Solution for preserving cells

Abstract

The invention relates to a solution for conserving or preserving cells in blood samples or other body fluids. The solution comprises a cell membrane-permeant proton carrier and is substantially free from chaotropic substances, alcohols and detergents. The solution as per the invention also has a pH that generates an intracellular cell pH of less than 6. A solution of this kind is used advantageously in sample tubes, blood collection syringes or blood collection tubes.

Description

[0001] 201534PEP-1

[0002] Solution for cell storage

[0003] The present invention relates generally to the stabilization of blood samples and, in particular, to the preservation of cells, especially circulating tumor cells, in blood samples or other body fluids. The invention specifically relates to a preservation solution and its use for the rapid, permanent interruption of cell metabolism to stabilize the gene expression state, especially of the transcriptome, while preserving morphology, thus enabling subsequent molecular analysis of the transcriptome. Furthermore, the invention relates to the use of preservation solutions as well as sample tubes or blood collection tubes or syringes containing a solution according to the invention.

[0004] Background of the invention

[0005] Biomolecules in a healthy, metabolic cell are subject to constant turnover. Depending on factors such as environmental conditions, the stage of differentiation, or other conditions influencing metabolism, this turnover occurs through both new synthesis and degradation. Changes in the biomolecular composition within cells are specific, controlled, and targeted. Outside of cells, however, biomolecules undergo nonspecific, uncontrolled, and random degradation. To understand the biological functions of a molecule within a cell, it is crucial to understand its function in the context of other biomolecules present at the same time. Recently, single-cell transcriptome analysis (RNASeq) has clearly demonstrated that cells of the same type can exhibit quite different gene expression patterns.In particular, the analysis of tumor cells revealed that a solid tumor can be heterogeneous in gene expression. Therefore, the analysis of the transcriptome of individual cells is of general interest.

[0006] Analyzing the gene expression state of a cell is complicated by various factors. These include the instability of many biomolecules, particularly RNA, when they are outside of cells. However, the storage of intact cells until analysis can also significantly influence gene expression. It is common practice, for example, to temporarily store cells from cell culture at 4°C during harvesting, remove the medium and growth factors, and then repeatedly centrifuge them for preparation. This not only induces significant temperature fluctuations but also causes oscillating changes in cell density. It is known that cell density has a considerable influence on differentiation and cell division. A similar situation arises after tissue or tumor removal. Preparation alters nutrient supply, oxygen and CCh partial pressures, and the microenvironment.This is followed by a rapid cellular response in the form of altered expression patterns, which become most apparent at the transcriptome level. It is therefore important to preserve the transcriptome as a whole as much as possible and to protect it from manipulation-induced changes.

[0007] The difficulties of transcriptome analysis become clear when considering biobanks. Human tumor samples, which are submitted to biobanks after surgery, for example, are usually fixed with 10% formalin immediately after collection. However, this has the significant disadvantage that the transcriptome, which is crucial for analyzing the current state, becomes cross-linked. This considerably complicates molecular analysis, and isolating intact molecules is generally no longer possible. Therefore, the fresh samples are usually stored at room temperature until they can be processed by specialists. This processing typically involves carefully dividing the sample to freeze one portion for later molecular analysis and to keep another portion, after formalin fixation, for morphological analysis.The storage time between collection and further processing can sometimes be several hours and poses significant risks to the validity of biobanks and the stored samples. Furthermore, the subsequent thawing of the sample for molecular analysis leads to the induction of RNA degradation processes and thus to alterations in the transcriptome.

[0008] In recent years, tumor biology has demonstrated a high degree of heterogeneity in the molecular makeup of cells within a tumor. Circulating tumor cells (CTCs) are considered particularly important in this context, as their analysis allows for the identification of treatment options and the monitoring of disease and treatment progress. Circulating tumor cells are cells that have detached from a primary tumor. They can be found either in the lymphatic system or circulate in the bloodstream. At least some types of circulating tumor cells have the potential to settle in other organs or tissues and form new tumors or metastases. The majority of cancer-related deaths are not caused by primary tumors, but rather by metastases and secondary tumors derived from them.Diagnostic methods that allow for the analysis of circulating tumor cells (CTCs) can significantly contribute to the selection of personalized and effective treatment approaches. Furthermore, CTC analysis can be helpful in establishing a prognosis for patients at an early stage, as it is now known that these circulating cells exist and can be detected even in the early stages of disease. Analysis of CTCs is also of great importance for investigating the efficacy of medications.

[0009] However, CTCs are present in extremely small quantities in the respective body fluids and are also sensitive to manipulation during analysis. As mentioned above, all biomolecules in an intact, metabolic cell are subject to constant turnover. Depending on various influencing factors, such as environmental conditions, the stage of differentiation, or other conditions affecting metabolism, this turnover of molecules occurs through new synthesis and degradation. Changes in the biomolecular composition within cells occur specifically, in a controlled manner, and in a targeted way. To understand the biological functions of a molecule in a cell, it is crucial to observe its function in the context of the other biomolecules present at the same time.This requires the preservation of cells and the stabilization of their metabolic and morphological state.

[0010] Although the existence of circulating tumor cells (CTCs) has been known for a long time, they only recently became a focus of research aimed at improving treatment options for cancer patients. Several methods for preserving blood have been standard practice worldwide for decades. EDTA, citrate, or heparin additives prevent blood clotting and allow for the analysis of blood components. However, these additives are not very effective at stabilizing CTCs, as many normal blood functions are retained even after blood collection, i.e., ex vivo. For example, the immune system reacts and attempts to eliminate CTCs, and CTCs already affected by chemotherapy undergo necrosis or apoptosis.

[0011] The storage time between blood collection and processing can also be many hours and poses significant additional risks. The expression status of circulating tumor cells (CTCs) changes with temperature and environmental conditions, to which the CTCs respond with altered gene expression. This leads to an undesirable change in the molecular profile in subsequent analysis. It is also known that normal blood cells are subject to significant changes during storage. On the other hand, blood storage is part of routine diagnostic procedures, as certain processes and timeframes are prescribed in everyday hospital practice. Storage-related changes include not only alterations in the expression status of blood cells, but also, for example, changes in...a type of cell disintegration, which induces so-called debris, i.e. aggregates of surrounding cell material, which not only makes cell analysis difficult but can also cause invalid analyses (see also US 7,863,012 B2).

[0012] This highlights that not only maintaining cellular integrity, but also preserving the expression status of cells over extended periods is crucial for the analysis of CTCs. RNA stability is particularly important, as the transcriptome can provide crucial insights into tumor biology and therapeutic options.

[0013] Molecular analysis of the transcriptome refers to the investigation of cellular RNA using molecular biological methods. These include, for example, spectroscopic quantification, Northern hybridization, reverse transcription with or without biochip hybridization, amplification of individual transcripts by polymerase chain reaction, and RNASeq methods (total RNA, mRNA, amplicon sequencing). Such molecular biological analysis methods are well-known and are not themselves the subject of the present invention. Morphological analysis, as used here, refers to the analysis of individual or multiple cells, up to and including the analysis of a cell cluster in its natural context or environment, and relates to size, shape, granularity, etc. Morphometric analysis, as used here, refers to the analysis of one or more cellular characteristics of individual or multiple cells, for example...the analysis for the expression of a specific cell marker.

[0014] State of the art

[0015] Several methods for maintaining cellular integrity have been proposed in the literature. These are generally based on cross-linking agents such as formaldehyde, paraformaldehyde, and glutaraldehyde (e.g., EP 0214613 A2, DE 4039716 A1). US 4971783 A describes a method for tissue preparation. US patent US 5976829 A describes a formalin-based fixative that is also suitable for DNA / RNA analysis. Another approach is described in the literature, where methylol derivatives enable the fixation of blood components. Methylol is also used as a preservative in the cosmetics industry. The exact mechanism of action is unknown, but formaldehyde appears to play a significant role. However, the cross-linking nature of these fixatives, particularly the cross-linking of nucleic acids, complicates the analysis of cells at the molecular level.This problem is addressed, among other things, by US 5976829 A through a fixative comprising an aldehyde, an alcohol, a chelating agent and a buffer containing no amino groups.

[0016] In scientific fields, other methods are also used for fixing cells or molecules. These include, for example, freezing at low temperatures, the use of alcohol-based fixatives (such as methanol, ethanol, glycol), or chaotropic reagents (such as isothiocyanate), which enable the stabilization of molecules by disrupting morphological relationships.

[0017] Various solutions are also available in the prior art for fixing tissues without cross-linking while preserving their structure. EP 2 126542 B1, FR 2852 392 A1, WO 2013 / 131816 A1, and WO 03 / 029783 A1 describe mixtures for tissue fixation using organic solvents. Solutions are also available for stabilizing biomolecules without preserving their morphological context. Non-cross-linking methods for preserving cells and tissues are generally based on dehydration by incubation in organic solutions such as alcohol or acetone, preferably together with strong acids, e.g., a combination of methanol and glacial acetic acid. Nucleic acids are comparatively well protected. Acetone and glacial acetic acid fix somewhat more gently. However, analysis of molecules at the cellular level after alcohol or acetone fixation is not possible without rehydration.Nucleic acids, especially RNA, are exposed to unhindered degradation through the breakdown of compartmentalization, i.e., damage to cellular membrane systems (e.g., lysosomes) through rehydration.

[0018] Other publications describe further solutions for cell fixation via various mechanisms. For example, a corresponding solution disclosed in WO 2012 / 150479 A1 contains halogenated cyanoacetamides, and US 2015 / 0050689 A1 describes a combination of polyamides and acids that react to release aldehydes, particularly formaldehyde. In practice, biological preparations for RNA preservation are usually deep-frozen. To obtain the RNA, the samples are then lysed in the frozen state under strongly denaturing or chaotropic conditions. While the RNA isolated in this way is often of satisfactory quality, it is not possible to qualify the cell(s) desired for investigation by morphological or morphometric analysis, even though molecular analysis of a subpopulation is particularly desirable in practice.However, morphological or morphometric selection of such a subset prior to freezing requires crosslinking formalin fixation to preserve the expression status, which consequently complicates or prevents molecular analysis. The alternative of non-crosslinking, dehydrating fixation for isolating a subset of cells is also unfavorable, as subsequent selection of a subpopulation in the rehydrated state leads to rapid RNA degradation.

[0019] CN202011616524 teaches the stabilization of DNA in whole blood using a complex mixture of anticoagulants and stabilizers at neutral or near-neutral pH.

[0020] Another prior art proposal for the non-crosslinking fixation of cells and tissues is Hepes-mediated glutamic acid protection (DE10021390C2). This method proposes an amino acid-containing solution to morphologically preserve tissues through dehydrating paraffin embedding for long-term preservation. Although the dehydrating fixation is relatively mild, using acetone, the lack of crosslinking leads to structural changes in the tissue. The system is comparatively ineffective in stabilizing the transcriptome, even though the process is performed at low temperatures.

[0021] Ringwald et al. (Transfusion Medicine Reviews, 20(2), 2006) stabilize platelets with a mixture of various salts and acids. Acetic acid, in particular, is used to maintain platelet metabolism at neutral pH during storage for transfusion purposes. As cell-nosed "cells," platelets are transcriptionally inactive.

[0022] In a recently published method for RNAseq transcriptome analysis, cells are fixed with a mixture of water, methanol, acetic acid, and glycerol (ACME). This procedure alters cell structure and morphology, and it cannot be ruled out that the alcoholic reagents used may leach RNA molecules and protein markers from the cells. Furthermore, rehydration leads to the degradation of RNA transcripts. A similar approach is pursued in the teaching of PCT / EP2015 / 061678 (WO 2015 / 181220 A1).

[0023] Various chaotropic reagents are commercially available for stabilizing RNA by disrupting morphological relationships (e.g.,

[0024] RNAIater, Ambion; ProtectAII, Qiagen).

[0025] A modification without an organic solvent, but using bisulfites, is described in US 5,432,056 A. This proposes a bisulfite-containing acidic system for fixing tissue sections or other thin-layer samples. US 6,337,189 B1, on the other hand, describes a urea compound in combination with alcohol for the non-crosslinking fixation of cytological preparations. However, the morphological preservation of cellular structures is limited with all these methods.

[0026] Such methods are unsuitable for preserving whole blood for CTC analysis because they are either not suitable for routine diagnostics, destroy cellular integrity, or do not guarantee effective molecular stabilization.

[0027] However, US 10,091,984 B2 teaches that a formaldehyde-releasing urea compound, when the formaldehyde is simultaneously scavenged by glycine, is suitable for morphologically stabilizing CTCs. Even though this disclosure does not postulate a covalent modification of the CTC molecules, the role of formaldehyde remains unclear.

[0028] Current techniques have limitations regarding the methods for preserving the transcriptome of intact cells in its current state for later molecular analysis. These limitations are particularly pronounced when molecular analysis of the transcriptome of specific cells is required: (A) Cross-linking agents preserve the expression status relatively slowly, depending on the agent's diffusion rate, thus complicating molecular analysis; (B) Non-cross-linking fixation using organic solutions necessitates rehydration and, through uncontrolled molecular degradation, accelerates transcriptome alteration; (C) Lysis of a cell cluster using chaotropic reagents does not allow for the differentiation of individual cells. While chaotropic lysis of individual cells is theoretically possible, this would require prior disruption of their context, leading to transcriptome changes.The challenge was therefore to develop a solution that would allow the experimenter to interrupt metabolism in cells of a culture or tissue at a time specified by the experimenter, in order to stabilize the transcriptome at that specific time while simultaneously leaving the cells and cell compartments intact to prevent uncontrolled RNA degradation. The solution should also enable time-delayed transcriptome analysis for cell preservation or for conducting cell-qualifying morphological or morphometric analyses.

[0029] The task at hand also involved developing a method and suitable means to interrupt the metabolism or biomolecule turnover in blood cells, particularly circulating tumor cells (CTCs), or other body fluids. This would ensure the temporary, highly stabilized, non-crosslinked preservation of the current state of the genome, transcriptome, and proteome, preventing uncontrolled degradation of the molecules while simultaneously maintaining cell morphological integrity and preventing blood coagulation. The solution should also enable time-delayed processing for the isolation of CTCs and the performance of molecular and, if necessary, morphological analyses of the CTCs or other cells.

[0030] Summary of the invention

[0031] The aforementioned problems are solved by the present invention. A first aspect of the present invention is a solution for preserving cells, in particular at least one eukaryotic cell and especially tumor cells and circulating tumor cells in a blood sample or other body fluid, which is characterized in that it comprises a

[0032] a) contains a membrane-penetrating proton carrier,

[0033] b) is essentially free of chaotropic substances, alcohols and detergents, and

[0034] c) has a pH value that produces an intracellular pH of the cell or cells of less than 6.

[0035] The solution according to the invention is characterized in that it is an aqueous solution which

[0036] a. Acetic acid,

[0037] b. water-soluble oxalate, preferably ammonium oxalate, and

[0038] c. Hydrochloric acid

[0039] comprising, wherein the pH of the solution is equal to or less than 4, measured at 20°C. Furthermore, another aspect of the present invention lies in the use of such a solution according to the first aspect of the invention for the preservation, storage and / or fixation of cells, in particular at least one eukaryotic cell, especially tumor cells or circulating tumor cells, in a blood sample or other body fluid while largely preserving the cell morphology, in particular for a molecular analysis of the genome, the transcriptome and the proteome.

[0040] A third aspect of the present invention relates to sample tubes, blood collection syringes or blood collection tubes containing a solution according to the first aspect of the invention.

[0041] Advantages and details of the present invention will become apparent from the claims, the following detailed description and the exemplary embodiments.

[0042] Detailed description of the invention

[0043] Intact cell membranes are impermeable to passive proton transport, and proton transport across the cell membrane is highly controlled. The present invention is based on the understanding that an abrupt and rapid change (lowering) of the pH value within cells leads to a loss of function of the cellular machinery for the synthesis and degradation of molecules and, via the inactivation of enzymatic activity, to a cessation of metabolism without interfering with cellular compartmentalization. The essential feature of the solution according to the invention is therefore that the cessation of metabolic processes occurs very rapidly through a reduction in pH value, thus avoiding the environment-dependent change in the expression state.

[0044] According to the invention, this change in intracellular pH is achieved by membrane-permeable proton carriers contained in the solution. A proton carrier is understood to be a molecule that, under suitable conditions, is able to cross the cell membrane in its rheophilized form, preferably passively, and which dissociates within the cell into an anion and a proton. Suitable conditions are created, for example, by mixing cells containing a proton carrier with an acidic pKa in an acidic environment. In this case, the rheophilized form of the proton carrier passes through the cell membrane and dissociates within the cell due to the higher intracellular pH. As an ion, the proton carrier cannot leave the cell, so that at equilibrium, the intracellular pH is determined by the extracellular pH.

[0045] Furthermore, the present invention involves a comprehensive charge neutralization of nucleic acids. This leads, particularly in the case of RNA, to partial precipitation from the aqueous environment and thus to stabilization against both enzymatic and alkaline hydrolysis.

[0046] Crucially, within the scope of the present invention, and particularly for preserving cell morphology, the essentially complete absence of chaotropic substances, which are present in many corresponding preservative solutions in the prior art, is essential. Chaotropic substances such as perchlorates, like sodium perchlorate, thiocyanates, like guanidium thiocyanate, but also guanidinium hydrochloride and barium salts, are substances that break down ordered hydrogen bonds in water, disrupt the water structure, and cause an increase in entropy. They interfere with the hydration shell of biomolecules, leading to their denaturation and, at sufficient concentrations, to the complete dissolution of cells.

[0047] In the context of the present invention, the term "essentially complete absence" means that a substance may only be present in such a maximum amount that it does not disrupt the morphological integrity of the cells. In preferred embodiments, the solution according to the invention is completely free of chaotropic substances.

[0048] Analogous to the absence of chaotropic substances, the solution according to the invention is also characterized by a substantially complete absence of alcohols and detergents. In preferred embodiments, these substances are also completely excluded from the solution according to the invention.

[0049] Finally, the solution according to the invention is further characterized in that it has a pH value which produces an intracellular pH of the cell or cells of less than 6.

[0050] Advantageously, membrane-penetrating carboxylic acids are used as proton carriers within the scope of the present invention, namely primarily C2 to Cs carboxylic acids. As mentioned above, a key feature of the invention is the rapid cessation of metabolism while preserving the morphology of cells, particularly blood cells. Blood cells are understood to be all cells containing a nucleus. This cessation of metabolism is hereinafter also referred to as fixation or preservation, although the mechanism of action of the present invention differs from known cross-linking, dehydrating, or denaturing fixations. The cessation of metabolism is preferably achieved by a dissolved carboxylic acid in combination with a low pH. The rheumatized form of the carboxylic acid passes through the cell membrane and dissociates inside the cell. However, other membrane-penetrating acids in their rheumatized form can also be used.

[0051] The acids in the solution according to the invention are preferably present in the form of a carboxylic acid buffer system or an acid / base system, which establishes and stabilizes a pH in the desired acidic range. These buffer systems preferably comprise weak acids and their conjugate bases. Blood has a very high buffering capacity. According to the invention, this buffering capacity is titrated with the buffer system contained in the solution in order to create an acidic environment in the mixture and, in particular, within the cells, which in turn largely inhibits metabolic activities. According to the invention, a suitable buffer system is one that provides a sufficient amount of carboxylic acid for membrane passage and simultaneously releases sufficient H+ ions intracellularly.

[0052] Depending on the sample material used, it may be necessary to add a strong acid to adjust the intracellular pH of the solution according to the invention, so that the buffering capacity of the sample material is eliminated.

[0053] Suitable strong acids include, in particular, hydrochloric acid or other mineral acids. However, these acids are not membrane-penetrating and therefore do not constitute proton carriers within the scope of the present invention.

[0054] In particular, the selection of the proton carrier and the adjustment of the pH of the solution according to the invention are carried out such that, after mixing with cells, the blood sample, or another body fluid or tissue sample, a mixture with a pH between 4 and 6 is obtained. The person skilled in the art can easily make the corresponding adjustment, since the pH and buffering capacity of blood samples or other body fluids or tissue samples that are suitable as objects of investigation are known to them, and they can therefore preset the pH of the solution according to the desired mixing ratio. Common mixing ratios of blood samples to be analyzed and preservative solutions are also known to those skilled in the art and are, for example, 1:10 for citrated blood. The mixing ratios can be adjusted accordingly for other objects of investigation and are generally within the same range.

[0055] In the invention, acetic acid is used as the carboxylic acid. In the solution according to the invention, acetic acid is preferably present as an acetic acid / acetate system, and the pH of the solution is adjusted so that it is at least below 6. In further preferred embodiments, the pH and buffering capacity of the solution according to the invention are selected such that the resulting pH after mixing with the sample is below 6. In particular, the pH of the mixture (solution plus sample) is even between 2 and 6, more preferably between 4.8 and 5.8, and particularly preferably between 5.0 and 5.5, or between 5.1 and 5.3. In certain preferred embodiments, the pH of the mixture is 4.6–5.6, preferably 5.2–5.4. For this purpose, solutions according to the invention with a pH below 6, preferably below 5.8, and particularly equal to or below 5.3, will be particularly suitable.Depending on the intended mixing ratio and the body fluid to be preserved, such solutions exhibit a pH value in the acidic range, in particular equal to or less than 4, preferably equal to or less than 3, and, if, for example, intended for mixing with blood in a ratio of 1:5, preferably between about 2 and about 3, more preferably between 2 and 3, and more preferably between 2.0 and 2.5. Within the scope of the present invention, the pH values ​​given refer to a temperature of 20°C. As already explained above, the intracellular pH value is also determined based on the pH of the solution in which the cells are present and the buffering capacity of the sample material.

[0056] The acid / base system of the proton carrier of the present invention is typically present at a concentration of 1 to 400 mM, depending on whether it is a concentrated solution that is diluted with the blood sample and other aqueous solutions, or a ready-to-use solution that is added in excess, e.g., at 10 times the volume, to isolated cells. A key feature of the solution according to the invention is the possibility of formulating it as a (multiple) concentrate to fix blood without excessive dilution. For this purpose, a solution according to the invention is particularly envisaged in which the acid / base system of the proton carrier and its anticoagulant components are formulated as a 2- to 20-fold, preferably 5- to 10-fold, concentrate for preserving blood or another body fluid in order to achieve the desired intracellular pH as described above.In preferred embodiments of the invention, the acid / base system, and preferably an acetic acid / acetate system, is present in a concentrated solution according to the invention at a concentration of 10 mM to 400 mM, preferably 25 mM to 200 mM, and particularly preferably 50 mM to 150 mM. Particularly for a concentrate, it is preferred that the acetic acid is present at a concentration of 300 mM to 400 mM, preferably 330 mM to 360 mM. In contrast, for the ready-to-use solution, it is preferred that the solution contains the acid / base system at a concentration of 1 mM to 100 mM, preferably 5 mM to 50 mM, and particularly preferably 30 mM to 50 mM.

[0057] The solution according to the invention can, in addition to the aforementioned or other proton carriers, also contain additional substances, such as buffer substances or conventional excipients. The solution according to the invention can contain imidazole, but can also additionally contain dimethyl sulfoxide (DMSO). In preferred embodiments, the solution according to the invention contains imidazole. Imidazole has a slightly acidic pKa and, in its protonated or non-protonated form, can cross the cell membrane. Imidazole can initially mobilize H + -ions into the cell interior. A suitable final concentration of imidazole in the solution according to the invention is 30-70 mM, preferably 40-60 mM. For use of the solution according to the invention for the frozen storage of test specimens, it preferably contains DMSO in a final concentration of 5 to 15%, particularly preferably 10%.

[0058] Other suitable proton carriers that can be used as part of the preservation solution within the scope of the invention are cyclic peptides such as e.g.

[0059] Valinomycin or nigericin. Furthermore, the solution according to the invention may contain other ions, in particular Cl' ions.

[0060] The solution according to the invention may also contain further substances that contribute to preservation. For example, the addition of β-mercaptoethanol or dithiotreitol or similar reagents can further reduce the activity of RNases. It is obvious to those skilled in the art that the addition of other enzyme inhibitors, for example phosphatase inhibitors, can also positively influence the preservative effect of the solution according to the invention.

[0061] The solution according to the invention further comprises a water-soluble oxalate. "Water-soluble oxalates" according to the present invention are, in particular, ammonium oxalate and alkali oxalates, e.g., sodium oxalate, free oxalic acid, and mixtures thereof. Water-soluble oxalates such as ammonium oxalate or sodium oxalate, in particular, prevent blood coagulation when the solution according to the invention is added to a blood sample. In preferred embodiments, the solution according to the invention comprises sodium oxalate and / or ammonium oxalate, preferably ammonium oxalate, as the water-soluble oxalate. A suitable final concentration of water-soluble oxalate in the solution is 5–20 mM, preferably 5–12 mM. Water-soluble oxalates, in particular ammonium oxalate as an exemplary water-soluble oxalate, have the advantage over the standard anticoagulant compounds used in the prior art, citrate, EDTA, and heparin, that they stabilize the cells and their components.This can be of particular importance for the intended molecular analysis of a transcriptome.

[0062] In further preferred embodiments, the solution according to the invention additionally comprises at least one amino acid. In particular, this is an amino acid that occurs naturally in tissues or cells. The buffer system of the present invention utilizes the protective effect of amino acids. This effect is particularly pronounced when such amino acids are present in a total concentration of 0.1 M to 1 M, preferably in a total concentration of 100 mM to 300 mM. Preferably, the amino acids are one or more from the group consisting of glycine, alanine, proline, serine, threonine, glutamic acid, and aspartic acid.

[0063] In particular, the solution according to the invention may contain glycine and / or glutamic acid. A suitable final concentration of glycine in the solution is 30-70 mM, preferably 35-50 mM. A suitable final concentration of glutamic acid in the solution is 20-60 mM, preferably 32-40 mM.

[0064] A further embodiment of the invention is the combination of rapid pH-dependent fixation with an additional preservative agent from the group of formaldehyde donors. Such agents can be, for example, diazolidinyl urea or imidazolidinyl urea.

[0065] Preferably, the composition of the solution according to the invention is designed such that the osmolarity does not exceed 1 osmol. Preferably, the osmolarity of the solution is above 100 and below 500 mosmol, preferably between 250 and 350 mosmol. Particularly preferably, the solution is isotonic or forms an isotonic environment for the cell(s) to be preserved.

[0066] In some embodiments of the invention, the formulation of the solution according to the invention does not completely prevent blood coagulation. It is known to those skilled in the art that these coagulation phenomena can be suppressed by the addition of chelating agents, anticoagulants, or inhibitory antibodies. The presence of such chelating agents, anticoagulants, or antibodies in the solution according to the invention therefore leads to further preferred embodiments of the invention. In certain embodiments, the solution according to the invention may contain MgSCL, dabigatran, and / or plasminogen activator (t-PA) as anticoagulants.

[0067] Another aspect of the present invention is the use of the solution according to the invention for the preservation, storage and / or fixation of cells, in particular at least one eukaryotic cell, especially tumor cells and circulating tumor cells in a blood sample or other body fluid, preferably with subsequent or delayed molecular analysis.

[0068] This use according to the invention is characterized in that the solution is mixed with the blood sample or other body fluid, preferably directly after it has been taken from an organism, e.g., a mammal, preferably a human. The molecular analysis can relate to any substance or group of substances present in the cell, in particular the genome, the transcriptome, and the proteome. Preferably, a transcriptome analysis is performed as the molecular analysis on the at least one cell after treatment with the solution according to the invention.

[0069] A particular feature of the invention is the ability to qualify largely intact cells based on a single characteristic and to use this qualification for molecular analyses. Preferably, a mixture of different cells treated with the solution according to the invention is analyzed for a marker. This is preferably a morphological marker (e.g., analyzed by FSC / SSC in flow cytometry) or a morphometric marker (e.g., a labeled antibody for binding to a surface protein). The mixture can consist of two, three, or more different cell populations. This marker is preferably used to qualify at least one cell for analysis of its cellular components, particularly preferably the transcriptome. It may be preferred to add at least one tissue digestion enzyme to the solution according to the invention in order to release individual cells from a tissue mass.Preferably, at least one enzyme is a collagenase, a dispase, or a combination thereof.

[0070] It may be preferred to store the at least one cell treated with the solution according to the invention. It may be preferred to carry out this storage at 2-8°C. Alternatively, it may be preferred to carry out this storage in a frozen state.

[0071] The use according to the invention preferably comprises at least: an input step a) treatment of cells or tissue with a suitable solution and an output step f) the molecular analysis of the transcriptome of at least one cell.

[0072] Preferably, the method includes further steps that can be carried out individually or in combination between the input and output steps, depending on the subject and objective of the investigation, namely one or more of the following: b) qualification of at least one cell for transcriptome analysis by morphological or morphometric selection, c) storage of the cells in a frozen state, d) storage of the cells at 2-8°C, e) protease digestion of a tissue to generate individual cells.

[0073] Within the scope of these applications of the invention, it is particularly advantageous to pre-fill the solution in a sample tube, blood collection tube or syringe, or another suitable container, such as a blood collection syringe. This allows, in particular, the direct mixing of the body fluid with the solution according to the invention after collection, thus preventing falsification of the analysis by, for example, enzymatic activity. Corresponding sample tubes, blood collection tubes or syringes, or other reaction vessels containing the solution according to the invention, are also further aspects of the present invention.

[0074] It is particularly preferred to present the solution according to the invention in concentrated form to avoid excessive dilution of the sample. Alternatively, instead of presenting the solution in sample tubes or the like, it can be mixed immediately after the blood sample or body fluid has been taken. The sample tubes, blood collection tubes or syringes, as well as other containers containing the solution according to the invention, can be used for the purposes described in the invention. Like the solution according to the invention itself, these items are also suitable for carrying out other procedures, such as cell-free DNA / RNA analyses. Use for such purposes is also encompassed by the present invention.

[0075] All explanations given above for the solution according to the invention also apply to the use of this solution according to the invention and likewise to the pre-made sample tubes or blood collection tubes containing the solution according to the invention.

[0076] The present invention is further characterized by the following points.

[0077] 1. Solution for the preservation or storage of nucleated cells in a blood sample or other body fluid,

[0078] characterized by the fact that it is an aqueous solution which

[0079] a. Acetic acid,

[0080] b. a water-soluble oxalate, preferably ammonium oxalate, and

[0081] c. Hydrochloric acid

[0082] includes, where the pH of the solution is equal to or less than 4, measured at 20°C.

[0083] 2. Solution according to point 1, characterized in that the solution further

[0084] d. Imidazole,

[0085] e. Glycine, and / or

[0086] f. Glutamic acid

[0087] includes.

[0088] 3. Solution according to point 2, characterized in that the solution contains all components of the.

[0089] up to f.

[0090] 4. Solution according to one of the preceding points, characterized in that the solution further

[0091] g. Aspartic acid,

[0092] h. Proline, i. Hepes,

[0093] J. Serin,

[0094] k. Threonine,

[0095] l. Alanine, and / or

[0096] m. Glucose

[0097] includes.

[0098] Solution according to point 4, characterized in that the solution includes all components g. to m.

[0099] A solution according to one of the preceding points, characterized in that the solution includes all components a. to m.

[0100] A solution according to any of the preceding points, characterized in that the pH value of the solution is equal to or less than 3, preferably from about 2 to about 3, more preferably from 2 to 3, more preferably from 2.0 to 2.5, in each case measured at 20°C.

[0101] Solution according to any of the preceding points, characterized in that the final concentration of acetic acid in the solution is from 300 mM to 400 mM, preferably from 330 mM to 360 mM.

[0102] Solution according to one of the preceding points, characterized in that the final concentration of water-soluble oxalate in the solution is from 5 mM to 20 mM, preferably from 5 to 12 mM, preferably characterized in that the final concentration of ammonium oxalate in the solution is from 5 mM to 20 mM, preferably from 5 to 12 mM.

[0103] A solution according to any of the preceding points, characterized in that the final concentration of hydrochloric acid in the solution is from about 120 mM to about 155 mM.

[0104] Solution according to any one of the preceding points, characterized in that the final concentration of imidazole in the solution is from 30 mM to 70 mM, preferably from 40 to 60 mM. 12. Solution according to any one of points 2-11, characterized in that the final concentration of glycine in the solution is from 30 mM to 70 mM, preferably from 35 to 50 mM.

[0105] 13. Solution according to one of points 2-12, characterized in that the final concentration of glutamic acid in the solution is from 20 mM to 60 mM, preferably from 32 mM to 40 mM.

[0106] 14. Solution according to any one of points 3-13, characterized in that the final concentration of aspartic acid in the solution is from 1 mM to 12 mM, preferably from 3 mM to 8 mM.

[0107] 15. Solution according to one of points 3-14, characterized in that the final concentration of proline in the solution is from 5 mM to 20 mM, preferably from 7 mM to 12 mM.

[0108] 16. Solution according to one of points 3-15, characterized in that the final concentration of Hepes in the solution is from 5 mM to 20 mM, preferably from 8 mM to 13 mM.

[0109] 17. Solution according to one of points 3-16, characterized in that the final concentration of serine in the solution is from 10 mM to 35 mM, preferably from 15 mM to 24 mM.

[0110] 18. Solution according to one of points 3-17, characterized in that the final concentration of threonine in the solution is from 1 mM to 10 mM, preferably from 2 mM to 7 mM.

[0111] 19. Solution according to one of points 3-18, characterized in that the final concentration of alanine in the solution is from 1 mM to 10 mM, preferably from 2 mM to 6 mM.

[0112] 20. Solution according to one of points 3-19, characterized in that the final concentration of glucose in the solution is from 10 mM to 30 mM, preferably from 12 mM to 22 mM.

[0113] 21. Solution according to any one of points 1-20, characterized in that the solution comprises: a. 330-360 mM acetic acid,

[0114] b. 5-12 mM water-soluble oxalate, preferably sodium oxalate and / or ammonium oxalate, more preferably ammonium oxalate, and

[0115] c. Hydrochloric acid to pH 2.0-2.2, measured at 20°C,

[0116] where the concentration in mM is the final concentration in the solution.

[0117] Solution according to one of points 1-21, characterized in that the solution includes:

[0118] a. 330-360 mM acetic acid,

[0119] b. 5-12 mM water-soluble oxalate, preferably sodium oxalate and / or ammonium oxalate, more preferably ammonium oxalate,

[0120] c. Hydrochloric acid to pH 2.0-2.2, measured at 20°C,

[0121] d. 40-60 mM imidazole,

[0122] e.g. 35-50 mM glycine, and

[0123] f. 32-40 mM glutamic acid,

[0124] where the concentration in mM is the final concentration in the solution and the pH value is the final pH value of the solution.

[0125] Solution according to one of the points 1-22, characterized in that the solution consists of:

[0126] a. 330-360 mM acetic acid,

[0127] b. 5-12 mM water-soluble oxalate, preferably sodium oxalate and / or ammonium oxalate, more preferably ammonium oxalate,

[0128] c. Hydrochloric acid to pH 2.0-2.2, measured at 20°C,

[0129] d. 40-60 mM imidazole,

[0130] e.g. 35-50 mM glycine,

[0131] f. 32-40 mM glutamic acid,

[0132] g. 3-8 mM aspartic acid,

[0133] h. 7-12 mM proline,

[0134] i. 8-13 mM Hepes,

[0135] j. 15-24 mM Serine,

[0136] k. 2-7 mM Threonine,

[0137] l. 2-6 mM alanine, and

[0138] m. 12-22 mM glucose,

[0139] where the concentration in mM is the final concentration in the solution, and the pH value is the final pH value of the solution. Solution according to any one of points 1-23, characterized in that the solution consists of:

[0140] a. 330-360 mM acetic acid,

[0141] b. 5-12 mM ammonium oxalate,

[0142] c. Hydrochloric acid to pH 2.0-2.2, measured at 20°C,

[0143] d. 40-60 mM imidazole,

[0144] e.g. 35-50 mM glycine,

[0145] f. 32-40 mM glutamic acid,

[0146] g. 3-8 mM aspartic acid,

[0147] h. 7-12 mM proline,

[0148] i. 8-13 mM Hepes,

[0149] j. 15-24 mM Serine,

[0150] k. 2-7 mM Threonine,

[0151] l. 2-6 mM alanine, and

[0152] m. 12-22 mM glucose,

[0153] where the concentration in mM is the final concentration in the solution, and the pH value is the final pH value of the solution.

[0154] Solution according to one of the preceding points, characterized in that the solution is formulated as a multiple concentrate, preferably as a 2-10-fold multiple concentrate, particularly preferably as a 5-fold concentrate.

[0155] Use of a solution according to any of points 1 to 25 for the preservation, storage and / or fixation of at least one nucleated eukaryotic cell, in particular tumor cells and circulating tumor cells, in a blood sample or other body fluid, wherein in particular the solution is mixed with the blood sample or body fluid directly after collection and / or the solution is placed in a sample tube, a blood collection tube or a blood collection syringe and the blood sample, body fluid, is added to this container.

[0156] 28. Use of a solution according to any one of points 1 to 25 for the preservation, storage, and / or fixation of tumor cells, in particular circulating tumor cells, in a blood sample, characterized in that the solution is mixed with the blood sample directly after collection. 29. Use of a solution according to any one of points 1 to 25 for the preservation, storage, and / or fixation of tumor cells, in particular circulating tumor cells, in a blood sample, characterized in that the solution is placed in a sample tube, a blood collection tube, or a blood collection syringe, and the blood sample is added to this container.

[0157] 29. Use according to any of points 26 to 28, characterized in that after mixing the solution with the blood sample or other body fluid, the pH value of the mixture is 4.6-5.6, preferably 5.2-5.4.

[0158] 30. Use according to any of points 26 to 29, in which the cell or cells remain substantially intact and RNA from at least one cell is used for transcriptome analysis.

[0159] 31. Use according to any of points 26 to 30, wherein a morphological analysis of at least one marker protein of the cell or cells is used to qualify at least one cell for transcriptome analysis.

[0160] 32. Use according to any of points 26 to 31, comprising an input step a) mixing cells in liquid or tissue with the solution according to any of points 1 to 25 and an output step f) molecular analysis of the transcriptome of at least one cell.

[0161] 33. Use according to point 32, comprising one or more intermediate steps selected from: b) quantification of at least one cell for transcriptome analysis by morphological or morphometric selection; c) storage of the cells in a frozen state; d) storage of the cells at 2 to 8 °C; e) protease digestion of a tissue to generate single cells.

[0162] 34. Sample tube, blood collection syringe or blood collection tube,

[0163] which are characterized by the fact that they contain a solution according to one of the points 1 to 25.

Claims

Claims 1. Solution for the preservation or storage of nucleated cells in a blood sample or other body fluid, characterized by the fact that it is an aqueous solution which a. Acetic acid, b. a water-soluble oxalate, preferably ammonium oxalate, and c. Hydrochloric acid includes, where the pH of the solution is equal to or less than 4, measured at 20°C.

2. Solution according to claim 1, characterized in that the solution further d. Imidazole, e. Glycine, and / or f. Glutamic acid includes.

3. Solution according to claim 1 or 2, characterized in that the solution further comprises g. aspartic acid, h. Proline, i. Hepes, J. Serin, k. Threonine, l. Alanine, and / or m. Glucose includes.

4. Solution according to claim 2 or 3, characterized in that the solution comprises all components d. to f. or g. to m.

5. Solution according to one of the preceding claims, characterized in that the solution comprises all components a. to m.

6. Solution according to one of the preceding claims, characterized in that the pH value of the solution is equal to or less than 3, preferably from about 2 to approximately 3, preferably from 2 to 3, more preferably from 2.0 to 2.5, each measured at 20°C.

7. Solution according to one of the preceding claims, characterized in that the final concentration of acetic acid in the solution is from 300 mM to 400 mM, preferably from 330 mM to 360 mM, and / or that the final concentration of water-soluble oxalate, preferably ammonium oxalate, in the solution is from 5 mM to 20 mM, preferably from 5 to 12 mM, and / or that the final concentration of hydrochloric acid in the solution is from about 120 mM to about 155 mM.

8. Solution according to any one of claims 2-7, characterized in that the final concentration of imidazole in the solution is from 30 mM to 70 mM, preferably from 40 to 60 mM, and / or that the final concentration of glycine in the solution is from 30 mM to 70 mM, preferably from 35 to 50 mM, and / or that the final concentration of glutamic acid in the solution is from 20 mM to 60 mM, preferably from 32 mM to 40 mM.

9. Solution according to any one of claims 3-8, characterized in that the final concentration of aspartic acid in the solution is from 1 mM to 12 mM, preferably from 3 mM to 8 mM, and / or that the final concentration of proline in the solution is from 5 mM to 20 mM, preferably from 7 mM to 12 mM, and / or that the final concentration of Hepes in the solution is from 5 mM to 20 mM, preferably from 8 mM to 13 mM, and / or that the final concentration of serine in the solution is from 10 mM to 35 mM, preferably from 15 mM to 24 mM, and / or that the final concentration of threonine in the solution is from 1 mM to 10 mM, preferably from 2 mM to 7 mM, and / or that the final concentration of alanine in the solution is from 1 mM to 10 mM, preferably from 2 mM to 6 mM, and / or that the final concentration of glucose in the solution is from 10 mM to 30 mM, preferably from 12 mM to 22 mM.

10. Solution according to any one of claims 1-9, characterized in that the solution comprises: a. 330-360 mM acetic acid, b. 40-60 mM imidazole, c. 5-12 mM water-soluble oxalate, preferably sodium oxalate and / or ammonium oxalate, more preferably ammonium oxalate, and d. Hydrochloric acid to pH 2.0-2.2, measured at 20°C, where the concentration in mM is the final concentration in the solution.

11. Solution according to one of claims 1-10, characterized in that the solution comprises: a. 330-360 mM acetic acid, b. 40-60 mM imidazole, c. 5-12 mM water-soluble oxalate, preferably sodium oxalate and / or ammonium oxalate, more preferably ammonium oxalate, d. Hydrochloric acid to pH 2.0-2.2, measured at 20°C, e.g. 35-50 mM glycine, and f. 32-40 mM glutamic acid, where the concentration in mM is the final concentration in the solution and the pH value is the final pH value of the solution.

12. Solution according to one of claims 1-11, characterized in that the solution consists of: a. 330-360 mM acetic acid, b. 40-60 mM imidazole, c. 5-12 mM ammonium oxalate, d. Hydrochloric acid to pH 2.0-2.2, measured at 20°C, e.g. 35-50 mM glycine, f. 32-40 mM glutamic acid, g. 3-8 mM aspartic acid, h. 7-12 mM proline, i. 8-13 mM Hepes, j. 15-24 mM Serine, k. 2-7 mM Threonine, l. 2-6 mM alanine, and m. 12-22 mM glucose, where the concentration in mM is the final concentration in the solution, and the pH value is the final pH value of the solution.

13. Use of a solution according to any one of claims 1 to 12 for preserving, storing or / and fixing at least one nucleated eukaryotic cell, in particular tumor cells and circulating tumor cells, in a blood sample or other body fluid, wherein in particular the solution is mixed with the blood sample or body fluid directly after collection and / or the solution is placed in a sample tube, a blood collection tube or a blood collection syringe and the blood sample or body fluid is added to this vessel, and after mixing the solution with the blood sample or the other body fluid the pH value of the mixture is 4.6-5.6, preferably 5.2-5.

4.

14. Use of a solution according to any one of claims 1 to 12 for preserving, storing or / and fixing tumor cells, in particular circulating tumor cells, in a blood sample, characterized in that the solution is mixed with the blood sample directly after collection, or that the solution is placed in a sample tube, a blood collection tube or a blood collection syringe and the blood sample is added to this vessel, and after mixing the solution with the blood sample the pH value of the mixture is 4.6-5.6, preferably 5.2-5.

4.

15. Sample tube, blood collection syringe or blood collection tube, which are characterized in that they contain a solution according to one of claims 1 to 12.