Methods of producing multi-layered tubular tissue constructs

Patent metadata

Field	Value
Publication number	US-12258585-B2
Application number	US-201816631677-A
Country	US
Kind code	B2
Filing date	Jul 20, 2018
Priority date	Jul 21, 2017
Publication date	Mar 25, 2025
Grant date	Mar 25, 2025

Abstract

Described are methods for producing multi-layered tubular tissue structures, tissue structures produced by the methods, and their use.

First claim

The invention claimed is:

1. A method of producing a perfusable multi-layered tubular tissue construct, comprising:
depositing on a substrate one or more cell-laden filaments, each cell-laden filament comprising:
a plurality of concentric and coaxial cell-laden ink layers, each cell-laden ink layer comprising one or more predetermined cell types and extending at least a portion of the length of the cell-laden filament, wherein the one or more predetermined cell types are cell aggregates or clusters of cells, and
a core comprising a fugitive ink, wherein the fugitive ink serves as a template for an open perfusable lumen within the cell-laden filament;

removing the fugitive ink to create the open perfusable lumen;
seeding the lumen with endothelial cells by:
providing the endothelial cells with the fugitive ink, wherein the endothelial cells remain in the open perfusable lumen after the fugitive ink is removed; and/or
injecting a suspension of endothelial cells into the open perfusable lumen after removing the fugitive ink; and

exposing the one or more cell-laden filaments to fluid perfusion to induce cell proliferation and development, thereby producing the perfusable multi-layered tubular tissue construct.

2. The method of claim 1 , wherein the step of depositing on a substrate one or more cell-laden filaments comprises:
flowing the fugitive ink through a first extrusion tube;
flowing a first cell-laden ink comprising one or more predetermined cell types through a second extrusion tube overlaying the first extrusion tube, the first cell-laden ink flowing around and enclosing the fugitive ink;
flowing a second cell-laden ink comprising one or more predetermined cell types through a third extrusion tube overlaying the second extrusion tube, the second cell-laden ink flowing around and enclosing the first cell-laden ink,
thereby forming the core comprising the fugitive ink surrounded by an inner layer comprising a first cell-laden ink layer and an outer layer comprising a second cell-laden ink layer.

3. The method of claim 2 , further comprising providing an extrusion head including the first, second, and third extrusion tubes arranged in a concentric configuration, wherein the extrusion head is moved relative to the substrate during the flowing of the fugitive ink, the first and the second cell-laden inks, the cell-laden filament being deposited on the substrate in a predetermined configuration.

4. The method of claim 1 , wherein each cell-laden ink comprises a different type of viable cells.

5. The method of claim 1 , wherein each cell-laden ink comprises overlapping populations of viable cells.

6. The method of claim 1 , wherein the cell types are selected from the group consisting of smooth muscle cells, mesenchymal cells, pericytes, endothelial cells, and epithelial cells.

7. The method of claim 2 , wherein the first cell-laden ink comprises smooth muscle cells and the second cell-laden ink comprises fibroblast cells.

8. The method of claim 1 , wherein the cell-laden ink layers form a medial layer and an adventitial layer of a blood vessel.

9. The method of claim 1 , wherein the perfusable multi-layered tubular construct is a printed blood vessel.

10. The method of claim 1 , wherein the multi-layered tubular construct is a branched multi-layered tubular construct.

11. The method of claim 1 , further comprising at least partially surrounding the one or more cell-laden filaments with an extracellular matrix composition, wherein the extracellular matrix composition comprises one or more of gelatin, fibrin, fibrinogen, transglutaminase, thrombin and gelatin methacrylate, collagen, collagen-acrylate, a solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, poly lactic-co-glycolic acid (PLGA), alginate, or chitosan.

12. The method of claim 11 , further comprising depositing one or more sacrificial filaments on the substrate prior to at least partially surrounding the one or more cell-laden filaments with the extracellular matrix composition to form a sacrificial filament network interpenetrating the one or more cell-laden filaments, each of the sacrificial filaments comprising a fugitive ink.

13. The method of claim 12 , wherein the network comprises flow channels in fluid communication with the cell-laden filaments for perfusion thereof after removal of the fugitive ink.

14. The method of claim 1 ,
wherein:
the cell-laden filaments comprise one or more functional chemical substances selected from the group consisting of: drugs, small molecules, toxins, proteins, growth factors, and hormones; and/or
each of the cell-laden ink layers comprises a cell concentration of from one cell/ml to about 10 9 cells/ml; and/or
the cell concentration is uniform throughout each of the cell-laden ink layers.

15. The method of claim 1 , wherein the step of exposing the one or more cell-laden filaments to fluid perfusion is under a fluid shear stress (FSS).

16. The method of claim 15 , wherein the FSS is pulsed to mimic blood pressure changes during regular heart beats.

17. The method of claim 1 , wherein:
the substrate is plastic, glass, or a solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, and
optionally, the substrate is plasma treated or coated with a layer of at least one of a solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, poly L-lysine, gelatin, fibrin, fibrinogen, nitogen, vitrogen, collage I, collagen IV, chitosan, alginate, glycosaminoglycans, or other biomaterial.

18. The method of claim 1 , wherein the cell-laden ink layers all have varying thickness.

19. The method of claim 1 , wherein each cell-laden filament further comprises one or more concentric and coaxial non-cellular fugitive ink layer.

20. The method of claim 19 , wherein the non-cellular fugitive ink layers comprise one or more materials that impart mechanical stability to the perfusable multi-layered tissue construct.

21. The method of claim 1 , wherein each cell-laden filament further comprises one or more concentric and coaxial layer comprising growth factors.

22. A perfusable multi-layered tubular tissue construct produced by the method of claim 1 .

23. The perfusable multi-layered tubular tissue construct of claim 22 , wherein the tubular structure is selected from the group consisting of an artery, an arteriole, a small scale vessel, and a vein.

24. A method of producing a blood vessel construct, comprising:
depositing on a substrate one or more filaments, each filament comprising: a first cell-laden ink layer and a second cell-laden ink layer, the first and the second cell-laden layers being concentric and extending at least a portion of the length of the filament, the first cell-laden ink layer comprising a smooth-muscle cell (SMC)-containing cell-laden ink and the second cell-laden ink layer comprising a fibroblast-containing cell laden ink, and within the cell-laden ink layers a core comprising a fugitive ink, wherein the fugitive ink serves as a template for an open perfusable lumen within the filament;
removing the fugitive ink to create the open perfusable lumen;
after removing the fugitive ink, injecting a suspension of endothelial cells into the open perfusable lumen; and
exposing the one or more filaments to fluid perfusion to induce cell proliferation and maturation thereby producing the blood vessel

Assignees

• Harvard College

Inventors

• Kroll Katharina Theresa
• Homan Kimberly A
• Skylar-Scott Mark A
• Uzel Sebastien G M
• Kolesky David B
• Lustenberger Patrick
• Lewis Jennifer A

Classifications

• C12N5/069

  Vascular Endothelial cells · CPC title

• C12N2533/90

  Substrates of biological origin, e.g. extracellular matrix, decellularised tissue · CPC title

• C12N2533/56

  Fibrin; Thrombin · CPC title

• C12N2533/54

  Collagen; Gelatin · CPC title

• C12N2513/00

  3D culture · CPC title