EP1615999A2 - Procede nouveau pour detacher des cellules confluentes de microsupports bidimensionnels et son application pour preparer des transplantations - Google Patents

Procede nouveau pour detacher des cellules confluentes de microsupports bidimensionnels et son application pour preparer des transplantations

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Publication number
EP1615999A2
EP1615999A2 EP04717723A EP04717723A EP1615999A2 EP 1615999 A2 EP1615999 A2 EP 1615999A2 EP 04717723 A EP04717723 A EP 04717723A EP 04717723 A EP04717723 A EP 04717723A EP 1615999 A2 EP1615999 A2 EP 1615999A2
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Prior art keywords
cells
adcs
microsupport
organoid
derived
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German (de)
English (en)
Inventor
Alain Miller
Christophe Henry
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CILBIOTECH S A
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CILBIOTECH S A
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Publication of EP1615999A2 publication Critical patent/EP1615999A2/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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using microcarriers
    • 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • 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
    • C12N2539/00—Supports and/or coatings for cell culture characterised by properties
    • C12N2539/10—Coating allowing for selective detachment of cells, e.g. thermoreactive coating

Definitions

  • the present invention relates to a process for detaching confluent anchorage- dependent cells (ADCs') from two-dimensional microsupports (2D-MS) onto which these ADCs' are cultivated, particularly ADCs' which can be cultivated in a state suitable for transplantation, such as ADCs' derived from pancreatic cells.
  • the invention further relates to a process for preparing reconstituted organoids from such detached confluent ADCs', which can be immortalized, according to the invention.
  • the invention also relates to the use of organoids obtained by the. process according to the invention for screening cell activity modulator or as therapeutic transplant for their administration in patients in need of such treatment.
  • the present invention relates to a method for preventing or treating diabetes wherein pancreatic cells-derived organoids according to the invention are transplanted in a patient in need of such treatment.
  • ADCs' are dependent on adhesion to a support for their proliferation and for retaining their cellular functions and viability.
  • the majority of vertebrate cell cultures in vitro are grown as monolayers on an artificial substrate bathed in nutrient medium.
  • the nature of the substrate on which the monolayers grow may be solid, such as plastics, or semisolid gels, such as collagen or agar.
  • disposable plastics have become the preferred substrate used in tissue and cell culture.
  • a number of solid microsupports used for the mass cultivation of ADCs' are often characterized by a three-dimensional (3D) geometry such as spherical geometry microbeads (i.e. CytodexTM Pharmacia (UPPSALA, Sweden)).
  • 3D-MS three-dimensional geometry
  • 2D-MS two-dimensional geometry
  • each single cell spreads into a starlike shape and then divides to finally form a "cobblestone" appearance at confluence at which stages, cells are joined together by intercellular junctions.
  • the method used to recover the monolayer cells sheet attached to its microsupport, particularly on 2D-MS, while retaining the biological and physiological properties of the cells is one of the limitations of this type of technology on microsupports.
  • the monolayers are detached from the microsupports onto which they are formed using enzymatic and/or chelating agents treatments, such as for example trypsin and collagenase enzymatic digestion , along with a chelating agent such as ethylene diamme tetraacetate (EDTA), all treatments which can damage the cell functions and particularly the intercellular bonds or intercellular junctions which are necessary to keep their properties.
  • enzymatic and/or chelating agents treatments such as for example trypsin and collagenase enzymatic digestion
  • EDTA ethylene diamme tetraacetate
  • pancreatic cells such as Langerhans islets-derived cells are transplanted into a patient suffering from diabetes in order to provide the controlled amounts of insulin necessary for the patient.
  • Diabetes mellitus (usually referred to simply as diabetes) is a complex disease characterized by a grossly abnormal pattern of carbohydrate metabolism (especially a permanent elevated blood glucose concentration) resulting from impaired insulin secretion and/or effectiveness.
  • the incidence of diabetes in industrialized countries is about 10 %. Indeed, diabetes is the most common serious metabolic disease in the world, it affects hundreds of millions of people.
  • Islets of Langerhans transplants named hereinafter PILS (Pseudo Islets of Langerhans), containing cells in a suitable state for transplantation and which could differentiate when transplanted into either insulin- or glucagon- producing cells.
  • the present inventors have discovered that a dispase enzymatic treatment to detach monolayers from solid microsupports onto which they are cultivated allows one to obtain monolayers wherein cell junctions and viability of these proliferative ADCs' are not impaired and wherein said monolayers can be organized in a suitable state for transplantation.
  • the present inventors have also discovered that monolayers derived from pancreatic cells and detached by a dispase enzymatic treatment from 2D-MS, such as MicroHexTM, allows them to obtain spontaneously-formed Pseudo Islets of Langerhans organoid (PILS) containing cells in a suitable state for transplantation and which could differentiate upon transplantation into either insulin- and/or glucagon-synthesizing cells.
  • 2D-MS such as MicroHexTM
  • the present inventions is directed towards a method to detach a confluent anchorage-dependent cell (ADC) monolayer from the flat microsupport onto which said ADCs' are cultured, characterized in that said method comprises an enzymatic treatment with an enzyme which cleaves fibronectin and type
  • proteases such as dispase, collagenase, trypsin or trypsin-EDTA, liberase, elastage, pronase, papain and accutase can be cited.
  • said enzyme is a dispase.
  • said dispase is a dispase II.
  • Dispase particularly Dispase II, has proven to be an effective but gentle neutral protease for separating intact epidermis from the dermis (Reenstra et al., Anat. Rec, 232(3):340-8, 1992) or to separate porcine keratinocytes primary culture in logarithmic growth phase from the floor of the petri dishes onto which they are cultivated (Cao et al., Zhonghua Wai Ke Za Zhi 2002, 40:24-26). Dispase II cleaves fibronectin and type IV collagen whereas it degrades type I collagen only minimally (Stenn et al., J. Invest. Dermatol., 1989, 93(2):287-290).
  • the invention is directed towards a method for detaching monolayers from their flat microsupport according to the present invention wherein the dispase II is added to a flask containing the microsupport suspension onto which the anchorage-dependent cells have reached confluence.
  • the invention is directed towards a method for detaching monolayers from their flat microsupport according to the present invention wherein about 5 mg/ml (5 ⁇ 1 mg/ml, preferred is 5 ⁇ 0.5 rhg/ml, most preferred is 5 ⁇ 0.1 mg/ml) of dispase II is added to the flask.
  • the invention is directed towards a method for detaching monolayers from their microsupport according to the present invention wherein the dispase II treatment is carried out during at least about 30 min (30 ⁇ 5min), preferably during a time comprised between about 35-45 min (30 ⁇ 5min - 45 ⁇ 5min), more preferably at about 37°C (37 ⁇ 5°C).
  • the invention is directed towards a method for detaching monolayers from their flat microsupport according to the present invention wherein said solid microsupport is a two-dimensional geometry microsupport (2D-MS).
  • two-dimensional geometry means that the thickness of these microsupports tends to become infinitesimal and negligible compared with the dimensions of the cultivated cells. This reduction in thickness is such that there is no possibility of cell growth on the microsupport's edges, but only on the two apposed faces of the 2D-MS.
  • Such 2D-MS offer the principal advantage of an anchorage surface per unit volume that is higher that of 3D microsupports.
  • the invention is directed towards a method for detaching monolayers from their 2D-MS according to the present invention wherein said microsupport is the two-dimensional geometry microsupports MicroHexTM (provided by Nunc, Roskilde, Denmark).
  • the invention encompasses a method for detaching monolayers from their 2D-MS according to the present invention wherein said ADCs' contain one type cell, such as ADCs' derived from one cell clone, or wherein said ADCs' contain at least two type cells such as derived from a co-culture of least two cell clones or derived from a mix of two distinct cell cultures or derived from a tissue containing two distinct cell type (e.g. pancreatic cells containing ⁇ - and ⁇ - cells).
  • ADCs' contain one type cell, such as ADCs' derived from one cell clone, or wherein said ADCs' contain at least two type cells such as derived from a co-culture of least two cell clones or derived from a mix of two distinct cell cultures or derived from a tissue containing two distinct cell type (e.g. pancreatic cells containing ⁇ - and ⁇ - cells).
  • the invention is directed to a method for detaching monolayers from their microsupports according to the present invention wherein said ADCs' can be derived from mammalian tissues, non-human or human mammal.
  • Any mammalian cell may be used in the present invention.
  • mammals cells those which may be grown in the three dimensional culture systems.
  • the most preferred are brain and other neural tissue, pancreatic, bone marrow, skin, liver, pancreas, kidney, heart, mucosal epithelium, neuroepithelial haemopoietic, endothelial, adenocarcinoma or melanoma tissue, or stem cells.
  • stem cell it is intented here to designate a pluripotent progenitor cell that has been characterized as a cell which can self-replicate and has multilineage potential, optionally that is transplantable.
  • the invention is directed towards a method for detaching monolayers from their microsupports according to the present invention wherein said ADCs' are derived from foetal or neonatal tissue, preferably from human foetal or neonatal tissue.
  • the invention is directed towards a method for detaching monolayers from their microsupports according to the present invention wherein said ADCs' are derived from pancreas.
  • the invention pertains to a method for detaching monolayers from their solid microsupports according to the present invention wherein said ADCs' are derived from pancreatic cells, preferably from human or pig pancreas.
  • the invention pertains to a method for detaching monolayers from their solid microsupports according to the present invention wherein said ADCs' are derived from foetal cells, preferably neural foetal cells.
  • the invention encompasses a method for detaching monolayers from their solid microsupports according to the present invention wherein said ADCs' are immortalized, preferably by a method comprising the incorporation of a myc gene (here named "myc-immortalized”).
  • myc-immortalized a myc gene
  • the international PCT patent application document published on September 13, 2001 under the number WO 01/66781 (Sinden et al, Reneuron LTD, GB) which discloses immortal cells including an exogenous polynucleotide introduced into the cell that encodes a member of the myc oncogene family and a conditionally-inducible oncogene which can be regulated under certain conditions.
  • the oncogene will undergo expression when permissive conditions are applied.
  • the oncogene that can be used could be a non-DNA binding, temperature-sensitive, mutant of the SV40 large T-antigen gene, e.g.
  • Suitable alternatives are also known and include the oncogene of the polyoma Tantigen adeno virus EIA and HP VI 6 or 18E7. Suitable cDNA variants may also be used.
  • C-myc, N-myc, L-myc, B-myc, V-myc and Gag-myc can be suitable for usein the present method for preparing the so-called myc-immortalized cells, although C-myc is preferred.
  • a review of the myc family is provided by Alt et al., Cold Spring Harbour Symp. Quant.
  • C-myc, N-myc, Lmyc and B-myc have a similar gene structure and encode nuclear phosphoproteins with homologous amino acid sequences (Legouy et al, EMBO J, 1987, 6:3359-3366; and Ingvarsson et al., Mol. Cell Biol., 1988, 8:3168-3174).
  • a preferred embodiment would permit control over the function of the Myc protein, such that the immortalising protein was not functional after transplanting the cells. This would reduce the risk of overgrowth or tumour growth by the transplanted cells.
  • a preferred conditional form of Myc is a fusion between Myc and the hormone binding domain of a modified estrogen receptor (Littlewood, T., Hancock, D., Danielian, P., Parker, M., and Evan, G., 1995.
  • a modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins. Nuc. Acids.
  • conditionally-inducible oncogene and the polynucleotide encoding the myc gene may be comprised in a recombinant DNA or retroviral vector or construct to transduce/infect the cells.
  • the two components may be incorporated into one vector or each may be comprised in a separate vector which may further comprise a suitable promoter region to initiate transcription of DNA and a selectable marker which may be used to identify those cells that have undergone transduction/infection. Regulation of expression may be carried out by methods known to the skilled person.
  • regulation may be effected using the long terminal repeat (LTR) promoter.
  • LTR long terminal repeat
  • Alternative promoters will be apparent to the skilled person.
  • regulation may be effected using the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • the CMV promoter is a very strong promoter, and may be preferred when the cells are neural cells, e.g. neuroepithelial stem cells.
  • Retroviral vectors including the Zen vectors (Harilaran et al, Oncogene Research, 1988, 3:387-399) may be utilised.
  • the oncogene and the myc gene are incorporated into the cell during the early culture phase, usually within the first 10 cell divisions.
  • the order of incorporating the oncogene and myc gene is not critical, although it is preferred that the myc gene is introduced first because introducing the myc gene first provides better assurance for achieving a diploid cell line.
  • the transduced or infected cells may be cultured under conditions known to those skilled in the art. It is preferable that the cells are cultured under non-stressed conditions. A skilled person will appreciate the conditions suitable for each particular cell type, based on conventional culture techniques.
  • Constructs including an oncogene and the myc gene may be used to transduce suitable cells to produce conditionally-immortalised recombinant cells that have improved stability during passaging.
  • the myc gene may activate directly the catalytic subunit of telomerase (Wu et al., Nature Genetics, 1999, 21:220-224). This may maintain the chromosomes during cell replication.
  • the present invention is directed towards a method for preparing isolated organoids characterized in that said method comprises the following steps of: a) detaching a confluent monolayers made out of ADCs' from the solid microsupport onto which said ADCs' are cultivated by the method according to the invention; b) separating the spontaneously formed organoids from the solid microsupports, said organoids being spontaneously formed from the monolayer detached from their solid microsupports.
  • Two-dimensional geometry microsupports such as MicroHexTM have been developed in order to dispose of a new microcarrier for the culture of anchorage- dependent cells at high cell concentration.
  • the production of isolated organoids according to the present invention consists in attaching cells onto, for example, MicroHexTM until they form a monolayer. When the confluence is reached, the cells are then detached by an enzymatic dispase or enzyme having a dispase type activity treatment according to the present invention which preserves, particularly, the intercellular cell junctions. The cell sheet, free from the microcarrier, then re-organises itself spontaneously into an organoid. This technology is thus of great help to provide both a culture system for the scale-up of precursor cells as well as means to sustain the cell differentiation and functionality by providing a 3D organisation close to that of the real organ.
  • these small spheres can be compared with the small endocrine organoids dispersed in the pancreas, called islets of Langerhans. If pancreatic cells are cultivated onto MicroHexTM the organoids which consequently form, are called PILS (Pseudo Islets of Langerhans).
  • the invention is directed towards a method for preparing isolated organoids according to the present invention, wherein the step b) of separating the organoids from the microsupports is carried out by filtration.
  • the invention is directed towards a method for preparing isolated organoids according to the present invention, wherein the step of filtration occurs through a nylon screen.
  • the invention is directed towards a method for preparing isolated organoids according to the present invention, wherein the step of filtration occurs through a nylon screen of 100 ⁇ m average porosity.
  • the present invention is directed towards a method for preparing islets of Langerhans transplants containing cells in a suitable state for transplantation and capable of differentiating, upon transplantation, into either insulin- and/or glucagon-synthesizing cells, characterized in that said method comprises a step of preparing pancreas-derived organoid, preferably islets of Langerhans-derived organoid, more preferably all pancreas-derived cells from neonatal (3 days-old) piglets organoid, said step of preparing pancreas-derived organoid comprising the following steps of: a) detaching a confluent monolayers made out of ADCs' pancreas-derived cells from the solid microsupport onto which said ADCs' are cultivated, preferably by the method for detaching monolayers from their solid microsupports according to the present invention and wherein said ADCs' are derived from pancreatic cells, preferably from human or pig pancreas,
  • the resulting detached monolayers from their microsupport according to the present invention or the resulting isolated organoids according to the present invention may be used as model systems for the study of physiologic or pathologic conditions.
  • these detached monolayers or isolated organoids derived therefrom may be used as a model to study the protein secretion of these ADCs', their receptors ligands, the signal induced by the ligand/receptor interaction, their proliferation or differentiation, notably in presence of compounds of interest.
  • the present invention is also directed to a method for preparing islets of
  • Langerhans transplants containing cells in a suitable state for transplantation and capable of differentiating when transplanted into either insulin or glucagon cells characterized in that said method comprises the step of: a) preparing pancreas-derived organoid by a method according to the invention claims, wherein the solid microsupport used is a two-dimensional geometry microsupport (2D-
  • said detached monolayer cells cultured on the microsupport are pancreas cells from neonatal piglets or/and the solid microsupport used is the MicroHexTM microsupport.
  • the present invention also pertains to the use of detached monolayer or organoids obtained by the method according to the invention as an in vitro model system for the study of physiologic or pathologic conditions of cells which grown in a three dimensional system.
  • the present invention also relates to an in vitro use of detached monolayer or organoids obtained by the method according to the invention for the screening of compounds that can modulate the activity of cells which grown in a three dimensional system, comprising a step of contacting said detached cell monolayer or organoids with the compound to be tested and selecting the compound if the activity of the cell which is desired to modulate is modulated by the tested compound.
  • the present invention also relates to an in vitro use of detached monolayer or organoids obtained by the method according to the invention as bioreactors for the production of biological materials in vitro.
  • the in vitro method for the production of biological material comprises the following steps of: a) culturing in appropriate conditions and medium the detached monolayer or organoids obtained by the method according to the invention; and b) recuperating from the cultured cells or from the cultured organoid obtained in step a), or from culture medium thereof, the biological material which is desired to produce.
  • said biological material is a protein and said cells of said cultured cells or of said organoid in step a) produce naturally or by recombinant route the protein which is desired to produce.
  • the present invention concerns an in vitro method for the production of a detached cells monolayer or organoid proportionally enriched in ⁇ -cells from anchorage-dependent cells (ADC) which are cultured onto a flat microsupport, said ADCs' being derived from pancreatic cells, containing an initial proportion of ⁇ - and ⁇ -cells, said method comprising the following steps of detaching the cells monolayer obtained by the methods according to the present invention or preparing pancreas-derived organoid by a method according to the present invention.
  • ADC anchorage-dependent cells
  • the present invention is directed towards the use of the organoids obtained by the methods according to the present invention as a transplant, preferably a therapeutic transplant, preferably for the treatment of disease requiring the transplantation of tissue capable of proliferating in vivo.
  • a transplant preferably a therapeutic transplant
  • the cells are differentiated cells, detached monolayers from their microsupport according to the present invention or the resulting isolated organoids according to the present invention may be used for the preparation of a therapeutic transplant for the prevention or the treatment of organ defect such as cancer, or a genetic disease associated to a disease causing mutation wherein the mutation of said cells is prior corrected by an ex vivo homologous recombination method well known by the skilled person.
  • these cells may be used for the prevention or the treatment of diabetes or for the treatment of pancreatic cancer, or liver cancer in case the cells are hepatocytes, etc.
  • Endothelial cells may be used for the revascularisation of the leg, heart and other organs.
  • Human neuroepithelial stem cell may be used in neural transplantation to repair cell loss or damage and correct behavioural or psychological deficits.
  • Neuroepithelial cells may be used in the treatment of Alzheimer's disease, Parkinson's disease, stroke and other forms of cerebral ischaemia, cerebral palsy, multiple sclerosis, Huntingdon's disease and Creuzfeld- Jacob's disease.
  • This detached cell monolayer, recombinant cell monolayer or organoids spontaneously formed by this detached (recombinant) cell monolayer according to the present invention may have use in therapy.
  • Methods for the preparation of formulations for delivery to a patient will be apparent to the skilled person. Suitable excipients, diluents etc., will again be apparent based on current practice in preparing cell-based therapies.
  • the amount of cells or organoids required for delivery will vary depending on the form of treatment, the severity of the disease/damage, and the need for applying multiple doses over a treatment period. The skilled person can readily determine the appropriate treatment based on existing cell transplantation therapies.
  • the cells may be administered using conventional techniques.
  • the present invention is directed towards a method for the treatment of disease requiring the transplantation of tissue wherein said tissue is an organoid obtained by the method according to the present invention and wherein said organoid is transplanted in the patient in need of such treatment.
  • the present invention is directed to a method for the treatment of diabetes wherein an organoid obtained by the method according to the present invention is transplanted in a patient in need of such treatment.
  • FIGURES Figure 1 Cells isolated from the pancreas of 3 days-old piglets incubated (20.10 6 cells/0.14 g MicroHexTM) with MicroHexTM (R&D grade-950 cm 2 /g) at a ratio of 256:1.
  • Figure 3A 3 days-old piglet pancreatic cells on MicroHexTM 7 days after inoculation in a TechneTM spinner flask (inoculation at 256 cells/microsupport); cells are stained with DAPI and picture is taken under UV-fluorescence.
  • Figure 3B PILS next to MicroHexTM, 7 days after inoculation of pancreatic cells in a TechneTM spinner flask (256 cells/microsupports) after detachment of the monolayer with Dispase II.
  • Figures 5 A to 5D Light micrographs of native neonatal (l-3d old) porcine pancreas (Figure 5A), fresh Neonatal Islet Cluster (NIC) ( Figure 5B), cells isolated following dissociation of NIC ( Figure 5C) and PILS ( Figure 5D). Sections (5 microns apart) were counterstained with hematoxylin then immunohistochemically stained for insulin.
  • Figure 6 Electron micrograph of PILS after 7 days of culture. Cells show zymogen- like granules (arrowhead) and micro villi at their membrane (arrow).
  • Figure 7 Blood glucose levels of transplanted diabetic nude mice with 20,000 PILS under the kidney capsule, non-transplanted control and diabetic non-transplanted control.
  • Figure 8 Body weight of transplanted diabetic nude mice with 20,000 PILS under the kidney capsule, non-transplanted control and diabetic non-transplanted control.
  • Figures 9A and 9B Light micrographs of PILS 21 days after transplantation under the kidney capsule of streptozotocined diabetic nude mice. Sections (5 microns apart) were counterstained with hematoxylin then iinmunohistochemically stained for insulin (A) or glucagon (B).
  • Figure 10A shows the majority of the MicroHexTM to be confluent with some aggregation of cell-laden microsupport beginning already to appear.
  • Addition of Dispase II solution to the 60 ml culture and incubation during 35 min. (Figure 10B) or 55 min. (Figure 10D) at 37°C makes the cells to quantitatively detach from the microsupport as films which reorganize themselves into aggregates. Aggregation is somewhat better when after 35 min. incubation at 37°C, the aggregates-naked microsupport suspension is left to stand without agitation ( Figure 10C). Cells in such aggregates are bound together firmly enough to resist a 20 min. exposure to PBS-EDTA (05 mM) at 37°C ( Figure 10E).
  • DAPI diamidinophenylindole
  • a fluorescent nucleic acid stain was purchased from Sigma.
  • Mowiol used to fix the cells on MicroHexTM under DAPI staining is composed of: Tris pH 8.5 (12 ml), Glycerol (6 grams), H 2 O (6ml), Polyvinylalcohol (2.4 grams) and after warming at 50°C, 2.5 % of DABCO (l,4-diazobicyclo-(2,2,2)-octane).
  • Primary antibodies mouse anti-insulin and anti-glucagon were from Novo Nordisk (Copenhagen, Denmark), secondary sheep antibodies anti-mouse IgG biotinylated were from Amersham (Bergrand, Nederland). Streptavidin-Peroxidase was from Roche (Indianapolis, U.S.A.).
  • mice Female, inbred, athymic nude swiss mice (age 6-8 wk) were used as recipients of the PILS (Iffa Credo, Lyon, France). Mice were made diabetic by intravenous injection of (250 mgkg) body wt steptozotocine (Sigma, St. Louis, U.S.A.; freshly dissolved in citrate buffer pH 6.4) 4-5 d before transplantation. Normoglycemic, age-matched mice served as normal controls. All animals entering this study exhibited blood glucose levels above 300 mg/dl. Blood samples were obtained from the tail vein for glucose assay (Glucotouch, Lifescan, Neckargem ⁇ nd, Germany).
  • the aggregates were then transferred to sterile tubes containing 0.2 mg/ml Dnase I and 1 mg/ml Dispase II and aspirated during 6 minutes through a 19G needle at 37°C.
  • the isolated cells were then centrifuged on a percoll gradient (density 1.06 g/1) at 450 g during 15 minutes.
  • the cell pellet was washed three times with Earle-HEPES and the cells then being suspended in Ham's F10 culture medium (supplemented as described above). 20 millions cells were mixed together with 0.140 grams MicroHexTM R&D grade in 125 ml TechneTM flasks with pendulum stirring at 37°C; gassed with 95%/5% O 2 /CO 2 mixture incubator.
  • Dispase II (Roche) were then dissolved into 10 ml of medium and filtered directly into the flask through a 0.22 ⁇ m filter and stirring was resumed at 25 rpm. Cells were maintained together with the enzyme at 37°C for 35-45 minutes. PILS were then separated from MicroHexTM by filtration through a 100 ⁇ m nylon screen. Before transplantation, PILS were washed three times with Earle-HEPES (450 g; 5 min.).
  • Immunohistochemistry Tissue, isolated cells or cell cluster were fixed overnight in Bouin's solution then washed 24 H in H 2 O. Fixed cells and tissues were then dehydrated, embedded in paraffin and tissue sections were cut (5 ⁇ m). Immunohistochemistry was performed to localise glucagon and insulin using a modified avidin-biotin peroxidase method (Petrik et al., Endocrinology 1998, 139:2994-3004). Slides were incubated overnight at 4°C in a humidified chamber with either mouse anti- insulin (1:6000 dilution) or mouse anti-glucagon (1:6000 dilution) (Novo Nordisk, Copenhagen, Denmark).
  • pancreatic cells After being isolated from the pancreas of 3 days-old piglets, pancreatic cells were cultivated in TechneTM spinner flasks. Following the first 24 hours of culture (interrupted stirring mode) approximately 5 % of the cells attached to the microsupport. For an optimised ratio of 256 cells/microsupports, the extent of colonisation (% of MicroHexTM bearing a minimum of one cell) of the culture is around 75 %; this proportion did not change during the culture even when the stirring became continuous 48 hours after the inoculation ( Figure 1).
  • Pancreatic cells proliferated with a population doubling time of 2 days and confluence was reached after 7 days of culture. At that time the monolayer contained a mean number of 29 ⁇ 2 cells (Figure 2).
  • the diameter of these organoids is heterogenous ranging from 40- to 100 ⁇ m ( Figure 4) with a mean diameter of 69 ⁇ 8 ⁇ m.
  • the neonate pancreas can not be considered as a mature organ: its endocrine cells are dispersed as small clusters in the pancreatic tissue; no islet-like structure is observed at this stage (Figure 5A).
  • CoUagenase digestion produces pieces (Neonatal Islet Cluster - NIC) simultaneously preserving the insulin cell content of the whole organ ( Figure 5B).
  • NICs' are dissociated and isolated cells appear. These cells are a mixture of all pancreatic cells types including insulin-and glucagon synthesizing beta cells and alpha cells respectively (Figure 5C). After 7 days of culture, the ratio of cells stained for insulin in the PILS is 5.2 ⁇
  • PILSs' functionality was assessed in an in vivo model using streptozotocined nude mice.
  • the animals made diabetic by injection of streptozotocine were transplanted with 20,000 PILS under the left kidney capsule.
  • Their blood glycemia ( Figure 7) and their weight ( Figure 8) were followed weekly.
  • mice All the transplanted mice have shown their glycemia return to normal value
  • PILS appear to be well conserved in terms of size and structure. They are surrounded by a fibroblastic tissue and neo-vascularisation can be observed in the graft area. Staining for insulin even if it cannot be quantified, appears to be much more intense than in the PILS before transplantation. Some cells are simultaneously stained for insulin and glucagon but the majority of them are stained either for insulin or for glucagon ( Figure 9).
  • Example 4 Culture of myc-immortalized human fetal neural stem cells derived from cortex on laminin-coated MicroHexTM
  • EM/CTX myc-immortalized cloned human fetal cortex cell line
  • EM/CTX myc-immortalized cloned human fetal cortex cell line
  • - inoculation 25,000 EM/CTX cells/cm 2 microsupport
  • MicroHexTM in TechneTM spinner flask on laminin-coated (0.25 ⁇ g laminin/cm 2 MicroHexTM - 10 cm 2 microsupport/ml) in 100 ml culture volume of serum-free DMEM/F12-based growth medium (Gibco cat. 21331-020) enriched with Gibco B27 supplement (cat.
  • Example 5 Formation of aggregates from confluent EM/CTX monolayers. Stability of the aggregates
  • the cell-laden microsupport are washed with growth medium and resuspended in 60 ml growth medium.
  • Ten milliliters of a Dispase II solution in growth medium is added and the culture then incubated for 35-45 min. at 37°C.
  • Dispase Il-mediated detachment 35 min at 37°C with continuous agitation at

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Abstract

L'invention concerne un procédé pour détacher des cellules confluentes dépendantes de l'ancrage (ADC's) de microsupports bidimensionnels (2D-MS) sur lesquels ces ADC's sont cultivées, notamment des ADC's qui peuvent être cultivées dans un état approprié pour transplantation, telles que des ADC's dérivées de cellules pancréatiques. L'invention concerne également un procédé de préparation d'organoïdes reconstitués à partir de ces ADC's confluentes détachées qui peuvent être immortalisées selon l'invention. L'invention concerne en outre l'utilisation des organoïdes obtenus par le processus selon l'invention pour dépister des modulateurs de l'activité cellulaire ou comme un transplant thérapeutique pour leur administration à des patients nécessitant un tel traitement. Finalement, l'invention concerne un procédé de prévention ou de traitement du diabète, selon lequel des organoïdes dérivés de cellules pancréatiques selon l'invention sont transplantés dans un patient nécessitant un tel traitement.
EP04717723A 2003-03-05 2004-03-05 Procede nouveau pour detacher des cellules confluentes de microsupports bidimensionnels et son application pour preparer des transplantations Withdrawn EP1615999A2 (fr)

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