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WO1999015625A1 - Milieu favorisant la survie des cellules insulaires - Google Patents

Milieu favorisant la survie des cellules insulaires Download PDF

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Publication number
WO1999015625A1
WO1999015625A1 PCT/CA1998/000887 CA9800887W WO9915625A1 WO 1999015625 A1 WO1999015625 A1 WO 1999015625A1 CA 9800887 W CA9800887 W CA 9800887W WO 9915625 A1 WO9915625 A1 WO 9915625A1
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WO
WIPO (PCT)
Prior art keywords
islets
culture medium
islet
medium
cells
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Application number
PCT/CA1998/000887
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English (en)
Inventor
Lawrence Rosenberg
Dusica Maysinger
Original Assignee
Mcgill University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mcgill University filed Critical Mcgill University
Priority to AU91498/98A priority Critical patent/AU9149898A/en
Priority to CA002304381A priority patent/CA2304381A1/fr
Publication of WO1999015625A1 publication Critical patent/WO1999015625A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/105—Insulin-like growth factors [IGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/22—Coculture with; Conditioned medium produced by pancreatic cells

Definitions

  • the invention relates to a culture medium which promote islet cell survival, which may be transplanted to reverse hyperglyce ia .
  • Adequate numbers of isogeneic islets transplanted into a reliable implantation site can only reverse the metabolic abnormalities m diabetic recipients m the shor term. In those that were normoglycemic post -transplant , hyperglycemia recurred within 3-12 months. The return of the diabetic state that occurs with time has been attributed either to the ectopic location of the islets, to a disruption of the enteromsular axis, or to the transplantation of an inadequate islet cell mass.
  • pancreatic fragments may actually have an inherent survival advantage compared to purified islets.
  • insulin-independence has been obtained after mtraportal injection of unpurified autologous islets. Fewer impure islets were more successful than many more purified ones.
  • a similar experience was repeated with allotransplants, with unpurified islets from a single human pancreas successfully reversing hyperglycemia (Gores PF et al . , Lancet, 1993, 341:19). From these and other reports, it is apparent that unpurified islets from one pancreas may survive as well as pure islets prepared from multiple donors.
  • Tissue transglutammase (TG) expression is a fundamental event m the induction of apoptosis.
  • TG is a calcium-dependent enzyme whose activity is well established m many mammalian tissues, including pancreas. It is involved m the cross- linking of intracellular proteins that precedes the irreversible ultrastructural changes characterizing W wO 9 ⁇ 9/ / 1 i 5 3 6o2z5:> PCT/CA98/00887
  • One aim of the present invention is to provide a culture medium which promotes the survival islet cell, which may be transplanted to reverse hyperglycemia.
  • a culture medium for promoting the survival of mammalian islet cells which comprises at least an effective amount of one or more growth factors having anti-apoptosis effect on islet cells in a physiologically acceptable culture medium.
  • a culture medium herein referred to as duct-conditioned medium (DCM)
  • DCM duct-conditioned medium
  • IGF- 11 34 ng/ l
  • NGF 4 ng/ml
  • the preferred growth factors include, without limitation, NGF, IGF-II and insulin.
  • Other growth factors include TGF ⁇ , IGF- 1 and HGF .
  • the preferred NGF concentration is between about 10 to about lOOng/ml of medium.
  • the preferred IGF-II concentration is between about 10 to about lOOng/ml of medium.
  • the culture medium of the present invention includes an immunosuppressant .
  • the preferred lmmunosuppressants are selected from the group consisting of FK506, a peptide having NGF potentiating effect, and therefore possibly a trophic effect on islet cells.
  • the preferred immunosuppressant concentration is about 1 micromolar.
  • the culture medium of the present invention further include insulin.
  • the preferred insulin concentration is from about 10 to about lOOng/ml of medium.
  • the culture medium of the present invention further include a soluble matrix molecule called fibronectin and type I collagen.
  • physiologically acceptable culture medium is intended to mean any commercially available culture medium including, without limitation, CMRL 1066, RAM 1640 and DMEM/F12.
  • the culture medium of the present invention may also be used to isolate islet cells or to irrigate the site of transplant to promote m si tu islet cell survival .
  • Fig. 1 illustrates an Agarose gel demonstrating DNA laddering m islets incubated m a control medium (control lane) , compared to islets incubated with insulin or NGF;
  • Fig. 2 illustrates TUNEL staining of porcine islets, (A) islets incubated m the standard medium and (B) islets incubated with insulin;
  • Fig. 3 illustrates a graph of the stimulation of insulin secretion by a glucose challenge in vi tro
  • Fig. 4 illustrates activation and expression of p38 m canine islets
  • Fig. 5 illustrates activation and expression of p38 m canine islets
  • Fig. 6 illustrates activation and expression of erk-1 and erk-2 m isolated islets m absence or presence of growth factors
  • Fig. 7 illustrates the effect of p38-MAPK inhibitor and FK506 on cell survival (PC-12 cells)
  • Fig. 8 illustrates the effect of ductal epithelium and duct -conditioned medium on islet necrosis development
  • Fig. 9 illustrates the effect of ductal epithelium and duct-conditioned medium on DNA fragmentation
  • Fig. 10 illustrates the effect of duct- conditioned medium and IGF-II on DNA fragmentation.
  • HGF Hepatocyte growth factor
  • IGFs Insulm-like growth factors
  • reg a gene that is over- expressed within pancreatic cells after pancreatitis or resection, and particularly during islet cell regeneration.
  • TGF Transforming Growth Factor
  • TGF- ⁇ is also a potent regulator of cell proliferation, but its major activity is to stimulate the synthesis and deposition of various ECM proteins and to increase the expression of mtegrins .
  • TGF- ⁇ expression in vivo may be important m the re- establishment of basement membrane following its loss during islet isolation, and hence in islet cell survival .
  • Growth factors may also be inhibitory in almost all situations in which apoptosis, or programmed cell death, is positively stimulated. This suggests that regulation of growth factor levels is not only important in the control of cell proliferation but also in maintaining viability of cells susceptible to apoptosis. These data suggest, that for the success of islet transplantation, the activity of survival factors may be fundamental to the long term maintenance of graft function.
  • the preferred Islet Survival Medium of the present invention takes advantage of (1) newly recognized effects of known growth factors on inhibiting the induction of apoptosis in islet cells, (2) the combination of these factors to achieve a synergistic beneficial effect on islet cell survival, and (3) the potentiation of this synergistic effect by the addition of the drug FK506 (ProGraf , Fujisawa) .
  • NGF nerve growth factor
  • IGF-I insulin-like growth factor-1
  • insulin insulin-like growth factor-1
  • FK506 is a macrolide immunosuppressive agent which acts by inhibiting T-cell activation.
  • FK506 is a W wO ⁇ 9 y 9 y / / 1 1 5 3 6o25 3 PCT/C A98/00887
  • FK506-FKBP12 binds several targets, one of which is the calcium calmodulin dependent phosphatase, calcineurin. FK506 has been shown to potentiate the neurotropic effects of NGF. The exact mechanism by which FK506 facilitates NGF activity is still unclear.
  • CMRL 1066 medium a conventional islet culture medium supplemented with the above substances can significantly reduce the amount of apoptotic cell death sustained by insulin- producing ⁇ -cells following islet isolation and purification.
  • the evidence is based on four assays.
  • TUNEL staining (another technique to visualize apoptotic events) of actual isolated islets, demonstrates a significant reduction in the number of apoptotic cells (Fig. 2).
  • p38 MAP kinase Signaling through the p38 MAP kinase is one pathway whose downstream effectors have been implicated in cell (Figs. 4 and 5) . Suppression of p38 activation is necessary but not sufficient for the survival of islet cells (Figs. 4 and 5) .
  • Serum alone can suppress p38 activation, but it is not sufficient to enhance islet survival (Figs. 4 and 5) .
  • Trophic factor (s) NGF, IGF-II
  • NGF Trophic factor
  • Example I which described the in vi tro results of islet culture; Fig. 4) . Not clearly seen on this graph, is the syneergism between IGF-II and NGF.
  • growth factors can modulate the degree of activation.
  • p38 and associated signaling cascades
  • subsequent intervention with the appropriate growth factors may not be effective. Therefore, these factors may need to be provided "up front" during the digestion and purification phases, and not just during culture.
  • fibronectin for human islets
  • collagen for canine islets
  • the data for p38 activation and its modulation by collagen represents the effect of 200 ⁇ g/ml of collagen type 1.
  • collagen represents the effect of 200 ⁇ g/ml of collagen type 1.
  • collagen in a concentration of 400 ⁇ g/ml will decrease p38 activation by over 50% .
  • NGF is the prototype of a family of neurotrophic factors that are important in the differentiation, maintenance and survival of neurons. Beta cells and neuronal cells share many characteristics and several markers initially thought to be specific for the nervous system have been found in beta cells. Of note, is the recent finding that beta cells express NGF receptors. The large number of similarities suggests that these two cell types could also share the same survival factors.
  • PC-12 cells are a cell line of neuronal origin used for the above studies.
  • Fig. 7 illustrates data from human islets which supports a similar role for FK506 and insulin islet survival.
  • PD169316 is a specific p38 inhibitor.
  • pancreatic ductal epithelium Effect of pancreatic ductal epithelium on islet cell survival in vitro, and characterize for the first time the specific growth factors involved in an islet-duct paracrine interaction.
  • Group 1 control
  • Group 2 islets plus ducts
  • Group 3 islets in 25% DCM
  • the hamster pancreatic duct isolation was performed with collagenase XI solution (1 mg/ml) (Sigma, St. Louis, USA) . Digestion was carried out in a stationary water bath at 37°C for 30 minutes, followed by a 10 sec dispersion by vortex. After washing, the digest was filtered through a 94 ⁇ m steel mesh filter (Bellco Glass, Vineland, NJ, USA) . The retained fragments were harvested by rinsing the inverted filter. The tissues were embedded into the neutralized rat tail collagen according to Richards (Richards et al .
  • Dulbecco's modified Eagle medium/F12 (Sigma, St. Louis, USA) supplemented with 10% NuSerum, insulin (1 g/ml , Eli Lilly, Toronto, Ontario, Canada) , dexamethasone (1 g/L, Sigma, St. Louis, USA), soybean trypsin inhibitor (0.1 mg/ml, Gibco, Burlington, Ontario, Canada), cholera toxin (CT) (100 ng/ml, Sigma, St. Louis, USA), epidermal growth factor (EGF) (10 ng/ml, Sigma, St.
  • DEM/F12 Dulbecco's modified Eagle medium/F12
  • DCM Duct-Conditioned Medium
  • DCM was prepared from purified tertiary ducts incubated at 37 °C for three days in DMEM/F12 without NuSerum, CT, EGF, or insulin. The conditioned media was collected and stored at -80°C. Jslet Isolation
  • Islets were isolated from hamster pancreata according to a method previously established in our laboratory (Metrakos et al . , 1993, Surgery 114:423-427; Metrakos et al . , 1992, Transplantation Proceedings 24:2830-2831; Gray & Morris, 1987, Transplantation 43 321-331) .
  • 4 ml of collagenase P (Boehringer Mannheim, Laval, Canada) solution at 0.7 mg/ml was slowly introduced into the common bile duct after occlusion of the distal end just proximal to the duodenum.
  • the distended pancreas was excised and the digestion was performed in a waterbath at 37 °C for 30 min. Islet purification was achieved using a two-step, discontinuous density gradient of bovine serum albumin (BSA) (Sigma, St. Louis, USA). Islets were collected from the interface between 1.000 and 1.081 g/ml layer.
  • BSA bovine serum album
  • the tissue was fixed m 4% paraformaldehyde and embedded m paraffin.
  • Serial sections (5 ⁇ thick and 50 ⁇ apart) were cut from each block, stained with haematoxylm and eosm (H&E) or toluidme blue, and then processed further for routine light microscopy.
  • H&E haematoxylm and eosm
  • Consecutive 5 ⁇ sections cut from the tissue block were immunostained for islet cell hormones (anti- human insulin antibody or rabbit polyclonal anti- glucagon or anti-somatostatm antibodies at a dilution of 1:100, Dako Corp., California), using the Streptavidm Biotm Complex method as described previously (Wang et al . , 1997).
  • the islet hormones are detected using N7AMP/ Fast Red as chromagen to obtain a red reaction product.
  • Negative controls are tissues processed with non- immune serum substituting for the 1° antibodies. To identify the presence of apoptotic cells, the tissues were co-processed for the TUNEL reaction (see below) . Detection of Apoptotic Cell Death
  • TUNEL To identify which cells in the islet were undergoing apoptosis, a TUNEL was employed. Islets were fixed in 4% paraformaldehyde and embedded in paraffin. 5 ⁇ m paraffin sections were cut and pretreated with 0.1 % Trypsin buffer in 37° for 5 min. The In Situ Cell Death Detection Kit (Boehringer Mannheim, Laval, Quebec) was used for the labeling of apoptotic cells and the sections were developed with DAB (Sigma, St Louis, MO) . To reduce non-specific labeling the POD antibody conjugate was diluted 1:5 in TBS buffer. Approximately 800 cells from 10 islets were counted in each group, and an apoptotic index (% of labeled cells) was calculated. Analysis of Culture Medium for Growth Factors
  • the islets were embedded in rat tail collagen and cultured in DMEM/F12 supplemented with 10% NuSerum. After three days in culture, three experimental groups were designed for this study: Group 1 (control) , islets cultured in DMEM/F12; Group 2, islets cultured in DMEM/F12+25%DCM; and Group 3, islets cultured in DMEM/F12+34 ng/ml IGF-II. The islets were retrieved from the collagen following 9 days in culture with this media .
  • Statistical Analysis Analysis
  • Islet diameter ranged from 50 to 300 ⁇ m, with the majority measuring 100 to 200 ⁇ m. Under the inverted microscope, freshly isolated islets had a smooth appearance with slightly irregular borders. After overnight incubation, the islets recovered a more regular spherical shape with well defined smooth borders .
  • the mean islet diameter ( ⁇ m) was similar in each of the three treatment groups- islets alone (161 ⁇ 34 ⁇ m) vs. islets+ducts (164 ⁇ 61 ⁇ m) vs. islets+DCM (155 ⁇ 51 ⁇ m) .
  • Islets cultured alone had a significant increase in central necrosis (p ⁇ 0.001) at all time points when compared to islets in the other two groups (Fig. 8) . Data are mean + SEM, (n 15) . * P ⁇ 0.001 (Student's t test) .
  • the apoptotic index (% TUNEL positive cells) demonstrated a significant difference (p ⁇ 0.001) between islets cultured alone (60.8 ⁇ 3.9%) compared to those co-cultured with ducts (29.8 + 8.3%) or in DCM supplemented medium (38.3 ⁇ 1.5%). Over 80% of TUNEL-positive cells were in the inner 80% of the islet, suggesting that these were primarily beta cells. Double labeling of cells for islet cell hormones confirmed this impression, although as anticipated, many TUNEL-positive cells in fact did not show immunoreactivity for any islet cell hormone.
  • duct conditioned medium was analyzed for growth factors that might be expected to be secretory products of pancreatic ductal epithelium, based on previous literature reports.
  • Our analysis demonstrated the presence of a small amount NGF (4 ng/ml) and much larger amount of IGF-II (34 ng/ml) . IGF-I was not identified.
  • IGF-II could account for the beneficial effect of DCM (p ⁇ 0.004) with respect to limiting apoptosis in cultured islets (islet alone- 4.9 ⁇ 0.7; islets+DCM- 2.7 ⁇ 0.4; islets+IGF- 2.5 ⁇ 1.0 ; Fig. 10).
  • islet cells When maintained under standard in vitro conditions, islet cells, predominantly in the islet core, underwent necrotic cell death. This finding is in keeping with a number of previous studies that have also documented the occurrence of central necrosis in islets maintained in vi tro. In the present study, however, when islets were co-localized with duct epithelium or with the secretory products of duct cells, the occurrence of central islet necrosis was diminished significantly.
  • pancreatic ductal epithelium promotes islet cell survival. This effect appears to be mediated in a paracrine manner by the release of IGF-II from cells in the ductal epithelium.

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Abstract

La présente invention porte sur un milieu de culture favorisant la survie des cellules insulaires mammaliennes, ce milieu comprenant au moins une quantité efficace d'un ou plusieurs facteurs de croissance ayant un effet anti-apoptose sur les cellules insulaires d'un milieu de culture physiologiquement acceptable.
PCT/CA1998/000887 1997-09-19 1998-09-17 Milieu favorisant la survie des cellules insulaires WO1999015625A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU91498/98A AU9149898A (en) 1997-09-19 1998-09-17 A medium to promote islet cell survival
CA002304381A CA2304381A1 (fr) 1997-09-19 1998-09-17 Milieu favorisant la survie des cellules insulaires

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CA2,216,055 1997-09-19
CA 2216055 CA2216055A1 (fr) 1997-09-19 1997-09-19 Milieu favorisant la survie des cellules secretrices d'insuline

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US09508822 A-371-Of-International 2000-09-12
US10/138,517 Continuation-In-Part US6562620B2 (en) 1997-09-19 2002-05-06 Medium to promote islet cell survival

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EP1248640B1 (fr) * 2000-01-20 2006-10-04 Diabcell Pty Limited Preparation et xenotransplantation d'ilots de porc

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995029989A1 (fr) * 1994-04-29 1995-11-09 The United States Of America, Represented By The Secretary, Department Of Health And Human Services COMPOSITIONS ET METHODES DE SIMULATION DE LA PROLIFERATION ET DE LA DIFFERENCIATION DE CELLULES PANCREATIQUES DU FOETUS ET DE L'ADULTE HUMAIN $i(EX VIVO)
WO1997016436A1 (fr) * 1995-10-27 1997-05-09 Bayer Aktiengesellschaft Derives 3-aryl-5-halogeno-pyrone utilises comme pesticides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995029989A1 (fr) * 1994-04-29 1995-11-09 The United States Of America, Represented By The Secretary, Department Of Health And Human Services COMPOSITIONS ET METHODES DE SIMULATION DE LA PROLIFERATION ET DE LA DIFFERENCIATION DE CELLULES PANCREATIQUES DU FOETUS ET DE L'ADULTE HUMAIN $i(EX VIVO)
WO1997016436A1 (fr) * 1995-10-27 1997-05-09 Bayer Aktiengesellschaft Derives 3-aryl-5-halogeno-pyrone utilises comme pesticides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CLARK SA ET AL: "*Islet* cell culture in defined serum-free *medium*.", ENDOCRINOLOGY, APR 1990, 126 (4) P1895-903, UNITED STATES, XP002090018 *
OBERG-WELSH C ET AL: "Effects of certain growth factors on in vitro maturation of rat fetal *islet*-like structures.", PANCREAS, MAY 1996, 12 (4) P334-9, UNITED STATES, XP002090017 *

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CA2216055A1 (fr) 1999-03-19
AU9149898A (en) 1999-04-12

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