Effects of growth factors on hair follicle cell proliferation and release of collagenolytic factors

• US 5,616,471 A
• Filed: 02/04/1991
• Issued: 04/01/1997
• Est. Priority Date: 05/06/1987
• Status: Expired due to Term

• Current Assignee: Health and Human Services United States of America As Represented By The SEC
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Yuspa, Stuart H.
• Primary Examiner(s): Czaja, Donald E.
• Assistant Examiner(s): WILLIAMS, JANE
• Application Number:
  US07/650,572
• Time in Patent Office: 2,248 Days
• Field of Search: 435/29, 435/240.23, 435/240.21, 435/240.243, 435/260, 435/243, 435/244, 514/880, 514/21, 530/356, 623/15
• US Class Current: 435/29
• CPC Class Codes: G01N 2333/485 Epidermal growth factor [EGF] (urogastrone)
• CPC Class Codes: G01N 2333/495 Transforming growth factor [TGF]
• CPC Class Codes: G01N 2333/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Abstract

The present invention relates to methods for detecting the effects of selected growth factors and pharmaceuticals on the growth and development of hair follicles by assaying hair follicle cell proliferation and cellular collagenolytic factor secretion. In particular, isolated hair follicle cells maintained as intact functioning folliculoids are embedded in a three-dimensional culture system, and exposed to the chemical agent of choice. The effect of this agent can then be determined with respect to its ability to modify cellular proliferation or secretion of proteolytic factors. Each determination involves the use of a separate assay.

Claims

1. A method for determining hair follicle cell proliferation in response to a chemical agent comprising the steps of:

i) isolating hair follicles from dermis;

ii) plating a layer of semi-solid medium on a support;

iii) mixing said follicles with a semi-solid medium, and plating said mixture over the layer of step (ii), thereby creating a three-dimensional culture system;

iv) adding a chemical agent to a liquid medium placed over the semi-solid medium of step (iii);

v) pulsing said three-dimensional culture system with a radio-labeled compound or reagent which can detect DNA synthesis;

vi) releasing cells from said follicles from the layer in which they are embedded, by enzymatic digestion; and

vii) measuring the amount of cellular proliferation by determining the amount of said radio-labeled compound or reagent incorporated into the DNA of said cells.

2. The method of claim 1 wherein said chemical agent is a growth factor or pharmaceutical.

3. The method of claim 2, wherein said growth factor is selected from the group consisting of EGF and cholera toxin.

4. The method of claim 2 wherein said growth factor is selected from the group consisting of TGF-α

, TGF-β

1, and TGF-β

2.

5. The method of claim 1 wherein said radio-labeled compound or reagent is a DNA precursor.

6. The method of claim 5 wherein said radio-labeled compound or reagent is tritiated thymidine.

7. The method of claim 1 wherein said layer of step (ii) comprises collagen.

8. A method for detecting the effect of a chemical agent on the release of lytic factors from hair follicle cells comprising the steps of:

i) isolating hair follicles from dermis;

ii) plating a layer of semi-solid medium, comprising a radio-labeled agent, on a support;

iii) mixing said follicles with a second semi-solid medium, and plating said mixture over the layer of step (ii), thereby creating a three-dimensional culture system;

iv) adding said chemical agent to a liquid placed over the semi-solid medium of step (iii); and

v) determining the percentage of the radio-labeled agent released from said layer of step (ii), thereby determining the ability of said follicle cells to release lytic factors.

9. The method of claim 8 wherein said chemical agent is a growth factor or pharmaceutical.

10. The method of claim 9 wherein said growth factor is selected from the group consisting of EGF and cholera toxin.

11. The method of claim 9 wherein said growth factor is selected from the group consisting of TGF-α

, TGF-β

1, and TGF-β

2.

12. The method of claim 8 wherein said radio-labeled agent of step (ii) is tritiated collagen.

13. The method of claim 8 wherein said layer of step (ii) comprises collagen.

14. The method of claim 8 wherein the lytic factors of step (v) are collagenolytic.

15. A method for determining non-human hair follicle cell proliferation in response to a chemical agent comprising the steps of:

i) isolating hair follicles from non-human mammalian dermis;

ii) plating a layer of semi-solid medium on a support;

iii) mixing said hair follicles with a semi-solid medium, and plating said mixture over the layer of step (ii), thereby creating a three-dimensional culture system;

iv) adding a chemical agent to a liquid medium placed over the semi-solid medium of step (iii);

v) pulsing said three-dimensional culture system with a radio-labeled compound or reagent which can detect DNA synthesis;

vi) releasing said hair follicle cells from the layer in which they are embedded, by enzymatic digestion; and

vii) measuring the amount of cellular proliferation by determining the amount of said radio-labeled compound or reagent incorporated into the DNA of said cells.

16. The method of claim 15 wherein said chemical agent is a growth factor or pharmaceutical.

17. The method of claim 16 wherein said growth factor is selected from the group consisting of EGF and cholera toxin.

18. The method of claim 16 wherein said growth factor is selected from the group consisting of TGF-α

, TGF-β

1, and TGF-β

2.

19. The method of claim 15 wherein said radio-labeled compound is a DNA precursor.

20. The method of claim 15 wherein said layer of step (ii) comprises collagen.

21. A method for detecting the effect of a chemical agent on the release of lytic factors from non-human hair follicle cells comprising the steps of:

i) isolating hair follicles from non-human mammalian dermis;

ii) plating a layer of semi-solid medium, comprising a detectable agent, on a support;

iii) mixing said hair follicles with a semi-solid medium, and plating said mixture over the layer of step (ii), thereby creating a three-dimensional culture system;

iv) adding said chemical agent to a liquid placed over the semi-solid medium of step (iii); and

(v) determining the percentage of the detectable agent released from said layer of step (ii), thereby determining the ability of said hair follicle cells to release lytic factors.

22. The method of claim 21 wherein said chemical agent is a growth factor or pharmaceutical.

23. The method of claim 22 wherein said growth factor is selected from the group consisting of EGF and cholera toxin.

24. The method of claim 22 whereon said growth factor is selected from the group consisting of TGF-α

, TGF-β

1, and TGF-β

2.

25. The method of claim 21 wherein said detectable agent is a radio-labeled nucleotide.

26. The method of claim 21 wherein said layer of step (ii) comprises collagen.

27. The method of claim 21 wherein the lytic factor of step (v) is collagenolytic.