Method of immobilizing a cell on a support using compounds comprising a polyethylene glycol moiety

Patent metadata

Field	Value
Publication number	US-10890585-B2
Application number	US-201615186581-A
Country	US
Kind code	B2
Filing date	Jun 20, 2016
Priority date	Dec 20, 2013
Publication date	Jan 12, 2021
Grant date	Jan 12, 2021

Abstract

The present invention relates to a method of immobilizing a cell on a support, the method comprising a) providing a compound or salt thereof comprising, preferably consisting of, one or more hydrophobic domains attached to a hydrophilic domain, wherein the one or more hydrophobic domains are covalently bound to said hydrophilic domain, and wherein the one or more hydrophobic domains each comprise a linear lipid, a steroid or a hydrophobic vitamin, and wherein the hydrophilic domain comprises a polyethylene glycol (PEG) moiety, and wherein the compound comprises a linking group; b) contacting a cell with the compound under conditions allowing the interaction of the compound with the membrane of the cell, thereby immobilizing the linking group on the surface of the cell; and c) contacting the linking group immobilized on the cell with a support capable of binding the linking group, thereby immobilizing the cell on the support.

First claim

The invention claimed is:

1. A method of immobilizing an animal cell on a support, the method comprising
a) providing a compound or salt thereof comprising two, three, or four hydrophobic domains attached to a hydrophilic domain,
wherein the hydrophobic domains are covalently bound to said hydrophilic domain, and
wherein the hydrophobic domains each comprise a steroid, and wherein the hydrophilic domain comprises a polyethylene glycol (PEG) moiety, and
wherein the compound comprises a linking group comprising biotin;

b) contacting an animal cell with the compound under conditions allowing the interaction of the compound with the membrane of the cell, thereby immobilizing the linking group on the surface of the cell; and
c) contacting the linking group immobilized on the cell with a support capable of binding the linking group, thereby immobilizing the cell on the support, and wherein the hydrophilic domain comprises a structure of Formula (I):
X1-[A1-(L1)] k1 -Z-[A2-(L1)] k2 -X2  (I),

wherein
Z is linear polyethylene glycol (PEG) moiety containing 1 to 100 —O—CH 2 —CH 2 — moieties, wherein the polyethylene glycol moiety optionally comprises 1 or more phosphate spacer moieties SP connecting two —O—CH 2 —CH 2 — moieties, and wherein the linear PEG moiety optionally comprises a linker moiety L3 at one or both ends,
each L1 is a phosphate moiety,
A1 are trifunctional moieties, and A2 are bifunctional or trifunctional moieties selected independently from each other, and at least one A2 is trifunctional,
k1 and k2 are integers between 1 and 5, selected independently from each other,
X1 and X2 are independently selected from hydrogen or a protecting group,
L3 is independently selected from a linear alkyl or alkenyl chain with 1 to 10 C atoms, which is optionally (i) interrupted by 1 to 3 N, O or S atoms, and/or (ii) substituted by 1 to 4 hydroxyl, carbonyl, amino or thiol groups,
and
wherein each of the hydrophobic domains comprise the steroid covalently bound to the trifunctional moiety A1 via a linker moiety L2, wherein L2 comprises a moiety —[[O—CH 2 —CH 2 ]y2-(SP)n]m1-,
y2 is an integer from 1 to 30, m1 is an integer from 1 to 10, n is either 0 or 1,
the linking group comprising biotin is covalently bound via the trifunctional moiety A2, the trifunctional moieties A1 have 1 to 10 C atoms and comprise at least one —OH, —SH and/or at least one NH 2 group,
the moieties A2 are independently selected from a bifunctional group selected from the group consisting of a phosphate group, carbamate group, amide group, a moiety comprising a nucleobase, and a linear alkyl group having 1 to 10 C atoms and which alkyl chain contains functional groups at the terminal C-atoms, and a trifunctional moiety having 1 to 10 C atoms and comprising at least one —OH, —SH and/or at least one —NH 2 group, or a salt thereof.

2. The method of claim 1 , wherein Z in Formula (I) has the following structure:
-(L3) n2 -[[O—CH 2 —CH 2 ] y -(SP) n1 ] m -[O—CH 2 —CH 2 ] y1 -(L3) n2 ,

wherein
SP is a phosphate spacer moiety,
each n1 is either 0 or 1, selected independently for each m moieties,
each n2 is either 0 or 1, selected independently of each other,
m is an integer from 1 to 100,
y is an integer from 1 to 100,
y1 is an integer from 0 to 30,
with the proviso that ym+y1≤100,
L3 is independently selected from a linear alkyl or alkenyl chain with 1 to 10 C atoms,
which is optionally (i) interrupted by 1 to 3 N, O or S atoms, and/or (ii) substituted by 1 to 4 hydroxyl, carbonyl, or thiol groups.

3. The method of claim 2 , wherein
(a) n1 is identical for the m moieties —[O—CH 2 —CH 2 ] y -(SP) n1 ]-, and/or
(b) y1 is 0, and/or
(c) y is 4, 5, or 6, and n1 is 1, and/or
(d) n2 is both 0, or
(e) one or both n2=1, and L3 is an alkyl group with 1 to 10 C atoms which optionally contains an amide group, carbonyl group, carbamate, and/or NH group.

4. The method of claim 1 , wherein X1 or X2 is the protecting group.

5. The method of claim 1 , wherein
(a) the steroid is a sterol, or
(b) wherein the steroid is selected from the group consisting of cholesterol; a steroid hormone; an ecdysteroid; a phytosterol; a brassinosteroid; a hopanoid; and an ergosterol.

6. The method of claim 1 , wherein each of the hybrophobic domains comprises a steroid bound to a trifunctional moiety A1 via the linker moiety tetraethylenglycol (TEG).

7. The method of claim 1 , wherein the compound further comprises a label moiety, wherein the label moiety is covalently bound via a trifunctional moiety A2, and/or wherein the moiety(ies) A2 are a label moiety, and/or wherein the label moiety is a fluorescent label.

8. A method of immobilizing a population of animal cells comprising a cell of interest on a support, the method comprising
a) providing the compound as defined in claim 1 ;
b) contacting the population of cells with the compound under conditions allowing the interaction of the compound with the membrane of the cells, thereby immobilizing the linking group on the surface of the cells;
c) contacting the linking group immobilized on the cell with a support capable of binding the linking group, thereby immobilizing the cells on the support; and
d) optionally detecting the cell of interest immobilized on the support.

9. The method of claim 8 wherein the cell of interest is a white blood cell, a rare cell such as a circulating tumor cell, endothelial cell or epithelial cell.

10. The method of claim 1 , wherein the cell is a cell in suspension and/or wherein the cell is human cell, a vertebrate cell, or a mammalian cell.

Assignees

• Roche Diagnostics Operations Inc

Inventors

• Josel Hans-Peter
• Heindl Dieter
• Froehlich Thomas
• Froehner Stefanie

Classifications

• C12N11/089 Primary

  obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds · CPC title

• C12N2533/20

  Small organic molecules · CPC title

• C12N5/0068

  General culture methods using substrates (for specific animal cell type C12N5/06) · CPC title

• G01N33/54353

  with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393) · CPC title

• C12N11/14

  Enzymes or microbial cells immobilised on or in an inorganic carrier · CPC title