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Macrophage-based therapy

A technology of macrophages and cells, applied in the field of autologous macrophages and non-polarized human macrophages, which can solve the problems of macrophage function and interaction complexity

Pending Publication Date: 2021-01-12
THE UNIV COURT OF THE UNIV OF EDINBURGH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Many macrophage-directed therapeutic approaches for liver diseases have been reported (e.g. reviewed in Tacke et al. J. Hepatology 2017;66:1300-1312), but due to the complexity, they face various challenges

Method used

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  • Macrophage-based therapy
  • Macrophage-based therapy
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116]Example 1 Optimization of GMP-compliant monocyte culture

[0117]Isolate cells from healthy donors

[0118]The donor buffy coat, which is a source of healthy donor monocytes, is provided by the National Blood Transfusion Service of Scotland (SNBTS) Blood Donation Centre (Edinburgh, UK) under SNBTS Sample Management 13-12 and 14-02. Choose from CD14 in PBMC: Use Histopaque 1077 (Sigma) to separate PBMC from normal donor buffy coat by density centrifugation. After washing, CD14+ monocytes were separated from the mononuclear cell fraction using CliniMACS GMP grade CD14 microbeads and LS separation magnetic column (Miltenyi Biotec). Briefly, resuspend the cells in PEA buffer (phosphate buffered saline [PBS] plus 2.5 mmol / L ethylenediaminetetraacetic acid [EDTA] and human serum albumin [0.5% final volume of Alburex 20%, Octopharma]) Medium to the appropriate concentration, incubate with CliniMACS CD14 beads according to the manufacturer's instructions, then wash and pass through a magnet...

Embodiment 2

[0132]Example 2 Extended function analysis: healthy donor macrophages

[0133]Phagocytic uptake by monocytes and macrophages

[0134]The functional characterization of normal macrophages investigated the ability of cells to absorb particles. The phagocytic uptake of monocytes and macrophages from buffy coat CD14 cells was tested using pHRodo beads that fluoresce only when entering acidic endosomes. In short, monocytes or macrophages were cultured with 1-2 uL of pHRodo E. coli bioparticles (Life Technologies, Thermo Fisher) for 1 h, and then the medium was removed and the cells were washed to remove unphagocytosed particles. An EVOS microscope (Thermo Fisher) was used to assess phagocytosis, and ImageJ software (NIH free software, https: / / imagej.nih.gov / ij) was used to capture images and quantify the cellular uptake of the beads. The use of pHRodo beads in these studies can give an accurate assessment because the beads are transparent before being swallowed and will fluoresce once exposed ...

Embodiment 3

[0139]Example 3 Process Verification of Patients with Cirrhosis

[0140]Culture monocytes from patient samples

[0141]The cultured monocytes from Prodigy separation and leukopenia were measured at 2×10 per square centimeter and per milliliter.6One monocyte was cultured in a culture bag (MACS GMP differentiation bag, Miltenyi) with GMP grade TexMACS (Miltenyi) and 100 ng / mL M-CSF. Monocytes were cultured with 100ng / mL GMP-compliant recombinant human M-CSF (R&D Systems). The cells were cultured in a humidified atmosphere with 5% CO2 at 37°C for 7 days. During the culture period (day 2 and day 4), 50% volume of medium was supplemented twice, 50% of the medium was removed, and then fresh medium supplemented with 200ng / mL M-CSF (with Restore the final concentration to 100ng / mL).

[0142]Process verification

[0143]The cell culture and identification data are used to design the GMP process and then validate it. A set of markers was selected from an extensive phenotypic analysis group to be used as ...

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Abstract

The present invention relate to autologous isolated unpolarized human macrophages for use in the treatment of liver disease and macrophages for use in a method of treating fibrosis in a human in needthereof.

Description

Background technique[0001]Cirrhosis of the liver is a major health problem in the Western world. In the United Kingdom, liver disease is the fifth leading cause of death, and it is estimated that more than 1 million people died of cirrhosis in 2010. Due to progressive tissue damage, fibrotic scars, and loss of liver function, the disease is associated with a high incidence, and the only treatment option for end-stage disease is liver transplantation. However, the availability of donor organs cannot meet the demand, and patients with end-stage liver disease are usually not eligible for transplantation. Those who receive transplants require lifelong immunosuppression, which increases health risks. In 2015, it was reported that 611 patients in the UK were included in the active liver transplant list. Patients may have to wait up to two years to receive a liver transplant. One year after registration, 17% of patients are still waiting on the liver transplant list. Similarly, in the Unit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/28A61P1/16
CPCA61K35/28A61P1/16A61K39/464A61K2239/38C12N5/0645A61K39/4622A61K39/4614A61K9/0019C12N2501/22C12N2506/02C12N2506/115C12N2506/1353C12N2506/45
Inventor 斯图尔特·福布斯约翰·坎贝尔尼尔·W·A·麦高文阿拉斯代尔·R·弗雷泽
Owner THE UNIV COURT OF THE UNIV OF EDINBURGH
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