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Assays for cell-based therapies or treatments

A cellular, therapeutic technique, applied in the field of assays for cell-based therapy or therapy, that addresses difficulties, time consuming, lack of quantitative results, etc.

Pending Publication Date: 2021-06-04
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, testing these cell-based therapies and treatments, and more generally for a wide variety of diseases, including cancer and autoimmune conditions, remains difficult because of the cost of in vivo assays in humans or model organisms. High, time-consuming and often lacks quantitative results

Method used

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  • Assays for cell-based therapies or treatments
  • Assays for cell-based therapies or treatments
  • Assays for cell-based therapies or treatments

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1 - Coated and Uncoated Substrates

[0099] Different coating agents were tested to determine suitable substrates for the methods of the present disclosure.

[0100] Opaque 96-well assay plates were uncoated or coated with poly-D-lysine or coated with human fibronectin. Plates were coated with poly-D-lysine by incubating 25 μl to 50 μl of a 200 μg / ml poly-D-lysine solution in each well for five minutes to two hours at room temperature. Alternatively, 250 μl of a 200 μg / ml poly-D-lysine solution was incubated in each well for one hour at room temperature. After incubation, the plates were washed with ddH 2 O rinse and then left to air dry for two hours. Plates were coated with human fibronectin by incubating 300 μl of a 20 μg / ml human fibronectin solution overnight at 37°C. After overnight incubation, plates were rinsed with Advanced DMEM / F12.

[0101] Human retinoblastoma (hRB) Y79 ( HTB-18 TM ) cells were added to three different assay plates. In the c...

Embodiment 2

[0102] Example 2 - Fluorescence-based cell viability assay

[0103] Fluorescence-based assays that measure cell viability were tested for use in the methods of the present disclosure.

[0104] First, the 12.5×10 3 , 2.5×10 4 , 5.0×10 4 , 1.0×10 5 and 2.0×10 5 Individual human retinoblastoma (hRB) Y79( HTB-18 TM ) cells were seeded in five separate wells of a 96-well assay plate and incubated in 50 μl of SM. After allowing the cells to settle for 30 minutes to two hours, the cells were then incubated after being thoroughly mixed with 250 μl of human retinoblastocyte conditioned medium. Then 200 μl of medium was withdrawn and 20 μl of diluted resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide sodium salt) was added to the cells at 37°C Reagents (1:4 in DPBS) lasted 1, 2, 3 or 4 hours. Resazurin is a compound that reduces to a compound called resorufin when internalized by living cells. Resorufin is highly fluorescent, meaning that the fluorescent signal produced can b...

Embodiment 3

[0106] Example 3 - Determining the potency of human retinoblastoid conditioned medium

[0107] The methods of the present disclosure were used to test the potency of human retinoblastocyte conditioned medium.

[0108] First, take a 2.5×10 4 hRB cells were seeded on the assay plate at a density of 2 cells / well. Cells were then incubated with human retinoblastocyte conditioned medium (hRPC CM) or standard medium (SM; standard human retinoblastoid medium that has not been exposed to human retinal progenitor cells). Media were not supplemented with sodium butyrate, supplemented with 2 mM sodium butyrate, 4 mM sodium butyrate or 8 mM sodium butyrate. Freshly made 200 mM sodium butyrate was added to 250 μl of medium to generate various sodium butyrate concentrations. Use 9.0×10 6 Individual human retinal progenitor cells generate hRPC CMs.

[0109] hRB cells were incubated in 300 μl of each medium for one hour or 72 hours. After incubation, use as described above Reagents to...

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Abstract

The present disclosure provides in vitro methods for determining the potency of a cell-based therapy or treatment. In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, comprising (or comprising use of) quantitative in vitro assays for determining the potency of cell-based therapies or treatments, including those used in the treatment of retinal degeneration.

Description

technical field [0001] related application [0002] This application claims priority and benefit to U.S. Provisional Application No. 62 / 737,359, filed September 27, 2018, the contents of which are incorporated herein by reference in their entirety. [0003] The present invention generally relates to cell-based assays and therapies. In alternative embodiments, there are provided compositions, including articles of manufacture and kits, for determining the efficacy of cell-based therapies or treatments, including those used to treat retinal degeneration; and methods, comprising (or comprising using) Quantitative in vitro assay. Background technique [0004] Retinal degeneration refers to degeneration or degeneration caused by progressive and irreversible decline and death of photoreceptor cells in the retina. The death of photoreceptor cells can cause blindness. Stem cells and other pluripotent cells have been considered for the treatment of patients with retinal degenerat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00C12Q1/00G01N33/50
CPCC12N5/0621C12N2502/085C12N2533/32C12N2533/52C12N2501/065G01N33/5014C12Q1/025G01N21/64C12N5/0602C12N2500/30G01N33/502G01N33/582G01N2496/00G01N2800/52
Inventor 杨静C·G·向亨利·克拉森
Owner RGT UNIV OF CALIFORNIA
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