA synthesis as function of cultivation time is currently a still
unsolved problem, which was also observed by other authors using neonatal and adult islet cells (King et al., 1978; Ono et aI., 1979; De Clercq et al., 1980). It is
HS might have a generally beneficial influence on growth and function of endocrine pancreatic tissue under in vitro
conditions. Therefore, in our opinion HS seems to be a more suitable supplement for the cultivation of pancreatic islets before their use for transplantation. Acknowledgements. The authors wish to thank Mrs. G. Strauch,
Mrs. M. Henkel and Ms. K, Gumm for skillful technical assistance.
References Andersson, A.; Hellerström, C.: Metabolic characteristics of isolated pancreatic islets in tissue culture. Diabetes 21 (1972) 546-554. Andersson, A.; Gunnarsson, R.; Hellerström, C.: Long-
term effects of glucose on insulin release and glucose oxidation by mouse pancreatic islets maintained in tissue culture. Biochem. J. 140 (1974) 377-382. Andersson, A.; Gunnarsson, R.; Hellerström, C.: Longterm effects of a low extracellular glucose concentration on glucose metabolism and insulin biosynthesis and release of mouse pancreatic islets maintained in tissue culture. Acta Endocrinol. (KBH) 82 (1976) 3 18-329. Andersson, A.: Isolated mouse pancreatic islets in culture: Effects of serum and different culture media on the insulin production of the islets. Diabetologia 14 (1987) 397-404. Barnes, G.; Sato, G.: Methods for growth of cultured cells in serum-free medium. Anal. Biochim. 102 (1980)
255-280. Besch, W.; Woltanski, K. P.; Keilacker, H.; Diaz-Alon-
so, J. M.; Schulz, B.; Amendt, P.; Kohnert, K. D.; Ziegler, M.: Measurement of insulin in human sera using a new RIA kit. 1. Insulin determination in the absence of insulin antibodies - conventional assay and micro modifi-
cation. Exp. Clin. Endocrinol. 90 (3), (1987) 264-270.
[71 Buitrago, A.; Gylfe, E.; Hellman, B.; Idahl, L. A.; Johansson, M.: Function of microdissected pancreatic islets cultured in a chemically defined medium. I. Insulin content and release. Diahetologia 11(1975) 535-540.
[81 Chick, W. L.: Beta cell replication in rat pancreatic monolayer cultures. Effects of glucose, tolbutamide, glucocorticoid, growth hormone and glucagon. Diabetes (1973) 687-693. [91 De Clercq, L.; Schmidt, G.; De Laere, P.; Remade, C.: Nuclear events in B-cells of young and senescent rat islets in organ culture. Cell Biol. Int. Rep. 4 (1980) 817-827. [10] Federlin, K.; Bretzel, R. G.: Islet isolation, culture and cryopreservation. In: Intern. Workshop, Giessen 1980. Eds. Federlin, K., Bretzel, R. G., Stuttgart u. a.; Thieme
u.a., 1981.
Downloaded by: Universite Laval. Copyrighted material.
Discussion
Exp. Clin. Endocrino!. 99 (1992) 1
38
E.: Protection of the pancreatic beta-ce]! glucoreceptor mechanism for insulin secretion during cul-
Federlin, K.; Bretzel, R. G., Stuttgart: Thieme 1981, pp.
tuie in chemically defined medium. Biochem. J. (1978) 959-964.
Ono, J.; Takaki, R.; Okano, H.; Fukuma, M.: Long-term culture of pancreatic islet with special reference to the Bcell function. In Vitro 15 (1979) 95-102.
174
Hahn, H. J.; Hellman, B.; Lernmark, B.; Täljedahl, I. B.: Insulinsekretion isolierter Veränderung der Langerhans'scher Inseln durch Behandlung mit Neuraminidase. Acta Bio].
Med. Germ. 32 (1974)
375-383. Hahn, H. J.: Die isolierte Langerhans'sche Insel - ein Modell zur Untersuchung der insulinsekretion in vitro. Endokrinologie 71(1978) 308-324. Hales, C. N.; Milner, D. G.: Cations and the secretion of insulin from rabbit pancreas in vitro. J. Physiol. 199 (1968) 177-187. King, D. L.; Chick, W. L.: Pancreatic beta cell replication
- effects of hexose sugars. Endocrino]. 99 (1976) 1003-1009. King, D. L.; Chick, W. L.; Kitchen, K. C.: Pancreatic
69-80.
Pace, C. S.; Clements, R. S.: Myo-inositol and the maintenance of 3-cell function in cultured rat pancreatic islets. Diabetes 30 (1981) 621-625. Swenne, 1.; Bone, A. J.; Howell, S. L.; Hellerström, C.: Effects of glucose and aminoacids on the biosynthesis of DNA and insulin in fetal rat islets maintained in tissue culture. Diabetes 29 (1980) 686-692. Swenne, I.: The role of glucose in the in vitro regulation of cell cycle kinetics and proliferation of fetal pancreatic 13cells. Diabetes 31(1982) 754-760.
Ziegler, B.; Kohnert, K. D.; Noack, S.; Hahn, H. J.: Effects of 3-isobutyl-1-methyl-xanthine on secretory response, cAMP accumulation and DNA synthesis of islets from postnatal and adult Wistar rats. Acta Bio!. Med.
von Wasieiewski, E.; Chick, E. W., Excepta Medica,
Germ. 41(1982)1171-1177. Ziegler, B.; Wilke, B.; Woltanski, K. P.; Knospe, S.;
Amsterdam 1977, p. 109.
Hahn, H. J.: Protection of insulin secretion by magnesium
King, D. L.; Kitchen, K. C.; Chick, W. L.: Pancreatic
during islet culture: Independence of cAMP or insulin
beta cell replication - relation to insulin secretion. Endocrino]. 103 (1978) 1321-1327. Kissane, J. M.; Robins, E.: The fluorometric measure-
accumulation.
beta cell replication. In: Pancreatic Beta Cell Culture. Eds.
Exp.
Clin.
Endocrino].
82
(1983)
199-207.
ment of deoxyribonucleic acid in animal tissues with
Accepted: 2 October 1990
special reference to the central nervous system. J. Biol. Chem. 233 (1958) 184-188. Nielsen, J. H.: Human pancreatic islets in tissue culture. In: Islet isolation, culture and cryopreservation. Eds.
Correspondence address: Dr. Annemarie Dunger, Institut für Diabetes "Gerhardt Katsch", Greifswalder Str. 11 a, O-2201 Karlsburg, BRD
Downloaded by: Universite Laval. Copyrighted material.
[li] Gylfe,
