WO1994004169A1 - Ilots de langerhans sous forme pure - Google Patents

Ilots de langerhans sous forme pure Download PDF

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Publication number
WO1994004169A1
WO1994004169A1 PCT/EP1993/002178 EP9302178W WO9404169A1 WO 1994004169 A1 WO1994004169 A1 WO 1994004169A1 EP 9302178 W EP9302178 W EP 9302178W WO 9404169 A1 WO9404169 A1 WO 9404169A1
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WO
WIPO (PCT)
Prior art keywords
langerhans
islands
islets
cell material
different
Prior art date
Application number
PCT/EP1993/002178
Other languages
German (de)
English (en)
Inventor
Bernhard J. Hering
Original Assignee
Hering Bernhard J
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 Hering Bernhard J filed Critical Hering Bernhard J
Priority to JP6505891A priority Critical patent/JPH07500504A/ja
Priority to AU47099/93A priority patent/AU680828B2/en
Priority to EP93917795A priority patent/EP0610473A1/fr
Publication of WO1994004169A1 publication Critical patent/WO1994004169A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis

Definitions

  • the invention relates to a preparation of isolated Langerhans ' islands with a degree of purity of more than 98%, based on the volume of the purified cellular particles, process for the preparation of this preparation and the diagnostic and / or therapeutic use of this preparation on the human or animal body .
  • the object of the present invention is to produce a preparation of isolated Langerhans' islands with a particularly high degree of purity and to reduce the immunogenicity which can be demonstrated in vitro using a standard test.
  • This object is achieved by the present invention, which relates to a preparation of isolated Langerhans' islands with a degree of purity of more than 98%, based on the volume of the purified cell material, and to the production process for this preparation.
  • Diameter of the majority of the cleaned cell material larger than 100 ⁇ m
  • amylase content of less than 0.05% compared to the island suspension after removal from the tissue structure of the distended pancreas and before the application of subsequent separation measures.
  • the preparation also contains physiologically acceptable carrier liquid.
  • cell material encompasses cellular fragments or particles, whole cells and agglomerations of whole cells up to agglomerations of cells, which are also called cell clusters.
  • the preparation of isolated Langerhans' islets is obtainable from the animal or human donor organ of the pancreas using the method described below and can preferably be applied to humans but also to animals for diagnostic and / or therapeutic methods.
  • the preparation of the isolated Langerhans ' islets from the human pancreas is preferred.
  • animal pancreas, especially pork is also preferred.
  • the preparation of isolated animal Langerhans' islands, in particular from pigs is preferred.
  • the preparation of isolated Langerhans ' islands from the human pancreas for diagnostic use is also the subject of the invention.
  • the preparation is preferably in the form of a suspension of the isolated Langerhans' islands in a physiologically acceptable carrier liquid
  • REPLACEMENT LEAF before preferably parenterally in the vena portae (portal vein of the liver), but also in the vena lienalis, in the splenic pulp, under the kidney capsule, in an omentum pocket, in the large network ("epiplpoic flap"), intraperitonea intramuscularly, subcutaneously, is injectable intracerabrally or intratesticularly.
  • physiologically acceptable carrier liquid defines all aqueous media which are described in the literature as being suitable for cleaning, diagnostic and transplantation processes from Langerhans' islands.
  • Preferred media are media which use the terms Hank's Balanced Sali Solution (Hank's solution), Minimum Essential Medium, Medium 199, RP 1640, CMRL 1066, University of Wisconsin Solution etc. are common.
  • the present invention also relates to the production process for the preparation of the isolated Langerhans' see inse described above with a degree of purity of more than 98% of the cell material.
  • This process is characterized by the following process features: a) the pancreas is dislodged with a physiologically acceptable carrier liquid, which may contain collagenase and / or other proteases, and Langerhans' see islands with additional cell material are extracted from the tissue, b) the detached Langerhans ' see islands from the additional cell material by at least one separation measure based primarily on a different sedimentation of cell assemblies with different diameters and if desired c) subject the available Langerhans' islands to a treatment that reduces their potential immunogenicity / antigenicity and / or preserve the treating islands .
  • This process step is known per se and is generally used in the known insulation and cleaning processes from Langerhans' islands.
  • distension means expansion.
  • the pancreatic duct e.g. From humans or pigs, the digestive enzymes formed in the exocrine part of the pancreas are released into the small intestine via the body, and mostly 2 Hank solutions with approx. 0.1 - 1.0% (w / v) collagenase are passed through this cannula per gram of pancreas retrograde i injected the pancreatic duct.
  • This process step is preferably carried out according to d by Lacy P.E. et al. in Diabetes 16: 35-39 (1967) and by Gray D.W.R. et al. i Diabetes 33: 1055-1061 (1984).
  • digestion means digestion. During digestion, the pancreas is dissociated into Langerhans' islets and cell material under enzymatic and mechanical influence. This measure is preferably carried out according to d by Ricordi C. et al .: Diabetes 37: 413-420, 1988 and in the method described in PCT application W 88/09667.
  • protease includes the enzymes known under the name neutral protease, clostripain, trypsin, elastase, dispase, etc.
  • separation measure based primarily on different sedimentation of cell material with different diameters, defines a separation method which is known per se, the use of which for isolating and cleaning Langerhans' islands is still new and is therefore the subject of the present invention.
  • This separation measure is referred to in international publications as velocity sedimentation, which includes following process variants include: sedimentation at 1 g, countercurrent centrifugation (elutriation), centrifugation in reorienting zonal rotors and isokinetic gradient centrifugation.
  • process variant known under the customary name "isokinetic gradient centrifugation" is preferred.
  • isokinetic gradient centrifugation is combined with a further separation measure based predominantly on different sedimentation of cell assemblies in different densities.
  • separation measure based primarily on sedimentation of cell assemblies with different densities defines a further separation process which is known per se and has also been used for the isolation and cleaning of Langerhans' islands, see publication by Londo N.J.M., loc. cit., page 5. This separation measure is also described in international publications under the term isopycnic gradient centrifugation. D preferred methodology is explained in more detail in the example.
  • the particularly preferred embodiment of the method is characterized in that b) the extracted Langerhans ' islands are separated from additional cell material by successive separation measures based primarily on a different sedimentation of cell groups with different densities and different diameters.
  • the procedure can be reversed by combining the separation measures based primarily on different sedimentation of cell assemblies with different diameters and different densities. You can also combine both separation measures in a continuous process.
  • aqueous medium with linear density gradient performed.
  • a medium is labeled "Gradient: " and is formed, for example, in a so-called gradient mixer or formed during centrifugation itself.
  • the gradient has the greatest density at the bottom of the centrifuge tube and decreases with the height of density.
  • Suitable Media for forming gradients are known, for example, under the names Ficoll R , Percoll R (Pharmacia, Freiburg) and can contain auxiliaries such as polysucrose, polyvinylpyrrolidone, albumin or dextran.
  • the migration of the sedimenting cell material is characterized by a gradient, the maximum density of which is not greater than that of the least dense sedimenting material During centrifugation, the sedimenting cell material moves at a largely constant speed through the gradient Acceptance is complete when the first cellular particles reach the bottom of the centrifuge tube.
  • This methodology is suitable for separating cell materials, which differ mainly in their diameter, but less in their density.
  • Treatment reducing potential immunogenicity / antigenicity e.g. Cultivation, UV radiation, gamma radiation, incubation with antibodies or encapsulation relates to customary methods of working up cell material in cultures for the applications described above, which are also described in the publications cited in this application.
  • the cryopreservation can preferably follow as a preservation measure. Due to the special purity of the available islands, an improved result is achieved after use with all measures that follow the separation measures, which improves the therapeutic and / or diagnostic findings.
  • the pancreas is freed in a slaughterhouse at a breeding sow (German federal hybrid breeding program, 240 kg, 34 months) after Bolzenschu bleeding and removal of the intestinal site while protecting the organ capsule from the surrounding adipose tissue under sterile cauldrons.
  • D Slaughtering process requires a warm ischemia time of 10 minutes.
  • the removed organ is transported to the island laboratory in a 4 ° cold Eurocollins R solution (Fresenius, Ba Homburg).
  • the cold ischemia time before isolation begins is 30 min.
  • the pancreatic duct ductus pancreaticus
  • the pancreatic duct is located at the cut surface between the spleen and the right leg, cannulated with a 19G cannula and the cannula is fixed with a surgical thread.
  • the weight of the pancreatic limb near the spleen determines the volume of the distension solution.
  • 2 ml of distension solution i.e. a total of 240 ml, retrograded i injected the pancreatic duct.
  • University o Wisconsin (UW) solution DuPont, Bad Homburg
  • 0.4% wt / vol collagenase (Serva, Heidelberg, catalog no. 17449)
  • the temperature of the distension solution is 22 °.
  • the cannula, other surrounding adipose and connective tissue, the organ capsule, and undissolved parenchyma will be removed (a total of 15 g without the cannula, i.e. the weight of the pancreas used is 105 g).
  • the dilated pancreatic segment is placed in the lower part of a 450 ml digestion chamber, the arrangement of which is described in PCT application WO 88/09667, together with seven glass spheres.
  • the hose system is filled with additional Hank solution, the pump is switched on to recirculate the solution and the flow rate is set to 200 ml / min.
  • the digestion chamber with the dilated pancreas and the glass balls is moved in the vertical axis at a frequency of 300 oscillations per minute. and an amplitude of 10 mm.
  • the temperature measured in the digestion chamber is increased by 3 ° / min using a heat exchanger. to 32-34 ° and keeps the temperature constant at this value during the recirculation phase (phase I).
  • the tissue suspension is taken up in cooled (8 °) 250 ml centrifuge beakers with a conical bottom, into which 35 ml of Hank's solution are placed.
  • This centrifuge beaker filled with tissue suspension is centrifuged for 2 min. long at 4 ° in a gravitational field of 120 x g, discards the supernatant, fills the entire pellet to 200 ml with UW solution (4 °), resuspends and stores at 8 ° C.
  • Representative samples are taken to determine the number of islands, the island volume, the DNA content, the insulin concentration and the amylase activity.
  • This solution is prepared by combining 500 ml Seromed R -Ficoll release agent (1.090 g / cm-3, Biochrom, Berlin), 12 ml HEPES IM (Gibco, Eggenstein), 5 ml penicillin streptomycin (10,000 lU / ml P., 10,000 ⁇ g / ml S., Flow, Irvine, GB), 1 ml 1 N NaOH and 50 ml medium 199 (lx, Biochrom, Berlin).
  • the contents of the beaker (bottom layer) are transferred to a 500 ml blood transport bag (model Biopack, Biotrans, Dreieich), this blood transport bag is connected to a previously in the blood cell washing device 2991 (Cobe, Kirchheim / Kunststoff)
  • REPLACEMENT LEAF according to the inserted disposable system (Blocd Call Processor Processin Set, Cobe, catalog no. 912-647-819) and fills the contents of the blood transport bag into the disposable system.
  • the centrifuge of the blood cell washing machine is accelerated for 2 min. long to 2000 rpm (rotations pe minute).
  • the one-time system is then deaerated and the feed tube is closed.
  • the following fractions from the "Processing Set" are transferred to the centrifuge beaker at the same speed using an integrated "gradient unloader":
  • Fraction IV 50 ml in a beaker.
  • the fractions are suspended in medium 199 with 10% NCS and 5 min. centrifuged long at 250 x g at room temperature. The supernatant is discarded, the pellet of the fractions is resuspended separately in Leibovitz medium (Biochrom, Berlin) with 10% fetal calf serum (FCS, Biochrom, Berlin), 1% penicillin-streptomycin solution and 2 mM glucyl-glutamine ( Biochrom, Berlin), transfers the suspensions into culture bottles and assesses the suspensions with regard to their island purity (percentage of the surface covered with island tissue). The fractions, which contain the mass of the volume of the islets, are almost without exocrine contamination and are then fed to isokinetic gradient centrifugation.
  • Representative samples are taken to determine the number of islands, the island volume, the DNA content, the insulin concentration, the amylase activity, the glucose-dependent insulin secretion in vitro and the immunogenicity.
  • REPLACEMENT LEAF 15 mm, a density of 1.026 g / cm 3 and a viscosity of 3.5 cP at 16 ° consisting of FICOLL and Leibovitz medium.
  • a continuous gradient with a volume of 7 ml and a height of 120 mm, a density range from 1.026 to 1.01 g / cm 3 and a viscosity range from 3.5 cP to is applied to the bottom layer (the cushion) with the aid of a two-chamber gradient former 2.0 cP at 16 ° consisting of FICOLL and Leibovitz medium piled up.
  • a 5 ml sample corresponds to a height of 8 mm and up to 50,000 island equivalents (one island equivalent is a volume-corrected island number, one island equivalent represents an island with a diameter of 150 ⁇ m, see Ricordi et al. Acta Diabetol Lat 27: 185-195, 1990) and cellular residual mass in Leibovit medium.
  • the centrifuge tubes with bottom layer (cushion), gradient and sample are positioned in a centrifuge in such a way that the distance between the boundary layer between cushion and gradient is 22.2 cm and the distance between the boundary layer between sample and gradient is 10.2 cm from the center of the Hettich Rotixa R Centrifuge (Hettich, Tuttlingen), which is equipped with a swing-out rotor type 5094 and suitable hangers and reducers for holding 100 ml centrifuge tubes. Opposite centrifuge tubes of the same content or balance tubes are positioned.
  • the centrifuge tubes are accelerated to 490 rpm for 30 seconds. At this speed, a centrifugal force of 27.4 x g acts on the boundary layer between sample and gradient and 59.6 x g on the boundary layer between the bottom layer and gradient.
  • the mixture is centrifuged at 16 ° for 60 seconds and decelerated to 0 rpm in 30 seconds.
  • the centrifuge tubes are removed from the centrifuge, the first fraction consisting of 80 ml and the second fraction consisting of 5 ml are aspirated and diluted with Leibovitz medium and washed again by centrifugation for min. at 120 x g and 16 °. The supernatant is discarded and the remaining cell suspension is resuspended with Leibovitz medium or other culture media and conventional additives.
  • Representative samples are taken to determine the number of islands, the island volume, the DNA content, the insulin concentration, the amylase activity, the glucose-dependent insulin secretion in vitro and the immunogenicity.
  • the available island suspension is used for further use, e.g. Culture, fed.
  • the islet number, the islet volume, the insulin concentration, the microfluorometric viability test, the glucose-dependent insulin secretion in vitro are determined in the individual samples according to international standards (Ricordi C. et al. Acta Diabetol. Lat., Loc. Cit.).
  • the DNA content is determined by the method of Labarca C. and Paigen K., Anal Biochem 102: 344-352,
  • the amylase activity of the individual samples is determined using the ⁇ -amylase EPS R kit (Boehringer Mannheim, Mannheim).
  • a mixed lymphocyte-island culture is carried out to determine the immunogenicity, see Ulrichs K. et al .: Horm. Metabol. Res. 25 / S: 123-127, 1990).
  • 100 pig islands are cultured together with 10-5 human lymphocytes for three days at 37 °, then after the addition of 10 ⁇ Ci ⁇ H-thymidine coculti vation for 20 h. long islets and xenogeneic lymphocytes.
  • the lymphocyte proliferation correlates with the ⁇ H-thymidine incorporation and is expressed with a ß-counter as "counts per minute" and taking into account control values after conversion as a stimulation index.
  • IPGZ isopycnic centrifugation

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Diabetes (AREA)
  • Veterinary Medicine (AREA)
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  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
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  • Obesity (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des préparations d'ilôts de Langerhans transplantables, sous forme particulièrement pure, un nouveau procédé de préparation, ainsi qu'une application thérapeutique et/ou diagnostique des ilôts.
PCT/EP1993/002178 1992-08-16 1993-08-16 Ilots de langerhans sous forme pure WO1994004169A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP6505891A JPH07500504A (ja) 1992-08-16 1993-08-16 純粋な形態におけるランゲルハンス島
AU47099/93A AU680828B2 (en) 1992-08-16 1993-08-16 Islets of Langerhan's in pure form
EP93917795A EP0610473A1 (fr) 1992-08-16 1993-08-16 Ilots de langerhans sous forme pure

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE4227066A DE4227066A1 (de) 1992-08-16 1992-08-16 Langerhans'sche Inseln in reiner Form
DEP4227066.9 1992-08-16

Publications (1)

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WO1994004169A1 true WO1994004169A1 (fr) 1994-03-03

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PCT/EP1993/002178 WO1994004169A1 (fr) 1992-08-16 1993-08-16 Ilots de langerhans sous forme pure

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EP (1) EP0610473A1 (fr)
JP (1) JPH07500504A (fr)
AU (1) AU680828B2 (fr)
CA (1) CA2120724A1 (fr)
DE (1) DE4227066A1 (fr)
WO (1) WO1994004169A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11033666B2 (en) 2016-11-15 2021-06-15 Giner Life Sciences, Inc. Percutaneous gas diffusion device suitable for use with a subcutaneous implant
US11642501B2 (en) 2017-05-04 2023-05-09 Giner, Inc. Robust, implantable gas delivery device and methods, systems and devices including same
US11701215B2 (en) 2013-09-24 2023-07-18 Giner, Inc. System for gas treatment of a cell implant

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU715177B2 (en) * 1994-04-13 2000-01-20 Research Corporation Technologies, Inc. Methods of treating disease using sertoli cells and allografts or xenografts
JP5763894B2 (ja) * 2009-07-01 2015-08-12 タカラバイオ株式会社 細胞の分離方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0191613A2 (fr) * 1985-02-07 1986-08-20 McDONNELL DOUGLAS CORPORATION Procédé d'isolation d'îlot

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3468611A (en) * 1966-05-10 1969-09-23 Lawrence T Ward Liquid applicator
JPS6345334U (fr) * 1986-09-12 1988-03-26
JPH04119898A (ja) * 1990-09-11 1992-04-21 Jiro Hori マーカ

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0191613A2 (fr) * 1985-02-07 1986-08-20 McDONNELL DOUGLAS CORPORATION Procédé d'isolation d'îlot

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANTONIO BUITRAGO, ET AL.: "RAPID ISOLATION OF PANCREATIC ISLETS FROM COLLAGENASE DIGESTED PANCREAS BY SEDIMENTATION THROUGH PERCOLL TM AT UNIT GRAVITY", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS., vol. 79, no. 3, 1977, DULUTH, MINNESOTA US, pages 823 - 828, XP024772291, DOI: doi:10.1016/0006-291X(77)91185-8 *
BIOLOGICAL ABSTRACTS, vol. 92, 1991, Philadelphia, PA, US; abstract no. 98540, MARCHETTI, PIERO ET AL.: "AUTOMATED LARGE-SCALE ISOLATION, IN VITRO FUNCTION AND XENOTRANSPLANTATION OF PORCINE ISLETS OF LANGERHANS" *
GARTH L. WARNOCK ET AL.: "VIABLE PURIFIED ISLETS OF LANGERHANS FROM COLLAGENASE-PERFUSED HUMAN PANCREAS", DIABETES, vol. 38, no. SUP1, January 1989 (1989-01-01), pages 136 - 139 *
TRANSPLANTATION, vol. 52, no. 2, 1991, pages 209 - 213 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11701215B2 (en) 2013-09-24 2023-07-18 Giner, Inc. System for gas treatment of a cell implant
US11033666B2 (en) 2016-11-15 2021-06-15 Giner Life Sciences, Inc. Percutaneous gas diffusion device suitable for use with a subcutaneous implant
US11642501B2 (en) 2017-05-04 2023-05-09 Giner, Inc. Robust, implantable gas delivery device and methods, systems and devices including same

Also Published As

Publication number Publication date
CA2120724A1 (fr) 1994-03-03
AU680828B2 (en) 1997-08-14
EP0610473A1 (fr) 1994-08-17
AU4709993A (en) 1994-03-15
JPH07500504A (ja) 1995-01-19
DE4227066A1 (de) 1994-02-17

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