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Last Updated: January 26, 2022

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Claims for Patent: 4,937,187

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Summary for Patent: 4,937,187
Title: Methods for separating malignant cells from clinical specimens
Abstract:Fragments of a biopsy sample on the order of about 50 to 5000 cells are preferred for establishing viable tumor cell cultures for purposes such as establishing cell lines, chemotherapeutic assays and the like. Such fragments retain the three-dimensional cellular structure or organization of the original tumor and, therefore, can be cultured more readily. To obtain such fragments suitable for culturing, the biopsy sample can be enzymatically digested in a proteolytic or nucleolytic enzyme, such as collagenase, or by mechanical dissociation, or both where necessary. The fragments can then be suspended in an aqueous medium so that non-aggregated cells (e.g., red blood cells, lymphocytes, macrophages) and cellular debris will form a supernatant while the remaining fragments containing aggregated tumor cells are deposited in a sediment layer. Preferably, the medium is an isotonic tissue culture medium and decantation is conducted at least twice; first in a serum-containing medium and then, secondly, in a serum-free medium. Fragments containing living tumor cells can be selected by fluorochromasia, that is, by contacting the sedimented layer with a fluorogenic substrate such that viable tumor cells take up and hydrolyse the substrate, and then exhibit fluorescence. Cytotoxicity assay protocols employing tumor cell aggregates prepared by the present techniques are also disclosed.
Inventor(s): Rotman; M. Boris (Jamestown, RI)
Assignee: Brown University Research Foundation (Providence, RI)
Application Number:07/011,219
Patent Claims:1. A method for separating viable malignant cell aggregates from a tumor biopsy sample, the method comprising:

separating the tumor biopsy sample into discrete fragments;

suspending the fragments in an aqueous medium such that non-aggregated cells and cellular debris form a supernatant, while aggregates of tumor cells exhibiting a multicellular organization form a sediment; and

recovering viable tumor cell aggregates from the sediment.

2. The method of claim 1 wherein the step of separating the sample further includes mechanically cleaving the sample into fragments.

3. The method of claim 1 wherein the step of separating the sample further includes chemically treating the sample with an enzymatic reagent.

4. The method of claim 3 wherein the enzymatic reagent is selected from the group consisting of collaganese, trypsin, chymotrypsin, DNAase, and RNAase.

5. The method of claim 4 wherein the enzymatic reagent is collagenase.

6. The method of claim 1 wherein the step of suspending the fragments in an aqueous medium further includes suspending the fragments in a first, serum-containing medium; decanting the supernatant and first medium; and then resuspending the sediment in a second, serum-free medium.

7. The method of claim 1 wherein the method further comprises contacting the sediment with a fluorogenic substrate whereby aggregates containing viable cells will take up the substrate and exhibit fluorescence, and then recovering the aggregates that exhibit fluorescence.

8. The method of claim 1 wherein the method further comprises recovering tumor cell aggregates that retain the structure and organization of the original tumor, and culturing the recovered tumor cell aggregates to establish a cell culture.

9. A method of separating visible malignant cell aggregates from a tumor biopsy sample, the method comprising:

separating the tumor biopsy sample into discrete fragments;

suspending the fragments in an aqueous medium such that non-aggregated cells and cellular debris form a supernatant, while aggregates of tumor cells exhibiting a multi-cellular organization form a sediment;

contacting the sediment with a fluorogenic substrate whereby aggregates containing viable cells will take up the substrate and exhibit fluorescence; and

recovering viable tumor cell aggregates which exhibit fluorescence from the sediment.

10. The method of claim 9 wherein the method of separating further includes mechanically cleaving the sample into fragments.

11. The method of claim 10 wherein the step of cleaving the sample into fragments further comprises cleaving the sample into fragments ranging from about 0.3 cubic millimeters to about 5.0 cubic millimeters in size.

12. The method of claim 10 wherein the mechanically cleaved fragments are further dissociated by shearing.

13. The method of claim 9 wherein the step of separating the sample further includes chemically treating the sample with an enzymatic reagent.

14. The method of claim 13 wherein the enzymatic reagent is chosen from the group of collagenase, trypsin, chymotrypsin, DNAase, and RNAase.

15. The method of claim 14 wherein the enzymatic reagent is collagenase.

16. The method of claim 9 wherein the step of suspending the fragments in an aqueous medium further includes suspending the fragments in an isotonic aqueous medium.

17. The method of claim 6 wherein the isotonic medium is an isotonic tissue culture medium.

18. The method of claim 9 wherein the step of suspending the fragments in an aqueous medium further includes suspending the fragments in a first, serum-containing medium; decanting the supernatant and first medium; and then resuspending the sediment in a second, serum-free medium.

19. A method of assaying the sensitivity of biopsied tumor cells to therapeutic agents, the method comprising:

separating biopsy sample from a tumor into discrete fragments;

suspending the fragments in an aqueous medium such that non-aggregated cells and cellular debris form a supernatant, while aggregates of tumor cells exhibiting a multi-cellular organization form a sediment,

recovering viable tumor cell aggregates that retain the structure and organization of the original tumor from the sediment,

culturing the recovered aggregates to establish a cell culture;

exposing the cell culture to a therapeutic agent; and

measuring changes in the cell culture following exposure to said agent as an indicator of the sensitivity of the cells to the agent.

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