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Method for Separating Cells

a cell and cell technology, applied in the field of cell separation methods, can solve the problems of multiple steps, incurring cell loss, limited detection ability of metastatic cells, etc., and achieve the effect of increasing recovery and more effective cell separation

Inactive Publication Date: 2008-05-08
STEMCELL TECHNOLOGIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention describes a method for separating different populations of cells from a mixed cell suspension using dense particles and discontinuous density gradients. This method allows for more effective cell separation without affecting the pelleting of the targeted cells. The method involves linking dense particles to the first population of cells and layering the sample over a density separation medium with a higher density than the second population of cells. The cells then settle to the interface between the density separation medium and the sample, resulting in the isolation of each population of cells. The method can be used for both positive and negative selection of cells. Centrifugation can accelerate the settling of cells. The invention also includes kits for performing the methods described.

Problems solved by technology

Furthermore, serious complications and indeed the success of a transplant procedure is to a large degree dependent on the effectiveness of the procedures that are used for the removal of cells in the transplant that pose a risk to the transplant recipient.
The ability to detect such metastatic cells is limited by the effectiveness of tissue or fluid sampling and the sensitivity of tumor detection methods.
These multiple steps require time and incur cell loss.
All density separation techniques have the same basic limitation; they can not separate subpopulations of cells with overlapping density distributions such as human lymphocyte subsets.
Simple density separation techniques do not offer the high cell specificity offered by antibody-mediated techniques.

Method used

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  • Method for Separating Cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Density Separation Media (DSM)

[0056] Density separation media (DSM) were prepared at different densities by mixing hetastarch, iodixanol and water in different proportions such that the desired density was obtained and the osmolarity was between 270 and 300 mOsm. The hetastarch and iodixanol components of this DSM serve the same function as the polysaccharide and metrizoate components respectively in both Ficoll-Paque® and Lymphoprep®.

example 2

Method to Negatively Select Cells from Whole Human Peripheral Blood Using the Method of the Invention with Red Blood Cells as Dense Particles

Preparation of Tetramers

[0057] In order to prepare a tetrameric antibody complex for use in the method of the present invention, the following protocol may be used: (a) take 1 mg of antibody specific for cells to be bound for red blood cells (e.g. anti-CD2, CD3, CD4, CD8, CD14, CD16, CD19 etc.); (b) add 3 mg anti-Glycophorin A antibody (against red blood cells); mix well (c) then add 4.0 mg of P9 antibody or 2.72 mg of the P9 F(ab′)2 antibody fragment. Incubate overnight at 37° C. The P9 antibody binds the Fc portion of the antibodies added in steps (a) and (b) resulting in a tetrameric antibody complex. For more information on the preparation of tetramers see U.S. Pat. No. 4,868,109 to Lansdorp, which is incorporated herein by reference. Tetrameric antibody complexes incorporating different antibodies to antigens expressed on nucleated cell...

example 3

Method to Positively Select Cells from Whole Human Peripheral Blood Using the Method of the Invention with Red Blood Cells as Dense Particles

Preparation of Tetramers

[0067] Tetrameric antibody complexes were prepared as described in Example 2. The antibody compositions are one or more types of tetrameric antibody complexes depending on which cells one wishes to select.

Separation Method

[0068] A positive selection protocol for separating cells from peripheral blood is set out below. [0069] 1. Add 100 μL antibody composition per mL of peripheral blood. [0070] 2. Incubate 20 minutes at room temperature. [0071] 3. Dilute sample with an equal volume of phosphate buffered saline (PBS)+2% fetal calf serum (FCS) and mix gently. [0072] 4. Layer the diluted sample on top of the DSM. [0073] 5. Centrifuge for 20 minutes at 1200×g, room temperature, with the brake off. [0074] 6. Remove the desired cells from the pellet. [0075] 7. Wash desired cells with 5-10× volume of PBS+2% FCS. [0076] 8. ...

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Abstract

The present invention is related to a method for separating a first population of cells from a second population of cells in a sample by discontinuous density gradient separation using dense particles to target the first population of cells and a density separation medium (DSM) that is at least about 0.001 g / cm3 higher than the density of the second population of cells.

Description

FIELD OF THE INVENTION [0001] The present invention relates to methods for separating cells. In particular the invention relates to methods for separating cells using dense particles and discontinuous density gradient centrifugation. BACKGROUND OF THE INVENTION [0002] In many applications it is desirable to enrich, or alternatively deplete, certain cell populations in a biological sample. For example, the separation of specific cell types from peripheral blood, bone marrow, spleen, thymus and fetal liver is key to research in the fields of haematology, immunology and oncology, as well as diagnostics and therapy for certain malignancies and immune disorders. [0003] Most cell separation techniques require that the input sample be a single cell suspension. For this reason, blood has historically been the most common tissue used for cell separations. Purified populations of immune cells such as T cells and antigen presenting cells are necessary for the study of immune function and are u...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01N1/02C12N5/00C12N5/02C12N5/06C12N5/08
CPCG01N35/00Y10S436/824Y10T436/25Y10T436/25375Y10S435/803
Inventor WOODSIDE, STEVEN M.
Owner STEMCELL TECHNOLOGIES
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