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Therapeutic lung repair by inhalation of lung spheroid cell-secreted factors

A technology of cell secretion and spheres, which is applied in the direction of respiratory/lung cells, animal cells, vertebrate cells, etc., and can solve problems of labor, high cost, and large quantities

Pending Publication Date: 2021-02-26
NORTH CAROLINA STATE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although stem cells have beneficial effects, their clinical application faces many challenges, including labor-intensive, high cost, and safety concerns
Cell-based therapies have certain tumorigenicity and immunogenicity risks, as well as cell stability issues, as both stem and progenitor cells have an inherent risk of transformation during long-term in vitro cell culture

Method used

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  • Therapeutic lung repair by inhalation of lung spheroid cell-secreted factors
  • Therapeutic lung repair by inhalation of lung spheroid cell-secreted factors
  • Therapeutic lung repair by inhalation of lung spheroid cell-secreted factors

Examples

Experimental program
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Effect test

Embodiment 1

[0236] Cell Culture: Human LSCs were generated from whole lung samples and expanded as described by Dinh et al., (2017) RespiratoryResearch. Human LSCs were prepared from healthy whole lung donors and expanded as described in Figure 5. Passage 2-5 LSCs were used for all in vitro and in vivo experiments. Cells were also analyzed by flow cytometry for appropriate markers (CD90+, CD105+, CCSP+, AQP5+, ProSPC+, Epcam+, CD31-, CD34- and CD45-) before use.

Embodiment 2

[0238] Collection and preparation of conditioned media: Human LSCs and MSCs were cultured to approximately 75% confluency before serum-containing media was removed and replaced with serum-free media (IMDM). The next day, cells were washed 6 times with fresh IMDM for 30 min each to deplete albumin on cells prior to media conditioning, as albumin interferes with some experiments (especially LC / MS / MS analyze). The medium (IMDM) was allowed to condition for three days before it was harvested and filtered through a 0.22 μm filter to remove any cells and cell debris. Store filtered CM at -80 °C for at least 24 h, or until solid. Freeze dry the frozen CM vials overnight or until the samples are dehydrated using the LABCONCO FreeZone 2.5 L Freeze Drying System. Once samples were lyophilized, they were stored at -20°C until ready to use.

Embodiment 3

[0240] Isolation and Characterization of Exosomes: Exosomes were collected and purified from human LSC-CMs by ultrafiltration. LSC-CMs are first filtered through a 0.22 μm filter to remove any cells or cell debris. The filtered medium was then placed on a 100 kDa MWCO ultrafiltration filter (Millipore) and centrifuged at 5000 RCF for 10-15 minutes depending on the volume. Any media content or small proteins were removed by filter centrifugation, and the remaining exosomes were suspended in PBS, then filtered and washed. Before use, all exosome samples were size-appropriately analyzed by nanoparticle tracking analysis (NTA; NanoSight, Malvern) and morphologically analyzed by transmission electron microscopy (TEM). Furthermore, successful exosome isolation was confirmed by immunoblotting for known exosomal markers (CD63 and CD81).

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Abstract

Idiopathic pulmonary fibrosis (IPF) is currently the most fatal form of idiopathic interstitial lung disease in which persistent lung injuries result in scar tissue formation. Provided are methods andcompositions for the treatment of pulmonary conditions such as fibrosis. Lung spheroid cell-derived conditioned media (LSC-CM) and exosomes (LSC-EXO) are used to treat different models of lung injury. Inhalation treatment with LSC-CM and LSC-EXO-derived compositions can attenuate and / or reverse bleomycin- and silica-induced fibrosis, reestablish normal alveolar structure and decrease extracellular matrix accumulation.

Description

[0001] Cross References to Related Applications [0002] This application claims priority and benefit to U.S. Provisional Application 62 / 691,811, filed June 29, 2018, entitled "Therapeutic Lung Repair by Inhalation of Cell-Secreted Factors from Lung Spheroids," the entire disclosure of which is available through This reference is incorporated herein in its entirety. technical field [0003] The present disclosure generally relates to methods of treating pathological conditions of the lung responsive to a stem cell-derived secretome or portion thereof. Background technique [0004] The entire human body and all its organs are covered with a protective layer of epithelial cells. Epithelial cells in the lungs not only facilitate oxygen exchange, but also protect against continuous daily exposure to inhaled irritants and toxins. Fortunately, the lung has the capacity to undergo facultative regeneration due to the resident population of stem and progenitor cells that is critica...

Claims

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Application Information

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IPC IPC(8): A61K35/42A61K9/72A61K9/19C12N5/071A61P11/00
CPCA61K35/42A61K38/00A61K9/0075A61K9/19C12N2513/00C12N5/0689A61K31/713A61P11/00C12N2310/141C12N2330/10A61K31/7105
Inventor 丁芳渊程珂
Owner NORTH CAROLINA STATE UNIV
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