Adult rat pancreatic islet cells adherent to microcarrier beads: Evaluation of function and morphology

Summary Dispersed adult rat pancreatic islet cells were incubated with Cytodex-3 microcarrier beads for 72 h, during which time single cells adhered firmly to bead surfaces. Electron microscopy revealed well-preserved ultrastructure of attached A, B, and D cells. Perifusion of these cultures showed stable basal insulin release, brisk, biphasic insulin responses to 30-min glucose stimulation, and consistent, monophasic spikes of insulin release in response to repeated, brief pulses of glucose. These results indicate that adult rat islet cells attach to microcarriers and remain viable in culture. This preparation offers advantages for studies of hormone secretory dynamics of differentiated single islet cells, free from cell-to-cell interactions. This study was supported by grants from the Medical Research Council of New Zealand. D.W.H. was the recipient of a Novo Diabetes Research Scholarship.     

Acute pancreatitis,expression of inducible nitric oxide synthase and defective insulin secretion

A high level of nitric oxide (NO) produced by inducible NO synthase (iNOS) is involved in pancreatic beta-cell dysfunction and apoptosis. In the present study, we examined whether iNOS is also expressed in beta cells after induction of acute pancreatitis (AP) in the rat. Pancreatic islets taken from AP animals and incubated for 60 min in the presence of 20.0 mmol/l glucose showed a decreased insulin secretory response to glucose. The basal insulin release at 1.0 mmol/l glucose was also moderately reduced. Experiments on the dynamics of insulin secretion from perfused pancreas revealed an impairment of both first and second phase of glucose-stimulated insulin release after the induction of AP. Confocal microscopy demonstrated that most of the beta cells in pancreas of rat with AP expressed strong immunoreactivity for iNOS. This was further confirmed by biochemical and Western blot analysis that showed a marked increase in iNOS protein expression and enzyme activity concomitant with a modest reduction in the cNOS protein and activity. Although the mechanisms underlying the defective insulin secretory response of beta cells seen during the early stage of AP are complex, the present finding suggests that the expression of iNOS and a marked iNOS-derived NO production in the beta cells may play at least a contributory role in the impairment of beta-cell function.This study was supported by the Swedish Medical Research Council (14X-4286), the Swedish Diabetes Association, and the Crafoord, Albert Påhlsson and Magnus Bergvall Foundations     

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.     

On the ultrastructural localization of noradrenaline in the central nervous system of the rat

Summary Isolated cells were prepared from microdissected pancreatic islets of guinea-pigs. Phase-contrast microscopy of the fresh islet cells suspended in a balanced salt solution displayed a number of cellular details, including cytoplasmic secretion granules. There was morphologic evidence of the survival of islet cells in monolayer cultures for up to 3 weeks. A moderate proliferation of cells occurred during the first 2 weeks after explantation. Different types of islet cells could not be distinguished in the phase-contrast microscope.Research carried out with the financial support of the Swedish Medical Research Council (B 67-12 X-109-03), the Medical Faculty of Uppsala University and the U. S. Public Health Service (Grant AM-05759-05). We thank Assistant Professor Björn Sandström, M. D., for much advice on the tissue-culture method.     

Effect of horse serum on neural cell differentiation in tissue culture

Summary The effects of various concentrations of horse serum on dissociated mouse glial precursor cells in colony cultures were evaluated. High concentrations (20% or more) favored cell attachment but inhibited cell proliferation and differentiation, whereas lower concentrations (5% to 10%) favored cell proliferation and differentiation. In fetal bovine serum the cells did not attach to culture surfaces to the same degree nor did they achieve the same level of differentiation as in corresponding concentrations of horse serum. Portions of this work were presented at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 10–14, 1978 (1). This investigation was supported by Grants MA and MT 4235 from the Medical Research Council of Canada.     

A human gallbladder adenocarcinoma cell line

Summary A continuous cell line, COLO 346, was established from a liver metastasis in a patient with adenocarcinoma of the gallbladder. COLO 346 grew as an adherent monolayer of pleomorphic epithelioid cells. COLO 346 cells produced esterone, but no estradiol, progesterone, or cortisol. No adrenocorticotropic hormone, β-subunit of human chorionic gonadotropin, carcinoembryonic antigen, or α-fetoprotein production by the cells was detected. Cell doubling time was 36 h. Seven allelic isozymes were assayed. COLO 346 had a chromosome mode of 74 at 21 months postestablishment with 6 marker chromosomes present in 100% of the cells analyzed. COLO 346 has been in continuous culture for over 2 yr and is available to other investigators for their studies. This paper was presented in part at the 31 st Annual Meeting of the Tissue Culture Association, June 1–5, 1980. The work was supported by Grants CA15018 and CA29514 from the National Cancer Institute, and by the Mary B. and L. H. Marshall Fund.     

Functional examination of microencapsulated bioengineered insulin-secreting beta-cells.

Clonal insulin-secreting BRIN-BD11 cells engineered by electrofusion were encapsulated inside natrium alginate beads and cultured in RPMI 1640 culture media. Acute insulin secretory responses to glucose and amino acids were compared between microencapsulated cells and non-encapsulated cells maintained in monolayer culture. Encapsulated cells exhibited a 1.5-fold, 2.9-fold and 4.2-fold increase (P< 0.001) in insulin release in response to 16.7 mmol/l glucose, 10 mmol/l L-arginine and 10 mmol/l L-alanine respectively. Insulin output by non-encapsulated cells was approximately 30% greater but the relative magnitudes of responses were similar. This is the first study to demonstrate the stability of cellular engineered insulin-secreting cells encapsulated in alginate beads, illustrating the utility of this approach for cellular engineering and potential transplantation in diabetes.     

Formation of islet cell monolayers from fetal human and neonatal rat pancreatic islets: protein biosynthesis, insulin secretion, and binding of islet cell antibodies

The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) has been used to promote monolayer formation in cultured islets isolated from fetal human or neonatal rat pancreas. Immunofluorescence with specific antiserum to insulin revealed B-cells in the outgrown monolayers. The rat islet cells were further characterized by their secretory and biosynthetic response to the action of IBMX. Both glucose-stimulated insulin release and recoverable insulin, i.e. intracellular insulin plus insulin secreted, were increased by the addition of IBMX (0.1 mmol/l) to the medium containing 10 mmol/l glucose. 3H-leucine incorporation into (pro)insulin was significantly higher following culture in 10 mmol/l glucose plus IBMX (0.1 and 1.0 mmol/l) than after cultivation with glucose alone. However, the percentage of (pro)insulin synthesized in relation to total protein synthesis was increased to a lesser extent after an acute incubation for 3 h at 1.5 mmol/l or in the absence of glucose. Moreover, the culture system employed in the present study proved to be useful for detecting islet cell antibodies bound to human as well as rat islet cells after exposure to islet cell antibody-positive sera.     

B.M.A. Annual Meeting,Sydney, 10–16 August: Report of Proceedings

The One Hundred and Thirty-sixth Annual Meeting of the British Medical Association was held in Sydney from 10 to 16 August jointly with the Seventh Annual Meeting of the Australian Medical Association. Both meetings were associated with the Third Australian Medical Congress. It was the second Annual Meeting of the Association to be held in Australia, the previous one having been in Melbourne in 1935. Four plenary sessions were held on successive mornings, while meetings of various sections were held in the afternoons. An account of the first part of the Meeting is given below. The remainder will be reported next week.     

Electroncytochemical and biochemical demonstration of guanylate cyclase activity in the pancreatic islet

Summary With a cytochemical method using guanylyl imidodiphosphate as a substrate, the guanylate cyclase activity was localized on the plasma membrane of A, B and D cells of islets of Langerhans isolated from the rat. Adequate control experiments were performed by a double-blind method. Parallel biochemical assay showed that guanylate cyclase activity was not completely lost after fixation with 1% glutaraldehyde and incubation with 4 mM lead nitrate. Furthermore, the depressed activity was still stimulatable with acetylcholine.This investigation was supported by grants from the Bickell Foundation and the Medical Research Council of Canada (MA-868). Part of this work was presented at the 35th Annual Meeting of the American Diabetes Association on June 16, 1975     

Pure gelatin microcarriers: Synthesis and use in cell attachment and growth of fibroblast and endothelial cells

Summary A new type of microcarrier was described using bead emulsion-polymerization techniques. An aqueous solution of gelatin and glutaraldehyde was dispersed in a hydrophobic phase of mineral oil, using Triton X-114 as an emulsifier, and polymerization was initiated. The resultant spherical beads, composed entirely of gelatin, showed excellent mechanical stability to ethanol drying, sterilization, and long-term use in microcarrier spinner cultures. The solid gelatin microcarriers supported the growth of L-929 fibroblast, swine aorta endothelial, human umbilical endothelial, and HeLa-S₃ cultures with no adverse effects on cell morphology or growth. The beads were transparent in growth medium and attached cells were clearly visualized without staining. The beads were also compatible with techniques for scanning electron microscopy. Collagenase could be used to entirely digest the gelatin beads, leaving the cells free from microcarriers and suspended in solution while retaining 98% cell viability. The results further showed that after collagenase treatment the cells would populate fresh gelatin microcarriers and grow to confluence. Cell attachment kinetics revealed that the endothelial cells attached to the gelatin beads at the same rate as to tissue culture plates, whereas the fibroblast cells attached to the beads more slowly. However, once the fibroblast cells were attached to the gelatin microcarriers they spread and grew normally. This research was supported in part by the National Institutes of Health (GN 29127) and Ventrex Laboratories, Portland, Maine.     

Characteristics of rat normal mammary epithelial cells and dimethylbenzanthracene-induced mammary adenocarcinoma cells grown in monolayer culture

Summary The characteristics of normal mammary epithelial and 7,12-dimethylbenz[a]anthracene (DMBA)-induced adenocarcinoma cells derived from rats and grown in monolayer culture were compared. Normal mammary epithelial cells exhibited different morphology and agglutinability by plant lectins, slower growth rate, and lower saturation density and cloning efficiency. In addition, the normal cells were sensitive to the toxic effect of DMBA, and were unable to grow in soft agar or to form tumors, when inoculated into newborn Sparague-Dawley rats. The converse was true in each case for the adenocarcinoma cells. Supported by Public Health Service Research Grant CA 01237603 from the National Cancer Institute Portions of this paper were presented at the 65th Annual Meeting of the American Association for Cancer Research at Houston, Texas, 1974.     

Regulation of insulin synthesis in an insulin-producing cell line (RINm5F): Long-term experiments

Summary The insulin-producing cell line RINm5F, has been used in short-term experiments to evaluate insulin secretion. We sought to maintain the responsiveness of these cells to stimuli for up to 2 days. We examined the course of new insulin synthesis over this period by measuring at intervals immunoreactive insulin (IRI) in two parts: IRI in the medium (M) and IRI extracted from the cells (C). Control cells were incubated in RPMI 1640/2.8 mM glucose/10% fetal bovine serum/200 μg/ml bacitracin (to prevent insulin degradation). The addition of dibutyryl cAMP 10 mM to the experimental dishes significantly increased total (M+C) IRI at 48 hr to 37% above the insulin content of the control dishes (p<0.01). Theophylline 10 mM increased total (M+C) IRI by 24% over control (p<0.05) after 24 hrs. Glucose, glyceraldehyde, leucine, arginine, glucagon and tolbutamide, other stimulants of insulin production, had no effect. Under the experimental conditions reported here, including the use of bacitracin, IRI synthesis can be studied for up to 48 hr. Portions of this study have been published in abstract form for the 47th Annual Meeting of the American Diabetes Association, Indianapolis, Indiana, 1987. Supported in part by the American Diabetic Association, Maryland Affiliate.     

Sustained high-yield production of recombinant proteins in transiently transfected COS-7 cells grown on trimethylamine-coated (hillex) microcarrier beads

The present study shows that COS-7 cells transiently transfected and maintained on positively charged (trimethylamine-coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high-level synthesis was observed with secreted chimeric proteins (murine E-selectin- and P-selectin-human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte-macrophage colony-stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine-coated microcarriers than to polystyrene flasks. After 10-14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5-fold increase in protein production from a single transfection over two weeks. Thus, microcarrier-based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.     

Angiotensin I-converting enzyme localization on cultured fibroblasts by immunofluorescence

Summary Angiotensin I-converting enzyme is responsible for the activation of angiotensin I and the inactivation of bradykinin. It has been localized by immunofluorescence on the endothelium of a variety of tissues and has been considered to be a specific marker for endothelial cells in culture. The present paper demonstrates, by immunofluorescence, the presence of angiotensin I-converting enzyme in monolayer cultures of fibroblasts derived from adult rat lung, bovine calf pulmonary artery, and human foreskin (CF-3 cells). Fluorescent localization of angiotensin I-converting enzyme was observed over the cytoplasm of adult rat lung and bovine calf pulmonary artery fibroblasts and as distinct areas overlying the nuclei of human foreskin fibroblasts. Determination of angiotensin I-converting enzyme activity by fluorimetric assay in parallel studies confirmed the presence of angiotensin I-converting enzyme activity in cultured fibroblasts. Immunofluorescent studies with antibody to Factor VIII demonstrated the presence of Factor VIII on cultured endothelial cells but not on fibroblasts. These results indicate that angiotensin I-converting enzyme is not confined to endothelial cells, and thus may not serve as a specific marker for endothelial cells in culture. Factor VIII may be a more specific marker for these cells. Presented in part at the 31st Annual Meeting of the Histochemical Society, April 11–15, 1980, New Orleans, Louisiana. Wendy Baur and Ms. Jane Aghajanian for expert assistance in the preparation of the cell cultures. This work was supported by Research Grant HL 14456 and Training Grant HL 07053 from the National Institutes of Health, Bethesda, MD.     

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.     

On the ultrastructure of follicles and isolated follicular granulosa cells of porcine ovary

Summary The endoplasmic reticulum in granulosa cells of primary, secondary, and small tertiary follicles of the porcine ovary is sparse and largely of the granular type.In granulosa cells of large tertiary follicles the endoplasmic reticulum shows distinct signs of proliferation. Some cells even contain whorls of endoplasmic reticulum membranes, essentially of the agranular variety.Direct continuity between endoplasmic reticulum membranes of the granular and agranular type as well as the continuous increase in agranular membranes suggest that these membranes may originate from the granular membranes.Granulosa cells isolated from large tertiary follicles by microdissection and keptin vitro show essentially the same ultrastructure as granulosa cells of intact large tertiary follicles.Some lipid droplets appear to be localized in cavities of the endoplasmic reticulum. It is suggested that the droplets contain precursor material for steroid hormone synthesis.Finally, the development of the agranular endoplasmic reticulum including the appearance of whorls in some granulosa cells of large tertiary follicles indicates that steroid synthesis may occur in such follicular granulosa cells.Read at the Meeting of the Swedish Society for Pathology in Umeå, September 25, 1965 (Bjersing, 1966).This investigation was supported by grants from the Swedish Medical Research Council (Projects No. 13 X-78-01, 12 X-78-02, and 12 X-78-03).     

Growth of Fish Cell Lines on Microcarriers

Microcarrier beads were evaluated as substrates for the propagation of five anchorage-dependent fish cell lines. Growth of rainbow trout gonad (RTG-2) and Atlantic salmon cells was limited on microcarriers maintained in suspension. However, stationary microcarriers were suitable substrates for the growth of RTG-2, AS, Chinook salmon embryo (CHSE-214), and fathead minnow cells. Cell yields ranged from 2 × 10⁶ to 2.9 × 10⁶ cells per ml, representing 7- to 10-fold increases over the initial cell concentrations. The yield of new RTG-2 cells per unit volume of growth medium was 2.8 times greater in microcarrier cultures than in standard monolayer cultures. Northern pike cells failed to grow on microcarriers. Yields of infectious pancreatic necrosis virus propagated in microcarrier cultures of RTG-2 cells were more than twice the yields in standard monolayer cultures. The greater economy of microcarrier cultures in terms of growth vessel and medium requirements holds great promise for the large-scale production of anchorage-dependent fish cell cultures and fish viruses.     

The duct-ligated pancreas transplant and its effect on the islet cellular composition of the host pancreas

Summary Rats rendered diabetic by streptozotocin were subjected to pancreas transplantation. After twenty weeks, the duct-ligated pancreas transplant was studied morphometrically to determine the effect of duct occlusion on the various cell populations of the islets. Concomitantly, the streptozotocin-treated host pancreas was examined for a possible influence of the graft on the diabetic pattern of islet cell population. Twenty weeks after pancreas transplantation, the volume fractions of insulin, glucagon, somatostatin and pancreatic polypeptide cells in the graft islets did not differ from those of the normal control pancreas. In the pancreas of nontransplanted diabetic rats, insulin-positive B cells were reduced from 60–65% to less than 10% of the islet volume, whereas non-B cells were significantly increased in volume density. The changes in fractional volume of the various islet cells correlated fairly well with changes in plasma concentration of the corresponding pancreas hormones. In the recipient's own pancreas, the relative volumes of glucagon and somatostatin cells were unaffected by the pancreas transplant. However, the insulin cell mass was significantly increased, and comprised about 20% of the islet volume, while cells containing pancreatic polypeptide were found only sporadically.Supported by Nordic Insulin Fund, The Swedish Diabetes Association, and MFR, proj. no. 4499. The technical assistance by M. Maxe and M. Carlesson is gratefully acknowledged     

Intrafollicular control of oocyte maturation in the PIG

Summary The mammalian oocyte becomes arrested at the diplotene stage of the first meiotic division during prenatal or early postnatal life. It remains arrested in meiosis until shortly before ovulation when the surge of gonadotropin induces resumption and completion of meiosis to the metaphase II stage. When oocytes are harvested from medium-sized or large follicles of pig and other species and cultured, they resume meiosis spontaneously indicating that the follicles exert an inhibitory influence on meiosis. To analyze the control of meiosis by follicular components, culture of isolated pig oocytes in the presence of follicular cells or follicular fluid (FF1) has been used as a model in this laboratory. An oocyte maturation inhibitor (OMI) has been isolated and partially purified by ultrafiltration and gel chromatography of FF1 and shown to be a polypetide with a molecular weight in the order of 2000 daltons. Physiological characterization has shown that the effect of OMI in vitro is reversible and that it can be overcome by luteinizing hormone (LH). The action of OMI requires the presence of cumulus cells surrounding the oocyte since it was found that denuded oocytes, stripped of cumulus cells, do not respond to OMI. Furthermore, when cumulus-enclosed oocytes were cultured, OMI inhibited the differentiation of the cumulus cells in terms of morphology and progesterone secretion in a dose-related manner. The inhibition of cumulus differentiation by OMI was reversible and could be overcome by LH. The results indicate that the effect of partially purified OMI upon meiosis may be mediated by the cumulus cells. Presented in the formal symposium on Sexual Differentiation in Vitro and in Vivo at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 4–8, 1978. This study was supported by Grants 760–0530 from the Ford Foundation (to C.P.C.), and Grant B78-14F-5158-01 from the Swedish Medical Research Council (to T.H.).