Novel methods and devices for high-throughput quantification, detection and temporal profiling of cellular secretions, and compositions identified using same

Patent metadata

Field	Value
Publication number	US-2017336399-A1
Application number	US-201515525328-A
Country	US
Kind code	A1
Filing date	Nov 13, 2015
Priority date	Nov 14, 2014
Publication date	Nov 23, 2017
Grant date	—

Abstract

The present invention relates to the unexpected discovery of methods and devices that can be used for high-throughput precise quantification, detection and/or temporal profiling of cellular secretions. In various embodiments, the methods of the invention allow for high-throughput absolute detection of secretions of cells, identification of the nature of the secreted molecules, and/or the nature of the secreting cells. Further, the present invention includes a device combining microfluidics and antibody printing, wherein the device can be used to detect protein secretion signature of cells in a high-throughput manner. Further, the present invention includes compositions comprising molecules that can be used to reduce cell death and to implement cell-less therapies. Further, the present invention includes a method for training an algorithm to predict temporal profile of cellular secretion.

First claim

What is claimed is:

1 . A device for the temporal high-throughput measurement of one or more molecules or compounds secreted by a cell using quantitative enzyme linked immunosorbant assay (qELISA), the device comprising an experimental chamber and an observational chamber, wherein the experimental chamber and the observational chamber are separated by a permeable barrier, wherein the permeable barrier is selected so that movement of the one or more molecules or compounds across the permeable barrier is hindered when the observational chamber comprises air and/or is free of liquid.

2 . The device of claim 1 , wherein the device further comprises one or more standardization chambers, one or more experimental chambers, and/or one or more detection chambers.

3 . The device of claim 1 , wherein the experimental chamber allows the adhesion of the cell.

4 . The device of claim 1 , wherein the observational chamber comprises rows of molecule or compound detection location, wherein each row is arranged transversely to the experimental chamber and comprises an antibody that selectively binds a biological molecule.

5 . The device of claim 1 , wherein the one or more molecules or compounds secreted by the cell migrate from the experimental chamber to the observational chamber through diffusion based movement.

6 . A method of calculating an intensity of a cellular secretion using the device of claim 1 , the method comprising:
a) contacting cells with the experimental chamber; b) exposing the cells to experimental conditions to induce secretion of the one or more molecules or compounds; c) moving the one or more molecules or compounds from the experimental chamber into the observation chamber; d) binding the one or more molecules or compounds to one or more molecule or compound detection locations in the observational chamber; and e) calculating an intensity of the one or more molecules or compounds.

7 . A method of generating a temporal intensity profile of one or more molecules or compounds secreted from a cell, the method comprising:
a) calculating an estimated intensity of the one or more molecules or compounds at a distinct molecule or compound detection location and time based on diffusion of the one or more molecules or compounds to the detection location (g[x,t]); b) calculating an observed intensity at the detection location due to an adsorption and binding of the one or more molecules or compounds to the molecule or compound detection location at an observed time (s[x,t]); c) calculating a difference between b) and a) (s[x,t]−g[x,t]) to obtain a loss function; d) updating the estimated intensity to minimize the loss function; e) generating the intensity profile for the one or more molecules or compounds at the molecule or compound detection location; and f) repeating steps a) through e) for a plurality of molecule or compound detection locations,
thereby training a function minimization algorithm to generate the temporal intensity profile of the one or more molecules or compounds secreted from a cell.

8 . A method of generating a temporal concentration profile of one or more molecules or compounds secreted from a cell, the method comprising:
a) calculating an estimated concentration of the one or more molecules or compounds at a distinct molecule or compound detection location and time based on diffusion of the one or more molecules or compounds to the molecule or compound detection location (c[t]); b) proposing a deviation (d[t]) from the estimated concentration (c[t]+d[t]); c) calculating an observed concentration at the molecule or compound detection location due to an adsorption and binding of the one or more molecules or compounds to the molecule or compound detection location at an observed time (s[x,t]); d) calculating a difference between b) and c) (c[t]+d[t]−s[x,t]) to obtain a posterior probability of the deviation; e) accepting or rejecting the proposed deviation of d[t] based on the ratio of the posterior probability of (d) compared to the estimated concentration a); f) generating the concentration profile for the one or more molecules or compounds at the molecule or compound detection location; and g) repeating steps a) through f) for a plurality of molecule or compound detection location,
thereby training a function minimization algorithm to generate the temporal concentration profile of the one or more molecules or compounds secreted from a cell.

9 . A method of detecting a secretion, and/or level of secretion, of a molecule or compound by a cell isolated from a subject, the method comprising measuring and determining temporal intensity profile and/or temporal concentration profile of the molecule or compound using the device of claim 1 .

10 . The method of claim 9 , wherein the subject is a mammal.

11 . The method of claim 9 , wherein the cell is an adherent cell selected from the group consisting of fibroblasts, immune cells, cancer cell lines, primary cancer cells, stem cells, progenitor cells, stromal cells, pluripotent stem cells, somatic cells derived from pluripotent stem cells, and somatic cells derived from adult stem cells.

12 . The method of claim 9 , wherein the cell is a non-adherent cell.

13 . The method of claim 9 , wherein the cell is derived from healthy or diseased heart tissue, connective tissue, vasculature, brain tissue, tumor environment and/or metastatic tumor environment.

14 . The method in claim 9 , wherein the cell is derived from a tissue explant that is placed in the experimental chamber from healthy or diseased heart, vasculature, brain, tumor, liver, pancreas, spleen, bone marrow, cartilage, adipose tissue, and/or connective tissue.

15 . The method of claim 9 , wherein the cell is pretreated by a stimulus.

16 . The method of claim 15 , wherein the stimulus comprises at least one from the group consisting of a drug, cytokine, growth factor, hypoxia, pathogen load, physical, chemical, mechanical, and biological stimulus.

17 . The method of claim 9 , wherein the cell is cultured in a biologically mimicking environment.

18 . The method of claim 9 , wherein the cell is co-cultured in a system selected from the group consisting of cancer cell in the presence of immune cells, immune cell in the presence of cancer cells, stem cell in the presence of immune cells, stem cell in the presence of stromal cells, stromal cell in the presence of stem cells, endothelial cell in the response to cancer cells, cancer cell in the response to endothelial cells, and cancer cell in the presence of other cancer cells.

19 . A method of identifying a cell isolated from a subject, the method comprising measuring and/or determining a temporal intensity profile and/or temporal concentration profile of one or more molecules or compounds using the device of claim 1 , wherein the profiles identify at least one selected from the group consisting of cell type, cell state, and cell response to a biological stimuli.

20 . The method of claim 18 , wherein the cell state comprises cell signaling, cell fate, cell age, and/or cell cycle.

21 . A method of treating a disease or disorder in a subject in need thereof, wherein the treatment is cell-free, the method comprising the steps of:
a) identifying a first temporal intensity profile and/or temporal concentration profile for one or more molecules or compounds secreted by a cell that is used for treating the disease or disorder, wherein the first profiles comprise one or more biolo

Assignees

• Univ Yale
• Univ Johns Hopkins
• Univ Johns Hopkins

Inventors

• Levchenko Andre
• Gupta Kshitiz
• Ellison David D
• Suhail Yasir
• Afzal Junaid

Classifications

• G01N33/557

  using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction · CPC title

• G01N33/54366 Primary

  Apparatus specially adapted for solid-phase testing · CPC title

• G06F19/24

  Physics · mapped topic

• G01N33/56966

  Animal cells · CPC title

• G01N2570/00

  Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes · CPC title