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Last Updated: March 28, 2024

Claims for Patent: 7,642,048


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Summary for Patent: 7,642,048
Title:Chemo-sensitivity assays using tumor cells exhibiting persistent phenotypic characteristics
Abstract: The assays, methods, tools and systems discussed herein represent an improved and unified system for monitoring the progression of an individual patient malignancy. The assays, methods, tools and systems discussed herein represent an improved and unified system for monitoring and for identifying cellular and secreted markers, for screening cells to detect phenotypic and genotypic drift and for predicting chemotherapeutic response of patient tumor cells to at least one therapeutic agent. The assays, methods, tools and systems discussed herein also represent an improved and unified system for monitoring and for screening multiple pharmaceutical agents for efficacy and long term effect as to a specific patient.
Inventor(s): Gabrin; Michael (Pittsburgh, PA), Brower; Stacey (Pittsburgh, PA), McDonald; Sean (Pittsburgh, PA), Gallion; Holly (Pittsburgh, PA), Nanavati; Payal (Pittsburgh, PA), Rice; Shara Dawn (Pittsburgh, PA), Chattopadhyay; Anuja (Pittsburgh, PA)
Assignee: Precision Therapeutics Inc. (Pittsburgh, PA)
Application Number:11/785,984
Patent Claims:1. A method for testing sensitivity of a tumor to a chemotherapeutic agent or combination, comprising: mincing an epithelial tumor specimen to prepare a plurality of multicellular tissue explants, and placing the explants in liquid medium in one or more containers; physically agitating the explants, culturing said explants to form one or more non-confluent monolayers, and suspending the non-confluent monolayer(s) in culture medium to obtain from about 4,000 to about 12,000 cells per ml; transferring the cells to a plurality of wells at about 100 to about 1000 cells per well; adding a serial dilution of at least one chemotherapeutic agent or combination across the wells; and determining sensitivity of the cells to the chemotherapeutic agent or combination.

2. The method of claim 1, wherein the explants are physically agitated by swirling, shaking, or rocking the container, or striking the container against a solid object.

3. The method of claim 1, wherein the cells are incubated in the plurality of wells for about 4 to 30 hours before the chemotherapeutic agent or combination is added.

4. The method of claim 1, wherein chemosensitivity is determined by quantifying cell death.

5. The method of claim 4, further comprising, preparing a dose response curve.

6. The method of claim 1, wherein the tumor is a breast, colorectal, or ovarian tumor.

7. The method of claim 1, wherein the epithelial tumor specimen contains from 15 to 35 mg of tissue.

8. The method of claim 1, wherein the explants each measure from 0.25 mm.sup.3 to 1.5 mm.sup.3.

9. The method of claim 1, wherein the explants are treated with one or more of Collagenase and DNase.

10. The method of claim 1, wherein the tumor specimen is an ovarian or colorectal tumor specimen, and the explants are treated with one or more of Collagenase and DNase.

11. The method of claim 1, wherein the explants are removed from culture when the monolayer is at about 10% to about 50% confluency.

12. The method of claim 1, further comprising monitoring the morphology and/or growth rate of the monolayer cells.

13. The method of claim 12, wherein the monolayer cells are monitored by phase contrast microscopy.

14. The method of claim 1, wherein the cells are suspended at about 8,000 cells/ml.

15. The method of claim 1, wherein about 200 to 400 cells are seeded per well.

16. The method of claim 15, wherein the cells are incubated in the wells for about 24 hours prior to contact with the chemotherapeutic agent or combination.

17. The method of claim 1, wherein the cells are contacted with the chemotherapeutic agent or combination for about 25 to 200 hours.

18. The method of claim 1, wherein cell death is quantified by fixing and staining the cells, and visualizing stained cells.

19. A method for testing sensitivity of a tumor to a chemotherapeutic agent or combination, comprising: disaggregating an ovarian or colorectal tumor specimen to prepare a plurality of explants; treating the explants with DNase and Collagenase while disturbing the explants with a swirling, shaking, or rocking motion so as to reduce the size of the explants, wherein the treated explants are multicellular tissue explants; culturing the multicellular tissue explants to form one or more non-confluent monolayers comprising malignant cells; suspending the non-confluent monolayer(s) in culture medium at about 4,000 to 12,000 cells per ml, and transferring the cells to a plurality of wells at about 100 to 1000 cells per well; incubating the cells for about 4 to 30 hours, and then adding a serial dilution of the chemotherapeutic agent or combination across the wells; quantifying cell death in each well as a measure of chemosensitivity, and preparing a dose response curve for the chemotherapeutic agent or combination.

20. The method of claim 19, wherein the explants are treated with a DNase and Collagenase II cocktail for from 3 minutes to one hour, the cocktail comprising from about 0.01% to about 0.6% of Collagenase II and from about 0.0007% to about 0.005% of DNase.

21. The method of claim 20, wherein the explants are removed from culture when the monolayer is at about 10% to about 50% confluency.

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