Rat islet culture in serum-free medium containing silk protein sericin

Background

The development of islet cultures is desirable for successful clinical islet transplantation. Fetal bovine serum (FBS) has been used as a supplement in islet culture medium, but it may be an unsuitable supplement due recent animal health problems. We have evaluated the use of the silk protein, sericin, derived from Bombyx mori as a replacement for FBS in islet culture medium.

Methods

Twenty rat islets were cultured in medium containing either sericin or FBS, or no supplement, for 14 days, during which time viable islets were counted in order to evaluate islet survival. Insulin secretion was measured in vitro by static incubation on days 3 and 7. In vivo function of cultured islets was tested by syngeneic transplantation. The islets were evaluated histologically and immunohistochemically after culture and transplantation.

Results

Ninety-five percent of islets were viable after culture for 14 days in culture medium supplemented with either FBS or sericin, while no islets survived beyond 7 days in culture without supplement. No significant differences in stimulated insulin secretion were noted between two groups of islets grown on supplemented media. Following transplantation, islets cultured in FBS or sericin rapidly reversed hyperglycemia and maintained normal glycemic control. Histologically, islets cultured with sericin displayed a well-preserved structure and strong insulin staining before and after transplantation.

Conclusion

Serum-free medium containing sericin appears to be useful for islet culture.     

Monolayer culture of adult rat pancreatic islets on extracellular matrix: long term maintenance of differentiated B-cell function

Fragmented islets, obtained by mild overdigestion of the adult rat pancreas with collagenase, readily formed monolayer cultures on dishes coated with extracellular matrix derived from bovine corneal endothelial cells. Contaminating fibroblasts were removed by treatment with sodium ethylmercurithiosalicylate. The cultured islets remained functional for over 6 weeks in primary culture and up to 9 weeks in secondary culture, as indicated by their substantial insulin response to an acute glucose stimulus. Insulin secretion from islet monolayers showed biphasic kinetics. The functional competence of the monolayers was further evaluated by studying glucose-stimulated insulin release in the presence of various modulators of B-cell function. The response to physiological agents such as somatostatin, epinephrine, glucagon, and arginine was retained for at least 4 weeks in culture. The sensitivity to inhibition by somatostatin and epinephrine (ID50 = 10 ng/ml) and that to stimulation by glucagon (ED50 = 3 ng/ml) were similar to or better than those for freshly isolated islets. We have thus obtained a fibroblast-free monolayer culture of pancreatic islets from adult rats containing B-cells that retain normal function for long periods. This experimental system appears ideally suited for studying chronic modulations of islet cell function under controlled in vitro conditions, which can allow the stimulation of normal and diabetic environments.     

Supplementation of islet culture medium with insulin may have a beneficial effect on islet secretory function

Recent reports suggest that apoptosis resulting from the disruption of the normal cell-matrix relationship (anoikis) during islet isolation could lead to a loss of islet tissue in culture. Insulin is known to have a role in cell growth and survival, and this study was undertaken to assess any beneficial effect on islets by supplementing the islet culture medium with insulin. Human and porcine islets were cultured in medium supplemented with 0, 10, 100, and 1,000 ng x mL(-1) insulin. Secretory function was assessed by perifusion at days 1 and 8. The results demonstrated a significant variation in stimulation index between isolations for human islets, but there was no effect relating to the concentration of insulin in the medium or time in culture. For porcine islets, there was a significant (p < 0.001) improvement in secretory function for islets cultured in 10 and 100 ng x mL(-1) insulin, relative to 0 and 1,000 ng x mL(-1) insulin. There was no interisolation variation or effect of time in culture. In conclusion, the secretory function of porcine islets benefited from the addition of 10 to 100 ng x mL(-1) insulin to the culture medium, but interisolation variation in human islet secretory function did not allow any specific effect of the insulin to be determined.     

Islet cell culture in defined serum-free medium

A serum-free, hormone- and factor-supplemented, defined medium was developed which maintains functional activity of primary cultures of adult islet cells and continuous islet cell lines. Medium supplements examined included proteose peptone (PP), transferrin (TrFe), insulin-like growth factor-I (IGF-I), an insulinotropic fragment of human GH [hGH-(6-13)], ethanolamine (EA), phosphoethanolamine (PEA), and human serum albumin (HSA). Glucose-stimulated insulin secretion from islet monolayers was determined after culture in serum-free supplemented medium by either 2- to 3-h static incubations or in a superfusion system with either low (2.8-8.3 mM) or stimulatory (16.7-19.4 mM) glucose concentrations. Glucose-induced secretion was not sustained after 3-4 days of culture in medium supplemented with PP (0.5 mg/ml) and TrFe (10 micrograms/ml) alone. Addition of T3 did not restore glucose-induced secretion, although a combination of T3 and IGF-I or of T3, IGF-I, and PRL (10(-10)-10(-9) M) maintained glucose-induced insulin secretion for 1 month. No beneficial effects were noted with hGH-(6-13). The beta-cell lines HIT-T15 and RINr 1046-38 were used to screen for a potential replacement for PP, the undefined component of the serum-free medium. A combination of HSA (1 mg/ml), EA (50 microM), and PEA (50 microM) provided a replacement for PP. In fact, insulin secretion from HIT-T15 cells was significantly better after culture in medium supplemented with HSA, EA, PEA, TrFe, T3, IGF-I, and PRL than in medium with PP, TrFe, T3, IGF-I, and PRL. HSA (1 mg/ml), EA (50 microM), and PEA (50 microM) in combination with TrFe (10 micrograms/ml), T3 (0.1 nM), IGF-I (0.65 nM), and PRL (1 nM) were used in studies with primary islet monolayers. After 3 weeks of culture islet monolayers were superfused, and the biphasic glucose-induced insulin secretion of cells maintained in defined medium was indistinguishable from the insulin secretion of cells maintained in medium with 5% fetal bovine serum. These studies indicate that adult rat beta-cells retain biphasic glucose-induced insulin secretion after extended culture in defined serum-free medium. The defined medium was also useful for cultures of RINr 1046-38 and HIT-T15 cells and should provide a basis for formulating media for islet cells from higher mammals, including man.     

Autocrine insulin action activates Akt and increases survival of isolated human islets

Aims/hypothesis The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in promoting the survival of pancreatic beta cells. Akt becomes activated in isolated human islets following overnight culture despite significant levels of cell death. The aim of the current study was to identify the cause of the observed increase in Akt phosphorylation in isolated islets. We hypothesised that a factor secreted by the islets in culture was acting in an autocrine manner to activate Akt.Methods In order to identify the stimulus of the PI3K/Akt pathway in culture, we examined the effects of different culture conditions on Akt phosphorylation and islet survival during the immediate post-isolation period.Results We demonstrated that islet-conditioned medium induced Akt phosphorylation in freshly isolated human islets, whereas frequent medium replacement decreased Akt phosphorylation. Following overnight culture, islet-conditioned medium contained significantly elevated levels of insulin, indicating that insulin may be responsible for the observed increase in Akt phosphorylation. Indeed, treatment with an anti-insulin antibody or with inhibitors of insulin receptor/IGF receptor 1 kinase activity suppressed Akt phosphorylation, leading to decreased islet survival. In addition, dispersion of islets into single cells also suppressed Akt phosphorylation and induced islet cell death, indicating that islet integrity is also required for maximal Akt phosphorylation.Conclusions/interpretation Our findings demonstrate that insulin acts in an autocrine manner to activate Akt and mediate the survival of isolated human islets. These findings provide new information on how culturing islets prior to transplantation may be beneficial to their survival by allowing for autocrine activation of the pro-survival Akt pathway.     

Effects of growth hormone, prolactin, and placental lactogen on insulin content and release, and deoxyribonucleic acid synthesis in cultured pancreatic islets

The direct effects of human GH (hGH), ovine pituitary PRL (oPRL), and human chorionic somatomammotropin [placental lactogen (hPL)] on the endocrine pancreas were studied in isolated pancreatic islets maintained in tissue culture. Islets of Langerhans were isolated by collagenase treatment of pancreatic tissue obtained from adult NMRI mice and adult or newborn Wistar rats. The islets were maintained for up to 3 weeks in petri dishes containing tissue culture medium RPMI 1640 supplemented with newborn calf serum or normal human serum. The release of insulin during culture and the islet content of insulin, glucagon, and DNA after culture were determined. The DNA synthesis in the newborn rat islets was evaluated by the incorporation of [methyl-3H]thymidine into islet cell DNA. In mouse islets, 1 micrograms/ml hGH, oPRL, or hPL markedly stimulated insulin release during a 2-week culture period and caused a significant increase in the insulin content in the islets after culture. While hGH did not affect the DNA content in adult mouse islets, an increase was observed in adult rat islets after 2-3 weeks of culture. In islets isolated from 3- to 5-day-old rats cultured for 2 weeks with hGH, there was a 30-40% higher DNA content than that found without hGH. Correspondingly, a significant stimulation of the incorporation of [methyl-3H]thymidine could be demonstrated 24 h after the addition of hGH, oPRL, or hPL. hCG and porcine ACTH had no effect. In conclusion, these results indicate that GH and related hormones have a direct stimulatory effect on both the insulin production and DNA synthesis in isolated islets of Langerhans. Whether the effect is directly on the beta-cell or mediated via locally produced growth factors remains to be determined.     

Preservation of Beta cell function in adult human pancreatic islets for several months in vitro

Summary Islets of Langerhans were isolated from four human kidney donors, aged 16 to 21 years, by the collagenase method described for isolation of rodent islets. So far the human islets have been kept in tissue culture, without attachment, in medium RPMI 1640 supplemented with 10% calf serum for more than 9 months, with preservation of the ability to release insulin in response to glucose stimulation. Replacement of calf serum with serum from normal human subjects did not affect B-cell survival, but resulted in elevated insulin values partly due to lower insulin degrading activity. Thus the described technique presents a valuable tool for studying chronic effects of metabolites and hormones on islet function, as well as for islet storage prior to transplantation into humans.     

Effect of the silk protein sericin on cryopreserved rat islets

Background/purpose

Cryopreservation is necessary for the long-term storage of islet cells and to increase the practicality of clinical islet transplantation. Fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) is generally used as a freezing medium for islet cells. However, FBS should ideally be avoided in cell culture and transplantation because of recent animal health problems, such as bovine spongiform encephalopathy and viral infections. The aim of this study was to develop a new serum-free freezing medium by examining the effectiveness of the silk protein sericin, which is produced by Bombyx mori.

Methods

Islets prepared from Lewis rats by collagenase digestion and Histopaque gradient centrifugation, followed by culture in medium containing 0.1% sericin for 3?days, were cryopreserved using 0.1, 0.5, 1, 2, and 5% sericin or FBS. DMSO (1, 4, 7, 10, and 15%) was added to the medium as a cryoprotectant. After thawing, on days 1, 4, 7, and 14, viable islets were counted in order to evaluate their survival. Insulin secretion was measured in vitro by a static incubation test on day 4. The in vivo function of cultured islets was tested by syngeneic transplantation. Islets were evaluated histologically and immunohistochemically after transplantation.

Results

There were no significant differences between freezing medium containing 1% sericin and that containing 10% FBS with regard to the survival rate of islets and stimulated insulin secretion. Following transplantation, islets rapidly reversed hyperglycemia and maintained normal glycemic control. In addition, the use of 7% DMSO as a cryoprotectant with sericin showed the same results as higher DMSO concentrations with FBS.

Conclusion

The present results showed that serum-free medium containing sericin is useful for both cryopreservation and cell culture.     

Stimulatory effect of serum from diabetic patients on insulin release from mouse pancreatic islets maintained in tissue culture

Summary Islets of Langerhans from NMRI-mice were kept for one week in tissue culture in medium supplemented with human serum obtained from either normal healthy subjects or newly diagnosed juvenile diabetic patients before insulin treatment. Islets cultured in diabetic serum released more inslin than islets cultured in normal serum, whether tissue culture medium 199 with 5.5–8.3 mmol/1 glucose and 10% serum, or culture medium RPMI1 640 with 11 mmol/1 glucose and 0.5% serum were used. Islets kept for one week in culture with diabetic serum did not show any decrease in DNA content or glucose induced insulin secretion and biosynthesis. It is concluded that serum from newly diagnosed insulin dependent diabetic patients stimulates insulin release from isolated mouse islets kept in tissue culture. The underlying mechanism is unknown.     

Dispersed adult rat pancreatic islet cells in culture: A, B, and D cell function

The availability of suitably characterized dispersed islet cell preparations may assist in studies of islet function. Since freshly dispersed adult rat islet cells failed to respond appropriately to secretagogues (no alteration in insulin, glucagon, or somatostatin release after glucose change; modest response to IBMX), these cells were established in primary monolayer culture. We then tested the hypothesis that islet function is at least partially determined by islet structure. B cells which had attached to Petri dishes during a culture period of four days were well preserved at the ultrastructural level, with mitochondria clustered at the cell face attached to the Petri dish and secretory granules concentrated towards the portion of the cell facing the medium. Since it was not possible to estimate cellular hormone content or hormone release as a function of the number of specific types of cells, fractional rates of release and hormone content ratios were compared with those for intact islets maintained in culture in parallel. Whereas the ratio of somatostatin:insulin content was similar for islets and cells (approximately 0.7:100), the dispersed cell population appeared depleted in glucagon (glucagon:insulin ratios being 17:100 for islets and 4:100 for cells) reflecting either degranulation or relative loss of A cells. In contrast to the lack of responsiveness seen with freshly dispersed islet cells, the cultured cells released insulin in response to glucose and glucose plus IBMX in a fashion comparable to that seen with cultured islets. Proinsulin biosynthesis (incorporation of [3H] leucine) was higher in cultured cells than islets. Somatostatin release was lower from dispersed cells than from islets while the opposite was true for glucagon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monolayer culture of neonatal pig pancreatic islet cells

Pancreatic islet cells from pigs (3-12 days old) were maintained in monolayer culture at 37 degrees C for greater than 30 days. Cultures were maintained in enriched Medium 199 with 5% fetal calf serum and with the glucose concentration being cycled between 11.10 and 27.75 mmol/l every 2 days. The presence of B cells in the cultures was demonstrated by insulin secretion into the culture medium, cell staining with aldehyde fuchsin and immunofluorescence. A cells in the cultures were monitored by glucagon secretion into the medium. The absence of exocrine cells was determined by the inability to detect alpha-amylase in the culture medium. Insulin secretion determined over a 34-day period, was found to diminish after 12 days in culture. The fall in levels of extractable B cell insulin with time correlated with the levels of insulin secreted into the medium. Response to short term glucose stimulation (2 h in serum-free medium) was determined on days 6, 10 and 12 in culture. Morphological changes appeared after 25 days in culture, although on day 33 insulin secretion was 62.5 microU X culture-1 X day-1. DNA synthesis (determined by 3H-thymidine incorporation and autoradiography) was demonstrated (day 12, 39.4 +/- 0.9 labelled cells/culture). These findings suggest that pig islet cell monolayer cultures are a useful tool for morphological and biochemical studies and that neonatal pig islets are a potential source of material for transplantation.     

Inhibitory effects of interleukin 1 on insulin secretion, insulin biosynthesis, and oxidative metabolism of isolated rat pancreatic islets

Recent observations suggest a role for interleukin 1 (IL-1), a macrophage-derived cytokine, in the autoimmune B cell destruction, which is observed in type 1 diabetes. In the present study we have investigated the effects of IL-1 and two other cytokines, namely tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma) on the pancreatic B cell paying particular attention to insulin production and glucose metabolism. Rat pancreatic islets were isolated and kept in tissue culture for 5 days. The islets were subsequently transferred to media containing medium RPMI 1640 plus 0.5% human serum with or without additions of human recombinant preparations of either IL-1 (25 U/ml), TNF (1000 U/ml), or IFN-gamma (500 U/ml), and cultured for another 48 h. After the culture period the islets were subjected to light microscope examination and different functional tests in short-term incubations in the absence of cytokines. IL-1 was found to reduce insulin release in culture and totally inhibit glucose-stimulated insulin release in short-term incubations. Islet (pro)insulin biosynthesis, glucose oxidation, and oxygen uptake at 16.7 mM glucose were partially inhibited by IL-1. The DNA content of islets cultured with IL-1 was decreased and may partly explain these latter findings. However, inhibition of glucose oxidation could not be seen in islets exposed to IL-1 in short-term experiments only. By light microscopy there were marked signs of degeneration in IL-1 treated islets. TNF and IFN-gamma were essentially without effect on islet morphology or function. The results of this study indicate that IL-1 may be cytotoxic to islet B cells. The primary toxic action of IL-1 seems to involve factors other than an impaired islet glucose metabolism.     

Perturbation of hormone storage and release induced by cyproheptadine in rat pancreatic islets in vitro.

The effects of cyproheptadine (CPH) added in vitro were studied in rat pancreatic islets maintained in culture medium. CPH added over 6 days resulted in either an increase (5 X 10(-7) M CPH) or a marked, but reversible decrease (5 X 10(-5 M) in insulin content of islets when related to that of controls. At both concentrations, however, total recoverable insulin from islets, cells detached from islets, and medium was decreased relative to control cultures. The increased insulin content observed after 6 days with 5 X 10(-7) M CPH may be explained by the partial inhibition of insulin release, preventing the normally occurring early drop in insulin content of control islets. The decreased total recoverable insulin in the culture system with 5 X 10(-5) M CPH (17% of the initial insulin content of the islets placed into the CPH-containing culture medium) was not acounted for by the combined effects of insulin degradation in the culture medium and inhibition of insulin biosynthesis. Together and by exclusion these data suggest increased insulin degradation within beta-cells as a result of exposure to 5 X 10(-5) M CPH. Since increased intracellular insulin degradation was not found at 5 X 10(-7) M CPH, the data suggest that only severe inhibition of insulin release (5 X 10(-5) M CPH) increases intracellular insulin degradation. CPH added in vitro irreversibly decreased islet glucagon content; the data suggest that these effects are due to alterations in the physical properties of the peripheral cell layers of isolated islets. Studies with 5 X 10(-5) M CPH on the biosynthesis of insulin immunoreactive material failed to link the appearance of flocculent material in dilated cisternae of the rough endoplasmic reticulum (observed by electron microscopy) with accumulation of an immunoreactive biosynthetic precursor for insulin.     

Effects of stimulation of adenylate cyclase and protein kinase-C on cultured fetal rat B-cells

To study the maturation of fetal pancreatic B-cells, cell suspensions of pancreas from 21.5-day-old fetuses were cultured in RPMI medium containing 10 mM glucose. Forskolin (1 microM), used to stimulate adenylate cyclase, moderately delayed the neoformation of islets, slightly accelerated the proliferation of endocrine cells, and considerably increased insulin release by the cultures. The latter increase was not completely compensated for by the stimulation of insulin biosynthesis, so that the islet insulin content was decreased. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 25 nM), used to stimulate protein kinase-C, had little effect on the evolution of the cultures, but increased insulin release. This increase was almost compensated for by the stimulation of insulin biosynthesis. After 9-10 days of culture, insulin release in response to 15 mM glucose or 10 mM leucine was studied with perifused islets. In control islets, glucose produced a sustained increase in insulin release, which, however, was 6-fold smaller than that produced by leucine. Addition of forskolin or TPA to the perifusion medium markedly amplified the response to glucose without causing a biphasic pattern of release. In islets cultured with forskolin, the insulin response to glucose or leucine was decreased, largely owing to the lower insulin stores. In islets cultured with TPA, the insulin response to glucose or leucine was also decreased, but these differences cannot be explained simply by changes in insulin content. Neither treatment affected the kinetics of release. In conclusion, acute stimulation of adenylate cyclase or protein kinase-C markedly increased insulin release from fetal islets without causing an adult-like biphasic pattern of secretion. Chronic stimulation did not accelerate maturation of B-cells.     

In vitro restoration of insulin production in islets from adult rats treated neonatally with streptozotocin

The aim of this study was to evaluate if the impaired insulin production of the beta-cell deficient islet organ of neonatally streptozotocin (SZ) injected rats is caused by exposure of the beta-cells to a long-lasting functional demand in vivo or a persistent toxic effect of the drug. For this purpose islets were isolated from adult rats which had received an ip injection of SZ (100 mg/kg body weight) on postnatal day 1 or from control rats receiving the solvent only. The islets used were either fresh or after culture for 2, 7, or 14 days in RPMI 1640 supplemented with 5.6, 11.1, or 16.7 mM glucose. After the various culture periods determinations were performed of the islet contents of insulin and insulin mRNA and the rates of (pro)insulin biosynthesis and insulin release. Freshly isolated islets from SZ-treated rats exhibited lower contents of insulin and insulin mRNA, a lower rate of (pro)insulin biosynthesis, and an impaired glucose-sensitive insulin release. Similar results were obtained after 2 days of culture, in each of the glucose concentrations. After 7 days of culture, however, the content of insulin mRNA and the rate of (pro)insulin biosynthesis of the SZ islets were restored to the control levels. When such islets were cultured for 7 days in 5.6 mM glucose, they exhibited a glucose-sensitive insulin release similar to that of the control islets. A difference in the insulin release between the two groups nevertheless persisted after culture for 7 days at either 11.1 and 16.7 mM glucose. Also, after 14 days of culture at 16.7 mM glucose there was an impaired glucose-sensitive insulin release from SZ islets, while islets cultured at 11.1 mM glucose showed a glucose-stimulated insulin release similar to that of the controls. The present data indicate that, as far as storage and biosynthesis of insulin is concerned, the functional aberrations observed in the freshly isolated SZ-islets did not reflect a permanent cytotoxic damage. The persistent impairment of insulin release after culture at 16.7 mM glucose may reflect either an injurious effect of the mildly diabetic metabolism in vivo or of the neonatal streptozotocin injection.     

Co-localization of nestin and insulin and expression of islet cell markers in long-term human pancreatic nestin-positive cell cultures

Strategies to differentiate progenitor cells into beta cells in vitro have been considered as an alternative to increase beta cell availability prior to transplantation. It has recently been suggested that nestin-positive cells could be multipotential stem cells capable of expressing endocrine markers upon specific stimulation; however, this issue still remains controversial. Here, we characterized short- and long-term islet cell cultures derived from three different human islet preparations, with respect to expression of nestin and islet cell markers, using confocal microscopy and semi-quantitative RT-PCR. The number of nestin-positive cells was found to be strikingly high in long-term cultures. In addition, a large proportion (49.7%) of these nestin-positive cells, present in long-term culture, are shown to be proliferative, as judged by BrdU incorporation. The proportion of insulin-positive cells was found to be high in short-term (up to 28 days) cultures and declined thereafter, when cells were maintained in the presence of 10% serum, concomitantly with the decrease in insulin and PDX-1 expression. Interestingly, insulin and nestin co-expression was observed as a rare event in a small proportion of cells present in freshly isolated human islets as well as in purified islet cells cultured in vitro for long periods of time. In addition, upon long-term subculturing of nestin-positive cells in 10% serum, we observed reappearance of insulin expression at the mRNA level; when these cultures were shifted to 1% serum for a month, expression of insulin, glucagon and somatostatin was also detected, indicating that manipulating the culture conditions can be used to modulate the nestin-positive cell's fate. Attempts to induce cell differentiation by plating nestin-positive cells onto Matrigel revealed that these cells tend to aggregate to form islet-like clusters, but this is not sufficient to increase insulin expression upon short-term culture. Our data corroborate previous findings indicating that, at least in vitro, nestin-positive cells may undergo the early stages of differentiation to an islet cell phenotype and that long-term cultures of nestin-positive human islet cells may be considered as a potential source of precursor cells to generate fully differentiated/ functional beta cells.     

Combination therapy with epidermal growth factor and gastrin induces neogenesis of human islet {beta}-cells from pancreatic duct cells and an increase in functional {beta}-cell mass

Pancreatic islet transplantation is a viable treatment for type 1 diabetes, but is limited by human donor tissue availability. The combination of epidermal growth factor (EGF) and gastrin induces islet beta-cell neogenesis from pancreatic exocrine duct cells in rodents. In this study we investigated whether EGF and gastrin could expand the beta-cell mass in adult human isolated islets that contain duct as well as endocrine cells. Human islet cells were cultured for 4 wk in serum-free medium (control) or in medium with EGF (0.3 mug/ml), gastrin (1.0 mug/ml), or the combination of EGF and gastrin. beta-Cell numbers were increased in cultures with EGF plus gastrin (+118%) and with EGF (+81%), but not in cultures with gastrin (-3%) or control medium (-62%). After withdrawal of EGF and gastrin and an additional 4 wk in control medium, beta-cell numbers continued to increase only in cultures previously incubated with both EGF and gastrin (+232%). EGF plus gastrin also significantly increased cytokeratin 19-positive duct cells (+678%) in the cultures. Gastrin, alone or in combination with EGF, but not EGF alone, increased the expression of pancreatic and duodenal homeobox factor-1 as well as insulin and C peptide in the cytokeratin 19-positive duct cells. Also, EGF plus gastrin significantly increased beta-cells and insulin content in human islets implanted in immunodeficient nonobese diabetic-severe combined immune deficiency mice as well as insulin secretory responses of the human islet grafts to glucose challenge. In conclusion, combination therapy with EGF and gastrin increases beta-cell mass in adult human pancreatic islets in vitro and in vivo, and this appears to result from the induction of beta-cell neogenesis from pancreatic exocrine duct cells.     

Use of non-human plasma for in vitro cultivation and antimalarial drug susceptibility testing of Plasmodium falciparum

Viability, growth rate, and chemotherapeutic susceptibility of the CDC/Indochina III, CDC/Sierra Leone I, and FCR-3 (Subline F-86) isolates of Plasmodium falciparum grown continuously in RPMI 1640 medium supplemented with goat, horse, porcine, bovine, or ovine plasma were evaluated. Results were compared to those obtained from parallel cultures maintained in medium supplemented with non-immune human plasma. Only media supplemented with goat or horse plasma supported significant continuous multiplication of the isolates. Medium supplemented with either ovine or porcine plasma supported continuous multiplication of the CDC/Indochina III isolate, but not the FCR-3 isolate. Medium supplemented with bovine plasma did not support continuous growth of any of the isolates tested. The light microscopic appearance of the isolates during and after continuous culture in medium supplemented with either goat or horse plasma was identical to that of the control parasites maintained in medium supplemented with human plasma. There were no statistically significant differences in the susceptibility to antimalarial drugs of the culture lines maintained in medium supplemented with either human or goat plasma.     

Dynamic perifusion to maintain and assess isolated pancreatic islets

Advances in human islet transplant techniques are hampered by the inability to assess the quality of isolated islets. A flow culture system was developed to perifuse isolated pancreatic islets or cultured beta-cell lines in order to continuously and noninvasively assess cell function and viability with high kinetic resolution. Continuous perifusion of large amounts of islet tissue as isolated from human pancreata enables the use of noninvasive measurement technologies not previously applied to islets. To compare dynamic perifusion of tissue at high density with conventional static cultures, we measured glucose-stimulated insulin secretion and O2 consumption of large amounts of INS-1 cells (45-65 x 10(6)) to confirm that perifused cells were adequately supplied with oxygen and nutrients and remained functionally responsive. Isolated human and monkey islets that were perifused for 18 h showed robust biphasic insulin secretion in response to a step increase in glucose, demonstrating the ability to maintain islets and the high kinetic resolution of the system. As an example of the system's ability to resolve multiple indicator dilution experiments, the retention of [3H]-glibenclamide was kinetically distinguished from that of an extracellular marker. In summary, the perifusion system is able to maintain healthy cells, assess insulin secretion and metabolite fluxes such as oxygen consumption and lactate production, and characterize the kinetics of the interaction between radiopharmaceuticals and islet cells. The ability to systematically assess the metabolic and functional viability of islets will facilitate the optimization of islet isolation procedures, islet transplantation studies, and islet storage methodologies.