Artificial immune system: methods for making and use
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 interaction with the immune system.
1. An in vitro two culture cellular system comprising:
a vaccination site (VS) culture comprising 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 three-dimensional artificial lymphoid tissue equivalent (LTE) culture comprising a second matrix and a plurality of lymphocytes attached to said second matrix, for receiving dendritic cells matured in said VS culture.
2. The in vitro cellular system of claim 1 , wherein said plurality of cells forms a vascular endothelium on one side of said first matrix.
3. The in vitro cellular system of claim 1 , wherein said plurality of cells forms a vascular endothelium on both sides of said first matrix.
4. 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 an opposing side of said first matrix.
5. The in vitro cellular system of claim 1 , wherein said first matrix comprises a natural biopolymer.
6. The in vitro cellular system of claim 5 , wherein said biopolymer is selected from the group consisting of type I rat tail collagen, bovine type I collagen, and chitosan.
7. The in vitro cellular system of claim 1 , wherein said first matrix comprises bovine type I collagen matrix deposited and congealed on a nylon mesh.
8. The in vitro cellular system of claim 1 , wherein said first matrix is selected from the group consisting of a xenographic extracellular matrix (ECM) sheet, natively polymerized human amniotic connective tissue, reconstituted collagen matrix, and chitosan/collagen membrane scaffolds.
9. The in vitro cellular system of claim 1 , wherein said plurality of cells attached to said first matrix comprises human cells.
10. The in vitro cellular system of claim 9 , wherein said human cells comprise human vascular endothelial cells (HUVECs).
11. The in vitro cellular system of claim 1 , wherein said vaccination site culture site further comprises a chemokine.
12. The in vitro cellular system of claim 1 , wherein said endothelial cells are human skin-derived vascular endothelial cells.
13. The in vitro cellular system of claim 1 , wherein said endothelial cells are vascular and lymphatic endothelial cells.
14. The in vitro cellular system of claim 1 , wherein said vaccination site culture further comprises integrated vascular tubes lined with vascular and lymphatic endothelium.
15. The in vitro cellular system of claim 14 , wherein said integrated vascular tubes comprise epidermal epithelium.
16. The in vitro cellular system of claim 1 , wherein said plurality of lymphocytes comprises T cells and B cells.
17. The in vitro cellular system of claim 16 , wherein said plurality of lymphocytes further comprises dendritic cells.
18. The in vitro cellular system of claim 1 , wherein said plurality of lymphocytes comprises naive T cells and naive B cells.
19. The in vitro cellular system of claim 1 , wherein said plurality of lymphocytes comprises memory T cells and memory B cells.
20. The in vitro cellular system of claim 1 , wherein said second matrix comprises synthetic extracellular matrix (ECM) materials.
21. The in vitro cellular system of claim 1 , wherein said second matrix comprises natural ECM materials.
22. The in vitro cellular system of claim 1 , wherein said second matrix comprises segregated T cell and B cell zones.
23. The in vitro cellular system of claim 22 , wherein said T cell zone comprises naive T cells and collagen fibers.
24. The in vitro cellular system of claim 23 , wherein said collagen fibers comprise collagen I, collagen III or fibronectin.
25. The in vitro cellular system of claim 22 , wherein said T cell zone comprises fibroblastic reticular cells.
26. The in vitro cellular system of claim 22 , wherein said segregated T cell and B cell zones are fabricated by combined action of digital printing and controlled release of chemoattractants.
27. The in vitro cellular system of claim 22 , wherein said segregated T cell and B cell zones are fabricated by digital printing.
28. The in vitro cellular system of claim 22 , wherein said segregated T cell and B cell zones are fabricated by controlled release of chemoattractants.
29. The in vitro cellular system of claim 1 , wherein said second matrix comprises synthetic lymphoid ECM-derived hydrogel.
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 lymphocytes further comprises lymphoid stromal cells.
32. The in vitro cellular system of claim 31 , wherein said lymphoid stromal cells are derived from lymph node fragments, lymph node, spleen, or tonsil.
33. The in vitro cellular system of claim 1 , wherein said second matrix comprises synthetic ordered macroporous hydrogel, wherein said hydrogel comprises poly(ethylene glycol) (PEG) dimethacrylate, PEG peptide PEG block copolymers and ordered colloidal crystal of poly (methyl methacrylate) latex microspheres.
34. The in vitro cellular system of claim 1 , wherein said artificial lymphoid tissue equivalent culture further comprises a chemokine.
35. The in vitro cellular system of claim 34 , wherein said chemokine is selected from the group consisting of CXCL-13, CCL-21 and MIP3.
36. The in vitro cellular system of claim 1 , wherein said second matrix comprises microbeads, wherein said microbeads comprise lymphoid ECM.
37. The in vitro cellular system of claim 30 , wherein said lymphoid ECM is porcine lymphoid ECM.
38. The in vitro cellular system of claim 1 , wherein said plurality of lymphocytes comprises B cells and T cells negatively selected from peripheral blood lymphocytes.
39. The in vitro cellular system of claim 1 , wherein said artificial lymphoid tissue equivalent culture further comprises heparin.
40. The in vitro cellular system of claim 1 , wherein said plurality of cells attached to said first matrix consists of endothelial and/or epithelial cells derived from embryonic stem cells.
41. The in vitro cellular system of claim 1 , wherein said plurality of lymphocytes attached to said second matrix comprises cells derived from embryonic stem cells.
42. The in vitro cellular system of claim 1 , further comprising a vascular network connecting said vaccination site culture to said artificial lymphoid tissue equivalent culture.
43. The in vitro cellular system of claim 42 , wherein said vascular network comprises a lymphatic vessel and a blood vessel.
44. The in vitro cellular system of claim 42 , wherein said vascular network comprises inserts or laminates having integrated microchannels.
45. The in vitro cellular system of claim 42 , further comprising a cell source.
46. The in vitro cellular system of claim 42 , further comprising magnetic microbeads or magnetic nanobeads.