Composition and Method for Assessing Regulation of Mast Cells
The present invention relates to mast cell cultures that are derived from hematopoietic progenitors and the use thereof. The invention describes a method for generating in-vitro cultures of human mast cells with functional phenotype of connective tissue-type mast cells. By monitoring the levels of chemokines released into the medium, such mast cell cultures can be used as a cell-based assay to assess regulation of mast cell functions and pharmacological activities of tryptase inhibitors.
1 . A mast cell culture product made by the process comprising (a) treating CD34 + progenitor cells under hypoxic condition in a medium comprising stem cell factor (SCF), interleukin-6 (IL-6) and interleukin-3 (IL-3) to generate an initial-stage cell culture; (b) treating the initial-stage cells under hypoxic condition in a medium comprising SCF, IL-6 and interleukin-9 (IL-9) to form an intermediate stage cell culture; (c) treating the intermediate-stage cells under normoxic condition in a medium comprising SCF and IL-6, thereby generating an immature mast cell culture.
2 . The product of claim 1 wherein the immature mast cell is further treated in step (d), comprising treating the immature mast cells under normoxic condition in a medium comprising SCF, IL-6 and interleukin-4 (IL-4), thereby generating a mature mast cell culture.
3 . The product of claims 1 or 2 wherein the CD34 + progenitor cells are derived from human.
4 . The product of claims 1 or 2 wherein the CD34 + progenitor cells are treated under hypoxic condition for about 1 week in step (a).
5 . The product of claims 1 or 2 wherein the initial-stage cell culture is treated under hypoxic condition for 0.5-2.5 weeks in step (b).
6 . The product of claim 5 wherein the initial-stage cell culture is treated under hypoxic condition for 1 week in step (b).
7 . The product of claims 1 or 2 wherein the intermediate-stage cell culture is treated under normoxic condition for 2.5-3.5 weeks in step (c).
8 . The product of claim 7 wherein the intermediate-stage cell culture is treated under normoxic condition for 3 weeks in step (c).
9 . The product of claim 2 wherein the immature mast cell culture is treated under normoxic condition for 1 week in step (d).
10 . The product of claims 1 or 2 wherein the concentration of IL-3 in the medium in step (a) is 0.1-4 ng/ml.
11 . The product of claim 10 wherein the concentration of IL-3 in the medium in step (a) is 1 ng/ml.
12 . The product of claims 1 or 2 wherein the concentration of IL-9 in the medium in step (b) is 15 ng/ml.
13 . The product of claims 1 or 2 wherein the concentration of SCF in the media in steps (a), (b) and (c) is 100-500 ng/ml.
14 . The product of claim 2 wherein the concentration of SCF in the medium in step (d) is 100-500 ng/ml.
15 . The product of claims 13 or 14 wherein the concentration of SCF is 200 ng/ml.
16 . The product of claims 1 or 2 wherein the concentration of IL-6 in the media in steps (a), (b) and (c) is 50-500 ng/ml.
17 . The product of claim 2 wherein the concentration of IL-6 in the medium in step (d) is 50-500 ng/ml.
18 . The product of claims 16 or 17 wherein the concentration of IL-6 is 100 ng/ml.
19 . The product of claim 2 wherein the concentration of IL4 in the medium in step (d) is 10 ng/ml.
20 . A method of culturing mast cells comprising the steps of (a) treating CD34 + progenitor cells under hypoxic condition for about 1 week in a medium comprising SCF, IL-6 and IL-3 to generate an initial-stage cell culture; (b) treating the initial-stage cell culture under hypoxic condition for about 0.5-2.5 weeks in a medium comprising SCF, IL-6 and IL-9 to form an intermediate stage cell culture; (c) treating the intermediate-stage cell culture under normoxic condition for 2.5-3.5 weeks in a medium comprising SCF and IL-6 to generate an immature mast cell culture.
21 . The method of claim 20 further comprising the step of (d) treating the immature mast cell under normoxic condition in a medium comprising SCF, IL-6 and IL-4.
22 . The method of claims 20 or 21 wherein the CD34 + progenitor cells are derived from human.
23 . The method of claims 20 or 21 wherein, the initial-stage cell culture is treated under hypoxic condition for 1 week in step (b).
24 . The method of claims 20 or 21 wherein the intermediate-stage cell culture is treated under normoxic condition for 3 weeks in step (c).
25 . The method of claims 20 or 21 wherein the concentration of IL-3 in the medium in step (a) is 0.1-4 ng/ml.
26 . The method of claim 25 wherein the concentration of IL-3 in the medium in step (a) is 1 ng/ml.
27 . The method of claims 20 or 21 wherein the concentration of IL-9 in the medium in step (b) is 15 ng/ml.
28 . The method of claims 20 or 21 wherein the concentration of SCF in the media in steps (a), (b) and (c) is 100-500 ng/ml.
29 . The method of claim 21 where the concentration of SCF in the medium in step (d) is 100-500 ng/ml.
30 . The method of claims 28 or 29 wherein the concentration of SCF is 200 ng/ml.
31 . The method of claims 20 or 21 wherein the concentration of IL-6 in the media in steps (a), (b) and (c) is 50-500 ng/ml.
32 . The method of claim 31 wherein the concentration of IL-6 in the medium in step (d) is 50-500 ng/ml.
33 . The method of claims 31 or 32 wherein the concentration of IL-6 is 100 ng/ml.
34 . The method of claim 21 wherein the concentration of IL-4 in the medium in step (d) is 10 ng/ml.
35 . A method of assessing regulation of mast cell functions by measuring the level of MCP-1 wherein the MCP-1 is secreted by mast cells over a period of time in a subject.
36 . The method of claim 35 wherein the regulation is a dysregulatioin of mast cell functions related to an inflammation-related condition, a disease or a disorder in a subject.
37 . The method of claim 35 wherein the mast cells are stimulated by an agent selected from the group consisting of anti-immunoglobulin E (IgE) and aggregated immunoglobulin G1 (IgG 1 ).
38 . The method of claim 35 wherein the level of MCP-1 is measured over 6-22 hours after stimulation.
39 . A method of assessing tryptase-inhibitory activity of an agent comprising the steps of (a) stimulating mast cells in the presence of the agent and; (b) measuring level of MCP-1 secreted by mast cells over a period of time.
40 . The method of claim 39 wherein the mast cells are stimulated by anti-IgE or aggregated IgG 1 .
41 . The method of claim 39 wherein the level of MCP-1 secreted by mast cells is measured over 6-22 hours.
42 . A cell culture of functionally mature human mast cells generated from progenitor cells expressing the CD34 antigen comprising said human mast cells showing phenotypic and functional maturity at six weeks in culture.
43 . The cell culture of claim 42 wherein the cells express both chymase and tryptase.
44 . The cell culture of claim 42 wherein the cells exhibit functional activation in response to stimulation by compound 48/80 or ionophores.
45 . The cell culture of claim 42 wherein the cells exhibit functional activation in response to activation of cell surface receptors on the cells.
46 . The cell culture of claim 45 wherein the cell surface receptors are selected from high-affinity IgE receptors, high-affinity IgG receptors, Toll-like receptors and Substance P receptors.