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Single cell-derived organoids

An organoid, cell technology, applied in the field of cell culture media and kits, in vitro prostate organoids, and cell culture media supplemented with bovine pituitary extract and epinephrine, capable of addressing the inability to mimic the complexity of the prostate cancer microenvironment or reproduce therapy resistance, etc.

Active Publication Date: 2017-04-19
RUTGERS THE STATE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, cell lines grown in monolayers and genetically engineered mouse models cannot mimic the complexity of the prostate cancer microenvironment or reproduce the multiple mechanisms of therapy resistance

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Organoids containing epithelial and mesenchymal tissues were prepared from single primary prostate cancer cells. Using Matrigel with hypoxic conditions TM The chamber and 1-2 weeks in the first step use growth factors that promote self-renewal and factors that induce differentiation at the same time to enrich the stem cell population and induce differentiation. The concentration of growth factors and the timing of growth factor addition determine the commitment of cells to self-renewal or cell differentiation programs. The second step involves additional growth factors that promote self-renewal. Within 14 days of culture in the second step, single-cell-derived organoid structures were formed from more than 14 patient-derived samples.

[0045] Specifically, surgically resected tissue is subjected to mechanical dissociation, collagenase treatment, filtration, and then propagation in differentiation medium for 1-2 weeks. After the "differentiation period", cells are har...

Embodiment 2

[0049] Prostate cancer tissue was isolated from prostatectomy specimens according to Institutional Review Board-approved methods. Deidentified tissue was obtained within 15 minutes of surgery, dissociated and treated in Matrigel simulating the basal layer of a normal prostate using the conditions described in Example 1 above. TM Use this tissue in the 3D droplet chamber to prepare organoids. This process produced multiple prostate organoids that survived for several months and could be driven again to regenerate a range of organoids. Using the conditions described in Example 1 above, organoids were obtained from 21 of 24 attempted prostate cancer samples with an efficiency of approximately 87%. Tissue was from high-risk prostate cancer using prostatectomy tissue or lymph node biopsy and normal adjacent tissue (NAT) cells.

[0050] To demonstrate the result that organoids are derived from single stem cell clones rather than cell aggregates, patient-derived prostate cancer cel...

Embodiment 3

[0055] The following studies were performed to determine whether stromal cell co-cultures could enhance cell growth, migration, and invasion of prostate cancer cells grown as 3D organoids compared to single tumor cell 3D cultures. Epithelial and stromal cells were labeled with different lentiviruses expressing luciferase and a fluorescent reporter. The effect of monolayer co-culture of EGFP-labeled prostate cancer cells and normal bone marrow-derived stromal cells labeled with mCherry on the morphology, migration and proliferation of tumor cells was studied. In parallel, the effect of 3D co-culture of labeled prostate cancer organoids derived from tumor-initiating cells with bone marrow-derived stromal cells labeled with mCherry on the same parameters used for monolayer culture studies was investigated. Cell viability analysis showed that 3D co-culture increased proliferation and cell viability approximately 3-fold compared to monoculture controls, suggesting that 3D cells are...

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PUM

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Abstract

The present invention relates to organoids derived from a single cell, such as a prostate cancer cell, and methods and compositions relating to the production and use thereof, including cell culture medium for producing organoids and methods of personalized treatment for prostate cancer. The invention further provides a humanized mouse comprising a prostate organoid derived from a patient's prostate cell.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 62 / 015,020, filed June 20, 2014, the disclosure of which is incorporated herein by reference in its entirety. [0003] Background of the invention [0004] The inability to proliferate primary tissues constitutes a major obstacle to understanding the mechanisms of regeneration and the balance of differentiated and stem cells in adult organisms. There is a need for a better understanding of the development of primary human pathological conditions such as tumors. For cancer research, current cancer models do not adequately represent the molecular and cellular diversity of human cancers. Existing human cancer cell lines lack clear and detailed information on the clinical manifestations of cancer and have inherent limitations in explaining mechanisms of therapy resistance. Therefore, new methods for maintaining cancer nascent tissue and novel drug develop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0683C12N2500/25C12N2500/84C12N2501/11C12N2501/115C12N2501/39C12N2501/395C12N2501/81C12N2503/02C12N2513/00C12N5/0693A01K67/0278A01K2207/12A01K2207/15A01K2227/105A01K2267/0331C12N2500/38C12N2502/13C12N2506/03G01N33/5011G01N33/5082
Inventor H·萨巴韦M·巴图奇
Owner RUTGERS THE STATE UNIV
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