Enzymes

Abstract

The present invention relates to methods and compositions for the dissociation of biological tissue with the aim of obtaining single cells for analysis. More particularly, the compositions of the present invention are enzyme compositions comprising proteinase K. The inventive compositions can be used in methods that effectively dissociate tissue at lower temperatures for application in high-throughput single cell omics research.

Description

[0001] ENZYMES

[0002] Technical Field

[0003] The invention relates to compositions comprising proteinase K and methods fortissue dissociation.

[0004] Enzymes are useful tools for dissociating cells from solid tissues for various applications in research, industry and medicine. Reichard and Asosingh et al. Cytometry A. 2019 95(2): 219-226 describes examples of enzymes useful for solid tissue dissociation. Combinations of different enzymes (also referred to as enzyme “cocktails”) can be used fortissue dissociation. However, the specific enzyme(s) have to be selected carefully since not all are suitable when used alone or in combination. For example, Reichard and Asosingh et al. 2019 reports that trypsin is not suitable for preparing single cells due to the inherent risk of cleaving cell surface receptors.

[0005] Moreover, due to the heat-sensitive nature of many enzymes, tissue dissociation is typically performed at a temperature which results in maximum activity and efficiency of the enzyme. Since many enzymes have an optimum working temperature of around 37°C (such as trypsin, TrypLE™, 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 usually performed at 37°C.

[0006] There are a number of commercial products for dissociating kidney tissue, such as Miltenyi Biotec's Multi Tissue Dissociation Kit 2, which can be used in conjunction with a specialised device such as the gentleMACS™ Dissociator (Miltenyi Biotec Multi Tissue Dissociation Kit 2 accessed: 5 May 2023 [https: / / static.miltenyibiotec.com / asset / 150655405641 / document_phpdh7tnkl2it4bi11qp1 IOo6o?content- disposition=inline]; Miltenyi Biotec Dissociation of mouse kidney using the Multi Tissue Dissociation Kit 2 accessed: 5 May 2023 [https: / / www.miltenyibiotec.com / upload / assets / IM0015569.PDF]). Mouse kidneys can also be dissociated with more general tissue dissociation enzymes at 37°C, one such example being the protocol used in Adam et al. 2017.

[0007] More recently, it has been discovered that dissociation of tissues at 37°C results in artificial changes to the cells such as the induction of stress response genes. Such changes are particularly problematic in the field of high-throughput single cell omics research (genomics, proteomics, metabolomics). As a consequence, methods for dissociating tissue are ideally performed at a lower temperature. Adam et al. 2017 describes one such method which utilises subtilisin A. Subtilisin A is a serine endopeptidase expressed in Bacillus licheniformis, a soil bacterium isolated from Himalayan glaciers, which is known to be effective at lower temperatures. O’Flanagan et al. Genome Biology. 2019 20(210) describes the use of subtilisin A as a protease in an enzyme cocktail to reduce the stress response on kidney cells during dissociation. In view of the above, there remains a need for further enzymes which can effectively dissociate tissue at lower temperatures for application in high-throughput single cell omics research.

[0008] Summary

[0009] In a first aspect, the invention provides a composition comprising: proteinase K; a buffering agent, wherein the buffering agent comprises Hanks' Balanced Salt Solution (HBSS); a DNase; and a source of Ca2+.

[0010] In a second aspect, the invention provides use of proteinase K forthe dissociation of tissue at a temperature of less than 20°C.

[0011] In a third aspect, the invention provides an ex vivo method of dissociating tissue comprising: a) contacting a tissue with a composition comprising proteinase K; b) incubating the tissue with the composition for a first period; and c) obtaining a cell suspension.

[0012] In a fourth aspect, the invention provides a system configured to carry out the method according to the third aspect of the invention.

[0013] In a fifth aspect, the invention provides a kit comprising the composition according to the first aspect of the invention and instructions for use.

[0014] The present invention relates to the unexpected discovery that proteinase K can be used as a tool for the effective dissociation of tissue into single cells. The single cells exhibit beneficial properties which make them suitable for high-throughput single cell omics research, particularly single cell RNA sequencing analyses such as single cell RNA-seq (scRNA-seq). Further, it was discovered that the tissue dissociation can be performed at lower temperatures (e.g., 4°C) to avoid artifacts relating to heat-induced stress response. Unexpectedly, it was also discovered that proteinase K can dissociate tissue at lower temperatures at least as effectively as subtilisin A (an enzyme already known to work at lower temperatures). This was especially surprising since proteinase K is known to have an optimum temperature range of between 20 to 65°C, with a maximum activity at 37°C (Bajorath Biochim Biophys Acta. 1988 954(2):176-182). Further, it was observed that in comparison to known enzyme compositions, the inventive proteinase K-containing composition does not cleave certain cell surface markers.

[0015] Proteinase K (EC 3.4.21.64), also referred to as Peptidase K, Endoproteinase K and Endopeptidase K, is a non-specific serine endopeptidase in the family S8 (subfamily S8A). It is commonly used for breaking down keratin, unwanted free proteins in molecular biology and nucleases (DNases & RNases). It is also used in prion research (Petrotchenko et al. Molecular & Cellular Proteomics. 2012 11 (7)), endotoxin / Limulus amebocyte lysate (LAL) testing (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). Proteinase K is stable over a pH range of approximately 4.0 to 12.5, with an optimum of pH 8.0.

[0016] Detailed Description

[0017] Terms and definitions

[0018] Unless specific definitions are provided, the nomenclatures utilised in connection with, and the laboratory procedures and techniques described herein, are those 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 identical meaning. Standard techniques may be used for formulating compositions and testing them. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art.

[0019] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention claimed. As used herein, the use of the singular includes the plural unless specifically stated otherwise. The section headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described.

[0020] As used herein, “proteinase K” refers to the amino acid sequence of SEQ ID NO: 1 (UniProt reference P06873 as of 16 May 2023), homologues in other species or variants thereof. The amino acid residues of proteinase K used herein are numbered relative to SEQ ID NO: 1 .

[0021] SEQ ID NO: 1 (Parengyodontium album (Tritirachium album), 384 amino acids, UniProt reference P06873 as of 16 May 2023):

[0022] MRLSVLLSLLPLALGAPAVEQRSEAAPLIEARGEMVANKYIVKFKEGSALSALDAAMEKISGKPDHVYKNV FSGFAATLDENMVRVLRAHPDVEYIEQDAVVTINAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVY VIDTGIEASHPEFEGRAQMVKTYYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQY STIIAGMDFVASDKNNRNCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNNADARNYSPA SEPSVCTVGASDRYDRRSSFSNYGSVLDIFGPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGK TTAASAC RYI ADTAN KGDLSN I PFGTVN LLAYN N YQA

[0023] As used herein, “or” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “includes,” and “included,” is not limiting.

[0024] “About” as used herein means that a number referred to as “about” comprises the recited number plus or minus 1-5% of that recited number. For example, “about” 100 degrees can mean 95-105 degrees or as few as 99-101 degrees depending on the context. Whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; i.e., meaning only 1 , only 2, only 3, etc., up to and including only 20. As used herein, the term "composition" includes products, formulations, and mixtures, as well as devices, apparatus, assemblies, kits, and so forth. Similarly, the term "method" includes processes, procedures, steps, and so forth.

[0025] As used throughout this disclosure, the words "can" and "may" 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," variants thereof (e.g., "includes," "has," and "involves," "contains," etc.), and similar terms as used herein, including the claims, shall be inclusive and / or open-ended, shall have the same meaning as the word "comprising" and variants thereof (e.g., "comprise" and "comprises"), and do not exclude additional, un-recited elements or method steps, illustratively.

[0026] It is noted that embodiments of the present disclosure can comprise one or more combinations of two or more of the features described herein. As used herein, "feature(s)" and similar terms can include, for example, compositions, ingredients, components, elements, members, parts, portions, systems, methods, configurations, parameters, properties, and so forth. Embodiments can include any of the features, options, and / or possibilities set out elsewhere in the present disclosure, including in other aspects or embodiments of the present disclosure. It is also noted that each of the foregoing, following, and / or other features described herein represents a distinct embodiment of the present disclosure. Features can also be combined and / or combinable with another one or more other features in any suitable combination and / or order, with or without one or more additional features included therewith or performed therebetween, to form unique embodiments, each of which is contemplated in the present disclosure. Such combinations of any two or more of such features represent distinct embodiments of the present disclosure. Accordingly, the present disclosure is not limited to the specific combinations of exemplary embodiments described in detail herein and disclosure of certain features relative to a specific embodiment of the present disclosure should not be construed as limiting application or inclusion of said features to the specific embodiment.

[0027] In addition, unless a feature is described as being requiring in a particular embodiment, features described in the various embodiments can be optional and may not be included in other embodiments of the present disclosure. Moreover, unless a feature is described as requiring another feature in combination therewith, any feature herein may be combined with any other feature of a same or different embodiment disclosed herein. Likewise, any steps recited in any method described herein and / or recited in the claims can be executed in any suitable order and are not necessarily limited to the order described and / or recited, unless otherwise stated (explicitly or implicitly). Such steps can, however, also be required to be performed in a particular order in certain embodiments of the present disclosure.

[0028] It will also be appreciated that where two or more values, or a range of values (e.g., less than, greaterthan, at least, and / or up to a certain value, and / or between two recited values) is disclosed or recited, any specific value or range of values falling within the disclosed values or range of values is likewise specifically disclosed and contemplated herein. Thus, disclosure of an illustrative measurement (e.g., length, width, thickness, etc.) that is less than or equal to about 10 units or between 0 and 10 units includes, illustratively, a specific disclosure of: (i) a measurement of 9 units, 5 units, 1 units, or any other value between 0 and 10 units, including 0 units and / or 10 units; and / or (ii) a measurement between 5 9 units and 1 units, between 8 units and 2 units, between 6 units and 4 units, and / or any other range of values between 0 and 10 units.

[0029] Embodiments of the invention

[0030] The following description of embodiments includes disclosure that is relevant to one or more embodiments of the present disclosure. Accordingly, some embodiments can include features disclosed in the following examples without departing from the scope of the present disclosure. In other words, features disclosed in the following examples can be included and / or incorporated into any one or more of the embodiments disclosed herein.

[0031] The composition of the present invention comprises proteinase K, which is the active peptidase that cleaves the amino acids present in the proteins that hold cells together.

[0032] Proteinase K is commercially available from a number of manufacturers e.g., Qiagen™ (including Blirt™). Preferably, the proteinase K in the composition of the present invention is Blirt™ catalogue number RP107B as of 17 May 2023. It is also possible to produce recombinant proteinase K using a host cell.

[0033] The host cell may be selected from group consisting of: Pichia species, Hansenula species such as Hansenula polymorpha, Saccharomyces species, Schizosaccharomyces species, Yarrowia species such as Yarrowia lipolytica, Kluyveromyces species and Aspergillus species. Preferably, Pichia pastoris is used as the host cell.

[0034] Preferably, proteinase K in the composition of the present invention comprises the amino acid sequence of SEQ ID NO: 1 . More preferably, proteinase K in the composition of the present invention consists of the amino acid sequence of SEQ ID NO: 1.

[0035] The amino acid sequence of a variant of proteinase K may share at least about 85% sequence identity to SEQ ID NO: 1 and may therefore 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 to SEQ ID NO: 1 . Preferably said sequence identity is at least 90% or at least 95%. More preferably, said sequence identity is 100%. Preferably, the variant of proteinase K substantially retains the enzymatic activity associated with the sequence of SEQ ID NO: 1 .

[0036] The amino acid sequence of a variant proteinase K may be altered by substitution, addition or deletion of an appropriate number of amino acids in the sequences of SEQ ID NO: 1 , for example 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. By "substitution, addition or deletion" is included combinations of substitutions, additions and deletions. Preferably, the variant of proteinase K substantially retains the enzymatic activity associated with the sequence of SEQ ID NO: 1 .

[0037] When a sequence is modified by substitution of a particular amino acid residue, the substitution may 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 important for the structure or function of a polypeptide may be expected to affect polypeptide structure / function less than a non-conservative amino acid substitution at the same position.

[0038] Families of amino acid residues having 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), non-polar 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 may be considered to be a substitution in which a particular amino acid residue is substituted for a different amino acid in the same family. However, a substitution of an epitope residue may equally be a non-conservative substitution, in which one amino acid is substituted for another with a side-chain belonging to a different family.

[0039] The proteinase K may be present in the composition of the invention 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. The proteinase K may be present in the composition of the invention 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 invention is 1 ±1 % mg / mL. Where “±x%” is used herein, it refers to ±x% of the stated value.

[0040] The proteinase K may be present in the composition of the invention at a concentration of about 1 mg / mL. Preferably, the proteinase K is present in the composition of the invention at a concentration of 1 mg / mL.

[0041] The proteinase K may be present in the composition of the invention 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.

[0042] The proteinase K may be present in the composition of the invention at a concentration from 0.1 to 10mg / mL, from 0.2 to 9mg / mL, from 0.3 to 8mg / mL, from 0.4 to 7mg / mL, from 0.5 to 6mg / mL, from 0.6 to 5mg / mL, from 0.7 to 4mg / mL, from 0.8 to 3mg / mL or from 0.9 to 2mg / mL. Preferably, proteinase K is present in the composition of the invention at a concentration from 0.1 mg / mL to 5 mg / mL. The amount of proteinase K in the composition may be defined in term of enzyme units. The following definition of an enzyme unit can be used: proteinase K hydrolyses urea-denatured haemoglobin producing colour equivalent of 1 pmol tyrosine per 1 min at 37°C and pH 7.5 (Folin & Ciocalteu’s method), 1 U = 1 mAnsonU. For example, when using 20mg / mL (minimum), and 800U / mL (minimum) stock, the final concentration is 1 mg / mL and the activity in the solution would be 40U / mL.

[0043] The proteinase K may be present in the composition of the invention 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. The proteinase K may be present in the composition of the invention at a concentration of 400 U / mL, 200 U / mL or 80 U / mL. Preferably, the proteinase K is present in the composition of the invention at a concentration of 40±1 % U / mL.

[0044] The proteinase K may be present in the composition of the invention at a concentration of about 40 U / mL. The proteinase K may be present in the composition of the invention at a concentration of 40 U / mL.

[0045] The proteinase K may be present in the composition of the invention at a concentration from 4 to 400U / mL, from 8 to 360 U / mL, from 12 to 320 U / mL, from 16 to 280 U / mL, from 20 to 240 U / mL, from 24 to 200 U / mL, from 28 to 160 U / mL, from 32 to 120 U / mL, from 36 to 80 U / mL, from 37 to 70 U / mL, from 38 to 60 U / mL and from 39 to 50 U / mL.

[0046] The composition of the present invention also comprises a DNase. DNase (particularly dsDNase) has been described as being useful in reducing cell aggregation / clumping caused by cell free DNA, resulting in a good quality sample for single cell RNA sequencing (Reichard and Asosingh et al. 2019).

[0047] Even though 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).

[0048] The DNase is commercially available from a number of manufacturers e.g., Qiagen™ (including Blirt™).

[0049] The DNase may be present in the composition of the invention 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. The DNase may be present in the composition of the invention at a concentration of 40 U / mL, 20 U / mL or 8 U / mL. Preferably, the DNase is present in the composition of the invention at a concentration of 4±1 % U / mL.

[0050] The DNase may be present in the composition of the invention at a concentration of about 4U / mL. The DNase may be present in the composition of the invention at a concentration of 4U / mL.

[0051] The DNase may be present in the composition of the invention at a concentration from 0.4 to 40U / mL, from 0.8 to 36U / mL, from 1 .2 to 32U / mL, from 1 .6 to 28U / mL, from 2 to 24U / mL, from 2.4 to 20U / mL, from 2.8 to 16U / mL, from 3.2 to 12U / mL, from 3.6 to 8U / mL, from 3.7 to 7U / mL, from 3.8 to 6U / mL and from 3.9 to 5U / mL. The following definition of an enzyme unit for the DNase can be used: 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 a substrate.

[0052] Preferably, the DNase is a double stranded DNase (dsDNase). Preferably, the dsDNase in the composition of the present invention is Blirt™ catalogue number EN33 as of 17 May 2023.

[0053] The DNase can be a member of the DNase I family selected from the group consisting of DNase I, DNasel L1 , DNase 1 L2 and DNasel L3. Preferably, the DNase is DNase I.

[0054] The DNase can be a member of the DNase II family selected from the group consisting of: DNase Ila and DNase lip.

[0055] The composition of the invention comprises a source of Ca2+, 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 source of Ca2+can comprise calcium chloride. The calcium chloride can be solid or aqueous calcium chloride. Preferably, the calcium chloride is aqueous calcium chloride.

[0056] The calcium chloride can be present in the composition of the invention 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 invention at a concentration of 100 mM, 50 mM or 20mM. Preferably, the calcium chloride is present in the composition of the invention at a concentration of 10±1 % mM.

[0057] Preferably, the calcium chloride is present in the composition of the invention at a concentration of between 1 1 and 12 mM, more preferably 11 .3 mM.

[0058] The calcium chloride can be present in the composition of the invention at a concentration of about 11 mM. Preferably, the calcium chloride is present in the composition of the invention at a concentration of 11 mM.

[0059] The calcium chloride can be present in the composition of the invention at a concentration of between 1 and 100mM, between 2 and 90mM, between 3 and 80mM, between 4 and 70mM, between 5 and 60mM, between 6 and 50mM, between 7 and 40mM, between 8 and 30mM and between 9 and 20mM.

[0060] The composition of the invention may comprise one or more buffering agents. The method or use of the invention may require one of more buffering agents. Suitable buffering agents of the present invention help support cell viability and yield, as well as reduce cell aggregation. The buffering agent may 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), Minimum 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- aminoethanesulphonic acid; 2-[bis(2- Hydroxyethyl)amino]-ethanesulfonic acid), Earle's Balanced Salt Solution (EBSS), MOPSO (2-Hydroxy-3-morpholinopropanesulfonic 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 water, ethylenediaminetetraacetic acid (EDTA), bovine serum albumin (BSA), and ethyleneglycol- bis(p-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA).

[0061] Preferably, the buffering agent is HBSS. HBSS is the buffering agent which is commonly used in cell culture and contains a balance of essential inorganic ions for cells to thrive and maintains the correct pH and osmolarity. HBSS is commercially available from a number of manufacturers e.g., Cytiva™. Preferably, the HBSS in the composition of the present invention is Cytiva™ catalogue number SH30268.01 as of 17 May 2023. The composition of Cytiva™ catalogue number SH30268.01 is shown in the table below. The components shown in the table below may vary by ±75%, ±50%, ±20%, ±10%, ±5% or ±1 % of the values shown. Calcium chloride (anhydrous) and / or magnesium sulfate (anhydrous) may be omitted.

[0062] 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.

[0063] More preferably, the composition of the invention comprises: 1 mg / mL proteinase K; 4 U / mL dsDNase; Hanks' Balanced Salt Solution (HBSS); and 10±15% mM calcium chloride.

[0064] In a further aspect, the invention provides a composition comprising: a buffering agent; proteinase K; a DNase; and a source of Ca2+.

[0065] In a preferred embodiment, the ex vivo method of dissociating tissue of the invention 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; and c) obtaining a cell suspension.

[0066] In a more preferred embodiment, the ex vivo method of dissociating tissue of the invention comprises: a) contacting a tissue with a composition comprising: 1 mg / mL proteinase K; 4U / mL dsDNase; Hanks' Balanced Salt Solution (HBSS); and 10±15% mM calcium chloride; b) incubating the tissue with the composition for a first period; c) obtaining a cell suspension; d) passing the cell suspension through a first filter; e) quenching the proteinase K, preferably with 10% foetal bovine serum (FBS) in phosphate buffered saline (PBS); and preferably f) passing the cell suspension through a second filter, preferably a 40 or 70pm filter.

[0067] The compositions of the invention may also comprise one or more additional agents such as other enzymes (particularly dissociation enzymes).

[0068] Alternatively, the composition of the invention may also be devoid of one or more additional agents such as other enzymes (particularly dissociation enzymes).

[0069] The one or more additional agents may be selected from the group consisting of: accutase, TrypLE™, trypsin, chymotrypsin, pronase, papain, liberase, collagenase, elastase, dispase, thermolysin, hyaluronidase, clostripain and neutral protease from Clostridium histolyticum, pronase, pepsin, lysozyme, chelating agents for bivalent ions (like EDTA or citrate) and combinations thereof.

[0070] The method of the invention may further comprise passing the cell suspension through a first filter and / or a second filter.

[0071] The size of the filter may 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.

[0072] Preferably, the size of the filter is 40 or 70pm.

[0073] The method of the invention may further comprise quenching the proteinase K to terminate the dissociation reaction. Quenching may be with a buffering agent described herein. Quenching may be with foetal bovine serum (FBS) in phosphate buffered saline (PBS), or alternatively, in another buffering agent described herein. The concentration of FBS may be from 1 to 100% v / v, from 2 to 90% v / v, from 3 to 80% v / v, from 4 to 70% v / v, from 5 to 60% v / v, from 6 to 50% v / v, from 7 to 40% v / v, from 8 to 30% v / v, from 9 to 20% v / v. Preferably, the concentration of FBS is 10%.

[0074] Quenching may alternatively or additionally be with a source of Cu2+. The source of Cu2+may be present in the composition of the invention in a buffering agent described herein. Quenching with FBS is preferable to quenching with Cu2+in view of the Examples below which demonstrate that quenching with FBS significantly improves viability and yield of dissociated cells compared to quenching with Cu2+.

[0075] Following dissociation, cell yield, viability and aggregation may be assessed by any suitable method known in the art. Preferably, cell yield, viability and aggregation are quantified using an automated cell counter, preferably the Nucleocounter® NC-200™ using a Via2-Cassette™ (Chemometec). Other automated cell counters may also be used (e.g., products by Nexcelom and Thermofisher). Cell yield, viability and aggregation may also be quantified by using a haemocytometer with trypan blue. Yield may be calculated as 1000((live cells per mL x dilution factor for counting x total volume in mL) / (mass in mg)) = live cells per gram of tissue.

[0076] The method of the invention may further comprise removing the supernatant. Removing the supernatant may comprise centrifugation.

[0077] The method of the invention may further comprise resuspending the cells in a buffering agent described herein. The buffering agent may comprise bovine serum albumin (BSA) and / or ethylenediaminetetraacetic acid (EDTA). The concentration of BSA may 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 may be between 0.04 and 4%, between 0.08 and 3.6%, between 0.12 and 3.2%, between 0.16 and 2.8%, between 0.2 and 2.4%, between 0.24 and 2%, between 0.28 and 1 .6%, between 0.32 and 1 .2% or between 3.6 and 0.8%. The concentration of EDTA may 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 may be between 0.01 and 1 mM, between 0.02 and 0.9mM, between 0.03 and 0.8mM, between 0.04 and 0.7mM, between 0.05 and 0.6mM, between 0.06 and 0.5mM, between 0.07 and 0.4mM, between 0.08 and 0.3mM or between 0.09 and 0.2mM. Preferably, the buffering agent comprises 0.4% BSA and 0.1 mM EDTA.

[0078] The method of the invention may further comprise mechanical dissociation, which may be carried out by a suitable device (preferably, an automated tissue dissociation system), for example, selected from the group consisting of: VIA Extractor™ tissue disaggregator, gentleMACS™ Dissociator, gentleMACS™ Octo Dissociator, Singulator™100 and Singulator™ 200. Preferably, the method of the invention is carried out using the VIA Extractor™ tissue disaggregator.

[0079] The composition, method or system of the invention may be suitable for use with the VIA Extractor™ Tissue Disaggregator.

[0080] The first period of the method of the invention may 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 period of the method of the invention may be for no longer than 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 21 , 22, 23, 24 or 25 minutes, preferably 20, more preferably 15. The first period of the method of the invention may be between 1 .5 and 150 minutes, between 3 and 135 minutes, between 4.5 and 120 minutes, between 6 and 105 minutes, between 7.5 and 90 minutes, between 9 and 75 minutes, between 10.5 and 60 minutes, between 12 and 45 minutes or between 13.5 and 30 minutes. Preferably, the first period of the method of the invention is about 15 minutes, more preferably 15 minutes.

[0081] The method or use of the invention may be performed at less than, or about, 50, 45, 40, 35, 30, 25, 30, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 and 1 °C. The method or use of the invention may be performed between 0.4 and 40°C, between 0.8 and 36°C, between 1 .2 and 32°C, between 1 .6 and 28°C, between 2 and 24°C, between 2.4 and 20°C, between 2.8 and 16°C, between 3.2 and 12°C or between 3.6 and 8°C.

[0082] The tissue for dissociation is preferably solid tissue, and may be selected from the group consisting of: spleen, heart, liver, brain and other neural tissues, kidney, lung, pancreas, breast, umbilical cord, skin, placenta, ovary, oviduct, uterus, prostate, tonsil, thymus, stomach, testis, trachea, cartilage, tendon, bone, skeletal muscle, smooth muscle, gut, colon, intestine, bladder, urethra, eye, gall bladder, organoids from cell cultures and tumours. Preferably, the tissue is kidney tissue, more preferably whole kidney tissue, even more preferably whole kidney tissue weighing less than 300mg.

[0083] The tissue may be obtained from a mammal, such as from the group consisting of: mice, rats, fowls such as chicken, ruminants such as cows, goat, 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 a mouse, more preferably a female mouse.

[0084] In another aspect, the invention provides a cell suspension obtained by the method of the third aspect of the invention.

[0085] A composition of the invention may be administered to a tissue using a device, such one described in EP3171152 A1 .

[0086] The kit of the invention may further comprise one or more containers. The various components of the composition of the invention may be present in the kit in one or more of the containers. For example, the components may all be present in one container or in separate containers.

[0087] Various modifications of the invention in addition to those shown and described herein 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 are as for each of the other aspects mutatis mutandis. The documents referred to herein are incorporated by reference to the fullest extent permitted by law.

[0088] Examples The invention will now be further described by way of reference to the following Examples and Figures which are included for the purposes of reference only and are not to be construed as being limitations on the invention.

[0089] Figure 7: visible dissociation of mouse kidneys with different concentrations of proteinase K (0.1 mg / mL, 1 mg / mL and 5 mg / mL) followed by quenching with Cu2+.

[0090] Figure 2 quantification of yield, viability and aggregation following dissociation of mouse kidneys with different concentrations of proteinase K (0.1 mg / mL, 1 mg / mL and 5 mg / mL) followed by quenching with Cu2+.

[0091] Figure 3: quantification of yield, viability and aggregation following dissociation of mouse kidneys at 4°C (“cold”) and 37°C (“warm”) with different concentrations of proteinase K (0.1 mg / mL, 0.5 mg / mL and 1 mg / mL) followed by quenching with FBS.

[0092] Figure 4: results of experiments in triplicate with proteinase K incubation times of 15 and 20 mins.

[0093] Figure 5 comparison of results following dissociation using i) VIA Extractor™ Tissue Disaggregator with subtilisin A at 4°C (left) ii) VIA Extractor™ Tissue Disaggregator with proteinase K at 4°C (middle) and iii) Miltenyi Multi Tissue Dissociation Kit 2 with the gentleMACS™ Octo Dissociator with Miltenyi enzymes at 37°C (right).

[0094] Figure 6: UMAP plots comparing the kidney cells clusters detected after mouse kidney was dissociated with proteinase K and subtilisin A using the VIA Extractor™ Tissue Disaggregator at 4°C.

[0095] Figure 7: UMAP plots comparing the cell clusters detected after mouse kidney was dissociated with proteinase K at 4°C (“cold”) and Miltenyi Multi Tissue Dissociation Kit 2 at 37°C using VIA Extractor™ Tissue Disaggregator.

[0096] Figure 8: stress marker comparison of kidney cells dissociated with proteinase K at 4°C and Miltenyi Multi Tissue Dissociation Kit 2 using VIA Extractor™ Tissue Disaggregator.

[0097] EXAMPLE 1

[0098] The following composition was prepared for use as a reagent for tissue dissociation.

[0099] • 1 mg / mL recombinant proteinase K (Blirt™ / QIAGEN™);

[0100] • 4U / mL dsDNase (Blirt™ / QIAGEN™); and

[0101] • HBSS buffer (Cytiva™) with added 10mM CaCI2(Thermofisher™). EXAMPLE 2 - a method for dissociating mouse kidney cells compatible with the VIA Extractor™ Tissue Disaggregator and the VIA Freeze™ Uno

[0102] A method of dissociating mouse kidney tissue was developed using the composition prepared in Example 1 . The method was tested on <300mg whole kidneys from female mice. All steps were routinely performed on ice.

[0103] The criteria for assessing the suitability of the cells following dissociation for, inter alia, single cell sequencing was based on 3 parameters: yield (>1x107live cells / g), viability (>80% live cells) and aggregation (<10%). However, other parameters for assessing such suitability can be used.

[0104] Viability and aggregation were assessed using the Nucleocounter® NC-200™ and Via2-Cassette™ (Chemometec). Yield was calculated as 1000((live cells per mL x dilution factor for counting x total volume in mL) / (mass in mg)) = live cells per gram of tissue.

[0105] The VIA Freeze Uno™ was set to cool to 2°C (to allow the VIA Extractor™ Tissue Disaggregator to reach 4°C), dwell for 60 minutes, at a speed of 200rpm and to hold until ready. The kidney tissue was placed in the Omics pouch using the Omics applicator. The composition was prepared according to Example 1 and 5mL was applied per sample / kidney / pouch portion.

[0106] The VIA Extractor™ Tissue Disaggregator was then set to run for 15 minutes. After which, the sample was mostly dissociated and could be dissociated further if necessary. The resulting cell suspension was then passed through a 100pm cell strainer, and the proteinase K was quenched using an excess of phosphate buffered saline (PBS) with 10% foetal bovine serum (FBS). The sample was centrifuged at 300xg 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).

[0107] Additional clean-up steps, such as further filtration using 70pm and 40pm cell strainers and / or red blood cell (RBC) lysis could then take place, depending on requirements.

[0108] Sample quality was then determined after the prior clean-up steps by using a cell counter to measure the parameters mentioned above.

[0109] EXAMPLE 3 - pilot experiment to determine optimum proteinase K concentration

[0110] The first pilot experiment tested the dissociation of mouse kidney tissue using three compositions comprising proteinase K with different concentrations of the enzyme (0.1 mg / mL, 1 mg / mL, 5mg / mL). A Cu2+quenching step was used to quench the protease activity. The results are shown in Figure 1 . The kidney tissue was effectively dissociated. The assessment of the parameters discussed in Example 2 is shown in Figure 2. 1 mg / mL was shown to be the optimal concentration.

[0111] EXAMPLE 4 - pilot experiment to assess mouse kidney cell aggregation, yield and viability following dissociation with different proteinase K concentrations either at 4°C (“cold”) or 37° C (“warm”) with FBS quenching

[0112] An alternative proteinase K quenching step was tested by replacing Cu2+quenching with FBS quenching. The results are shown in Figure 3. Surprisingly, quenching proteinase K activity with FBS was shown to significantly improve viability and yield compared to quenching with Cu2+.

[0113] EXAMPLE 5 - pilot experiment to determine optimum proteinase K incubation time for use with the VIA Extractor™ Tissue Disaggregator

[0114] Experiments were performed in triplicate with incubation times of 15 and 20 mins to confirm the optimal incubation time and to allow for confirmatory statistical analyses to be carried out. The results are shown in Figure 4.

[0115] With the most effective incubation time with proteinase K was found to be 15 mins.

[0116] EXAMPLE 6 - comparing tissue dissociation between VIA Extractor™ Tissue Disaggregator and Miltenyi Multi Tissue Dissociation Kit 2 with the gentleMACS™ Octo Dissociator

[0117] Experiments were carried out to compare the tissue dissociation of a composition prepared according to Example 8 using the VIA Extractor™ Tissue Disaggregator and a commercially available kit: the Miltenyi Multi Tissue Dissociation Kit 2 using the gentleMACS™ Octo Dissociator. The results are shown in Figure 5.

[0118] The viability was improved, and the aggregates were significantly lower, when using the proteinase K composition according to Example 8 with the VIA Extractor™ Tissue Disaggregator in comparison to the Miltenyi Multi Tissue Dissociation Kit 2 with the gentleMACS™ Octo Dissociator.

[0119] Although the nature of the DNase used is different to that of Example 1 , comparison between the dissociative ability of the VIA Extractor™ Tissue Disaggregator with the active protease, proteinase K, at 4°C and Miltenyi Multi Tissue Dissociation Kit 2 with the gentleMACS™ Octo Dissociator at 37°C can nevertheless be made. A statistical comparison was made as in Example 8.

[0120] EXAMPLE 7 - characterisation of single cell RNA-seq (scRNA-seq) data obtained via dissociation with proteinase K Following the dissociation and clean up method according to Example 2, scRNA-seq was performed using the resulting cell suspensions, following the same method as in Example 9 below. As a comparison, a composition comprising 5mM CaCh in HBSS, 125U / mL DNase I (Sigma), 5mg / mL and subtilisin A (a dissociation enzyme known to be effective at low temperature) was used. It was confirmed there is no difference between the identified clusters when the sample is dissociated using subtilisin A or proteinase K as the main protease (see Figure 6).

[0121] The results therefore showed that proteinase K is at least as suitable as subtilisin A for dissociation of tissue at lower temperatures.

[0122] EXAMPLE 8 - comparing the dissociation of kidney tissue when using proteinase K and subtilisin A

[0123] The following experiments allow a comparison to be made between the dissociation of tissue by proteinases K with subtilisin A (a dissociation enzyme known to be effective at low temperature). The experimental method was performed as follows.

[0124] For the dissociation with subtilisin A, all reagents were prepared, including: PBS + 10% FBS, HBSS + 5mM CaCh, 10mg / mL DNase I in HBSS with 5mM CaCh, 1X RBC lysis buffer (Miltenyi Biotec), DBPS + 0.4% BSA + 0.1 mM EDTA and set VIA to 4°C 200 RPM. All steps were completed on ice.

[0125] For the dissociation with proteinase K, all reagents were prepared, including: PBS + 10% FBS, HBSS + 10mM CaCh, 10mg / mL DNase I with 10mM CaCh, 1X RBC lysis buffer (Miltenyi Biotec), DBPS + 0.4% BSA + 0.1 mM EDTA and set VIA to 4°C 200 RPM. All steps were completed on ice.

[0126] Although the concentrations of calcium chloride were different, a comparison between the dissociative function of the proteases can be made. A statistical comparison was made by performing a t-test between both sets of Miltenyi results and found the differences insignificant, the data was then pooled together and averaged the result treating them as 6 replicates, a one-way ANOVA was done with multiple comparisons between the results of the dissociations using the Miltenyi process, proteinase K and subtilisin A.

[0127] 1. The tissue was weighed and rinsed with PBS. The tissue was placed in a pouch, heat sealed, and a clamp was added to the pouch.

[0128] 2. For the dissociation using proteinase K, an enzyme master mix was prepared (1 mg / mL proteinase K + 125U / mL DNase I; tot. = 15mL): 750pL proteinase K 20mg / mL, 201.9pL DNase I 10mg / mL, 14.22mL HBSS + 10mM Ca2+and 5mL was added to each pouch portion.

[0129] 3. For the dissociation using subtilisin, an enzyme master mix was prepared (5mg / mL subtilisin A + 125U / mL DNase I; tot. = 15mL): 750pL subtilisin A 100mg / mL, 201.9pL DNase I 10mg / mL, 14.22mL HBSS + 10mM Ca2+and 5mL was added to each pouch portion. 4. The tissue was left to dissociate for 15 minutes.

[0130] 5. The resulting cell suspension was transferred to a 50mL tube with 1 mL wetted 100pm strainer, the pouch was rinsed with 5mL PBS + FBS.

[0131] 6. The cell suspension was centrifuged at 300xg for 10 mins, the supernatant was removed and resuspended in 1 mL DPBS + BSA + EDTA. 14mL of RBC lysis solution was when added and the sample inverted and incubated for 5 mins.

[0132] 7. The cell suspension was then centrifuged at 300xg for 10 mins to remove supernatant. 3mL of DPBS + BSA + EDTA was added and mixed thoroughly.

[0133] 8. Optionally, the above straining step was repeated with 70pm + 40pm strainers.

[0134] 9. The resulting sample was then assessed based on the parameters in Example 2.

[0135] The results are shown in Figure 5. The results shown in the middle relate to using proteinase K with the VIA Extractor™ Tissue Disaggregator. The results on the left relate to using subtilisin A with the VIA Extractor™ Tissue Disaggregator. The results on the right relate to using Miltenyi Multi Tissue Dissociation Kit 2 with the gentleMACS™ Octo Dissociator.

[0136] It can be seen from the data that the viability score was similar between all three test conditions. In terms of aggregation, proteinase K performed at least as well as subtilisin A in the final sample and significantly better than the Miltenyi Multi Tissue Dissociation Kit 2 with the gentleMACS™ Octo Dissociator.

[0137] The above experiments demonstrate that i) proteinase K can be used as a tool for the effective dissociation of tissue into single cells, ii) dissociation using proteinase K can be performed effectively at lower temperatures and iii) proteinase K can dissociate tissue at lower temperatures at least as effectively as subtilisin A.

[0138] EXAMPLE 9 - comparison of single cell RNA-seq (scRNA-seq) profiles following dissociation with either proteinase K at 4°C or Miltenyi Multi Tissue Dissociation Kit at 37°C using the VIA Extractor™ Tissue Disaggregator

[0139] Following dissociation of mouse kidney using the composition outlined in Example 1 , a scRNA-seq workflow was performed and outlined below.

[0140] For single cell sequencing, cells from samples were quantified to ascertain mean count. The manufacturer’s instructions for 10X Genomics Chromium Next GEM Single Cell 3’ dual index kit v3.1 were followed with an aim to sequence 1000 cells for each sample. A 10X Genomics Chromium Controller was used to capture the cells into gel beads in emulsion (GEMs). Libraries were sequenced on a NextSeq 550 Base (Illumina Inc.) using NextSeq 550 high output kit v2.5 (Illumina Inc.). Two sequencing runs were performed to achieve depth. The scRNA matrix data were analysed using UMAP (Becht et al. Nature Biotechnology 2018 37(1):38-44) in Seurat (Hao et al. Cell. 2021 184(13)). Each sample was analysed individually and filtered to remove duplicates and include cells with feature RNA between 200 and 4000, and to keep all cells with a mitochondrial gene expression percentage less than 50%. Once all samples were filtered and clustered, the data from each sample were combined into a single dataset to allow comparison using UMAP (Becht et al. Nature Biotechnology 2018 37(1):38-44) in Seurat (Hao et al. Cell. 2021 184(13)). Cell types representing each cluster were identified using Seurat and marker genes identified by He et al. Nature Communications. 2021 9;12(1 ) and Chung et al. J Am Soc Nephrol. 2020 10;31 (10):2341-54. Genes lists from cell clusters with differential gene expression profiles were further analysed using gene ontology software package PANTHER (Muller (2017) PANTHER.db: A set of annotation maps describing the entire PANTHER Gene Ontology. R package version 1.O.4.). The results are shown in Figures 7 and 8.

[0141] Figure 7 demonstrates that kidney tissue dissociated with proteinase K at 4°C exhibits a similar scRNA-seq profile to that dissociated with Miltenyi Multi Tissue Dissociation Kit 2 at 37°C.

[0142] Figure 8 shows stress marker gene expression between dissociation of kidney tissue with Miltenyi Multi Tissue Dissociation Kit 2 at 37°C and proteinase K at 4°C. The dot plots show the percent of cells in each cluster expressing “stress” genes listed in O’Flanagan et al. Genome Biology. 2019 20(210). The dark dots represent Miltenyi Multi Tissue Dissociation Kit 2 and lights dots represent proteinase K. Dissociation at 4°C with proteinase K demonstrates reduced stress gene expression and therefore improved suitability for application in high-throughput single cell omics research.

[0143] It will be appreciated that certain embodiments (e.g., compositions, kits, method, etc.) may 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. Accordingly, the various features of one embodiment can be compatible with, combined with, included in, and / or incorporated into other embodiments of the present disclosure. Disclosure of certain features relative to one embodiment of the present disclosure should not be construed as limiting application or inclusion of said features to the specific embodiment. Rather, it will be appreciated that other embodiments can also include said 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 may be combined with any other feature of a same or different embodiment disclosed herein.

[0144] The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope. Various alterations and / or modifications and additional applications of the features illustrated herein which would occurto one skilled in the relevant art and having possession of this disclosure, can be made to the illustrated embodiments without departing from the spirit and scope of the invention as defined by the claims, and are to be considered within the scope of this disclosure. While various features and embodiments have been disclosed herein, other features and embodiments are contemplated. For instance, well-known features and embodiments are not described herein in particular detail in order to avoid obscuring aspects of the described embodiments. Such features and embodiments are, however, also contemplated herein.

Claims

Claims1 . A composition comprising: a buffering agent; proteinase K; a DNase; and a source of Ca2+.

2. The composition according to claim 1 wherein the buffering agent 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), Minimum 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- aminoethanesulphonic acid; 2-[bis(2- Hydroxyethyl)amino]-ethanesulfonic acid), Earle's Balanced Salt Solution (EBSS), MOPSO (2-Hydroxy-3-morpholinopropanesulfonic 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 water, ethylenediaminetetraacetic acid (EDTA), bovine serum albumin (BSA), and ethyleneglycol- bis(p-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA).

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

4. The composition according to any proceeding claim, wherein the DNase is a double stranded DNase (dsDNase); optionally wherein the DNase is DNase I.

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

6. The composition according to any proceeding claim, wherein the source of Ca2+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 proceeding claim 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 the dissociation of tissue at a temperature of less than 20°C; optionally less than 10°C; optionally around 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 to 7.

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

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

12. The method according to 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 according to claims 11 or 12, wherein the composition further comprises a source of Ca2+.

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

15. The method according to any one of claims 11 to 14, where the composition is the composition according to any one of claims 1 to 7.

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

17. The method according to any one of claims 11 to 16, wherein the method further comprises quenching the proteinase K, optionally with 10% foetal bovine serum (FBS) in phosphate buffered saline (PBS) and / or with a source of Cu2+.

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

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

20. The method according to any one of claims 1 1 to 19, wherein the method further comprises mechanical dissociation, optionally wherein the mechanical dissociation is carried out by the VIA Extractor™ Tissue Disaggregator.21 . The method according to any one of claims 11 to 20, wherein the first period is at least 15 minutes.

22. The method according to any one of claims 1 1 to 21 , wherein the method is performed at temperature of less than 20°C; optionally less than 15°C; optionally less than 10°C; optionally less than 5°C; optionally around 4°C.

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

24. The use according to any one of claims 8 to 10 or claim 23, or the method according to any one of claims 10 to 22, wherein the tissue is obtained from the group consisting of: mice, rats, fowls such as chicken, ruminants such as cows, goat, deer, sheep, horses and other animals such as pigs, cats, dogs and primates such as humans, chimpanzees, gorillas and monkeys; optionally mice; optionally female mice.

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

26. A system configured to carry out the method according to any one of claims 11 to 25.

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

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