Self-assembling multicellular bodies and methods of producing a three-dimensional biological structure using the same

• US 9,556,415 B2
• Filed: 04/03/2014
• Issued: 01/31/2017
• Est. Priority Date: 06/24/2008
• Status: Active Grant

• Current Assignee: Curators of the University of Missouri (University of Missouri)
• Original Assignee: Curators of the University of Missouri (University of Missouri)
• Inventors: Forgacs, Gabor, Marga, Francoise Suzanne, Norotte, Cyrille
• Primary Examiner(s): Ware, Debbie K
• Assistant Examiner(s): Srivastava, Kailash C
• Application Number:
  US14/244,679
• Publication Number:
  US 20140220685A1
• Time in Patent Office: 1,034 Days
• Field of Search: 435/325, 435/395, 435/397, 435/174
• US Class Current: 1/1
• CPC Class Codes: A61L 27/222 Gelatin
• CPC Class Codes: A61L 27/225 Fibrin; Fibrinogen
• CPC Class Codes: A61L 27/3625 Vascular tissue, e.g. heart valves
• CPC Class Codes: A61L 27/3691 characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
• CPC Class Codes: A61L 27/3886 comprising two or more cell types
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: B33Y 80/00 Products made by additive manufacturing
• CPC Class Codes: C12M 21/08 for producing artificial tissue or for ex-vivo cultivation of tissue prostheses A61F2/00, grafts A61L27/00
• CPC Class Codes: C12M 23/10 Petri dish crystallising dishes B01L3/06
• CPC Class Codes: C12M 25/06 Plates; Walls; Drawers; Multilayer plates
• CPC Class Codes: C12M 25/14 Scaffolds; Matrices in general C12N5/0068
• CPC Class Codes: C12M 33/00 Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00
• CPC Class Codes: C12M 35/08 Chemical, biochemical or biological means, e.g. plasma jet, co-culture
• CPC Class Codes: C12N 2506/1307 from adult fibroblasts
• CPC Class Codes: C12N 2506/1338 from smooth muscle cells
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2533/56 Fibrin; Thrombin
• CPC Class Codes: C12N 2533/76 Agarose, agar-agar
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture
• CPC Class Codes: C12N 5/0068 : General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0602 : Vertebrate cells
• CPC Class Codes: C12N 5/0691 : Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels

Abstract

Structures and methods for tissue engineering include a multicellular body including a plurality of living cells. A plurality of multicellular bodies can be arranged in a pattern and allowed to fuse to form an engineered tissue. The arrangement can include filler bodies including a biocompatible material that resists migration and ingrowth of cells from the multicellular bodies and that is resistant to adherence of cells to it. Three-dimensional constructs can be assembled by printing or otherwise stacking the multicellular bodies and filler bodies such that there is direct contact between adjoining multicellular bodies, suitably along a contact area that has a substantial length. The direct contact between the multicellular bodies promotes efficient and reliable fusion. The increased contact area between adjoining multicellular bodies also promotes efficient and reliable fusion. Methods of producing multicellular bodies having characteristics that facilitate assembly of the three-dimensional constructs are also provided.

Claims

1. A method of producing a three-dimensional biological engineered tissue, the method comprising:

arranging a plurality of elongate multicellular bodies on a substrate according to a pattern such that each of the multicellular bodies contacts at least one other multicellular body, wherein each multicellular body comprises a plurality of living cells,

arranging one or more filler bodies in the pattern with the multicellular bodies so that each filler body contacts at least one multicellular body or another filler body, each filler body comprising a biocompatible material that resists migration and ingrowth of cells from the multicellular bodies into the filler bodies and resists adherence of cells in the multicellular bodies to the filler bodies,

allowing at least one of the multicellular bodies to fuse with at least one other multicellular body to produce a three-dimensional biological engineered tissue; and

removing the one or more filler bodies.

2. The method of claim 1, wherein the arranging comprises arranging the multicellular bodies and the filler bodies to form a three-dimensional structure having a plurality of spaces that are not occupied by the multicellular bodies and that are not occupied by the filler bodies.

3. The method of claim 1, further comprising filling at least some of the plurality of spaces with tissue culture medium.

4. The method of claim 1, wherein arranging the filler bodies comprises arranging filler bodies which are permeable to nutrients.

5. The method of claim 1, wherein arranging the multicellular bodies comprises arranging multicellular bodies consisting essentially of cells of a single cell type.

6. The method of claim 1, wherein arranging the multicellular bodies comprises arranging multicellular bodies wherein each multicellular body includes living cells of a first type and at least about 90 percent of the cells in each multicellular body are cells of the first cell type.

7. The method of claim 1, wherein arranging the multicellular bodies comprises arranging one or more multicellular bodies comprising a plurality of living cells of a first cell type and a plurality of living cells of a second cell type, the second cell type being different from the first cell type.

8. The method of claim 7, wherein the living cells of the first type are endothelial cells and the living cells of the second type are smooth muscle cells.

9. The method of claim 7, wherein the living cells of the first type are smooth muscle cells and the living cells of the second type are fibroblasts.

10. The method of claim 7, wherein the living cells of the first type are endothelial cells and the living cells of the second type are fibroblasts.

11. The method of claim 1, wherein one or more of the multicellular bodies comprises a plurality of living cells of a first cell type, a plurality of living cells of a second type, and a plurality of living cells of a third cell type, wherein each of the first, second and third cell types are different from the others of the first, second, and third cell types.

12. The method of claim 11, wherein, the living cells of the first cell type are endothelial cells, the living cells of the second cell type are smooth muscle cells, and the living cells of the third cell type are fibroblasts.

13. The method of claim 1, wherein arranging the multicellular bodies comprises arranging multicellular bodies that are substantially uniform in shape.

14. The method of claim 1, wherein arranging the filler bodies comprises arranging filler bodies that are substantially uniform in shape.

15. The method of claim 1, wherein arranging the multicellular bodies and filler bodies comprises arranging multicellular bodies and filler bodies that are substantially identical in shape.

16. The method of claim 1, wherein arranging the filler bodies comprises arranging filler bodies that are elongate in shape.

17. The method of claim 1, wherein arranging the multicellular bodies comprises arranging at least some of the multicellular bodies to form a sheet.

18. The method of claim 1, wherein arranging the multicellular bodies comprises arranging multicellular bodies wherein the cohesion of the cells in the multicellular body is sufficiently strong to allow the multicellular body to retain a three-dimensional shape while supporting itself on a flat surface, said three-dimensional shape including a width and a height, the height being at least 50 percent of the width.

19. The method of claim 1, wherein arranging the multicellular bodies comprises arranging multicellular bodies wherein the cohesion of the cells in the multicellular body is sufficiently strong to allow the multicellular body to support the weight of another multicellular body that is substantially identical to the multicellular body when said other multicellular body is on top of the multicellular body.

20. The method of claim 1, wherein arranging the multicellular bodies comprises arranging multicellular bodies wherein the cohesion of the cells in the multicellular body is sufficiently strong to allow the multicellular body to be picked up by an implement.

21. The method of claim 1, wherein the arranging is automated.

22. The method of claim 1, wherein the one or more filler bodies is removed by dissolving the filler bodies.

23. The method of claim 22, wherein the one or more filler bodies is dissolved by change in temperature or light.

24. The method of claim 1, wherein the plurality of living cells comprises one or more of:

endothelial cells, muscle cells, fibroblasts, liver cells, kidney cells, neural cells, bone cells, cartilage cells, epithelial cells, and undifferentiated cells.