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Use of bone marrow cells for long term culture of pancreatic islet cells

A technology of bone marrow cells and islet cells, applied in bone/connective tissue cells, pancreatic cells, tissue culture, etc., can solve problems such as difficulty in maintaining viable islets and expanding islet tissue

Inactive Publication Date: 2010-02-17
ROGER WILLIAMS GEN HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Key issues with the use of β-cell islet transplantation protocols relate to the difficulty of maintaining viable islets in vitro or in vivo and the inability to expand islet tissue in vitro

Method used

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  • Use of bone marrow cells for long term culture of pancreatic islet cells
  • Use of bone marrow cells for long term culture of pancreatic islet cells
  • Use of bone marrow cells for long term culture of pancreatic islet cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 - cell staining method

[0056] Methods of staining cells and immunofluorescence using fluorescent and chemical dyes are well known to those skilled in the art. Numerous examples include such dyeing methods. Exemplary cell staining methods are provided. Cells grown on chamber slides were fixed with 3% paraformaldehyde and then exposed to 10% normal goat serum. Slides were blotted without washing, primary antibody mix was applied, and slides were incubated overnight at 4°C in a humidified chamber. Slides were washed 3 times before exposure to secondary antibody for 45 minutes at room temperature. After washing, diluted secondary antibody was applied and slides were incubated for 15 minutes. The slides are then washed thoroughly with PBS, and the above process is optionally repeated with a second fluorescent color (eg, DAPI) and / or a third antigen detection antibody. At the end of the process, cover the slides with fluorescent mounting medium and a coversli...

Embodiment 2

[0057] Example 2 - Co-cultivation of human bone marrow and human islet β cells

[0058] Human islet tissue from normal donors was obtained from the Islet Resource Centers of the ICR Basic Science Islet Distribution Program (ICR Basic Science Islet Distribution Program, Human Islet Laboratory, University of Pennsylvania (Philadelphia, PA)). (ICR)), Joslin Diabetes Center (Boston, MA) and City of Hope National Medical Center (Duarte, CA). Use of these cells was approved by the IRB and ICR committees of Roger Williams Hospital.

[0059] Human bone marrow from normal donors was obtained after signing an appropriate informed consent form that had been approved by the Roger Williams Hospital Institutional Review Committee (IRB). Pass through Ficoll-Paque according to the manufacturer's directions TM Plus (Amersham Biosciences; Amersham, UK) isolated bone marrow mononuclear cells. Cells were then washed twice with 5% FCS / PBS and resuspended in medium (see below). Cell viability w...

Embodiment 3

[0069] Example 3 - Evaluation of islet function in an insulin release assay

[0070] Islet cell function was assessed by measuring insulin release with and without glucose challenge. Media was collected twice a week and stored at -80°C until basal insulin release was measured by ELISA.

[0071] The high glucose challenge assay was performed weekly as follows: media was collected and cultured cells were washed once with RPMI media. The medium was then replaced with high glucose (20 mM) RPMI 1640 for 15 and 30 minutes. Media was finally collected and stored at -80°C until insulin assay by ELISA.

[0072] Insulin concentrations in specimens (cell culture medium or tissue extracts) were detected using a human insulin ELISA kit (Linco Research, St. Charles, MO) according to the manufacturer's instructions. Briefly, insulin standards and appropriately diluted (1:50-1:500) samples were added to insulin antibody-coated 96-well microtiter plates and incubated at 4°C for 2 hours. Af...

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Abstract

Bone marrow cells have been demonstrated to be useful in the maintenance of pancreatic islet ss cell viability, structure, and / or function in culture for a sustained period. Bone marrow cells were also found to promote ss cell growth while reducing inflammatory cytokine release and reduce apoptosis. Moreover, islet cells co-cultured with bone marrow cells were shown to retain insulin response function and to function in an islet cell transplant in a mouse model of diabetes to restore normal insulin secretion. Cord blood cells and isolated peripheral CD34+ blood cells were unable to support ssislet cell growth or increase survival.

Description

[0001] References to related applications [0002] This application claims priority to US Provisional Patent Application Serial No. 60 / 860,637, filed November 21, 2006, which is hereby incorporated by reference in its entirety. [0003] Statement Regarding Commonwealth Supported Research [0004] This invention was made with US Government support through National Institutes of Health (NIH) grant number P20RR018757. Accordingly, the US Government has certain rights in this invention. field of invention [0005] The present invention relates generally to the field of islet cell culture. More specifically, the present invention relates to the use of bone marrow cells for the long-term culture and possible expansion of islet cells prior to transplantation. The invention also includes the use of islet cells cultured in the presence of bone marrow cells to replace human islet function in a patient in vivo. Background of the invention [0006] Diabetes is a major and growing gl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/02C12N5/06C12N5/08A61K35/12C12N5/071
CPCC12N2502/1394A61K35/12C12N5/0676A61P43/00A61P3/10
Inventor 罗履广
Owner ROGER WILLIAMS GEN HOSPITAL
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