Method for lysing cells and tissues by using polymers and use of polymers for this method - Patent Application 20070122997

Abstract

Methods for lysing cells and tissues by using certain polymers and the use of these polymers to completely or partially lyse cells and tissues are described.

Description

[Technical Field]

[0001] The present invention relates to a method for lysing cells and / or tissues by using polymers, and the use of polymers for this method. [Background technology]

[0002] Cell lysis is an inevitable step for analytical approaches of biomolecules, such as soluble and membrane proteins, nucleic acids, and other molecules, in all kinds of downstream applications. To achieve reproducible and optimal results, the native conformation of extracted proteins is a major advantage. Membrane-associated interaction partners, protein complexes, and receptor-ligand couples benefit from the native folds present in the cell membrane. State-of-the-art approaches are often based on lysis buffers containing NP-40, SDS, and deoxycholate, which, combined with mechanical procedures, induce a non-native state without the essential lipid environment, thereby reliably enhancing cell lysis. These methods, such as French press and sonication, are also often used.

[0003] For DNA preparation in particular, alkaline lysis has been established for approximately 40 years. This procedure applies alkaline solution and SDS under moderate mechanical stress to purify plasmid DNA without genomic DNA impurities.

[0004] For protein extraction from mammalian cells, a mixture of RIPA buffer, SDS, sodium chloride, sodium deoxycholate, octylphenoxypolyethoxyethanol, and Tris buffer has been established. However, this procedure has many drawbacks because proteins are denatured by SDS and sodium deoxycholate. Therefore, this method cannot be used for applications requiring intact, active, native proteins.

[0005] Although mammalian cells can be lysed by applying mild detergents such as DDM, even under these conditions, native proteins cannot be prepared.

[0006] Although digitonin can solubilize mammalian proteins and obtain proteins in their native state, the solubilization procedure remains tedious due to the limited solubility of digitonin. Digitonin is a natural product that varies from one charge to the next, but its most important drawback is the need for additional solubilization, whether mechanical or chemical. DETAILED DESCRIPTION OF THE INVENTION

[0007] The technical problem underlying the present invention is to provide a method for lysing cells and tissues that is fast and easy to perform, highly effective, and reliably lyses cells and tissues.

[0008] This is achieved by the subject matter as defined in the independent claims. Preferred embodiments are defined in the dependent claims.

[0009] According to the present invention, there is provided a method for lysing cells and tissues, wherein the cells and tissues are contacted with a polymer.

[0010] The term "lysis", as used in accordance with the present invention, refers to the disintegration of cells caused by damage to their cell (outer) membrane. It is noted that the term "lysis" as used in accordance with the present invention contemplates the lysis of at least a portion of the cells of a cell pool, up to all of the cells of the cell pool. According to the method of the present invention, this is caused by a specific polymer, as defined below.

[0011] The term "cells and / or tissues," as used in accordance with the present invention, refers to cells, tissues, and mixtures of cells and tissues that can be dissolved by the use of certain polymers. Tissues are a biological organization level between cells and complete organs. Tissues are groups of similar cells from the same origin and their extracellular matrix that together perform a specific function.

[0012] According to the method of the present invention, cells and / or tissues are contacted with a particular polymer. This step can be carried out in a medium, e.g., an aqueous medium such as saline or a buffer. The specific composition of the medium is selected so that neither the cells or tissues nor the polymer are adversely affected to effect lysis.

[0013] In one embodiment, the cells are eukaryotic cells. Surprisingly, it has been found that no lysis of bacterial cells (gram positive / -negative) and yeast could be observed.

[0014] The polymer may be a homopolymer or a copolymer. In one embodiment, the polymer may have hydrophilic groups, such as COOH, maleimide, OH, amine, ammonium salt, zwitterion such as phosphocholine, and hydrophobic groups, such as polymerized styrene groups, polymerized diisobutylene groups, or linear C1-C16 (such as methyl and ethyl) aliphatic groups, branched C1-C16 (such as isopropyl or t-butyl) aliphatic groups, and cyclic C5-C12 aliphatic or aromatic groups.

[0015] In one embodiment, the polymer is selected from the group consisting of diisobutylene / maleic acid copolymer, styrene maleic acid copolymer, (acrylic acid-co-styrene) copolymer, and amphipol, which are particularly suitable for lysing cells or tissues.

[0016] The molecular weight of the polymer used according to the method of the present invention may be from 1900 to 20000, for example from 2000 to 18000, or from 2000 to 15000, or from 4000 to 16000, or from 4000 to 13000, or from 5000 to 14000. The molecular weight may be measured by gel permeation chromatography and mass spectrometry.

[0017] Examples of polymers can be, but are not limited to, styrene / maleic acid copolymers sold under the trade name "SMA," derivatives of styrene / maleic acid copolymers such as SMA 200 and 300, and styrene / maleimide copolymers such as SMA 502. These materials can also be functionalized on the COOH groups to amides with amines such as ethanolamine or ethylenediamine, or to esters with alcohols such as glycerol. Polymers can also be functionalized to esters with polyethylene glycol and to amides with aminated polyethylene glycol.

[0018] The polymer can be a diisobutylidene / maleic acid copolymer, such as DIBMA 10 and DIBMA 12 from Cube Biotech, derivatives of diisobutylidene / maleic acid copolymers such as DIBMA Gly, DIBMA Glu, Glyco DIBMA, and diisobutylidene / maleimide copolymers. DIBMA copolymers can be functionalized with the same molecules as SMA.

[0019] Further polymers may be copolymers of styrene and acrylic acid, in particular those with molecular weights of 5,500 and 11,000, with an acrylic acid / styrene ratio of 45% / 55% to 55% / 45%, sold under the name "AASTY".

[0020] Modified polymers from polyacrylic acid can be used in which 10-90% of the carboxylic acid groups can be modified to amides with cyclooctylamine, 2-cyclohexyl-ethylamine, etc. These substances are sold under the name "Amphipol Ultrasolve".

[0021] In addition to the polymer disclosure above, the polymers are further described below.

[0022] Examples of hydrophilic groups may include, but are not limited to, acrylic and methacrylic acid, maleic acid, polymers of common carboxylic acid groups, α,ω alkylenediamines, ω-hydroxyalkylamines and ω-aminoalkylthiols, amides with trimethylammonio-alkylamines, amides from carboxylic acid groups with amino-glycerol tris or bis-tris, maltosamine, glucosamine, mannosamine, and other amino-functionalized carbohydrates, amides with taurine.

[0023] Mention may also be made of esters of carboxylic acid groups with polyethylene glycols, diols, triols, polyols, and carbohydrates.

[0024] Other examples may be maleimides with the nitrogen atom functionalized with an alkyl chain bearing an alcohol, thiol, amine, ammonium salt, and the like.

[0025] Alternatively, a zwitterionic molecule consisting of an ammonium and a phosphate group can be linked to a carboxylic acid group, as described in US2020 / 281855A1 or US2021 / 171673A1.

[0026] Examples of hydrophobic groups may include, but are not limited to, polymerized styrene and derivatives such as methylstyrene, diisobutylene, and straight and branched chain alkanes such as 2-propyl, hexyl, octyl, or decyl, linked to a carboxylic acid group via an ester or amide functionality. Also suitable are maleimide groups bearing alkyl or aryl groups on the amino functionality.

[0027] An example of the synthesis of styrene-maleic acid copolymers can be found in Shintaro Sugai, Nobumichi Ohno, Conformational transitions of the hydrophobic polyacids, Biophysical Chemistry, Volume 11, Issues 3-4, June 1980, Pages 387-395.

[0028] The use of SMA to construct complexes with lipids is described in WO2006 / 129127 and references therein.

[0029] SMA can be purchased as SMALP 140, SMALP 200, or SMALP 300 from Orbiscope or Cube Biotech. DIBMA:

[0030] The synthesis of copolymers from diisobutylene and maleic anhydride is described in US 4,250,289 by BASF. The hydrolysis of anhydride copolymers to diisobutylene-co-maleic acid is described by Lee, Nature Protocols Vol. 11, No. 7, 2016, pp. 1149-1162, which is described for SMA copolymers but can be applied to DIBMA without any problems.

[0031] The synthesis of DIBMA polymers with glucosamine functionalization on 50% of all carboxyl groups can be found in B. Danielczak, M. Rasche, J. Lenz, E. Perez Patallo, S. Weyrauch, F. Mahler, M. Tope Agbadaola, A. Meister, J. Oyebamiji Babalola, C. Vargas, C. Kolar and S. Keller, A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs†, DOI: 10.1039 / D1NR03811G (Paper) Nanoscale, 2022, 14, 1855-1867.

[0032] DIBMA can be purchased from Cube Biotech as DIBMA 10 and DIBMA 12.

[0033] The preparation of poly(acrylic acid-co-styrene) copolymers is described in WO 2020 / 257637 and S. Harrisson, F. Ercole and BW Muir, "Living spontaneous gradient copolymers of acrylic acid and styrene: one-pot synthesis of pH-responsive amphiphiles," Polym. Chem., 2010, 1, 326-332.

[0034] In some cases, the copolymer is a copolymer of styrene and acrylic acid, or a copolymer of styrene and an acrylic acid derivative. Any copolymer derivative can be used in the subject copolymer. Examples of derivatives include acrylates, methacrylates, acrylic esters, acrylamides, and N-substituted acrylamides. In certain cases, the acrylic esters or acrylamides are substituted with zwitterionic species, as described in U.S. Patent Application Publication No. 2019 / 0062469A1, the disclosure of which is incorporated herein by reference.

[0035] In certain embodiments, the copolymer contains an acrylic acid or acrylic acid derivative content of 30% to 70%, 35 to 65%, or 40 to 60%.

[0036] The synthesis of amphipols is described in WO 115083 and Marconnet, A., Michon, B., Le Bon, C., Giusti, F., Tribet, C., & Zoonens, M. (2020). Solubilization and stabilization of membrane proteins by cycloalkane-modified amphiphilic polymers. Biomacromolecules. doi:10.1021 / acs.biomac.0c00929.

[0037] Additional polyacrylates modified with alkyl groups such as pentyl, hexyl, and tert-butyl are described in US2020 / 0383918.

[0038] A polymethacrylate containing about 0.52 butyl methacrylate (BMA) in the copolymer and about 0.48 methylacryloxycholine (MAC) in the copolymer, with a degree of polymerization (DP) of about 39.00, is sold by Avanti Polar Lipid under the brand name Polymethacrylate Copolymer (N-C4-52-6.9). Other polymethacrylates are described in Yasuhara K, Arakida J, Ravula T, Ramadugu SK, Sahoo B, Kikuchi JI, Ramamoorthy A. 2017. Spontaneous Lipid Nanodisc Formation by Amphiphilic Polymethacrylate Copolymers. J Am Chem Soc. 139(51):18657-18663.

[0039] Polyacrylate polymers modified with alkanes such as n-butyl, t-butyl, pentyl, neopentyl, and hexyl are described in N.Z. Hardin, T. Ravula, G. Di Mauro, A. Ramamoorthy, Hydrophobic Functionalization of Polyacrylic Acid as a Versatile Platform for the Development of Polymer Lipid Nanodisks, Small. 2019 March;15(9):e1804813. doi:10.1002 / smll.201804813, and US2020 / 383918A1.

[0040] Alternatively, linear carbohydrates with a degree of polymerization of less than 100 functionalized with hydrophobic groups are described in US 2022 / 093587A. An example of the linear carbohydrate is inulin, and examples of the hydrophobic group are alkyl, alkenyl, alkynyl, cycloalkyl, or heteroalkyl having 1 to 3 heteroatoms. The hydrophobic group is attached to the carbohydrate via an ether, ester, or amide group.

[0041] In another embodiment, the concentration (v / v) of the polymer is from about 0.01% to about 5%, particularly from about 0.1% to about 2.5%.

[0042] Solutions of polymers such as DIBMA, SMA, AASTY, Ultrasolute Amphipol can dissolve cells and tissues very quickly.

[0043] In a further embodiment, the cells and / or tissues are contacted with the polymer for between about 10 seconds and about 5 minutes. It has been found that even after such short contact times, the cells and tissues are sufficiently lysed.

[0044] As noted above, eukaryotic cells are selectively lysed by the methods of the present invention, while lysis of prokaryotic cells and yeast has not been observed. This surprising discovery enables, in one embodiment, the methods of the present invention to be used to separate eukaryotic cells or cellular components from prokaryotic cells or cellular components. This can be achieved, for example, by lysing the cells and then separating intact prokaryotic or yeast cells from the lysed fragments of the eukaryotic cells by known methods.

[0045] The present invention further relates to the use of the polymers defined above for lysing cells and tissues, for details of this use see the above description of materials such as cells and polymers and the overall method steps thereof.

[0046] The present invention, particularly in the above-described embodiments, offers several advantages. First, it provides an easy-to-use, highly effective, rapid, and reliable lysis method that can stabilize membrane proteins in their unique lipid environment. This can be achieved by utilizing polymers at different concentrations (v / v) ranging from 0.1% to 5% in the lysis buffer. The lysis buffer (polymer present in the buffer) used ensures time-efficient and stable solubilization and stabilization of target proteins for studying protein-protein interactions, native membrane protein activity, and other functional studies. In addition to membrane proteins, soluble proteins can also be prepared in their native state. Native proteins can be used for interaction analyses such as immunoprecipitation and mass spectrometry, binding to specific antibodies, analysis of enzymatic activity, regulation of metabolic processes, and other functional studies.

[0047] Proteomics is the study of the proteome, i.e., the totality of all proteins present in a cell or organism under defined conditions and at a defined time. In contrast to the (somewhat) static genome, the proteome is (highly) dynamic and therefore can change in its qualitative and quantitative protein composition due to changing conditions (environmental factors, temperature, gene expression, drug administration, etc.). The polymers of the present invention allow for efficient and mild dissolution to obtain a variety of proteins in their native state, making these compounds ideally suited for use in proteomics.

[0048] Higher copolymer concentrations (1%-5%) result in effective cell lysis and membrane solubilization in less than 10 minutes, leading to the release of intact long-chain DNA and RNA, a process that does not require mechanical assistance that can disrupt nucleic acid strands.

[0049] This is a powerful advantage for "next-generation sequencing" approaches where long read lengths are desired. The long DNA or RNA obtained by the process of the present invention can be used in sequencing methods such as nanopore sequencing, which are optimized for high read lengths. Alternative sequencing technologies that can use the nucleic acids obtained herein include single-molecule real-time sequencing (SMRT), pyrosequencing, reversible dye-terminator sequencing, and sequencing by ligation mediated by ligase enzymes.

[0050] In a further embodiment, lower copolymer concentrations (0.01-0.1%) result in mild but effective cell lysis without affecting nuclei, facilitating sample processing in less than 5 minutes. In this case, RNA fragments such as mRNA, tRNA, and rRNA can be purified and prepared without major genomic DNA impurities. Therefore, downstream applications of RNA fractions, proteins, and other biomolecules, such as metabolites, obtained from the cytosol are faster and more efficient.

[0051] The present invention also relates to the preparation of DNA and RNA after cell lysis.Nucleic acid, especially DNA, is not attacked and remains intact in long chains due to the mild lysis conditions.The DNA and RNA thus obtained are hardly subjected to mechanical shearing due to the mild lysis, and therefore are less broken and more intact than those obtained by conventional lysis methods.

[0052] The method of the present invention is not limited to the preparation of nucleic acids. After lysis, proteins and other biomolecules can also be prepared. The mild lysis method of the present invention allows the recovery of a large number of native proteins for proteomics applications, such as MALDI mass spectrometry, affinity proteomics, and microarrays. Protein samples can be used, for example, for expression proteomics and drug discovery.

[0053] Furthermore, it is possible to lyse the cell envelope while leaving the nucleus intact, which is an important advantage in nucleic acid purification: eukaryotic cells are lysed, but bacterial cells (E. coli, gram-positive) remain intact. This advantage allows for easy separation of bacterial and eukaryotic cell components.

[0054] In further embodiments, even lower copolymer concentrations (0.005-0.05%) result in mild cell perforation while leaving the majority of cells viable. The polymer concentration and incubation time can be adjusted to ensure that more than 75% of the cells remain, preferably more than 90% of the cells remain, and more preferably more than 95% of the cells remain. This can be used for many applications, such as the uptake of active agents into cells.

[0055] In cell biology, transfection is the introduction of foreign DNA or RNA into animal and possibly other eukaryotic cells. Methods for transfection include coating or stabilizing nucleic acids to facilitate uptake with substances such as cyclodextrins, liposomes, dendrimers, or cationic polymers. Physical methods involve the use of some physical means to deliver the transfected material into the nucleus of the target cell. This sometimes works in conjunction with disruption of the cell membrane, e.g., electroporation, or magnetofection.

[0056] Thus, in a further embodiment, low concentrations (0.005-0.05%) of the copolymer result in disruption of the cell membrane without complete lysis and death of the cell, allowing the naked or modified form of nucleic acid to enter the cell. The polymers of the invention can also be used in conjunction with substances that aid in the entry of nucleic acids into cells, such as lipids, cyclodextrins, dendrimers, or functionalized polymers.

[0057] In diagnostics, biological components such as DNA, RNA, proteins, and metabolites are examined qualitatively and quantitatively. This provides information about disease, genetic predisposition, or health conditions. Diagnostic tests can be performed by medical professionals, but can also be performed by individuals. Due to their ability to gently lyse cells and tissues, preserve nucleic acids with low levels of fragmentation, and preserve proteins in their native state, the polymers of the present invention are particularly suitable for diagnostic tests.

[0058] In a further aspect of the invention, the polymers may also be used in kit form to facilitate customer use of these materials.

[0059] Other possible components of such a kit include: The reducing agent contained in the reagent can be any reducing agent known to reduce disulfide bonds in cellular proteins. Examples of such reducing agents are mercaptans, and preferred mercaptans are DTT (dithiothreitol), beta-mercaptoethanol, and tris(2-carboxyethyl)phosphine (TCEP). Dithiothreitol and 3-mercaptoethanol are particularly preferred. The concentration of the reducing agent in the reagent according to the present invention is about 1 mM to about 10 mM, preferably about 1 mM to about 5 mM. A chelating agent that disrupts the secondary and tertiary structure of RNA can be included. A preferred concentration is 0.2 mM to 5 mM, and more preferably about 1 to 2 mM. Preferred chelating agents can be EDTA (ethylenediaminetetraacetic acid), EGTA (ethyleneglycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), NTA (nitrilotriacetic acid), DTPA (diethylenetriaminepentaacetic acid), citric acid, histidine, and 8-hydroxyquinol. Buffer components, such as HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES, 2-(N-morpholino)ethanesulfonic acid, tris[tris(hydroxymethyl)aminomethane], or bis-tris-2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol], stabilize the pH of the solution in the physiological range, or 6.5 to 8.0, preferably 7.5 to 8.0. The buffer concentration is 1 mM to 250 mM, preferably 25 mM to 100 mM. Lithium, sodium, or potassium salts, or mixtures thereof, are preferred to maintain the ionic strength of the mixture, which helps stabilize proteins, nucleic acids, and other biomolecules. The concentration of the alkali halide should be between 1 mM and 200 mM, preferably between 10 and 100 mM, and more preferably between 20 and 50 mM.

[0060] The present invention is further illustrated by the following examples and figures, which it is emphasized that the examples and figures should not be construed as limiting the invention thereto. [Brief explanation of the drawings]

[0061] [Figure 1] Lysis of HEK cells after treatment with either RIPA buffer, Ultrasolute Amphipol 0.1% / 1% / 2.5%, or control buffer for 1 min / 10 min is shown. [Figure 2] Detectable dead HEK cells after 1 min / 10 min / 30 min treatment are shown. [Figure 3] SDS PAGE analysis of HEK cell supernatants after treatment for 1 min / 10 min / 30 min is shown. [Figure 4] 1 shows SDS PAGE analysis of eukaryotic cell lysis after different lysis times with different copolymers. [Figure 5] FIG. 1 shows eukaryotic cell lysis by AASTY 50, a comparison of RIPA and AASTY 6-50, and protein amount (RIPA: 100%). [Figure 6]Figure 1 shows detectable dead HEK293 cells after treatment with either RIPA buffer, Ultrasolute Amphipol, SMA, AASTY, DIBMA, or control buffer for up to 30 minutes. 100%: amount of cells provided. [Figure 7] Figure 1 shows SDS PAGE analysis of prokaryotic cell lysis by different copolymers and protein gels in which no protein was detected. [Figure 8] 1 shows an SDS PAGE analysis of cardiac tissue lysis by Ultrasolute Amphipol 18. [Figure 9] Figure 1 shows SDS PAGE analysis of eukaryotic cell lysis by different copolymers and protein gels with and without lysed cell nuclei. [Example]

[0062] Example 1: Total number of countable HEK cells after treatment with either RIPA buffer, Ultrasolute Amphipol 0.1%, 1%, or 2.5%, or control buffer for 1 minute or 10 minutes. Fewer cells equate to fully lysed cells. The fewer countable cells, the more fully lysed the cells are (no detectable organelles—release of intact long-chain DNA). A 1-minute incubation with 0.1% Ultrasolute Amphipol 18 results in a comparable number of fully lysed cells compared to RIPA buffer. A longer 10-minute incubation time results in a 50% reduction in detectable cells, thus increasing the number of fully lysed cells compared to the shorter 1-minute incubation. However, longer incubation times in RIPA buffer do not show a change in cell number. Increasing the concentration of Amphipol Ultrasolute 18 solution further increases the number of completely lysed cells (4.4-fold increase at 1% / 15.7-fold increase at 2.5% compared to RIPA buffer at 10 min).

[0063] Example 2: Percentage of detectable dead HEK cells after 1, 10, or 30 minutes of treatment with either RIPA buffer, Ultrasolute Amphipol 0.1%, or control buffer. Already after 1 minute of incubation, 61% of the cells treated with 0.1% Ultrasolute Amphipol are found to be dead. After 10 minutes, 69% are found to be dead, and after 30 minutes, 88% are found to be dead. RIPA buffer and buffer control show dead cell counts of 2-5%.

[0064] Example 3: SDS-PAGE analysis of HEK cell supernatants after treatment with either RIPA buffer, Ultrasolute Amphipol 0.1%, or control buffer for 1, 10, or 30 minutes. After incubation, intact cells and cell debris were centrifuged, and equal volumes of supernatant were run on SDS-PAGE and detected via Coomassie staining. Compared to the buffer control, cells treated with Ultrasolute Amphipol 0.1% were successfully lysed after just 1 minute in a comparable amount of cells to those treated with RIPA buffer. Longer incubation times of 10 to 30 minutes resulted in better cell lysis compared to RIPA buffer, as indicated by the stronger corresponding bands on SDS-PAGE. Ultrasolute Amphipol already outperformed RIPA buffer at low concentrations (0.1%) after only 10 minutes of incubation.

[0065] Example 4: Eukaryotic cell lysis by different copolymers To determine solubilization efficiency, SDS-PAGE analysis was performed on HEK293 cell supernatants after treatment with either RIPA buffer (Thermo Fisher, Waltham, MA, USA), Ultrasolute Amphipol 18, SMALP 200, AASTY 6-50, or DIBMA 10 (all from Cube Biotech, Monheim, Germany) for 1 to 30 minutes, or control buffer. Fewer cells equate to fully lysed cells. The fewer countable cells, the more complete the cell lysis (no detectable organelles—release of intact long-chain DNA). After incubation, intact cells and cell debris were centrifuged, and equal volumes of supernatant were run on SDS-PAGE and detected via Coomassie staining (Figure 4).

[0066] Quantification was performed by SDS-PAGE analysis of HEK293 / T. ni. cell supernatants after 10 min of treatment with either RIPA buffer, Ultrasolute Amphipol, SMALP, AASTY, DIBMA, or control buffer. After incubation, intact cells and cell debris were centrifuged, and equal volumes of supernatants were run through SDS-PAGE and detected via Coomassie staining.

[0067] The polymers (ASTY 6-50 or Ultrasolute Amphipol 18) increased the protein content by approximately 20% (HEK293) or 50% (T. ni.) by SDS-PAGE band analysis. RIPA was used as a standard, set at 100% (Figure 5).

[0068] Figure 6 shows detectable dead HEK293 cells after treatment with either RIPA buffer, Ultrasolute Amphipol, SMA, AASTY, DIBMA, or control buffer for up to 30 minutes. 100%: amount of cells provided.

[0069] Example 5: Prokaryotic cell lysis by different copolymers To test solubilization efficiency, SDS-PAGE analysis of E. coli / B. subtilis cell supernatants after treatment with either RIPA buffer, Ultrasolute Amphipol 18, SMALP200, AASTY 6-50, DIBMA 10, or control buffer for 1 min to 16 h was performed (Figure 7). After incubation, intact cells and cell debris were centrifuged, and equal volumes of supernatants were run through SDS-PAGE and detected via Coomassie staining. Compared to eukaryotic cells, no distinct protein bands were detected, only a smear. Treated cells were still able to form colonies when plated on agar plates. - 200 μl of bacterial suspension was taken from a flask with an OD600 of 1. The reaction tube was centrifuged at 2500 g for 5 minutes at RT. The resulting clear supernatant was discarded. The resulting pellet was resuspended in 200 μl of buffer containing buffer / RIPA / 2.5% (w / v) polymer solution. - The reaction tubes were incubated on ice for different amounts of time. A centrifugation step at 14000 g for 15 min at RT was performed. -25 μl of the supernatant was added to SDS sample buffer and 20 μl of the sample was loaded onto SDS PAGE.

[0070] Example 6: Cardiac tissue lysis with Ultrasolute Amphipol 18 Blue Native Page analysis of bovine heart tissue treated with either Ultrasolute Amphipol 18 or buffer control. Both samples were incubated with either buffer control or 1% (w / v) Ultrasolute Amphipol solution for 1 h at RT, followed by a 100 kg ultracentrifugation step for 1 h at 4°C. 5 μl of the resulting supernatant was applied to Blue Native Page. Treatment with Ultrasolute Amphipol 18 resulted in the solubilization of a wide range of proteins / protein complexes of different sizes, whereas the buffer control showed only one clear band (Figure 7).

[0071] Example 7: Eukaryotic cell lysis by different copolymers, leaving the cell nucleus intact To test the solubilization efficiency, SDS-PAGE analysis of HEK293 and Sf9 cell supernatants after 10 min of treatment with 0.01% to 2.5% RIPA buffer, Ultrasolute Amphipol 18, SMALP 200, AASTY 6-50, DIBMA 10, or control buffer was performed (Figure 9). After incubation, intact cells and cell debris were centrifuged to obtain equal volumes of supernatant. When 0.01% to 0.05% was used for the 10 min incubation period, there was no increase in viscosity. When a 2.5% polymer concentration was used for the 10 min incubation period, the lysate became a highly viscous fluid. The high viscosity indicated free DNA and, therefore, lysed cell nuclei. After digestion with Benzonase (catalog no. 71206, Merck Millipore, 4 °C, 30 min, used as described in the manufacturer's protocol), the viscosity decreases, and as a result, the solution can be pipetted with a standard pipette, demonstrating the presence of DNA in the lysate. When using copolymer concentrations of 0.01%–0.05% with a 10 min incubation time, cytoplasmic proteins are detectable via SDS-PAGE, with no increase in viscosity. Therefore, by using lower polymer concentrations, membrane solubilization can be achieved while leaving the nucleus intact.

Claims

1. A method for lysing cells and tissues, comprising contacting said cells and / or said tissues with a polymer.

2. The method of claim 1 , wherein the cell is a eukaryotic cell.

3. 10. The method of any one of the preceding claims, wherein the polymer is a polymer capable of solubilizing and stabilizing membrane proteins and GPCRs.

4. 4. The method of claim 3, wherein the polymer has hydrophilic groups, such as COOH, maleimide, OH, amines, ammonium salts, zwitterions such as phosphocholine, and hydrophobic groups, such as polymerized styrene groups, polymerized diisobutylene groups, or linear C1-C16 aliphatic groups, branched C1-C16 aliphatic groups, and cyclic C5-C12 aliphatic groups or C5-C12 aromatic groups.

5. 6. The method of any one of claims 3 to 5, wherein the polymer is selected from the group consisting of polyacrylic acid, diisobutylene / maleic acid copolymer, styrene / maleic acid copolymer, and acrylic acid / styrene copolymer, optionally with some of the carboxylic acid groups bonded with cyclic amines to form amides.

6. The method according to any one of claims 3 to 5, wherein the molecular weight of the polymer is from 1,900 to 20,000.

7. 10. The method of any one of the preceding claims, wherein the concentration of the polymer is from 0.01% to 5%.

8. 10. The method of any one of the preceding claims, wherein the cells and / or tissue are contacted with the polymer for less than 5 minutes.

9. 10. The method of any one of the preceding claims, wherein the cells and / or tissue are contacted with the polymer for less than 1 minute.

10. 10. The method of any one of the preceding claims, wherein the cells and / or tissue are contacted with the polymer for less than 30 seconds.

11. 10. The method according to any one of the preceding claims, wherein the method is used to separate eukaryotic cells or cellular components from prokaryotic cells or cellular components.

12. 10. The method according to any one of the preceding claims, wherein the method is used for perforation of cell walls while leaving the cell nucleus intact.

13. 10. A method according to any one of the preceding claims, wherein the method is used for perforation of cell walls while leaving the majority of the cells viable.

14. 10. The method according to any one of the preceding claims, wherein said method is used to introduce foreign DNA or RNA into human, animal and other eukaryotic cells.

15. Use of a polymer according to any one of claims 3 to 6 for completely or partially lysing cells and tissues.

16. Use of the polymer according to any one of claims 3 to 6 for lysing muscle tissue, heart tissue, brain tissue, liver tissue, kidney tissue, lung tissue or blood cells.

17. Use of a polymer according to any one of claims 3 to 6 in a nucleic acid sequencing procedure.

18. Use of the polymer of any one of claims 3 to 6 in long read sequencing procedures.

19. Use of a polymer according to any one of claims 3 to 6 for the preparation of a stable and active membrane protein.

20. Use of a polymer according to any one of claims 3 to 6 for the preparation of an active soluble protein.

21. Use of the polymer according to any one of claims 3 to 6 in proteomics.

22. Use of the method according to any one of claims 1 to 14 in drug discovery and expression proteomics.

23. Use of the method according to any one of claims 1 to 14 for subsequent sequencing applications.

24. 10. A kit for cell lysis containing one or more polymers according to any one of the preceding claims.

25. The list below: - reducing agents such as DTT (dithiothreitol) β-mercaptoethanol or TCEP (tris(2-carboxyethyl)phosphine); buffer components such as HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES 2-(N-morpholino)ethanesulfonic acid, tris[tris(hydroxymethyl)aminomethane], or bis-tris 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol]; chelating agents such as EDTA (ethylenediaminetetraacetic acid), EGTA (ethyleneglycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid), NTA (nitrilotriacetic acid) or DTPA (diethylenetriaminepentaacetic acid), - one or more components from the group of lithium-sodium or potassium salts.

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