Liver Tissue Model Constructs and Methods for Providing the Same

1 . A liver tissue model construct comprising at least one bioprinted structure, wherein the structure comprises a Bioink A mixed with at least one Cell A, and a Bioink B mixed with at least one Cell B,

wherein at least one of Bioink A and Bioink B, independently of each other, is based on methacrylated gelatin, collagen, nanocellulose and/or alginate;

wherein at least one of Bioink A and Bioink B comprises liver-specific ECM components;

optionally wherein at least one of Bioink A or Bioink B comprises a thickening agent, wherein the thickening agent is a natural polysaccharide selected from the group consisting of: xanthan gum, glucomannan and nanocellulose;

wherein at least one of Bioink A or Bioink B comprises a Factor A;

wherein Cell A is a hepatic cell line of human or animal origin; and

wherein Cell B is a non-parenchymal cell of human or animal origin from cell lines, primary cells, or derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), wherein the non-parenchymal cells are selected from the group consisting of: liver sinusoidal endothelial cells, Kupffer cells, biliary cells, lymphocytes, and hepatic stellate cells in normal and abnormal physiology.

2 . The liver tissue model construct according to claim 1 , wherein the Bioink A comprises methacrylated gelatin or collagen with a liver specific ECM component.

3 . The liver tissue model construct according to claim 1 , wherein Bioink B comprises liver specific components.

4 .- 6 . (canceled)

7 . The liver tissue model construct according to claim 1 , wherein the Factor A is selected from the group consisting of: TGF-β, free fatty acids, cytokines, interleukins, tumor necrosis factors, and lipopolysaccharide in combination with allyl alcohol.

8 . The liver tissue model construct according to claim 1 , wherein Bioink A and Bioink B, each and independently of each other, comprises:

methacrylated gelatin 0-90%;

alginate 0-90%;

liver specific ECM components 0-50%;

thickening agents 0-50%;

a photo initiator 0-10%; and

a growth factor specific to parenchymal cells 0-20%.

9 . The liver tissue model construct according to claim 1 , wherein Cell A and Cell B are used at different ratios in relation to each other, in order to simulate a pathology in vitro.

10 . The liver tissue model construct according to claim 1 , wherein the liver tissue model construct is suitable for use within science, medicine, tissue engineering, pharmaceutical therapies, regenerative medicine, stem cell research, and in vitro models.

11 . Method of bioprinting the liver tissue model construct of claim 1 , comprising 3D bioprinting the liver tissue model construct with an extrusion based bioprinting device.

12 . The liver tissue model construct according to claim 1 , wherein the liver tissue model construct is suitable for use in:

i) Developmental biology in order to gain understanding of cellular activities within a 3D environment such as cellular distribution, migration, proliferation, matrix production, interactions with other cells and the surrounding environment;

ii) Pharmaceutical applications for drug discovery, target validation, toxicity studies, metabolic studies, cellular differentiation/maturation, spheroid differentiation/maturation, and organoid differentiation/maturation;

iii) Tissue regeneration applications such as tissue remodeling, cellular proliferation, cellular metabolism, cellular differentiation/maturation, cell-cell interaction, cell-matrix interaction, cellular crosstalk/signaling, and vascularization; and/or

iv) Stem cell research with focus on cellular differentiation and maturation as dispersed cells, spheroids, and organoids.

13 . Method of 3D bioprinting of a liver tissue model construct of claim 1 , comprising:

i) mixing cells with the bioinks, according to Cell A with Bioink A, and Cell B with Bioink B, in order to obtain a cell ladened Bioink A and a cell ladened Bioink B,

ii) transferring each of the obtained cell ladened Bioinks A and B to bioprinting cartridges A and B, respectively;

iii) mounting the bioprinting cartridges on printheads of a 3D bioprinter device;

iv) performing extrusion based bioprinting of the cell ladened bioinks, thereby obtaining at least one tissue construct comprising at least one structure, wherein the structure comprises Bioink A mixed with at least one Cell A, and Bioink B mixed with at least one Cell B;

v) crosslinking of the bioprinted tissue to polymerize and/or gelate the tissue construct for culturing; and

vi) culturing the tissue construct and optionally adding Factor A for stimulating or inducing abnormal changes.

14 . Method of providing a tissue for implantation into a subject, said method comprising 3D bioprinting of a liver tissue model construct of claim 1 , wherein the method further comprises at least 48 hours of culturing the 3D bioprinted tissue in order to obtain a tissue that may be implanted into the subject.

15 . Method of treatment of a subject, said method comprising implanting a liver tissue obtained according to the method of claim 14 into the subject.

16 . A kit for use in 3D bioprinting for generating a liver tissue model construct according to claim 1 , said kit comprising:

i) a Bioink A, and

ii) a Bioink B,

wherein at least one of Bioink A and Bioink B comprises methacrylated gelatin, collagen, alginate or nanocellulose;

wherein at least one of Bioink A and Bioink B comprises liver-specific ECM components;

optionally wherein at least one of Bioink A or Bioink B comprises a thickening agent, wherein the thickening agent is a natural polysaccharide selected from the group consisting of: xanthan gum, glucomannan and nanocellulose;

wherein at least one of Bioink A or Bioink B comprises a Factor A;

and optionally other components selected from the group consisting of: Cell A and/or Cell B, and/or suitable antibodies, for use together with Bioink A and Bioink B.

17 . (canceled)

18 . The liver tissue model construct according to claim 1 , wherein (1) at least one of Bioink A and Bioink B is based on methacrylated gelatin with no addition of thickening agent, or (2) at least one of Bioink A and Bioink B is based on alginate with addition of nanocellulose as thickening agent.

19 . The liver tissue model construct according to claim 1 , comprising at least two 3D bioprinted structures, wherein the first structure comprises the Bioink A mixed with at least one Cell A, and the second structure comprises the Bioink B mixed with at least one Cell B.

20 . The method according to claim 13 , wherein iv) comprises obtaining at least one tissue construct comprising at least two structures, wherein the first structure comprises the Bioink A mixed with at least one Cell A, and the second structure comprises the Bioink B mixed with at least one Cell B.

21 . The liver tissue model construct of claim 1 , wherein the liver-specific ECM components are selected from the group consisting of: laminins, fibronectin, and whole organ digest extracellular matrix (ECM) from animal or human origin.

22 . The liver tissue model construct of claim 2 , wherein the liver-specific ECM component is selected from the group consisting of: laminins, fibronectin, and whole organ digest ECM from animal or human origin.

23 . The liver tissue model construct of claim 3 , wherein the liver-specific components are selected from the group consisting of: laminins, fibronectin, and whole organ digest ECM from animal or human origin.

24 . The liver tissue model construct of claim 9 , wherein the different ratios are in an interval from 2:1 to 4:1 (Cell A:Cell B).

25 . The kit of claim 16 , wherein the liver-specific ECM components are selected from the group consisting of: laminins, fibronectin, and whole organ digest ECM from animal or human origin; and wherein the Factor A is selected from the group consisting of: TGF-β, free fatty acids, cytokines, interleukins, tumor necrosis factors, and lipopolysaccharide in combination with allyl alcohol.