Methods and devices for cellular analysis
Embodiments of the present invention are directed to improved methods and devices for analyzing a cell, aggregated cells, or a solid tumor. Such methods and devices are, for example, useful in the field of pathology and can provide improved cell processing and analytical results.
1. A method for processing cancer cells from a solid tumor sample from a subject comprising the following steps in sequential order:
(a) disaggregating and dispersing an aqueous solution containing live aggregated cancer cells into at least one test aliquot in a first isolated chamber, wherein a predetermined amount of laminar fluid shear force to disrupt aggregates of the cancer cells without killing the cancer cells and triggering only a minimal stress response or no stress response in the cancer cells is used;
(b) optionally purifying the aliquot to increase the percentage of target cancer cells relative to other contaminating cell types by removing the contaminating cells;
(c) distributing the optionally purified live cancer cells into one or more second isolated chambers for analysis;
(d) contacting the distributed live cancer cells ex vivo with at least one agent to produce a measurable quantitatively or qualitative effect on a target ex vivo biomarker or biomolecules in a cellular pathway;
(e) stabilizing the target ex vivo biomarker or biomolecule of the cancer cells within about one to four hours by lysing or fixing the cancer cells on a solid support using a solution comprising a polymer, thereby killing the cancer cells; and
(f) measuring the changes in levels of the target ex vivo biomarker or biomolecule in the cellular pathway to assess the response of the target cancer cells to the at least one agent.
2. The method of claim 1 , wherein the subject is a human and the method is performed at the point of care.
3. The method of claim 1 , wherein the cancer cells are obtained from the subject as a solid tumor biopsy.
4. The method of claim 1 , wherein the solid tumor sample is obtained using a fine needle aspiration technique.
5. The method of claim 1 , in which the total number of the live aggregated cancer cells processed is between about 1000 and 10×10 6 .
6. The method of claim 1 , wherein the disaggregation step comprises passing the fluid comprising the cancer cells from the solid tumor sample through a needle or pipette tip of a predetermined size.
7. The method of claim 1 , wherein the cancer cells are dispersed by the shear force of between about 100 to about 800 dyne/cm 2 .
8. The method of claim 1 , wherein the optional purification comprises immunodepletion.
9. The method of claim 1 , wherein the one or more second isolated chambers contain less than about 1,000,000 of the purified cancer cells.
10. The method of claim 1 , wherein the distribution step is done manually or using an automated system.
11. The method of claim 1 , wherein the distributed live cancer cells have over about 75% viability as compared to the number of viable cancer cells in the fluid prior to the distribution.
12. The method of claim 1 , wherein the distributed live cancer cells are divided into at least two aliquots and wherein each aliquot is contacted with a different at least one agent in step (d).
13. The method of claim 1 , wherein the at least one agent is selected from the group consisting of a pharmaceutical agent, an agent for stimulating a cell, a polypeptide, a polynucleotide, an antibody, an Fab fragment, an Fc fragment, RNA, siRNA and a phosphoprotein.
14. The method of claim 1 , wherein the cellular pathway is selected from the group consisting of a metabolic pathway, a replication pathway, a cellular signaling pathway, an oncogenic signaling pathway, an apoptotic pathway, and a pro-angiogenic pathway.
15. The method of claim 1 , wherein the agent is an epidermal growth factor (EGF).
16. The method of claim 1 , wherein the quantitative or qualitative effect measured is the expression level of a gene selected from the group consisting of an immediate or delayed early gene family.
17. The method of claim 1 , wherein the target ex vivo biomarker or biomolecule is selected from the group consisting of ions, enzymes, lipids, and post-translationally modified proteins.
18. The method of claim 13 , wherein the at least one agent is preloaded into one or more second isolated chambers before the purified live cancer cells are distributed into the one or more second isolated chambers.
19. The method of claim 1 , wherein the at least one agent is a detectable agent selected from the group consisting of: an enzyme, fluorescent material, luminescent material, bioluminescent material, radioactive material, positron emitting metal using a positron emission tomography, and nonradioactive paramagnetic metal ion.
20. The method of claim 1 , wherein the solid support is a glass slide.