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Ex vivo generation of functional leukemia cells in a three-dimensional bioreactor

A leukemia cell and leukemia technology, applied in the field of cell culture, can solve the problems of lack of cell culture system

Inactive Publication Date: 2004-01-28
UNIVERSITY OF ROCHESTER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0021] A consistent cell culture system for culturing leukemia cells has been lacking

Method used

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  • Ex vivo generation of functional leukemia cells in a three-dimensional bioreactor
  • Ex vivo generation of functional leukemia cells in a three-dimensional bioreactor
  • Ex vivo generation of functional leukemia cells in a three-dimensional bioreactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 - Preparation of bioreactor

[0085] Bioreactors were fabricated with polycarbonate plates ( Figure 1A ). The culture chamber (3 / 16" high x 5 / 16" wide x 5 / 16" long) is filled with 0.01 g of porous microcarriers. The microcarrier packing layer is covered with medium. The medium chamber (1 / 2" high x 5 / 16" "W x 12 / 16" L) contained 0.6 ml of medium. Use Teflon TM The membrane (50 μm thick) facilitates gas exchange.

[0086] Use Cellsnow throughout the experiment TM -EX, L-type (low ionic charge) macroporous cellulose microcarriers (Kirin, Japan; 1-2 mm in diameter; 100-200 μm pore size; 95% porosity) were used as artificial scaffolds for human bone marrow cells. ( Figure 1B )

Embodiment 2

[0087] Embodiment 2-human bone marrow preparation

[0088] Bone marrow was aspirated from the iliac crest of volunteers, diluted 1:1 with McCoy's 5A medium (Gibco, Grand Island, New York), and plated onto Ficol / Paque according to the instructions of the University of Rochester Research Project Review Committee. (Pharmacia, Piscataway, New York, density 1.027 g / ml) and centrifuged at 200 g for 30 minutes. The mononuclear cell layer was collected, washed three times, and used to seed into bioreactors. Set aside a portion of cells as needed for various analyses.

Embodiment 3- 3

[0089] Example 3 - Three-dimensional long-term human bone marrow culture

[0090] Pipette an appropriate amount (4-6×10 6 cells) were transferred to the porous microcarrier section of the bioreactor to inoculate the culture. cultures in humid CO 2 incubator (with 5% CO 2 ) were cultured at 37°C. The LTBMC medium (replaced daily) was made of 70% (v / v) McCoy's 5A medium (Gibco), 1×10 -6M hydrocortisone (Sigma, St. Louis, MO), 50 μ / ml penicillin (Sigma), 50 mg / ml streptomycin (Sigma), 0.2 mM L-glutamine (Gibco), 0.045% carbonic acid sodium hydrogen (Sigma), 1xMEM sodium pyruvate (Gibco), 1xMEM vitamin solution (Gibco), 0.4xMEM amino acid solution (Gibco), 12.5% ​​(v / v) heat-inactivated horse serum (Gibco), and 12.5% ​​heat-inactivated Live FBS (Gibco) composition. For the first two weeks, the culture was grown with complete medium every two days. After the second week, the culture was depleted (50 microliters / well) by gently agitating and mixing the porous microsphere laye...

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Abstract

The present invention provides cultured leukemia cells. The method comprises isolating mononuclear cells, which contain leukemia cells, culturing the leukemia cells in a chamber having a scaffolding covered or surrounded with culture medium, where the scaffolding allows for leukemia cells to have cell to cell contacts in three dimensions. The subject leukemia cells are useful for screening compounds which inhibit or stimulate leukemia cell function or formation.

Description

[0001] This application is based on provisional application serial no. 60 / 186,564 filed March 2, 2000. [0002] The invention described herein was made with US Government support under National Science Foundation Contract No. BES-963160 and is therefore subject to certain US Government rights. field of invention [0003] The present invention relates to the field of cell culture, and more particularly to methods and compositions for culturing leukemia cells. Background of the invention [0004] Leukemia is a form of cancer that starts in blood-making cells in the bone marrow—the soft central part of bone. Leukemia—literally the Greek word for "white blood"—occurs when there is an excess of abnormal white blood cells in the blood. These cells, called leukocytes, are so abundant in some individuals that the blood does have a whitish tinge. [0005] Normally, blood-forming cells in the bone marrow make white blood cells to protect the body against infection by organisms like ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N5/09G01N33/50
CPCC12N2501/23G01N33/5011C12N2501/39C12N2501/22C12N2533/76C12N5/0694C12N2533/78C12N2531/00C12N2503/00
Inventor J·H·D·吴A·曼塔拉里斯N·帕诺斯卡尔蒂斯
Owner UNIVERSITY OF ROCHESTER