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WO2019106685A1 - Néo-organe endocrinien humanisé mis au point par bio-ingénierie utilisant des matrices de rate décellularisée - Google Patents

Néo-organe endocrinien humanisé mis au point par bio-ingénierie utilisant des matrices de rate décellularisée Download PDF

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Publication number
WO2019106685A1
WO2019106685A1 PCT/IN2018/050183 IN2018050183W WO2019106685A1 WO 2019106685 A1 WO2019106685 A1 WO 2019106685A1 IN 2018050183 W IN2018050183 W IN 2018050183W WO 2019106685 A1 WO2019106685 A1 WO 2019106685A1
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Prior art keywords
organ
neo
endocrine
spleen
humanized
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PCT/IN2018/050183
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English (en)
Inventor
Sandeep Kumar Vishwakarma
Avinash Bardia
Md. Aejaz Habeeb
Syed Ameer Basha Paspala
Aleem Ahmed Khan
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Dr Habeebullah Life Sciences Limited
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Publication of WO2019106685A1 publication Critical patent/WO2019106685A1/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
    • C12N5/0679—Cells of the gastro-intestinal tract
    • 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
    • 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
    • A61K35/26—Lymph; Lymph nodes; Thymus; Spleen; Splenocytes; Thymocytes
    • 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
    • C12N5/067—Hepatocytes
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated 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
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C12N5/0672—Stem cells; Progenitor cells; Precursor cells; Oval 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
    • 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 described herein relates with the generation of functional humanized endocrine neo-organ.
  • This invention is comprised of an unique strategy of utilizing decellularized organ scaffold to bioengineer functional biologically tolerable heterografts.
  • the invention disclosed herein further discloses a method of preparing the insulin producing heterograft humanized endocrine neo-organ of claim 1 comprising of preparing ex vivo acellularized/decellularized spleen matrices (DSM) using detergents/chemicals through one more perfusions, and repopulating the acellularized/decellularized spleen matrices with hHPC’s.
  • DSM ex vivo acellularized/decellularized spleen matrices
  • the decellularization process as described herein is simple, faster and efficient which maintains intact organ architecture, extracellular matrix, vasculature and mechanical properties and provides enhanced level of engraftment, long-term survival and function of repopulated cells.
  • FIG. 1 illustrates the functional analysis of endocrine nature of repopulated DSM under hyperglycaemic stimulation.
  • A Representative quantification of dynamics for insulin secretion under sequential hyperglycaemic challenge showing increased levels at each challenge
  • B Fluorescence images of un-induced and hyperglycemia induced cells within the DSM at different time points
  • C-D Comparison of released insulin by repopulated DSM and 2D-cultured cells on collagen under continuous hyperglycaemic challenge. The insulin level was comparatively higher in DSM than 2D-cultured cells.
  • the invention describes a process for the repopulation of prepared decellularized spleen matrices (DSM) with trans-differentiated human hepatic progenitor cells (hHPCs) which would be triggered into insulin producing cells (InPCs) under hyperglycemic challenge.
  • DSM decellularized spleen matrices
  • hHPCs trans-differentiated human hepatic progenitor cells
  • InPCs insulin producing cells
  • the endocrine response of repopulated cells was evaluated with hyperglycemic challenge in time dependent manner and compared with the conventional two-dimensional culture system.
  • bio/immune compatibility and in vivo functional response of humanized bioengineered neo-endocrine organ was identified by intraperitoneal transplantation in rats.
  • hHPCs in DSM through splenic artery demonstrated flow of cell perfusate inside the vascular network form proximal to distal and further distributed into periphery region of the organ. Few cells were found to leak out from the vessels and entered into the decellularized parenchyma matrix, while others remained into the decellularized vascular networks of the spleen. Since only limited numbers of hHPCs (3.0 X 106) were infused, the majority of the organ space remained empty as observed after 4 h of repopulation (Fig. 4A). Further these repopulated cells were cultured in high glucose medium (23mM) and allowed to proliferate for 7 days within the DSM.
  • high glucose medium 23mM
  • Glucose induced activation of pancreatic transcription factors in hHPCs within DSM was identified using immunofluorescence staining of repopulated DSM at day 1, day 3 and day 14.
  • Antibodies specific to beta cell specific protein Nkx-6.1 and insulin related protein C-peptide was used along with DAPI to identify the expression of pancreatic markers which is required to trigger trans-differentiation of hHPCs into insulin producing cells (InPCs) as reported in our earlier studies (Khan et al. 2010; Vishwakarma et al. 2014b).
  • Fig. 4 clearly demonstrates highly induced co-expression of both Nkx-6.1 and C-peptide in trans-differentiated cells within the DSM. The highest co-expression for both the markers was observed at day 7.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
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  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne le développement d'un néo-organe humanisé produisant de l'insuline à l'aide de la rate entière décellularisée repeuplée avec des cellules sensibles au glucose. Le néo-organe produisant de l'insuline survit et conserve la capacité de produire de l'insuline à la suite d'une greffe intra-omentale. L'invention concerne également un procédé de préparation dudit néo-organe humanisé produisant de l'insuline à l'aide de la rate entière décellularisée.
PCT/IN2018/050183 2017-11-29 2018-03-31 Néo-organe endocrinien humanisé mis au point par bio-ingénierie utilisant des matrices de rate décellularisée WO2019106685A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN201741042838 2017-11-29
IN201741042838 2017-11-29

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WO2019106685A1 true WO2019106685A1 (fr) 2019-06-06

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009039185A1 (fr) * 2007-09-17 2009-03-26 The Trustees Of Columbia University In The City Of New York Utilisations d'un échafaudage modifié immunologiquement pour la prévascularisation d'un tissu et la transplantation de cellules
US20120064537A1 (en) * 2010-06-30 2012-03-15 Miromatrix Medical Inc. Use of perfusion decellularized organs for matched recellularization
US20130084266A1 (en) * 2009-10-29 2013-04-04 The General Hospital Corporation Methods for generating pancreatic tissue

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009039185A1 (fr) * 2007-09-17 2009-03-26 The Trustees Of Columbia University In The City Of New York Utilisations d'un échafaudage modifié immunologiquement pour la prévascularisation d'un tissu et la transplantation de cellules
US20130084266A1 (en) * 2009-10-29 2013-04-04 The General Hospital Corporation Methods for generating pancreatic tissue
US20120064537A1 (en) * 2010-06-30 2012-03-15 Miromatrix Medical Inc. Use of perfusion decellularized organs for matched recellularization

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