Multisort cell separation method

Patent metadata

Field	Value
Publication number	US-2016186165-A1
Application number	US-201514944991-A
Country	US
Kind code	A1
Filing date	Nov 18, 2015
Priority date	Dec 27, 2014
Publication date	Jun 30, 2016
Grant date	—

Abstract

The invention is directed to a method for enriching target cells from a sample of cells characterized by:
a) contacting the sample with a cell aggregation agent and first magnetic particles having an iron content of 0.1 pg to 5000 pg, coupled to a first antigen recognizing moiety; and second magnetic particles having an iron content of 0.05 fg to 100 fg and coupled to a second antigen recognizing moiety to obtain mixture a) b) applying a first magnetic field gradient to the mixture a) thereby removing the cells bound to the first antigen recognizing moiety coupled to the first magnetic particles, to obtain a mixture b) and obtaining an agglomerate comprising the cells of mixture a) bound to the cell aggregation agent c) applying a second magnetic field gradient to the mixture b) thereby immobilizing the cells bound to the second antigen recognizing moiety d) recovering the immobilized cells from the second magnetic field gradient as target cells.

First claim

What is claimed is:

1 ) A method for enriching target cells from a sample of cells, comprising:
a) contacting the sample with a cell aggregation agent and first magnetic particles having an iron content of 0.1 pg to 5000 pg, coupled to a first antigen recognizing moiety; and second magnetic particles having an iron content of 0.05 fg to 100 fg and coupled to a second antigen recognizing moiety to obtain mixture a) b) applying a first magnetic field gradient to the mixture a) thereby removing the cells bound to the first antigen recognizing moiety coupled to the first magnetic particles, to obtain a mixture b) and obtaining an agglomerate comprising the cells of mixture a) bound to the cell aggregation agent c) applying a second magnetic field gradient to the mixture b) thereby immobilizing the cells bound to the second antigen recognizing moiety coupled to the second magnetic particles d) recovering the immobilized cells from the second magnetic field gradient as target cells.

2 ) The method according to claim 1 , wherein the agglomerate comprising the cells of the mixture a) bound to the cell aggregation agent are separated from mixture b).

3 ) The method according to claim 1 , wherein the second magnetic field gradient is higher than the first magnetic field gradient.

4 ) The method according to claim 1 , wherein the first and second magnetic field gradient is applied by subjecting mixture b) and mixture c) on ferromagnetic separation means into first and second magnetic field.

5 ) The method according to claim 1 , wherein the first magnetic particles have a cv of diameter of less than 15%.

6 ) The method according to claim 1 , wherein the cell aggregation reagent is selected from the group consisting of proteins like fibrinogen and immunoglobulins, dextran, hydroxyethyl starch, polyvinyl pyrrolidone (PVP), methylcellulose or hydroxypropylmethylcellulose (HPMC).

7 ) The method according to claim 1 , characterized in that the first magnetic particles are coupled to a plurality of different first antigen recognizing moieties.

8 ) The method according to claim 1 , wherein the second magnetic particles are coupled to a plurality of different second antigen recognizing moieties.

9 ) The method according to claim 1 , wherein the first and/or second antigen recognizing moieties are antibodies against antigens selected from the group CD3, CD4, CD8, CD14, CD15, CD19, CD34, CD36, CD61, CD123, CD193, CD235a, anti-IgE and anti-TCRab

10 ) The method according to claim 1 , wherein the sample of cells is a sample of whole blood, Buffy-Coat or peripheral blood.

11 ) The method according to claim 1 , wherein the first magnetic particles have a mean diameter of 1-5 μm

12 ) The method according to claim 1 , wherein the second magnetic particles have a mean diameter of 10-250 nm.

13 ) The method according to claim 1 , wherein the cell aggregation agent aggregates erythrocytes.

Assignees

• Miltenyi Biotec Gmbh

Inventors

• Dose Christian
• Huppert Volker
• Kauling Burgund
• Steinbrück Philipp

Classifications

• C12N5/0087

  Purging against subsets of blood cells, e.g. purging alloreactive T cells · CPC title

• G01N33/54333

  Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction · CPC title

• G01N2001/4038

  electric methods, e.g. electromigration, electrophoresis, ionisation · CPC title

• C12N5/0646

  Natural killers cells [NK], NKT cells · CPC title

• B03C2201/26

  for use in medical or biological applications · CPC title