Method for measuring sensory irritation in vitro

• US 5,811,256 A
• Filed: 10/10/1995
• Issued: 09/22/1998
• Est. Priority Date: 10/10/1995
• Status: Expired due to Fees

• Current Assignee: Monell Chemical Senses Center (Main Line Health System)
• Original Assignee: Monell Chemical Senses Center (Main Line Health System)
• Inventors: Bryant, Bruce P.
• Primary Examiner(s): LEARY, LOUISE N
• Application Number:
  US08/541,641
• Time in Patent Office: 1,078 Days
• Field of Search: 435/29, 435/7.2, 435/7.21, 435/284, 435/287, 435/300, 435/968, 435/975, 435/284.1, 435/287.1, 435/288.2, 435/304.2, 435/305.1, 435/305.2, 435/288.4
• US Class Current: 435/29
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5044 involving specific cell types
• CPC Class Codes: G01N 33/5047 Cells of the immune system
• CPC Class Codes: G01N 33/5058 Neurological cells
• CPC Class Codes: G01N 33/5061 Muscle cells
• CPC Class Codes: G01N 33/5064 Endothelial cells
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/975 Kit

Abstract

Methods for ascertaining the sensory irritation of chemicals in vitro are described. The methods include the cultivation of neuronal cells alone, with target tissue cells, and with target tissue cells and mast cells; the introduction of a chemical to be tested; and the measuring of neuronal response in the form of ion uptake or change in membrane potential. A co-culture system of neuronal and target tissue cells for performing said methods is also described.

Claims

1. A method for assessing the sensory irritation of chemicals in vitro comprising:

a) cultivating neonatal neurons isolated from sensory ganglia in vitro in a culture medium containing nerve growth factor for a period of from about 2 to about 10 days;

b) introducing Ca⁺⁺ or cobalt ions to said medium;

c) adding a chemical to be tested to said culture medium and incubating for a period of up to about 1 hour;

d) adding a means for measuring the uptake of Ca⁺⁺ or cobalt by said neurons; and

e) comparing said uptake with that of a control culture of neurons to which the chemical has not been added.

2. The method of claim 1 wherein the Ca⁺⁺ or cobalt ions are introduced simultaneously with said chemical.

3. The method of claim 1 wherein the uptake of Ca⁺⁺ is measured using fluorescent staining.

4. A method for assessing the sensory irritation of chemicals on a particular target tissue in vitro comprising:

a) cultivating neonatal neurons isolated from sensory ganglia in a first chamber and target tissue cells in a second chamber, said first and second chambers being on parallel planes and separated by a material permitting said neurons to innervate said target tissue cells, wherein said neurons and said target tissue cells are cultured in a culture medium containing nerve growth factor for a period of from about 2 to about 10 days;

b) introducing Ca⁺⁺ or cobalt ions to medium in said first chamber;

c) adding a chemical to be tested to said culture medium in said second chamber and incubating for a period of from about 1 minute to about 1 hour;

d) adding a means for measuring the uptake of Ca⁺⁺ or cobalt by said neurons to said first chamber; and

e) comparing said uptake with that of a control culture of neurons to which the chemical has not been added.

5. The method of claim 4 wherein said target tissue cells are selected from the group consisting of epithelial, endothelial, visceral organ, and muscle cells.

6. The method of claim 4 further comprising mast cells at a density representative of the density of said mast cells in said target tissue in vivo in said second chamber.

7. The method of claim 4 wherein the uptake of Ca⁺⁺ is measured using fluorescent microscopy.

8. A method for assessing the sensory irritation of chemicals on a particular target tissue in vitro comprising:

a) cultivating neonatal neurons isolated from sensory ganglia in a first chamber and target tissue cells in a second chamber, said first and second chambers being on parallel planes separated by a material permitting said neurons to innervate said target tissue cells, wherein said neurons and said target tissue cells are cultured in a culture medium containing nerve growth factor for a period of from about 2 to about 10 days;

b) adding a voltage-sensitive fluorescent dye to said first chamber;

c) adding a chemical to be tested to said culture medium in said second chamber and incubating for a period of up to about 20 minutes; and

d) measuring changes in membrane potential.

9. The method of claim 8 wherein said target tissue cells are selected from the group consisting of epithelial, endothelial, visceral organ and muscle cells.

10. The method of claim 8 further comprising mast cells at a density representative of the density of mast cells in said target tissue in vivo in said second chamber.

11. A method for assessing the sensory irritation of chemicals in vitro comprising:

a) cultivating neonatal neurons isolated from sensory ganglion in vitro in a culture medium containing nerve growth factor for a period of from about 2 to about 10 days;

b) adding a first stimulus to said neurons and incubating for a period of up to about 2 days;

c) introducing Ca⁺⁺ or cobalt ions to said medium;

d) adding a second stimulus to said neurons and incubating for a period of up to about 20 minutes;

e) adding a means for measuring the uptake of Ca⁺⁺ or Co⁺ by said neurons after said second stimulus has been added; and

f) comparing said uptake with that of a control culture of neurons to which the first stimulus has not been added, a control culture to which the second stimulus has not been added, or a control culture to which neither stimulus has been added.

12. The method of claim 11 wherein the first stimulus is selected from the group consisting of thermal, photic, or chemical stimulus.

13. The method of claim 11 wherein the second stimulus is selected from the group consisting of thermal, photic, or chemical stimulus.

14. The method of claim 11 wherein said Ca⁺⁺ or cobalt ions are introduced prior to said second chemical.

15. A method for assessing the sensory irritation of chemicals on a particular target tissue in vitro comprising:

a) cultivating neonatal neurons isolated from sensory ganglia in a first chamber and target tissue cells in a second chamber, said first and second chambers being on parallel planes separated by a material permitting said neurons to innervate said target tissue cells, wherein said neurons and said target tissue cells are cultivated in a culture medium containing nerve growth factor for a period of from 2 to about 10 days;

b) adding a first stimulus to said target tissue cells in said second chamber and incubating for a period of up to about 2 days;

c) introducing Ca⁺⁺ or cobalt ions to the medium in said first chamber;

d) adding a second stimulus to said target tissue cells in said second chamber and incubating for a period of up to about 20 minutes;

e) adding a means for measuring the uptake of Ca⁺⁺ or cobalt ions by said neurons after said second stimulus has been added; and

f) comparing said uptake with that of a control culture of neurons to which the first stimulus has not been added, a control culture to which the second stimulus has not been added, or a control culture to which neither stimulus has been added.

16. The method of claim 15 wherein the first stimulus is selected from the group consisting of thermal, photic, or chemical stimulus.

17. The method of claim 15 wherein the second stimulus is selected from the group consisting of thermal, photic, or chemical stimulus.

18. The method of claim 15 wherein said target tissue cells are selected from the group consisting of epithelial, endothelial, visceral organ and muscle cells.

19. The method of claim 15 further comprising mast cells at a density representative of the density of said mast cells in said target tissue in vivo in said second chamber.

20. The method of claim 15 wherein Ca⁺⁺ or cobalt ions are introduced simultaneously with said second stimulus.

21. The method of claim 15 wherein the uptake of Ca⁺⁺ is measured using fluorescent imaging technology.

22. An assay kit for assessing the sensory irritation of chemicals in vitro comprising:

a) a tissue culture medium containing nerve growth factor;

b) tissue culture plates;

c) inserts sized to fit in individual wells of said tissue culture plates;

d) a source of Ca⁺⁺ or cobalt ions; and

e) a means for measuring the uptake of Ca⁺⁺ or cobalt ions by cells cultured in said medium.

23. The kit of claim 22 wherein said means for measuring the uptake of Ca⁺⁺ is a change in fluorescence intensity.

24. The kit of claim 22 wherein said means for measuring the uptake of Ca⁺⁺ is a change in membrane voltage by fluorescence intensity.

25. The kit of claim 22 further comprising neuronal cells.

26. The kit of claim 22 further comprising target tissue cells.

27. A co-culture system for testing the sensory irritation of chemicals in vitro comprising target tissue cells cultured in a plane separate from and parallel to neuronal cells, said target tissue cells being separated from said neuronal cells by a material permitting innervation by neurites of the neuronal cells into the target tissue cells, wherein said neurites are not directly exposed to chemicals applied only to the target tissue cells.

28. The system of claim 27 wherein said target tissue is selected from the group consisting of skin, respiratory, hepatic, gastrointestinal, nasal, corneal, and oral tissue.

29. The system of claim 27, further comprising mast cells at a density typical for said target tissue in vivo.