Methods to produce mast cell culture
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 method to make a mast cell culture comprising steps of (a) treating CD34 + progenitor cells under hypoxic condition for 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 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, wherein a concentration of IL-3 in the medium in step (a) is 0.1-4 ng/ml, a concentration of IL-6 in the media in steps (a), (b) and (c) is 50-500 ng/ml, and a concentration of IL-9 in the medium in step (b) is 1-50 ng/ml; and (d) treating the immature mast cell under normoxic condition in a medium comprising SCF, IL-6 and IL-4, wherein a concentration of IL-6 in the medium in step (d) is 50-500 ng/ml, a concentration of IL-4 in the medium in step (d) is 1-100 ng/ml and a concentration of SCF in the media in steps (a), (b), (c), and (d) is 100-500 ng/ml.
2. The method of claim 1 , wherein the concentration of SCF is 200 ng/ml.
3. The method of claim 1 , wherein the concentration of IL-6 is 100 ng/ml in steps (a), (b) and (c).
4. The method of claim 1 , wherein the concentration of IL-4 in the medium in step (d) is 10 ng/ml.
5. The method of claim 1 , wherein the CD34 + progenitor cells are human CD34 + progenitor cells.
6. The method of claim 1 , wherein the initial-stage cell culture is treated under hypoxic condition for 1 week in step (b).
7. The method of claim 1 , wherein the intermediate-stage cell culture is treated under normoxic condition for 3 weeks in step (c).
8. The method of claim 1 , wherein the concentration of IL-3 in the medium in step (a) is 1 ng/ml.
9. The method of claim 1 , wherein the concentration of IL-9 in the medium in step (b) is 15 ng/ml.
10. The method of claim 1 , wherein the concentration of IL-6 is 100 ng/ml in step (d).