System for separating a cell sample by centrifugation and column chromatography while maintaining sterility

Patent metadata

Field	Value
Publication number	US-9625463-B2
Application number	US-201414181023-A
Country	US
Kind code	B2
Filing date	Feb 14, 2014
Priority date	Dec 7, 2007
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

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

The invention claimed is:

1. An apparatus configured for fractionating a blood or bone marrow sample in a sterile tubing set installed on the apparatus, wherein the tubing set comprises:
a) a first connecting member configured to receive the sample and deliver the sample to a rotatable container,
b) said rotatable container installed in a centrifuge in the apparatus, wherein the rotatable container contains at least one processing chamber and is configured to rotate about an axis and thereby to apply centrifugal force to a blood or bone marrow sample in the chamber so as to separate cells from the sample according to a first cell characteristic;
c) a second connecting member configured to receive a selected subset of cells that has been separated from the sample by the rotatable container and to deliver the subset to a cell separation column;
d) said separation column installed in a column holder in the apparatus, which is configured such that cells can be delivered to the cell separation column by the second connecting member; and
e) a third connecting member configured to recover cells that have been selected by the cell separation column in the holder according to a second cell characteristic and to deliver the selected cells to a product collection container;
wherein the apparatus is configured to operate so that a sample is processed from the first connecting member of the tubing set through the rotatable container in the centrifuge and through the cell separation column in the column holder and a product thereof is delivered into the product collection container while maintaining sterility of the product throughout all the aforelisted components of the tubing set.

2. The apparatus of claim 1 , wherein the rotatable container comprises a detection window that is positioned substantially perpendicular to the axis of rotation such that a detector can monitor separation of cells in the chamber radially through and along the detection window.

3. The apparatus of claim 2 , wherein a prism or a mirror is positioned radially along the detection window.

4. The apparatus of claim 1 , wherein the first connecting member is connected to the processing chamber of the rotatable container by way of a central line that passes through rotational seals and along the axis.

5. The apparatus of claim 1 , wherein the first and the second connecting members are connected to the processing chamber of the rotatable container by way of an inner line and an outer line that both pass through rotational seals and along the axis.

6. The apparatus of claim 2 , further comprising a detector that is positioned so as to receive light that has been provided by a light source and has passed through a portion of the sample along a radial cross-section of the rotatable container.

7. The apparatus of claim 6 , configured so that upon processing of a blood sample in the rotatable container, the detector can detect an outer ring of erythrocytes and granulocytes, a ring of peripheral blood mononuclear cells, and an inner ring of platelets.

8. The apparatus 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 without stopping the centrifuge.

9. The apparatus of claim 1 , wherein the rotatable container comprises a plurality of parallel surfaces that are perpendicular to the rotational axis, wherein the surfaces are configured so that cells can be cultured upon them.

10. The apparatus of claim 1 , wherein the first characteristic is cell density.

11. The apparatus of claim 9 , wherein the first characteristic is affinity for a surface, and the rotatable container is constructed such that cells adhering to the surfaces may be separated while the chamber is rotating from cells with lesser affinity for the surfaces.

12. The apparatus of claim 1 , comprising fluid circuitry configured to distribute a plurality of different cell fractions from the rotatable container and/or the cell separation column each to a different cell collection container.

13. The apparatus of claim 1 , comprising fluid circuitry configured to pass cells multiple times over a cell separation column in the column holder.

14. The apparatus of claim 1 , wherein the cell separation column is configured to separate hematopoietic cells from the rotatable container according to a characteristic of a subset of cells in a sample, or a label that is attached selectively to a subset of cells in the sample.

15. The apparatus of claim 14 , wherein the cell separation column is a magnetic separation column configured so that cells labeled with a magnetic label will be retained by the column, and cells that are not so labeled will pass through the column.

16. The apparatus of claim 14 , wherein the cell separation column is configured with a ferromagnetic matrix that can be rendered magnetic to retain cells labeled with a magnetic particle, and then can be rendered non-magnetic to release cells that have been retained.

17. An apparatus configured for fractionating a cell sample, the apparatus comprising:
(a) a sample processing unit that includes a first holder sized and shaped to reversibly secure a rotatable container, and a drive configured to rotate such a rotatable container when secured in the first holder, thereby applying centrifugal force to a blood or bone marrow sample in the container so as to separate different cell types in the sample according to a first cell characteristic;
(b) a sample separation unit that includes a second holder sized and shaped to reversibly secure a cell separation column, the sample separation unit being configured to operate such a cell separation column secured in the second holder so as to separate different cell types according to a second cell characteristic;
wherein the apparatus is configured to be operatively installed with a sterile tubing set that comprises:
(i) a first connector configured for sterile connection of a sample container;
(ii) a rotatable container that operatively fits in the sample processing unit;
(iii) a first inlet port configured to deliver a first cell population into the rotatable container for separation;
(iv) a first outlet port configured to receive a selected subset of cells from the first cell population that have been separated by the rotatable container;
(v) a second inlet port configured to deliver a second cell population onto a cell separation column secured in the second holder;
(vi) said cell separation column which operatively fits in the sample separation unit;
(vii) a second outlet port configured to receive a subset of cells that has been selected from the second cell population by the cell separation column; and
(viii) a second connector configured for sterile connection to a product collection container;
so that when a cell sample is supplied into the tubing set through the first connector, the sample can be processed by the apparatus in the rotatable container secured in the first holder and processed by the apparatus in the cell separation column secured in the second holder, following which a product therefrom can be delivered by the apparatus though the second connector into a product collection container, whereby cells in the product have been kept sterile during processing in the sterile tubing set.

18. A system comprising the apparatus of claim 17 installed with the sterile tubing set such that the rotatable container is secured by and connected to the first holder and the ce

Assignees

• Miltenyi Biotec Gmbh

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