HK1228499A1 - Use of compounds comprising two or more hydrophobic domains and a hydrophilic domain comprising peg moieties for stabilization of a cell - Google Patents

Description

Use of a compound comprising two or more hydrophobic domains and a hydrophilic domain comprising a PEG moiety for stabilizing a cell

The present invention relates to the use of compounds comprising two or more hydrophobic domains and a hydrophilic domain comprising a polyethylene glycol (PEG) moiety for stabilizing cells and methods related thereto.

Octavidi T. et al (Biotechnology and Bioengineering 199036: 911-920) describe the effect of the shear protectant Pluronic F-68 (Poloxamer 188), a non-ionic surfactant, on hybridomas growing under hydrodynamic stress. It is disclosed in the paper that shear sensitivity of mammalian cells may be a problem preventing the development of large-scale animal cell cultures. Octavidio et al investigated the relationship between plasma membrane fluidity, shear sensitivity, and the effect of a shear protectant added to the media. They have shown that plasma membrane fluidity is reduced by adding cholesterol to the medium and they show that cell survival of cells subjected to a selected shear rate is higher when cholesterol is added to the medium than in the control group. Pluronic F-68 has shown the same effect.

Tomeczekowski J. et al 1993; Enzyme and microbial technology 15: 849-.

Laura A. et al (Enzyme and microbiological Technology 200026: 324-331) describe Pluronic F-68 as a shear protectant for animal cells from hydrodynamic stress and they investigated the mechanism of action of Pluronic F-68. A review by Laura et al shows different other publications showing that Pluronic F-68 shows two protective mechanisms (physical and biological/cellular). Pluronic F-68 reduces the level of frequency of forces experienced by the cells, for example, it stabilizes the foam layer and reduces the rate of growth of bubbles (rising velocity), thereby reducing shear forces. At the cellular level, Pluronic F-68 reduced plasma membrane fluidity.

Similar disclosures are found in Thomas C. et al (Advances in Bioprocess Engineering 1998: 137-.

However, cholesterol is a hydrophobic molecule and therefore it must be dissolved in solvents such as DMSO or alcohol which exhibit cytotoxicity at concentrations above 1%, resulting in limited cholesterol concentrations which can be used to stabilize cells.

Furthermore, monovalent molecules such as cholesterol or Pluronic F-68 have been shown to have lower shear protection properties than divalent molecules. Therefore, the concentration of monovalent protective agents (as disclosed) needs to be higher than that of divalent molecules.

Eventually, monovalent molecules may internalize inside the cell and thus alter cellular physiology.

Accordingly, there is a need for new uses and methods employing compounds and compositions that are capable of binding cells without affecting viability and/or stabilizing the cells. For example, such uses and methods should stabilize cells exposed to stress, such as shear stress.

The use and method of the present invention solves this problem and overcomes the disadvantages of the prior art. The use and method of the invention are particularly effective in stabilizing cells, particularly against shear stress.

In one embodiment, the present invention relates to the use of a compound comprising, preferably consisting of, two or more hydrophobic domains linked to a hydrophilic domain, wherein said two or more hydrophobic domains are covalently bound to said hydrophilic domain, and wherein said two or more hydrophobic domains each comprise a linear lipid, a steroid or a hydrophobic vitamin, and wherein said hydrophilic domain comprises a polyethylene glycol (PEG) moiety, for stabilizing a cell.

Polyethylene glycol (PEG) moieties are understood to be linear or branched moieties, preferably linear moieties, comprising at least one-O-CH₂-CH₂-a fraction, preferably 1-50, more preferably 4-30-O-CH₂-CH₂-a moiety.

The rationale for stabilization is assumed to be that the terminal hydrophobic part of the compound is anchored into the lipid bilayer of the cell membrane. This hydrophobic molecule immobilization decreases plasma membrane fluidity and thus stabilizes the cells.

Stabilization and in particular shear protection is demonstrated in particular for cholesterol, myristic acid and stearic acid as hydrophobic moieties in the compounds useful in the method of the invention (see example 5).

By "stable cells" according to the invention is understood higher viability of the cells compared to the viability of control cells not applying the method of the invention or the compound used according to the invention under defined conditions, preferably shear stress conditions, such as centrifugation, e.g. cells centrifuged at 500g for 20 min. Stabilization is typically determined on a population of cells, e.g., 2, 10, 100 or more cells, and the respective average viability of the cell populations is compared. A higher average viability of the treated cell population compared to the average viability of the control population indicates a stabilizing effect. Viability may be determined by measuring cell morphology, cell viability and/or cell recovery (cell recovery). Methods for determining cell morphology, cell viability and cell recovery are known in the art. Specifically, the method as described in example 5 can be used. For determining cell morphology, a visual inspection by microscopy can be performed. For measuring cell viability, a cell viability test using the WST-1 proliferation kit (RAS) can be performed. In particular, a viability that is at least 5%, more preferably 10%, even more preferably 20% higher than the viability of the control cell or control cell population indicates a stabilizing effect and thus a stabilization of the cell or cell population.

In a preferred embodiment of the invention, the stabilization is stabilization during exposure of the cells to shear forces.

In a further preferred embodiment, the stabilization is stabilization of the cells during exposure to shear forces due to centrifugation, large scale cell culture, flow cytometry, fluorescence activated cell sorting and/or bead based cell separation.

In a further preferred embodiment, stabilization is stabilization of cells exposed to centrifugation, large scale cell culture, flow cytometry, fluorescence activated cell sorting, and/or bead based cell separation processes.

"shear stress" or "shear force" is understood to be defined as the component of stress that is coplanar with a cross-section of a substance. Shear stress results from a force vector component parallel to the transverse plane. Such stresses are applied to the cells during centrifugation, large-scale cell culture, flow cytometry, fluorescence activated cell sorting, and/or bead-based cell separation.

Centrifugation is a method used in industrial and laboratory environments that involves the use of centrifugal force with a centrifuge to settle a mixture. The cells may be centrifuged, e.g., at 100g, 200g, 500g, 1000g or more for 5 minutes or more, e.g., 1 hour or 5 hours.

Flow cytometry is a laser-based biophysical technique for cell counting, cell sorting, biomarker detection, and protein engineering by suspending cells in a fluid stream and passing them through an electronic detection device. It allows simultaneous multiparametric analysis of physical and chemical characteristics of up to thousands of particles per second.

Fluorescence Activated Cell Sorting (FACS) is a specialized type of flow cytometry. It provides a method of sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence characteristics of each cell.

In bead-based cell separation methods, beads, such as magnetic beads, are used, which are typically coated with a member of a bioaffinity binding pair, such as an antibody. Using such beads, cells of interest carrying markers recognized by such binding pair members can be bound and subsequently isolated, e.g., using magnetic properties. The separation step applies a shear force to the cells bound to the beads.

Large scale cell culture is understood to be the cultivation of cells in a volume of more than 10 ml, 50ml, 100 ml or 1l of liquid medium or more, especially as batch culture involving stirring. Such agitation also implies shear stress on the cells to be cultured.

The compounds used in the present invention comprise, preferably consist of, two or more hydrophobic domains and one hydrophilic domain.

Preferably, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hydrophobic domains are covalently bound to the hydrophilic domain.

For stabilization it was found to be advantageous that the compounds used according to the invention preferably comprise 2 or 3 or more, more preferably 2 or 3 hydrophobic domains.

Of particular advantage, in a particular embodiment, at least one of the lipid hydrophobic domains comprises a steroid, even more preferably cholesterol.

In a preferred embodiment of the invention, 2 or 3 hydrophobic moiety hydrophobic domains are covalently bound to the hydrophilic domain.

For the general understanding herein, a "hydrophobic moiety" is comprised in and forms a major part of a "hydrophobic domain" and thus determines its hydrophobic character.

The hydrophobic moieties of the compounds comprising 2 or more hydrophobic moieties may be the same or may be different. For example, a compound comprising two hydrophobic domains may comprise 2 myristic acid moieties, or one myristic acid moiety or one cholesteryl moiety.

The cholesterol moiety is a particularly preferred hydrophobic moiety of the compounds used in the present invention.

In a preferred embodiment of the use, the compound comprises, preferably consists of,

wherein the two or more hydrophobic domains are covalently bound to the hydrophilic domain, and

wherein the two or more hydrophobic domains each comprise a linear lipid, a steroid, or a hydrophobic vitamin, and

wherein the hydrophilic domain comprises a compound of formula (I):

X1-[A1 -(L1)_n]_k1-Z - [A2 -(L1)_n]_k2- X2 (I),

wherein

Z is a group containing 1 to 100, preferably 1 to 50, more preferably 4 to 30-O-CH₂-CH₂-a linear polyethylene glycol (PEG) moiety of a moiety, wherein said polyethylene glycol moiety optionally comprises one or more linkages connecting two-O-CH₂-CH₂-a spacer portion SP of the portion,

and wherein said linear PEG moiety optionally comprises a linker moiety L3 at one or both termini,

each L1 is a linker moiety selected independently of the other,

each n is 0 or 1, independently selected from each other,

a1 and A2 are difunctional or trifunctional moieties selected independently of one another, with the proviso that at least one of A1 or A2 is trifunctional,

k1 and k2 are integers from 0 to 10, selected independently of each other, with the proviso that at least one of k1 and k2 is not 0,

x1 and X2 are independently selected from hydrogen or a protecting group,

l3 is independently selected from a linear alkyl or alkenyl chain having 1 to 10C atoms, which is optionally (i) interrupted by 1 to 3N, O or S atoms, and/or (ii) substituted by 1 to 4 hydroxy, carbonyl, amino or mercapto groups,

and is

Wherein the two or more hydrophobic domains are covalently bound to the hydrophilic domain via the trifunctional domain,

or a salt thereof.

In a preferred embodiment, k1+ k2 ≧ 2.

The lipid is a hydrophobic small molecule selected from the group consisting of: fats, waxes, sterols, soluble fats, hydrophobic vitamins such as vitamin A, D, E and K, fatty acid monoglycerides, diglycerides, triglycerides and phospholipids.

The hydrophobic domains each comprise, preferably consist of, a linear lipid, a steroid or a hydrophobic vitamin.

The linear lipid, steroid or hydrophobic vitamin may be bound directly to the trifunctional moiety or to the trifunctional moiety via linker L2. An example of a compound in which the linear lipid is directly bound to the trifunctional moiety is the compound myristic-myristic acid- (spacer C18) 7-Fluos-biotin-TEG. An example of a compound in which the steroid is bound to a trifunctional moiety via linker L2 is the compound cholesteryl-TEG- (spacer C18) 7-Fluos-biotin-TEG. In this latter example, TEG (tetraethyleneglycol) is the linker L2.

In a preferred embodiment, the hydrophobic domains each consist of a linear lipid, a steroid or a hydrophobic vitamin. In this case, it is clear that the hydrophobic domain is hydrophobic, more preferably lipophilic, since linear lipids, steroids or hydrophobic vitamins are hydrophobic, more preferably lipophilic.

Hydrophobic parts are understood to be parts which repel water (a mass of water). Preferably, the moiety is lipophilic; i.e. it tends to be soluble in other non-polar lipophilic substances such as fats or fatty acids.

In another preferred embodiment, the hydrophobic domains each comprise a linear lipid, a steroid or a hydrophobic vitamin and one or more other moieties. In this embodiment, the hydrophobic moiety as a whole is hydrophobic, more preferably lipophilic.

In an even more preferred embodiment, the hydrophobic domain of the invention comprising a linear lipid, a steroid or a hydrophobic vitamin is capable of insertion into the cell membrane. This can be determined by methods known in the art.

In a preferred embodiment, 2 or 3 or more hydrophobic moieties of the compounds used according to the invention are different hydrophobic domains, or in the case of 3 or more hydrophobic moieties, two or more are different or all are different from each other.

In a preferred embodiment of the invention, the first hydrophobic domain comprises, preferably consists of, saturated fatty acids, especially myristic, stearic or behenic acid, especially myristic acid; and/or the second hydrophobic domain comprises, preferably consists of cholesterol. In the case of the third hydrophobic domain, this domain preferably comprises cholesterol or a saturated fatty acid, in particular myristic, stearic or behenic acid, in particular myristic acid, preferably consists of the above, and/or is identical to the first or second hydrophobic domain. In case of more than 4 hydrophobic domains, the same preferred embodiment for the third hydrophobic domain applies.

In particular, compounds comprising at least one cholesterol moiety, especially compounds of 1, 2 or 3 cholesterol moieties are especially preferred.

For stabilization, the compounds used in the present invention comprise 2 or 3 or more, more preferably 2 or 3 hydrophobic domains.

This conformation shows a higher binding affinity for cells than a monovalent molecule (i.e., a molecule of the invention comprising one hydrophobic moiety). Thus, lower concentrations of the compounds used in the present invention are required to achieve shear protection compared to monovalent molecules.

The hydrophilic portion of the molecule inhibits internalization of the compounds used in the present invention and the shear protection is induced by incorporation of the hydrophobic portion into the outer plasma membrane. Experiments with labeled compounds used in the present invention have confirmed that the compounds just incorporated into the outer plasma membrane without affecting the cell interior.

Furthermore, the compounds used according to the invention surprisingly exhibit a favourable binding or immobilization on cells, as shown in detail in the examples. Compounds further exhibiting immobilization comprise a linking group. The compound may further comprise a labeling moiety. Such compounds may additionally be used to target and/or detect cells.

For further applications of cell labeling and immobilization, compounds having a hydrophobic moiety and further comprising a linker and/or a labeling moiety were found in the examples to show targeting and tight retention of all cell types (see in particular example 2). In particular, cholesterol, myristic acid, stearic acid and behenic acid moieties have been found to be particularly useful for this purpose. With exemplary advantages and the possibility of quantitative cell targeting, compound 5' -cholesteryl TEG-PEG2000-Fluos-3 (internal reference: BMO 29.891133) represents a preferred compound for use in the present invention.

Furthermore, compounds containing two or three hydrophobic moieties further comprising a linker group were found to be useful for quantitative cell fixation in experiments.

According to the invention, a "cholesterol-di-linker molecule" is understood to be a compound used according to the invention comprising two hydrophobic moieties, both of which are cholesterol. Thus, a "myristic acid-triple linker molecule" is understood as a compound used according to the invention comprising three hydrophobic moieties, which are all myristic acid.

The use of compounds comprising two hydrophobic moieties, both of which are cholesterol, is particularly preferred.

Furthermore, the use of compounds comprising two hydrophobic moieties, both of which are myristic acid, is especially preferred.

Such compounds are effective in stabilizing cells according to example 5.

According to the present invention, an "asymmetric double linker molecule" is understood to be a compound for use according to the present invention comprising two hydrophobic moieties, wherein the two hydrophobic moieties are different from each other.

The compounds used in the present invention are described in the examples, mostly in this modular, schematic form.

According to the invention, the compound "cholesteryl-TEG- (spacer C18) 7-Fluos-biotin-TEG" is understood as indicated in 6A) as a compound in which two cholesterol moieties as hydrophobic moieties are bound to a trifunctional moiety via TEG (tetraethylene glycol).

According to fig. 6, which shows a modular description of the compounds used in the present invention together with the chemical formula, "(spacer C18)" is understood to be a PEG moiety of 18 atoms in length followed by a phosphate moiety as spacer moiety. Accordingly, - (spacer C18) 7-is understood to be a moiety consisting of 7 "(spacer C18)" moieties.

According to the invention, "Fluos" is understood to be the fluorescein moiety directly bound to the trifunctional moiety a 2.

According to the invention, "biotin-TEG" is understood as the biotin moiety bound to the trifunctional moiety a2 via a linker TEG.

In the case of the compounds used in the invention disclosed in this schematic way, the trifunctional moiety a1 is typically glycerol, which is a TEG-bound hydrophobic moiety (see fig. 6A). Furthermore, embodiments are also disclosed in which serinol or 6- [ (2-hydroxyethyl) amino ] -1-hexanol is used instead of glycerol as the trifunctional moiety. Other alternatives for such trifunctional moieties are available to the skilled person.

Trifunctional moiety a1 is serinol for the compound of fig. 6A, wherein the hydrophobic moiety is directly bound to trifunctional moiety a 1.

In an even more schematic way, "cholesteryl-TEG- (spacer C18) 7-Fluos-biotin-TEG" can be described as the structure "5 '-XXYYYYYYYFZ-3'", wherein Y = is a PEG + spacer moiety, X is a hydrophobic moiety bound to the hydrophilic moiety via a trifunctional linker, F is a fluorescent tag fluorescein, and Z is a linker (biotin). 5 'and 3' represent the synthetic orientations by automated synthesis similar to nucleotides as shown in the examples.

Similarly, -PEG 2000-is understood to be a PEG2000 moiety; i.e. from 45C₂H₆O₂A polyethylene glycol (PEG) chain composed of subunits.

In the compounds used according to the invention described in the experimental part, L1 is present (n = 1) and is a phosphate ester if not explicitly stated otherwise.

"spacer" in the context of the specifically disclosed compounds used in the present invention in the examples is to be understood as a PEG moiety comprising a phosphate moiety. The length of the PEG moiety is determined by e.g. C9 or C12, which means that the PEG moiety has a length of 9 or 12 atoms, respectively.

"dT" is understood to be thymidine, as exemplified in fig. 6B). This moiety dT can be used to determine the concentration of the compound by absorption and is a bifunctional moiety according to the present invention.

Furthermore, the compounds used in the present invention comprising 2 or 3 hydrophobic molecules covalently bound to hydrophilic domains exhibit tight binding of cells, potentially exploiting cooperative binding effects. The binding of such molecules to cells is 100-fold stronger than that using compounds containing only one hydrophobic molecule.

Furthermore, it is preferred in one embodiment that 2, 3 or more, especially 2 or 3 hydrophobic moieties are spatially separated by use of a linker moiety L1.

In such preferred embodiments, n =1, and thus L1 is present.

The hydrophilic domain of the compounds used in the present invention comprises a PEG moiety and is therefore flexible.

The terminal hydrophobic part of the compounds used in the present invention is then a long flexible hydrophilic domain.

The hydrophilic domain allows flexible folding around the target cell, which is required for safe embedding of the cell, resulting in a cell-friendly, hydrogel-like environment, which is important for maintaining the cell morphology and function effectively and thereby stabilizing the cell.

Different linear PEG moieties can be used, which are phosphorus in length and/or comprise spacer moieties, e.g., between PEG moietiesThe acid esters differ to achieve flexible hydrophilic domains. For example, a combination of 45C's as described above may be used₂H₆O₂A polyethylene glycol (PEG) chain of subunits (PEG 2000) (see example 6B) or a PEG moiety with a phosphate spacer such as- (spacer C18) 7-.

Suitable protecting groups are known in the art. Suitable protecting groups for phosphoramidite chemistry are, for example, (4,4' -Dimethoxytrityl (DMT) and fluorenylmethoxycarbonyl (Fmoc).

Various salts of the compounds used in the present invention can be used, such as Na of the compounds used in the present invention⁺And/or TEA⁺Salt, as shown in figure 1.

Other salts are also possible and known to the skilled person. Preferably, salts are used that do not affect or substantially affect cell viability or function.

In a preferred embodiment of the invention, moiety Z has the following structure:

-(L3)_n2- [O-CH₂-CH₂]_y- (SP)_n1]_m-[O-CH₂-CH₂]_y1-(L3)_n2- ，

wherein

SP is a spacer moiety that is a spacer moiety,

each spacer moiety SP is selected independently of the other,

each n1 is 0 or 1, independently selected for each m portion,

each n2 is 0 or 1, selected independently of each other,

m is an integer of 1 to 100, preferably 1 to 50, more preferably 4 to 30,

y is an integer from 1 to 100, preferably from 1 to 50, more preferably from 4 to 30,

y1 is an integer from 0 to 30, preferably from 0 to 10, more preferably from 0 to 4,

provided that y m + y1 is 100 or less

And wherein L3 is as defined above.

In a further preferred embodiment of the invention, n1 is for m moieties- [ O-CH₂-CH₂]_y- (SP)_n1]-are identical.

As can be seen from the examples, n1 is generally either always 0 in the compounds used according to the invention or always 1 in the compounds used according to the invention.

An exemplary compound where n1=1 is cholesteryl-TEG-spacer C12-cholesteryl-TEG- (spacer C18) 7-Fluos-biotin-TEG.

An exemplary compound where n1=0 is cholesteryl-TEG-PEG 2000-Fluos-biotin-TEG.

In a further preferred embodiment of the invention y1 is 0.

An exemplary compound where y1=0 is cholesteryl-TEG-spacer C12-cholesteryl-TEG- (spacer C18) 7-Fluos-biotin-TEG.

In a further embodiment of the invention y1 is 1.

An exemplary compound where y1=1 is 5 '-cholesteryl TEG- (spacer C18) 7-spacer C3-dT-biotin TEG-3'.

In a further preferred embodiment of the invention y is 3, 4, 5 or 6 and n1 is 1. Even more preferably, m is 3, 4, 5, 6, 7, 8, 9 or 10.

In a further preferred embodiment of the present invention, the spacer moieties SP are independently from each other selected from the group consisting of phosphate and bifunctional moieties.

Preferably all spacer moieties SP are identical. Even more preferably, all fractions SP are phosphate esters.

A bifunctional moiety according to the present invention is understood to be a moiety comprising two functional groups prior to the synthesis of the compound used according to the present invention. Such bifunctional moieties are therefore suitable for the synthesis of linear compounds. Suitable bifunctional groups are preferably selected from phosphate groups, carbamate groups, amide groups, nucleobase containing moieties, even more preferably dT, and straight chain alkyl groups having 1-10C atoms, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 atoms, and said alkyl chain comprising a functional group at the terminal C atom, in particular independently selected from amine, carbonyl, hydroxyl, thiol, carbonate groups. Examples of suitable straight chain alkyl groups having terminal functional groups are diaminoalkyl moieties such as H₂N-(CH₂)₅-NH₂Or hydroxy-carbonyl moieties such as-C (O) - (CH2)₄-O-。

A trifunctional moiety according to the invention is understood to be a moiety comprising three functional groups prior to the synthesis of the compound used according to the invention. Such trifunctional moieties are therefore suitable for the synthesis of branched compounds. Suitable trifunctional moieties are preferably selected from trifunctional moieties having 1-10C atoms (in particular 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10C atoms) and comprising at least one-OH, -SH and/or at least one-NH 2 group, more preferably from amino acids such as lysine or serine, serinol, -O-CH2-CH ((CH) s-H₂)₄-NH₂) -CH2-, glycerol and 1, 3-diaminoglycerol moieties.

In a further preferred embodiment of the invention, X1 and/or X2, preferably X1 or X2, are replaced by a hydrophobic binding domain. In such embodiments, k1+ k2 may be 1. An exemplary compound in which X1 is replaced by a hydrophobic domain is biotin-PEG-Lys- (C18)2, as shown in the examples.

In a further preferred embodiment of the invention, n2 are both 0. In such embodiments, the central linear PEG moiety is directly attached to the moieties X1- [ A1- (L1) n ] k1 and [ A2- (L1) n ] k 2-X2.

In a further preferred embodiment of the invention, one or bothn2=1, and L3 is an alkyl group having 1-10C atoms, optionally containing an amide, carbonyl, carbamate, and/or NH group. In a further preferred embodiment of the present invention, L3 is an alkyl group having 1 to 10C atoms, optionally comprising an amide, carbonyl, carbamate and/or NH group. For example, one L3 may be-NH-CH₂-CH₂NHCO-CH₂-CH₂-, the compound of the invention biotin-PEG 2000-Lys- (C18)₂In (1).

In a further preferred embodiment of the invention, the linear lipid is

(a) Saturated or unsaturated fatty acids, and/or

(b) Fatty acids having 8 to 26C atoms, preferably 12 to 22C atoms, more preferably 14 to 18C atoms.

Fatty acids are carboxylic acids with long aliphatic tails, which are saturated or unsaturated. Most naturally occurring fatty acids have chains with an even number of carbon atoms (4-28).

Examples of saturated fatty acids are caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidonic acid, behenic acid, lignoceric acid and cerotic acid.

Examples of suitable unsaturated fatty acids are:

in an even more preferred embodiment, the linear lipid is selected from oleic acid, myristic acid, stearic acid and behenic acid, more preferably from myristic acid and oleic acid.

Hydrophobic vitamins are small molecules selected from the group consisting of vitamin A, D, E and K. In a more preferred embodiment, the hydrophilic vitamin is alpha-tocopherol. An exemplary compound for use in the present invention comprising alpha-tocopherol is 5 '-alpha-tocopherol TEG-PEG 2000-Fluos-3'.

In a further preferred embodiment, steroids may be used as the hydrophobic moiety.

Steroids are a class of organic compounds that contain a characteristic arrangement of four cycloalkane rings linked to one another. The core of a steroid consists of seventeen carbon atoms bonded together, in the form of four fused rings: three cyclohexane rings (designated as rings A, B and C) and one cyclopentane ring (ring D). Steroids differ by the functional group bound to the tetracyclic core and by the oxidation state of the ring. Sterols are a specific form of steroid with a hydroxyl group at position 3 and a backbone derived from cholestanes.

In a further preferred embodiment of the present invention,

(a) the steroid is a sterol, or

(b) The steroid is selected from cholesterol; a steroid hormone, preferably a gonadal steroid, more preferably an androgen such as a anabolic steroid, androstenedione, dehydroepiandrosterone, dihydrotestosterone, or testosterone, an estrogen such as estradiol, estriol, or estrone; progestogens, such as progesterone or progestin (progestine), corticosteroids, in particular glucocorticoids or mineralocorticoids; ecdysteroids such as ecdysterone; a phytosterol; brassinosteroids; hopanoids (hopanoids); and an ergosterol, and a salt of ergosterol,

more preferably the steroid is cholesterol, or

(c) The hydrophobic vitamin is alpha-tocopherol.

In further preferred embodiments of the invention, two, three or four, preferably two or three, hydrophobic domains are covalently bound to a hydrophilic domain.

In a further preferred embodiment of the invention, the two or more hydrophobic domains covalently bound to the hydrophilic binding structure are different or identical.

In a further preferred embodiment of the invention, the hydrophobic domain consists of a linear lipid, a steroid or a hydrophobic vitamin.

In a further preferred embodiment of the invention, the hydrophobic domain comprises (preferably consists of): a linear lipid, steroid or hydrophobic vitamin covalently bound to the trifunctional moiety a1 via a linker moiety L2.

Such bifunctional and trifunctional moieties are successfully used in the compounds used in the present invention for binding of hydrophobic moieties either directly or via linker L2.

Linker L2 is independently any linker moiety suitable for covalently bonding a hydrophobic moiety to a hydrophilic moiety, and the linker has a length of 50, 30 or 20 atoms or less between the hydrophobic moiety and a1 or a2, respectively.

In a preferred embodiment, linker L2 comprises, preferably consists of: phosphate group, moiety- [ O-CH₂-CH₂]_y2- (SP)_n]_m1-, wherein SP and n are as defined above, preferably n =0, y2 is an integer from 1 to 30, preferably from 3 to 10, and m1 is an integer from 1 to 10, preferably from 1 to 3, a glycerol moiety, a carbamate group, an amide group, a straight chain alkyl group having from 1 to 10C atoms (in particular 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 atoms) and said alkyl chain comprising a functional group at the terminal C atom, in particular independently selected from amine, carbonyl, hydroxyl, mercapto, carbonate groups, said alkyl group may optionally be substituted by 1, 2, 3, 4 or 5 moieties R1, wherein R1 is independently C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 aminoalkyl, C1-C4 cyanoalkyl, hydroxyl, mercapto, amino or a carbonyl moiety. Examples of suitable straight chain alkyl groups having terminal functional groups are diaminoalkyl moieties such as H₂N-(CH₂)₅-NH₂Or hydroxy-carbonyl moieties such as-C (O) - (CH2) 4-O-. Preferably, the straight chain alkyl group is unsubstituted. Even more preferably, the linear lipid, steroid or hydrophobic vitamin is conjugated to the trifunctional moiety A1 via a linker moiety- (O-CH2-CH2) j-wherein j is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably j is 3, in particular tetraethylene glycol (TEG), a phosphate moiety or comprises TEG, glycerol, andpart of a phosphate moiety, or comprising-TEG-glyceryl-phosphate-O- (CH)₂)₄-c (o) -or a part consisting thereof.

In a more preferred embodiment, the compounds used in the present invention comprise a linear lipid, steroid or hydrophobic vitamin covalently bound to a trifunctional moiety a1 via a linker moiety L2, preferably wherein L2 is selected from the group consisting of a phosphate, amide, carbamate, ester group or moiety

- [O-CH₂-CH₂]y₂- (SP)_n]_m1-,

Wherein

SP and n are as defined above, preferably n =0,

y2 is an integer from 1 to 30, preferably from 3 to 10, and

m1 is an integer from 1 to 10, preferably from 1 to 3,

more preferably wherein the linear lipid, steroid or hydrophobic vitamin is bound to the trifunctional moiety a1 via a linker moiety tetraethylene glycol (TEG) or phosphate.

In a further preferred embodiment of the invention k1 is 1, 2, 3, 4 or 5, preferably 1, 2 or 3.

In a particularly preferred embodiment of the invention, the hydrophobic domain is via the trifunctional moiety A1 only or via the domain X1- [ A1- (L1) described above_n]_k1Covalently bound to the hydrophilic domain. For such embodiments, further preferred embodiments of the compounds used according to the invention also apply. In such compounds, the hydrophobic domain is located exclusively at one terminal portion of the molecule, while other groups, such as a linker or a label moiety (if present), are located at the other terminal portion, spatially separated therefrom.

In a further preferred embodiment of the invention k1 is 1, 2, 3, 4, 5 or 6, preferably 1, 2 or 3.

In case the compounds used in the present invention comprise a dT moiety as bifunctional moiety a2, k2 is preferably 3, 4, 5 or 6.

In another preferred embodiment of the invention k1 is 0 and X1 is replaced by a hydrophobic domain, preferably comprising a steroid, more preferably cholesterol. In a particularly preferred embodiment, Z is a moiety- (L3)_n2-TEG(L3)_n2-, wherein n2 is independently 0 or 1. In an even more preferred embodiment of the invention, k2 is 1, 2, 3, 4, 5 or 6, preferably 3, 4, 5 or 6. One or more (especially one) further hydrophobic moiety binding moieties- [ A2- (L1) n]k2-X2, wherein the further hydrophobic moiety comprises a steroid, more preferably cholesterol. Even more preferably, L2 is the linker moiety tetraethylene glycol (TEG), phosphate or a moiety comprising TEG, glycerol and phosphate moieties, or a moiety comprising or consisting of-TEG-glyceryl-phosphate-O- (CH2)4-c (O) -. An exemplary compound for use in the present invention is Chol-TEG-Chol-TEG-doublet (Doubler) -biotin-dT as shown in FIG. 12.

In case the compounds used in the present invention comprise a dT moiety as bifunctional moiety a2, k2 is preferably 3, 4, 5 or 6.

In a further preferred embodiment of the invention, the compounds used according to the invention further comprise a labeling moiety and/or a linking group.

In still even further preferred embodiments of the present invention, the compound further comprises a labeling moiety.

Such compounds are furthermore useful for cell labeling purposes. An exemplary compound for use in the present invention is 5 '-cholesteryl TEG-PEG 2000-Fluos-3'.

In one such preferred embodiment, the compound does not further comprise a linking group.

In another even further preferred embodiment of the present invention, the compound further comprises a linking group. An exemplary compound is 5' -cholesteryl TEG-PEG 2000-biotin TEG-3.

In a preferred embodiment, the compound does not further comprise a labeling moiety.

In another more preferred embodiment of the invention, the compound further comprises a labeling moiety and a linking group.

Such compounds are particularly suitable for applications where, in addition to stabilization, it is also desirable to achieve both fixation and detection of cells, e.g. for the localization of fixed cells and/or for cell quantification. An example of such a compound is 5'- (cholesteryl-TEG) 2-PEG 2000-Fluos-biotin TEG-3', which was successfully used to immobilize cells to streptavidin coated plates and detect these cells.

Suitable label moieties are moieties suitable for in vitro detection and are known to the skilled person. The detection may be direct (as in the case of luminescence, especially fluorescence) or indirect (in the case of an enzyme or its substrate). Thus, label moieties suitable for indirect or indirect detection may be used.

As used herein, "label" or "label moiety" refers to any substance capable of generating a signal for direct or indirect detection. The label moiety may thus be detected directly or indirectly. For direct detection, the label moiety suitable for use in the present invention may be selected from any known set of detectable labels, such as chromogens, chemiluminescent groups (e.g., acridinium esters or dioxetanes), electrochemiluminescent compounds, dyes or fluorescent dyes (e.g., fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine and derivatives thereof), luminescent metal complexes, such as ruthenium or europium complexes, and radioisotopes.

In an indirect detection system, the first partner of a bioaffinity (bioaffine) binding pair is a labeling moiety for a compound used in the present invention; i.e. the first partner is covalently bound to a moiety of a compound used in the present invention. Examples of suitable binding pairs are haptens or antigens/antibodies, biotin or biotin analogues such as aminobiotin (aminobiotin), iminobiotin or desthiobiotin/avidin or streptavidin, sugars/lectins, nucleic acids or nucleic acid analogues/complementary nucleic acids, and receptors/ligands, e.g. steroid hormone receptors/steroid hormones. Preferred first binding pair members comprise a hapten, an antigen and a hormone. Haptens such as tags, digoxin and biotin and analogues thereof are also preferred. The second partner of such a binding pair, e.g., an antibody, streptavidin, etc., is typically labeled to allow direct detection, e.g., via a labeling moiety as described above.

Thus, in a preferred embodiment, the label moiety is a label moiety for direct labeling or for indirect labeling.

In a preferred embodiment, the labeling moiety is selected from (a) direct labeling moieties selected from: chromogens, chemiluminescent groups (e.g., acridinium esters or dioxetanes), electrochemiluminescent compounds, dyes or fluorescent dyes (e.g., fluorescein, coumarin, rhodamine, oxazines, resorufin, cyanines and their derivatives), luminescent metal complexes, such as ruthenium or europium complexes, and radioisotopes; (b) or one of the partners of an indirect detection system, preferably wherein the label moiety is one of the members of a binding pair selected from the group consisting of: (i) a hapten or antigen/antibody, (ii) biotin or biotin analogue such as aminobiotin (aminobiotin), iminobiotin or desthiobiotin/avidin or streptavidin, (iii) a sugar/lectin, (iv) a nucleic acid or nucleic acid analogue/complementary nucleic acid, and (v) a receptor or receptor fragment/ligand, e.g. steroid hormone receptor/steroid hormone.

Preferred first binding pair members that are labeled moieties suitable for indirect detection include haptens, antigens, and hormones. Haptens such as digoxin and biotin and analogues thereof are also preferred. The second partner of such a binding pair, e.g., an antibody, streptavidin, etc., is typically labeled to allow direct detection, e.g., by a directly labeled moiety as described above; however, it is also possible to use antibodies in the compounds used according to the invention and to use labelled antigens or haptens for detection.

In the above description of binding pair members, the term antibody is to be understood as encompassing both antibodies and antigen-binding fragments thereof.

In a preferred embodiment, the label moiety is a label moiety for direct labeling, even more preferably the label moiety is a fluorescent moiety or a dye.

Suitable fluorescent moieties (or dyes) are known in the art and include fluorescein, Cy3, Cy5, Cy5.5, Cy2, Cy3.5, Cy3b, Cy7, Alexa Fluor dyes, xanthene derivatives such as rhodamine, Oregon Green, eosin, or Texas Red, cyanine derivatives such as cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine, naphthalene derivatives such as dansyl and sodium fluorosilicate derivatives, coumarin derivatives, oxadiazole derivatives such as pyridinyloxazole, nitrobenzoxadiazole and benzoxadiazole, pyrene derivatives such as cascade blue, oxazine derivatives such as nile red, nile blue, cresyl violet, oxazine 170, acridine derivatives such as proflavine, acridine orange, acridine yellow, arylmethine (arylmethine) derivatives such as auramine, crystal violet, malachite green, tetrapyrrole derivatives such as porphine, phthalocyanine and bilirubin.

In the examples, fluorescein was used as a representative label. This allows for sensitive detection of the label, allowing for localization and/or quantification of the label. Fluorescent labels are particularly preferred marker moieties of the invention.

Suitable radioactive isotopes (radioactive isotopes) or radioisotopes for labelling and methods for labelling compounds for use in the present invention with such radioactive labels are known to the skilled person. For example, one of the following isotopes may be used:¹⁴C、³H、³²P、³³P、¹²³I、¹²⁵i and¹³¹I。

where an antibody or antigen-binding fragment is used as a member of an indirect system antibody/antigen or hapten, the antibody or antigen-binding fragment specific for the epitope or hapten can be part of the compound used in the invention, or the epitope or hapten can be part of the compound used in the invention. Thus, each other member may be directly labeled, for example, with a fluorescent label for subsequent detection. Suitable antibodies or antigen binding fragments are described in more detail below.

In a preferred embodiment of the invention, the linking group and/or the label binding moiety [ A2- (L1)_n]_k2-X2。

In a particularly preferred embodiment of the invention, the hydrophobic domain is via the trifunctional moiety A1 only (or via the domain X1- [ A1- (L1) described above)_n]_k1) Covalently bound to said hydrophilic domain, and a linking group and/or a label moiety binding moiety [ A2- (L1)_n]_k2-X2. This ensures that the hydrophobic domain inserted into the cell membrane and the moiety for immobilisation and/or labelling are spatially separated (if present).

Such compounds are suitable for immobilization in the presence of a linking group in addition to being stable.

Such compounds are suitable for labeling and detection in the presence of a labeling moiety in addition to being stable.

In a further particularly preferred embodiment of the invention, the hydrophobic domain is solely via the trifunctional moiety A1 (or via the domain X1- [ A1- (L1) described above)_n]_k1) Covalently bound to said hydrophilic domain, and a linking group and a label moiety binding moiety [ A2- (L1)_n]_k2-X2。

Such compounds further allow, in addition to being stable, both immobilization and labeling, detection and quantification.

In a still further particularly preferred embodiment of the invention, the hydrophobic domain is solely via the trifunctional moiety A1 (or via the domain X1- [ A1- (L1) described above)_n]_k1) Covalently bound to said hydrophilic domain, and a linker other than the label moiety binding moiety [ A2- (L1)_n]_k2-X2。

Such compounds may be used if only the fixation or only the labeling, detection and/or quantification of the cells to which binding is intended in addition to stabilization.

The linking group is a moiety suitable for reversibly or irreversibly, and/or covalently or noncovalently, immobilizing a compound to a support, particularly a solid support. In preferred embodiments, the linking group is an antibody or antigen-binding antibody fragment, a receptor or binding site thereof, a ligand for a receptor, an enzyme or binding site thereof, a substrate for an enzyme, a tag binding site, a tag, or a functional chemical group.

The functional chemical group may for example be a thiol group, which may be bound to the gold-coated substrate surface by forming a covalent, irreversible-S-bond.

Binding of biotin to streptavidin or an antibody or antigen-binding antibody fragment is non-covalent and reversible. Such a linker group utilizing non-covalent binding to a solid support is preferred in cases where-in addition to stabilization-the cells are intended to be separated again for further use, e.g. for administration in an animal model.

In a preferred embodiment, the linking group may be, for example, a biotin moiety that allows non-covalent binding to a streptavidin-coated surface, or a sulfhydryl group that may bind to a gold-coated substrate surface as a solid support.

In an even more preferred embodiment of the present invention, the compound used in the present invention comprises a label moiety and/or a linker group, wherein said label moiety is a fluorescent label and/or said linker group is biotin.

In an even more preferred embodiment, the compound used in the present invention comprises a label moiety and a linking group, wherein the label moiety is a fluorescent label and the linking group is biotin.

In an even more preferred embodiment, the compounds used in the present invention comprise a linking group, which is biotin.

The term "solid support" refers to a material in a solid phase that interacts with a reagent in a liquid phase through a heterogeneous reaction. The use of solid supports is well known in the fields of chemistry, biochemistry, pharmacy and molecular biology. Many types of solid supports have been developed according to the technical problem to be solved. Any of these may be used in the context of the present invention. For example, the solid support may comprise the following components: silica, cellulose acetate, cellulose nitrate, nylon, polyester, polyethersulfone, polyolefin or polyvinylidene fluoride, or combinations thereof. Further suitable solid supports include, but are not limited to, controlled pore glass, glass plates or slides, polystyrene, and activated dextran. In other aspects, synthetic organic polymers such as polyacrylamides, polymethacrylates, and polystyrene are also illustrative support surfaces. In addition, polysaccharides such as cellulose and dextran are further illustrative examples of support surfaces. Other support surfaces such as fibers are also possible.

The solid support can be contained in a container, wherein the container is a tube, such as a centrifuge or spin tube (spintube), a syringe, a cartridge, a chamber, a multi-well plate, or a test tube, or a combination thereof. The solid support may be pretreated or functionalized to allow cell immobilization. For example, the well plate may be pretreated with streptavidin, as shown in the examples. In one embodiment, the solid support may be fibrous or particulate, generally allowing for suitable contact. The size of the solid support suitable for use may vary. Cells may be bound to only one solid support (e.g., one container or multi-well plate) or may be bound to a plurality of solid supports (e.g., beads). The shape of the solid support suitable for use may be, for example, a sheet, a precut disc, a cylinder, a single fiber, or a solid support composed of fine particles. In a preferred embodiment, the solid support is flat, or substantially flat with a cavity. In one embodiment, the solid support may be fibrous or particulate. The size of the solid support may vary and may be selected according to the method or application to be performed.

In some embodiments, the solid phase is a test strip, a chip, in particular a microarray or nanoarray chip, a microtiter plate or a microparticle.

In a more preferred embodiment, the labeling moiety and/or linking group, when present, is covalently attached via the trifunctional moiety a2, as described above.

In another embodiment one or more moieties a2 are bifunctional or trifunctional labelling moieties or linkers, more preferably moiety a2 is a nucleobase containing moiety, even more preferably moiety a2 is dT (thymidine). Such compounds comprising dT are used for the determination of the concentration of the compound.

In a further preferred embodiment of the present invention, linker L1 is independently selected from the group consisting of phosphate, amide, carbamate, and ester groups.

In a further preferred embodiment of the present invention, the moieties a1 and a2 are independently selected from difunctional groups selected from: a phosphate group, a carbamate group, an amide group, a nucleobase-containing moiety (even more preferably dT), and a straight chain alkyl group having 1-10C atoms (and the alkyl chain comprising a functional group at the terminal C atom, in particular independently selected from amine, carbonyl, hydroxyl, sulfhydryl, carbonic acid groups), and a trifunctional moiety having 1-10C atoms and comprising at least one-OH, -SH and/or at least one-NH₂A group, preferably selected from lysine, serine, serinol, -O-CH₂-CH((CH₂)₄-NH₂)-CH₂-, glycerol, and 1,3 diaminoglycerol moieties.

In a further more preferred embodiment of the invention, linker L2 is independently selected from the group consisting of phosphate, amide, carbamate, ester group and moiety

- [O-CH₂-CH₂]_y2- (SP)_n]_m1-,

Wherein

SP and n are as defined above, preferably n =0,

y2 is an integer from 1 to 30, preferably from 3 to 10, and

m1 is an integer from 1 to 10, preferably from 1 to 3.

PEG-based linkers, i.e., TEG linkers, are shown to be useful in exemplary compounds used in the present invention. An exemplary compound is 5 '-cholesteryl TEG- (spacer C18) 7-Fluos-biotin TEG-3'.

The compounds used according to the invention and their intermediates can be prepared by methods known to the skilled worker. An exemplary synthesis of the compounds used in the present invention is shown in fig. 6C. Further, intermediates used in the synthesis of the compounds used in the present invention are shown in fig. 12. Furthermore, the general concept of the synthesis of the compounds is briefly described in example 1. The compounds can be prepared on solid phases analogously to phosphoramidite-based synthesis of nucleotides. The compounds may be synthesized by synthesis on a solid support such as CPG as described in the examples. In particular, the compounds can be synthesized by a subsequent coupling step and cleavage from a solid support (in the example: CPG (controlled pore glass)) under conditions known to the skilled person. Other solid supports such as macroporous polystyrene may also be used for the synthesis. The synthesis may be performed by retaining the protecting group or by cleaving the protecting group. Specifically, the compounds can be synthesized with or without DMT (DMT on), leaving the DMT molecule at the end of the molecule referred to as the 3' end, or by cleaving off the DMT group. The compound is optionally further purified, for example by dialysis.

The synthesis of biotin-PEG-Lys- (C18)2 is described in detail in FIG. 6C).

Other compounds for use in the present invention may be prepared in a similar manner according to methods known in the art.

In a still further embodiment, the present invention relates to a composition comprising at least one compound as described above bound to at least one cell, preferably a living cell. Such compositions are provided for fixed cells. Depending on the further presence of the label moiety and/or the linking group, the composition may further be used for detection and/or immobilization of the cell, respectively.

In a preferred embodiment, such compositions further comprise a solid support to which at least one compound used in the present invention is bound via a linking group. In such embodiments, at least one immobilized cell is immobilized to a solid support via a compound used in the invention. In case the compound further comprises a labeling moiety, it is possible to localize, detect and quantify the cells.

In another preferred embodiment, the composition comprising at least one compound for use according to the invention bound to at least one cell comprises an aqueous buffer solution, wherein the at least one cell to which the at least one compound for use according to the invention is bound is in suspension. Such compositions are suitable where the cells are sufficiently stable (e.g., in FACS or in centrifugation).

The compounds are suitable for binding to any cell comprising a lipid bilayer. Preferably, the cell is a eukaryotic cell, more preferably an animal cell, even more preferably a vertebrate cell, most preferably a human cell.

In a further preferred embodiment, the cell is a leukocyte, a rare cell, a tumor cell or a mutated cell, more preferably a vertebrate or human leukocyte, a rare cell, a tumor cell or a mutated cell.

In a further embodiment, the present invention relates to the use of a composition comprising one or more compounds as described above for stabilizing cells.

Thus, in another embodiment, the present invention relates to the use of a composition comprising at least three different compounds that can be used according to the present invention, wherein the different compounds differ at least in their hydrophobic domain, for stabilizing a cell.

By using a plurality of compounds used in the present invention, at least two of which differ in their hydrophobic domains, a composition can be obtained that effectively binds and stabilizes all cell types.

In an even more preferred embodiment, the composition thus comprises at least four, five, six, seven, eight, nine or ten different compounds for use according to the invention. In even more preferred embodiments, two, three, four, five, six, seven, eight, nine, ten or all compounds of such compositions differ at least in their hydrophobic domains.

Suitable preferred hydrophobic domains are those as defined above. For example, a composition comprising 5 '-cholesteryl TEG-PEG 2000-Fluos-3' may be used.

In a more preferred embodiment, one or all of the hydrophobic domains of at least one compound comprise, preferably consist of: saturated fatty acids, especially myristic, stearic or behenic acid, especially myristic acid; and/or one or all of the hydrophobic domains of at least one compound comprises, preferably consists of: steroids, in particular cholesterol, or hydrophobic vitamins, in particular alpha-tocopherol.

In a preferred embodiment, the invention relates to the use of an aqueous solution comprising one or more compounds for stabilizing cells.

The aqueous solution is preferably buffered. For example, the solution of the present invention may be Phosphate Buffered Saline (PBS), Tirs, and/or Hepes buffered solutions.

The pH of the solutions of the present invention is preferably from about 5.5 to 8.5, more preferably from 6.5 to 7.5.

In a further embodiment, the present invention relates to a method of stabilizing a cell, the method comprising:

a) providing a compound as defined above; and

b) contacting the cell with a composition under conditions that allow the compound to interact with the cell membrane, thereby stabilizing the cell, and

c) optionally, a shear force is applied to the cells.

In a preferred embodiment of the invention, the stabilization is stabilization during exposure of the cells to shear forces.

In a further preferred embodiment, the stabilization is stabilization of the cells during exposure to shear forces due to centrifugation, large scale cell culture, flow cytometry, fluorescence activated cell sorting and/or bead based cell separation.

In a further preferred embodiment, stabilization is stabilization of cells exposed to centrifugation, large scale cell culture, flow cytometry, fluorescence activated cell sorting, and/or bead based cell separation processes.

For compounds that can be used in such processes, the same embodiments apply with respect to the uses described above. The provision of the compounds to be used in the process of the invention is described above.

Compositions comprising two or more compounds as described above may also be used in the methods of the invention. Such compounds and their use according to the invention are described above.

The compounds may be contacted with the cells in step b) as an aqueous solution comprising one or more compounds used according to the invention. Such solutions are suitable for pipetting or otherwise adding to cells. The aqueous solution is preferably buffered. For example, the solution of the present invention may be Phosphate Buffered Saline (PBS), Tirs, and/or Hepes buffered solutions or solutions containing culture media. The pH of the solution is preferably about 5.5 to 8.5, more preferably 6.5 to 7.5.

Since stabilizing cells is accomplished with live cells or cells that may be viable, the cells are typically present in an aqueous solution (which is preferably buffered and/or contains nutrients), e.g., the cells are suspended in PBS or culture medium. The compounds used in the present invention can be added to the cells by methods known in the art such as pipetting, for example in the form of a solution, for example as an aqueous solution.

The pH of the cell suspension is preferably about 5.5-8.5, more preferably 6.5-7.5.

Mixing can be done gently to maintain cell viability.

The compound is contacted with the cell in step b) of the method of the invention. Typically, more than one cell will be present and contacted with a compound of the invention. Thus, it is preferred to add the compound to a composition comprising a plurality of cells, for example 2 or more, 10 or more, 50 or more, 100 or more, or 1000 or more cells. Preferably, such cell population is suspension cells. Thus, a solution comprising a compound as disclosed herein may be added to a cell suspension to be stabilized.

The cell population may be cells of the same or different cell types. For example, a leukocyte population comprising different cell types may be used, as in the examples (see example 5).

Typically, the contacting typically occurs at a temperature of about 1 ℃ to 45 ℃, preferably 10 ℃ to 30 ℃, more preferably 22 ℃ to 38 ℃.

Moreover, the contacting typically occurs at a pressure of about 900-.

Furthermore, the cells are preferably incubated with the compound for a sufficient time to allow binding of the cells. Generally, the cells are preferably incubated with the compound for 1 minute to 3 days, preferably 5 minutes to 24 hours, even more preferably 10 minutes to 8 hours.

Furthermore, the aqueous solution is typically selected so as not to affect the integrity and/or viability of the cells. Therefore, the solution preferably does not contain cytotoxic compounds.

Such conditions allow the interaction of the compound with the cell membrane, thereby stabilizing the cell.

The stabilization already takes place upon interaction without further application of shear stress. Thus, in one embodiment, no shear stress is applied to the cells after step b) of the method of the invention.

However, in a preferred embodiment, shear forces or shear stresses are applied to the cells after step b) of the method of the invention.

In a preferred embodiment of the cells, the shear force is applied to the cells by centrifugation, large scale cell culture, flow cytometry, fluorescence activated cell sorting and/or bead based cell separation, as described above. Thus, in a preferred embodiment, after step b) of the method of the invention, the cells are centrifuged, cultured on a large scale, subjected to flow cytometry, subjected to fluorescence activated cell sorting and/or separated using beads.

In a still further preferred embodiment of the invention the cell is a suspension cell and/or the cell is an animal or human cell, in particular a vertebrate cell, especially a mammalian cell. Such cells are particularly sensitive to shear forces and are therefore difficult to handle and manipulate without affecting viability. Even more preferably, the cells are suspended animal or human cells, in particular suspended vertebrate cells, especially suspended mammalian cells.

In an even more preferred embodiment, the cell or cell population is a leukocyte or leukocytes, respectively, which are even more preferably human leukocytes. The method of the invention effectively stabilizes such cells (example 5).

The method of the invention is carried out during centrifugation for cell stabilization.

Such centrifugation steps are for example performed for separating cells from surrounding liquid, such as culture medium. The cells must be centrifuged and therefore exposed to shear stress. Very sensitive and fragile cell populations may be damaged by such processes. The methods and uses of the invention improve the handling of such cell populations. In a preferred embodiment, the cells are centrifuged after step b) of the method of the invention. Preferably, they are centrifuged for 5 minutes or more, e.g. at 100g, 200g, 500g, 1000g or more, e.g. 1 hour or 5 hours.

The methods of the invention can also be used for biotechnological purposes such as cell stabilization in large-scale animal cell culture: it has been disclosed that shear sensitivity of mammalian cells can be a related problem complicating the development of large-scale animal cell cultures. The methods and uses of the present invention reduce these problems.

Thus, the invention also relates to the method of the invention for stabilizing cells in large scale animal cell culture. In a preferred embodiment, the cells are cultured after step b) of the method of the invention in large scale, for example by batch culture and/or in volumes of more than 10 ml, 50ml, 100 ml or 1l of liquid medium.

The methods of the invention can also be used for cell stabilization in flow cytometry and/or fluorescence activated cell sorting:

flow cytometry is a very common method of isolating specific cell populations. In this method, the cells are exposed to high shear stress depending on the flow rate. The method of the present invention reduces this shear stress.

Thus, the present invention also relates to a method of the invention for stabilizing cells in flow cytometry and/or fluorescence activated cell sorting or wherein the cells are exposed to flow cytometry and/or fluorescence activated cell sorting after step b) of the method of the invention.

The method of the invention can also be used for cell stabilization in bead-based cell separation methods:

cell populations with different phenotypes can be isolated by specific antibodies coupled to magnetic beads. In this method, cells are exposed to high shear stress depending on the bead size. The method of the present invention reduces this shear stress.

Thus, the methods of the invention can also be performed for cell stabilization in bead-based cell separation methods. In a preferred embodiment, the cells are coupled to beads, in particular magnetic beads, and separated, in particular magnetically separated, after step b) of the method of the invention.

In a still further embodiment, the present invention relates to the use of a kit comprising at least one compound or composition as described above for stabilizing cells.

The kit may further comprise two or more compounds for use in the invention stored separately, e.g. in containers or syringes. They may be stored in dry form, e.g. lyophilized or dried, or as a solution, or in frozen form, e.g. as a frozen solution.

In case the compound further comprises a labeling moiety and/or a linker group, the stable cell to which the compound binds may furthermore be used for detection and/or characterization of rare cells, preferably for one rare cell characterization.

In such uses, nucleated cells isolated from leukocytes can be immobilized on a defined surface using the compounds used in the invention on an array, preferably a microarray or a nanoarray. Rare cells in the population of nucleated cells, for example in a population of White Blood Cells (WBCs), such as circulating tumor cells, endothelial cells, or epithelial cells, can be quantitatively bound to the surface and identified via antibodies or specific binding molecules directed against an antigen or biochemical characteristic specific to the rare cell population. This enables accurate positioning and repositioning for further characterization steps (if required).

The compounds further comprising a linking group may further be used for immobilizing suspension cells, preferably for screening, even more preferably for screening with antibodies or antibody fragments binding to antigens or other forms of binding molecules. Screening of cultured cell lines for antibodies or antigen-binding fragments thereof is a common application in antibody development. One application involves the binding of antibodies to specific receptor molecules on the cell surface. Using secondary antibodies (sandwich effect), the characteristics of the primary antibody can be studied. Such experiments are difficult to perform using suspended cells. The developed compounds for cell fixation allow careful fixation of suspended cells without losing any physiological cellular properties and can thus be used to perform such screening assays. In addition, suspension cells can be immobilized for functional cell assays using the compounds used in the present invention. Assays to study cell function in vitro or in vivo are important: functional cell assays are commonly used in pharmaceutical, pesticide, and biotechnology research and development to study small molecule compounds or biologics or to identify classes of small molecules in high throughput screening. Some functional assays are based on surface-dependent assays and are therefore typically performed with adherent cells.

In a preferred embodiment, the uses and methods of the invention are in vitro uses and methods.

Compounds further comprising a linking group are further useful for binding living cells to a solid surface, followed by detachment from the surface and implantation into a mouse model. These types of functional assays are of particular importance, for example, for studying the tumor-inducing potential of circulating abnormal cells.

The compounds are further useful in lab-on-a-chip (a lab on a chip): to study the cell morphology or cell function of a small number of cells, e.g. 2-50 cells or single cells, the surface may optionally or systematically be spotted (spotted) with compounds further comprising a linker group for use in the present invention. Such spotting allows targeted fixation of a few cells or single cells on such spots. This allows direct molecular analysis on the surface (chip). The chip may be an array, in particular a microarray or a nanoarray.

The compounds used in the present invention further comprising a linking group may be bound to a solid substrate. Such solid substrates may be particles such as nanoparticles, in particular magnetic nanoparticles, columns, or flat substrates, arrays or well plates, in particular oligo-well plates or multi-well plates.

Disclosed is a method of labeling a cell, the method comprising:

a) providing a compound for use in the present invention, wherein said compound comprises a labeling moiety; and

b) contacting the cell with the compound under conditions that allow the compound to interact with the cell membrane, thereby immobilizing the label on the cell; and

c) optionally detecting the label.

As shown in the examples, the compounds used in the present invention (wherein the compounds comprise a labeling moiety) are contacted with a cell. Since labeling is preferably accomplished with live cells or potentially live cells, the cells are typically present in an aqueous solution (which is preferably buffered and/or contains nutrients), e.g., cells suspended in PBS. The labelled compounds used in the present invention may be added to the cells by methods known in the art such as pipetting, for example in the form of a solution, for example as an aqueous solution.

Typically, the contacting occurs at a temperature of about 1 ℃ to 45 ℃, preferably 10 ℃ to 30 ℃, more preferably 22 ℃ to 38 ℃.

Moreover, the contacting occurs at a pressure of about 900-.

Furthermore, the cells are preferably incubated with the compound for a sufficient time to allow binding. Generally, the cells are preferably incubated with the compound for 1 minute to 3 days, preferably 5 minutes to 24 hours, even more preferably 10 minutes to 8 hours.

Furthermore, the aqueous solution is typically selected so as not to affect the integrity and/or viability of the cells.

Such conditions allow the interaction of the compound with the cell membrane. Whereby the label moiety is immobilized on the cell.

The label moiety, and hence the cell, can be detected as described above, depending on the label moiety selected. In the case of direct labeling, detection may occur directly, for example by detecting fluorescence of fluorescein or absorbance of dT, as shown in the examples.

In the case of an indirect detection system, the second member of the binding pair may be detected. For example, a biotin-labeled compound used in the present invention may be used. For detection, streptavidin may be used, which in turn is labeled with a directly detectable label. Thus, depending on the further step, biotin may represent a linker group or a labeling moiety of the invention.

For cell-independent labeling, certain compounds of the invention described above may be used.

Disclosed is a method for labeling a cell, the method comprising

a) Providing a composition comprising at least three different compounds for use according to the invention, wherein the different compounds differ at least in their hydrophobic domain and wherein the different compounds comprise a labeling moiety,

b) contacting the cell with the composition under conditions that allow the compound to interact with the cell membrane, thereby labeling the cell; and

c) optionally detecting the label.

Thus, the composition is preferably a solution, more preferably an aqueous solution comprising the compound used in the present invention.

A method of immobilizing a linking group on the surface of a cell is disclosed, the method comprising

a) Providing a compound for use in the present invention, wherein the compound comprises a linking group; and

b) contacting the cell with the compound under conditions that allow the compound to interact with the cell membrane, thereby immobilizing the linking group.

With respect to antibodies and antibody fragments that bind to antigens, the skilled artisan is aware of such molecules: naturally occurring antibodies are globular plasma proteins (-150 kDa (http:// en. wikipedia. org/wiki/Dalton _ unit)), also known as immunoglobulins, which have a basic structure. They are glycoproteins because they have sugar chains added to amino acid residues. The basic functional unit of each antibody is an immunoglobulin (Ig) monomer (comprising only one Ig unit); secreted antibodies may also be dimerized with two Ig units, such as IgA, tetramerized with four Ig units, such as teleost IgM, or pentamerized with five Ig units, such as mammalian IgM. In the present invention, examples of suitable formats include formats of naturally occurring antibodies including antibody isotypes known as IgA, IgD, IgE, IgG and IgM.

In addition to naturally occurring antibodies, artificial antibody formats have also been developed that include antibody fragments. Some of which are described below.

Although the general structure of all antibodies is very similar, the unique properties of a given antibody are determined by the variable (V) region, as detailed above. More specifically, the variable loops (three each on the light (VL) chain and three on the heavy (VH) chain) are responsible for binding to the antigen, i.e. for its antigen specificity. These loops are called Complementarity Determining Regions (CDRs). Since the CDRs from both the VH and VL domains form the antigen binding site, it is the combination of the heavy and light chains, not either one alone, that determines the final antigen specificity.

Thus, the term "antibody" as used herein means any polypeptide having structural similarity to naturally occurring antibodies and capable of specifically binding to the respective target, wherein the binding specificity is determined by the CDRs. Thus, an "antibody" is intended to refer to an immunoglobulin-derived structure that binds to each target, including, but not limited to, a full-length or intact antibody, an antigen-binding fragment (a fragment derived (physically or conceptually) from an antibody structure), a derivative of any of the former, a chimeric molecule, a fusion of any of the former with another polypeptide, or any alternative structure/composition that selectively binds to each target. The antibody or functionally active portion thereof may be any polypeptide comprising at least one antigen binding fragment. An antigen-binding fragment consists of at least the variable domain of the heavy chain and the variable domain of the light chain, arranged in such a way that both domains are simultaneously capable of binding a specific antigen.

A "full-length" or "complete" antibody refers to a protein comprising two heavy (H) and two light (L) chains interconnected by disulfide bonds, comprising: (1) in the heavy chain aspect, the variable region and the heavy chain constant region comprising three domains CH1, CH2, and CH 3; and (2) in the light chain aspect, a light chain variable region and a light chain constant region comprising one domain CL.

An "antigen-binding antibody fragment" or "antigen-binding fragment thereof" also comprises at least one antigen-binding fragment as defined above and exhibits substantially the same function and binding specificity as a full antibody from which the functionally active portion (or fragment) is derived. Limited proteolytic digestion with papain cleaves the Ig prototype into three fragments. Two identical amino-terminal fragments, each comprising one complete L chain and about half an H chain, are antigen-binding fragments (Fab). The third fragment, which is similarly sized but contains the carboxy-terminal half of the two heavy chains with their interchain disulfide bonds, is the crystallizable fragment (Fc). The Fc comprises a carbohydrate, a complement binding site, and an FcR binding site. Limited pepsin digestion produces a single F (ab')2 fragment containing both a Fab fragment and a hinge region, including the H-H interchain disulfide bond. F (ab')2 is bivalent for antigen binding. The disulfide bond of F (ab ')2 can be cleaved to obtain Fab'. In addition, the variable regions of the heavy and light chains may be fused together to form a single chain variable fragment (scFv).

Variable domains (Fv) are the smallest fragments with an entire antigen-binding domain consisting of one VL and one VH. Such fragments, having only binding domains, can be produced by enzymatic methods or by expression of relevant gene fragments (e.g., in bacterial and eukaryotic cells). Different approaches can be used, such as Fv fragments alone or 'Fab' fragments comprising one of the upper arms of a "Y" comprising Fv plus a first constant domain. These fragments are usually stabilized by introducing a polypeptide linkage between the two chains, which results in the production of single chain fv (scFv). Alternatively, disulfide-linked fv (dsfv) fragments may be used. The binding domains of the fragments may be combined with any constant domain to produce full length antibodies or may be fused to other proteins and polypeptides.

The recombinant antibody fragment is a single chain fv (scfv) fragment. Dissociation of the scFv results in monomeric scFv that can complex into dimers (diabodies), trimers (triabodies), or larger aggregates such as TandAbs and Flexibodies.

Antibodies with two binding domains can be generated via binding of two scfvs with a simple polypeptide linker (scFv) 2 or via dimerization of two monomers (diabodies). The simplest design is a diabody with two functional antigen-binding domains, which can be identical, similar (bivalent diabody) or have specificity for different antigens (bispecific diabody).

Furthermore, antibody formats comprising four variable domains of the heavy chain and four variable domains of the light chain have been developed. Examples of these include tetravalent bispecific antibodies (TandAbs and Flexibodies, affected Therapeutics AG, heidelberg. Flexibodies are a combination of scFv and diabody multimer motifs, resulting in multivalent molecules with high flexibility for linking two molecules very distant from each other on the cell surface. An antibody is multispecific if more than two functional antigen-binding domains are present and if they are specific for different antigens.

In general, specific immunoglobulin classes that represent antibodies or antigen-binding fragments thereof include, but are not limited to, the following antibodies: fab (monovalent fragment with variable light chain (VL), variable heavy chain (VH), constant light Chain (CL) and constant heavy chain 1 (CHl) domains), F (ab')2 (bivalent fragment comprising two Fab fragments linked by a disulfide bond or optionally at the hinge region), Fv (VL and VH domains), scFv (single chain Fv wherein VL and VH are linked by a linker, e.g., a peptide linker), bispecific antibody molecules (antibody molecules with specificity as described herein and linked to a second functional moiety with different binding specificity than the antibody, including but not limited to another peptide or protein such as an antibody or receptor ligand), bispecific single chain Fv dimers, diabodies, triabodies, tetrabodies, minibodies (scFv linked to CH 3).

The antibody may be a monoclonal antibody, a chimeric antibody or a humanized antibody.

The tag is a peptide motif used for identification in biotechnology. As is well knownThe tag is His tag (6 x histidine), which can bind Ni²⁺And (3) a column.

In the case of nucleic acids or nucleic acid analogues/complementary nucleic acids as binding pairs, any nucleic acid sequence and its complement may be used.

Lectins are carbohydrate-binding proteins that are highly specific for sugar moieties. As suitable lectins concanavalin a can be used, which binds to alpha-D-mannosyl and alpha-D-glucosyl residues, branched alpha-mannoside (mannosidic) structures (high alpha-mannose type, or hybrid type and biantennary complex type N-glycans.

As receptor/ligand binding pairs, for example, steroid hormone receptors/steroid hormones may be used. For example, estrogens and their receptors can be used as the corresponding binding partners.

Drawings

FIG. 1: plate used in the experiment of example 6: streptavidin-treated MTP (Microcoat), 12 well, NUNC, MC ID: 604176, lot number 1665C 2

FIG. 2: the experimental design of example 6 is shown. 4 x. Line A: 200 μ l of PBS was introduced, 1nmol of compound was added thereto, respectively, mixed, incubated for about 30min, 2 × PBS was washed, 800 μ l of PBS was introduced, and 300.000 WBC (untreated) was added. Line B: 800 μ l PBS was introduced and 300.000 WBC (untreated) was added. Line C: 10x10^6 WBC in 1ml were incubated with 10nmol of the compound of the invention for 10min, 800 μ l PBS/well, 300.000 treated WBC respectively. After 30min the first MTP plate was washed 2x with PBS, covered with hchst and incubated for 15 min. Measurement > Cellavista (operators 9s 5). The second plate was measured after 90 min. The third panel was measured after 150 min.

FIG. 3: the results of example 6 are shown after 30, 90 or 120 min incubation.

FIG. 4: the results of example 6 after 30, 90 or 120 min incubation are shown as a graph.

FIG. 5: the plates of example 6 are shown after 30, 90 or 150 minutes incubation.

FIG. 6: chemical structures of exemplary compounds used in the present invention and by-products of the synthesis. And C, synthesizing biotin-PEG-Lys- (C18)2 and synthesizing byproducts.

FIG. 7: cells were shown stained with a) a cholesteryl group containing compound having internal reference number 29.891180, B) a myristic acid containing compound having internal reference number 29.891194. C) MDA-MB468 was stained. D) And E): different exposure times for staining cells with different compounds used in the invention are illustrated. Representative pictures according to example 3.

FIG. 8: a) And B) shows the results of an xCelelligence experiment with Jurkat cells according to example 3. B) 1, PBS + biotin linker; PBS +10% FCS + biotin linker; PBS +1% FCS + biotin linker; PBS without (w/o) biotin linker; PBS +10% FCS No (w/o) Biotin linker; PBS +1% FCS No (w/o) Biotin linker; 7: PBS + biotin linker without (w/o) SA, 8: PBS without biotin linker without SA.

FIG. 9: the results of the xCelligence experiment with WBC cells according to example 3 are shown. B) 1, PBS + biotin linker; PBS +10% FCS + biotin linker; PBS +1% FCS + biotin linker; PBS without (w/o) biotin linker; PBS +10% FCS No (w/o) Biotin linker; PBS +1% FCS No (w/o) Biotin linker.

FIG. 10: staining of the fixed cells according to example 3 is shown. Left column: DA-MB 468-antibody: k5/8. Middle fence: MDA-MB 468-antibody: EpCAM Miltenyi FITC. Right column: MDA-MB 468-antibody: an EGFR.

FIG. 11: staining of the fixed cells according to example 3 is shown. Left column: MDA-MB 468-antibody: EpCAMBiolegend. Middle fence: MDA-MB 468-antibody: EpCAM Miltenys APC. Right column: WBCs-antibodies: CD45 Biolegend.

FIG. 12: structures of further and reference compounds and intermediates used in the present invention are shown.

FIG. 13: WBC recovery after centrifugation and fixation with different molecular cells is shown. Molecular probes HH1749, HH1750, and HH1755 (biotin-PEG-cytolytic (Lysin) - (C18)2) showed different performance on recovery after centrifugation: the higher the molecular concentration, the higher the cell recovery after centrifugation. Centrifugal characteristics: 10min, 300 x g.

FIG. 14: WBC recovery after centrifugation and fixation with different molecular cells is shown. Molecular probes HH1749, HH1750, and HH1755 showed different properties with respect to cell immobilization rates at different concentrations. The higher the compound concentration, the higher the cell fixation rate.

FIG. 15: WBC recovery after centrifugation using different compounds at different time points is shown. Molecules A and B (A: cholesteryl-TEG- (spacer C18) 7-Fluos-biotin-TEG; B: biotin-PEG-cytolytic enzyme- (C18)2) showed different properties with respect to recovery after centrifugation. Each left column: no compound of the invention; each left column: 0.35 nmol of molecule a; third column on each left: 100 nmol of molecule B; each right column: 0.5 nmol of molecule B. The higher the molecular concentration, the higher the cell recovery after centrifugation. Molecule B was able to fix the cells within 3.5 hours. Centrifugal characteristics: 10min, 300 x g.

FIG. 16: WBC recovery after centrifugation is shown for different experimenters. Each of the left, center and right bars of each assay represents a different experimenter 1, 2 and 3. The higher the molecular concentration, the higher the cell recovery after centrifugation. Furthermore, cell stability was independent of the experimenter. Centrifugal characteristics: 10min, 300 x g. Molecule (a): cholesteryl-TEG- (spacer C18) 7-Fluos-biotin-TEG.

FIG. 17: WBC recovery after centrifugation at different time points and at centrifugation settings is shown. The following molecules were tested: 1234:5'- (cholesteryl-TEG) 2-spacer C18-dT-biotin-TEG-3'; 1248:3'- (cholesteryl-TEG) 2-PEG 2000-Fluos-biotin-TEG-5' INVERS; 1254:3'- (cholesteryl-TEG) 2-spacer C18-Fluos-biotin-TEG-5' INVERS; 1255:3'- (myristic acid) 2-PEG 2000-dT-biotin-TEG-5' INVERS. All molecules were able to be cell fixed within 2 hours. WBCs in PBS were damaged during centrifugation at 300 x g for 20 min. Molecule 1234 showed the best performance, followed by compounds 1255 and 1254. Centrifugal characteristics: 20 min,300 x g. Each left column: incubate with molecules for 10 min. Each center pillar: incubate with molecules for 1 h. Each right column: incubate with molecules for 2 hours.

FIG. 18: WBC recovery after centrifugation at different time points and at centrifugation settings is shown. The following molecules were tested: 1255:3'- (myristic acid) 2-PEG 2000-dT-biotin-TEG-5' INVERS, 1234:5'- (cholesteryl-TEG) 2-spacer C18-dT-biotin-TEG-3', 1248:3'- (cholesteryl-TEG) 2-PEG 2000-Fluos-biotin-TEG-5' INVERS, 1254:3'- (cholesteryl-TEG) 2-spacer C18-Fluos-biotin-TEG-5' INVERS. All molecules were able to be cell fixed within 2 hours. WBCs in PBS were damaged during centrifugation at 500 x g for 20 min. Molecule 1234 showed the best performance, followed by molecules 1255 and 1254. Centrifugal characteristics: 20 min, 500 x g. Each left column: incubate with molecules for 10 min. Each center pillar: incubate with molecules for 1 h. Each right column: incubate with molecules for 3 hours.

FIG. 19: WBC recovery after centrifugation at different time points and at centrifugation settings is shown. The following molecules were tested: 1255:3'- (myristic acid) 2-PEG 2000-dT-biotin-TEG-5' INVERS, 1234:5'- (cholesteryl-TEG) 2-spacer C18-dT-biotin-TEG-3', 1248:3'- (cholesteryl-TEG) 2-PEG 2000-Fluos-biotin-TEG-5' INVERS, 1254:3'- (cholesteryl-TEG) 2-spacer C18-Fluos-biotin-TEG-5' INVERS. All molecules were able to be cell fixed within 2 hours. WBCs in PBS were damaged during centrifugation at 1000 x g for 20 min. Centrifugal characteristics: 20 min, 1000 x g. Each left column: incubate with molecules for 10 min. Each center pillar: incubate with molecules for 1 h. Each right column: incubate with molecules for 2 hours.

FIG. 20: recovery of Jurkat cells after centrifugation at different time points is shown. Counting columns from the left: 1, incubation with the molecule for 10 min. 2 incubation with molecules for 1 hour. 3 incubation with molecules for 3.5 hours. 4, incubation with the molecule for 5.5 h min. The following molecules were tested: 1255 3'- (myristic acid) 2-PEG 2000-dT-biotin-TEG-5' INVERS. 1234, 5'- (cholesteryl-TEG) 2-spacer C18-dT-biotin-TEG-3', 1248, 3'- (cholesteryl-TEG) 2-PEG 2000-Fluos-biotin-TEG-5' INVERS, 1254, 3'- (cholesteryl-TEG) 2-spacer C18-Fluos-biotin-TEG-5' INVERS. Jurkat cultured cells were stable in PBS during centrifugation and within 5.5 hours using different molecules. Centrifugal characteristics: 20 min, 500 x g.

FIG. 21: the trifunctional linker moiety was shown not to affect cell viability. Cell viability assays using the WST-1 proliferation kit (RAS) were performed using different compounds used in the present invention, which differ in the trifunctional linker moiety. Different linkers did not appear to affect cell viability during the 4 hour linker incubation time. A) 2 hours and B) viability test after 4 hours.

FIG. 22: the trifunctional linker moiety was shown not to affect cell viability. Test compound found to be used in the present invention, i.e. a): no. 1244 and B as compounds having a cholesterol moiety): 1274, which is a compound with stearic acid moieties, did not affect cell morphology during the 4.5 hour linker incubation time. Left panel: incubate for 1 hour. The middle graph is as follows: incubate for 2.5 hours. Right panel: incubate for 4.5 hours. The upper diagram: bright field. The following figures: DAPI.

FIG. 23: shows the morphology of cells incubated without linker at different time points. Without the addition of the compounds used according to the invention, the cells spread out during an incubation time of 4.5 hours (dispersion away). Cell morphology in the left cells was unaffected during the incubation time. Left panel: incubate for 1 hour. The middle graph is as follows: incubate for 2.5 hours. Right panel: incubate for 4.5 hours. The upper diagram: bright field. The following figures: DAPI.

FIG. 24: showing recovery of MDA-MB468 cells after centrifugation at different time points. Counting columns from the left: 1, incubation with the molecule for 10 min. 2 incubation with molecules for 1 hour. 3 incubation with molecules for 3 hours. And 4, incubating the mixture with the molecules for 5 h. The following compounds used in the present invention were tested: 5'- (cholesteryl-TEG) 2-spacer C18-dT-biotin-TEG-3' 1234, 3'- (myristic acid) 2-PEG 2000-dT-biotin-TEG-5' INVERS. Cells cultured in PBS during centrifugation and using different molecules used in the present invention were stable within 5 hours of MDA-MB 468. Centrifugal characteristics: 20 min, 500 x g.

Example 5: stabilization of cells using compounds useful in the methods of the invention

The effect of compounds useful in the methods of the invention on stabilizing cells and on immobilization is determined.

A)WBC recovery after centrifugation and fixation with different molecular cells

As shown in figure 14, molecular probes HH1749, HH1750, and HH1755 (, biotin-PEG-cytolytic- (C18)2) showed different properties with respect to recovery after centrifugation: the higher the molecular concentration, the higher the cell recovery after centrifugation. Centrifugal characteristics: 10min, 300 x g. As can be seen from fig. 15, molecular probes HH1749, HH1750, and HH1755 showed different properties with respect to cell immobilization rates at different concentrations. The higher the linker concentration, the higher the cell fixation rate.

B)WBC recovery after centrifugation with different Compounds-different time points

As can be seen from FIG. 16, molecules A and B (A: cholesteryl-TEG- (spacer C18) 7-Fluos-biotin-TEG; B: biotin-PEG-cytolytic enzyme- (C18)2) show different properties with respect to recovery after centrifugation. The higher the molecular concentration, the higher the cell recovery after centrifugation. Molecule B was able to fix the cells within 3.5 hours. Centrifugal characteristics: 10min, 300 Xg, A cholesteryl-TEG- (spacer C18) 7-Fluos-biotin-TEG. B Biotin-PEG-cytolytic enzyme- (C18)2

C)WBC recovery after centrifugation-different experiments

As seen in fig. 17, the higher the concentration of molecules, the higher the cell recovery after centrifugation. Furthermore, cell stability was independent of the experimenter. Centrifugal characteristics: 10min, 300 x g. Molecule (a): cholesteryl-TEG- (spacer C18) 7-Fluos-biotin-TEG.

D)WBC recovery after centrifugation-different time points and centrifugation settings

The results of the first experiment are shown in fig. 18. The following molecules were tested:

5'- (cholesteryl-TEG) 2-spacer C18-dT-Biotin-TEG-3'

3'- (Cholesterol-TEG) 2-PEG 2000-Fluos-Biotin-TEG-5' INVERS 1254:3'- (Cholesterol-TEG) 2-spacer C18-Fluos-Biotin-TEG-5' INVERS

1255 '- (myristic acid) 2-PEG 2000-dT-Biotin-TEG-5' INVERS

All molecules were able to cell fix within 2 hours.

WBC-compromised wounds in PBS during centrifugation at 300 x g for 20 min

Molecule 1234 showed the best performance followed by compounds 1255 and 1254.

Centrifugal characteristics: 20 min,300 x g.

The results of the second experiment in this context are shown in fig. 19. The following molecules were tested:

1255:3'- (myristic acid) 2-PEG 2000-dT-biotin-TEG-5' INVERS 1234:5'- (cholesteryl-TEG) 2-spacer C18-dT-biotin-TEG-3'

1248 3'- (Cholesterol-TEG) 2-PEG 2000-Fluos-Biotin-TEG-5' INVERS

1254 '- (Cholesterol-TEG) 2-spacer C18-Fluos-Biotin-TEG-5' INVERS

The results are as follows:

all molecules were able to cell fix within 2 hours.

WBC-injured in PBS during centrifugation at 500 x g for 20 min

Molecule 1234 showed the best performance, followed by compounds 1255 and 1254

Centrifugal characteristics: 20 min, 500 x g. The results of the third experiment in this context are shown in fig. 20. The following molecules were tested:

1255 '- (myristic acid) 2-PEG 2000-dT-Biotin-TEG-5' INVERS

5'- (cholesteryl-TEG) 2-spacer C18-dT-Biotin-TEG-3'

1248 '- (Cholesterol-TEG) 2-PEG 2000-Fluos-Biotin-TEG-5' INVERS

1254 '- (cholesteryl-TEG) 2-spacer C18-Fluos-Biotin-TEG-5' INVERS

The results are as follows:

all molecules were able to cell fix within 2 hours.

WBC-injured in PBS during centrifugation at 1000 x g for 20 min

Centrifugal characteristics: 20 min, 500 x g.

E)Jurkat recovery after centrifugation-different time points

The results of this experiment are shown in fig. 21. The following molecules were tested: :

1255 '- (myristic acid) 2-PEG 2000-dT-Biotin-TEG-5' INVERS

5'- (cholesteryl-TEG) 2-spacer C18-dT-Biotin-TEG-3'

1248 '- (Cholesterol-TEG) 2-PEG 2000-Fluos-Biotin-TEG-5' INVERS

1254 '- (cholesteryl-TEG) 2-spacer C18-Fluos-Biotin-TEG-5' INVERS

The results are as follows:

jurkat cultured cells were stable in PBS during centrifugation and within 5.5 hours using different molecules.

Centrifugal characteristics: 20 min, 500 x g.

F)The trifunctional linker moiety does not affect cell viability

The results of the first experiment in this context are shown in fig. 22A and B.

Cell viability assays using the WST-1 proliferation kit (RAS) were performed using different molecules that differ in the trifunctional linker moiety that can be used in the methods of the invention.

Different linkers did not affect cell viability during the 4 hour linker incubation time, as can be seen in fig. 22.

The results of the second experiment in this context are shown in fig. 23A and B. It was found that the molecules tested that can be used in the method of the invention (nos. 1244 and 1274) do not affect the cell morphology during the 4.5 hour linker incubation time.

G)Cell morphology without incubation with molecules useful in the methods of the invention-different time points

The results of this experiment are shown in fig. 24. The following were found:

cells expand during the 4.5 hour incubation time (use away) when no molecules are added that can be used in the method of the invention.

Cell morphology in the left cells was unaffected during the incubation time.

H)MDA-MB468 recovery after centrifugation-at different time points

The results of this experiment are shown in fig. 25. The following compounds were tested for use in the methods of the invention:

5'- (cholesteryl-TEG) 2-spacer C18-dT-Biotin-TEG-3'

1255 '- (myristic acid) 2-PEG 2000-dT-Biotin-TEG-5' INVERS

The following were found:

cells cultured in PBS during centrifugation and using different molecules useful in the methods of the invention were stable within 5 hours.

Centrifugal characteristics: 20 min, 500 x g.

Example 6: SA plates incubated with Compounds useful in the methods of the invention (streptavidin plates) in comparison to Comparison of WBC (white blood cells) incubated with Compounds useful in the methods of the invention

Starting material 5'- (cholesteryl-TEG) 2-PEG 2000-Fluos-Biotin-TEG-3' INVERS was used

(14530pmol/μ l) (internal reference: 29.891250) and streptavidin-treated MTP (Microcoat), 12-well plate.

Erythrocyte lysis was performed as follows:

EDTA-Whole blood 59.4236.400 WBC/μ l (Amblanz Roche)

Lysis buffer: 100mM NH4Cl + 5mM Hepes + 0.5mM KHCO3 + 0.1mM EDTA-K

Ca 1x8ml whole blood was loaded into 50ml Falcon tubes with lysis buffer and incubated for 10 minutes at room temperature.

Centrifuge at 250g for 15 minutes, resuspend pellet by pipetting in lysis buffer; add up to 50ml with lysis buffer

Centrifugation at 250g for 15 min, resuspension of the pellet with PBS, addition to 50ml with PBS, centrifugation at 250g for 15 min, addition to 50ml with PBS.

WBC measured in Sysmex

1:37.100 WBC / µl

The experimental design of the plates is explained below (see fig. 2):

3x 12 well MTP treatment of WBCs with compounds useful in the methods of the invention:

4x determination:

line A: introducing 200 μ l PBS, adding 1nmol compound to the PBS, mixing, incubating for about 30min, washing 2 × PBS,

800 μ l PBS was introduced and 300.000 WBC (untreated) was added.

Line B: 800 μ l PBS was introduced and 300.000 WBC (untreated) was added.

Line C: 1ml of 10x10^6 WBC incubated with 10nmol of a compound useful in the methods of the invention for 10 min.

800 μ l PBS/well, 300.000 treated WBC were introduced, respectively.

After-30 min the first MTP plate was washed 2x with PBS, covered with hchst and incubated for 15 min. Measurement > Cellavista (operators 9s 5).

The second plate was measured after-90 min.

The third panel was measured after-150 min.

The calculation results are shown in fig. 3. A graph representing these results is depicted in fig. 4. The experimental plate is shown in figure 5.

The process of the invention was found to be clearly and surprisingly advantageous.

Claims (15)

1. Use of a compound comprising, preferably consisting of, two or more hydrophobic domains linked to a hydrophilic domain for stabilizing a cell, wherein the two or more hydrophobic domains are covalently bound to the hydrophilic domain, and wherein the two 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.

2. The use according to claim 1, wherein the compound comprises, preferably consists of, two or more hydrophobic domains and one hydrophilic domain,

wherein the two or more hydrophobic domains are covalently bound to the hydrophilic domain, and

wherein the two or more hydrophobic domains each comprise a linear lipid, a steroid, or a hydrophobic vitamin, and

wherein the hydrophilic domain comprises a compound of formula (I):

X1-[A1 -(L1)_n]_k1-Z - [A2 -(L1)_n]_k2- X2 (I)，

wherein

Z is a group containing 1 to 100, preferably 1 to 50, more preferably 4 to 30-O-CH₂-CH₂-a linear polyethylene glycol (PEG) moiety of a moiety, wherein said polyethylene glycol moiety optionally comprises one or more linkages connecting two-O-CH₂-CH₂-a spacer portion SP of the portion,

and wherein said linear PEG moiety optionally comprises a linker moiety L3 at one or both termini,

each L1 is a linker moiety selected independently of the other,

each n is 0 or 1, independently selected from each other,

a1 and A2 are difunctional or trifunctional moieties selected independently of one another, with the proviso that at least one of A1 or A2 is trifunctional,

k1 and k2 are integers from 0 to 10, selected independently of each other, with the proviso that at least one of k1 and k2 is not 0,

x1 and X2 are independently selected from hydrogen or a protecting group,

l3 is independently selected from a linear alkyl or alkenyl chain having 1 to 10C atoms, which is optionally (i) interrupted by 1 to 3N, O or S atoms, and/or (ii) substituted by 1 to 4 hydroxy, carbonyl, amino or mercapto groups,

and is

Wherein the two or more hydrophobic domains are covalently bound to the hydrophilic domain via the trifunctional domain,

or a salt thereof.

3. The use of claim 2, wherein Z in formula (I) has the following structure:

-(L3)_n2- [O-CH₂-CH₂]_y- (SP)_n1]_m-[O-CH₂-CH₂]_y1-(L3)_n2，

wherein

SP is a spacer moiety that is a spacer moiety,

each spacer moiety SP is selected independently of the other,

each n1 is 0 or 1, independently selected for each m portion,

each n2 is 0 or 1, selected independently of each other,

m is an integer of 1 to 100, preferably 1 to 50, more preferably 4 to 30,

y is an integer from 1 to 100, preferably from 1 to 50, more preferably from 4 to 30,

y1 is an integer from 0 to 30, preferably from 0 to 10, more preferably from 0 to 4,

provided that y m + y1 is 100 or less,

l3 is independently selected from a linear alkyl or alkenyl chain having 1 to 10C atoms, which is optionally (i) interrupted by 1 to 3N, O or S atoms, and/or (ii) substituted by 1 to 4 hydroxy, carbonyl, or mercapto groups.

4. The method of claim 3, wherein

(a) n1 for m parts- [ O-CH₂-CH₂]_y- (SP)_n1]Are identical, and/or

(b) y1 is 0, and/or

(c) y is 4, 5 or 6 and n1 is 1, or

(d) y is 4, 5 or 6 and n1 is 1, and/or

(e) The spacer moieties SP are independently from each other selected from the group consisting of phosphate and bifunctional moieties, and/or

(f) n2 are all 0, or

(g) One or two n2=1, and L3 is an alkyl group having 1-10C atoms, optionally containing an amide, carbonyl, carbamate, and/or NH group.

5. The use of any one of claims 2-4, wherein X1 or X2 is substituted with a hydrophobic domain.

6. The method of any one of claims 1-5, wherein the linear lipid is

(a) Saturated or unsaturated fatty acids, and/or

(b) Fatty acids having 8 to 26C atoms, preferably 12 to 22C atoms,

more preferably, the linear lipid is selected from oleic acid, myristic acid, stearic acid and behenic acid, more preferably from myristic acid and oleic acid.

7. Use according to any one of claims 1 to 5, wherein

(a) The steroid is a sterol, or

(b) The steroid is selected from cholesterol; a steroid hormone, preferably a gonadal steroid, more preferably an androgen such as a anabolic steroid, androstenedione, dehydroepiandrosterone, dihydrotestosterone, or testosterone, an estrogen such as estradiol, estriol, or estrone; progestogens, such as progesterone or progestin, corticosteroids, especially glucocorticoids or mineralocorticoids; ecdysteroids such as ecdysterone; a phytosterol; brassinosteroids; hopane species; and an ergosterol, and a salt of ergosterol,

more preferably wherein the steroid is cholesterol, or

(c) The hydrophobic vitamin is alpha-tocopherol.

8. Use according to any one of claims 1 to 7, wherein

(a) Two, three or four, preferably two or three hydrophobic domains are covalently bound to the hydrophilic domain, and/or

(b) The two or more hydrophobic domains covalently bound to the hydrophilic domain are different or identical, preferably identical.

9. Use according to any one of claims 1 to 8, wherein the hydrophobic domain

(a) Consisting of linear lipids, steroids or hydrophobic vitamins, or

(b) Comprising a linear lipid, steroid or hydrophobic vitamin covalently bound to a trifunctional moiety a1 via a linker moiety L2, wherein L2 comprises, preferably consists of: phosphate group, moiety

- [O-CH₂-CH₂]_y2- (SP)_n]-，

Wherein SP and n are as defined above, preferably n =0, y2 is an integer from 1 to 30, preferably from 3 to 10, and m1 is an integer from 1 to 10, preferably from 1 to 3, a glycerol moiety, a carbamate group, an amide group, a linear alkyl group having from 1 to 10C atoms, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 atoms, and said alkyl chain comprising a functional group at the terminal C atom, in particular being independently selected from amine, carbonyl, hydroxyl, mercapto, carbonate groups, said alkyl group being optionally substituted by 1, 2, 3, 4 or 5 moieties R1, wherein R1 is independently C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 aminoalkyl, C1-C4 cyanoalkyl, hydroxyl, mercapto, amino or carbonyl moiety, more preferably a C1-C4 alkyl, C-4 hydroxyalkyl, C1-C4 aminoalkyl, C1-C4 cyanoalkyl

L2 is selected from the group consisting of phosphate, amide, carbamate, ester group and moiety

- [O-CH₂-CH₂]y₂- (SP)_n]_m1-，

Wherein

SP and n are as defined above, preferably n =0,

y2 is an integer from 1 to 30, preferably from 3 to 10, and

m1 is an integer from 1 to 10, preferably from 1 to 3,

more preferably wherein the linear lipid, steroid or hydrophobic vitamin is bound to the trifunctional moiety a1 via a linker moiety tetraethylene glycol (TEG) or phosphate.

10. Use according to any one of claims 1 to 9, wherein

(a) k1 is 1, 2, 3, 4 or 5, preferably 2 or 3, and/or

(b) The hydrophobic domain is covalently bound to the hydrophilic domain only through the trifunctional moiety A1, and/or

(c) k2 is 1, 2, 3, 4, 5 or 6, preferably 1, 2 or 3.

11. The use of any one of claims 1-10, wherein the compound further comprises a labeling moiety and/or a linking group,

preferably

Wherein the labelling moiety and/or the linking group is covalently bound via a trifunctional moiety A2, and/or

Wherein one or more of moieties A2 is a label moiety or linker, more preferably wherein moiety A2 is a nucleobase-containing moiety, even more preferably wherein moiety A2 is dT,

even more preferably wherein the label moiety is a fluorescent label and/or wherein the linking group is biotin.

12. The method of any one of claims 1-11, wherein

(a) Linker L1 is independently selected from the group consisting of phosphate, amide, carbamate, and ester groups, and/or

(b) The moieties a1 and a2 are independently selected from difunctional groups selected from: phosphate groups, carbamate groups, amide groups, nucleobase-containing moieties even more preferably dT, and straight chain alkyl groups having 1-10C atoms, and said alkyl chain comprising a functional group at the terminal C atom, in particular independently selected from amine, carbonyl, hydroxyl, sulfhydryl, carbonic acid groups, and trifunctional moieties having 1-10C atoms and comprising at least one-OH, -SH and/or at least one-NH 2 group, preferably selected from lysine, serine, serinol, -O-CH2-CH ((CH2)4-NH2) -CH2-, glycerol, and 1, 3-diaminoglycerol moieties, and/or straight chain alkyl groups having 1-10C atoms

(c) Linker L2 is independently selected from the group consisting of phosphates, amides, carbamates, ester groups and moieties

- [O-CH₂-CH₂]_y2- (SP)_n]_m1-，

Wherein

SP and n are as defined above, preferably n =0,

y2 is an integer from 1 to 30, preferably from 3 to 10, and

m1 is an integer from 1 to 10, preferably from 1 to 3.

13. A method of stabilizing a cell, the method comprising

(a) Providing a compound as defined in any one of claims 1 to 12; and

(b) contacting a cell with a compound under conditions that allow the compound to interact with the cell membrane, thereby stabilizing the cell, and

(c) optionally, a shear force is applied to the cells.

14. The method of claim 13, wherein the shear force is applied to the cells by centrifugation, large scale cell culture, flow cytometry, fluorescence activated cell sorting, and/or bead based cell separation.

15. The use of any one of claims 1 to 12 or the method of claim 13 or 14, wherein the cell is a suspension cell and/or wherein the cell is an animal or human cell, in particular a vertebrate cell, especially a mammalian cell.