Enzymes

Abstract

The present invention relates to methods and compositions for dissociating biological tissue with the aim of obtaining single cells for analysis. More specifically, the compositions of the invention are enzyme compositions comprising protease K. The compositions of the invention are useful in methods for efficiently dissociating tissue at lower temperatures for application in high throughput unicellular omics studies.

Description

TECHNICAL FIELD

[0001] The present invention relates to compositions comprising proteinase K and methods for tissue dissociation. BACKGROUND

[0002] Enzymes are useful tools for dissociating cells from solid tissue for various applications in research, industry, and medicine. Reichard and Asosingh et al. Cytometry A . 2019 95(2): 219-226 describe examples of enzymes that can be used for solid tissue dissociation. Combinations of different enzymes, also known as enzyme “cocktails”, can be used for tissue dissociation. However, the specific enzymes must be carefully chosen because not all enzymes are suitable when used alone or in combination. For example, Reichard and Asosingh et al . 2019 report that trypsin is not suitable for preparing single cells due to the inherent risk of cleaving cell surface receptors.

[0003] Furthermore, due to the heat-sensitive nature of many enzymes, tissue dissociation is often performed at temperatures that result in maximum activity and efficiency of the enzymes. Because many enzymes have an optimal working temperature of around 37°C (e.g., trypsin, TrypLE TM , pronase, collagenase, liberase, and dispase, as discussed in Adam et al . Development. 2017 144(19): 3625-3632, methods for enzymatic tissue dissociation are routinely performed at this temperature. For example, kidney tissue dissociation is often performed at 37°C.

[0004] There are many commercial products for dissociating kidney tissue, such as the Multi Tissue Dissociation Kit 2 by Miltenyi Biotec, which can be used in combination with a specialized device such as the gentleMACS™ Dissociator (Miltenyi Biotec Multi-tissue dissociation Kit 2 Retrieved: May 5, 2023 [https: / / static.miltenyibiotec.com / asset / 150655405641 / document_phpdh7tnkl2it4bi11qp1l0o6o content-disposition=inline]; Miltenyi Biotec Dissociation of mouse kidney using Multi-tissue dissociation kit 2Accessed: May 5, 2023 [https: / / www.miltenyibiotec.com / upload / assets / IM0015569.PDF]). Mouse kidneys can also be dissociated at 37°C using more general tissue dissociation enzymes; one such example is in Adam. et al The scheme used in 2017.

[0005] Recently, it has been found that tissue dissociation at 37°C leads to artificial changes in cells, such as the induction of stress-response genes. Such changes are particularly problematic in high-throughput single-cell omics studies (genomics, proteomics, metabolomics). Therefore, methods for tissue dissociation are ideally performed at lower temperatures. et al A method described in 2017 utilizes Bacillus subtilis protease A. Bacillus subtilis protease A is a protease found in Bacillus licheniformis, a soil bacterium isolated from Himalayan glaciers. Bacillus licheniformis The serine endopeptidase expressed in O'Flanagan is known to be effective at lower temperatures. et al . Genome Biology 2019 20(210) describes the use of subtilisin A as a protease in an enzyme mixture to reduce stress response in kidney cells during dissociation.

[0006] Based on the above, more enzymes are still needed that can effectively dissociate tissues at lower temperatures for use in high-throughput single-cell omics studies. Summary of the Invention

[0007] In a first aspect, the present invention provides a composition comprising: proteinase K; a buffer, wherein the buffer comprises Hanks' balanced salt solution (HBSS); DNase; and Ca. 2+ source.

[0008] In a second aspect, the present invention provides the use of proteinase K for dissociating tissues at temperatures below 20°C.

[0009] In a third aspect, the present invention provides an in vitro method for dissociating tissue, comprising: a) contacting the tissue with a composition containing proteinase K; b) incubating the tissue with the composition for a first time period; and c) obtaining a cell suspension.

[0010] In a fourth aspect, the present invention provides a system configured to perform the method according to the third aspect of the invention.

[0011] In a fifth aspect, the present invention provides a kit comprising a composition according to the first aspect of the invention and instructions for use.

[0012] The present invention relates to the surprising discovery that proteinase K can be used as a tool to efficiently dissociate tissue into single cells. The single cells show advantageous properties that make them suitable for use in high-throughput single cell omics studies, in particular single cell RNA sequencing analysis such as single cell RNA-seq (scRNA-seq). Furthermore, it was found that tissue dissociation can be performed at lower temperatures (e.g., 4°C) to avoid artifacts associated with heat-induced stress responses. Surprisingly, it was also found that proteinase K can dissociate tissue at lower temperatures with at least the same effect as subtilisin A, an enzyme known to work at lower temperatures. This is particularly surprising because proteinase K is known to have an optimum temperature range of 20 to 65°C with maximum activity at 37°C (Bajorath Biochim Biophys Acta . 1988 954(2): 176-182). Furthermore, it was found that the proteinase K containing compositions of the present invention do not cleave certain cell surface markers compared to known enzyme compositions.

[0013] Proteinase K (EC 3.4.21.64), also known as Peptidase K, Endoproteinase K, and Endopeptidase K, is a non-specific serine endopeptidase in family S8 (subfamily S8A). It is commonly used to break down keratin (free proteins not needed in molecular biology) and nucleases (DNase and RNase). It is also used in prion research (Petrochenko et al . Molecular & Cellular Proteomics . 2012 11(7)), endotoxin / lipopolysaccharide limulus amoebocyte lysate (LAL) test (Petsch et al . Analytical Biochemistry . 1998 259(1): 42-47), protease footprinting (Hori and Carey Journal of Biological Chemistry . 1997 272(2): 1180-1187A) and isolating nucleic acids (Hilz et al . European Journal of Biochemistry 1975 56(1): 103-108).

[0014] Proteinase K is stable in a pH range of about 4.0 to 12.5, with an optimum at pH 8.0. DETAILED DESCRIPTION

[0015] Terms and definitions Unless otherwise provided, nomenclature used herein and laboratory procedures and techniques described herein are known in the art. Standard chemical symbols are used interchangeably with the full names represented by such symbols. Thus, for example, the terms "hydrogen" and "H" are understood to have the same meaning. Standard techniques can be used for formulation of compositions and testing of compositions. The foregoing techniques and procedures can be generally performed according to conventional methods well-known in the art.

[0016] It should be understood that the general description above and the following detailed description are only exemplary and explanatory and are not restrictive of the application as claimed. As used herein, the use of the singular includes the plural, unless specifically stated otherwise. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0017] As used herein, "proteinase K" refers to the amino acid sequence of SEQ ID NO: 1 (UniProt Reference No. P06873 as of May 16, 2023), homologs in other species, or variants thereof. Amino acid residues of proteinase K used herein are numbered relative to SEQ ID NO: 1.

[0018] SEQ ID NO: 1 ( Parengyodontium album Tritirachium album ), 384 amino acids, UniProt Reference No. P06873 as of May 16, 2023): MRLSVLLSLLPLALGAPAVEQRSEAAPLIEARGEMVANKYIVKFKEGSALSALDAAMEKISGKPDHVYKNVFSGFAATLDENMVRVLRAHPDVEYIEQDAVVTINAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQYSTIIAGMDFVASDKNNRNCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNNADARNYSPASEPSVCTVGASDRYDRRSSFSNYGSVLDIFGPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACRYIADTANKGDLSNIPFGTVNLLAYNNYQA ​As used herein, "or" means "and / or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms such as "include", "includes" and "included" is not limiting.

[0019] As used herein, "about" means the value "about" the referenced number contains the referenced number plus or minus 1-5% of the referenced number. For example, "about" 100 degrees can mean 95-105 degrees or as little as 99-101 degrees, depending on the context. When a range is expressed, for example, "1 to 20," it is meant to refer to every integer within the given range; that is, it means only 1, only 2, only 3, and so forth, up to and including only 20.

[0020] As used herein, the term "composition" includes products, formulations, and mixtures, as well as devices, instruments, assemblies, kits, and the like. Similarly, the term "method" includes procedures, protocols, steps, and the like.

[0021] As used throughout this disclosure, the words "may" and "can" are used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Additionally, the terms "including," "having," "involving," "containing," "characterised by," their variants (e.g., "includes," "has" and "involves," "contains," etc.) and like terms, as used herein (including the claims) are intended to be inclusive and / or open-ended and have the same meaning as the term "comprising" and its variants (e.g., "comprise" and "comprises") and, for example, do not exclude additional, unrecited elements or method steps.

[0022] It should be noted that embodiments of the present disclosure can include one or more combinations of two or more of the features described herein. As used herein, "feature" and like terms can include, for example, compositions, ingredients, components, elements, members, parts, portions, systems, methods, configurations, parameters, properties, and the like. Embodiments can include any of the features, options, and / or possibilities set forth elsewhere in the present disclosure, including those set forth in other aspects or embodiments of the present disclosure. It should also be noted that each of the foregoing, following, and / or other features described herein represents a different embodiment of the present disclosure. The features can also be combined in any suitable combination and / or order, with or without one or more additional features included or intervening therebetween, with one or more other features, and / or are combinable, to form unique embodiments, each of which is contemplated in the present disclosure. Such combinations of any two or more such features represent a different embodiment of the present disclosure. Thus, the present disclosure is not limited to the particular combinations of exemplary embodiments described herein and the disclosure of certain features with respect to a particular embodiment of the present disclosure should not be construed as limiting of the application or inclusion of that feature to the particular embodiment.

[0023] Further, unless a feature is described as requiring in a particular embodiment, a feature described in one embodiment can be optional and can not be included in other embodiments of the present disclosure. Further, unless a feature is described as requiring combination with another feature, any feature herein can be combined with any other feature of the same or different embodiments disclosed herein. Likewise, any steps recited in any method described herein and / or recited in a claim can be performed in any suitable order, and are not necessarily limited to the order described and / or recited, unless otherwise indicated (either explicitly or implicitly). However, such steps can also require, in certain embodiments of the present disclosure, to be performed in a particular order.

[0024] It will also be understood that, in the disclosure or where a range of values or value ranges are disclosed (e.g., less than, greater than, at least, and / or at most a certain value, and / or between two stated values), any particular value or value range falling within the disclosed value or value range is also specifically disclosed. Thus, for example, disclosure of an exemplary measurement (e.g., length, width, thickness, etc.) of less than or equal to about 10 units, or between 0 and 10 units, includes, for example, the following particular disclosure: (i) a measurement of 9 units, 5 units, 1 unit, or any other value between 0 and 10 units, including 0 units and / or 10 units; and / or (ii) a measurement of any other value range between 9 units and 1 unit, between 8 units and 2 units, between 6 units and 4 units, and / or between 0 and 10 units.

[0025] Embodiments of the invention The following embodiments describe include the disclosure related to one or more embodiments of the present disclosure. Therefore, some embodiments can include features disclosed in the subsequent examples without departing from the scope of the present disclosure. In other words, the features disclosed in the subsequent examples can be included in and / or incorporated into any one or more embodiments disclosed herein.

[0026] The composition of the present invention comprises proteinase K, which is an active peptidase that cleaves amino acids present in proteins that hold cells together.

[0027] Proteinase K is commercially available from many manufacturers, such as Qiagen TM (including Blirt TM ). Preferably, the proteinase K in the composition of the present invention is Blirt TM Cat. No. RP107B as of May 17, 2023. It is also possible to produce recombinant proteinase K using host cells.

[0028] The host cell can be selected from the group consisting of Pichia sp., Hansenula sp. such as H. polymorpha, Saccharomyces sp., Schizosaccharomyces sp., Yarrowia sp. such as Y. lipolytica, Kluyveromyces sp., and Aspergillus sp. Preferably, Pichia pastoris is used as the host cell.

[0029] Preferably, the proteinase K in the composition of the present invention comprises the amino acid sequence of SEQ ID NO: 1. More preferably, the proteinase K in the composition of the present invention consists of the amino acid sequence of SEQ ID NO: 1.

[0030] The amino acid sequence of the variant of proteinase K can share at least about 85% sequence identity with SEQ ID NO: 1, and thus can share at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1. Preferably, the sequence identity is at least 90% or at least 95%. More preferably, the sequence identity is 100%. Preferably, the variant of proteinase K substantially retains the enzymatic activity associated with the sequence of SEQ ID NO: 1.

[0031] The amino acid sequence of the variant proteinase K can be altered by substituting, adding or deleting a suitable number of amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 residues in the sequence of SEQ ID NO: 1. By "substitution, addition or deletion" is meant a combination of substitution, addition and deletion. Preferably, the variant of proteinase K substantially retains the enzymatic activity associated with the sequence of SEQ ID NO: 1.

[0032] When a sequence is modified by substitution of a particular amino acid residue, the substitution can be a conservative amino acid substitution. The term "conservative amino acid substitution" as used herein refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Amino acids with similar side chains tend to have similar properties, and thus a conservative substitution of an amino acid that is important to the structure or function of a polypeptide can be expected to affect the polypeptide structure / function less than a non-conservative amino acid substitution at the same position.

[0033] Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. asparagine, glutamine, serine, threonine, tyrosine), nonpolar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Thus, a conservative amino acid substitution can be considered to be a substitution in which a particular amino acid residue is replaced with a different amino acid in the same family. However, a substitution of an epitope residue can be equivalent to a non-conservative substitution in which one amino acid is replaced with another amino acid having a side chain belonging to a different family.

[0034] Proteinase K can be present in the composition of the application at a concentration of 1 ± 75% mg / mL, 1 ± 50% mg / mL, 1 ± 10% mg / mL, 1 ± 5% mg / mL or 1 ± 1% mg / mL. Proteinase K can be present in the composition of the application at a concentration of 10 mg / mL, 5 mg / mL or 2 mg / mL. Preferably, the concentration of proteinase K in the composition of the application is 1 ± 1% mg / mL. When "± x%" is used herein, it means ± x% of the stated value.

[0035] Proteinase K can be present in the composition of the application at a concentration of about 1 mg / mL. Preferably, proteinase K is present in the composition of the application at a concentration of 1 mg / mL.

[0036] Protease K can be present in the composition of the application at a concentration of 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, or 1.5 mg / mL.

[0037] Protease K can be present in the composition of the application at a concentration of 0.1 to 10 mg / mL, 0.2 to 9 mg / mL, 0.3 to 8 mg / mL, 0.4 to 7 mg / mL, 0.5 to 6 mg / mL, 0.6 to 5 mg / mL, 0.7 to 4 mg / mL, 0.8 to 3 mg / mL, or 0.9 to 2 mg / mL. Preferably, protease K is present in the composition of the application at a concentration of 0.1 mg / mL to 5 mg / mL.

[0038] The amount of protease K in the composition can be defined in terms of enzyme units. The following enzyme unit definition can be used: Protease K hydrolyzes urea-denatured hemoglobin to produce 1 μmol of color equivalent of tyrosine per 1 minute at 37°C and pH 7.5 (Folin & Ciocalteu method), 1 U = 1 mAnson U. For example, when using a 20 mg / mL (minimum) and 800 U / mL (minimum) stock, the final concentration is 1 mg / mL and the activity in solution will be 40 U / mL.

[0039] Protease K can be present in the composition of the application at a concentration of 40 ± 75% U / mL, 40 ± 50% U / mL, 40 ± 10% U / mL, 40 ± 5% U / mL, or 40 ± 1% U / mL. Protease K can be present in the composition of the application at a concentration of 400 U / mL, 200 U / mL, or 80 U / mL. Preferably, protease K is present in the composition of the application at a concentration of 40 ± 1% U / mL.

[0040] Protease K can be present in the composition of the application at a concentration of about 40 U / mL. Protease K can be present in the composition of the application at a concentration of 40 U / mL.

[0041] Protease K can be present in the composition of the application at a concentration of 4 to 400 U / mL, 8 to 360 U / mL, 12 to 320 U / mL, 16 to 280 U / mL, 20 to 240 U / mL, 24 to 200 U / mL, 28 to 160 U / mL, 32 to 120 U / mL, 36 to 80 U / mL, 37 to 70 U / mL, 38 to 60 U / mL, and 39 to 50 U / mL.

[0042] The composition of the present application also comprises DNase. DNase (in particular dsDNase) has been described as useful to reduce cell clustering / aggregation caused by cell-free DNA, leading to good quality samples for single-cell RNA sequencing (Reichard and Asosingh et al . 2019).

[0043] Although proteinase K is known to break down nucleases, such as DNase, the presence of Ca2+ions in solution is known to have a protective effect on DNase (Tullis and Rubin Analytical Biochemistry . 1980 107(1): 260-264).

[0044] DNase is commercially available from many manufacturers, such as Qiagen TM (including Blirt TM ). DNase can be present in the composition of the present application at a concentration of 4 ± 75% U / mL, 4 ± 50% U / mL, 4 ± 10% U / mL, 4 ± 5% U / mL or 4 ± 1% U / mL. DNase can be present in the composition of the present application at a concentration of 40 U / mL, 20 U / mL or 8 U / mL. Preferably, DNase is present in the composition of the present application at a concentration of 4 ± 1% U / mL.

[0045] DNase can be present in the composition of the present application at a concentration of about 4 U / mL. DNase can be present in the composition of the present application at a concentration of 4 U / mL.

[0046] DNase can be present in the composition of the present application at a concentration of 0.4 to 40 U / mL, 0.8 to 36 U / mL, 1.2 to 32 U / mL, 1.6 to 28 U / mL, 2 to 24 U / mL, 2.4 to 20 U / mL, 2.8 to 16 U / mL, 3.2 to 12 U / mL, 3.6 to 8 U / mL, 3.7 to 7 U / mL, 3.8 to 6 U / mL and 3.9 to 5 U / mL.

[0047] The following enzyme unit definition can be used for DNase: One unit is defined as an increase in absorbance at 260 nm of 1.0 in 30 minutes at 37°C and pH 8.0 with herring sperm DNA as substrate.

[0048] Preferably, the DNase is double-stranded DNase (dsDNase). Preferably, the dsDNase in the composition of the present application is Blirt TM Cat. No. EN33 as of May 17, 2023.

[0049] The DNase can be a member of the DNase I family selected from DNase I, DNase1L1, DNase 1L2 and DNase1L3. Preferably, the DNase is DNase I.

[0050] The DNase can be a member of the DNase II family selected from DNase IIa and DNase IIb.

[0051] The composition of the present application comprises Ca 2+ source, which can be calcium chloride. The purpose of the additional calcium ions is to protect the DNase and its activity from degradation by proteinase K (Tullis and Rubin 1980). The Ca 2+ source can comprise calcium chloride. The calcium chloride can be solid or aqueous calcium chloride. Preferably, the calcium chloride is aqueous calcium chloride.

[0052] The calcium chloride can be present in the composition of the present application at a concentration of 10 ± 75% mM, 10 ± 50% mM, 10 ± 10% mM, 10 ± 15% mM, 10 ± 5% mM or 10 ± 1% mM. The calcium chloride can be present in the composition of the present application at a concentration of 100 mM, 50 mM or 20 mM. Preferably, the calcium chloride is present in the composition of the present application at a concentration of 10 ± 1% mM.

[0053] Preferably, the calcium chloride is present in the composition of the present application at a concentration of 11 to 12 mM, more preferably 11.3 mM.

[0054] The calcium chloride can be present in the composition of the present application at a concentration of about 11 mM. Preferably, the calcium chloride is present in the composition of the present application at a concentration of 11 mM.

[0055] The calcium chloride can be present in the composition of the present application at a concentration of 1 to 100 mM, 2 to 90 mM, 3 to 80 mM, 4 to 70 mM, 5 to 60 mM, 6 to 50 mM, 7 to 40 mM, 8 to 30 mM and 9 to 20 mM.

[0056] The composition of the invention can comprise one or more buffers. The method or use of the invention can require one or more buffers. Suitable buffers of the invention help support cell viability and yield, as well as reduce cell aggregation. The buffer can be selected from the group consisting of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Hanks’ Balanced Salt Solution (HBSS), MOPS (3-(N-morpholino)propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), Minimal Essential Medium (MEM), Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640, Iscove’s Modified Dulbecco’s Medium (IMDM), BES (NN-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid; 2-[bis(2-hydroxyethyl)amino]-ethanesulfonic acid), Earle’s Balanced Salt Solution (EBSS), MOPSO (2-hydroxy-3-morpholino propanesulfonic acid), ACES (N--2-aminoethanesulfonic acid), fetal bovine serum (FBS), TAPS (([tris(hydroxymethyl)methylamino]propanesulfonic acid), phosphate buffered saline (PBS), Dulbecco’s phosphate buffered saline (DPBS), bicine, tricine, cell culture grade water, ethylenediaminetetraacetic acid (EDTA), bovine serum albumin (BSA), and ethylene glycol-bis(β-aminoethyl)-N,N,Nʹ,Nʹ-tetraacetic acid (EGTA).

[0057] Preferably, the buffer is HBSS. HBSS is a buffer commonly used for cell culture and contains a balance of essential inorganic ions for cell growth, as well as to maintain the correct pH and osmotic pressure. HBSS is commercially available from many manufacturers, for example Cytiva TM . Preferably, the HBSS in the composition of the invention is Cytiva TM Cat. No. SH30268.01 as of May 17, 2023. The composition of Cytiva TM Cat. No. SH30268.01 is shown in the table below. The components shown in the table below can vary by ±75%, ±50%, ±20%, ±10%, ±5%, or ±1% of the values shown. Calcium chloride (anhydrous) and / or magnesium sulfate (anhydrous) can be omitted. Component description mg / L mmol / L Calcium chloride (anhydrous) 140 1.2615 Potassium chloride 400 5.3655 Potassium dihydrogen phosphate (anhydrous) 60 0.4409 Magnesium sulfate (anhydrous) 97.67 0.8112 Sodium chloride 8000 136.8925 Sodium hydrogen phosphate (anhydrous) 47.68 0.3359 D-glucose (anhydrous) 1000 5.5506 Sodium bicarbonate 350 4.1662

[0058] Preferably, the composition of the invention comprises: 1 ±10% mg / mL Proteinase K; 4 ±10% U / mL dsDNase; Hanks’ Balanced Salt Solution (HBSS); and 10 ±15% mM calcium chloride.

[0059] More preferably, the composition of the present application comprises: 1 mg / mL proteinase K; 4 U / mL dsDNase; Hanks' Balanced Salt Solution (HBSS); and 10 ± 15% mM calcium chloride.

[0060] In a further aspect, the present application provides a composition comprising: a buffer; proteinase K; DNase; and Ca 2+ source.

[0061] In a preferred embodiment, the ex vivo method of dissociating a tissue of the present application comprises: a) contacting a tissue with a composition comprising: 1 mg / mL proteinase K; 4 U / mL dsDNase; Hanks' Balanced Salt Solution (HBSS); and 10 ± 15% mM calcium chloride; b) incubating the tissue with the composition for a first period of time; and c) obtaining a cell suspension.

[0062] In a more preferred embodiment, the ex vivo method of dissociating a tissue of the present application comprises: a) contacting a tissue with a composition comprising: 1 mg / mL proteinase K; 4 U / mL dsDNase; Hanks' Balanced Salt Solution (HBSS); and 10 ± 15% mM calcium chloride; b) incubating the tissue with the composition for a first period of time; c) obtaining a cell suspension; d) passing the cell suspension through a first filter; e) quenching the proteinase K, preferably with 10% fetal bovine serum (FBS) in phosphate buffered saline (PBS); and preferably f) passing the cell suspension through a second filter, preferably a 40 or 70 pm filter.

[0063] The composition of the present application can also comprise one or more additional agents, such as other enzymes (in particular dissociation enzymes).

[0064] Alternatively, the composition of the present application can also lack one or more additional agents, such as other enzymes (in particular dissociation enzymes).

[0065] The one or more additional agents can be selected from the group consisting of: accutase, TrypLE TM , trypsin, chymotrypsin, pronase, papain, liberase, collagenase, elastase, dispase, thermolysin, hyaluronidase, clostridial protease from clostridium histolyticum and neutral protease, pronase, pepsin, lysozyme, chelators of divalent ions (such as EDTA or citrate), and combinations thereof.

[0066] The method of the present application can further comprise passing the cell suspension through a first filter and / or a second filter.

[0067] The size of the filter can be selected from the group consisting of: 100, 95, 80, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2 and 1 pm.

[0068] Preferably, the size of the filter is 40 or 70 pm.

[0069] The method of the application can further comprise quenching the proteinase K to terminate the dissociation reaction. The quenching can be performed with a buffer described herein. The quenching can be performed with fetal bovine serum (FBS) in phosphate buffered saline (PBS) or in another buffer described herein. The concentration of FBS can be 1 to 100% v / v, 2 to 90% v / v, 3 to 80% v / v, 4 to 70% v / v, 5 to 60% v / v, 6 to 50% v / v, 7 to 40% v / v, 8 to 30% v / v, 9 to 20% v / v. Preferably, the concentration of FBS is 10%.

[0070] Alternatively or in addition, the quenching can be performed with a Cu 2+ source. The Cu 2+ source can be present in the composition of the application in a buffer described herein.

[0071] According to the examples below, quenching with FBS improves significantly the viability and yield of dissociated cells compared to quenching with Cu 2+ source. The examples below demonstrate that quenching with FBS is more suitable than quenching with Cu 2+ source. The examples below demonstrate that quenching with FBS improves significantly the viability and yield of dissociated cells compared to quenching with Cu

[0072] After dissociation, cell yield, viability and aggregation can be evaluated by any suitable method known in the art. Preferably, cell yield, viability and aggregation are evaluated using an automated cell counter, preferably a Nucleocounter ® NC-200™ using Via2-Cassette™ (Chemometec). Other automated cell counters can also be used (e.g. products from Nexcelom and Thermofisher). Cell yield, viability and aggregation can also be quantified by using a hemocytometer with trypan blue. Yield can be calculated as 1000 ((viable cells per mL x dilution factor used for counting x total volume in mL) / (mass in mg)) = viable cells per gram of tissue.

[0073] The method of the application can further comprise removing the supernatant. Removing the supernatant can comprise centrifugation.

[0074] The method of the application can further comprise resuspending the cells in a buffer described herein. The buffer can comprise Bovine Serum Albumin (BSA) and / or Ethylenediaminetetraacetic acid (EDTA). The concentration of BSA can be selected from the group consisting of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.9, 1, 2, 3, 4 and 5%. The concentration of BSA can be 0.04 to 4%, 0.08 to 3.6%, 0.12 to 3.2%, 0.16 to 2.8%, 0.2 to 2.4%, 0.24 to 2%, 0.28 to 1.6%, 0.32 to 1.2% or 3.6 to 0.8%. The concentration of EDTA can be selected from the group consisting of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mM. The concentration of EDTA can be 0.01 to 1 mM, 0.02 to 0.9 mM, 0.03 to 0.8 mM, 0.04 to 0.7 mM, 0.05 to 0.6 mM, 0.06 to 0.5 mM, 0.07 to 0.4 mM, 0.08 to 0.3 mM or 0.09 to 0.2 mM. Preferably, the buffer comprises 0.4% BSA and 0.1 mM EDTA.

[0075] The method of the application can further comprise mechanical dissociation, which can be performed by a suitable device, preferably an automated tissue dissociation system, for example selected from the group consisting of: VIA Extractor™ tissue dissociator, gentleMACS™ Dissociator, gentleMACS™ Octo Dissociator, Singulator™ 100 and Singulator™ 200. Preferably, the method of the application is performed using a VIA Extractor™ tissue dissociator.

[0076] The composition, method or system of the application can be suitable for use with a VIA Extractor™ tissue dissociator.

[0077] The first time period of the method of the present invention can be at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 minutes. The first time period of the method of the present invention can be no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 minutes, preferably 20 minutes, more preferably 15 minutes. The first time period of the method of the present invention can be 1.5 to 150 minutes, 3 to 135 minutes, 4.5 to 120 minutes, 6 to 105 minutes, 7.5 to 90 minutes, 9 to 75 minutes, 10.5 to 60 minutes, 12 to 45 minutes, or 13.5 to 30 minutes. Preferably, the first time period of the method of the present invention is about 15 minutes, more preferably 15 minutes.

[0078] The methods or uses of the present invention can be carried out at temperatures below or about 50, 45, 40, 35, 30, 25, 30, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1°C. The methods or uses of the present invention can also be carried out at temperatures from 0.4 to 40°C, 0.8 to 36°C, 1.2 to 32°C, 1.6 to 28°C, 2 to 24°C, 2.4 to 20°C, 2.8 to 16°C, 3.2 to 12°C, or 3.6 to 8°C.

[0079] The tissue used for dissection is preferably a solid tissue and may be selected from: spleen, heart, liver, brain and other nerve tissues, kidney, lung, pancreas, breast, umbilical cord, skin, placenta, ovary, fallopian tube, uterus, prostate, tonsils, thymus, stomach, testis, trachea, cartilage, tendon, bone, skeletal muscle, smooth muscle, digestive tract, colon, intestine, bladder, urethra, eye, gallbladder, organoids derived from cell culture, and tumors. Preferably, the tissue is kidney tissue, more preferably intact kidney tissue, and even more preferably intact kidney tissue weighing less than 300 mg.

[0080] The tissue may be obtained from mammals, for example, from the group consisting of: mice, rats, birds such as chickens, ruminants such as cattle, goats, deer, sheep, horses, and other animals such as pigs, cats, dogs, and primates such as humans, chimpanzees, gorillas, and monkeys. Preferably, the tissue is obtained from mice, more preferably from female mice.

[0081] In another aspect, the present invention provides a cell suspension obtained by the method of the third aspect of the present invention.

[0082] The compositions of the present invention can be administered to tissues using a device, such as the device described in EP3171152 A1.

[0083] The kit of the present invention may further comprise one or more containers. Various components of the compositions of the present invention may be present in the kit within one or more of said containers. For example, the components may be present entirely in one container or in separate containers.

[0084] In addition to those shown and described herein, various modifications of the invention will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. Preferred features of each aspect of the invention, when suitably adapted, are equally applicable to each of the other aspects. References herein are incorporated by way of reference to the fullest extent permitted by law. Example

[0085] The present invention will now be further described with reference to the following embodiments and accompanying drawings, which are included for reference only and should not be construed as limiting the invention.

[0086] Figure 1 Different concentrations of proteinase K (0.1 mg / mL, 1 mg / mL, and 5 mg / mL) were followed by Cu. 2+ Quenching of visible cleavage in mouse kidneys.

[0087] Figure 2 Different concentrations of proteinase K (0.1 mg / mL, 1 mg / mL, and 5 mg / mL) were followed by Cu. 2+ Quantification of yield, activity and aggregation of mouse kidneys after quenching.

[0088] Figure 3 The production, activity, and aggregation of mouse kidneys after dissociation were quantified by using different concentrations of proteinase K (0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL) followed by FBS quenching at 4°C (“cold”) and 37°C (“hot”).

[0089] Figure 4 Triple copies of experimental results obtained by incubation with proteinase K for 15 and 20 minutes.

[0090] Figure 5 Comparison of dissociation results using i) VIA Extractor™ Tissue Dissociator with Bacillus subtilis protein A at 4°C (left), ii) VIA Extractor™ Tissue Dissociator with proteinase K at 4°C (middle), and iii) Miltenyi Multi-Tissue Dissociation Kit 2 with gentleMACS™ Octo Dissociator with Miltenyi enzyme at 37°C (right).

[0091] Figure 6 : UMAP plot of kidney cell clusters detected after dissociation of mouse kidney with proteinase K and subtilisin A at 4°C using a VIA Extractor™ tissue dissociator.

[0092] Figure 7 : UMAP plot of cell clusters detected after dissociation of mouse kidney with proteinase K at 4°C (“cold”) and Miltenyi Multi Tissue Dissociation Kit 2 at 37°C using a VIA Extractor™ tissue dissociator.

[0093] Figure 8 : Stress marker comparison of kidney cells dissociated with proteinase K at 4°C and Miltenyi Multi Tissue Dissociation Kit 2 using a VIA Extractor™ tissue dissociator.

[0094] Example 1 The following composition was prepared for use as a reagent for tissue dissociation.

[0095] • 1 mg / mL recombinant proteinase K (Blirt TM / QIAGEN TM ); • 4 U / mL dsDNase (Blirt TM / QIAGEN TM ); and • HBSS buffer (Cytiva TM ) with 10 mM CaCl2 (Thermofisher TM ) added.

[0096] Example 2 - Pre-experiment to determine optimal protease K concentration Figure 1 A method for dissociating mouse kidney tissue was developed using the composition prepared in Example 1. The method was tested on <300 mg intact kidney from a female mouse. All steps were routinely performed on ice.

[0097] The criteria for evaluating the suitability of the dissociated cells, in particular for single cell sequencing, were performed according to 3 parameters: yield (>1 x 10 7 live cells / g), viability (>80% live cells) and aggregation (<10%). However, other parameters for evaluating such suitability can be used.

[0098] Nucleocounter ®NC-200™ and Via2-Cassette™ (Chemometec) to evaluate viability and aggregation. Yield was calculated as 1000 ((viable cells per mL x dilution factor used for counting x total volume in mL) / (mass in mg)) = viable cells per gram of tissue.

[0099] The VIA Freeze Uno™ was set to cool to 2°C (to allow the VIA Extractor™ tissue dissociator to reach 4°C), run continuously at a speed of 200 rpm for 60 minutes and hold until ready for use. The kidney tissue was placed in the Omics bag using the Omics applicator. The composition was prepared according to Example 1 and 5 mL was applied per sample / kidney / bag portion.

[0100] The VIA Extractor™ tissue dissociator was then set to run for 15 minutes. After this time, the sample was mostly dissociated and could be further dissociated if needed. The resulting cell suspension was then passed through a 100 pm cell strainer and the protease K was quenched using an excess of phosphate buffered saline (PBS) containing 10% fetal bovine serum (FBS). The sample was centrifuged at 300 x g for 10 minutes, the supernatant was removed and the pellet was resuspended in a buffer consisting of PBS + 0.4% bovine serum albumin (BSA) + 0.1 mM ethylenediaminetetraacetic acid (EDTA).

[0101] Further purification steps could then be performed as needed, for example further filtration using 70 pm and 40 pm cell strainers and / or red blood cell (RBC) lysis.

[0102] The sample quality was then determined after the previous purification steps by measuring the parameters mentioned above using a cell counter.

[0103] Figure 2 A first pre-experiment tested the dissociation of mouse kidney tissue using three compositions containing different enzyme concentrations (0.1 mg / mL, 1 mg / mL, 5 mg / mL) of protease K. Cu 2+ The quenching step was used to quench the protease activity. The results are shown in Example 4 - Pre-experiment to evaluate mouse kidney cell aggregation, yield and viability after dissociation with different protease K concentrations at 4°C ("cold") or 37°C ("warm") with FBS quenching The kidney tissue was effectively dissociated.

[0104] The evaluation of the parameters discussed in Example 2 is shown in Figure 3 1 mg / mL showed to be the optimal concentration.

[0105] Example 5 - Pre-experiment to determine optimal protease K incubation time for use with the VIA Extractor™ tissue dissociator Figure 4 The Cu 2+ quenching test replaced the protease K quenching step. The results are shown in Example 6 - Comparison of tissue dissociation between the VIA Extractor™ tissue dissociator and the Miltenyi Multi-tissue dissociation kit 2 with the gentleMACS™ Octo Dissociatorsurprisingly, with Cu 2+ Quenching of proteinase K activity with FBS showed a significant improvement in viability and yield compared to quenching.

[0106] Figure 5 Example 7 - Characterization of single cell RNA-seq (scRNA-seq) data obtained by dissociation with protease K Experiments were performed in triplicate with 15 and 20 minutes incubation times to confirm the optimal incubation time and to allow the performance of definitive statistical analysis. Results are shown in Figure 6 .

[0107] The most efficient incubation time with proteinase K was found to be 15 minutes.

[0108] Example 8 - Comparison of kidney tissue dissociation when using protease K and subtilisin A Figure 5 Experiments were performed to compare tissue dissociation of the composition prepared according to Example 8 using the VIA Extractor™ tissue dissociator and commercially available kits: Miltenyi Multi Tissue Dissociation Kit 2 using the gentleMACS™ Octo Dissociator. Results are shown in Example 9 - Comparison of single cell RNA-seq (scRNA-seq) profiles after dissociation with protease K at 4°C or the Miltenyi Multi-tissue dissociation kit at 37°C using the VIA Extractor™ tissue dissociator .

[0109] When using the proteinase K composition according to Example 8 with the VIA Extractor™ tissue dissociator, viability was improved and aggregates were significantly lower compared to the Miltenyi Multi Tissue Dissociation Kit 2 with the gentleMACS™ Octo Dissociator.

[0110] Although the nature of the DNase used was different from Example 1, it was possible to compare the dissociation capacity between the VIA Extractor™ tissue dissociator with an active protease, i.e. proteinase K at 4°C and the Miltenyi Multi Tissue Dissociation Kit 2 with the gentleMACS™ Octo Dissociator at 37°C. Statistical comparison was performed as in Example 8.

[0111] t al. Following the dissociation and purification method according to Example 2, the resulting cell suspension was used to perform scRNA-seq following the same method as in Example 9 below. As a comparison, a composition comprising 5 mM CaCl2, 125 U / mL DNase I (Sigma), 5 mg / mL and Subtilisin A (a dissociation enzyme known to be efficient at low temperature) in HBSS was used. It was confirmed that there was no difference between the clusters identified when dissociating the sample using Subtilisin A or proteinase K as the main protease (see et al. ).

[0112] Thus, the results show that Proteinase K is at least as suitable for dissociating tissue at lower temperatures as Subtilisin A.

[0113] et al. The following experiment allowed for a comparison between dissociating tissue by Proteinase K and Subtilisin A, a known efficient dissociation enzyme at low temperatures. The experimental method was performed as follows.

[0114] For dissociation with Subtilisin A, all reagents were prepared including: PBS + 10% FBS, HBSS + 5 mM CaCl2, 10 mg / mL DNase I in HBSS with 5 mM CaCl2, IX RBC lysis buffer (Miltenyi Biotec), DBPS + 0.4% BSA + 0.1 mM EDTA, and set the VIA to 4°C 200 RPM. All steps were completed on ice.

[0115] For dissociation with Proteinase K, all reagents were prepared including: PBS + 10% FBS, HBSS + 10 mM CaCl2, 10 mg / mL DNase I in 10 mM CaCl2, IX RBC lysis buffer (Miltenyi Biotec), DBPS + 0.4% BSA + 0.1 mM EDTA, and set the VIA to 4°C 200 RPM. All steps were completed on ice.

[0116] A comparison between the dissociation function of the proteases can be made despite the different concentrations of calcium chloride. Statistical comparison was made between the two sets of Miltenyi results by performing a t-test and found that the difference was not significant, then the data was combined together and the results averaged, treating them as 6 replicates, a one-way ANOVA was performed and multiple comparisons between the dissociation results using the Miltenyi process, Proteinase K, and Subtilisin A.

[0117] 1. Tissue was weighed and rinsed with PBS. The tissue was placed in a bag, heat sealed and a clip was added to the bag.

[0118] 2. For dissociation using Proteinase K, the enzyme master mix was prepared (1 mg / mL Proteinase K + 125 U / mL DNase I; total = 15 mL): 750 µL Proteinase K 20 mg / mL, 201.9 µL DNase I 10 mg / mL, 14.22 mL HBSS + 10 mM Ca2+, and 5 mL was added to each bag portion.

[0119] 3. For dissociation using subtilisin, prepare the enzyme master mix (5 mg / mL subtilisin A + 125 U / mL DNase I; total = 15 mL): 750 μL subtilisin A 100 mg / mL, 201.9 μL DNase I 10 mg / mL, 14.22 mL HBSS + 10 mM Ca2+, and add 5 mL to each bag portion.

[0120] 4. Place the tissue in the dissociation for 15 minutes.

[0121] 5. Transfer the resulting cell suspension to a 50 mL tube with a 1 mL wetted 100 μm filter, the bag is rinsed with 5 mL PBS + FBS.

[0122] 6. Centrifuge the cell suspension at 300 x g for 10 minutes, remove the supernatant and resuspend in 1 mL DPBS + BSA + EDTA. Then add 14 mL of RBC lysis solution and invert the sample and incubate for 5 minutes.

[0123] 7. Then centrifuge the cell suspension at 300 x g for 10 minutes to remove the supernatant. Add 3 mL of DPBS + BSA + EDTA and mix well.

[0124] 8. Optionally, the above filtration step is repeated with a 70 μm + 40 μm filter.

[0125] 9. Then evaluate the resulting sample according to the parameters in Example 2.

[0126] The results are shown in et al. . The results shown in the middle relate to the use of proteinase K and the VIA Extractor™ tissue dissociator. The results on the left relate to the use of subtilisin A and the VIA Extractor™ tissue dissociator. The results on the right relate to the use of the Miltenyi Multi Tissue Dissociation Kit 2 and the gentleMACS™ Octo Dissociator.

[0127] From the data it can be found that the viability score is similar between all three test conditions. In terms of aggregation, proteinase K performs at least as well as subtilisin A and significantly better than the Miltenyi Multi Tissue Dissociation Kit 2 with the gentleMACS™ Octo Dissociator in the final sample.

[0128] The above experiment demonstrates that i) proteinase K can be used as an effective tool to dissociate tissue into single cells, ii) dissociation using proteinase K can be effectively performed at lower temperatures, and iii) proteinase K can dissociate tissue at lower temperatures at least as effectively as subtilisin A.

[0129] et al. et al. Following dissociation of mouse kidneys using the composition outlined in Example 1, the scRNA-seq workflow was performed and outlined as follows.

[0130] For single cell sequencing, cells from samples were quantified to determine average counts. Following manufacturer’s instructions for the 10X Genomics Chromium Next GEM Single Cell 3’ dual index kit v3.1, the goal was to sequence 1000 cells for each sample. The 10X Genomics Chromium Controller was used to capture cells into gel beads in emulsion (GEMs). Libraries were sequenced on a NextSeq 550 Base (Illumina Inc.) using a NextSeq 550 high output kit v2.5 (Illumina Inc.). Two sequencing runs were performed to obtain depth. In Seurat (Hao et al. 2019 Cell 177(7):1883-1902), the data was processed and analyzed. The data was filtered to remove doublets and cells with less than 200 features and mitochondrial gene expression percentage less than 50%. The data was then normalized and scaled. The data was then clustered and visualized using UMAP (Becht et al. 2019 Cell 184(13):3578-3590) and tSNE (Wolinsky et al. 2019 Cell 177(7):1883-1902). Figure 7 Cell. 2021 184(13)) using UMAP (Becht Figure 7 Nature Biotechnology 2018 37(1):38-44) was performed. Using Seurat and by He Figure 8 Cell. 2021 184(13)) using UMAP (Becht et al Nature Biotechnology 2018 37(1):38-44) was performed. Using Seurat and by He Genome Nature Communications. 2021 9;12(1) and Chung Biology.J Am Soc Nephrol. 2020 10;31(10):2341-54 Marker genes identified to identify cell types representative of each cluster. Gene lists from cell clusters with differential gene expression profiles were further analyzed using the Gene Ontology software package PANTHER (Muller (2017) PANTHER.db: A set of annotation maps describing the entire PANTHER Gene Ontology. R package version 1.0.4.). Results are shown in ​ and 8 .

[0131] ​ Kidney tissue dissociated with proteinase K at 4°C demonstrated similar scRNA-seq profiles to kidney tissue dissociated with Miltenyi Multi Tissue Dissociation Kit 2 at 37°C.

[0132] ​ Stress marker gene expression between kidney tissue dissociated with Miltenyi Multi Tissue Dissociation Kit 2 at 37°C and proteinase K at 4°C is shown. Dot plots show expression of O’Flanagan ​ . ​ ​ Cell percentage of “Stress” genes listed in J Am Soc Nephrol. 2019 20(210). Dark dots represent Miltenyi Multi Tissue Dissociation Kit 2 and light dots represent proteinase K. Dissociation with proteinase K at 4°C demonstrated reduced stress gene expression, thus improving suitability for application in high-throughput single cell omics studies.

[0133] It will be understood that certain embodiments (e.g., compositions, kits, methods, etc.) can include, incorporate, or otherwise comprise features (e.g., properties, components, ingredients, elements, parts, portions, steps, etc.) described in other embodiments disclosed and / or described herein. Thus, individual features in one embodiment can be compatible with, combined with, included within, and / or incorporated into other embodiments of the present disclosure. Disclosure of certain features in relation to one embodiment of the present disclosure should not be interpreted as limiting application or inclusion of the features to the particular embodiment. Rather, it will be understood that other embodiments can also include the features without necessarily departing from the scope of the present disclosure. Moreover, unless a feature is described as requiring another feature in combination therewith, any feature described herein can be combined with any other feature of the same or different embodiments disclosed herein.

[0134] The described embodiments are to be considered in all respects only as illustrative and not restrictive in character, as the scope of the application is indicated by the appended claims rather than by the foregoing description. All changes that come within the meaning of the claims and equivalents thereof are to be embraced within their scope. Various features and embodiments of the present disclosure are described herein, and it is to be understood that features and embodiments from one example can be combined with features and embodiments from another example. Various changes and / or modifications and further applications of the examples set forth herein can be apparent to persons skilled in the relevant art, such as changes in the function and / or structure of the described features and embodiments, and in addition, other applications that are within the scope of the present disclosure. Such changes, modifications and further applications can be accomplished without departing from the spirit and scope of the underlying disclosure and, in addition, any result from the application of the principles of the described embodiments to the production of any further applications that are within the scope of the present disclosure.

Claims

1. A composition comprising: a buffer; proteinase K; DNase; and Ca 2+ source.

2. The composition according to claim 1, wherein the buffer is selected from the group consisting of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Hanks’ Balanced Salt Solution (HBSS), MOPS (3-(N-morpholino)propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), Minimal Essential Medium (MEM), Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640, Iscove’s Modified Dulbecco’s Medium (IMDM), BES (NN-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid; 2-[bis(2-hydroxyethyl)amino]-ethanesulfonic acid), Earle’s Balanced Salt Solution (EBSS), MOPSO (2-hydroxy-3-morpholino propanesulfonic acid), ACES (N--2-aminoethanesulfonic acid), fetal bovine serum (FBS), TAPS (([tris(hydroxymethyl)methylamino]propanesulfonic acid), phosphate buffered saline (PBS), Dulbecco’s phosphate buffered saline (DPBS), bicine, tricine, cell culture grade water, ethylenediaminetetraacetic acid (EDTA), bovine serum albumin (BSA), and ethylene glycol-bis(β-aminoethyl)-N,N,Nʹ,Nʹ-tetraacetic acid (EGTA).

3. The composition according to claim 1 or 2, wherein the proteinase K is present in the composition at a concentration of 0.1 mg / mL to 5 mg / mL, preferably at a concentration of 1 ± 10% mg / mL.

4. The composition according to any one of the preceding claims, wherein the DNase is double-stranded DNase (dsDNase); optionally wherein the DNase is DNase I.

5. The composition according to any one of the preceding claims, wherein the DNase is present in the composition at a concentration of 4 ± 10% U / mL.

6. The composition of any one of the preceding claims, wherein the Ca 2+ source comprises calcium chloride; optionally wherein the calcium chloride is present in the composition at a concentration of 10 ± 15% mM.

7. The composition according to any one of the preceding claims, comprising: 1 ± 10% mg / mL proteinase K; 4 ± 10% U / mL dsDNase; Hanks’ Balanced Salt Solution (HBSS); and 10 ± 15% mM calcium chloride.

8. Use of proteinase K for dissociating a tissue at a temperature below 20°C, optionally below 10°C, optionally about 4°C.

9. The use according to claim 8, wherein the proteinase K is present in a composition, wherein the composition is according to any one of claims 1-7.

10. The use according to claim 8 or 9, wherein the proteinase K is incubated with the tissue for no more than 20 minutes, optionally no more than 15 minutes.

11. An ex vivo method of dissociating a tissue, comprising: a) contacting a tissue with a composition comprising proteinase K; b) incubating the tissue with the composition for a first period of time; and c) obtaining a cell suspension.

12. The method of claim 11, wherein the composition further comprises a DNase; optionally wherein the DNase is a double-stranded DNase (dsDNase); optionally wherein the DNase is DNase I.

13. The method of claim 11 or 12, wherein the composition further comprises Ca 2+ source.

14. The method of any one of claims 11-13, wherein the composition further comprises Hanks’ Balanced Salt Solution (HBSS).

15. The method of any one of claims 11-14, wherein the composition is the composition of any one of claims 1-7.

16. The method of any one of claims 11-15, wherein the method further comprises passing the cell suspension through a first filter, optionally a 100 pm filter.

17. The method of any one of claims 11-16, wherein the method further comprises quenching the proteinase K, optionally with 10% fetal bovine serum (FBS) in phosphate buffered saline (PBS) and / or with Cu 2+ source to quench the proteinase K.

18. The method of any one of claims 11-17, wherein the method further comprises removing supernatant, optionally by centrifugation, and resuspending the cells in a buffer, optionally in bovine serum albumin (BSA) and ethylenediaminetetraacetic acid (EDTA), optionally in 0.4% bovine serum albumin (BSA) and 0.1 mM ethylenediaminetetraacetic acid (EDTA).

19. The method of any one of claims 11-18, wherein the method further comprises passing the cell suspension through a second filter, optionally a 40 or 70 pm filter.

20. The method of any one of claims 11-19, wherein the method further comprises mechanical dissociation, optionally wherein the mechanical dissociation is performed by a VIA Extractor™ tissue dissociator.

21. The method of any one of claims 11-20, wherein the first time period is at least 15 minutes.

22. The method of any one of claims 11-21, wherein the method is performed at a temperature of less than 20 °C, optionally less than 15 °C, optionally less than 10 °C, optionally less than 5 °C, optionally about 4 °C.

23. The use of any one of claims 8-10 or the method of any one of claims 11-22, wherein the tissue is selected from the group consisting of: spleen, heart, liver, brain and other neural tissue, kidney, lung, pancreas, breast, umbilical cord, skin, placenta, ovary, fallopian tube, uterus, prostate, tonsil, thymus, stomach, testis, trachea, cartilage, tendon, bone, skeletal muscle, smooth muscle, digestive tract, colon, intestine, bladder, urethra, eye, gall bladder, organoids and tumors from cell culture.

24. The use of any one of claims 8-10 or 23 or the method of any one of claims 10-22, wherein the tissue is obtained from the group consisting of: mouse, rat, avian such as chicken, ruminant such as cattle, goat, deer, sheep, horse and other animals such as pig, cat, dog and primates such as human, chimpanzee, gorilla and monkey; optionally mouse; optionally female mouse.

25. The use according to any one of claims 8-10, claim 23 or claim 24 or the method according to any one of claims 11-24, wherein the tissue is kidney tissue, optionally whole kidney tissue and / or weighs less than 300 mg.

26. A system configured to perform the method according to any one of claims 11-25.

27. A kit comprising the composition according to any one of claims 1-7 and instructions for use.

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