Preparing antigen-specific T cells using a self-enclosed processing system that contains both a centrifuge and a magnetic separation column

Patent metadata

Field	Value
Publication number	US-10620212-B2
Application number	US-201715706517-A
Country	US
Kind code	B2
Filing date	Sep 15, 2017
Priority date	Dec 7, 2007
Publication date	Apr 14, 2020
Grant date	Apr 14, 2020

Abstract

The invention relates to a system, comprising: a) a sample processing unit, comprising an input port and an output port coupled to a rotating container having at least one sample chamber, the sample processing unit configured provide a first processing step to a sample or to rotate the container so as to apply a centrifugal force to a sample deposited in the chamber and separate at least a first component and a second component of the deposited sample; and b) a sample separation unit coupled to the output port of the sample processing unit, the cell separation unit comprising separation column holder (42), a pump (64) and a plurality of valves (1-11) configured to at least partially control fluid flow through a fluid circuitry and a separation column (40) positioned in the holder, the separation column configured to separate labeled and unlabeled components of sample flowed through the column.

First claim

What is claimed is:

1. A method of enriching a defined population of target cells from a mixed population of blood cells using an apparatus that is configured for processing such cells using an interconnected assembly of disposable cell separation elements (optionally referred to as a “tubing set”);
wherein the interconnected assembly comprises:
(a) a first connecting member configured to receive and deliver the sample to a rotatable container,
(b) said rotatable container, which is configured to rotate about an axis and thereby to apply centrifugal force to a sample contained in a processing chamber in the container so as to separate cells from the sample from other components of the sample;
(c) a second connecting member configured to transfer cells processed by the rotatable container to a cell separation column;
(d) said separation column, which is configured to separate labeled cells from unlabeled cells; and
(e) a third connecting member configured to transfer cells separated by the cell separation column to a product collection container;
wherein the method comprises:
(1) delivering the sample through the first connecting member into the interconnected assembly; and
(2) operating the apparatus such that the sample is processed by the rotatable container and by the separation column in the interconnected assembly whereby the target cells are delivered into the product collection container while maintaining sterility of the target cells throughout the aforelisted components of the assembly.

2. The method of claim 1 , wherein the mixed population of cells has been obtained from human whole blood, apheresis harvest, or bone marrow.

3. The method of claim 1 , wherein the target cells delivered into the product collection container are T cells.

4. The method of claim 1 , wherein the target cells delivered into the product collection container are antigen-specific T cells.

5. The method of claim 1 , whereby the rotatable container comprises an inlet and an outlet port configured for exchange of air or gas inside the chamber so as to support culturing of cells in the chamber under sterile conditions.

6. The method of claim 4 , comprising culturing cells from the mixed population in the chamber with a particular antigen so as to re-stimulate T cells that are specific for the antigen.

7. The method of claim 4 , comprising incubating cells from the mixed population in the chamber for release of cytokines.

8. The method of claim 3 , comprising genetically altering the T cells in the interconnected assembly before delivering them to the product collection container.

9. The method of claim 1 , comprising washing and adjusting the volume of a suspension of cells from the sample by centrifugation in the rotatable container.

10. The method of claim 1 , comprising specifically labeling a proportion of cells from the sample with magnetic particles before delivering cells from the sample to the cell separation column.

11. The method of claim 10 , comprising separating cells labeled with the magnetic particles from cells not labeled with the magnetic particles using the separation column.

12. The method of claim 1 , comprising eluting the target cells from the separation column and delivering the target cells into the product container in a small volume suitable for injection into a subject in need thereof.

13. The method of claim 1 , wherein the target cells are stem cells or progenitor grafts, and the method includes eluting CD34 or CD133 positive cells from the cell separation column.

14. The method of claim 1 , wherein the target cells are dendritic cells, and the method includes eluting CD14, CD304, or CD1c positive cells from the cell separation column.

15. The method of claim 1 , wherein the target cells are natural killer cells, and the method includes depleting CD3 positive cells and enriching CD56 positive cells from the mixed population.

16. The method of claim 1 , wherein the target cells delivered to the product container are characterized as being enriched for cells having surface markers CD34 or CD133 or as being depleted for CD3, CD3 and CD19, CD6, CD4 and CD8, T cell receptor alpha and beta, or CD3, CD19, CD16, and CD14.

17. The method of claim 1 , wherein the apparatus comprises:
a sample processing unit that includes a first holder that is sized and shaped to reversibly secure the rotatable container of the interconnected assembly, and a drive configured to rotate the rotatable container when secured in the first holder, thereby applying centrifugal force to a mixed population of cells in the container so as to separate different cell types in the mixed cell population; and
a sample separation unit that includes a second holder that is sized and shaped to reversibly secure the cell separation column of the interconnected assembly, the sample separation unit being configured to operate the cell separation column when secured in the second holder so as to separate cells labeled with magnetic particles from cells not labeled with magnetic particles.

18. A method of preparing T cells specific for a particular antigen from a sample of human whole blood or apheresis harvest using an apparatus that is configured for processing such cells using an interconnected assembly of disposable cell separation elements;
wherein the interconnected assembly comprises:
(a) a first connecting member configured to receive and deliver the sample to a rotatable container,
(b) said rotatable container, which is configured to support culturing of cells in a processing chamber in the container, and is also configured to rotate about an axis, thereby applying centrifugal force to a sample in the chamber so as to separate cells from the sample from other components of the sample;
(c) a second connecting member configured to transfer cells processed by the rotatable container to a cell separation column;
(d) said separation column, which is configured to separate cells labeled with magnetic particles from cells not labeled with magnetic particles; and
(e) a third connecting member configured to transfer cells separated by the cell separation column to a product collection container;
wherein the method comprises:
(1) delivering the sample through the first connecting member into the interconnected assembly; and
(2) operating the apparatus such that the sample is processed by the rotatable container and by the separation column in the interconnected assembly whereby said antigen-specific T cells are delivered into the product collection container while maintaining sterility of the T cells throughout the processing.

19. The method of claim 18 , which comprises:
delivering the sample through the first connecting member into the interconnected assembly along with said antigen and a media supplement;
culturing cell from the sample with the antigen in the chamber so as to stimulate said antigen specific T cells;
incubating cells in the chamber for release of cytokines;
magnetically labelling said antigen specific T cells; and
enriching the magnetically labeled T cells in the cell separation column.

20. The method of claim 18 , which comprises:
installing said interconnected assembly on the apparatus so that both the rotatable container and the cell separation column are operable by the apparatus;
delivering a sample through the first connecting member into the rotatable container, causing the rotatable container to rotate, thereby applying centrifugal force to the sample so as to separate different cell types from the sa

Assignees

• Miltenyi Biotec Gmbh
• Miltenyi Biotec Bv & Co Kg

Inventors

• Miltenyi Stefan
• Schimmelpfennig Winfried
• Lantow Holger
• Neuschaefer Niklas Elmar
• Biehl Martin
• Kabaha Eiad
• Schulz Juergen

Classifications

• C12N2501/998

  Proteins not provided for elsewhere · CPC title

• C12N5/0018

  Culture media for cell or tissue culture (media for specific animal cell type C12N5/06) · CPC title

• B01L3/5021

  Test tubes specially adapted for centrifugation purposes · CPC title

• C12N2500/34

  Sugars · CPC title

• B01D21/262

  by using a centrifuge · CPC title