GB2607706A

GB2607706A - Enzyme kit for dispersing biological tissue and method for obtaining single-cell suspension using same

Info

Publication number: GB2607706A
Application number: GB2205828.3A
Authority: GB
Inventors: Li Xiangyao; Wu Cheng; Liu Li; Sheng Tao; Wang Jinghua; Lian Yanna
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-04-22
Filing date: 2022-04-21
Publication date: 2022-12-14
Anticipated expiration: 2042-04-21
Also published as: GB2607706B; CN113388599A; CN113388599B; GB202205828D0

Abstract

The enzyme kit includes a first reagent and a second reagent, where the first reagent includes pronase, and the second reagent includes papain. The single-cell suspension obtained by the method of the present disclosure has a high survival rate and a low aggregation rate, which is conducive to subsequent sorting and single-cell sequencing. The first or second reagents may also include HAG medium, made of Hibernate A, Actinomycin D, and GlutaMAX 1. The method may include dividing the tissue sample into two parts, digesting the first part with the first reagent and the second part with the second reagent, and mixing the first and second digested solution in the same HAG medium. There may be additional filtration and centrifugation steps.

Description

ENZYME MT FOR DISPERSING BIOLOGICAL TISSUE AND METHOD FOR

OBTA INING SINGLE-CELE SUSPENSION USING SAME

TECHNICAL HELD

100011 The present disclosure relates to the field of cell biology, and in particular, to an enzyme kit for dispersing biological tissue and a method for obtaining a single-cell suspension using the same.

BACKGROUND ART

100021 Single-cell sequencing technology can be used to observe changes in genes at a single-cell level, enabling better classify cells of tissues and seeking changes in gene expression in specific cell types. This technology is widely used in research in the field ot'celi biology. A key to single-cell sequencing technology is to obtain high-quality single-cell suspensions. Since components involved in cell adhesion and cell junction varies in different tissues, there is a highly complicated process in obtaining the single-cell suspensions. Even in the same tissue, single cells obtained by different preparation methods may vary greatly in surface properties, hormone response states, and material and energy metabolisms. Therefore, it is essential to develop suitable preparation methods to obtain high-quality single-cell suspensions.

100031 In the prior art, the single-cell suspension is generally prepared by enzymatic digestion. In enzymatic digestion, proteins in tight junctions between cells are hydrolyzed mainly by pronase to achieve cell dissociation. However, the single-cell suspension prepared by this method has disadvantages of a large amount of cell debris, high cell aggregation rate, and relatively poor cell viability. Especially in the separation step of brain tissue cells, due to a. complex connection between nerve cells and other types of cells in the brain, traditional pronase cannot disperse the complex brain tissue cells based on ensuring a high survival rate and low aggregation rate. Accordingly, a single-cell suspension with massive cell debris is generally obtained, not conducive to subsequent sorting and single-cell sequencing. Given this, it is imperative to develop an enzyme kit for dispersing biological tissue and to develop a method for separating tissue cells of high quality to obtain a single-cell suspension with a high survival and low aggregation rate.

SUALMARY

100041 An objective of the present disclosure is to provide an enzyme kit for dispersing a biological tissue and a method for obtaining a single-cell suspension using the same. The single-cell suspension obtained by the method has a high survival rate and a low aggregation rate, conducive to subsequent sorting and single-cell sequencing.

100051 To achieve the above purpose, the present disclosure provides an enzyme kit for dispersing, a biological tissue, including a first reagent and a second reagent, wherein the first reagent includes pronase" and the second reagent includes papain.

100061 in some embodiments, the biological tissue may be preferably a mammalian brain tissue or a mammalian cochlear vestibular tissue.

100071 Based on the beneficial effects of further reducing intercellular adhesion and promoting digestion of the biological tissue, in some embodiments, the first reagent may further include DNase and/or bovine serum albumin BSA); and the DNase is preferably DNase I. 10008] Based on the beneficial effects of further reducing intercellular adhesion and promoting digestion of the biological tissue, in some embodiments, the second reagent may further include the DNase and/or the BSA; and the DNase is preferably the DNase 1.

100091 In sonic embodiments, the first reagent may iiirther include a HAG medium.

100101 In some embodiments, the second reagent may further include the HAG medium, 100111 In some ernbodiments, the first reagent may include the HAG medium, the DNase 10% BSA and pronase; and the second reagent may include the HAG medium, the DNase, the 10% BSA and papain.

100121 In some embodiments, per milliliter of the first reagent may include: 10013] DNase (0.5-1.4) VA' % (0.9), 100141 10% BSA (5.0-14.0) VA' 8b (9), 100151 promise (0.5-5.0) mgtml_..

100161 In some embodiments, the first reagent may include the HAG medium, the DNase" the 10% BSA and the promise.

100171 In some embodiments, per milliliter of the econc reagent may include: 100181 DNase (0.5-1.4) Vilit"N" 10019] 10% BSA (5.0-14.0) 'WV °)J, 10020] papain (0.5-5.0) mg/m1_,.

100211 In some embodiments, per milliliter of the first reagent mayinclude: 100221 DNase (0.8-1.0) WV °A) (0.9), 100231 10% BSA (8.0-10.0) WV % (9), 100241 pronase mg/min. $

[0025] In some embodiments, per milliliter of the second reagent may include: [0026] DNase (0.8-1.0) VN % (0 9), 100271 10% BSA (8.0-10.0) VAT % (9), 100281 papain ( 1-3) m &MTh.

[0029] Specifically, in some embodiments, the enzyme kit may be prepared by the following method: [0030] the first reagent, adding 100 ul of the 10% BSA, 10 ul of DNase, and 1-3 mg of pronase into each milliliter of the HAG medium; and [0031] the second reagent: adding 100 ill of 10% BSA, 10 pl of DNase, and 1-3 ing of papain into each milliliter of the HAG medium.

100321 A method for obtaining a single-cell suspension using the enzyme above-mentioned, which includes the following steps: [0033] (1) dividing tissue fragments to be separated into two parts; digesting one part with the first reagent to obtain a first digested solution and digesting the other part with the second o obtain a second digested solution; and 100341 (2) mixing the first digested solution and the second digested solution obtained in step (1) in the same HAG medium, dispersing, and collecting a supernatant to obtain a single-cell suspension.

[0035] Based on the beneficial effects of further removing cell debris from the cell suspension and obtaining a purer single-cell suspension, in some embodiments, the method may further include step (3) after step (2): filtering the supernatant obtained in step (2) through a cell strainer to obtain a filtrate.

10036.1 Based on the beneficial effects of further isolating single cells and obtaining, a purer single-cell suspension, in some embodiments, the method may further include step (4) after step (3): performing gradient centrifugation on the filtrate obtained in step (3), collecting cells in a lower layer, and mixing the cells in a liquid medium-well.

100371 In some embodiments, the method may further include step (5) after step (4): filtering the cells mix.ed well in the liquid medium in step (4) through a cell strainer, collecting a filtrate, centrifuging to discard supernatant, and mixing obtained cells in the liquid medium. [0038] Based on the beneficial effects of fully digesting the fragments of a tissue, in some embodiments, in step (1), 0.03 g to 0.1 g of the fragments of tissue may be preferably added per milliliter of the first reagent; [0039] based on the beneficial effects of fully digesting the fragments of a tissue, in some embodiments, in step (1), 0.03 g to 0.1 g of the fragments of tissue may be preferably added per milliliter of the second reagent; 11)(1401 in some embodiments, in step (1), the digesting may be conducted preferably on a constant-temperature shaker; 100411 based on the beneficial effects of hilly digesting the fragments of a tissue, in some embodiments, in step (1), the digesting may be conducted for preferably 15 min to 30 min; 100421 in sortie embodiments, in step (2), a volume ratio of the total volume of the first digested solution and the second digested solution to a volume of the HAG medium may preferably be I:(0.7-2), I:1; 100431 in some embodiments, in step (2), the dispersing may be conducted by preferably pipetting the cells with an air syringe, for example, pipetting the cells several times with a 1 mL pipette until the cells are dispersed; [0044] in some embodiments, in step (3), the supernatrmt may be filtered twice through cell strainers of 70 um and 40 pm in sequence; 10045] in some embodiments, in step (4), the gradient centrifligation may he conducted at preferably 600 g to 1,000g. for example, 800 g; 100461 in some embodiments, in step (4), the gradient centrifugation may be conducted for preferably 10 min to 20 min, for example, 15 min. [0047] Based on the beneficial effect of further removing cell debris from the cell suspension and sufficiently retaining a desired number and viability of single cells in the single-cell suspension, in some embodiments, in step (5), the cells may be filtered three times through cell strainers of 70 pm, 40 um, and 10 um in sequence. After a detailed experimental study, it is found that finer cell debris in the cell suspension cannot be sufficiently removed only through the cell strainers of 70 um and 40 um in sequence; and the number of single cells required in the cell suspension is greatly reduced if a pore size of the cell strainer is less than 10 urn 100481 In some embodiments, the cencrifugation may be conducted preferably more than one time, more preferably two times.

[0049] On the basis of conforming to common knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred embodiments of the present disclosure, [0050] The reagents and raw materials used in the present disclosure are all commercially available.

[0051] Compared with the prior art, the s-nt disclosure has at least the following advantages:

S

100521 (1) In the present disclosure, the enzyme kit for dispersing a biological tissue has the advantages of simple components" convenient preparation. It can be used to digest various tissues to obtain a single cell suspension with a high survival rate and a low aggregation rare. [0053] (2) The enzyme kit for dispersing a biological tissue can be used for preparing a single cell suspension of difficult-to-disperse brain tissues.

[0054] (3) The method for obtaining a single cell suspensto a obtain a single cell suspension with a higher survival rate and a lower aggregation which is conducive to subsequent sorting and single-cell sequencing.

[0055] (4) A single-cell suspension with less cell debris, an extremely high survival rate of nerve cells, and an extremely low aggregation rate can be obtained by using the method for preparing a single cell suspension from difficult-to-disperse brain tissues.

BRIEF DESCRIPTION OF THE DRAW INGS

[0056] One or more examples are exemplified by the accompanying drawings, and these exemplified descriptions do not to be constituted limiting the examples.

[0057) FIG. 1 is a schematic diagram showing a microscopic examination result of a cell suspension obtained in Example 2 of the present disclosure; [0058] FIG. 2 is a schematic diagram showing a microscopic examination -suit of a cell suspension obtained in Example 3 of the present disclosure; [0059] FiG. 3 is a schematic diagram showing a microscopic examination result of a cell suspension obtained in Example 4 of the present disclosure; [0060] FIG. 4 is a schematic diagram showing a microscopic examination result of a -ell suspension obtained in Comparative Example 1 of the present disclosure; [0061] FIG. 5 is a schematic diagram showing a microscopic examination esult of a cell suspension obtained in Comparative Example 2 of the present disclosure; [0062] FIG. 6 is a schematic diagram showing a microscopic examination result of a cell suspension obtained in Comparative Example 3 of the present disclosure; [0063] FIG. 7 is a schematic diagram showing a microscopic examination result of a cell suspension obtained in Comparative Example 4 of the present disclosure; [0064] FIG. 8 is a schematic diagram showing a microscopic examination result of a cell suspension obtained in Comparative Example 5 of the present disclosure; [0065] FiG. 9 is a schematic diagram showing a microscopic examination result of a cell suspension obtained in Comparative Example 6 of the present disclosure; [0066] FIG. 10. is a schematic diagram showing a microscopic examination result of a cell suspension obtained in Comparative Example 7 of the present disclosure; [0067] FIG. H is a schematic diagram showing a microscopic examination result of a cell suspension obtained in Comparative Example 8 of the present disclosure; [0068] FIG. 12 is a schematic diagram showing a microscopic examination result of a cell suspension obtained in Comparative Example 9 of the present disclosure; [0069] FIG. 13 is a schematic diagram showing a NeuN immunotluorescmce staining result of the cell suspension obtained in Example 2 of the present disclosure; [0070] FIG. 14 is a schematic diagram showing a NeuN immunofluorescence staining result of the cell suspension obtained in Example 3 of the present disclosure; and 100711 FIG. 15 is a schematic: diagram showing a NeuN immunothiorescence staining result of the cell suspension obtained in Comparative Example 9 of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0072] In order to make the objectives, technical solutions and advantages of the examples of the present disclosure Clearer, the embodiments of the present disclosure are described in detail 'below regarding the examples. Those of ordinary skill in the art should understand that many technical details are proposed in the examples of the present disclosure to make the present application better understood. However, even without these technical details and various changes and modifications made based on the following examples, the technical solutions claimed in the present disclosure may still he realized.

[0073] In the present disclosure, through a large number of experimental studies, it is found that, compared with the prior art, an enzyme kit including independently a pronase reagent and a papain reagent can disperse a biological tissue (such as brain tissue) in a higher quality to obtain a single cell suspension with higher survival rate and lower clustering rate, which is helpful for subsequent cell sorting and single-cell sequencing.

[0074] Example 1 Preparation of an enzyme kit 1.0075_1 In this example, a Hibernate A medium was purchased from Gibco, with an item number of A1247501; [0076] a GlutaMAX-1 (100x) was purchased from Elbe') with an item number of 35050-061; 100771 an Actinomycin D was purchased from APExBIO, with an item number of A4448; [0078] 10% BSA was purchased from Amreseo, with an item number of 9048-46-8; [0079] DNase I was purchased from Sigma-Aldrich with an item number of D5025; [0080] pronase was purchased from Sigma-Aldrich, with an hem number of P69 11-10; and 100811 papain was purchased from Worthington with an item number of kS003126.

[0082] (1) Preparation of a HAG medium: 75 pl of the GlutaMAX-1 (1 00x), 3.3 pi of 45, Actinomycin D were added to 30 ml of the Hibernate A medium to obtain a HAG medium. [0083] (2) Preparation of a first reagent: I ml of the HAG medium was measured and mixed well with 100 pi of the 10% BSA, 10 pi of the DNase I and 1-3 mg of the pronase to obtain the first reagent, and [0084] preparation of a second reagent: 1 ni of medium was measured and mixed well with 100 pl of the 10% BSA, 10 pi of the DINiase I and 10-50 pi of the papain (42 mg.P/ini) to obtain the second reagent.

100851 Example 2 Preparation of a single cell suspension of mouse prefrontal cortex tissue cells [0086] (1) 0.03-0.1 g of mouse prefrontal cortex tissue was chopped up and divided into two equal parts, where one part was placed in the first reagent, and the other part was placed in second reagent; tissue blocks were gently crushed by tweezers" and placed on a constant-temperature shaker to digest at 37°C and 200 rpm/min for 15 min to 30 min. [0087] (2) Two tubes of digested tissue fluid were added into the same HAG medium (2 ml), the cells were pipetted 12 times with a 1 ml pipette tip and allowed to stand for 2 nun; supernatant was collected and placed in a. new centrifuge tube, and 2 ml. of the HAG medium was added to the original tube; the above steps were repeated twice to collect a cell suspension.

100881 (3) The collected cell suspension was filtered with sterile cell strainers of 70 pm and 40 tim in sequence, and a filtrate was collected.

100891 (4) The collected filtrate was performed gradient centrifugation at 800 g for 15 mm, and fragments at an upper layer were removed after the centrifugation.

[0090] (5) Cells at a lower layer were collected into 5 mi of the 1% BSA and mixed well; the cells were filtered through sterile cell strainers of 70 pm, 40 pin and 10 pm, respectively; collected filtrate was centrifuged at 200 g for 5 min, supernatant was discarded, and the cells were collected.

[0091] (6) 1 ml of the 1% BSA was added to the collected cells, pipetted gently, the mixture was centrifuged at 200 g for 5 min, and a supernatant was discarded; 50 pi of the 1% BSA was added to resuspend the cells to obtain the cell suspension.

100921 Example 3 Preparation of a single cell suspension of partial temporal lobe tissue cells or ad tilts [0093] (1) 0.03-0.1 g of partial temporal lobe tissue of adults was chopped up and di e into two equal pans where one part was placed in the first reagent, and the other part was placed in the second reagent; tissue blocks were gently crushed by tweezers, and placed on a constant-temperature shaker to digest at 37°C and 200 rpm/min for 15 min to 30 min. 100941 (2) Two tubes of digested tissue fluid were added into the same HAG medium raft the cells were pipetted 12 times with a 1 ml pipette tip and allowed to stand for 2 min, a supernatant was collected and placed in a new centrifuge tube, and 2 ml of the HAG medium was added to the original tube; the above steps were repeated twice to collect a cell suspension.

100951 (3) The collected cell suspension was filtered with sterile cell strainers of 70 pm and 40 pm in sequence, and a filtrate was collected 10096] (4) The collected filtrate was performed gradient ce rifiagation at 800 g for 15 min, and fragments at an upper layer were removed after the centrifugation.

10097] (5) Cells at a lower layer were collected into 5 ml of the 1% BSA and mixed well; the cells were filtered through sterile cell strainers of 70 prm 40 pm and 10 tun, respectively; a collected filtrate was centrifuged at 200 g for 5 min, supernatant was discarded, and the cells were collected.

100981 (6) 1 ml of the 1% :BSA was added to the collected cells, pipetted gently, the mixture was centrifuged at 200 g for 5 min, and a supernatant was discarded; 50 pl of the 1% BSA was added to resuspend the cells to obtain the cell suspension.

100991 Example 4 Preparation of a single cell suspension of cochlear vestibular cells 1001001 (1) 0.03-0.1 g of cochlear vestibular tissue was chopped up and divided into two equal pans, where one pan was placed in the first reagent, and the other part was placed in the second reagent; tissue blocks were gently crushed by tweezers, and placed on a constant-temperature shaker to digest at 37°C and 200 rpm/min flir 15 min to 30 min. 100101] (2) Two tubes of digested tissue fluid were added into the same HAG medium (2 ml); the cells were pipetted 12 limes with a 1 ml pipette tip and allowed to stand for 2 min; a supernatant was collected and placed in a new centrifuge tube; and 2 ml of the HAG medium was added to the original tube; the above steps were repeated twice to collect a cell suspension.

1001021 (3) The collected cell suspension was filtered with sterile cell strainers of 70 pm and 40 pm in sequence; and a filtrate was collected.

1001031 (4) The collected filtrate was performed to gradient centrifugation at $OO g for 15 min, and fragments at a.n upper layer were removed after the centrifugation.

100104_1 (5) Cells at a lower layer were collected into 5 ml of the 1% BSA and mixed well; the cells were filtered -through sterile cell strainers of /0 pm, 40 um and 10 pm, respectively; a collected filtrate was centrifuged at 200 g for 5 min, supernatant was discarded, and the cells were collected_ [00105] (6) 1 ml of the 1% BSA was added to the collected cells, pipetted gently, the mixture was centrifuged at 200 g for 5 min, and a supernatant was discarded; 50 pi of the 1% BSA was added to resuspend the cells to obtain the cell suspension.

[00106] Comparative Example 1 Digestion of brain tissue cells by chondroitinase [00107] 0.03-0.1 g of mouse prefrontal cortex tissue from the same source as in Example 2 was chopped up and put into a chondroititaase solution (2 units/m0 for digestion, followed by conducting lysis fully on a constant-temperature shaker at 37°C and 200 rpinimin for 30 mm; the brain tissue cells were performed density gradient centrifugation and filtration in the same manner as that in Example 2 to remove fragments and clumps; the cells were washed twice in the same manner as that in Example 2, and then the cells were resuspended to obtain a cell suspension.

1001081 Comparative Example 2 Digestion of brain tissue cells by pronase 10010910.03-0. I g of mouse prefrontal cortex tissue from the same source as in Example 2 was chopped up and put into a. promise solution (1. ingimi to 3 mg/m1) for digestion, followed by conducting lysis fully on a constant-temperature shaker at 37°C and 200 rprnlinin for 30 min; the brain tissue cells were performed density gradient centrifugation and filtration in the same manner as that in Example 2 to remove fragments and clumps; the cells were washed twice in the same manner as that in Example 2, and then the cells were resuspended to obtain a cell suspension.

1001101Com para five Example 3 Digestion of brain tissue cells by papain [0011110.03-0.1 g of mouse prefrontal cortex tissue from the same source as in Example 2 was chopped up and put into a papain solution (10 utl 42 mgP/m1 to 50 pl 42 mgP/m1) for digestion, followed by conducting lysis fully on a constant-temperature shaker at 37°C and 200 rpm/min for 30 min; the brain tissue cells were performed density gradient centrifugation and filtration in the same manner as that in Example 2 to remove fragments and clumps; the cells were washed twice in the same manner as that in Example 2, and then the cells were resuspended to obtain a cell suspension.

1001121 Comparative Example 4 Digestion of brain tissue cells by hyaluronidase 10011310.03-0.1 g of mouse prefrontal cortex tissue from the same source as in Example was chopped up and put into a hyaluronidase solution (1 mg/mi to 3 mg/ml) for digestion, followed by conducting lysis fully on a constant-temperature shaker at 37°C and 200 rpm/trin for 30 it he brain tissue cells were performed density gradient centrifugation and filtration in the same manner as that in Example 2 to remove fragments and clumps; the cells were washed twice in a same manner as that in Example 2, and then the cells were resuspended to obtain a cell suspension.

100114Womparative Example 5 Digestion of brain tissue cells by a mixture of pronase and impala 10011510.03-0.1 g of mouse prefrontal cortex tissue from the same source as in Example 2 was chopped up and put into a mixture of pronase and papain (including I mg/m1 to 3 mg/m1 of the pronase and 10 pl 42 mgP/mi to 50 Ed 42 mgP/m1 of the papain) for digestion, followed by conducting lysis fully on a constant-temperature shaker at 37°C and 200 rprnimin for 30 min; the brain tissue cells were performed density gradient centrifugation and filtration in the same mariner as that in Example 2 to remove fragments and clumps; the cells were washed twice in the same manner-as that in Example 2, and then the cells were resuspended to obtain a cell suspension.

1001161Comparative Example 6 Digestion of brain tissue cells by a tissue dissociation solution from GEXCOPE 10011710.03-0.1 g of mouse prefrontal cortex tissue from the same source as in Example 2 was chopped up and put into a tissue dissociation solution from GEXCOPE for digestion, followed by conducting lysis fully on a constant-temperature shaker at 37°C and 200 rpinimin for 30 min; the brain tissue cells were performed density gradient centrifugation and filtration in the same manner as that in Example 2 to remove fragments and clumps; the cells were washed twice in the same manner as that in Example 2, and then the cells were resuspended to obtain a cell suspension.

100118I Comparative Example? Filtration without a 10 pm sterile cell strainer [00119] A single-cell suspension of mouse prefrontal cortex tissue in Comparative Example 7 was obtained by steps roughly the same as those in Example 2, except that: in step (5), the cells were filtered through sterile cell strainers of only 70 tun and 40 urn, without a sterile cell strainer of 10 tun.

100120] Comparative Example 8 Digestion of brain tissue cells by Hibernate-Ca ± papain [00121]0.03-0.1 g of mouse prefrontal cortex tissue from the same source as in Example 2 was chopped up and placed in a liquid medium, and then the cells were fully rinsed with 0.2 ml of warm HA-Ca. The cells were separated by centrifugation at 200 g at 22°C (at 1100 rpm in a bucket centrifuge), supernatant was discarded, and the tube was flicked to disperse cell pellets. The cells were resuspended in 0.2 ml of Hibernate-Ca a-papain. The resuspended cells were added to an original medium to which papain was added. The cells were incubated at an ambient temperature of 37°C in CO2 (not 5%) for 10 min. The cells were Opened 12 times using a -1 nil pipette tip; after allowing standing for 2 mim supernatant was collected into a new centrifage tube to resuspend the cells to obtain a cell suspension.

1001221Comparative Example 9 Digestion of brain tissue cells by pronase and.Ityaluronidase independently 100123] A single-cell suspension of a mouse prefrontal cortex tissue in Comparative Example 9 was obtained by steps roughly the same as those in Example 2, except that: in step enzyme added in the second reagent is hyaluronidase instead of papain.

1001241Test Example 1 Test of cell viability and aggregation rate of a cell suspension of brain tissues [00125]The cell suspensions of mouse prefrontal cortex tissue obtainer in Example Example 3, Comparative Example I, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 7, and Comparative Example 8 were respectively added with trypan blue staining solution and mixed; and the cell viability and aggregation rate of the prefrontal cortex of mice were counted through microscopic examination with a hernocytometer. The microscopic examination results of each example and comparative example are shown in Table I. 1001261Table Groin Celt viability l Example 2 95% A enation rate 1.Act!!Y.!!!RL.

I

<10% FIG. I i

Example 3 96% 1

Example 4 98% <10% FIG, 3 Comparative Example 1 34.2% 9.9% 1 FIG. 4 c 7 lp c 67.5% 1 _____Pompara. I 9.2% 1 JIGS 1 Comparative Example 3 69.1% 1 17.8% FIG-. 6 Comparative Example 4 46% ) FIG. 7 1 ComparatVetxample 5 50% 50% 1 FIG._8__ Comparative Example 6 69.5% I 23.4% FIG-. 9 i Comparative Example? 64% 23.5% FIG. 10

_______ _________

___ 1.11%______ ____ j_ FIG 11 _____Compa i* e Example __ _ ____ ________ ____ % 10% FIG 12.

1001281Test Example 2 NetEN iimminofluoreseenee staining experiment 100129] The cell suspensions obtained in Example 2, Example 3 and Comparative Example 9 were subjected to Neuron marker NeuN immunotluorescence staining experiment and a ratio of neurons in the cell suspension was tested (with a scale bar of 20 um, 561 nm channel-labelled Neurons, and 405 nm channel-labelled nuclei). The results are shown in Table 2, FIG. 13, FIG. 14 and FIG 15.

10013211n neurobiology, neurons have a low survival rate during separation due to extreme fragility. Therefore, counting the ratio of neurons to the total number of cells can further prove that the cells obtained by obtaining a single cell suspension of the present disclosure have a high survival rate and can be used to separate fragile neurons.

100133111 can be seen from the table that the number of neurons accounts for less than 20% in the single-cell suspension of the mouse prefrontal cortex tissue obtained by the method in Comparative Example 9. In comparison, the number of neurons is not less than 40% in the single-cell suspension of mouse prefrontal cortex tissue obtained by the method of the present-disclosure (Example 2), with neuron viability increased by more than I time. When the method of the present disclosure is used to prepare a single cell suspension of human brain tissues, the neurons account for more than 70% of the total cells. This proves that the method for obtaining a single cell suspension of the present disclosure causes less damage to fragile cells, especially to nerve cells.

1001341 It will be understood by those skilled in the art that the above embodiments are specific examples for implementing the present application. In practical applications, various changes may be made to the above implementations in terms of form and details without departing from the spirit and scope of the present application.

100130] Table 2 Group

A ratio of the number of neurons to the total Schematic drawings of

____ ____

number of cells stainingtresults FIG, 13 FIG 14 FIG. 15 40.1%

Example 2

--------__

Comparative Example 9 71.74%

Claims

WHAT IS CLAIMED IS: 1.. An enzyme kit for dispersing a biological tissue, comprising a first reagent and a second reagent, wherein the first reagent comprises pronase, and the second reagent comprises papain.
2. The enzyme kit according to claim 1 wherein the first reagent further comprises DNase and/or bovine serum albumin (BSA); the DNase is preferably DNase I; and/or the second reagent further comprises DNase and/or BSA.
3. The enzyme kit according I_ wherein the first reagent further comprises a HAG medium; and/or the second reagent further cornpnses a HAG medium.
4. The enzyme kit according to claim 1, wherein per milliliter of the first reagent comprises: DNasc (0.5-1.4) WV 43- A 10% BSA pronase (5.0-14.0) VP1/4," (9), (0.5-5.0) ing/m.L. and/or per milliliter of the second reagent comprises: DNase (0.5-1.4) V/V 91), 10% BSA papal n (5.0-14.0)5,7V %, (0.5-5.0) mg/nil-
5. A method for obtaining a single-cell suspension using the enzyme kit according to ally one of claims 1 to 4, comprising the following steps: (I) dividing tissue fragments to be separated into two parts; digesting one part with the first reagent to obtain a first digested solution and digesting the other part with the second reagent to obtain a second digested solution; and (2) mixing the first digested solution and the second digested solution obtained p (1) in a same HAG medium, dispersing and collecting a supernatant to obtain a sin cell suspension.
6. The method for obtaining a single cell suspension according to claim 5, further comprising step (3) after step (2) filtering the supernatant obtained in step (2) through a cell strainer to obtain a filtrate.
T The method for obtaining a single cell suspension according to claim 6, fUrther comprising step (4) after step (3): perfol ruing gradient centrifitgation on die filtrate obtained in step (3), collecting cells in a lower layer, and mixing the cells in a liquid medium-welt
8. The method for obtaining a single cell suspension according to claim 7, further comprising step (5) after step (4): filtering the cells mixed well in the liquid medium in step (4) through a cell strainer, collecting a filtrate, centrifuging to discard a supernatant, and mixing obtained cells in the liquid medium.
9. The method for obtaining a single cell suspension accorcung to claim 5, where step (1), 0.03 g to 0.1 g of the fragments of a tissue are added to per milliliter of the first 1, and/or 0.03 g to 0.1 g of the fragments of a tissue are added to per milliliter of the second reagent; and/or the digesting is conducted on a constant-temperature shaker; and/or the digesting is conducted for 15 min to 30 min; and/or in step (2), volume ratio of a total volume of the first digested solution and the second digested solution to a volume of the HAG medium is 1:(0.7-2); and/or in step (2), the dispersing is conducted by pipetting the cells with an air syringe.
10. The method for obtaining a single cell suspension according to claim 8, wherein in step (5), the cells are filtered three times through cell strainers of 70 pm, 40 pm, and 10 Rm in sequence; and/or the centhfugation is conducted more than one time.