Fabrication of vascularized tissue using microfabricated two-dimensional molds

• US 20030003575A1
• Filed: 07/22/2002
• Published: 01/02/2003
• Est. Priority Date: 04/30/1999
• Status: Abandoned Application

• Current Assignee: Charles Stark Draper Laboratory Incorporated
• Original Assignee: Charles Stark Draper Laboratory Incorporated
• Inventors: Borenstein, Jeffrey T., Vacanti, Joseph P., Cunningham, Brian T., Pien, Homer
• Application Number:
  US10/200,955
• Publication Number:
  US 20030003575A1
• Time in Patent Office: Days
• US Class Current: 435/371
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• CPC Class Codes: A61L 27/3604 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
• CPC Class Codes: A61L 27/38 containing added animal cells organs or tissue containing native cells A61L27/36
• CPC Class Codes: A61L 27/3886 comprising two or more cell types
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: B33Y 70/00 Materials specially adapted for additive manufacturing
• CPC Class Codes: C12N 2502/14 hepatocytes
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 2535/10 Patterned coating
• CPC Class Codes: C12N 5/0671 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0697 Artificial constructs associating cells of different lineages, e.g. tissue equivalents blood vessels C12N5/0691

Abstract

A method and materials to create complex vascularized living tissue in three dimensions from a two-dimension microfabricated mold has been developed. The method involved creating a two dimensional surface having a branching structure etched into the surface. The pattern begins with one or more large channels which serially branch into a large array of channels as small as individual capillaries, then converge to one or more large channels. The etched surface serves a template within a mold formed with the etched surface for the circulation of an individual tissue or organ. Living vascular cells are then seeded onto the mold, where they form living vascular channels based on the pattern etched in the mold. Once formed and sustained by their own matrix, the top of the mold is removed. The organ or tissue specific cells are then added to the etched surface, where they attach and proliferate to form a thin, vascularized sheet of tissue. The tissue can then be gently lifted from the mold using techniques such as fluid flow and other supporting material, as necessary. The tissue can then be systematically folded and compacted into a three-dimensional vascularized structure. This structure can then be implanted into animals or paitents by directly connecting the blood vessels to flow into and out of the device. Immediate perfusion of oxygenated blood occurs, which allows survival and function of the entire living mass.

Claims

1. A system for creating thin tissue layers comprising:

(a) a mold comprising a support surface having a pattern of channels thereon, suitable for attachment and culturing of cells to form lumens within the channels, and suitable for attachment and culturing of a different type of cells in the areas on the surface surrounding the channels.

2. The mold of claim 1 wherein the system further comprises

(b) connections and means for circulation of culture fluid through the mold for culturing of the cells attached thereto.

3. The system of claim 1 wherein the mold has branched channels therein, beginning from one or more inlets, expanding into more channels, and then converging back into one or more outlets.

4. The system of claim 1 further comprising pumping means for circulating fluid through the mold.

5. The system of claim 1 wherein the mold is formed from a material selected from the group consisting of silicon, glass, natural cell substrates like hydroxyapatite, and polymer.

6. The system of claim 1 further comprising a coating on the channels which promotes adhesion and lifting of cells as intact sheets from the mold.

7. A method for making a mold comprising a support surface having a pattern of channels thereon, suitable for attachment and culturing of cells to form lumens within the channels, and suitable for attachment and culturing of a different type of cells in the areas on the surface surrounding the channels, comprising:

(a) selecting a material for forming the support surface from the group consisting of silicon, metals, polymers, and natural cell substrates like hydroxyapatite,

(b) patterning channels in the surface of the material to create lumens which can be seeded with cells to form tubular structures for fluid flow, and

(c) forming the mold so that cells can be seeded within the mold to form a thin layer of tissue surrounding the tubular structures.

8. The method of claim 7 wherein the patterning is done by a process selected from the group consisting of micromachining, lithography, etching, or molding.

9. A method for forming complex tissues comprising

(a) selecting a mold having a complex pattern of channels formed into at least one surface into which cells can be seeded;

(b) seeding cells into the channels to form blood vessels or other lumens;

(c) culturing the cells under conditions until they form vessels or lumens;

(d) seeding other type(s) of cells onto or into the mold so that a tissue is formed incorporating the vessels or lumens; and

(e) removing the tissue layer.

10. The method of claim 9 further comprising

(f) assembling multiple layers of the tissue layers until the desired complex structure is formed.

11. The method of claim 10 further comprising

(g) implanting into a body the complex structure and anastomizing the vessles with the blood supply or lumens in other organs or tissues within the body.

12. The method of claim 11 further comprising applying to the channel a coating on the channels which promotes adhesion and lifting of cells as intact sheets from the mold.

14. The structure of claim 13 wherein the vasculature comprises endothelial cells.

15. The structure of claim 14 wherein the other cells are selected from the group consisting of parenchymal cells, cells forming cartilage or bone, muscle cells, and nerve cells.

16. The structure of claim 15 wherein the parenchymal cells are derived from organs selected from the group consisting of heart, liver, pancreas, intestine, brain, kidney, reproductive tissues and lung.

13. A complex structure comprising multiple layers of tissue and vasculature formed by a method comprising

(a) selecting a mold having a complex pattern of channels formed into at least one surface into which cells can be seeded;

(b) seeding cells into the channels to form blood vessels or other lumens;

(c) culturing the cells under conditions until they form vessels or lumens;

(d) seeding other type(s) of cells onto or into the mold so that a tissue is formed incorporating the vessels or lumens; and

(e) removing the tissue layer.