Automatable artificial immune system (AIS)
The present invention relates to methods of constructing an integrated artificial immune system that comprises appropriate in vitro cellular and tissue constructs or their equivalents to mimic the normal tissues that interact with vaccines in mammals. The artificial immune system can be used to test the efficacy of vaccine candidates in vitro and thus, is useful to accelerate vaccine development and testing drug and chemical interactions with the immune system.
1. An in vitro two culture cellular system comprising:
a plurality of vaccination site (VS) cultures organized in a first multi-well format, wherein each of said VS cultures comprises a first substantially planar matrix, a plurality of cells consisting of endothelial cells and/or epithelial cells attached to said first matrix and an exogenous antigen of interest, for receiving a population of peripheral blood mononuclear cells (PBMCs) and for maturing dendritic cell precursors present in said population of PBMCs; and
a plurality of three-dimensional artificial lymphoid tissue equivalent (LTE) cultures organized in a second multi-well format, wherein each of said LTE cultures comprises a second matrix and a plurality of lymphocytes attached to said second matrix, for receiving dendritic cells matured in said VS cultures.
2. The in vitro cellular system of claim 1 , wherein said plurality of cells forms an endothelial layer on one side of said first matrix.
3. The in vitro cellular system of claim 1 , wherein said plurality of cells forms an endothelium on one side of said first matrix.
4. The in vitro cellular system of claim 1 , wherein said plurality of cells forms a vascular endothelial layer on one side of said first matrix.
5. The in vitro cellular system of claim 1 , wherein said plurality of cells forms a vascular endothelium on one side of said first matrix.
6. The in vitro cellular system of claim 1 , wherein said plurality of cells forms an endothelial layer on both sides of said first matrix.
7. The in vitro cellular system of claim 1 , wherein said plurality of cells forms an endothelium on both sides of said first matrix.
8. The in vitro cellular system of claim 1 , wherein said plurality of cells forms a vascular endothelium on both sides of said first matrix.
9. The in vitro cellular system of claim 1 , wherein said plurality of cells forms an endothelial layer on one side of said first matrix and an epithelial layer on the other side of said first matrix.
10. The in vitro cellular system of claim 1 , wherein said plurality of cells forms an endothelial layer on one side of said first matrix and an epithelium on the other side of said first matrix.
11. The in vitro cellular system of claim 1 , wherein said plurality of cells forms an endothelium on one side of said first matrix and an epithelium on the other side of said first matrix.
12. The in vitro cellular system of claim 1 , wherein said plurality of cells forms a vascular endothelium on one side of said first matrix and an epithelial layer on the other side of said first matrix.
13. The in vitro cellular system of claim 1 , wherein said plurality of cells forms an endothelium on one side of said first matrix and an epithelial layer on the other side of said first matrix.
14. The in vitro cellular system of claim 1 , wherein said plurality of cells forms a vascular endothelium on one side of said first matrix and an epithelium on the other side of said first matrix.
15. The in vitro cellular system of claim 1 , wherein said plurality of cells forms a vascular endothelial layer on one side of said first matrix and a lymphatic endothelial layer on the other side of said first matrix.
16. The in vitro cellular system of claim 1 , wherein said plurality of cells in the vaccination site cultures comprise human vascular endothelial cells (HUVECs).
17. The in vitro cellular system of claim 1 , wherein said plurality of cells in the vaccination site cultures comprises human dermal microvascular endothelial cells (HMVECs).
18. The in vitro cellular system of claim 1 , wherein said plurality of cells forms a vascular endothelium on one side of said first matrix and a lymphatic endothelium on the other side of said first matrix.
19. The in vitro cellular system of claim 1 , wherein said first matrix comprises a natural biopolymer.
20. The in vitro cellular system of claim 1 , wherein said first matrix comprises a natural biopolymer selected from the group consisting of xenographic extracellular matrix (ECM) sheet, reconstituted collagen matrix, and chitosan/collagen membrane scaffolds.
21. The in vitro cellular system of claim 1 , wherein said first matrix comprises the natural biopolymer bovine type I collagen on a nylon mesh.
22. The in vitro cellular system of claim 1 , wherein said first matrix comprises the natural biopolymer bovine type I collagen on a polycarbonate mesh.
23. The in vitro cellular system of claim 1 , wherein said second matrix comprises synthetic extracellular matrix (ECM) materials.
24. The in vitro cellular system of claim 1 , wherein said second matrix comprises synthetic extracellular matrix materials selected from the group consisting of hydrogels, poly(methyl methacrylate), poly(lactide-co-glycolide), polytetrafluoroethylene, poly(ethylene glycol dimethacrylate) hydrogels (PEGDA or PEGDMA), poly(ethylene oxide), and poly(propylene fumarate-co-ethylene glycol) (PPF-PEG).
25. The in vitro cellular system of claim 1 , wherein said second matrix comprises natural ECM material.
26. The in vitro cellular system of claim 1 , wherein said second matrix comprises a natural ECM material selected from the group consisting of collagen, hyaluronic acid hydrogels, calf skin gelatin, fibrinogen, thrombin, and decellularized ECM.
27. The in vitro cellular system of claim 26 , wherein said decellularized ECM is selected from the group consisting of intestine submucosa and urinary bladder mucosa.
28. The in vitro cellular system of claim 1 , wherein said second matrix comprises synthetic lymphoid ECM-derived hydrogel.
29. The in vitro cellular system of claim 1 , wherein said second matrix comprises natural lymphoid ECM-derived scaffolds.
30. The in vitro cellular system of claim 1 , wherein said second matrix comprises natural lymphoid ECM-derived hydrogel.
31. The in vitro cellular system of claim 1 , wherein said plurality of cells in said vaccination site cultures comprises human cells.
32. The in vitro cellular system of claim 1 , wherein said plurality of cells in said vaccination site cultures comprise human vascular endothelial cells (HUVECs).
33. The in vitro cellular system of claim 1 , wherein said plurality of cells in said vaccination site cultures comprise blood vessel endothelial cells.
34. The in vitro cellular system of claim 1 , wherein said plurality of lymphocytes and leukocytes comprises T cells and B cells.
35. The in vitro cellular system of claim 1 , wherein said plurality of lymphocytes and leukocytes comprises dendritic cells.
36. The in vitro cellular system of claim 1 , wherein said plurality of lymphocytes comprises naive T cells and naive B cells.
37. The in vitro cellular system of claim 1 , wherein said plurality of lymphocytes comprises memory T cells and memory B cells.
38. The in vitro cellular system of claim 1 , wherein the antigen is attached to the first matrix.
39. The in vitro cellular system of claim 1 , wherein the antigen is embedded in the first matrix.
40. The in vitro cellular system of claim 1 , wherein the second matrix is situated on a dialysis membrane.
41. The in vitro cellular system of claim 1 , wherein said second matrix and said cells attached thereto form a tissue scaffold.
42. The in vitro cellular system of claim 1 , wherein each of said vaccination site cultures comprises:
a first substantially planar matrix comprising an ECM membrane;
a layer of epithelial cells attached to a first side of the ECM membrane; and
a layer of endothelial cells attached to a second side of the ECM membrane.
43. The in vitro cellular system of claim 1 , wherein the exogenous antigen of interest is different in each of the vaccination site cultures.