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Integrated functional and molecular profiling of cells

A technology of cells and cell groups, applied in the direction of receptors/cell surface antigens/cell surface determinants, analysis materials, cancer antigen components, etc., can solve problems such as lack of integration capabilities

Pending Publication Date: 2018-02-06
UNIV HOUSTON SYST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Existing approaches to study cellular activity lack the ability to integrate dynamic cellular and molecular behavior at the single-cell level

Method used

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  • Integrated functional and molecular profiling of cells
  • Integrated functional and molecular profiling of cells
  • Integrated functional and molecular profiling of cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] Example 1. Individual Cell-Cell Detection by High-Throughput Nanowell In-Grid Time-Lapse Imaging Microscopy (TIMING) Automatic overview of the interactions

[0163] In this example, fluorescently labeled human T cells, natural killer cells (NK), and various target cells (NALM6, K562, EL4) were placed in PDMS arrays with sub-nanoliter pores (nanopores) co-incubated and detected by multi-channel time-lapse microscopy. A novel cell segmentation and tracking algorithm that takes into account cell variability and nanopore limitations increases the yield of correctly analyzed nanopores from 45% (current algorithm) to 98% for nanopores containing effectors and single targets. This enables reliable automated quantification of cell location, morphology, movement, interaction, and death. Automated analysis of recordings from 12 different experiments demonstrated that, with default parameters, automated nanowell delineation was more than 99% accurate despite differences in il...

Embodiment 11

[0176] Example 1.1. Sample preparation and imaging

[0177] The TIMING dataset was derived from an ongoing study in which human T cells (genetically engineered to express a chimeric antigen receptor CAR) and natural killer (NK) cells were used as effectors. Human leukemia cell lines NALM6, K562 or mouse EL4 cells expressing appropriate ligands were used as targets (T).

[0178] Both cell types were washed once in serum-free medium, suspended to approximately 2 million / mL and labeled with PKH67 green and PKH26 red dyes, respectively, according to the manufacturer's (Sigma-Aldrich) instructions. Approximately 100,000 effector (E) cells were loaded onto the nanowell array, followed by approximately 200,000 target cells. Cells were allowed to settle within the nanotubes for 5 minutes, then excess cells were washed away.

[0179] Next, 50 μL of annexin V-Alexa Fluor 647 (AnnV-AF647, Life Technologies (Life Technologies)) in 3 mL of complete medium (PMI-1640+10% FBS, excluding p...

Embodiment 12

[0181] Example 1.2. Automatic positioning of nanopores

[0182] Automated positioning of nanowells is preferably used to delineate cell-confined regions, correct for stage repositioning errors, and decompose the entire TIMING dataset into a large number of motion-corrected video sequences, one for each nanowell. Preferably, this manipulation is reliable, since a single well-detection error can render a nanowell unusable for analysis, reducing experimental yield. Preferably, the manipulation must be process robust against focus drift (taking into account shrinkage / expansion / irregularities of the polymer matrix), holes with compromised geometry, variations in illumination, ringing artifacts, and debris or air bubbles possible will be displaced, appearing / disappearing abruptly in time from the camera's field of view ( Figure 4A ).

[0183] Content-independent image registration methods like SIFT are neither sufficiently reliable nor practical for TIMING data. They require t...

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Abstract

Presented herein are methods of evaluating cellular activity by: placing a cell population on an area; assaying for a dynamic behavior of the cell population as a function of time; identifying cell(s)of interest based on the dynamic behavior; characterizing a molecular profile of the cell(s); and correlating the obtained information. The assayed dynamic behavior can include cellular activation, cellular inhibition, cellular interaction, protein expression, protein secretion, cellular proliferation, changes in cellular morphology, motility, cell death, cell cytotoxicity, cell lysis, and combinations thereof. Sensors associated with the area may be utilized to facilitate assaying. Molecular profiles of the cell(s) can then be characterized by various methods, such as DNA analysis, RNA analysis, and protein analysis. The dynamic behavior and molecular profile can then be correlated for various purposes, such as predicting clinical outcome of a treatment, screening cells, facilitating a treatment, diagnosing a disease, and monitoring cellular activity.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 138,813, filed March 26, 2015, and U.S. Provisional Patent Application No. 62 / 157,174, filed May 5, 2015. The entire contents of each of the aforementioned applications are hereby incorporated by reference. [0003] Statement Regarding Federally Funded Research [0004] This invention was made with government support under RO1 grant number CA174385 awarded by the National Institutes of Health (NIH) and a grant awarded by the Cancer Prevention and Research Institute of Texas (CPRIT) Grant No. RP130570 was completed. The government has certain rights in this invention. Background technique [0005] Existing methods for studying cellular activity lack the ability to integrate dynamic cellular and molecular behaviors at the single-cell level. The present disclosure addresses the above-mentioned deficiencies in the art. Contents of the inv...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/569
CPCC12Q1/68G01N33/56966A61K39/464838A61K39/4631A61K39/464412A61K39/4611G01N33/5005G01N2570/00C07K2319/03C07K14/7051C12Q1/6869
Inventor N·瓦拉达拉加G·罗曼I·利亚蒂V·G·桑德拉B·罗伊桑
Owner UNIV HOUSTON SYST
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