WO2017199034A1 - Nouveau modèle de maladie rénale - Google Patents

Nouveau modèle de maladie rénale Download PDF

Info

Publication number
WO2017199034A1
WO2017199034A1 PCT/GB2017/051386 GB2017051386W WO2017199034A1 WO 2017199034 A1 WO2017199034 A1 WO 2017199034A1 GB 2017051386 W GB2017051386 W GB 2017051386W WO 2017199034 A1 WO2017199034 A1 WO 2017199034A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
matrix
renal disease
podocytes
model
Prior art date
Application number
PCT/GB2017/051386
Other languages
English (en)
Inventor
John Waters
John Bradley
Yvonne RICHARDS
Original Assignee
Cambridge Enterprise Limited
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 Cambridge Enterprise Limited filed Critical Cambridge Enterprise Limited
Priority to GB1820111.1A priority Critical patent/GB2565957A/en
Publication of WO2017199034A1 publication Critical patent/WO2017199034A1/fr

Links

Classifications

    • 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
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • 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
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • 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
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • 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
    • C12N2502/25—Urinary tract cells, renal cells
    • C12N2502/256—Renal 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/28—Vascular endothelial 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
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • 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
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/04—Screening or testing on artificial tissues
    • 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
    • C12N2513/00—3D culture
    • 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
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin
    • 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
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the invention relates to a renal disease model comprising: a matrix which comprises glomerular endothelial cells, mesangial cells and podocytes; and an agent which induces renal disease morphology.
  • a renal disease model comprising: a matrix which comprises glomerular endothelial cells, mesangial cells and podocytes; and an agent which induces renal disease morphology.
  • This invention also relates to methods of making and using the same, and kits thereof.
  • Glomerulosclerosis is the fibrotic glomerular scarring that occurs in many primary renal and systemic chronic kidney diseases, including idiopathic focal segmental glomerulosclerosis (FSGS) and the nephropathy that commonly accompanies diabetes.
  • Progressive chronic kidney disease (CKD) leads to end stage renal failure (ESRF), which carries a high morbidity and mortality.
  • ESRF end stage renal failure
  • Renal replacement therapy with haemodialysis or peritoneal dialysis provides only 10-15% of normal renal function, and the relative risk of death on haemodialysis is 28.6 compared to the normal population at age 30-34 (NHS (2010) Kidney Disease: Key Facts And Figures). Transplantation is not available to all, carries risks associated with immunosuppression, and the current average lifespan of a renal graft is 10-15 years (Wolfe, R.A. ef al. (1999) N Engl J Med 341 , 1725-1730). Preventing or reversing glomerulosclerosis would dramatically improve the health and life expectancy of a large population of patients, and provide a major financial saving to healthcare providers. However, at present the mechanisms of
  • glomerulosclerosis are poorly understood and therapeutic strategies are limited.
  • mice are much more resistant to streptozotocin (Rees, D.A., and Alcolado, J.C. (2005) Diabet Med 22, 359-370).
  • the 5/6 nephrectomy model produces widespread glomerulosclerosis at 12 weeks, but its utility is hampered by a high rate of mortality from this point due to uraemia (Yang, H.C., Zuo, Y., and Fogo, A.B. (2010) Drug Discov Today Dis Models 7, 13-19).
  • the unilateral urethral obstruction model of CKD is not a usual cause of CKD, and only replicates the tubule-interstitial changes and not glomerulosclerosis (Chevalier, R.L., Forbes, M.S., and Thornhill, B.A (2009) Kidney Int 75, 1145-1152).
  • Cell culture studies have been of limited value in modelling glomerulosclerosis because the development of lesions is dependent on complex cellular networks and matrix interactions that are not established in typical cell culture models, often involving a single cell type in monolayer culture.
  • a renal disease model comprising
  • kits comprising the matrix as defined herein and an agent which induces renal disease, as a renal disease model.
  • a method of identifying an agent for the treatment of renal disease comprising contacting a test compound with the model as described herein, and determining if disease progression has been reduced or renal morphology is improved.
  • Figure 1 Human glomerular endothelial microvessels in the 3D matrix
  • Figure 2 Mesangial nodule formation in 3D.
  • FIG. 4 TGFp receptor changes in 3D culture of MCs in response to TGFp and B P7 & Phospho-SMAD expression in human chronic kidney disease tissue, (a)
  • Figure 5 3D culture of human podocytes.
  • (c) CTGF release by podocytes in 2D (black bars) and 3D (white bars) by quantitative reverse transcriptase PCR analysis in response TGFp (10ng/ml) and BMP7 (100ng/ml) stimulation and co-stimulation (fold change compared to control, n 3, standard deviation bars, 2-tailed Student's f test).
  • Figure 6 3D triculture of human GECs, MCs and Podocytes.
  • Insert shows high powered view of foot process with cord
  • FIG. 7 3D culture system analysis of idiopathic focal segmental glomerulosclerosis (FSGS). Actin changes within glomerular endothelial cells within the 3D system in response to urokinase-type-plasminogen-activator receptor treatment (b) versus control (a).
  • FSGS focal segmental glomerulosclerosis
  • a cell culture comprising glomerular endothelial cells, mesangial cells and podocytes.
  • the advantage of this triculture of glomerular cells is the formation of a glomerular vascular network.
  • signalling pathways can be studied in cell cultures of individual cell types
  • the current invention provides a cell culture comprising all three glomerular cells where the interactions between all three cell types can be studied.
  • the cell culture described herein has significant advantages, in particular in respect to modelling glomerular disease for use in identifying therapeutic targets.
  • references to the term "cell culture” as used herein, refer to maintaining cells in an artificial environment comprising the use of items selected from: a suitable vessel; a medium; growth factors; hormones; gasses; and a matrix, and includes 2-dimensiona! and 3 ⁇ dimensionai ceil culture.
  • techniques employed in cell culture include media changes, passaging cells, and fransfecting cells.
  • the glomerular endothelial cells and/or mesangial cells and/or podocytes are mammalian derived.
  • the glomerular endothelial cells and/or mesangial cells and/or podocytes are human derived.
  • glomerular endothelial cells include those which may be obtained from ScienCell Research Laboratories, San Diego, CA, USA as catalogue #4000.
  • mesangial cells includes those which may be obtained from ScienCell Research Laboratories, San Diego, CA as catalogue #4200.
  • podocytes includes those which may be obtained from Celprogen, Torrance, CA, USA as catalogue #36036-08-T25.
  • the cell culture comprises at least 100,000 mesangial cells, at least 20,000 glomerular endothelial cells and at least 5,000 podocytes.
  • the cell culture comprises at least 200,000 mesangial cells, at least 40,000 glomerular endothelial cells and at least 10,000 podocytes.
  • the cell culture comprises at least 300,000 mesangial cells, at least 50,000 glomerular endothelial cells and at least 20,000 podocytes.
  • the cell culture comprises 330,000-340,000 mesangial cells, 50,000-70,000 glomerular endothelial cells and 20,000-24,000 podocytes.
  • a cell culture comprising glomerular endothelial cells, mesangial cells and podocytes in a ratio of at least 1 : 1 : 1.
  • a cell culture comprising glomerular endothelial cells, mesangial cells and podocytes in a ratio of at least 1 :2: 1 or at least 1 :3:1.
  • a cell culture comprising glomerular endothelial cells, mesangial cells and podocytes in a ratio of at least 5: 1 : 1 or at least 10: 1 :1 or at least 5:2:1 or at least 10:2:1.
  • a cell culture comprising glomerular endothelial cells, mesangial cells and podocytes in a ratio of at least 16:3:1.
  • a cell culture comprising glomerular endothelial cells, mesangial cells and podocytes in a ratio of 16:3:1.
  • Optimisation of the media and cell ratios allowed triculture of the three glomerular cell types in 3-dimensions to create a structure that reflects the glomerulus.
  • glomerular endothelial cells surprisingly form vessels with lumens and fenestrae which associate intimately with podocytes, as presented in Figures 1 and 6.
  • a method of producing the cell culture as described herein which comprises combining the cells (i.e. the glomerular endothelial cells, mesangial cells and podocytes) in a medium.
  • the medium comprises components selected from: basal medium; serum; cell growth supplement; and antibiotics.
  • suitable serum include fetal bovine serum.
  • suitable antibiotics include penicillin and streptomycin.
  • the medium comprises RPMI 1640 (Gibco by Thermo Fisher, UK), fetal bovine serum, penicillin, streptomycin, insulin, apo-transferrin, sodium selenite, and EGCS (ScienCell Research Laboritories, San Diego, CA, USA).
  • RPMI 1640 Gibco by Thermo Fisher, UK
  • fetal bovine serum penicillin, streptomycin, insulin, apo-transferrin, sodium selenite
  • EGCS Cruz Chemical Cell Research Laboritories, San Diego, CA, USA
  • the cell culture will require maintenance through application of cell culture techniques.
  • the cell culture is maintained by periodic application of new medium.
  • the cell culture is maintained by application of new medium at least every 24 hours, such as every 48 hours.
  • the cells exhibit normal renal morphology.
  • References to the term "normal renal morphology" as used herein, may refer to inclusion of morphological features selected from any or all of the following features:
  • cord-like structures that lack lumens
  • cord-like structures that lack lumens formed by mesangial cells
  • lumenised cords formed by glomerular endothelial cells via vacuolisation to which podocytes associate; and lumenised cords formed by glomerular endothelial cells via vacuolisation with which podocytes associate and mesangial cells surround but which are not intimately associated.
  • the cells exhibit normal renal morphology within at least 12 hours, such as within at least 24 hours.
  • a matrix comprising glomerular endothelial cells, mesangial cells and podocytes.
  • Advantages provided by the use of the matrix as described herein, include the application of a 3-dimensional system where the signalling pathways between the three glomerular cells can be studied, such as the association of glomerular endothelial cells with podocytes presented in Figure 6.
  • References to the term "matrix” as used herein, refer to a platform used to facilitate the growth of 3-dimensional cellular structures including extracellular matrix and scaffold systems such as hydrogel matrices and solid scaffolds.
  • the matrix is an extracellular protein scaffold. In a further embodiment, the matrix is an extracellular protein scaffold wherein said extracellular protein scaffold comprises collagen and fibronectin. In still a further embodiment, the matrix is an extracellular protein scaffold wherein said extracellular protein scaffold comprises type 1 collagen and plasma fibronectin. An example of suitable type 1 collagen is rat tail type 1 collagen. An example of suitable plasma fibronectin is human plasma fibronectin. In still a further embodiment, the matrix comprises type 1 collagen, plasma fibronectin, HEPES, NaHCOs buffered M199 medium and HCI.
  • the matrix has a pH greater than 7, such as pH 7-8, in particular pH 7.4. As presented in the Examples described herein, favourable results were obtained at a pH 7.4.
  • polymerisation refers to the formation of polymer chains or 3-dimensional networks.
  • said polymerisation typically takes at least 10 minutes, such as 15-25 minutes, in particular 20 minutes.
  • said method is conducted at greater than 3°C, such as 3-10°C, in particular 4°C.
  • 3°C such as 3-10°C, in particular 4°C.
  • said polymerisation of step (b) comprises incubating the matrix at at least 25°C, such as 30-45°C, in particular 37°C.
  • the matrix comprising glomerular endothelial cells, mesangial cells and podocytes will require maintenance through application of cell culture techniques.
  • the matrix comprising glomerular endothelial cells, mesangial cells and podocytes is maintained by periodic application of new medium.
  • the matrix comprising glomerular endothelial cells, mesangial cells and podocytes is maintained by application of new medium at least every 24 hours, such as every 48 hours.
  • a renal disease model comprising:
  • a matrix which comprises or includes glomerular endothelial cells, mesangial cells and podocytes;
  • the renal disease model described herein has significant advantages over the current animal and monolayer cell culture models by forming glomerular vascular networks which provide an in vitro platform for the study of renal disease aetiology.
  • the renal disease model is a glomerular disease model. In a further embodiment, the renal disease model is a glomerulosclerosis model.
  • the renal disease model described herein has significant advantages as a glomerulosclerosis model because histological changes characteristic of glomerulosclerosis are seen ( Figure 6). References to the term "renal disease” as used herein, refer to any disease state where the kidneys fail to adequately filter waste products from the blood.
  • diseases include but are not limited to: nephropathy; nephritis; nephrosis; kidney failure; chronic kidney disease; primary renal chronic kidney disease; systemic chronic kidney diseases; renal insufficiency; end stage renal failure; nondiabetic renal failure; acute kidney injury; acute-on-chronic kidney failure; and associated pathophysiology.
  • glomerular disease refers to any disease state where the glomerular is affected. Examples of such diseases include but are not limited to: glomerulonephritis; and glomerulosclerosis.
  • the glomerular filtration unit is a multicellular structure consisting of a complex basement membrane partitioning glomerular endothelial cell (GEC)-lined capillaries and mesangial cells (MCs) on the side facing the blood from podocytes that face into Bowman's space (Suh, J.H., and Miner, J.H (2013) Nat Rev Nephrol 9, 470-477).
  • GEC basement membrane partitioning glomerular endothelial cell
  • MCs mesangial cells
  • the inventors have developed a 3-dimensional in vitro human glomerular cell culture model of glomerulosclerosis.
  • the inventors first used the 3D system to culture each of the three main human glomerular cells in isolation, and then refined the system to form an in vitro human glomerular vascular structure through 3- dimensional triculture of human glomerular endothelial cells, mesangial cells and podocytes.
  • the inventors demonstrate that TGFp induces a glomerulosclerotic phenotype in this model, which can be used to screen therapeutic targets.
  • the signalling pathways can be studied in culture of individual cell types, the mechanism involves an interaction between all three types and identification of therapeutic targets depends on the 3D culture of all three glomerular cells.
  • glomerulosclerosis is characterised by expansion of matrix within the mesangio-capillary compartment, initially "thickening" the basement membrane and eventually replacing the glomerulus entirely (Schlondorff, D., and Banas, B. (2009) J Am Soc Nephrol 20, 1179-1187) and may refer to nodular glomerulosclerosis, focal segmental glomerulosclerosis, idiopathic focal segmental glomerulosclerosis (FSGS) and associated renal fibrosis.
  • causes of glomerulosclerosis include but are not limited: glomerulonephritis (including but not limited to focal segmental glomerulosclerosis); diabetes; drug use;
  • glomerulosclerosis refers to fibrotic glomerular scarring, the pathophysiology of which includes features selected from but not limited to: a loss of the glomerular endothelial cell vascular structures; podocyte detachment; nodule formation; and matrix deposition.
  • model refers to an in vitro platform which emulates in vivo cells, such as animal models and ceil culture models, !n one embodiment, the model is a 3-dimens!onal cell culture which may be grown in a bioreactor, a small capsule in which the cells can grow into spheroids, or a 3-dimensional cell colony.
  • a method of producing the renal disease model as described herein which comprises:
  • step (c) incubating the product obtained in step (b) with an agent that induces renal disease.
  • step (b) is conducted for at least 12 hours, such as at least 24 hours, in particular 24 hours.
  • step (c) is conducted for at least 12 hours, such as at least 24 hours, in particular 24 hours. It will be apparent to the skilled person that the renal disease model will require maintenance through application of cell culture techniques. In a further embodiment, the renal disease model is maintained by periodic application of new medium. In still a further embodiment, the renal disease model is maintained by application of new medium at least every 24 hours, such as every 48 hours.
  • a method of identifying a compound for the treatment of renal disease comprising contacting a test compound with the model as described herein, and determining if disease progression has been reduced or renal morphology is improved.
  • renal disease morphology refers to morphological features which may be selected from any or all of the following features:
  • a method of identifying a compound for the treatment of renal disease comprising contacting a test compound with the model as described herein, and determining if disease progression is reduced or renal morphology is improved.
  • a compound as described herein for use in the treatment of renal disease, such as glomerular disease, in particular glomerulosclerosis.
  • renal disease such as glomerular disease, in particular glomerulosclerosis.
  • agents refer to a compound or substance capable of producing an effect. Therefore, the term “an agent which induces renal disease” as used herein, refers to a compound or substance, the presence of which results in renal disease or a more advanced form of renal disease.
  • the agent induces deviation from normal renal morphology. In a further embodiment, the agent induces renal disease morphology. In one embodiment, the agent is a member of the TGFp signalling pathway.
  • TGF- ⁇ has been identified as an important downstream mediator of glomerulosclerosis, and a potential therapeutic target for preventing the fibrotic changes that accompany many glomerular diseases.
  • BMP7 may act as an anti-fibrotic agent, counteracting the effects of TGF .
  • Border et al. Shihab, F.S. et al. (1995) J Am Soc Nephrol 6, 286-294; Yamamoto, T. et al. (1996) Kidney Int 49:461-469
  • TGFp signalling pathway refers to the gene regulatory pathway comprising TGFp superfamily ligands, type II receptors, type I receptors, receptor-regulated SMADs, coSMADs, ALK receptors and their regulators.
  • BMP7 expression is altered in glomerulosclerosis.
  • a study assessed BMP7 expression in biopsies from diabetic patients with glomerulosclerosis, and found a significant decrease in BMP7 expression compared to normal biopsies.
  • a larger study found no difference in BMP7 levels within the glomeruli of patients with CKD compared to controls, but did find a significant decrease within the interstitium (Bramlage, CP. et al. (2010) BMC Nephrol 11 , 31).
  • BMP7 Tail vein injection of BMP7 in streptozotocin treated mice reversed proteinuria, restored glomerular filtration rate (GFR), and decreased the percentage of sclerotic glomeruli (Wang, S. et al. (2003) Kidney Int 63, 2037-2049).
  • Studying BMP7 as an anti-fibrotic agent has been hampered due to technical challenges in producing large enough quantities of bioactive BMP7 (Nematollahi, L. et al. (2012) Avicenna J Med Biotechnol 4, 178-185). Sugimoto et al.
  • the agent increases mRNA for ALK5 and/or decreases mRNA for ALK1.
  • the agent decreases TGFpRII/ALK1/ALK5 trimerisation and/or increases TGFpRII/ALK5 dimerisation. In a further embodiment, the agent increases SMAD2/3 phosphorylation and/or decreases SMAD 1/5 phosphorylation.
  • the agent is TGFp.
  • TGFp induces a phenotype characteristic of glomerulosclerosis, therefore the renal disease model presented herein advantageously provides the possibility for in vitro study of glomerulosclerosis and related renal diseases.
  • the agent is present at a concentration of at least 1 ng/ml, such as at least 5 ng/ml, in particular at least 10 ng/ml, such as 10 ng/ml.
  • kits comprising the matrix as defined herein and an agent which induces renal disease, as a renal disease model.
  • a kit comprising the matrix as described herein and an agent which induces renal disease.
  • the kit as described herein as a renal disease model.
  • a matrix as described herein or a component thereof for use in the treatment of renal disease is provided.
  • the glomerular endothelial cells and/or mesangial cells and/or podocytes cells are used as explants for the treatment of renal disease.
  • a portion of the matrix comprising glomerular endothelial cells and/or mesangial cells and/or podocytes cells are used as explants for the treatment of renal disease.
  • Human GECs were cultured in medium supplied by the vendor consisting of 500 ml of basal medium, 25 ml of FBS, 5 ml of endothelial cell growth supplement (ECGS) and 5 ml of penicillin/streptomycin solution.
  • Human mesangial cells (MCs) were cultured in supplied medium consisting of 500 ml of basal medium, 10 ml of fetal bovine serum (FBS), 5 ml of mesangial cell growth supplement, and 5 ml of penicillin/streptomycin solution (glomerular endothelial cells (GECs), MCs, media and supplements were all from ScienCell Research Laboratories, San Diego, CA.
  • GECs, MCs and podocytes in monoculture or co-culture were suspended in a solution of rat tail type 1 collagen (1.5 mg/ml; BD, NJ, USA) and human plasma fibronectin (90 pg/ml; Millipore, MA, USA) in 25 mM HEPES and 1.5 mg/ml NaHC03 buffered M199 medium at
  • triculture media consisting of 100mls RPMI 1640 (GibcoTM by Thermo Fisher, UK), 2mls FBS, 1 ml penicillin/streptomycin, 1 ml insulin (1mg/ml), Apo-transferrin (1 mg/ml), sodium selenite (3.4 uM) (in ITS mix) and 1ml ECGS (supplements all from ScienCell Research Laboratories, San Diego, CA) were pipetted onto the top of the gel, and this media was changed every second day for cultures maintained longer than 24 hours.
  • Stimulation assays 3D stimulation assays to TGF3 and BMP7
  • TGF 10 ng/ml TGF (R&D Systems, MN, USA) or 100 ng/ml BMP7 (R&D
  • the cultures were followed in real time by phase contrast microscopy and at relevant timepoints by epifluorescence microscopy [using a Leica DM I 3000B manually inverted microscope with ImagePro software (MediaCybernetics, MD, USA)] or confocal microscopy [using a Leica TCS SP5 microscope with Leica Application Suite software, Wetzlar, Germany].
  • epifluorescence microscopy using a Leica DM I 3000B manually inverted microscope with ImagePro software (MediaCybernetics, MD, USA)] or confocal microscopy [using a Leica TCS SP5 microscope with Leica Application Suite software, Wetzlar, Germany].
  • For labelling GECs were either transfected with GFP or whole mount immunostained for Ulex europeus Agglutinin I (ULEX, Vector Laboratories, CA, USA).
  • GECs were transduced with green fluorescent protein (GFP) using lentiviral vector containing the human polypeptide chain elongation factor-1 (EF-1) promoter by using 10 ⁇ virus in 4000 ⁇ media per approximately 300,000 GECs.
  • EF-1 human polypeptide chain elongation factor-1
  • Cells were counted and plated into 6-well TC treated plates (Corning Incorporated, Corning, NY), left in culture 24 hours and then culture medium was replaced and cells were treated with lentivirus for 48 hours. After treatment GECs were ready to be counted and incorporated into 3D culture.
  • EF-1 human polypeptide chain elongation factor-1
  • the 3D matrices were washed in normal saline, fixed in 2% glutaraldehyde in 0.1 M PIPES buffer pH 7.4 in 0.1 M sodium cacodylate buffer for 14 hours, dehydrated with increasing concentrations of ethanol to 100%, frozen in liquid nitrogen, and fractured with a razor blade and hammer. They were then critical point dried, mounted on Cambridge SEM stubs with Silver DAG, sputter coated with 10nm of gold and viewed in an FEI XL30 FEG scanning electron microscope operated at 5keV (FEI, Oregon, USA).
  • siRNA knockdown of MCs a 20 ⁇ solution of lyophilised siRNA in 1x siRNA buffer (5x, Dharmacon CO, USA) was used. MCs plated on day one at 150,000 per well (in supplied medium consisting of 500 ml of basal medium, 10 ml of fetal bovine serum (FBS), 5 ml of mesangial cell growth supplement) were transfected on day three. Wells were first washed with 2ml / well of Optimem 1 (Invitrogen, CA, USA) for two hours and then changed to fresh Optimem 1.6ml / well.
  • Optimem 1 Invitrogen, CA, USA
  • SMAD2 and s ⁇ SMAD3 were made up as per manufacturer's instructions (Invitrogen, CA, USA). Lipoplexes were left on the cells for 4 hours, and then removed and normal growth medium added. They were then left overnight before use in stimulation assays. For SMAD3 knockdown the same method was also carried out using DharmaFECT 2 and 4. Knockdown was confirmed by immunoblotting for total SMAD2 and total SMAD 3.
  • CTGF neutralising antibody (ab109606, Abeam, Cambridge, UK) was used at a concentration of 8pg/ml.
  • GECs or MCs were grown in 6 cm dishes to ⁇ 80% confluence and treated with 10 ng/ml TGF6 (R&D Systems, MN, USA) and / or 100 ng/ml BMP7 (R&D Systems, MN, USA) in mesangial cell or endothelial cell medium as described above. At 6 hours the media was aspirated and the cells washed with ice cold PBS, which was then aspirated.
  • the dishes were then placed on ice and the cells scraped in 350 ⁇ RIPA buffer (ThermoFisher Scientific, MA, USA) with protease inhibitors (Roche, Switzerland), transferred to an Eppendorf tube and agitated on ice three times for ten seconds every two minutes, and centrifuged at 1200 rpm for 20 minutes at 4°C. The supernatant was aspirated to another Eppendorf tube for storage at -80°C. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, MA, USA). Proteins (15 g) in Laemmli sample buffer were separated by SDS-polyacrylamide gel (10%) electrophoresis and then transferred to polyvinylidene fluoride membrane.
  • Blots were blocked in 5% dried milk powder / 0.05% Tween 20 in PBS for one hour at room temperature or overnight at 4°C. After blocking they were immunoblotted with rabbit anti-pSMAD2 (3108, Cell Signalling, MA, USA), rabbit anti- pSMAD1/5 (9516, Cell Signalling, MA, USA), rabbit anti-pSMAD3 (1880-1 , Epitomics, Abeam, Cambridge, UK), rabbit anti-tSMAD2 (3122, Cell Signalling, MA, USA), rabbit anti- tSMAD3 (9513, Cell Signalling, MA, USA), or mouse anti- ⁇ actin (A5441 , Sigma-Aldrich, MO, USA) for two hours (40 minutes for actin) at room temperature or overnight at 4°C.
  • rabbit anti-pSMAD2 3108, Cell Signalling, MA, USA
  • rabbit anti- pSMAD1/5 9516, Cell Signalling, MA, USA
  • rabbit anti-pSMAD3 (1880-1 , Epitomics, Abeam, Cambridge,
  • RNA was isolated from 3D matrices by trizol extraction. 1 ml of trizol was added per 300 ⁇ of matrix and vortexed for 15 seconds, left at room temperature for 15 minutes, and vortexed for a further 15 seconds. Chloroform (0.2 mis per 1 ml of trizol used) was added, shaken vigorously for 5 seconds, left to stand for 3 minutes at room temperature, and then centrifuged at 12000g at 4°C for 15 minutes. The RNA (aqueous phase) was removed and a half volume of 100% ethanol added.
  • RNA isolation kit Mo Bio RNA Isolation kit, MoBio, CA, USA
  • RNA isolation kit Mo Bio RNA Isolation kit, MoBio, CA, USA
  • 45 ⁇ of tris-HCI (pH 7.5), sodium chloride and magnesium chloride solution and 5 ⁇ of DNase was added to the filter and incubated at room temperature for 15 minutes. 400 ⁇ of aqueous solution of guanidine thiocyanate was then added and centrifuged at 8000 rpm for 1 minute at 4°C.
  • qPCR reactions were prepared with 45 ng of cDNA using the SYBR® Green JumpstartTM Taq ReadymixTM (Sigma-Aldrich, MO, USA) containing 200 nM of the relevant sense and antisense primers and 10 nM fluorescein (Invitrogen, NY, USA). Reactions were amplified on an iCycler (Bio-Rad, CA, USA) using Quantitect Primers for COL1A 1, COL4A1, CTGF, MMP2, TGFBR2, ALK1, ALK5, ALK3, ALK6 and ALK2 (Qiagen, Germany). The relative expression of target mRNAs was normalized to the housekeeping genes B2M or GAPDH using the ⁇ method and expressed as the fold-change relative to the control.
  • MCs were plated onto non-treated plastic, tissue culture treated plastic, or plastic coated with collagen by incubation overnight with 1.5 mg / ml type I rat tail collagen (BD, Franklin Lakes, N.J., USA) in PBS at 37°C before removal by aspiration, or fibronectin [0.1 mg/ml human fibronectin Millipore, Billerica, Mass., USA in PBS], which was left on the dish overnight at 37°C before aspiration.
  • MCs were cultured in 6 cm culture dishes (Corning, NY, USA) at low density. 10 ng/ml of TGF$ in 3 ml of MC media (described above) was used to culture the cells, and this media was changed every 24 hours. The cells were then followed by phase contrast microscopy using a Leica DM I 3000B manually inverted microscope until they were 3 days post confluent. Staining of human collagen in MC 3D cultures
  • DAB tetrahydrochloride
  • Kidney tissue staining Kidney tissue from patients with diabetic nephropathy (n 3) or focal segmental
  • Quantification of cord networks and nodule counts within the 3-D matrix was performed by taking random images through the matrix with phase contrast or fluorescence microscopy at low power using an Axiovert 200 M Carl Zeiss microscope or Leica DM I 3000B manually inverted microscope. Images were analysed using Image J software (NIH, USA) to quantify cord length (total and average length), branching points (nodes), tube width and cord number as well as MC nodule number. Differences between treatments were analysed using an unpaired two-tailed Student t test. Experiments were repeated three times (biological replicates).
  • TGFp causes glomerular endothelial cell vascular network rarefication in 3D culture which is prevented by BMP7 Human GECs cultured on tissue culture treated plastic form a confluent monolayer with a cobblestone appearance (data not shown). GECs cultured in a 3D collagen and fibronectin matrix form networks of cords surviving up to 6 days (Fig. 1a). Narrow lumens appear to form by coalescence of intracellular vacuoles by about 24 h (Fig. 1a insert and 1 b) and fenestrae can be identified by electron microscopy (Fig. 1c). TGFp induces a loss of arborisation of vascular networks consistent with capillary rarification seen in
  • TGFp causes mesangial cell nodule formation with matrix deposition in 3D culture, which is not prevented by BMP7
  • MCs cultured on plastic coated with or without rat tail collagen type I- or fibronectin grow to confluence and do not alter their morphology in response to TGF ⁇ (data not shown).
  • 3D culture individual MCs randomly disperse throughout the matrix (Fig. 2a). Some MCs intimately associate with collagen fibres, forming cord-like structures that lack lumens (data not shown).
  • TGFp treatment of 3D MC cultures promotes, within 24 h, formation of large nodules (Fig. 2b, c), containing both cells and matrix proteins (Fig. 2b, c). Human collagen type I and IV mRNA was also increased in the TGFp-treated group compared to controls (Fig.
  • EXAMPLE 3 BMP7 modulates TGFp SMAD responses in glomerular endothelial cells but not mesangial cells
  • TGFp also decreased the BMP7 SMAD1/5 phosphorylation. In other words, BMP7 modulates TGFp SMAD responses in GECs but not MCs.
  • EXAMPLE 4 TGFp induced mesangial cell nodule formation 3D culture, which is prevented by ALK5 inhibition TGFp increased mRNA for ALK5 and decreased mRNA for ALK1 in 3D MC cultures (Fig. 4a). This is likely to decrease TGFpRII/ALK1/ALK5 trimerisation and increase
  • EXAMPLE 5 Podocytes exhibit characteristic morphology in 3D culture and upregulate CTGF in response to TGFp
  • Podocytes cultured within the 3D matrix were randomly dispersed and maintained characteristic foot processes (Fig. 5a, b). Treatment with TGFp did not alter morphology, but increased connective tissue growth factor (CTGF) mRNA, particularly in 3D culture (Fig. 5c).
  • CTGF connective tissue growth factor
  • TGFp leads to mesangial nodule formation with loss of glomerular networks and podocyte detachment in 3D triculture of glomerular cells and is prevented with combined ALK5 inhibition and CTGF neutralisation
  • FIG. 6a Optimisation of the media and cell ratios allowed triculture of the three glomerular cell types in 3D (Fig. 6a).
  • GECs formed lumenised cords via vacuolisation (Fig. 1 a insert) to which podocytes associated within 24 h (Fig. 6b, c).
  • MCs surrounded these networks but were not intimately associated with either the GECs or podocytes, which maintained their foot processes (Fig. 6c insert).
  • Fig. 6d treatment with TGFp induced nodule formation
  • Fig. 6e reduced interaction between GECs and podocytes, caused loss of GEC network arborisation
  • Fig. 6f increased collagen I and IV expression
  • FSGS 3D culture system analysis of idiopathic focal segmental glomerulosclerosis (FSGS) FSGS is an irreversible scarring disease of the kidneys with unknown cause.
  • FSGS urokinase-type- plasminogen-activator receptor
  • Glomerular endothelial cells and podocytes were cultured within the 3D system as described herein. They were cultured for 24 hrs. In the treatment group urokinase-type-plasminogen- activator receptor (UPAR, R & D systems) was added to the culture medium at a concentration of 20ng/ml. At the 24 hour time point the matrix was fixed for 30 minutes with 4% paraformaldehyde, washed with phosphate buffered solution-tween (PBST) three times for 5 mins each time. It was then blocked with 5% bovine serum albumin in PBST for 1 hour.
  • UPAR urokinase-type-plasminogen- activator receptor
  • the 3D culture of human glomerular cells described here reveals their ability to assemble into a glomerular vascular structure when cultured in a 3D matrix at optimised ratios and media.
  • GECs form vessels with lumens and fenestrae, which associate intimately with podocytes.
  • TGFp induces a phenotype characteristic of glomerulosclerosis in this model. This includes loss of GEC vascular structures, podocyte detachment, nodule formation and matrix deposition. These aspects provide quantifiable measures that can be used to assess signalling targets involving cross talk between cells, which cannot be assessed in traditional 2D monolayer cultures.
  • BMP7 prevented the GEC component of the glomerulosclerotic phenotype but not the MC component.
  • TGFp to modulate its own receptors. For example, high concentrations of TGFp downregulate TGFpRII and III receptors in human osteoblasts (Gebken, J., et al. (1999) J Endocrinol 161 , 503-510). Such changes are believed to form a feedback loop to dampen down responses in the presence of high concentrations of TGF (Gebken, J., et al. (1999) supra). In the MCs assayed here, TGFp increased the expression of ALK1 and decreased the expression of ALK5 in the 3D system. This was not preventable by BMP7.
  • the response of a cell to TGFp is influenced by the heterodimeric or trimeric receptor formed to accept the ligand.
  • TGFp associated with a heterodimer of TGFbRII and ALK5 signals through phosphorylation of SMAD2 and SMAD3
  • TGFp associated with a heterotrimer of TGFbRII, ALK1 and ALK5 signals thorough SMAD1 , SMAD5 and SMAD8 (Upton, P.D., and Morrell, N.W. (2009) Curr Opin Pharmacol 9, 274-280).

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Urology & Nephrology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un modèle de maladie rénale comprenant : une matrice qui comprend des cellules endothéliales glomérulaires, des cellules mésangiales et des podocytes; et un agent qui induit une morphologie de maladie rénale. Cette invention concerne également des méthodes de production et d'utilisation de celui-ci, et des kits associés.
PCT/GB2017/051386 2016-05-18 2017-05-18 Nouveau modèle de maladie rénale WO2017199034A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
GB1820111.1A GB2565957A (en) 2016-05-18 2017-05-18 Novel renal disease model

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1608764.5 2016-05-18
GBGB1608764.5A GB201608764D0 (en) 2016-05-18 2016-05-18 Novel cell culture

Publications (1)

Publication Number Publication Date
WO2017199034A1 true WO2017199034A1 (fr) 2017-11-23

Family

ID=56320617

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2017/051386 WO2017199034A1 (fr) 2016-05-18 2017-05-18 Nouveau modèle de maladie rénale

Country Status (2)

Country Link
GB (2) GB201608764D0 (fr)
WO (1) WO2017199034A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109456934A (zh) * 2018-10-31 2019-03-12 清华大学 一种三维肾小球模型的制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6492325B1 (en) * 1998-05-22 2002-12-10 Boys Town National Research Hospital Use of α1β1 integrin receptor inhibitors and TGF-β1 inhibitors in the treatment of kidney disease
WO2011073793A1 (fr) * 2009-12-17 2011-06-23 Fondazione Irccs Ca' Granda-Ospedale Maggiore Policlinico Procédé de coculture tridimensionnelle de podocytes et de cellules endothéliales et système de coculture in vitro correspondant

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6492325B1 (en) * 1998-05-22 2002-12-10 Boys Town National Research Hospital Use of α1β1 integrin receptor inhibitors and TGF-β1 inhibitors in the treatment of kidney disease
WO2011073793A1 (fr) * 2009-12-17 2011-06-23 Fondazione Irccs Ca' Granda-Ospedale Maggiore Policlinico Procédé de coculture tridimensionnelle de podocytes et de cellules endothéliales et système de coculture in vitro correspondant

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MOON KYUNG HYUN ET AL: "Kidney diseases and tissue engineering", METHODS, vol. 99, 29 June 2015 (2015-06-29), pages 112 - 119, XP029499263, ISSN: 1046-2023, DOI: 10.1016/J.YMETH.2015.06.020 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109456934A (zh) * 2018-10-31 2019-03-12 清华大学 一种三维肾小球模型的制备方法
CN109456934B (zh) * 2018-10-31 2021-05-11 清华大学 一种三维肾小球模型的制备方法

Also Published As

Publication number Publication date
GB201820111D0 (en) 2019-01-23
GB201608764D0 (en) 2016-06-29
GB2565957A (en) 2019-02-27

Similar Documents

Publication Publication Date Title
Simões et al. Macrophages directly contribute collagen to scar formation during zebrafish heart regeneration and mouse heart repair
KR102656200B1 (ko) 혈관 오가노이드, 상기 오가노이드의 제조 및 사용 방법
KR102460536B1 (ko) 신장 세포 모집단 및 이의 용도
Daniel et al. Nestin expression in repopulating mesangial cells promotes their proliferation
JP2021500929A (ja) 組成物および方法
Bugg et al. MBNL1 drives dynamic transitions between fibroblasts and myofibroblasts in cardiac wound healing
US20210386786A1 (en) Compositions comprising cell-delivered vesicles and uses thereof
Harari-Steinberg et al. Ex vivo expanded 3D human kidney spheres engraft long term and repair chronic renal injury in mice
Chen et al. Chondrocyte sheet in vivo cartilage regeneration technique using miR-193b-3p to target MMP16
Liu et al. Umbilical cord mesenchymal stem cell‐derived small extracellular vesicles deliver miR‐21 to promote corneal epithelial wound healing through PTEN/PI3K/Akt pathway
Belmokhtar et al. Regeneration of three layers vascular wall by using BMP2-treated MSC involving HIF-1α and Id1 expressions through JAK/STAT pathways
Waters et al. A 3D tri‐culture system reveals that activin receptor‐like kinase 5 and connective tissue growth factor drive human glomerulosclerosis
Kellenberger et al. Differential effects of Smad3 targeting in a murine model of chronic kidney disease
WO2017199034A1 (fr) Nouveau modèle de maladie rénale
CN107828878B (zh) Fkbp11基因在防治主动脉夹层中的应用
Chiang et al. The prediction of drug metabolism using scaffold-mediated enhancement of the induced cytochrome P450 activities in fibroblasts by hepatic transcriptional regulators
Wang et al. TLR4-SIRT3 mechanism modulates mitochondrial and redox homeostasis and promotes EPCs recruitment and survival
WO2022104120A1 (fr) Populations de cellules rénales bioactives enrichies, leurs caractéristiques et utilisations
WO2021212044A1 (fr) Procédés et compositions pour améliorer des cellules sc-bêta ou améliorer leur utilité
Lopera Higuita et al. Basement membrane of tissue engineered extracellular matrix scaffolds modulates rapid human endothelial cell recellularization and promote quiescent behavior after monolayer formation
WO2018164141A1 (fr) Inhibiteur de lypd1 et procédé de production de tissu biologique l'utilisant
WO2014173832A1 (fr) Procédé de culture de tissu
US20130108594A1 (en) Method for evaluating angiogenic potential
Azzarello et al. Age‐dependent vascular endothelial growth factor expression and angiogenic capability of bladder smooth muscle cells: implications for cell‐seeded technology in bladder tissue engineering
Sewell Modelling Human Glomerulosclerosis in vitro

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17726019

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 201820111

Country of ref document: GB

Kind code of ref document: A

Free format text: PCT FILING DATE = 20170518

122 Ep: pct application non-entry in european phase

Ref document number: 17726019

Country of ref document: EP

Kind code of ref document: A1