Nonenzymic in vitro isolation of perinatal islets of Langerhans

Summary We have developed a method to circumvent the use of exogenous proteolytic enzymes in the isolation of islets of Langerhans from the perinatal rodent pancreas. Advantage is taken of the propensity of fibroblastlike cells to attach and migrate on polystyrene at low-serum concentrations (5%). In contrast, at this serum level, rat islet epithelial cells tend not to adhere to the substrate. At 3 d of culture, islets are visible at the edges of the explants. With further fibroblast outgrowth the majority of islets are freefloating by 7 d. Simple agitation of the medium and centrifugation yields approximately 50 μg of islet tissue per perinatal pancreas. Further purification of the islets can be obtained by subculture. Rat islets can be maintained in this manner for several months in Medium F12 supplemented with 25% horse serum in an atmosphere of 5% CO₂ and air at 37° C. Hormone content of the islet tissue remains constant during prolonged subculture and such islets continue to exhibit appropriate insulin and glucagon responses to glucose and theophylline. The morphological integrity of the endocrine cells within the cultured islets was confirmed by immunocytochemistry and ultrastructural study. Nonendocrine cells are not identifiable within the long-term cultured islets. This research was supported in part by Grants AM 19899 and HD 00412 from the National Institutes of Health, Bethesda, MD, and grants from the American Diabetes Association, Minnesota Affiliate. Portions of this work were presented at the Thirty-third Annual Meeting of the Tissue Culture Association, held in San Diego, California, June 6–10, 1982.     

A,B, D cells and A fourth cell type in longterm cultures of fetal rat pancreas

Summary Whole pancreases from fetal rats of 13 days and 18 days gestation were explanted onto rayon grids and grown in organ culture. Cultures were fixed in Bouin’s fluid, sectioned and stained with the fluorescent antibody techniques for glucagon and insulin, aldehyde fuchsin for B cells, pseudoisocyanin for D cells and a silver technique for the fourth cell type. The 13-day explants were fixed after 10 days in culture. A, B and D and the fourth cell type were seen, indicating that precursors of all four endocrine cell types must be present in the fetal pancreas shortly after the formation of the pancreatic bud (11 days). Further, the presence of these four cell types in the walls of tubules in these cultures indicates the tubules as the site of origin of all the endocrine tissue. The 18-day explants were collected every other day of culture from 2 to 30 days in a long-term experiment. A number of large islets with well granulated B cells was still present after 30 days of culture. The relative abundance of cell types at different stages was estimated as follows: 18-day fetal controls, A>B=4>D; after 2 to 10 days in culture, B>A⩾4>D; after 18 to 30 days in culture, B>D>A>4.     

Monolayer culture of pancreatic islets from the syrian hamster

Summary Pancreatic islets of Langerhans of the Syrian hamster were maintained in culture for as long as 43 wk. Islets were prepared by collagenase/hyaluronidase digestion of minced pancreas. The islets quickly attached to the plastic culture flasks and lost their spherical form as they flattened out to form circular monolayers. Few fibroblastoid cells were observed. As outward migration continued, the islets became vacoulated with the ultimate formation of monolayer rings. Throughout the culture period the beta cells continued to synthesize and secrete insulin. Furthermore, the cells maintained a responsiveness to glucose stimulation with increased rates of hormone secretion in the presence of elevated concentrations of the sugar. These studies demonstrate the suitability of Syrian hamster islets for studies involving long-term culture. This work was supported by Grants CA26651 and AM31669 from the National Institutes of Health, DHHS: by Grant 82-10 from the Hospital for Sick Children Foundation (Toronto); and by the Ontario Cancer Treatment and Research Foundation. The authors acknowledge the expert technical assistance of Anoja Giles.     

Review of vitreous islet cryopreservation: Some practical issues and their resolution

Transplantation of pancreatic islets for the treatment of diabetes mellitus is widely anticipated to eventually provide a cure once a means for preventing rejection is found without reliance upon global immunosuppression. Long-term storage of islets is crucial for the organization of transplantation, islet banking, tissue matching, organ sharing, immuno-manipulation and multiple donor transplantation. Existing methods of cryopreservation involving freezing are known to be suboptimal providing only about 50% survival. The development of techniques for ice-free cryopreservation of mammalian tissues using both natural and synthetic ice blocking molecules, and the process of vitrification (formation of a glass as opposed to crystalline ice) has been a focus of research during recent years. These approaches have established in other tissues that vitrification can markedly improve survival by circumventing ice-induced injury. Here we review some of the underlying issues that impact the vitrification approach to islet cryopreservation and describe some initial studies to apply these new technologies to the long-term storage of pancreatic islets. These studies were designed to optimize both the pre-vitrification hypothermic exposure conditions using newly developed media and to compare new techniques for ice-free cryopreservation with conventional freezing protocols. Some practical constraints and feasible resolutions are discussed. Eventually the optimized techniques will be applied to clinical allografts and xenografts or genetically-modified islets designed to overcome immune responses in the diabetic host.     

Method for isolation of pancreatic blood vessels,their culture and coculture with islets of langerhans

The only cure available for Type 1 diabetes involves the transplantation of islets of Langerhans isolated from donor organs. However, success rates are relatively low. Disconnection from vasculature upon isolation and insufficient rate of revascularization upon transplantation are thought to be a major cause, as islet survival and function depend on extensive vascularization. Research has thus turned toward the development of pretransplantation culture techniques to enhance revascularization of islets, so far with limited success. With the aim to develop a technique to enhance islet revascularization, this work proposes a method to isolate and culture pancreas-derived blood vessels. Using a mild multistep digestion method, pancreatic blood vessels were retrieved from whole murine pancreata and cultured in collagen Type 1. After 8 days, 50% of tissue explants had formed anastomosed microvessels which extended up to 300 μm from the explant tissue and expressed endothelial cell marker CD31 but not ductal marker CK19. Cocultures with islets of Langerhans revealed survival of both tissues and insulin expression by islets up to 8 days post-embedding. Microvessels were frequently found to encapsulate islets, however no islet penetration could be detected. This study reports for the first time the isolation and culture of pancreatic blood vessels. The methods and results presented in this work provide a novel explant culture model for angiogenesis and tissue engineering research with relevance to islet biology. It opens the door for in vivo validation of the potential of these pancreatic blood vessel explants to improve islet transplantation therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2745, 2019.     

Mitochondrial respiration in heart, liver, and kidney of copper-deficient rats

Morphological observations in some tissues indicate that dietary copper deficiency results in structural damage to mitochondria. The purpose of this study was to determine whether mitochondrial function is impaired as well. Male, weanling Sprague-Dawley rats were fed diets deficient or sufficient in copper for 4 weeks. Mitochondria were isolated from heart, liver, kidney cortex, and kidney medulla. P/O ratio, state 3 and state 4 respiration rates (oxygen consumed in the presence and absence of ADP, respectively), and acceptor control index (ratio of state 3:state 4) were determined using succinate or pyruvate/malate as substrate. State 3 respiration rate in mitochondria from copper-deficient hearts and livers was lower than in mitochondria from copper-sufficient hearts. Copper deficiency reduced the state 4 respiration rate only in cardiac mitochondria. Neither respiration rate was affected by copper deficiency in mitochondria from kidney medulla or cortex. P/O ratio was not significantly affected by copper deficiency in any tissue examined. Acceptor control index was reduced only in liver mitochondria. The observed decreases in respiration rates are consistent with decreased cytochrome c oxidase activity, shown by others to occur in mitochondria isolated from hearts and livers of copper-deficient rats.     

Enzymatic,metabolic and secretory perturbations in pancreatic islets of thyroidectomized rats

In thyroidectomized rats, the activity of FAD-linked glycerophosphate dehydrogenase was severely diminished in liver homogenates but not affected significantly in pancreatic islet homogenates, whilst the activity of 2-ketoglutarate dehydrogenase was decreased modestly in both liver and islet homogenates. Likewise, in intact islets of thyroidectomized rats, the generation of³HOH from [2-³H]glycerol was not decreased, and the ratio between oxidative and total glycolysis not significantly lower than in islets from sham-operated rats, at least in the presence of a high concentration of D-glucose. Nevertheless impaired oxidation of both D-[3,4-¹⁴C]glucose and D-[6-¹⁴C]glucose was observed in islets of thyroidectomized rats, the relative magnitude of such a decrease being more pronounced at a low than at a high D-glucose concentration. Such metabolic anomalies coincided with a lower level of plasma insulin and a decreased output of insulin by islets incubated at low (2·8 mM ), but not higher, concentrations of D-glucose. It is concluded that hypothyroidism does not mimic the deficiency in islet FAD-linked glycerophosphate dehydrogenase activity found in rats with inherited or acquired non-insulin-dependent diabetes.     

Insulin, glucagon and somatostatin in fetal rat islets maintained free-floating in culture: immunocytochemistry and radioimmunoassay.

Fetal rat islets maintained free-floating in tissue culture represent a source of B-cells. Because we recently noted the occurrence of other cell types during long-term tissue culture, this in vitro model was used to examine the possible development of non B-cells. The changes in the numbers and percentages of B, A and D-cells in vitro were estimated by counting the hormone-positive cells after immunocytochemical staining. Insulin, glucagon, and somatostatin contents were determined in extracts of the cultured tissue. The experiments described here showed that the cultured islets maintained their viability over a two-week culture period, as evidenced by the increase of both the number of B-cells per islet and the DNA content per islet. During the first few days of culture, immunocytochemically stained free-floating islets indicated the presence of rare A- and D-cells at the periphery of B-cells; thereafter, numerous A- and D-cells were seen interdigitating with B-cells. Expressed per islet, the number of A- and D-cells increased during the culture; within the endocrine cell population, the percentage of these cells increased with time, at the expense of the percentage of B-cells. The glucagon and somatostatin contents of the free-floating islets were also increased. These converging observations suggest that additional non B-cells may have been produced by free-floating islets during long-term tissue culture.     

L-glutamine and palmitate catabolism in pancreatic islets from rats depleted in long-chain polyunsaturated omega 3 fatty acids

The catabolism of D-glucose was recently found to be impaired in pancreatic islets from rats depleted in long-chain polyunsaturated omega3 fatty acids. The specificity of this alteration was now investigated by characterizing the oxidative fate of endogenous nutrients in islets preincubated with either L-[U-14C]glutamine or [U-14C]palmitate and then incubated variously in the absence of D-glucose, presence of the hexose or presence of metabolic poisons. Relative to their radioactive content after preincubation, the production of 14CO2 by islets prelabelled with [U-14C]glutamine was higher in omega3-depleted rats than control animals. The enhancing action of D-glucose upon such production was impaired, however, in the omega3-depleted rats. The net uptake of 14C-palmitate and absolute value for 14CO2 output were both increased in omega3-depleted rats, whilst the ratio between 14CO2 output and islet radioactive content was decreased in the same animals. The inhibition of 14CO2 production by metabolic poisons was comparable in all cases. These results are consistent with recent findings on such items as the availability of endogenous amino acids and uptake of unesterified fatty acids in extrapancreatic sites of the omega3-depleted rats. They also support the view that the alteration of D-glucose metabolism in the islets of the latter animals may be attributable, in part at least, to alteration of glucokinase kinetics by high intracellular acyl-CoA levels.     

Entrapment of dispersed pancreatic islet cells in CultiSpher-S macroporous gelatin microcarriers: Preparation, in vitro characterization, and microencapsulation.

Immunoprotection of pancreatic islets for successful allo- or xenotransplantation without chronic immunosuppression is an attractive, but still elusive, approach for curing type 1 diabetes. It was recently shown that, even in the absence of fibrotic overgrowth, other factors, mainly insufficient nutrition to the core of the islets, represent a major barrier for long-term survival of intraperitoneal microencapsulated islet grafts. The use of dispersed cells might contribute to solve this problem due to the conceivably easier nutritional support to the cells. In the present study, purified bovine islets, prepared by collagenase digestion and density gradient purification, and dispersed bovine islet cells, obtained by trypsin and DNAsi (viability > 90%), were entrapped into either 2% (w/v) sodium alginate (commonly used for encapsulation purposes) or (dispersed islet cells only) macroporous gelatin microcarriers (CulthiSpher-S, commonly used for the production of biologicals by animal cells). Insulin release studies in response to glucose were performed within 1 week and after 1 month from preparation of the varying systems and showed no capability of dispersed bovine islet cells within sodium alginate microcapsules to sense glucose concentration changes. On the contrary, bovine islet cells entrapped in CulthiSpher-S microcarriers showed maintained capacity of increasing insulin secretion upon enhanced glucose concentration challenge. In this case, insulin release was approximately 60% of that from intact bovine islets within sodium alginate microcapsules. MTT and hematoxylineosin staining of islet cell-containing microcarriers showed the presence of viable and metabolically active cells throughout the study period. This encouraging functional data prompted us to test whether the microcarriers could be immunoisolated for potential use in transplantation. The microcarriers were embedded within 3% sodium alginate, which was then covered with a poly-L-lysine layer and a final outer alginate layer. Maintained insulin secretion function of this system was observed, which raises the possibility of using microencapsulated CulthiSpher-S microcarriers, containing dispersed pancreatic islet cells, in experimental transplantation studies.     

小鼠原代呼吸道上皮细胞分离培养的新方法

现有的呼吸道上皮细胞系大多来源于肿瘤组织或成系时与肿瘤细胞融合,其生物学行为与正常呼吸道上皮细胞差异较大.为更准确反映呼吸道疾病条件下该类细胞的生物学效应,本文对小鼠呼吸道上皮细胞分离的新技术和培养方法进行了探索.利用链霉蛋白酶消化法分离获得小鼠呼吸道上皮细胞,利用特殊的完全培养基和Ⅰ型胶原包被的培养皿进行原代培养.镜...     

Organ culture of the islets of Langerhans from young and senescent rats

Summary The B-cells of the endocrine pancreas constitute an adequate model for in vitro study of the aging process in highly differentiated cells. In the present study, collagenase-isolated islets of Langerhans from young and senescent rats were cultured up to 28 days. The response of the B-cells to the stimulatory conditions of the culture medium involved the nucleus, ribosomes, endoplasmic reticulum, Golgi apparatus, and secretory granules. Correlated data from light microscopy, electron microscopy, and insulin radioimmunoassay show that the differentiation and function of senescent B-cells are maintained in culture, as it has been proven for the B-cells of younger animals. On the other hand, signs of cytological deficiency not directly concerned with the specific function of B-cells were observed: abnormal mitochondria and lysosomes are more numerous in the senescent B-cells. The proliferative capacity of the B-cells of aged rats is reduced. Send offprint requests to: Université Catholique de Louvain, Unité de Morphologie animale, Place Croix du Sud, 5, B 1348 Louvain-la-Neuve, BelgiumM.C.M. is currently associated with the Unité d'Histologie, Université de Louvain     

Labelling of lipids by D-[1-14C]glucose, D-[6-14C]glucose and D-[3-3H]glucose in pancreatic islets from normal and GK rats

In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-¹⁴C]glucose than D-[6-¹⁴C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired ¹⁴C/³H ratio found in islets exposed to both D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose and D-[3-³H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-¹⁴C]glucose. The labelling of islet lipids by D-[6-¹⁴C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphate generated from the C₁-C₂-C₃ moiety of D-glucose than D-glyceraldehyde-3-phosphate produced from the C₄-C₅-C₆ moiety of the hexose, (iii) that only a limited amount of [3-³H]glycerone 3-phosphate generated from D-[3-³H]glucose is detritiated at the triose phosphate isomerase level before being converted to L-glycerol-3-phosphate, and (iv) that a rise in D-glucose concentration results in an increased labelling of islet lipids, this phenomenon being somewhat more pronounced in the case of D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose rather than D-[3-³H]glucose.     

Interference of D-mannoheptulose with D-glucose phosphorylation,metabolism and functional effects: Comparison between liver,parotid cells and pancreatic islets

D-mannoheptulose is currently used as a tool to inhibit, in a competitive manner, D-glucose phosphorylation, metabolism and functional effects in the pancreatic islet B-cell. In order to better understand the mode of action of the heptose, we have explored its effect upon D-glucose phosphorylation in liver, parotid cells and islet homogenates, this allowing to characterize the interference of the heptose with glucokinase and/or hexokinase. The effect of D-mannoheptulose upon the metabolism of D-glucose was also examined in both intact parotid cells and pancreatic islets. Last, the effect of D-mannoheptulose upon glucose-stimulated insulin release was reinvestigated over large concentration ranges of both the heptose and hexose. The experimental data revealed a mixed type of D-mannoheptulose inhibitory action upon D-glucose phosphorylation, predominantly of the non-competitive and competitive type, in liver and parotid homogenates, respectively. Despite efficient inhibition of hexose phosphorylation in both parotid cell and islet homogenates, the heptose suppressed the metabolic and functional responses to D-glucose only in pancreatic islets, whilst failing to affect adversely D-glucose catabolism in parotid cells. These findings suggest that factors such as the intracellular transport and availability of the heptose may interfere with the expression of its antagonistic action upon D-glucose metabolism.     

Golgi fractions from livers of control and ethanol-intoxicated rats. Enzymic and morphologic properties following rapid isolation

Following rapid isolation, it has been found that Golgi apparatus from ethanol-intoxicated animals contain high levels of galactosyltransferase but also detectable glucose-6-phosphatase and microsomal esterase, as well as 5′-nucleotidase activity. In experiments carried out in parallel on littermate animals but without intoxication, similar recoveries and specific activities of the four enzymes were observed. Morphologic analysis of Golgi fractions isolated from control animals demonstrated no striking morphologic difference to those from the ethanol-intoxicated animals. Indeed, using galloyl glucoselead staining techniques to mark the lipoprotein particles in situ, it was found that all Golgi apparatus of hepatocytes from control animals were marked by very low density lipoprotein particles. It is therefore concluded that within the limits of the present analyses, Golgi fractions isolated from control animals are as valid as those isolated from ethanol-intoxicated rats.     

Glucagon production by cultured pancreatic islets: Effects of different culture conditions and media

Summary Various conditions for tissue culture of collagenase-isolated mouse pancreatic islets were studied for their effects on the glucagon production of the cultured specimens. Culture media containing heat-treated bovine calf serum degraded [¹²⁵I]glucagon to a much less extent than those supplemented with untreated serum. Addition of aprotinin to the heattreated serum gave a further reduction of the [¹²⁵I]glucagon degradation in the culture medium. A similar supplementation of Medium 199, used for culture of isolated islets, resulted in the most extensive glucagon accumulation in the culture medium. Islets cultured free-floating or attached to the bottom of the culture dishes contained similar amounts of glucagon. However, the free-floating islets released less glucagon when tested in short-term experiments performed at the end of the 1 wk culture period. A comparison between different culture media showed that islets cultured in RPMI-1640 had the highest glucagon content and released most glucagon to the culture medium. Moreover, these islets responded most actively to an acute arginine challenge at the end of the culture period. The present data suggest that the optimal conditions for culture of isolated islets aimed at studies of glucagon production may be obtained by using a culture medium consisting of RPMI-1640 supplemented with both a proteinase inhibitor and heat-inactivated serum. This work was supported by grants from the Swedish Medical Research Council (12X-109), the Nordic Insulin Fund, and the Swedish Diabetes Association.     

Characterization of human islet-like structures generated from pancreatic precursor cells in culture

This study addresses the characterization of human islet-like structures generated from a newly discovered sparse population of precursor cells (Petropavlovskaia and Rosenberg, 2002) in the human pancreas. These cells may be progenitor cells capable of producing pancreatic cells suitable for the treatment of type 1 diabetes. The cells were cultured successfully in non-adherent stationary cultures and yielded, as an important first step, a 1.9-fold expansion in a serum-free medium developed specifically for this cell type. This expanded population grew as pancreatic cell aggregates, which were analyzed for islet-like characteristics. Specifically, through RT-PCR analyses and functionality assays, we show that cells within the population expressed all four of the endocrine hormone genes and proteins (insulin, glucagon, somatostatin, pancreatic polypeptide). As well, the expanded pancreatic precursor cell population exhibited glucose responsiveness although the produced cells appeared to be still primitive in nature.     

Effects of energy dissipation rate on islets of Langerhans: Implications for isolation and transplantation

Acute physical stresses can occur in the procurement and isolation process and potentially can contribute to islet death or malfunction upon transplantation. A contractional flow device, previously used to subject suspended cells to well‐defined hydrodynamic forces, has been modified and used to assess the vulnerability of porcine islets of Langerhans to hydrodynamic forces. The flow profiles and velocity gradients in this modified device were modeled using commercial CFD software and characterized, as in previous studies, with the scalar parameter, energy dissipation rate (EDR). Porcine islets were stressed in a single pass at various stress levels (i.e., values of EDR). Membrane integrity, oxygen uptake rate, caspase 3/7 activity, and insulin release were not affected by the levels of fluid stress tested up to an EDR of 2 × 10³ W/m³. Visual observation of the stressed islets suggested that cells at the islet exterior were peeled away at EDR greater than 10,000 W/m³, however, this observation could not be confirmed using image analysis software, which determined the ratio of surface perimeter to total area. The result of this study suggests an upper limit in fluid stress to which islets can be subjected. Such upper limits assist in the design and operation of future islet processing equipment and processes. Biotechnol. Bioeng. 2009;103: 413–423. © 2008 Wiley Periodicals, Inc.