enzyme

Abstract

This invention relates to a method and composition for dispersing biological tissues with the aim of obtaining single cells for analysis. More specifically, the composition of the present invention is an enzyme composition comprising proteinase K. The composition of the present invention can be used in a method for effectively dispersing tissues at low temperatures for applications in high-throughput single-cell omics research.

Description

[Technical Field]

[0001] The present invention relates to a composition containing proteinase K and a method for tissue dispersion. [Background technology]

[0002] Enzymes are a useful means of dispersing cells from solid tissues for various applications in research, industry, and medicine. Reichard and Asosingh et al., Cytometry A. 209 95(2):219~226, describe examples of enzymes useful for solid tissue dispersion. Various combinations of enzymes (also called enzyme "cocktails") can be used for tissue dispersion. However, not all enzymes are suitable when used alone or in combination, so specific enzymes must be carefully selected. For example, Reichard and Asosingh et al., 2019, reported that trypsin is not suitable for single-cell preparation because it carries the risk of cleaving cell surface receptors.

[0003] Furthermore, because many enzymes are heat-sensitive, tissue dispersion is typically performed at the temperature at which enzyme activity and efficiency are maximized. As discussed in Adam et al., Development. 2017 144(19):3625~3632, the optimal operating temperature for many enzymes is approximately 37°C (trypsin, TrypLE®, pronase, collagenase, liberase, and dispase, etc.), so methods for enzymatic tissue dispersion are routinely performed at this temperature. For example, kidney tissue dispersion is typically performed at 37°C.

[0004] Numerous commercially available products exist for dispersing kidney tissue, such as Miltenyi Biotec's Multi-Tissue Dispersion Kit 2, which can be used in combination with specialized equipment such as the gentleMACS® disperser (for details on Miltenyi Biotec's Multi-Tissue Dispersion Kit 2, see [https: / / static.miltenyibiotec.com / asset / 150655405641 / document_phpdh7tnkl2it4bi11qp1l0o6o?content-disposition=inline] dated May 5, 2023; for details on Miltenyi Biotec's dispersion of mouse kidneys using Multi-Tissue Dispersion Kit 2, see [https: / / www.miltenyibiotec.com / upload / assets / IM0015569.PDF] dated May 5, 2023). Mouse kidneys may also be dispersed at 37°C using more common tissue dispersing enzymes, one example of which is the procedure used by Adam et al. in 2017.

[0005] More recently, it has been discovered that dispersing tissues at 37°C induces artificial changes in 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 result, methods for dispersing tissues should ideally be carried out at low temperatures. Adam et al., 2017, describe one such method utilizing subtilisin A. Subtilisin A is a serine endopeptidase expressed by Bacillus licheniformis, a soil bacterium isolated from Himalayan glaciers, and is known to be effective at low temperatures. O'Flanagan et al., Genome Biology, 2019 20(210), describe the use of subtilisin A as a protease in an enzyme cocktail to mitigate the stress response of kidney cells during dispersal.

[0006] Considering the above, there is still a need for further enzymes that can effectively disperse tissues at low temperatures for application in high-throughput single-cell omics research. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] European Patent Application Publication No. 3171152 [Non-patent literature]

[0008] [Non-Patent Document 1] Reichard and Asosingh et al., Cytometry A. 209 95(2):219~226 [Non-Patent Document 2] Adam et al., Development.2017 144(19): 3625~3632 [Non-Patent Document 3] O'Flanagan et al., Genome Biology, 2019 20(210) [Non-Patent Document 4] Bajorath Biochim Biophys Acta.1988 954(2):176~182 [Non-Patent Document 5] Petrotchenko et al., Molecular & Cellular Proteomics. 2012 11(7) [Non-Patent Document 6] Petsch et al., Analytical Biochemistry.1998 259(1):42~47 [Non-Patent Document 7] Hori and Carey, Journal of Biological Chemistry, 1997, 272(2):1180-1187A [Non-Patent Document 8] Hilz et al., European Journal of Biochemistry 1975 56(1):103~108 [Non-Patent Document 9] Tullis and Rubin, Analytical Biochemistry. 1980, 107(1):260 - 264

Non - Patent Document 10

Non - Patent Document 11

Non - Patent Document 12

Non - Patent Document 13

Summary of the Invention

Means for Solving the Problems

[0009] In a first aspect, the present invention provides a composition comprising proteinase K and a buffer, and the buffer comprises Hanks' balanced salt solution (HBSS), DNase, and a Ca 2+ source.

[0010] In a second aspect, the present invention provides the use of proteinase K for the dispersion of tissues at a temperature below 20°C.

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

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

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

[0014] This invention relates to the unexpected discovery that proteinase K can be used as a means to effectively disperse tissue into single cells. These single cells exhibit beneficial properties suitable for high-throughput single-cell omics studies, particularly single-cell RNA sequencing analyses such as single-cell RNA-seq (scRNA-seq). Furthermore, it was discovered that tissue dispersion can be carried out at low temperatures (e.g., 4°C) to avoid artificial phenomena associated with heat-induced stress responses. Unexpectedly, it was also discovered that proteinase K can disperse tissue at low temperatures at least as effectively as subtilisin A (an enzyme already known to act at low temperatures). This was particularly surprising, as it is known that the optimal temperature range for proteinase K is 20–65°C, with maximum activity at 37°C (Bajorath Biochim Biophys Acta. 1988 954(2):176–182). Furthermore, compared to known enzyme compositions, compositions containing proteinase K of the present invention were observed not to cleave certain cell surface markers.

[0015] Proteinase K (EC 3.4.21.64), also known as peptidase K, endoproteinase K, or endopeptidase K, is a nonspecific serine endopeptidase of the S8 family (subfamily S8A). In molecular biology, it is commonly used to degrade keratin, a free protein that is not needed, and nucleases (DNases and RNases). It is also used in prion research (Petrotchenko et al., Molecular & Cellular Proteomics. 2012 11(7)), endotoxin / horseshoe crab blood cell extract (LAL) testing (Petsch et al., Analytical Biochemistry. 1998 259(1):42~47), protease footprint (Hori and Carey Journal of Biological Chemistry. 1997 272(2):1180~1187A), and nucleic acid isolation (Hilz et al., European Journal of Biochemistry 1975 56(1):103~108).

[0016] Proteinase K is stable in a pH range of approximately 4.0 to 12.5, with pH 8.0 being optimal. [Modes for carrying out the invention]

[0017] Terms and Definitions Unless otherwise specified, the nomenclature used in connection with this specification, as well as the experimental 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 the formulation of compositions and their testing. The techniques and procedures described above can generally be carried out in accordance with conventional methods well known in the art.

[0018] It should be understood that the general description above and the detailed description below are for illustrative and explanatory purposes only and do not limit the claimed invention. As used herein, unless otherwise specified, the singular form includes the plural. The headings used herein are for structural purposes only and should not be construed as limiting the subject matter described.

[0019] As used herein, “proteinase K” means the amino acid sequence of SEQ ID NO: 1 (UniProt reference number P06873 as of May 16, 2023), its homologs in other species, or their variants. The amino acid residues of proteinase K used herein are numbered relative to SEQ ID NO: 1.

[0020] Sequence ID 1 (Parengyodontium album (Tritirachium album)), 384 amino acids, UniProt reference number P06873 as of May 16, 2023):

[0021] [ka]

[0022] As used herein, unless otherwise stated, “or” means “and / or.” Furthermore, the use of the term “including,” as well as other forms such as “includes,” and “included,” is not limited to these.

[0023] As used herein, "approximately" means that the number referred to as "approximately" includes the stated number plus or minus 1-5% of that number. For example, "approximately" 100 degrees may mean only 95-105 degrees or 99-101 degrees, depending on the context. Wherever used herein, a numerical range such as "1-20" means each integer within that range, i.e., 1 only, 2 only, 3 only, etc., up to a maximum of 20.

[0024] As used herein, the term “composition” includes products, formulations, and mixtures, as well as apparatus, equipment, assemblies, kits, etc. Similarly, the term “method” includes methods, procedures, processes, etc.

[0025] As used throughout this disclosure, the words “can” and “may” are used in a permissive sense (i.e., possible) rather than a compulsory sense (i.e., must). Furthermore, the terms “including,” “having,” “involving,” “containing,” “characterized by,” and their variations (e.g., “includes,” “has,” “involves,” “contains,” etc.), as well as similar terms used herein, including in the claims, are comprehensive and / or open-ended and have the same meaning as the word “comprising” and its variations (e.g., “comprise” and “comprises”), without exception including additional, unlisted elements or process steps.

[0026] Please note that embodiments of the Disclosure may include one or more combinations of two or more features described herein. As used herein, “feature” and similar terms may include, for example, composition, component, element, member, part, section, system, method, configuration, parameter, property, etc. Embodiments may include any of the features, options, and / or possibilities described elsewhere in the Disclosure, including other aspects or embodiments of the Disclosure. Please also note that each of the features described herein, described above, and / or other features represents an individual embodiment of the Disclosure. Features can also be combined and / or combined with one or more other features in any appropriate combination and / or order, with or without additional features included with or implemented between them to form a specific embodiment envisioned in the Disclosure. Any two or more such combinations of features represents an individual embodiment of the Disclosure. Therefore, this disclosure is not limited to any particular combination of the exemplary embodiments described in detail herein, and any disclosure of certain features relating to a particular embodiment of this disclosure should not be construed as limiting the application or inclusion of such features to a particular embodiment.

[0027] Furthermore, unless otherwise stated, features described in various embodiments are optional and may not be included in other embodiments of this disclosure. Furthermore, unless otherwise stated, any feature in this specification may be combined with any other feature in the same or different embodiments disclosed herein. Similarly, any steps described herein and / or enumerated in any manner in the claims may be performed in any suitable order and are not necessarily limited to the order described and / or enumerated unless otherwise stated (expressly or implicitly). However, such steps may also need to be performed in a specific order in certain embodiments of this disclosure.

[0028] Where two or more values ​​or ranges of values ​​(for example, less than, greater than, at least a certain value, and / or up to a certain value, and / or between two enumerated values) are disclosed or enumerated, it will be understood that any specific value or range of values ​​that falls within the disclosed value or range of values ​​is also specifically disclosed and considered herein. Accordingly, disclosure of exemplary measurements (e.g., length, width, thickness, etc.) that are about 10 units or less or between 0 and 10 units includes, for example, specific disclosures of measurements of any other values ​​between 0 and 10 units, such as 9 units, 5 units, 1 unit, or 0 units and / or 10 units, and / or (ii) measurements of any other ranges of values ​​between 5, 9 units and 1 unit, between 8 units and 2 units, between 6 units and 4 units, and / or between 0 and 10 units.

[0029] Embodiments of the present invention The following description of embodiments includes disclosures relating to one or more embodiments of the present disclosure. Therefore, some embodiments may include features disclosed in the following embodiments without departing the scope of the present disclosure. In other words, features disclosed in the following embodiments may be included in and / or incorporated into any one or more embodiments disclosed herein.

[0030] The composition of the present invention contains proteinase K, an active peptidase that cleaves amino acids present in proteins that hold cells together.

[0031] Proteinase K is commercially available from several manufacturers, such as Qiagen (Blirt (trademark) and others). Preferably, the proteinase K in the composition of the present invention is Blirt (trademark) catalog number RP107B as of May 17, 2023. Recombinant proteinase K can also be produced using host cells.

[0032] The host cell may be selected from the 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.

[0033] Preferably, the proteinase K in the composition of the present invention contains 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.

[0034] The amino acid sequence of the proteinase K variant may share at least about 85% sequence identity with SEQ ID NO: 1, and therefore may 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 proteinase K variant substantially retains the enzymatic activity related to the sequence of SEQ ID NO: 1.

[0035] The amino acid sequence of variant proteinase K may be altered by substitution, addition, or deletion of an appropriate number of amino acids in the sequence 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. "Substitution, addition, or deletion" includes combinations of substitutions, additions, and deletions. Preferably, the variant proteinase K substantially retains the enzymatic activity associated with the sequence of SEQ ID NO: 1.

[0036] When a sequence is modified by the substitution of a specific amino acid residue, the substitution may be a conserved amino acid substitution. As used herein, the term “conserved amino acid substitution” means an amino acid substitution in which one amino acid residue is replaced by another amino acid residue having a similar side chain. Since amino acids with similar side chains tend to have similar properties, it can be predicted that a conservative substitution of an amino acid important to the structure or function of a polypeptide will have less impact on the polypeptide structure / function than a non-conservative amino acid substitution at the same position.

[0037] Families of amino acid residues having similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged 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 conserved amino acid substitution can be considered a substitution in which a particular amino acid is replaced by a different amino acid of the same family. However, epitope residue substitutions may also be non-conserved substitutions in which one amino acid is replaced by another having a side chain belonging to a different family.

[0038] Proteinase K may be present in the composition of the present 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. Proteinase K may also be present in the composition of the present 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 present invention is 1 ± 1% mg / mL. Where "±x%" is used herein, it means ±x% of the stated value.

[0039] Proteinase K may be present in the composition of the present invention at a concentration of about 1 mg / mL. Preferably, proteinase K is present in the composition of the present invention at a concentration of 1 mg / mL.

[0040] Proteinase K may be present in the composition of the present invention at concentrations 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.

[0041] Proteinase K may be present in the composition of the present invention at concentrations of 0.1-10 mg / mL, 0.2-9 mg / mL, 0.3-8 mg / mL, 0.4-7 mg / mL, 0.5-6 mg / mL, 0.6-5 mg / mL, 0.7-4 mg / mL, 0.8-3 mg / mL, or 0.9-2 mg / mL. Preferably, proteinase K is present in the composition of the present invention at a concentration of 0.1 mg / mL to 5 mg / mL.

[0042] The amount of proteinase K in a composition may be defined in enzyme units. The following definition of enzyme units may be used: Proteinase K hydrolyzes urea-modified hemoglobin, producing a color equivalent to 1 μmol of tyrosine per minute at 37°C and pH 7.5 (Folin & Ciocalteu method), 1 U = 1 mAnsonU. For example, when using 20 mg / mL (minimum) and 800 U / mL (minimum) storage solutions, the final concentration will be 1 mg / mL and the activity of the solution will be 40 U / mL.

[0043] Proteinase K may be present in the composition of the present invention at concentrations of 40±75% U / mL, 40±50% U / mL, 40±10% U / mL, 40±5% U / mL, or 40±1% U / mL. Proteinase K may also be present in the composition of the present invention at concentrations of 400 U / mL, 200 U / mL, or 80 U / mL. Preferably, proteinase K is present in the composition of the present invention at a concentration of 40±1% U / mL.

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

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

[0046] The composition of the present invention also comprises DNase. DNase (in particular dsDNase) has been described as useful in reducing cell aggregation / coagulation caused by cell-free DNA, resulting in high-quality samples for single-cell RNA sequencing (Reichard and Asosingh et al., 2019).

[0047] Proteinase K is known to degrade nucleases such as DNase, but Ca 2+ It is known that the presence of ions in solution provides a protective effect against DNases (Tullis and Rubin Analytical Biochemistry. 1980 107(1):260~264).

[0048] DNase is commercially available from several manufacturers, such as Qiagen (Blirt (trademark) and others). DNase may be present in the composition of the present invention at concentrations of 4±75% U / mL, 4±50% U / mL, 4±10% U / mL, 4±5% U / mL, or 4±1% U / mL. DNase may also be present in the composition of the present invention at concentrations of 40 U / mL, 20 U / mL, or 8 U / mL. Preferably, DNase is present in the composition of the present invention at a concentration of 4±1% U / mL.

[0049] DNase may be present in the composition of the present invention at a concentration of about 4 U / mL.

[0050] DNase may be present in the composition of the present invention at concentrations of 0.4-40 U / mL, 0.8-36 U / mL, 1.2-32 U / mL, 1.6-28 U / mL, 2-24 U / mL, 2.4-20 U / mL, 2.8-16 U / mL, 3.2-12 U / mL, 3.6-8 U / mL, 3.7-7 U / mL, 3.8-6 U / mL, and 3.9-5 U / mL.

[0051] The enzyme unit of DNase can be defined as follows: 1 unit is defined as an increase of 1.0 in absorbance at 260 nm over 30 minutes at 37°C and pH 8.0 using 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 EN33, as of May 17, 2023, in the Blirt® catalog.

[0053] The DNase may be a member of the DNaseI family, selected from the group consisting of DNaseI, DNase1L1, DNase1L2, and DNase1L3. Preferably, the DNase is DNaseI.

[0054] DNase may be a member of the DNase II family, selected from the group consisting of DNase IIα and DNase IIβ.

[0055] The composition of the present invention, Ca 2+ The source may include calcium chloride. The purpose of adding calcium ions is to protect DNase and its activity from degradation by proteinase K (Tullis and Rubin 1980). 2+ The source may contain calcium chloride. The calcium chloride may be solid or aqueous. Preferably, the calcium chloride is aqueous calcium chloride.

[0056] Calcium chloride may be present in the composition of the present invention at a concentration of 10±75%mM, 10±50%mM, 10±10%mM, 10±15%mM, 10±5%mM, or 10±1%mM. Calcium chloride may also be present in the composition of the present invention at a concentration of 100mM, 50mM, or 20mM. Preferably, calcium chloride is present in the composition of the present invention at a concentration of 10±1%mM.

[0057] Preferably, calcium chloride is present in the composition of the present invention at a concentration of 11 to 12 mM, more preferably 11.3 mM.

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

[0059] Calcium chloride may be present in the composition of the present invention at concentrations of 1-100 mM, 2-90 mM, 3-80 mM, 4-70 mM, 5-60 mM, 6-50 mM, 7-40 mM, 8-30 mM, and 9-20 mM.

[0060] The compositions of the present invention may contain one or more buffers. The methods or uses of the present invention may require one or more buffers. Buffers suitable for the present invention help support cell viability and yield and reduce cell aggregation. Buffers include HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Hanks' equilibrium 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, Iskov's Modified Dulbecco's Medium (IMDM), BES (NN-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid; 2-[bis(2-hydroxyethyl)amino]-ethanesulfonic acid), Earle's equilibrium salt solution (EBSS), MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid), and ACES. The following may be selected from the group consisting of (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 ethylene glycol-bis(β-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA).

[0061] Preferably, the buffer is HBSS. HBSS is a buffer commonly used in cell culture, containing a balanced amount of inorganic ions essential for cell growth and maintaining appropriate pH and osmotic pressure. HBSS is commercially available from several manufacturers, for example, Cytiva®. Preferably, the HBSS in the composition of the present invention is Cytiva® catalog number SH30268.01 as of May 17, 2023. The composition of Cytiva® catalog 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 indicated values. Calcium chloride (anhydrous) and / or magnesium sulfate (anhydrous) may be omitted.

[0062] [Table 1]

[0063] Preferably, the composition of the present invention comprises proteinase K 1±10% mg / mL; dsDNase 4±10% U / mL; Hanks' equilibrium salt solution (HBSS); and calcium chloride 10±15% mM.

[0064] More preferably, the composition of the present invention comprises proteinase K 1 mg / mL; dsDNase 4 U / mL; Hanks' equilibrium salt solution (HBSS); and calcium chloride 10 ± 15% mM.

[0065] In a further embodiment, the present invention relates to buffering agents; proteinase K; DNase; and Ca 2+ The present invention provides a composition containing a source.

[0066] In a preferred embodiment, the exovivo method for dispersing tissue according to the present invention comprises the steps of: a) contacting the tissue with a composition comprising proteinase K 1 mg / mL, dsDNase 4 U / mL, Hanks' equilibrium salt solution (HBSS), and calcium chloride 10 ± 15% mM; b) incubating the tissue and the composition for a first period of time; and c) obtaining a cell suspension.

[0067] In a more preferred embodiment, the exovivo method for dispersing tissue according to the present invention comprises the steps of: a) contacting the tissue with a composition comprising proteinase K 1 mg / mL, dsDNase 4 U / mL, Hanks' equilibrium salt solution (HBSS), and calcium chloride 10 ± 15% mM; b) incubating the tissue and composition for a first period; c) obtaining a cell suspension; d) passing the cell suspension through a first filter; e) stopping the reaction of proteinase K with 10% fetal bovine serum (FBS) preferably dissolved in phosphate-buffered saline (PBS); and preferably f) passing the cell suspension through a second filter, preferably a 40 or 70 μm filter.

[0068] The compositions of the present invention may also contain one or more additional agents, such as other enzymes (in particular, dispersing enzymes).

[0069] Alternatively, the composition of the present invention may also not contain one or more additional agents, such as other enzymes (in particular, dispersing enzymes).

[0070] 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, clostripine, and neutral proteases, pronase, pepsin, lysozyme, divalent ion chelating agents (such as EDTA or citrate), and combinations thereof.

[0071] The method of the present invention may further include the step of passing the cell suspension through a first filter and / or a second filter.

[0072] The filter size 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 μm.

[0073] Preferably, the filter size is 40 or 70 μm.

[0074] The method of the present invention may further include stopping the reaction of proteinase K to terminate the dispersion reaction. The reaction may be stopped with the buffer described herein. The reaction may be stopped with fetal bovine serum (FBS) dissolved in phosphate buffered saline (PBS) or another buffer described herein. The concentration of FBS may be 1-100% v / v, 2-90% v / v, 3-80% v / v, 4-70% v / v, 5-60% v / v, 6-50% v / v, 7-40% v / v, 8-30% v / v, 9-20% v / v. Preferably, the concentration of FBS is 10%.

[0075] The reaction may alternatively or additionally be stopped with a Cu 2+ source. The Cu 2+ source may be present in the composition of the present invention dissolved in the buffer described herein.

[0076] Considering the following examples demonstrating that reaction termination with FBS significantly improves the viability and yield of dispersed cells compared to reaction termination with Cu 2+ , reaction termination with FBS is preferred over reaction termination with Cu 2+ .

[0077] After dispersion, cell yield, viability, and aggregation can be evaluated by any suitable method known in the art. Preferably, cell yield, viability, and aggregation are quantified using an automated cell counter, preferably a Nucleocounter® NC-200™, using a 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 using a hemocytometer with trypan blue. The yield can be calculated as 1000((viable cells per mL × dilution factor for counting × total volume mL) / (mass mg)) = viable cells per gram of tissue.

[0078] The method of the present invention may further include a step of removing the supernatant. The removal of the supernatant may include centrifugation.

[0079] The method of the present invention may further include the step of resuspending cells in a buffer as described herein. The buffer may contain 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 0.04-4%, 0.08-3.6%, 0.12-3.2%, 0.16-2.8%, 0.2-2.4%, 0.24-2%, 0.28-1.6%, 0.32-1.2%, or 3.6-0.8%. The EDTA concentration 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 0.01-1 mM, 0.02-0.9 mM, 0.03-0.8 mM, 0.04-0.7 mM, 0.05-0.6 mM, 0.06-0.5 mM, 0.07-0.4 mM, 0.08-0.3 mM, or 0.09-0.2 mM. Preferably, the buffer contains 0.4% BSA and 0.1 mM EDTA.

[0080] The method of the present invention may further include mechanical dispersion and can be carried out by a suitable device (preferably an automated tissue dispersion system) selected from the group consisting of, for example, VIA Extractor® tissue dispersion device, gentleMACS® dispersion device, gentleMACS® Octo dispersion device, Singulator® 100, and Singulator® 200. Preferably, the method of the present invention is carried out using a VIA Extractor® tissue dispersion device.

[0081] The compositions, methods, or systems of the present invention may be suitable for use with the VIA Extractor® tissue dispersion apparatus.

[0082] The first period of the method of the present 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 present invention may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 minutes, preferably 20 minutes, more preferably 15 minutes or less. The first period of the method of the present invention may 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 period of the method of the present invention is about 15 minutes, more preferably 15 minutes.

[0083] The methods or uses of the present invention may be carried out at temperatures below 50, 45, 40, 35, 30, 25, 30, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1°C, or at approximately 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 may also be carried out at temperatures of 0.4-40°C, 0.8-36°C, 1.2-32°C, 1.6-28°C, 2-24°C, 2.4-20°C, 2.8-16°C, 3.2-12°C, or 3.6-8°C.

[0084] The tissue for dispersion is preferably a solid tissue and may be selected from the group consisting of spleen, heart, liver, brain and other nerve 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, intestine, colon, intestinal tract, bladder, urethra, eye, gallbladder, organoids from cell culture, and tumor. Preferably, the tissue is kidney tissue, more preferably whole kidney tissue, and even more preferably whole kidney tissue in a mass of less than 300 mg.

[0085] The tissue can be obtained from mammals such as mice, rats, poultry including chickens, ruminants including cattle, goats, deer, sheep, and horses, other animals including pigs, cats, and dogs, and primates including humans, chimpanzees, gorillas, and monkeys. Preferably, the tissue is obtained from mice, more preferably from female mice.

[0086] In another aspect, the present invention provides a cell suspension obtained by the method of a third aspect of the present invention.

[0087] The compositions of the present invention may be administered to tissue using an apparatus such as that described in European Patent Application Publication No. 3171152.

[0088] The kit of the present invention may further include one or more containers. Various components of the composition of the present invention may be present in one or more containers within the kit. For example, the components may all be present in one container or in separate containers.

[0089] In addition to those shown and described herein, various modifications of the invention will be apparent to those skilled in the art from the foregoing description and are included in the appended claims. Preferred features of each aspect of the invention are also applicable to other aspects. Documents referenced herein are incorporated by reference to the maximum extent permitted by law. [Examples]

[0090] The present invention will be further described herein with reference to the following embodiments and figures, which are included for reference purposes only and should not be construed as limiting the invention. [Brief explanation of the drawing]

[0091] [Figure 1] This figure visualizes mouse kidneys that were dispersed with proteinase K at various concentrations (0.1 mg / mL, 1 mg / mL, and 5 mg / mL) and then stopped with Cu2+. [Figure 2]This figure shows the yield, survival rate, and aggregation quantification of mouse kidneys dispersed with various concentrations of proteinase K (0.1 mg / mL, 1 mg / mL, and 5 mg / mL) and then stopped with Cu2+. [Figure 3] This figure shows the yield, survival rate, and aggregation quantification of mouse kidneys after dispersion with various concentrations of proteinase K (0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL) at 4°C (cold) and 37°C (warm), followed by reaction termination with FBS. [Figure 4] This figure shows the results of three experiments conducted with proteinase K incubation times of 15 and 20 minutes. [Figure 5] This figure compares the results after dispersion using the VIA Extractor™ tissue dispersion device with subtilisin A at 4°C (left), the VIA Extractor™ tissue dispersion device with proteinase K at 4°C (center), and the gentleMACS™ Octo dispersion device with Miltenyi enzyme using Miltenyi Multi-Tissue Dispersion Kit 2 at 37°C (right). [Figure 6] This figure shows a UMAP plot comparing the detected kidney cell populations after dispersing mouse kidneys with proteinase K and subtilisin A at 4°C using the VIA Extractor™ tissue dispersion device. [Figure 7] This figure shows a UMAP plot comparing the cell populations detected after dispersing mouse kidneys using the VIA Extractor™ tissue dispersion device with proteinase K at 4°C ("cold") and with the Miltenyi Multi-Tissue Dispersion Kit 2 at 37°C. [Figure 8] This figure compares stress markers in kidney cells dispersed using the VIA Extractor™ tissue dispersion device with proteinase K at 4°C, and with Miltenyi Multi-Tissue Dispersion Kit 2.

[0092] (Example 1) The following compositions were prepared for use as reagents for tissue dispersion. Recombinant proteinase K (Blirt Inc. / QIAGEN Inc.) 1 mg / mL; dsDNase (Blirt™ / QIAGEN™) 4U / mL; and HBSS buffer (Cytiva®) was mixed with 10 mM CaCl2 (Thermofisher®).

[0093] (Example 2) Method for dispersing mouse kidney cells compatible with VIA Extractor® tissue dispersion apparatus and VIA Freeze® Uno. A method for dispersing mouse kidney tissue was developed using the composition prepared in Example 1. This method was tested on whole kidneys of female mice, less than 300 mg in total. The entire process was typically carried out on ice.

[0094] The criteria for evaluating the suitability of dispersed cells, particularly for single-cell sequencing, are three parameters: yield (>1x10⁻¹). 7 This was based on live cells / g, viability (>80% live cells), and aggregation (<10%). However, other parameters can also be used to assess such compatibility.

[0095] Viability and aggregation were evaluated using Nucleocounter® NC-200® and Via2-Cassette® (Chemometec). Yield was calculated as 1000 ((live cells per mL × dilution factor for counting × total volume mL) / (mass mg)) = live cells per gram of tissue.

[0096] The VIA Freeze Uno® was set to cool to 2°C (so that the VIA Extractor® tissue dispersion device would reach 4°C) and kept idle at a speed of 200 rpm for 60 minutes to maintain the temperature until ready. Kidney tissue was placed in Omics pouches using an Omics applicator. The composition was prepared according to Example 1, with 5 mL applied per sample / kidney / pouch.

[0097] Next, the VIA Extractor® tissue dispersion device was set to operate for 15 minutes. The sample was then mostly dispersed, but further dispersion may be performed if necessary. The resulting cell suspension was then passed through a 100 μm cell strainer, and the reaction was stopped by using an excess of phosphate-buffered saline (PBS) containing 10% fetal bovine serum (FBS) to remove proteinase K. The sample was centrifuged at 300 × 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).

[0098] If necessary, further filtration using 70 μm and 40 μm cell strainers and / or additional washing steps such as red blood cell (RBC) lysis may be performed.

[0099] Next, sample quality was determined after the washing process using a cell counter, and the aforementioned parameters were measured.

[0100] (Example 3) Pilot experiment to determine the optimal concentration of proteinase K In the first pilot experiment, the dispersion of proteinase K in mouse kidney tissue was tested using three compositions containing proteinase K at various enzyme concentrations (0.1 mg / mL, 1 mg / mL, 5 mg / mL). 2+ The reaction termination step was used to stop the reaction by terminating the protease activity. The results are shown in Figure 1. The kidney tissue was effectively dispersed.

[0101] Figure 2 shows the evaluation of the parameters examined in Example 2. It was shown that 1 mg / mL was the optimal concentration.

[0102] (Example 4) A pilot experiment to evaluate the aggregation, yield, and viability of mouse kidney cells after dispersion at 4°C ("cold") or 37°C ("warm") with various proteinase K concentrations and reaction termination with FBS. The alternative proteinase K reaction termination step is Cu 2+ The test was conducted by replacing reaction arrest with FBS reaction arrest. The results are shown in Figure 3. Surprisingly, reaction arrest of proteinase K activity by FBS was observed for Cu 2+ It has been shown that this method significantly improves both survival rate and yield compared to reaction termination by other means.

[0103] (Example 5) Pilot experiment to determine the optimal incubation time for proteinase K for use with the VIA Extractor (trademark) tissue dispersion device. To determine the optimal incubation time and to conduct statistical analysis for confirmation, the experiment was conducted three times with incubation times of 15 minutes and 20 minutes. The results are shown in Figure 4.

[0104] The most effective incubation time using proteinase K was found to be 15 minutes.

[0105] (Example 6) Comparison of tissue dispersion between the VIA Extractor™ tissue dispersion device and the Miltenyi Multi-Tissue Dissociation Kit 2 using the gentleMACS™ Octo dispersion device. Experiments were conducted to compare the tissue dispersion of compositions prepared according to Example 8 using the VIA Extractor™ tissue dispersion device with that of the Miltenyi Multi-Tissue Dispersion Kit 2, a commercially available kit, using the gentleMACS™ Octo dispersion device. The results are shown in Figure 5.

[0106] Compared to Miltenyi Multi-Tissue Dispersion Kit 2 using the gentleMACS® Octo Dispersion Apparatus, using the VIA Extractor® Tissue Dispersion Apparatus with the Proteinase K composition according to Example 8 resulted in improved viability and significantly reduced aggregation.

[0107] Although the properties of the DNase used differ from those of Example 1, it is still possible to compare the dispersion capabilities of the VIA Extractor™ tissue dispersion device using an active protease, Proteinase K, at 4°C, and the Miltenyi Multi-Tissue Dispersion Kit 2 using the gentleMACS™ Octo dispersion device at 37°C. A statistical comparison was performed in Example 8.

[0108] (Example 7) Characterization of single-cell RNA-seq (scRNA-seq) data obtained by dispersion using proteinase K. Following the dispersion and washing method according to Example 2, scRNA-seq was performed using the obtained cell suspension according to the same method as in Example 9 below. For comparison, a composition containing 25 mM CaCl dissolved in HBSS, 125 U / mL DNase I (Sigma), and 5 mg / mL subtilisin A (a dispersion enzyme known to be effective at low temperatures) was used. When the samples were dispersed using subtilisin A or proteinase K as the primary protease, no difference was observed between the identified populations (see Figure 6).

[0109] Therefore, the results showed that proteinase K is at least as suitable as subtilisin A for tissue dispersion at low temperatures.

[0110] (Example 8) Comparison of kidney tissue dispersion using proteinase K and subtilisin A The following experiment allows for a comparison of tissue dispersion between proteinase K and subtilisin A (a dispersion enzyme known to be effective at low temperatures). The experimental method was carried out as follows.

[0111] For dispersion using subtilisin A, all reagents were prepared, including PBS + 10% FBS, HBSS + CaCl 25 mM, DNase I 10 mg / mL dissolved in HBSS containing CaCl 25 mM, 1× RBC lysis buffer (Miltenyi Biotec), DBPS + 0.4% BSA + EDTA 0.1 mM, and the viaa was set to 4°C at 200 RPM. The entire process was carried out on ice.

[0112] For the dispersion using proteinase K, all reagents were prepared, including PBS + 10% FBS, HBSS + CaCl2 10mM, DNase I 10 mg / mL containing CaCl2 10mM, 1× RBC lysis buffer (Miltenyi Biotec), DBPS + 0.4% BSA + EDTA 0.1mM, and the viaa was set to 4°C at 200 RPM. The entire process was carried out on ice.

[0113] Although the calcium chloride concentrations differed, it was possible to compare the dispersion functions of the proteases. A t-test was performed between the results of both Miltenyi groups to perform a statistical comparison, and no significant difference was found. Subsequently, the data were combined and processed as six copies, the results were averaged, and a one-way ANOVA was performed by performing multiple comparisons between the dispersion results using the Miltenyi method, proteinase K, and subtilisin A.

[0114] 1. The tissue mass was measured and washed with PBS. The tissue was placed in a pouch, heat-sealed, and a clasp was attached to the pouch. 2. For dispersion using proteinase K, enzyme master mix (proteinase K 1 mg / mL + DNase I 125 U / mL; total = 15 mL): 750 μL of proteinase K 20 mg / mL, 201.9 μL of DNase I 10 mg / mL, HBSS + Ca 2+ A 10 mM solution of 14.22 mL was prepared, and 5 mL was added to each pouch. 3. For dispersion using subtilisin, an enzyme master mix (subtilisin A 5 mg / mL + DNase I 125 U / mL; total = 15 mL): 750 μL of subtilisin A 100 mg / mL, 201.9 μL of DNase I 10 mg / mL, HBSS + Ca 2+ A 10 mM solution of 14.22 mL was prepared, and 5 mL was added to each pouch. 4. The organization remained dispersed for 15 minutes. 5. The obtained cell suspension was transferred to a 50 mL test tube using a 1 mL moist 100 μm strainer, and the pouch was washed with 5 mL of PBS + FBS. 6. The cell suspension was centrifuged at 300 × g for 10 minutes, the supernatant was removed, and the cell suspension was resuspended in 1 mL of DPBS + BSA + EDTA. After adding 14 mL of RBC lysis solution, the sample was inverted and incubated for 5 minutes. 7. Next, the cell suspension was centrifuged at 300 × g for 10 minutes, and the supernatant was removed. 3 mL of DPBS + BSA + EDTA was added and thoroughly mixed. 8. Optionally, the above filtration process was repeated using a 70 μm + 40 μm strainer. 9. Next, the obtained samples were evaluated based on the parameters of Example 2.

[0115] The results are shown in Figure 5. The results shown in the center relate to the use of proteinase K in the VIA Extractor® tissue dispersion device. The results shown on the left relate to the use of subtilisin A in the VIA Extractor® tissue dispersion device. The results shown on the right relate to the use of Miltenyi multi-tissue dispersion kit 2 in the gentleMACS® Octo dispersion device.

[0116] The data shows that the survival rate scores were similar across all three test conditions. Regarding aggregation, proteinase K performed at least as well as subtilisin A in the final sample, and significantly better than Miltenyi Multi-Tissue Dispersion Kit 2 using the gentleMACS® Octo dispersion device.

[0117] The above experiments demonstrate that i) proteinase K can be used as a means to effectively disperse tissue into single cells, ii) dispersion using proteinase K can be effectively carried out at low temperatures, and iii) proteinase K can disperse tissue at low temperatures at least as effectively as subtilisin A.

[0118] (Example 9) Comparison of single-cell RNA-seq (scRNA-seq) profiles after dispersion using the VIA Extractor™ tissue dispersion device with proteinase K at 4°C or with the Miltenyi Multi-Tissue Dispersion Kit at 37°C. After dispersing mouse kidneys using the composition outlined in Example 1, the scRNA-seq workflow was performed and outlined below.

[0119] For single-cell sequencing, the number of cells in each sample was quantified to determine the average number. Following the manufacturer's instructions, we aimed to sequence 1000 cells per sample using the 10X Genomics Chromium Next GEM Single Cell 3' Dual Index Kit v3.1. Cells were captured in gel beads (GEM) in an emulsion using a 10X Genomics Chromium controller. Libraries were sequenced using the NextSeq 550 High-Power Kit v2.5 (Illumina Inc.) on a NextSeq 550 Base (Illumina Inc.). Two sequencing runs were performed to achieve sufficient depth. ScRNA matrix data were analyzed in Seurat using UMAP (Becht et al., Nature Biotechnology 2018 37(1):38~44) (Hao et al., Cell. 2021 184(13)). Each sample was analyzed individually, duplicates were removed, and the data was filtered to retain all cells with mitochondrial gene expression rates less than 50%, including cells with 200–4000 characteristic RNAs. After all samples were filtered and clustered, the data from each sample were combined into a single dataset, which could then be compared using UMAP (Becht et al., Nature Biotechnology 2018 37(1):38–44) with Seurat (Hao et al., Cell. 2021 184(13)). Cell types representative of each population were identified using marker genes identified by Seurat, as well as He et al., Nature Communications. 2021 9;12(1) and Chung et al., J Am Soc Nephrol. 2020 10;31(10):2341–54. The gene lists of cell populations with different 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). The results are shown in Figures 7 and 8.

[0120] Figure 7 shows that kidney tissue dispersed with proteinase K at 4°C exhibits a similar scRNA-seq profile to kidney tissue dispersed with Miltenyi Multi-Tissue Dispersion Kit 2 at 37°C.

[0121] Figure 8 shows stress marker gene expression when kidney tissue was dispersed at 37°C using the Miltenyi Multi-Tissue Dispersion Kit 2 and when dispersed at 4°C using Proteinase K. The plotted points represent the percentage of cells in each population expressing the “stress” genes listed by O’Flanagan et al., Genome Biology. 2019 20(210). Dark points represent the Miltenyi Multi-Tissue Dispersion Kit, and bright points represent Proteinase K. Dispersion at 4°C with Proteinase K showed a decrease in stress gene expression, improving its suitability for application in high-throughput single-cell omics studies.

[0122] It will be understood that certain embodiments (e.g., compositions, kits, methods, etc.) may include, incorporate, or otherwise include features (e.g., properties, components, elements, parts, sections, processes, etc.) described in other embodiments disclosed and / or described herein. Therefore, various features of one embodiment may be adapted, combined, included, and / or incorporated in other embodiments of this disclosure. The disclosure of certain features relating to one embodiment of this disclosure should not be construed as limiting the application or inclusion of such features to that particular embodiment. Rather, it will be understood that other embodiments may also include such features without necessarily departing from the scope of this disclosure. Furthermore, any feature described herein may be combined with any other feature of the same or different embodiments disclosed herein, unless otherwise stated that a feature requires another feature to be combined with it.

[0123] The embodiments described herein are, in all respects, illustrative only and not limiting. Accordingly, the scope of the invention is defined not by the foregoing description but by the appended claims. Any modifications that fall within the meaning and scope equivalent to the claims are incorporated within the claims. Various modifications and / or modifications and additional applications of the features described herein, conceivable by a person skilled in the art and owner of this disclosure, can be made to the illustrative embodiments without departing from the spirit and scope of the invention as defined in the claims, and are considered within the scope of this disclosure. While various features and embodiments are disclosed herein, other features and embodiments are also considered. For example, well-known features and embodiments are not described in particular detail herein to avoid ambiguity of the aspects of the embodiments described. However, such features and embodiments are also considered herein.

Claims

1. cushioning material, Proteinase K, DNase, and Ca 2+ A composition containing a source.

2. The buffers mentioned above include HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Hanks' equilibrium 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, Iskov's Modified Dulbecco's Medium (IMDM), BES (NN-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid; 2-[bis(2-hydroxyethyl)amino]-ethanesulfonic acid), Earle's equilibrium salt solution (EBSS), MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid), ACES (N-2-aminoethanesulfonic acid), fetal bovine serum (FBS), and TAPS. The composition according to claim 1, selected from the group consisting of (([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 ethylene glycol-bis(β-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 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 claims 1 to 3, wherein the DNase is a double-stranded DNase (dsDNase), and optionally the DNase is DNase I.

5. The composition according to any one of claims 1 to 4, wherein the DNase is present in the composition at a concentration of 4 ± 10% U / mL.

6. The Ca 2+ The composition according to any one of claims 1 to 5, wherein the source contains calcium chloride, and optionally the calcium chloride is present in the composition at a concentration of 10 ± 15% mM.

7. Proteinase K 1±10% mg / mL dsDNase 4±10%U / mL Hanks' equilibrium salt solution (HBSS), and A composition according to any one of claims 1 to 6, comprising 10 ± 15% mM calcium chloride.

8. Use of proteinase K for tissue dispersion at temperatures below 20°C, optionally below 10°C, and optionally around 4°C.

9. The use according to claim 8, wherein the proteinase K is present in the composition and the composition is described in any one of claims 1 to 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, or optionally no more than 15 minutes.

11. a) A step of bringing the tissue into contact with a composition containing proteinase K, b) A step of incubating the tissue and the composition for a first period of time, c) An ex vivo method for dispersing tissue, comprising the step of obtaining a cell suspension.

12. The method according to claim 11, wherein the composition further comprises a DNase, optionally the DNase being a double-stranded DNase (dsDNase), and optionally the DNase being DNase I.

13. The composition is Ca 2+ The method according to claim 11 or 12, further comprising a source.

14. The method according to any one of claims 11 to 13, wherein the composition further comprises Hanks' equilibrium salt solution (HBSS).

15. The method according to any one of claims 11 to 14, wherein 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, further comprising the step of passing the cell suspension through a first filter, and optionally a 100 μm filter.

17. Optionally, 10% fetal bovine serum (FBS) and / or Cu dissolved in phosphate-buffered saline (PBS). 2+ The method according to any one of claims 11 to 16, further comprising the step of stopping the reaction of proteinase K at the source.

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

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

20. The method according to any one of claims 11 to 19, further comprising mechanical dispersion, wherein the mechanical dispersion is optionally carried out by a VIA Extractor® tissue dispersion apparatus.

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 11 to 21, which is carried out at a temperature of less than 20°C, optionally less than 15°C, optionally less than 10°C, optionally less than 5°C, or optionally about 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 the spleen, heart, liver, brain and other nerve 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, intestine, colon, intestinal tract, bladder, urethra, eye, gallbladder, organoids obtained by cell culture and tumors.

24. The use according to any one of claims 8 to 10 or 23, or the method according to any one of claims 10 to 22, wherein the tissue is obtained from a group consisting of poultry such as mice, rats, and chickens, ruminants such as cattle, goats, deer, sheep, and horses, as well as other animals such as pigs, cats, and dogs, as well as primates such as humans, chimpanzees, gorillas, and monkeys, optionally selected for mice, and optionally selected for female mice.

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

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

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

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