Cell culturing methods and apparatus

• US 4,734,372 A
• Filed: 06/21/1984
• Issued: 03/29/1988
• Est. Priority Date: 02/04/1983
• Status: Expired due to Fees

• Current Assignee: Nu-Tech Bio-Med, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Brown University
• Inventors: Rotman, M. Boris
• Primary Examiner(s): Scott, Samuel
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/623,183
• Time in Patent Office: 1,377 Days
• Field of Search: 435/3, 435/4, 435/29, 435/32, 435/34, 435/240, 435/241, 435/284, 435/287, 435/291, 435/808, 435/810, 435/811, 436/4, 436/34, 436/63, 436/68, 436/172, 436/178, 422/48, 422/61, 422/68
• US Class Current: 435/29
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/5014 for testing toxicity

Abstract

A system and protocol for performing cytotoxicity studies including apparatus and methods for culturing the biopsied cells, for providing oxygenated nutrients, for removing cell-waste products, for introducing a fluorogenic substrate, for introducing cytotoxic agents including anticancer drugs, for measuring the released fluorescence and for measuring intracellular accumulation of fluorescein. Cytotoxicity can be determined by measuring fluorescence in the efflux of the vessel and concommitantly by direct photometric comparisons of the cells in the vessel before and after exposure to the cytotoxic agent.

Claims

1. A cytotoxicity assay system comprising a cell culturing vessel having a cell enclosure into which cells can be introduced and cultured;

nutrient-transporting means for transporting nutrients to the cells, the nutrient-transporting means include a source of nutrients and at least one fluid permeable membrane, connecting said source of nutrients to said cells, across which nutrients can migrate to nourish the cells within the vessel;

fluorogenic substrate-transporting means for transporting a fluorogenic substrate to the cell compartment, so that living cells within the vessel will take up said substrate and accumulate a characteristic fluorescence;

means for introducing a therapeutic agent into the cell compartment; and

means for measuring cytotoxicity within the cell compartment, including means for detected changes in the fluorescence of the cells within the vessel.

2. The system of claim 1 wherein the vessel further includes at least one transparent surface through which fluorescence within the cell enclosure can be observed.

3. The system of claim 1 wherein the vessel further includes at least one sampling port for withdrawing an sample from the cell enclosure to measure the fluorescence of the sample.

4. The system of claim 1 wherein the fluid permeable membrane has a pore size ranging from about 5 to about 35 microns.

5. The system of claim 1 wherein the fluid permeable membrane is a synthetic capillary.

6. The system of claim 1 wherein the fluid permeable membrane is a fibrous cellulose material.

7. The system of claim 1 wherein the permeable membrane is polymeric mesh.

8. The system of claim 1 wherein the means for measuring fluorescence is a camera.

9. The system of claim 8 wherein the means for measuring fluorescence further includes a monochromatic light source and a matched filter.

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

a. culturing the cells in a vessel;

b. contacting the cells with a fluorogenic substrate whereby living cells accumulate a characteristic amount of fluorescence;

c. introducing the agent into the vessel; and

d. measuring changes in the fluorescence by photometrically analyzing the intensity of fluorescence within the vessel as an indicator of the sensitivity of the cells to the agent.

11. The method of claim 10 wherein the step of measuring changes in fluorescence further including photometrically recording the fluorescence with a photographic camera.

12. The method of claim 10 wherein the step of measuring changes in fluorescence further includes photometrically recording the fluorescence with a video camera.

13. The method of claim 10 wherein the step of measuring changes in fluorescence further includes illuminating the cultured cells with blue monochromatic light.

14. The method of claim 13 wherein the step of measuring changes in fluorescence further includes filtering light from the cultured cells with a matching blue filter.