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WO2019122351A1 - Bioencres humaines spécifiques d'un tissu pour la bio-impression 3d physiologique de tissus humains pour une culture in vitro et une transplantation - Google Patents

Bioencres humaines spécifiques d'un tissu pour la bio-impression 3d physiologique de tissus humains pour une culture in vitro et une transplantation Download PDF

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Publication number
WO2019122351A1
WO2019122351A1 PCT/EP2018/086632 EP2018086632W WO2019122351A1 WO 2019122351 A1 WO2019122351 A1 WO 2019122351A1 EP 2018086632 W EP2018086632 W EP 2018086632W WO 2019122351 A1 WO2019122351 A1 WO 2019122351A1
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WIPO (PCT)
Prior art keywords
cells
tissue
ecm
bioprinted
human
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Ceased
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PCT/EP2018/086632
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English (en)
Inventor
Luca FRENGUELLI
Hector Martinez
Erik Gatenholm
Giuseppe Mazza
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Engitix Ltd
Bico Group AB
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Engitix Ltd
Cellink AB
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Publication of WO2019122351A1 publication Critical patent/WO2019122351A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3633—Extracellular matrix [ECM]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing

Definitions

  • Tissue-specific human bioinks for the physiological 3D-bioprinting of human tissues for in vitro culture and transplantation
  • the present invention relates the emerging fields of 3D bioprinting and functional tissue engineering. More specifically it refers to the use of extracellular matrix material (ECM) for use in combination with a biocompatible bioink in order to bioprint human tissues and scaffolds for subsequent use in in vitro culture, transplantation, tissue development, and drug screening and development.
  • ECM extracellular matrix material
  • the authors used two syringes for constructing each structure, one containing PCL and maintained at 80°C, the other one loaded with cell-laden pre-gel and maintained at temperatures below 15°C.
  • the applied pneumatic pressure was in the range of 400-650 kPa for fabrication of the PCL framework.
  • pepsin i.e a protease, breaking down proteins into smaller peptides
  • Skardal et al. (“A hydrogel bioink toolkit for mimicking native tissue biochemical and mechanical properties in bioprinted tissue constructs", Acta Biomaterialia, 2015, 25, 24-34) discloses a hydrogel bioink toolkit, wherein the hydrogel bioink is based on hyaluronic acid and gelatin. It is disclosed that liver spheroids are bioprinted rather than single cells. Further, it is disclosed that tissue-specific ECM digest solutions from liver, cardiac and skeletal muscle can be prepared, and that primary liver spheroids in a liver-specific bioink is bioprinted to create in vitro liver constructs. Skardal et al describe a 2 stage cross-linking approach for the bioprintiong of ECM.
  • the present invention relies on the discovery that the combination of two biomaterials, human tissue-specific ECM (extracellular matrix) and a polysaccharide hydrogel-based, preferably cellulose-based, bioink, with or without cells, in the 3D-bioprinting of human tissues and scaffolds, results in excellent printability and improved cell function, viability and engraftment.
  • the present invention is based on the first report describing the bioprinting of human tissue, specifically hepatic tissue, using human ECM, specifically liver ECM, in combination with cellulose-based bioink.
  • hepatic tissue This is a key advance in the development of cell-instructive bioinks for the study of liver disease and for the development of 3D hepatic tissue for transplantation.
  • the results on hepatic tissue are applicable on many other tissues as well.
  • human tissue and/or cells can be printed by this approach, and alternatively a decellularised tissue in the form of a bioprinted scaffold can be repopulated with cells, and the field opens up for a wide range of applications.
  • the results of the present invention showed the relevance of providing the right natural environment to cells allowing cell survival, growth, proliferation and maintainance of biological functions with the capability of responding to pathological stimuli. Interactions of cells with extracellular matrix molecules play a crucial role in development and disease progression. There is continuous crosstalk between cells and molecules of the extracellular matrix which leads to development of patterns, morphogenesis, differentiation and maintenance of the differentiated phenotype or disease phenotype.
  • the invention relates to a composition
  • a composition comprising biocompatible and chemically-inert polysaccharide hydrogel, preferably nanocellulose hydrogel (e.g. nanofibrillated cellulose, microfibrillated cellulose, crystalline nanocellulose and bacterial nanocellulose), with or without cells, together with human tissue-specific ECM for physiological bioprinting of human tissue analogues and scaffolds.
  • growth factors including super affinity growth factors can be added in bioprinted scaffolds and/or bioprinted tissues.
  • the physiological conditions are related to both 3D bioprinting parameters which are cytocompatible (e.g. temperature, printing pressure, nozzle size, bioink gelation process) as well as to providing a physiological, human tissue environment for 3D cell culture (human ECM).
  • the combination of nanocellulose-based biomaterial together with human ECMs showed improvement in cell function, viability and engraftment compared to the nanocellulose only printed tissues.
  • the invention thus relates to products (e.g. human tissue specific bioinks) and methods (e.g. physiological printing conditions), as well as several applications.
  • the invention relates to a composition for use in 3D-bioprinting and culturing of human or animal tissue comprising
  • ECM proteins are provided, and the ECM solubilisation is protease-free.
  • the composition is provided with cells, preferably human cells.
  • the ratio based on volume of ECM-material versus bioink is in the interval from 5:95 to 95:5 (vol/vol), and preferably the ratio based on volume of ECM- material versus bioink is in the interval from 70:30 to 30:70 (vol/vol).
  • the polysaccharide hydrogel-based bioink is chosen from a bioink based upon at least one of the following constituents: cellulose derivatives, such as cellulose nanofibrils, hyaluronic acid, alginate, agar, pectin, chitosan, gellan gum and carrageenan, or a combination of these constituents.
  • the polysaccharide hydrogel-based bioink is chosen from cellulose nanofibril-based bioink.
  • the polysaccharide hydrogel-based bioink has a concentration in the interval from 0.5 to 40 % (w/v), preferably from 0,5 to 10 % (w/v). This concentration level is relevant both as initial and final concentration, and after dilution with other components of the composition.
  • the polysaccharide hydrogel-based bioink comprises additional biopolymers for cross-linking purposes and/or to contribute to rheological properties of the bioink, such as hydrocolloids or thickening and gelling agents.
  • the additional biopolymer, hydrocolloid or thickening and gelling agent is preferably chosen from the group comprising alginates, hyaluronic acid and its derivatives, agarose and its derivatives, chitosan, fibrin, gellan gum, nanofibrillated cellulose, microfibrillated cellulose, crystalline nanocellulose, bacterial nanocellulose, carrageenans, collagen and its derivatives as well as gelatin and its derivatives.
  • the ECM material obtained from decellularized tissues, is specific for human tissues chosen from healthy and pathological liver, intestine, pancreas, lung, kidney, heart, brain, muscle, skin, vessels, bile duct, adipose, corneal, bone, spleen, thymus, placenta, peritoneum, stomach, prostate, breast or cartilage.
  • the ECM material is non-pathological tissue, or pathological tissue displaying a pathology associated with damage or disease.
  • the concentration of the ECM material is in the interval from 0.001 to
  • the concentration of cells is in the interval from 0.1 million/ml to 150 million/ml.
  • the cells are of human or porcine origin, preferably human origin.
  • the cells are of the following cell types: healthy or diseased cells, such as human primary , stem cells, differentiated stem cells and cell line tissue cells.
  • Stem cells include but are not limited to iPSCs or cells derived from patient-specific iPSCs, embryonic stem cells (hESCs) or cells derived from hESC, mesenchymal stem cells (hMSC) or cells derived from hMSC, MSC, fetal stem cells (e.g. amniotic fluid stem cells) or cells derived from fetal stem cells, cancer stem cells, endothelial progenitor cells (EPC) and bipotent liver stem cells.
  • iPSCs embryonic stem cells
  • hMSC mesenchymal stem cells
  • fetal stem cells e.g. amniotic fluid stem cells
  • EPC endothelial progenitor cells
  • Tissue-specifc cells include but are not limited to (hepatic tissue) primary hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells, intrahepatic NK and T cells, Kupffer cells, liver metastatic cells or hepatocellular carcinoma cell and lines (intestinal tissue), intestinal stem cells, myofibroblasts, or Caco-2 cells; (pancreatic tissue) Islet-beta cells, endothelial cells, pancreatic stellate cells, pancreatic cancer cells, (kidney tissue) podocytes, tubule cells, or cancer cells; (heart tissue) cardiomyocytes, cardiac muscle cells, endothelial cells; (skin tissue) primary dermal fibroblast, keratinocytes, melanocytes; (muscle tissue)smooth muscle cells, skeletal muscle cells, myosatellite cells; (lung tissue)bronchial epithelial cells, macrophage, smooth muscle cells, lung cancer cells; (bone tissue) osteoblasts, osteo
  • the composition is provided in physiological conditions.
  • composition is provided so that at least one of the following conditions are met:
  • composition a. a pH-value for the composition in the interval from 5-8, preferably about 7; b. that the composition is essentially free from pepsin or other proteases;
  • the osmolarity of the composition is in the interval from 275 to 300 mOsm/kg, preferably about 295 mOsm/kg; or
  • the invention in a second aspect, relates to a method for 3D-bioprinting of human tissue comprising bioprinting the composition of the invention, thereby combining cellulose nanofibril-based bioink, and human tissue-specific extracellular matrix (ECM) material, with human cells.
  • ECM extracellular matrix
  • the invention in a third aspect, relates to a method for 3D-bioprinting of at least one scaffold comprising bioprinting the composition of the invention, thereby combining cellulose nanofibril-based bioink and human tissue-specific extracellular matrix (ECM) material.
  • ECM extracellular matrix
  • the method(s) for bioprinting of the invention is/are performed under physiological conditions.
  • At least one of the following conditions are met during 3D-bioprinting:
  • the temperature during the 3D-bioprinting is in the interval from 4°C to 40°C and most approporiately at 37°C;
  • the priniting pressure during the 3D bioprinting is in the interval from 1 to 200 kPa, and preferably below 50 kPa, and even more preferably in the interval from 6-10 kPa, when bioprinting with cells; or
  • cross-linking is performed in one step after the bioprinting.
  • An advantage with a fast, reproducible and accurate single cross-linking step, that is performed post bioprinting, is that the cell culturing and the cell viability is optimised.
  • the invention relates to a bioprinted tissue or organ prepared by the method for 3D-bioprinting with human cells according to invention.
  • the invention relates to the bioprinted tissue or organ according to the invention, for use in the therapeutic applications of e.g. liver diseases, metabolic diseases, diabetes, heart diseases, kidney diseases, skin defects, bone defects, bone and soft tissue sarcomas, lung diseases, vessels repair, intestinal diseases, retinal defects, bladder diseases, prostate diseases, tissue fibrosis (e.g liver, kidney, intestine, lung, skin), cancer in any tissue, such as hepatocellular carcinoma, metastases in any tissue, such as the liver, colon or pancreas, colon cancer, lung cancer, liver cancer, pancreatic cancer, and cancer in any other tissue.
  • liver diseases e.g. liver diseases, metabolic diseases, diabetes, heart diseases, kidney diseases, skin defects, bone defects, bone and soft tissue sarcomas
  • lung diseases vessels repair, intestinal diseases, retinal defects, bladder diseases, prostate diseases, tissue fibrosis (e.g liver, kidney, intestine, lung, skin), cancer in any tissue, such as hepatocellular carcinoma, metastases in any tissue,
  • the invention in another aspect, relates to a method for treating liver diseases, metabolic diseases, diabetes, heart diseases, kidney diseases, skin defects, bone defects, bone and soft tissue sarcomas, lung diseases, vessels repair, intestinal diseases, retinal defects, bladder diseases, prostate diseases, tissue fibrosis (e.g liver, kidney, intestine, lung, skin), cancer in any tissue, such as hepatocellular carcinoma, metastases in any tissue, such as the liver, colon or pancreas, colon cancer, lung cancer, liver cancer, pancreatic cancer, and cancer in any other tissue comprising using the bioprinted tissue or organ according to the invention.
  • tissue fibrosis e.g liver, kidney, intestine, lung, skin
  • cancer in any tissue such as hepatocellular carcinoma, metastases in any tissue, such as the liver, colon or pancreas, colon cancer, lung cancer, liver cancer, pancreatic cancer, and cancer in any other tissue comprising using the bioprinted tissue or organ according to the invention.
  • the invention relates to a method for culturing the bioprinted tissue or organ of the invention, wherein the bioprinted tissue or organ is cultured under physiological or pathological conditions.
  • at least two types of cells are co-cultured at different ratio. Ratio for cells in co-culture are chosen from: 1:1; 1:5, 1:10, 1:25, 1:50; 1:100, 1:150 and any range in between. In case of more than two cell types in culture the ratio is chosen from: 1:1:1; 1:1:5; 1:1:10; 1:1:50; 1:1:100 and any range in between.
  • the method of culturing is for the purpose of in vitro culture, disease modelling, drug screening, biomarker discovery, tissue models for drug
  • the invention relates to an in vitro culture prepared by the method for culturing according to the invention.
  • the invention relates to the use of the in vitro culture according to the invention for tissue development, disease development, drug screening and development and biomarkers.
  • the invention relates to a bioprinted scaffold prepared by the method for 3D-bioprinting according to the invention.
  • the invention relates to the use of the bioprinted scaffold according the invention for wound healing.
  • the invention relates to a method for preparing recellularised tissue, comprising repopulating the bioprinted scaffold of the invention.
  • the invention in another aspect relates to a recellularised bioprinted tissue, produced by repopulating the bioprinted scaffold of the invention with human cells.
  • the invention relates to a bioprinted tissue, scaffold or recellularised bioprinted tissue of the invention, further comprising growth factors.
  • the invention relates to a method for promoting tissue repair, comprising implanting the bioprinted tissue, scaffold or recellularised tissue comprising growth factors of the invention in a diseased tissue or organ.
  • the invention relates to a method of transplanting a bioprinted tissue, organ or scaffold of the invention, whereby the bioprinted scaffolds and/or tissues are implanted into the diseased tissue or organ, such as ectopically implanted subcutaneously or intra-omentum or directly as tissue-patches into the diseased tissue or organ.
  • the invention relates to a method of repairing a tissue or an organ, whereby the bioprinted scaffolds and/or tissues of the invention are implanted as tissue- patches for improving wound healing.
  • the invention in another aspect, relates to a method of treating a disease in a tissue or an organ, wherein a bioprinted tissue of the invention or a recellularised bioprinted tissue of the invention is applied to the tissue or organ, such as by injection, implantation,
  • the invention relates to a method for diagnosing a disease in a human individual comprising the steps of: a. providing a sample of a bioprinted scaffold of the invention from an individual;
  • the invention relates to a method for disease modelling, comprising the steps of: a. providing a bioprinted scaffold of the invention; b. determining the effect of a compound, drug, biological agent , device or therapeutic intervention on the scaffold or tissue.
  • the invention relates to a bioprinted tissue, scaffold or recellularised bioprinted tissue for use in a. implantation in a diseased tissue or organ;
  • Figure 1 discloses the process of providing HEP X bioink, i.e. a human tissue (liver) specific ECM bioink derived from decellularized human liver combined with Cellink Bioink.
  • Figure 2 discloses IHC (Immunohistochemistry) for ECM proteins, wherein HEP X bioink is shown to be positive to all ECM proteins analysed (Samples: LX2 at 7 Day).
  • Figure 3 The printablity tests with the INKREDIBLE 3D bioprinter confirmed the feasibility of printing human liver ECM in solution (hLECM) together with CELLINK bioink at different ratios (hLECM :CELLINK); 50:50 (2,65 mg/ml ECM), 30:70 (1,59 mg/ml ECM) and 20:80 (1,06 mg/ml ECM).
  • (A) Shows the printed filament diameter of the bioink composition 50:50 (hLECM :CELLINK) obtained when bioprinting with varying nozzle diameters of 22G, 25G and 27G and pressures (5, 10 and 15kPa, respectively).
  • (B) shows the printed filament diameter of the bioink composition 50:50 (hLECM :CELLINK) using a 27G nozzle, 15kPa pressure and 2000 mm/min printing speed.
  • Figure 4 discloses live/dead Hep G2.
  • - 7D Cellink condition only dead cells (red in original figure (here: dark grey));
  • - 7D ECM+Cellink mainly alive cells (green (here: light grey/grey));
  • - 14D Cellink condition no cells;
  • - 14D ECM+Cellink massive cell proliferation. Bioprinted tissue was fully packed of cells (still alive, green (light grey/grey)).
  • Figure 5 discloses live/dead Hep LX2.
  • - 7D Cellink condition both alive and dead cells. Good engraftment
  • - 7D ECM+Cellink more alive cells than dead cells. Higher metabolic activity considering the rapid change of the colour of the medium as a function of metabolic activity
  • - 14D Cellink condition only dead cells
  • - 14D ECM+Cellink both alive and dead cells. Most likely 1ml of medium was too low so we should add 2ml after 5-7 days of culture. Extensive proliferation and metabolic activity appreciated by medium colour change.
  • Figure 6 discloses viability at two weeks.
  • AlamarBlue is a proven cell viability indicator that uses the natural reducing power of living cells to convert resazurin to the fluorescent molecule, resorufin.
  • Viable and metabolic active cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viability— and cytotoxicity.
  • Both HepG2 and LX2 viability and metabolic activity was dramatically improved when bioprinted using HEP X (ECM+Cellink). This confirms the previous findings on both live/dead analysis and rapid change in medium colour (from red to yellow) during culture.
  • Figure 7 discloses histology for HepG2. - 7D Cellink condition: only few cells; - 7D
  • ECM+Cellink good distribution through the tissue
  • - 14D Cellink condition few cells
  • - 14D ECM+Cellink massive cell distribution/spheroids.
  • Figure 8 discloses histology for LX2. - 7D Cellink condition: not a lot of cells; - 7D
  • ECM+Cellink excellent distribution through the tissue
  • - 14D Cellink condition few aggregates
  • - 14D ECM+Cellink massive cell distribution/spheroids.
  • Figure 9 discloses histology for LX2 + TGF-beta.
  • Cellink + TGF-beta NO morphological change
  • HEP X + TGFbeta Seems to be some spindle like cells.
  • Figure 10 discloses RNA extraction.
  • RNA extraction values confirmed previous analyses in terms of cell engraftment at different time points.
  • HEP X bioink has a statistical significant effect in increasing cell number at different time points for both LX2 and Hepg2.
  • Figure 11 discloses LX2 gene expression.
  • Gene expression analysis showed that COLlal mRNA expression is downregulated in ECM+Cellink conditions thus suggesting a less activated status of stellate cells (conditions required for in vitro fibrosis model). This is up- regulated upon exposure to TGFbeta.
  • ACTA2 increases at 7days in ECM-cellink but there are no differences at prolonged culture conditions neither with TGFbeta treatment.
  • LOX liver fibrosis target
  • Figure 12 discloses Pro-Collagen 1 secretion.
  • Type-1 collagen is abundantly secreted during liver fibrosis progression.
  • TGF-beta is a central regulator in all stages of chronic liver diseases.
  • One of TGFbeta key function is to activate stellate cells and increases the expression of collagen 1.
  • Data were not normalized on the cell amount (e.g RNA values) because this would dramatically reduce the value for cell-ink only.
  • Figure 13 discloses HEPG2 gene expression. Gene expression analysis was performed only on HepG2 reseeded into ECM-Cellink because no RNA was extracted from Cellink only conditions. Gene expression showed a time-dependent increase in the mRNA expression of Albumin (suggesting a stronger metabolic-like phenotype) as well as AFP (hepatoblastoma marker linked to proliferation)
  • FIG 14 discloses albumin secretion. Albumin secretion was analysed only in ECM-Cellink conditions because HepG2 did not survive in Cellink only. Albumin secretion showed time-dependent statistical significant increase in ECM-Cellink conditions
  • Figure 15 discloses live/dead SNU-387 and SNU-449.
  • A+B 5 million cells/ml
  • C+D 15 million cells/ml.
  • Scale bars 100 pm, 20X magnification.
  • Figure 16 discloses histology for SNU-387 and SNU-449.
  • A+B 5 million cells/ml
  • C+D 15 million cells/ml. Histological analysis and comparison of 5 and 15 million cell densities. It is possible to appreciate an increase in spheroids and clusters formation in figures C,D representing 15 million cells per ml, when compared to figures A,D which represent densities of 5 million cells per ml.
  • Hematoxylin and Eosin were used to proceed with the staining of the samples after 7 days of culture. Scale bars: 100 pm, 20X magnification.
  • Figure 17 discloses SNU-387 and SNU-449 cell density assessment: viability test.
  • Figure 18 discloses a project overview for a pancreatic cancer model.
  • Figure 19 discloses selection of the best cell density for Panc-1. A comparison between 5 million, 10 million and 20 million/ml showed better cell metabolic activitity in 20 million/ml. Increased spatiotemporal proliferation is observed with increased cell density in live/dead and histology. Distinct cell morphology of spheroids and aggregates mimicking tumour development are observed more with 20 million/ml.
  • Figure 20 discloses live/dead staining for CACO-2; day 3, day 7 and day 13. Images showed a time dependent increasing of alive cells with the formation of spheroids at day 13.
  • Figure 21 discloses histology for CACO-2; day 3, day 7 and day 13. Images confirmed live/dead staining results and preservation of epithelial phenotype.
  • Figure 22 discloses co-culture of hepatic cell lines.
  • Fluorescent microscopy images demonstrate higher viable cells (green (here: light grey/grey) than dead cells (red (here: dark grey).
  • LX2 cells cluster within the HEP X bioink while HepG2 cells remain single cells after 14 days.
  • co-culture at 2:1 and 4:1 (hepatocytes: stellate cells) ratios small cluster formation was observed. Scale bars 100 pm.
  • Figure 23 discloses hepatocyte cells in HEP X bioink. Live/dead staining. Hepatocyte (yellow (here: white/light grey)) clustering was observed for up to 21 days of culture with
  • Figure 24 discloses primary stellate cells in HEP X bioink. Live/dead staining. After 3D bioprinting within HEP X, the stellate cells maintain their morphological characteristics (as seen in brightfield). Even only after 4 days, tunnel formation was observed with viable cells (live - green (light grey), dead - red (dark grey)). Scale bars 100 pm.
  • Figure 25 discloses primary stellate cells in HEP X bioink with TGF-b induction. Multiphoton image of 3D bioprinted construct with primary stellate cells. The constructs were cultured for 2 weeks, and then TGF-b (5 ng/ml) induction was performed. TGF-b induction was maintained for 9 days. Primary cells are autofluorescent (seen as yellow (white)) and demonstrated stretched morphology. Clusters of stellate cells exhibit extracellular matrix production (as seen in purple (medium/dark grey)). The extracellular matrix are fibrillary and intertwined within the clusters and cell network. Scale bars are 100 pm (a) and 50 pm (b).
  • Figure 26 discloses primary stellate cells in HEP X bioink with no TGF-b induction from the same experiment as in figure 24.
  • the controls with no TGF-b treatment show some extracellular network but visually not as high in quantity. Scale bars are 425 pm (a and b).
  • Figure 27 discloses printing of single cells in PanX; (a) sample 2 with 55 % viable cells; (b) 57 % viable cells. Images at 10X magnification. Live cells are green in original figure (here: light grey/grey) and dead cells are red (here: dark grey).
  • HEP X is a human tissue specific ECM bioink derived from decellularized human liver combined with Cellink bioink.
  • “Gut-Ink” refer to a human tissue specific ECM bioink derived from decellularized human intestine combined with Cellink bioink.
  • polysaccharide hydrogel-based bioink refers to a bioink having as one of its main constituents at least one polysaccharide component, such as cellulose nanofibrils, alginate, and chitosan, or a combination of these constituents, and that has the capacity to create a suitable microenvironment for combining it with tissue- specific ECM material so that 3D-bioprinting of the combined material, with or without cells, can be performed under conditions that results in excellent printability and improved cell function, viability and engraftment.
  • polysaccharide hydrogel-based bioink refers to a bioink having as one of its main constituents at least one polysaccharide component, such as cellulose nanofibrils, alginate, and chitosan, or a combination of these constituents, and that has the capacity to create a suitable microenvironment for combining it with tissue- specific ECM material so that 3D-bioprinting of the combined material, with or without cells, can be performed under conditions that results in excellent
  • Cellulose nanofibril based bioink refers to a dispersion of cellulose nanofibrils in a liquid media (see W02016/100856 for further definitions).
  • the cellulose nanofibrils have a length of about 1-100 microns and a width of about 10-30 nanometers; a viscosity of between 0.01 and 100 Pa ' s at 100 s "1 ; a solids content ranging from about 0.1-40%.
  • tissue-specific ECM material refers to extracellular matrix biomolecules, such as proteins, growth factors, etc. that are specific for a certain tissue, and are obtained from decellularized tissue, and allow the cells of the tissue to receive physiological and/or pathophysiological signals (see W02010/017474 and WO2015/185912 for further definitions).
  • Bioprinting refers to the utilization of 3D printing and 3D printing-like techniques to combine cells, growth factors, and biomaterials to fabricate biomedical parts that maximally imitate natural tissue characteristics.
  • 3D bioprinting utilizes the layer-by-layer method to deposit materials known as bioinks to create tissue-like structures that are later used in medical and tissue engineering fields.
  • physiological conditions are meant that that the culture or the cells are exposed to conditions (such as pH, osmolarity, temperature and printing pressure (which is equal to extrusion pressure in this context) that are typical to the normal environment for the culture or cells, such as, for human cells, a temperature around 37 °C, such as in the interval from 35-39 °C, a printing pressure in the interval from 1 kPa to 200 kPa, preferably about 6 kPa, a pH in the interval from 5-8, preferably about 7, and an osmolarity in the interval from 275 to 300 mOsm/kg, preferably aboput 295 mOsm/kg.
  • conditions such as pH, osmolarity, temperature and printing pressure (which is equal to extrusion pressure in this context) that are typical to the normal environment for the culture or cells, such as, for human cells, a temperature around 37 °C, such as in the interval from 35-39 °C, a printing pressure in the interval from 1 kPa
  • pathological conditions are meant that the culture or the cells are exposed to inflammatory and/or carcinogenic conditions, e.g. recapitulating the disease.
  • co-culturing cells is meant that cells of at least two types are cultured together.
  • bioprinted scaffold refers to bioprinted structure or tissue printed with a composition without cells.
  • bioprinted tissue refers to bioprinted structure or tissue printed with a composition with cells.
  • the invention relates to a composition
  • a composition comprising a combination of polysaccharide hydrogel-based bioink, such as nanocellulose-based bioink (CELLINK), and tissue-specific extracellular matrix material (ECM), obtained from decellularized tissue, with or without cells depending on application.
  • polysaccharide hydrogel-based bioink such as nanocellulose-based bioink (CELLINK)
  • ECM tissue-specific extracellular matrix material
  • Nanocellulose bioink material Embodiments of the present invention relate to biomaterial in liquid or gel form (e.g., dispersions) defined as a bioink which can be used for 3D bioprinting of scaffolds, tissues and organs. More particularly, embodiments of the invention include the use of the bioink from nanocellulose material with and without cells to bioprint 3D scaffolds, 3D cell culture models, tissues and organs.
  • biomaterial in liquid or gel form e.g., dispersions
  • Bioink, CELLINK ® as described in this invention is preferably composed of a nanofibrillated cellulose dispersion with preferable addition of a crosslinking component. Such bioink can be crosslinked preferably after printing or even during the 3D bioprinting operation. In some applications CELLINK ® can be used without a crosslinking agent. CELLINK ® as described in this invention has unique rheological and gelation properties. That is, it exhibits shear-thinning behavior, high zero-shear viscosity, a fast response to re-establish the high zero-shear viscosity after extrusion and a rapid gelation to avoid deformation of the bioprinted construct.
  • the viscosity of CELLINK ® can be tailor made by selecting a suitable concentration of cellulose nanofibrils, their length (aspect ratio), charge and additives. Desired cytotoxicity characteristics and cell viability characteristics have been developed by a purification process and adaptation of osmolarity of the dispersion in order to print CELLINK ® with living cells.
  • Embodiments of the invention include cellulose nanofibril bioink products prepared by the methods described and include using the products in 3D Bioprinting operations.
  • Cellulose can be generated from plants (such as annual plants), trees, fungi or bacteria, with preferred embodiments generated from bacteria such as from one or more of the genera Aerobacter, Acetobacter, Acromobacter, Agrobacterium, Alacaligenes, Azotobacter, Pseudomonas, Rhizobium, and/or Sarcina, specifically Gluconacetobacter xylinus, Acetobacter xylinum, Lactobacillus mali, Agrobacterium tumefaciens, Rhizobium leguminosarum bv.trifolii, Sarcina ventriculi, enterobacteriaceae Salmonella spp., Escherichia coli, Klebsiella pneu-moniae and several species of cyanobacteria.
  • Cellulose can be generated from any vascular plant species, which include those within the groups Tracheophyta and Tracheobionta.
  • Cellulose nanofibrils formed from cellulose producing bacteria most closely mimic the characteristics of collagen found in human and animal soft tissue.
  • the array of fibrils provides a porous yet durable and flexible material.
  • the nanofibrils allow nutrients, oxygen, proteins, growth factors and proteoglycans to pass through the space between the fibrils, allowing the scaffold to integrate with the implant and surrounding tissue.
  • the nanofibrils also provide the elasticity and strength needed to replace natural collagen.
  • the bacterial cellulose materials have been, after purification, homogenized and hydrolyzed to smooth dispersion.
  • W02016/100856 is hereby incorporated as a reference for the cellulose-based bioink material.
  • Wood-derived cellulose nanofibrils were selected as an alternative raw material for the preparation of cellulose nanofibrillated bioink. The difference is that they do not form three dimensional network and their width is lower (10-20 nanometers) and length is lower (1-20 micrometers).
  • the disadvantage of the wood derived cellulose nanofibrils can be the presence of other wood biopolymers such as hemicelluloses which can affect cells and cause foreign body reaction. These dispersions should preferably therefore be purified by an extraction process and removal of the water phase. It is a sensitive process since it can lead to agglomeration of fibrils which can result in bioink which tends to clog the 3D bioprinter printing nozzle.
  • cell culture media can be used to replace the water content in the nanocellulose bioink utilizing the same method of consecutive centrifugation and resuspention steps until all water content is replaced by cell culture media.
  • the sterilization procedure was performed using electron beam (EB) sterilization at 25 kGy. No effect on viscosity or stability of nanocellulose dispersion was observed after the sterilization process.
  • the nanocellulose bioink can be 3D bioprinted without addition of crosslinker or biopolymer acting as binder.
  • Embodiments are designed to allow cells to stay in the bioink and are able to support extracellular matrix production which results in tissue formation without contraction.
  • nanofibrillated bioinks can be non- degradable.
  • Most biologically occurring materials are degradable, meaning they will break down or deteriorate over time, which can be be problematic for use as disease models, for drug screening or for soft tissue repair.
  • a non-degradable biological material provides a biologically compatible scaffold that will tend to maintain structure and function, or maintain structure and/or function for a desired period of time (such as the length of anticipated testing).
  • materials with good mechanical properties are provided, which properties are desired for use of the constructs as implants.
  • bioink at least one additional biopolymer is added to the bioink, wherein the biopolymer gelling agent or hydrocolloid is chosen from the group comprising alginates, hyaluronic acid and its derivatives, agarose and its derivatives, chitosan, fibrin, gellan gum, crystalline nanocellulose, carrageenans, collagen and its derivatives as well as gelatin and its derivatives.
  • the biopolymer gelling agent or hydrocolloid is chosen from the group comprising alginates, hyaluronic acid and its derivatives, agarose and its derivatives, chitosan, fibrin, gellan gum, crystalline nanocellulose, carrageenans, collagen and its derivatives as well as gelatin and its derivatives.
  • these additional biopolymers are added to the bioink for cross- linking purposes and/or to contribute to rheological properties as hydrocolloids or thickening agents. Addition of crosslinker or binding biopolymers such as alginate can be used to improve printability but also provide mechanical stability after
  • the nanocellulose bioink is used as support material for printing of pH neutralized collagen solution in combination with extracellular matrix.
  • the nanocellulose component will help keep the 3D shape of the printed construct due to its shear-thinning behavior and high zero-shear viscosity. This allows for printing of a complex 3D support, which can, after gelation of collagen solution and/or extracellular matrix, be removed.
  • cellulose nanofibril hydrogels are the extreme shear thinning properties which is crucial for high printing fidelity.
  • High printing fidelity makes it possible to bioprint porous structures which can be spontaneously vascularized upon implantation.
  • Vascularization is a key to promote engraftment with the host tissue, since vascularization makes it possible for oxygen and nutrient transport throughout the bioprinted construct.
  • the cells can migrate through porosity to enhance tissue formation.
  • Another advantage of using cellulose nanofibrils is their large surface area and hydrophilic properties which make them an excellent binder and dispersing agent for organic and inorganic particles.
  • bioinks comprising at least one of the following constituents: cellulose derivatives, such as cellulose nanofibrils, hyaluronic acid, alginate, agar, pectin, chitosan, gellan gum and carrageenan, or a combination of these constituents, or alternatively a non-polysaccharide based bioink such as collagen or gelatin-based, combined with a polysaccharide-based thickening agent.
  • cellulose derivatives such as cellulose nanofibrils, hyaluronic acid, alginate, agar, pectin, chitosan, gellan gum and carrageenan, or a combination of these constituents
  • a non-polysaccharide based bioink such as collagen or gelatin-based, combined with a polysaccharide-based thickening agent.
  • bioinks can be used: CELLINK RGD, CELLINK BONE, CELLINK A, CELLINK A-RGD, CELLINK Collagen (in solution and fibrillary form), CELLINK CollMaGel, CELLINK GelMa, and chitosan-based bioinks.
  • CELLINK BONE bioink offers the same good printability properties and biologically relevant 3D environment as CELLINK bioink with an additional biofunctionalization of synthetic, osteoconductive particles for bone tissue engineering applications.
  • This bioink is composed of two of the main constituents of bone, i.e., calcium and phosphorous.
  • CELLINK BONE bioink can mixed with high concentration of cells with Cellink's CELLMIXER for a one-step bioprinting process.
  • CELLINK BONE bioink is simply cross-linked with Cellink's ionic binding agent after the bioprinting process.
  • CELLINK A Alginate
  • CELLINK A is a biodegradable bioink specifically developed for advanced 3D Bioprinting researchers.
  • CELLINK A offers excellent biocompatibility, easy handling, and works universally with a wide range of human cells.
  • CELLINK A is composed of highly purified sodium alginate and crosslinks with divalent cations. This is a widely used biomaterial known to be inert and biocompatible.
  • CELLINK A bioink can mixed with high concentration of cells with Cellink's CELLMIXER for a one-step bioprinting process. As with Cellink's other bioinks, CELLINK A is simply cross-linked with Cellink's ionic binding agent. Any of Cellink's sacrificial bioinks such as CELLINK Support and CELLINK Start can be utilized when bioprinting with CELLINK A to create porous structures.
  • CELLINK A-RGD bioink can mixed with high concentration of cells with our CELLMIXER for a one-step bioprinting process.
  • CELLINK A-RGD bioink is simply cross-linked with Cellink ' s ionic binding agent. Any of Cellink's sacrificial bioinks such as CELLINK Support can be utilized when bioprinting with CELLINK A-RGD to create porous structures.
  • CELLINK CollMaGel (Collagen methacryloyl) is a collagen l-based bioink that provides mammalian cells with a milieu close to their native environment. The naturally
  • derived collagen I is modified with methacryloyl substitution groups, which crosslink with the assistance of a photoinitiator (eg. Irgacure 2959 or lithium acylphosphinate salt, LAP) and exposure to UV light (eg. INKREDIBLE UV Curing System).
  • a photoinitiator eg. Irgacure 2959 or lithium acylphosphinate salt, LAP
  • UV light eg. INKREDIBLE UV Curing System
  • CELLINK GelMa (Gelatin Methacryloyl) is a gelatin-based bioink that provides mammalian cells with a milieu that resembles some essential properties of their native environment. GelMA is modified with methacryloyl substitution groups, which crosslink in the presence of a photoinitiator (eg. Irgacure 2959 or lithium acylphosphinate salt, LAP) and exposure to UV light (eg. INKREDIBLE UV Curing System), yielding a stable gel. Prior to bioprinting, CELLINK GelMa bioink can mixed with high concentration of cells with Cellink's CELLMIXER for a one- step bioprinting process.
  • a photoinitiator eg. Irgacure 2959 or lithium acylphosphinate salt, LAP
  • UV light eg. INKREDIBLE UV Curing System
  • a thickening agent e.g. in the form of a nanocellulose hydrogel (chosen from nanofibrillated cellulose, microfibrillated cellulose, crystalline nanocellulose and bacterial nanocellulose) to the polysaccharide-based bioink, in order to create an optimal microenvironment for the 3D-bioprinted material.
  • a polysaccharide-based thickening agent may also be combined with a collagen or gelatin-based bioink (i.e. a polypeptide- based bioink).
  • Extracellular matrix material ECM
  • Suitable tissue samples for the ECM material include liver, kidney, muscle, bone, adipose, cartilage, lung, bladder, cornea, skin, intestine, pancreas, prostate, breast, spleen, placenta and heart samples.
  • the tissue sample may include combination of different tissues, such as an animal tail.
  • the ECM material is obtained from decellularized tissue.
  • the tissue sample may be mammalian tissue, for example pig, sheep, rodent, non-human primate or human tissue.
  • the tissue sample is human tissue.
  • ECM material lacking pepsin is used.
  • the ECM material can easily be used for clinical applications, thereby avoiding the down-breaking effects of the animal enzyme pepsin.
  • Human tissue for decellularisation may be obtained from human organs that are unsuitable for clinical use in transplantation. Suitable organs may be obtained in accordance with relevant national laws and ethical guidelines. In addition to this, human tissue may be obtained from tissue resection after surgery. W02010/017474 and WO2015/185912 are hereby incorporated as references for the decellularisation.
  • the sampled tissue may be normal tissue which does not display pathology associated with damage or disease.
  • the sampled tissue may be pathological tissue which displays pathology associated with damage or disease.
  • sampled tissue may be fatty, fibrotic, cancerous, inflamed or display one or more other features associated with disease or damage.
  • pathological tissue may display pathology associated with acute or chronic disease, including viral infections, alcohol or toxin damage, fibrosis, amyloidosis and cancer.
  • pathological tissue examples include fibrotic, cirrhotic or cancerous liver tissue (from different etiologies), amyloidotic kidney tissue, amyloidotic heart tissue, fibrotic intestine tissue, for example from a patient with Crohn' s disease, ulcerative colitis or Inflammatory Bowel Disease (IBD) , cancerous pancreatic tissue, fibrotic lung tissue, fibrotic skin, fibrotic kidney, cancerous breast tissue, cancerous lung tissue, cancerous colon tissue, cancerous pancreatic tissue and cancerous prostate tissue.
  • IBD Inflammatory Bowel Disease
  • Decellularised scaffolds produced from pathological tissue samples may have a different structure and composition from scaffolds produced from healthy tissue samples.
  • the morphology of the pathological scaffold or the amounts or relative amounts of ECM components, such as collagen, tenascin and laminin may be altered in scaffolds from pathological tissue samples compared to healthy tissue samples.
  • Characteristic features of a disease e.g. amyloidogenic protein
  • Pathological tissue may be obtained from an individual with disease.
  • the scaffold may comprise a normal ECM or may be a disease modified ECM.
  • the scaffold may comprise one or more biochemical and biomechanical alterations that are characteristic of a disease or pathology in the tissue or organ.
  • the human ECM scaffolds allow effective attachment, migration, proliferation and three- dimensional organization of cells that are cultured in the scaffold.
  • the decellularised human scaffold may also provide bioactive molecules and bioinductive properties, which maintain cell phenotype and functional properties, and encourage production of tissue specific matrix.
  • a method may comprise bioprinting the scaffold with cells to produce engineered tissues. Suitable cells for bioprinting are listed below.
  • the cells to be added in the composition may be of any type that is suitable for the intended use.
  • the cells may originate from any suitable human or animal species, even though human cells typically will be used, and the cells may be of any tissue type for which the invention is useful and for which the invention works.
  • the cells are of the following cell types: healthy or diseased cells, such as human primary , stem cells, differentiated stem cells and cell line tissue cells.
  • Stem cells include but not limited to iPSCs or cells derived from patient-specific iPSCs, embryonic stem cells (hESCs) or cells derived from hESC, mesenchymal stem cells (hMSC) or cells derived from hMSC,
  • Tissue- specifc cells include but are not limited to (hepatic tissue) primary hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells, intrahepatic NK and T cells, Kupffer cells, liver metastatic cells or hepatocellular carcinoma cell and lines (intestinal tissue), intestinal stem cells, myofibroblasts, intestinal cancer cells, or Caco-2 cells; (pancreatic tissue) Islet-beta cells, endothelial cells, pancreatic stellate cells, pancreatic cancer cells, (kidney tissue) podocytes, tubule cells, or cancer cells; (heart tissue) cardiomyocytes, cardiac muscle cells, endothelial cells; (skin tissue) primary dermal fibroblast, keratinocytes, melan
  • the invention relates to a method for preparing bioprinted tissues or scaffolds that are suitable for use in the various products, uses and methods of the invention.
  • the method for 3D bioprinting of human tissue (with cells) or scaffolds (without cells) comprises combining nanocellulose-based bioink, (with or without human cells), and human tissue-specific extracellular matrix (ECM) material, wherein the 3D-bioprinting is performed under physiological conditions.
  • the 3D bioprinted tissue or scaffold can e.g. be in the form of a grid, drop, tissue-specific shapes like hepatic lobule for liver etc., or the like.
  • the 3D bioprinted tissue or scaffold can e.g.
  • the bioprinter apparatus can be of any commercially available type, such as the 3D Bioprinter INKREDIBLE from CELLINK AB.
  • the method for preparing bioprinted tissues or scaffolds is performed under physiological conditions, which could vary depending on the tissue and/or the cells that are printed. More specifically, the conditions and parameters during bioprinting varies within the following intervals:
  • a cross-linking reagent may be used during or after the bioprinting process (preferably lOOmM CaCI 2 solution). It is preferable to perform the cross-linking step quickly and in such way that reproducible and accurate results are obtained. E.g. the duration of the cross- linking step may be less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes or less than 1 minute. Also, it is important to use a cross-linking approach that will not affect the viability of any bioprinted tissue in a negative way. Therefore, it is for example preferable to perform the cross-linking in one step, and to exclude the use of a UV curing step, especially when viable cells are included in the bioprinting process.
  • Bioprinted tissues produced as described herein display the tissue-specific extracellular matrix protein composition of the source tissue sample.
  • Bioprinted tissues produced from fibrotic, cirrhotic or carcinogenic source tissue samples display the increased ECM components and disease-specific ECM proteins characteristic of the source tissue.
  • Bioprinted scaffold and use of bioprinted scaffold Another aspect of the invention provides a bioprinted human scaffold or tissue produced as described above for the use in e.g. tissue repair.
  • bioprinted scaffolds with or without cells and/or with or without known growth factors can be implanted in diseased-tissues or organs, e.g. as tissue-patches, in order to promote tissue repair.
  • tissue repair can be promoted by wound healing due to the capability of ECM to favour immunomodulation and therefore reducing tissue scarring in fibrotic diseases (e.g. liver fibrosis, intestinal fibrosis, fistulas, Chron's Disease, cartilage defects, etc.).
  • the invention relates to a method for embedding the cells in the bioink to obtain a homogenous cell distribution in order to produce an artificial bioprinted tissue.
  • the cells are of the following cell types: healthy or diseased cells, such as human primary , stem cells, differentiated stem cells and cell line tissue cells.
  • Stem cells include but not limited to iPSCs or cells derived from patient-specific iPSCs, embryonic stem cells (hESCs) or cells derived from hESC, mesenchymal stem cells (hMSC) or cells derived from hMSC,
  • Tissue- specifc cells include but are not limited to (hepatic tissue) primary hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells, intrahepatic NK and T cells, Kupffer cells, liver metastatic cells or hepatocellular carcinoma cell and lines (intestinal tissue), intestinal stem cells, myofibroblasts, or Caco-2 cells; (pancreatic tissue) Islet-beta cells, endothelial cells, pancreatic stellate cells, pancreatic cancer cells, (kidney tissue) podocytes, tubule cells, or cancer cells; (heart tissue) cardiomyocytes, cardiac muscle cells, endothelial cells; (skin tissue) primary dermal fibroblast, keratinocytes, melanocytes; (heart tissue) cardiomyocytes, cardiac muscle cells, endothelial cells; (skin tissue) primary dermal fibroblast, keratinocytes, melanocytes; (skin tissue) primary dermal fibroblast,
  • Bioprinted hepatic tissue may be printed with primary hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells or Kupffer cells.
  • Bioprinted intestinal tissue may be printed with liver cancer cells, liver metastatic cells , iPSCs or cells derived from patient-specific iPSCs, embryonic stem cells (hESCs), mesenchymal stem cells (hMSC) foetal stem cells (e.g. amniotic fluid stem cells) or bipotent liver stem cells.
  • Bioprinted intestinal tissue may be printed with epithelial cells, myofibroblast, endothelial cells, intestinal cancer cells, iPSCs or cells derived from patient-specific iPSCs, embryonic stem cells (hESCs), mesenchymal stem cells (hMSC) foetal stem cells (e.g. amniotic fluid stem cells) .
  • Bioprinted intestinal tissue may be printed with intestinal stem cells, myofibroblasts, or Caco-2 cells.
  • Bioprinted pancreatic tissue may be printed with Islet-beta cells, endothelial cells, pancreatic stellate cells, pancreatic cancer cells, iPSCs or cells derived from patient-specific iPSCs, embryonic stem cells (ESCs), mesenchymal stem cells (MSCs) or foetal stem cells (e.g. amniotic fluid stem cells).
  • Bioprinted kidney tissue may be printed with podocytes, tubule cells, MSC, iPSC or cells derived from patient-specific iPSCs, foetal stem cells (e.g. amniotic fluid stem cells) or cancer cells.
  • Bioprinted heart tissue may be printed with cardiomyocytes, endothelial cells, iPSC or cells derived from patient-specific iPSCs, foetal stem cells (e.g. amniotic fluid stem cells) or MSCs.
  • Bioprinted skin tissue may be bioprinted with primary dermal fibroblast, keratinocytes, melanocytes and immune cells.
  • Bioprinted muscle tissue may be bioprinted with e.g. smooth muscle cells, skeletal muscle cells or myosatellite cells.
  • Bioprinted lung tissue may be bioprinted with e.g.
  • bronchial epithelial cells macrophage, smooth muscle cells, lung cancer cells iPSCs or cells derived from patient-specific iPSCs, embryonic stem cells (ESCs), mesenchymal stem cells (MSCs) or foetal stem cells (e.g. amniotic fluid stem cells).
  • Bioprinted bone tissue may be bioprinted with e.g. osteoblasts, osteoclasts, osteocytes, MSC or MSC differentiated cells, osteoprogenitor cells.
  • Bioprinted cartilage may be bioprinted with e.g. MSC or MSC differentiated cells.
  • Bioprinted tissues can be used for testing the immune system including TCells, Bcells, NK cells, peripheral blood mononuclear cells (PBMC), dendritic cells, monocytes, engineered Tcells or NK cells as well as bioprinted directly with immune cells.
  • Bioprinted immune system tissue may be bioprinted with TCells, Bcells, NK cells, peripheral blood mononuclear cells (PBMC), dendritic cells, monocytes, engineered Tcells or NK cells.
  • Bioprinted tissues may be printed with single cells or with co-culture of cells at different ratio.
  • the bioprinted tissue may be printed with a cell-laden bioink and cultured under suitable conditions.
  • the cells may be directly mixed into the combination of ECM and nanocellulose-based bioink and/or dropped on the surface of the bioprinted scaffold.
  • the cell-laden and/or cell-seeded bioprinted tissue may be cultured under static conditions, for example in a well with culture medium, or under dynamic conditions, for example in a perfusion chamber and/or bioreactor.
  • the bioprinted tissue may be bioprinted with autologous human cells obtained from a patient, for example to produce autologus bioartificial tissue for implantation into the patient.
  • the bioprinted tissue may be bioprinted with allogeneic human cells i.e. cells derived from a different human individual, for example to produce allogenic bioartificial tissue for implantation into the patient.
  • the allogeneic human cells may be screened for immunocompatibility with the patient before implantation.
  • the bioprinted tissue may be bioprinted with non-immunogenic cells, for example cell that have been engineered to remove surface antigens, such as HLA, that might elicit an immune response in an individual.
  • Another aspect of the invention provides a bioprinted human scaffold or tissue produced as described above for the use in modeling human diseases, testing drugs and biomarker discovery.
  • a bioprinted tissue can be cultured in vitro at different time points in order to evaluate tissue development or disease progression.
  • Bioprinted tissue can be composed by one cell type or more cell types.
  • Bioprinted tissue can be exposed to external stimuli in order to recapitulate a specific feature of human disease and identify potential drug targets.
  • bioprinted tissues can be exposed to different concentration of pro-toxicity factors (e.g chemicals, paracetamol, etc.) pro-fatty factors (e.g free fatty acids).
  • bioprinted tissues can be exposed to different concentration of pro-fibrogenic factors (e.g TGFbeta) and/or pro-contractile factors (e.g endothelin-1).
  • pro-fibrogenic factors e.g TGFbeta
  • pro-contractile factors e.g endothelin-1
  • disease-specific ECM as bioink (e.g cirrhotic ECM for modeling hepatocellular carcinoma) or tissue-specific ECM bioink for modeling liver metastases (e.g liver ECM bioink for modeling colon and pancreatic liver metastases), pancreatic cancer (e.g PAN X, pancreatic ECM bioink), colon cancer (e.g intestine ECM bioink), lung cancer (e.g lung ECM bioink)
  • bioink e.g cirrhotic ECM for modeling hepatocellular carcinoma
  • tissue-specific ECM bioink for modeling liver metastases
  • pancreatic cancer e.g PAN X, pancreatic ECM bioink
  • colon cancer e.g intestine ECM bioink
  • lung cancer e.g lung ECM bioink
  • Bioprinted tissue can be used to screen drugs and/or cell-based therapies.
  • bioprinted tissue with cancer cells can be exposed to chemotherapy agents, immunotherapy and/or CAR-T , NK cells.
  • TGF beta treated conditions to model liver fibrosis.
  • Yet another aspect of the invention provides a bioprinted human scaffold or bioprinted human tissue produced as described above for use in the transplantation of a tissue or organ in an individual.
  • a bioprinted human scaffold or bioprinted human tissue may be transplanted to an individual to replace an organ or a tissue.
  • Another aspect of the invention provides a bioprinted human scaffold or bioprinted human tissue produced as described above for use in the treatment of disease or dysfunction in a tissue or organ in an individual.
  • a bioprinted human scaffold or bioprinted human tissue may be implanted in an individual to regenerate a complete new organ or to improve the repair of a damaged organ, or may support the organ function of the individual from outside the body.
  • the bioprinted scaffold or tissue may be useful in therapy, for example for the replacement or supplementation of tissue in an individual.
  • a method of treatment of a disease may comprise implanting a bioprinted human scaffold or bioprinted human tissue produced as described above into an individual in need thereof.
  • the implanted bioprinted scaffold or tissue may replace or supplement the existing tissue in the individual.
  • the bioprinted scaffold or tissue may be used for the treatment of any one of the diseases chosen from, but not limited to: liver diseases, metabolic diseases, diabetes, heart diseases, kidney diseases, lung disease, skin defects, muscle defects, bone defects, bone and soft tissue sarcomas, lung diseases, vessels repair, intestinal diseases, fistulas, cartilage defects, retinal defects, bladder diseases, prostate diseases, tissue fibrosis (e.g liver, kidney, intestine, lung, skin), cancer in any tissue, such as hepatocellular carcinoma, metastases in any tissue, such as the liver, colon or pancreas, colon cancer, lung cancer, liver cancer, pancreatic cancer, and cancer in any other tissue disclosed in this application, comprising using the bioprinted tissue, organ or scaffold.
  • diseases chosen from, but not limited to: liver diseases, metabolic diseases, diabetes, heart diseases, kidney diseases, lung disease, skin defects, muscle defects, bone defects, bone and soft tissue sarcomas, lung diseases, vessels repair, intestinal diseases, fistulas, cartilage defects, retinal defects,
  • the bioprinted tissue or bioprinted scaffold may be useful for disease modelling.
  • Suitable ECM source may be derived from normal tissue sample or pathological tissue sample, as described above.
  • a method of disease modelling may comprise; providing a bioprinted tissue or scaffold produced as described above, optionally bioprinting the tissue or scaffold with cells to produce a recellularised bioprinted tissue, and determining the effect of a compound, drug, biological agent, device or therapeutic intervention on the bioprinted scaffold or tissue or the cells therein.
  • tissue diseases or diseases affecting the tissue such as tissue fibrosis, tissue cancer and metastases, tissue drug toxicity, post- transplant immune responses, and autoimmune diseases.
  • Bioprinted scaffolds and tissues may be useful for the diagnosis of disease. Suitable bioprinted scaffolds and tissues may be derived from tissue from an individual suspected of having a disease in the tissue or organ.
  • a method of diagnosing disease in a human individual may comprise:
  • the presence and amount of scaffold proteins in the sample may be indicative of the presence of disease in the tissue or organ of the individual.
  • bioprinted scaffolds and tissues may also be useful for proteomics, biomarker discovery, and diagnostic applications.
  • the effect of a protease on the components, architecture or morphology of a bioprinted scaffold and tissue may be useful in the identification of biomarkers.
  • ECM solution preparation Decellularized human extracellular matrix (ECM) from Engitix Ltd (United Kingdom) was used for this study. Human decellularized ECM was lyophilized overnight and then placed in a rotary knife mill to create ECM powder. Particles less than 250 pm were collected by sieve through a #60 screen. 1 gram of ECM powder was solubilized in 250 ml of pre-cooled Acetic Acid 0.5 M (ECM concentration 8 mg/ml) and then solution was stirred at 4°C for 72 hours. To better dissolve ECM proteins, solution was sonicated once a day. For pH neutralization, ECM solution was mixed with NaOH 10M and resupsended in PBS lOx (1:9 ECM volume) for restoring the osmotic pressure.
  • ECM extracellular matrix
  • PBS lOx 1:9 ECM volume
  • CELLINK ® bioink preparation CELLINK ® bioink from CELLINK AB (Sweden) was used as bioink in this study.
  • CELLINK bioink was prepared in aseptic conditions and contains 2.5% (w/w) of plant-derived, sterile nanofibrillated cellulose (NFC) and 0.5% (w/w) sterile sodium alginate.
  • NFC dispersion produced by mechanical refinement and enzymatic treatment was used as raw material for bioink preparation. The NFC dispersion was purified using ultrafiltration followed by diafiltration with pyrogen-free water. The charge density of the NFC was determined to be 24 peq/g. The concentration of the NFC was adjusted to 3.0 % (w/w) by centrifugation and removal of excess supernatant.
  • the centrifugation was carried out at 4000 rpm for 10-20 minutes until the desired amount of supernatant was reached.
  • the concentrated NFC was then mixed intensely using a high-speed mixer for viscous materials (DAC 150.1FVZ-K SpeedMixer).
  • the osmolarity of the NFC was then adjusted for cells by dissolving of D-mannitol direcly in the nanocellulose hydrogel to a final concentration of 4.6% D-mannitol (w/v).
  • the sterilization procedure was performed using electron beam (EB) sterilization at 25 kGy. No effect on viscosity or stability of nanocellulose dispersion was observed after the sterilization process.
  • a concentration of 3% (w/w) sodium-alginate (sterile) was prepared in 4.6% D-mannitol (w/v) solution and mixed with the NFC dispersion at an optimal composition of 80:20 (NFCalginate) using the speedmixer device.
  • the resulting CELLINK ® bioink was then transferred to a 3ml cartridge under aseptic conditions and loaded in an INKREDIBLE 3D Bioprinter (CELLINK AB) for printing scaffolds under physiological conditions (23°C, 22kPa) using a 25G conical nozzle and printing speed of 600mm/min.
  • the resulting CELLINK ® bioink was combined with decellularized human ECM and then transferred to a 3ml cartridge under aseptic conditions and loaded in an INKREDIBLE 3D Bioprinter (CELLINK AB) for printing scaffolds under physiological conditions (23°C, 6-10kPa) using a 25G conical nozzle and printing speed of 600-1200mm/min.
  • Example 1 Printability tests: The printablity tests confirmed the feasibility of printing human liver ECM in solution (hLECM) together with CELLINK bioink at different ratios (hLECM :CELLINK); 50:50 (2,65 mg/ml ECM), 30:70 (1,59 mg/ml ECM), 20:80 (1,06 mg/ml ECM). The best ratio in terms of quality of the printed liver ECM seems to be (macroscopic appearance) the 30:70
  • HEP X bioink was prepared by combining Cellink+liver ECM at 50:50 ratio. The final concentration of ECM was 2,65 mg/ml. Human liver cells (HepG2 and Lx2) were mixed within the HEP X to a final cell concentration of 10 million/ml, utilizing the CELLMIXER ® device.
  • Hepg2 cell line is derived from human hepatoblastoma and it is characterized by epithelia phenotype.
  • LX2 is a human hepatic stellate cell line and therefore it is a stromal cell (e.g. fibroblast).
  • the cell-laden constructs were crosslinked with 100 mM buffered calcium chloride solution for 5 minutes.
  • the CaCI 2 solution was thereafter removed; the constructs were rinsed once in complete culture medium and thereafter kept in complete medium, replacing it every other day, depending on the metabolic activity of the cells.
  • the bioprinted tissue constructs were kept in culture up to 14 days and analysed at different time points (e.g 3,7,10, 14 days).
  • a)Cellink+ ECM+LX-2 snap-frozen 4 samples for gene expression and 3 for western blot; fixed 3 samples in formalin for histology and 1 in glutaraldehyde for SEM; 1 sample used for live/dead staining; 6 samples treated with TGF-b 5ng/ml.
  • b)Cellink +LX-2 snap-frozen 4 samples for gene expression and 3 for western blot; fixed 2 samples in formalin for histology and 1 in glutaraldehyde for SEM; 1 sample used for live/dead staining; 6 samples treated with TGF-b 5ng/ml.
  • d)Cellink + HepG2 snap-frozen 4 samples for gene expression; fixed 1 samples in formalin for histology and 1 in glutaraldehyde for SEM; 1 sample used for live/dead staining.
  • b)Cellink +LX-2 snap-frozen 4 samples for gene expression; fixed 2 samples in formalin for histology and 1 in glutaraldehyde for SEM; 1 sample used for live/dead staining and 3 samples used for viability test (alamar blue); collected supernatant from 5 samples treated with TGF-b 5ng/ml, snap-frozen 4 samples treated with TGF-b 5ng/ml and fixed in formalin for histology last 2 samples treated with TGF-b 5ng/ml .
  • c)Cellink + ECM + HepG2 snap-frozen 4 samples for gene expression and 4 for western blot; fixed 2 samples in formalin for histology and 1 in glutaraldehyde for SEM; 1 sample used for live/dead staining; 3 samples used for viability test (alamar blue).
  • d)Cellink + HepG2 snap-frozen 4 samples for gene expression and 4 for western blot; fixed 2 samples in formalin for histology and 1 in glutaraldehyde for SEM; 1 sample used for live/dead staining; 3 samples used for viability test (alamar blue).
  • Example 4 In vitro biocompatibility tests of cellink only vs Cellink+human liver ECM together with LX2 (human hepatic stellate cell line) or HepG2 (human hepatoblastoma cell line)
  • Decellularized human livers were lyophilized and solubilized using a protease-free protocol employing 0.5M acetic acid.
  • the solubilized ECM was mixed with cellulose-based bioink (CELLINK ® Bioink) as support for bioprinting.
  • Human hepatic cell lines (Hepg2 and LX2) were gently mixed with ECM bioink or CELLINK bioink (employed as control) using a CELLMIXER ® directly into a cartridge before bioprinting.
  • Tissue printing was performed in a INKREDIBLE 3D bioprinter under physiological conditions (low extrusion pressure at 6-10kPa and room temperature).
  • Bioprinted tissues were maintained in 3D culture up to 14 days and exposed to TGF i for 6 days in order to promote an in vitro fibrogenic process.
  • the resultant bioprinted liver tissue was analysed by histology, viability assay and gene and protein expression.
  • o 3D Bioprinted tissues IHC for ECM proteins.
  • HEP X bioinkl positive to all ECM proteins analysed (Samples: LX2 at 7 day) ( Figure 2).
  • o Live/dead staining showed improved viability in all tested cells when cells were cultured in Cellink+human Liver ECM.
  • Live/dead HepG2 and live/dead LX2 Figure 3 and 4
  • ⁇ LX2 collAl, ACTA2 and LOX (figure 11);
  • HEP X (ECM+Cellink) is the most suitable bioink for the culture of human hepatic cells. (-) does not satisfy the criteria; (o) does partially satisfy the criteria; (+) does satisfy the criteria.
  • Example 5 In vitro biocompatibility tests of Cellink + human liver ECM together with SNU- 387 or SNU-449 Two different cell densities were used in the bioprinting process for each cell line in order to find out the most suitable conditions for cell proliferation, availability and spheroid formation. The chosen densities based on previous experimental data were 5 and 15 million cells per ml of bioink bioprinted, which transkates to 3.22xl0 5 and lxlO 6 cells per sample respectively. Differences in cell proliferation, metabolic activity, cell viability and spheroid formation capability were assessed through Presto Blue, Live/Dead and H&E analysis.
  • Live/Dead fluorescent assay and histological analysis of the samples showed a clear difference in cell survival as well as the capacity of forming clusters and spheroids, being higher in the case of 15 million cells/ml after 13 days of culture ( Figure 15/16).
  • Presto Blue analysis of the samples after 13 days of culture corroborated the results obtained through Live/Dead and Histology demonstrating a disparity not only in the absolute values of the metabolic activity but also in the trends of the same during the progression of the experiment (from day 3 to day 13), showing better results in the case of 15 million cells/ml (Figure 17).
  • Decellularized human pancreas were lyophilized and solubilized using a protease-free protocol employing 0.5M acetic acid.
  • the solubilized ECM was mixed with cellulose-based bioink (CELLINK ® Bioink) as support for bioprinting.
  • Human pancreatic cancer cell line Panel
  • pancreatic ECM PAN X
  • Tissue printing was performed in a Inkredible printer under physiological conditions (low extrusion pressure at 6-10kPa and room temperature) (Schematic view, Figure 18).
  • Example 7 In vitro biocompatibility tests of Cellink + human intestine ECM together with Caco-2 Decellularized human intestine was lyophilized and solubilized using a protease-free protocol employing 0.5M acetic acid.
  • the solubilized ECM was mixed with cellulose-based bioink (CELLINK ® Bioink) as support for bioprinting.
  • Human colorectal cancer cell line (Caco2) was gently mixed with intestine ECM bioink using a CELLMIXER ® directly into a cartridge before bioprinting.
  • Tissue printing was performed in a Inkredible printer under physiological conditions (low extrusion pressure at 6-10kPa and room temperature).
  • Example 8 Testing other ECM (intestine) with tissue specific cells
  • Human ECM from other tissues will be solubilised as described above (e.g liver, pancreas and intestine ECM).
  • the ECM solution will be mixed with cellulose bioink.
  • human cells e.g myofibroblast or cancer cells
  • Viability, cell proliferation, gene expression and protein secretion will be assessed at different time points.
  • Expected results i) cell survival; ii) maintainance of cell phenotype; iii) modelling of tissue disease (e.g fibrosis or cancer) recapitulating human tissue diseases.
  • Example 9 Test primary cells (hepatocytes) and iPSC-Hep
  • 3D bioprinted liver tissue models can become an essential platform for drug development by providing drug metabolism.
  • the liver is responsible for metabolizing various compounds for distribution and activation.
  • 3D bioprinting technologies - bioprinters and bioink - can advance mimickry of the complex architecture of the cellular organization, vascular branches, and bile canaliculi network.
  • the inventors have demonstrated that co-culture or single culture of major live cells such as hepatocytes and stellate cells can be bioprinted into 3D constructs using the human liver ECM based HEP X bioink of the invention. Since liver cells are niche within their liver lobule unit, the liver ECM component will provide the environmental cues to maintain and improve specific cell functions. HEP X will also provide a favourable environment of primary hepatocytes, stellate cells and non-parenchymal cells. a) Co-culture of hepatic cell lines
  • HepG2 hepatocyte cell line
  • LX2 tellate-like cell line
  • the inventors have also demonstrated 3D bioprinting of primary human or stellate cells as in vitro assay models for drug development.
  • Human primary hepatocytes are sensitive cells; however, within the HEP X bioink (Fig. 23), the cells can survive 3D bioprinting (live - green, dead - read). Hepatocyte (yellow) clustering was observed for up to 21 days of culture with multiphoton imaging. Scale bars 100 pm or 25 pm as indicated.
  • Multiphoton image of 3D bioprinted construct with primary stellate cells (Fig. 25).
  • the constructs were cultured for 2 weeks, and then TGF-b (5 ng/ml) induction was performed. TGF-b induction was maintained for 9 days.
  • Primary cells are autofluorescent (seen as yellow) and demonstrated stretched morphology.
  • Clusters of stellate cells exhibit extracellular matrix production (as seen in purple). The extracellular matrix are fibrillary and intertwined within the clusters and cell network. Scale bars are 100 pm (a) and 50 pm (b).
  • PAN X bioink for culturing pancreatic cells.
  • the human pancreas has two main functions, an exocrine - for digestion and an endocrine - for regulating blood sugar levels.
  • a precise regulation of blood sugar levels is necessary for key organs including the brain, liver, and kidneys to function correctly.
  • 3D bioprinted beta cells can be used in research and development of new drugs/pharmaceutical substances for diabetes, as well as for transplantation studies and reduce the need for animal testing.
  • Aim The aim of the following project was to culture and evaluate the viability of iPS derived beta cells bioprinted in CELLINK PAN X ink.
  • iPS derived beta cells was purchased from Takara Bio Europe.
  • iPS derived beta cells have a number of advantages over using human islets. It can potentially serve as an unlimited source of starting material for a cell therapy product for the treatment of Type 1 diabetes, as well as for drug testing and research. It can also abolish the need for Islet donors (that are in shortage) and donor to donor variations.
  • Fig. 27 show live/dead staining using Thermofisher kit. Images at 10X magnification.
  • Example 12 Transplant the bioprinted scaffolds and tissues
  • bioprinted scaffolds the expected results are: i) biocompatibility; ii) host vascularization; iii) long-term function; iv) tissue remodelling; v) tissue repair; vi) restoring tissue function.

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Abstract

La présente invention repose sur la découverte que la combinaison de deux biomatériaux, une matrice extracellulaire (ECM) spécifique de tissu humain et une bioencre à base de cellulose, avec ou sans cellules, dans la bio-impression 3D de tissus et d'échafaudages humains, permet d'obtenir une excellente capacité d'impression et de meilleures fonction cellulaire, viabilité et prise de greffe. La présente invention est basée sur le premier rapport décrivant la bio-impression de tissu humain, en particulier de tissu hépatique, à l'aide d'ECM humaine, spécifiquement une ECM du foie, en combinaison avec de la bioencre à base de cellulose. Plus spécifiquement, la présente invention concerne une composition destinée à être utilisée dans la bio-impression 3D et la culture de tissu humain ou animal, comprenant (i) une bioencre à base de nanofibrilles de cellulose, et (ii) un matériau de matrice extracellulaire (ECM) spécifique d'un tissu humain ou animal. En outre, l'invention concerne un procédé de bio-impression 3D de tissu et/ou d'échafaudages humains comprenant la bio-impression de la composition de l'invention, ainsi que les tissus et/ou échafaudages bio-imprimés. De plus, l'invention concerne diverses applications utilisant les tissus et/ou échafaudages bio-imprimés.
PCT/EP2018/086632 2017-12-22 2018-12-21 Bioencres humaines spécifiques d'un tissu pour la bio-impression 3d physiologique de tissus humains pour une culture in vitro et une transplantation Ceased WO2019122351A1 (fr)

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WO2021175842A1 (fr) * 2020-03-02 2021-09-10 Universiteit Maastricht Normes de contrôle de qualité destinées à une imagerie par spectrométrie de masse
WO2021256995A1 (fr) * 2020-06-15 2021-12-23 National University Of Singapore Encre biologique
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WO2020264385A1 (fr) * 2019-06-28 2020-12-30 Arizona Board Of Regents On Behalf Of The University Of Arizona Systèmes et procédés biologiques sensibles autonomes
US20220267720A1 (en) * 2019-07-23 2022-08-25 Xylyx Bio, Inc. Fibrosis-specific cell culture substrate and methods of use
WO2021016489A3 (fr) * 2019-07-23 2021-03-25 Xylyx Bio, Inc. Substrat de culture cellulaire spécifique à la fibrose et procédés d'utilisation
WO2021175842A1 (fr) * 2020-03-02 2021-09-10 Universiteit Maastricht Normes de contrôle de qualité destinées à une imagerie par spectrométrie de masse
WO2021256995A1 (fr) * 2020-06-15 2021-12-23 National University Of Singapore Encre biologique
US12427229B2 (en) 2020-08-06 2025-09-30 PhosPrint P.C. Laser ablation/removal and laser induced forward transfer of biological material
DE102020004900A1 (de) 2020-08-12 2022-02-17 Universitätsmedizin Der Johannes Gutenberg-Universität Mainz Autologe prävaskulisierte 3D-Druckverfahren-erzeugte Brustgewebe-Konstrukte und Verfahren zu deren Herstellung
WO2022034058A1 (fr) 2020-08-12 2022-02-17 Universitätsmedizin Der Johannes Gutenberg-Universität Mainz Constructions de tissu mammaire autologues, prévascularisées produites dans un procédé d'impression 3d, et leurs procédés de production
US11918703B2 (en) 2020-08-13 2024-03-05 Universidad De Los Andes Extrudable photocrosslinkable hydrogel and method for its preparation
WO2022055559A1 (fr) * 2020-09-11 2022-03-17 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Procédés pour la préparation d'un hydrogel ou d'une suspension colloïdale, stérilisé(e) de manière terminale, issu(e) d'une matrice extracellulaire et utilisations correspondantes
WO2022073090A1 (fr) 2020-10-06 2022-04-14 Janaina De Andrea Dernowsek Ltda Procédé pour la production d'une composition de protéines de matrice extracellulaire et produit obtenu au moyen de ce procédé
EP4230726A4 (fr) * 2020-11-17 2025-02-12 Cellartgen Inc. Support dérivé de matrice extracellulaire pancréatique pour la culture et la transplantation d'organoïdes pancréatiques, et son procédé de préparation
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WO2024094890A1 (fr) * 2022-11-04 2024-05-10 The Provost, Fellows, Scholars And Other Members Of Board Of Trinity College Dublin Implant pour la réparation du cartilage et du ménisque
WO2024188290A1 (fr) * 2023-03-14 2024-09-19 The University Of Hong Kong Bio-impression 3d de tissus tumoraux pour l'étude mécanistique et le criblage de médicaments
CN117504004A (zh) * 2023-11-27 2024-02-06 中国医学科学院生物医学工程研究所 一种仿生型脱细胞基质水凝胶及其应用
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