Digestive juice, application of digestive juice, ligament tissue cell suspension and preparation method of ligament tissue cell suspension

Abstract

The invention relates to the technical field of biological cells, and discloses digestive juice and a preparation method and application thereof as well as cell suspension and a preparation method thereof. The digestive juice contains I-type collagenase, II-type collagenase, III-type collagenase, IV-type collagenase, hyaluronidase, elastase, neutral protease and deoxyribonuclease. The cell suspension prepared from the digestive juice has the advantages of high cell viability and large number of living cells, and the method has the advantages of high preparation speed and high efficiency.

Description

Technical Field

[0001] This invention relates to the field of biological cell technology, specifically to a digestive fluid and its application, and a ligament tissue cell suspension and its preparation method. Background Technology

[0002] In recent years, various single-cell analysis technologies have developed rapidly. Compared with traditional analysis methods, such as bulk RNA-seq, the advantage of single-cell analysis technology is that it can elucidate the heterogeneity of the genome, epigenome and transcriptome in the cell population, as well as the changes in these levels, rather than simply measuring the average transcript expression in a mixture of cells.

[0003] For single-cell analysis, the preparation of single-cell suspensions is the most crucial preliminary step, as any stress or damage can affect cell number and viability. A decrease in cell number may lead to the loss of rare cell types, while reduced cell viability will result in fewer captured live cells, thus adversely affecting the results. Due to the significant differences between different tissues and organoids, each type requires specific processing methods and conditions to obtain sufficiently effective single-cell suspensions.

[0004] Ligaments are important tissues in animals and have significant clinical research value. The human body has over 900 ligaments, most of which are located in the limbs. These ligaments function to stabilize joints and internal organs, connect bones and organs, and protect sensitive tissues such as blood vessels and glands. Currently, many questions remain regarding ligaments, and single-cell analysis, as an emerging technology, can advance related research. However, current methods for preparing ligament single-cell suspensions are not very effective and suffer from problems such as excessive time consumption, high cost, and the need for specific tissue dissociators.

[0005] CN108504627A discloses a method for extracting human ligamentum flavum stem cells. The method includes: 1) taking human ligamentum flavum tissue, rinsing, and mincing; 2) digesting the minced tissue with type I collagenase; 3) collecting the digested cells to obtain a single-cell suspension; 4) culturing the cells and passaged three times; and 5) screening the stem cells with a 10 μg / ml fibronectin solution. This method yields a large quantity of ligamentum flavum stem cells with high survival rates. However, the processing time is long, especially the digestion process, which may cause significant gene stress expression in the cells.

[0006] Therefore, developing a rapid and efficient method for preparing ligament single-cell suspensions with a large number of cells and high cell viability is crucial for the application of single-cell analysis in ligament tissues and will greatly assist current ligament-related research. Summary of the Invention

[0007] The purpose of this invention is to solve the problems of low cell viability, small number of live cells, and slow and inefficient preparation methods of single-cell suspensions in existing technologies.

[0008] To achieve the above objectives, a first aspect of the present invention provides a digestive solution for obtaining ligament cells, the digestive solution containing type I collagenase, type II collagenase, type III collagenase, type IV collagenase, hyaluronidase, elastase, neutral protease, and deoxyribonuclease.

[0009] Relative to 100 parts by weight of the type I collagenase, the content of the type II collagenase is 50-200 parts by weight, the content of the type III collagenase is 50-200 parts by weight, the content of the type IV collagenase is 50-200 parts by weight, the content of the hyaluronidase is 50-200 parts by weight, the content of the elastase is 50-200 parts by weight, the content of the neutral protease is 50-200 parts by weight, and the content of the deoxyribonuclease is 2.5-15 parts by weight.

[0010] A second aspect of the invention provides the application of the digestive fluid described in the first aspect for obtaining ligament cells in single-cell analysis.

[0011] A third aspect of the present invention provides a method for preparing a ligament tissue cell suspension, the method comprising:

[0012] (1) The ligament tissue was pretreated with pancreatic enzyme solution to obtain intermediate I;

[0013] (2) The intermediate I is digested with digestion liquid to obtain material I. The material I is then filtered and centrifuged in sequence to obtain the first cell precipitate.

[0014] (3) The first cell precipitate is subjected to a first resuspension treatment with the resuspension solution to obtain material II. Material II is subjected to red blood cell lysis treatment and second centrifugation treatment in sequence to obtain the second cell precipitate.

[0015] (4) The second cell precipitate is subjected to a second resuspension treatment with the resuspension to obtain a ligament tissue cell suspension;

[0016] In step (2), the digestive fluid is the digestive fluid for obtaining ligament cells as described in the first aspect.

[0017] A fourth aspect of the invention provides a ligament tissue cell suspension prepared by the method described in the third aspect.

[0018] Compared with the prior art, the present invention has at least the following advantages:

[0019] (1) The ligament tissue cell suspension prepared by the digestive fluid of the present invention has the advantages of high cell viability and a large number of live cells.

[0020] (2) The method for preparing ligament tissue cell suspension described in this invention has the advantages of being fast and efficient. Attached Figure Description

[0021] Figure 1 This is Embodiment 1 of the present invention. Figure 1 a) and Comparative Example 1 Figure 1 Scatter plot of flow cytometry apoptosis results of ligament tissue cell suspension prepared in b) of the diagram;

[0022] Figure 2 The results of AOPI staining and detection of the ligament tissue cell suspension prepared in Example 1 of the present invention on a CountStar cell counter are shown. Figure 2 In the image, 'a' represents a bright-field image (showing all objects in the suspension, including all cells and impurities) obtained from Example 1 after AOPI staining, detected using a CountStar cell counter. Figure 2 b in the image shows the fluorescence field of the cell suspension from Example 1 after AOPI staining, detected on a CountStar cell counter (showing all cells, with live cells showing green fluorescence and dead cells showing red fluorescence).

[0023] Figure 3 The results of AOPI staining and detection of the ligament tissue cell suspension prepared in Comparative Example 3 of this invention were obtained using a CountStar cell counter. Figure 3 In the image, 'a' represents the bright field image of the cell suspension detection results from Comparative Example 3. Figure 3 b in the figure represents the fluorescence field results of the cell suspension in Comparative Example 3;

[0024] Figure 4 The results of AOPI staining and detection of the ligament tissue cell suspension prepared in Example 2 of the present invention on a CountStar cell counter are shown. Figure 4 In the image, 'a' represents a bright-field image of the cell suspension detection results from Example 2. Figure 4 b in the figure represents the fluorescence field result of the cell suspension in Example 2;

[0025] Figure 5 The results of AOPI staining and detection of the ligament tissue cell suspension prepared in Example 3 of the present invention were obtained using a CountStar cell counter. Figure 5 In the image, 'a' represents a bright-field image of the cell suspension detection results from Example 3. Figure 5 b in the figure represents the fluorescence field results of the cell suspension in Example 3. Detailed Implementation

[0026] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0027] It should be noted that, in all aspects of the present invention, the same components in each aspect are described only once in one aspect and not repeatedly, and those skilled in the art should not understand this as a limitation of the present invention.

[0028] In this invention, FBS is fetal bovine serum, and PBS buffer is phosphate buffer solution.

[0029] As previously described, a first aspect of the present invention provides a digestive fluid for obtaining ligament cells, the digestive fluid containing type I collagenase, type II collagenase, type III collagenase, type IV collagenase, hyaluronidase, elastase, neutral protease and deoxyribonuclease.

[0030] Relative to 100 parts by weight of the type I collagenase, the content of the type II collagenase is 50-200 parts by weight, the content of the type III collagenase is 50-200 parts by weight, the content of the type IV collagenase is 50-200 parts by weight, the content of the hyaluronidase is 50-200 parts by weight, the content of the elastase is 50-200 parts by weight, the content of the neutral protease is 50-200 parts by weight, and the content of the deoxyribonuclease is 2.5-15 parts by weight.

[0031] The type II collagenase content mentioned in this invention refers to the content of type II collagenase on a dry basis, excluding any water content that may be present. Those skilled in the art can prepare type II collagenase solutions of different concentrations according to actual needs. Type I, type III, type IV collagenase, hyaluronidase, elastase, neutral protease, and deoxyribonuclease have similar definitions, that is, all refer to the content on a dry basis. These definitions will not be elaborated upon here, and those skilled in the art should not interpret them as limitations on this invention.

[0032] Preferably, the digestion solution also contains DMEM culture medium and FBS.

[0033] More preferably, in the digestive solution, the concentrations of type I collagenase, type II collagenase, type III collagenase, type IV collagenase, hyaluronidase, elastase, neutral protease, and deoxyribonuclease are 1-2 mg / mL and 0.05-0.15 mg / mL, respectively. The inventors of this invention have found that, under this preferred condition, the ligament tissue cell suspension of this invention has a higher cell viability and a greater number of viable cells.

[0034] In a preferred embodiment, the concentration of FBS in the digestion solution is 4.5-5.5 vol%, and the concentration of CaCl2 is 0.4-0.7 mg / mL. The inventors of this invention have discovered that, in this preferred embodiment, the ligament tissue cell suspension of this invention exhibits higher cell viability and a greater number of viable cells.

[0035] As previously stated, a second aspect of the present invention provides the application of the digestive fluid described in the first aspect in single-cell analysis.

[0036] As previously described, a third aspect of the present invention provides a method for preparing a ligament tissue cell suspension, the method comprising:

[0037] (1) The ligament tissue was pretreated with pancreatic enzyme solution to obtain intermediate I;

[0038] (2) The intermediate I is digested with digestion liquid to obtain material I. The material I is then filtered and centrifuged in sequence to obtain the first cell precipitate.

[0039] (3) The first cell precipitate is subjected to a first resuspension treatment with the resuspension solution to obtain material II. Material II is subjected to red blood cell lysis treatment and second centrifugation treatment in sequence to obtain the second cell precipitate.

[0040] (4) The second cell precipitate is subjected to a second resuspension treatment with the resuspension solution to obtain the ligament tissue cell suspension;

[0041] In step (2), the digestive fluid is the digestive fluid for obtaining ligament cells as described in the first aspect.

[0042] According to a particularly preferred embodiment, the method further includes: before performing step (1), cleaning and cutting the ligament tissue; and then pretreating the material obtained after cleaning and cutting with the pancreatic enzyme solution.

[0043] Preferably, the washing conditions include washing the ligament tissue in PBS buffer with a volume content of 4-6 vol% FBS.

[0044] Preferably, the shearing process includes: cutting the cleaned ligament tissue into pieces with a volume of 1-3 mm. 3 Fragments of ligament tissue.

[0045] Preferably, in step (1), the amount of pancreatic enzyme solution used is 3-10 μL relative to 1 mg of the ligament tissue.

[0046] Preferably, in step (1), the concentration of the pancreatic enzyme solution is 0.2wt%-0.3wt%.

[0047] Preferably, in step (2), the amount of digestion solution used is 10-50 μL relative to 1 mg of intermediate I on a dry basis.

[0048] Preferably, in step (2), the amount of type I collagenase used is such that the initial concentration of type I collagenase in the digestive solution is 1-2 mg / mL. The inventors of this invention have found that, under this preferred condition, the ligament tissue cell suspension of this invention has a higher cell viability and a greater number of live cells.

[0049] According to a particularly preferred embodiment, in step (2), the initial concentration of the type I collagenase is 1-2 mg / mL, the initial concentration of the type II collagenase is 1-2 mg / mL, the initial concentration of the type III collagenase is 1-2 mg / mL, the initial concentration of the type IV collagenase is 1-2 mg / mL, the initial concentration of the hyaluronidase is 1-2 mg / mL, the initial concentration of the elastase is 1-2 mg / mL, the initial concentration of the neutral protease is 1-2 mg / mL, and the initial concentration of the deoxyribonuclease is 0.05-0.15 mg / mL. The inventors of this invention have found that, under this preferred embodiment, the ligament tissue cell suspension of this invention has a higher cell viability and a greater number of viable cells.

[0050] More preferably, in the digestive solution, the initial concentration of FBS is 4.5-5.5 vol%, and the initial concentration of CaCl2 is 0.4-0.7 mg / mL. The inventors of this invention have discovered that, under this preferred condition, the ligament tissue cell suspension of this invention has a higher cell viability and a greater number of live cells.

[0051] According to a preferred embodiment, the method further includes: in step (2), the intermediate I is first sheared into a homogenate, and then digested with the digestion liquid to obtain the material I.

[0052] According to a preferred embodiment, in step (3), the amount of the resuspension is 0.5-1.5 mL relative to 1 mg of the first cell precipitate.

[0053] More preferably, in step (4), the amount of the resuspension is 0.5-1.5 mL relative to 1 mg of the second cell precipitate.

[0054] Preferably, the resuspension is a PBS buffer with a volume content of 4-6 vol% FBS.

[0055] Preferably, in step (1), the pretreatment conditions include: a temperature of 36-38°C, a time of 8-12 minutes, and a rotation speed of 70-90 rpm. The inventors of this invention have discovered that, under this preferred condition, the ligament tissue cell suspension of this invention has a higher cell viability and a greater number of live cells.

[0056] Preferably, in step (2), the digestion conditions include: a temperature of 36-38°C, a time of 40-50 min, and a rotation speed of 60-100 rpm.

[0057] It should be noted that, in the method for preparing ligament tissue cell suspension of the present invention, the methods of filtration, the first centrifugation, the second centrifugation, the red blood cell lysis, the first resuspension, and the second resuspension are not particularly limited, and those skilled in the art can select them according to known techniques in the art. However, in order to make the ligament tissue cell suspension of the present invention have a higher cell viability and a greater number of live cells, the present invention provides the following preferred embodiments.

[0058] According to a preferred embodiment, in step (1), the filtration process includes: filtering with a 40-100 μm cell filter membrane and rinsing the filter membrane with PBS buffer.

[0059] According to a preferred embodiment, the conditions for both the first and second centrifugation treatments include: a temperature of 3-5°C, a time of 5-10 min, and a rotation speed of 1200-1500 rpm.

[0060] According to a preferred embodiment, the conditions for the red blood cell lysis treatment include: a temperature of 25-35°C and a time of 5-10 min.

[0061] According to a preferred embodiment, the first resuspension treatment includes: agitating the first cell precipitate with the resuspension solution to completely disperse the first cell precipitate in the resuspension solution.

[0062] As previously described, the fourth aspect of the present invention provides a ligament tissue cell suspension prepared by the method described in the third aspect.

[0063] In the following examples, unless otherwise specified, all raw materials are commercially available products.

[0064] In this invention, each set of examples is set up with 3 parallel experiments, and the test results of each set of examples are the average of the 3 parallel experiments.

[0065] Pancreatic enzyme: Product number 25200072, purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0066] Type I collagenase: CAS No. 9001-12-1, Product No. C8140, purchased from Beijing Solarbio Technology Co., Ltd.

[0067] Type II collagenase: CAS No. 9001-12-1, Product No. C8150, purchased from Beijing Solarbio Technology Co., Ltd.

[0068] Type III collagenase: CAS No. 9001-12-1, Product No. C8490, purchased from Beijing Solarbio Technology Co., Ltd.

[0069] Type IV collagenase: CAS No. 9001-12-1, Product No. C8160, purchased from Beijing Solarbio Technology Co., Ltd.

[0070] Hyaluronidase: CAS No. 37326-33-3, Product No. H8030, purchased from Beijing Solarbio Technology Co., Ltd.

[0071] Elastase: CAS No. 39445-21-1, Product No. E8211, purchased from Beijing Solarbio Technology Co., Ltd.

[0072] Neutral protease: CAS No. 42613-33-2, Product No. D6430, purchased from Beijing Solarbio Technology Co., Ltd.

[0073] Deoxyribonuclease I: CAS No. 9003-98-9, Product No. D8070, purchased from Beijing Solarbio Technology Co., Ltd.

[0074] Digestive fluid I: Multi Tissue Dissociation Kit 1, purchased from Miltenyi Biotec.

[0075] Tissue preservation solution: Tissue Storage Solution, purchased from Miltenyi Biotec.

[0076] In the following examples, the method for calculating the number of viable cells (cells / mL) and cell viability in the ligament tissue cell suspension is as follows:

[0077] (1) Take 10 μL of the cell suspension to be tested and mix it thoroughly with 10 μL of AOPI to obtain a mixed liquid;

[0078] (2) Place 20 μL of the mixture in a CountStar cell counter and use the AOPI cell counting function to count the cells to obtain cell viability and number of live cells;

[0079] Total number of live cells = number of live cells × volume of cell suspension.

[0080] The CountStar cell counter and AOPI were both purchased from Shanghai Ruiyu Biotechnology Co., Ltd.

[0081] In the following examples, the rat ligament tissue used was sourced as follows:

[0082] Three 8-week-old SPF-grade SD rats with a weight difference of ±5g were selected from each group for ligament tissue dissection experiments to eliminate the differences in ligament tissue among different rats due to factors such as physical condition and environment.

[0083] Preparation Example 1

[0084] This preparation example is used to prepare the ligament tissue in Example 1;

[0085] (1) Three 8-week-old rats were euthanized by dislocation;

[0086] (2) Take fresh ligament tissue and wash it twice with PBS buffer;

[0087] (3) Cut the cleaned ligament tissue into pieces with a volume of 2 mm. 3 The fragment of ligament tissue was named Z1.

[0088] Preparation Example 2

[0089] This preparation example is used to prepare the ligament tissue in Example 2;

[0090] (1) Three 8-week-old rats were euthanized by dislocation;

[0091] (2) Take fresh ligament tissue, wash it twice with PBS buffer, and then transfer it to tissue preservation solution and store it at 4°C for 24 hours;

[0092] (3) The ligament tissue that had been stored for 24 hours was washed twice with PBS buffer.

[0093] (4) Cut the cleaned ligament tissue into pieces with a volume of 2 mm. 3The fragment of ligament tissue was named Z2.

[0094] Preparation Example 3

[0095] This preparation example is used to prepare the ligament tissue in Example 3;

[0096] (1) Three 8-week-old rats were euthanized by dislocation;

[0097] (2) Take fresh ligament tissue, wash it twice with PBS buffer, and then transfer it to tissue preservation solution and store it at 4°C for 48 hours;

[0098] (3) The ligament tissue stored for 48 hours was washed twice with PBS buffer.

[0099] (4) Cut the cleaned ligament tissue into pieces with a volume of 2 mm. 3 The fragment of ligament tissue was named Z3.

[0100] Example 1

[0101] (1) 0.3 g of Z1 was pretreated with 1 mL of trypsin solution (concentration of 0.25 wt%) to obtain intermediate I;

[0102] The pretreatment conditions include: a temperature of 37°C, a time of 10 min, and a rotation speed of 80 rpm;

[0103] (2) First, intermediate I was sheared into a homogenate. 0.2 g of intermediate I (dry basis) was digested with 5 mL of digestion solution to obtain material I. Material I was then passed through a 70 μm diameter cell filter membrane (model 130-098-462). SmartStrainers (purchased from Miltenyi Biotec) were used to wash the filter membrane with PBS buffer to obtain filtrate, which was then centrifuged to obtain the first cell pellet.

[0104] The digestion conditions include: a temperature of 37°C, a time of 45 min, and a rotation speed of 80 rpm.

[0105] The centrifugation conditions include: a temperature of 4°C, a time of 5 minutes, and a rotation speed of 1200 rpm.

[0106] (3) 1 mg of the first cell pellet was resuspended in 1 mL of 5 vol% FBS PBS buffer to obtain the first cell suspension; then 1 mL of the first cell suspension was lysed in 3 mL of red blood cell lysis buffer, and the lysis reaction was terminated with 8 mL of PBS buffer to obtain the second cell suspension; then the second cell suspension was centrifuged to obtain the second cell pellet.

[0107] The first resuspension treatment includes: agitating the first cell precipitate and the resuspension solution to completely disperse the first cell precipitate in the resuspension solution;

[0108] The formula of the digestive fluid is shown in Table 1.

[0109] Example 2

[0110] This embodiment uses a similar method and process conditions as Example 1 to prepare ligament tissue cell suspension. The difference is that the ligament tissue used in step (1) is Z2 obtained in Preparation Example 2.

[0111] Example 3

[0112] This embodiment uses a similar method and process conditions as Example 1 to prepare ligament tissue cell suspension. The difference is that the ligament tissue used in step (1) is Z3 obtained in Preparation Example 3.

[0113] Comparative Example 1

[0114] This comparative example uses a similar method and process conditions as Example 1 to prepare ligament tissue cell suspension, except that the trypsin solution pretreatment in step (1) is not performed. The specific method is as follows:

[0115] (1) 0.3 g of Z1 was digested with 5 mL of digestion solution to obtain material I. Material I was then passed through a cell filter membrane with a diameter of 70 μm (model 130-098-462). SmartStrainers (purchased from Miltenyi Biotec) were used to wash the filter membrane with PBS buffer to obtain filtrate, which was then centrifuged to obtain the first cell pellet.

[0116] The digestion conditions include: a temperature of 37°C, a time of 45 min, and a rotation speed of 80 rpm.

[0117] The centrifugation conditions include: a temperature of 4°C, a time of 5 minutes, and a rotation speed of 1200 rpm.

[0118] (2) 1 mg of the first cell pellet was resuspended in 1 mL of 5 vol% FBS PBS buffer to obtain the first cell suspension. Then, 1 mL of the first cell suspension was lysed in 3 mL of red blood cell lysis buffer and the lysis reaction was terminated with 8 mL of PBS buffer. The resulting second cell suspension was further centrifuged to obtain the second cell pellet.

[0119] The first resuspension treatment includes: agitating the first cell precipitate and the resuspension solution to completely disperse the first cell precipitate in the resuspension solution;

[0120] The formula of the digestive fluid is shown in Table 1.

[0121] Comparative Example 2

[0122] This comparative example uses a similar method and process conditions as Example 1 to prepare ligament tissue cell suspension, except that in step (2), the digestive solution is the aforementioned purchased digestive solution I.

[0123] Comparative Example 3

[0124] The ligament cell suspension was prepared using the method described in the literature "Ziji Zhang, Zhiwen Li, Shiyong Zhang et al. Identification of anterior cruciate ligament fibroblasts and their contribution for knee osteoarthritis progression by single-cell analyses, April 4, 2023, PREPRINT (Version 1) available at Research Square [https: / / doi.org / 10.21203 / rs.3.rs-2734014 / v1]". The specific method is as follows:

[0125] (1) Wash Z1 with PBS buffer and cut Z1 into 1 mm pieces. 3 Fragments of tissue;

[0126] (2) Wash the tissue fragments from step (1) three times with PBS solution containing penicillin (concentration of 100 IU / mL) and streptomycin (concentration of 100 μg / mL) to obtain tissue I;

[0127] (3) Tissue I was digested with a type I collagenase solution with a concentration of 1 mg / mL to obtain mixture I; the digestion temperature was 37℃ and the time was 8h.

[0128] (4) Mixture I was filtered through a 70 μm cell filter (BS70-XBS; Biosharp, Hefei, China) and centrifuged to obtain tissue II; the centrifugation speed was 1500 rpm and the time was 3 min.

[0129] (5) Tissue II was mixed with a 0.25 wt% trypsin solution to obtain mixture II; the mixing temperature was 37 °C and the time was 3 min;

[0130] (6) Filter mixture II using a 40 μm filter to obtain tissue III. Wash tissue III with PBS buffer (containing 0.02 vol% ethylene glycol tetraacetic acid) and then centrifuge and filter to obtain tissue IV.

[0131] (7) Tissue IV was resuspended in PBS buffer to obtain ligament cell suspension.

[0132] Comparative Example 4

[0133] This comparative example uses a similar method and process conditions as Comparative Example 3 to prepare ligament tissue cell suspension. The difference is that the ligament tissue used is Z2 obtained in Preparation Example 2.

[0134] Comparative Example 5

[0135] This comparative example uses a similar method and process conditions as Comparative Example 3 to prepare ligament tissue cell suspension. The difference is that the ligament tissue used is Z3 obtained in Preparation Example 3.

[0136] Comparative Example 6

[0137] This comparative example uses a similar method and process conditions as Comparative Example 3 to prepare ligament tissue cell suspension, except that in step (1), the pretreatment time is 20 min.

[0138] Table 1

[0139] raw material concentration Type I collagenase 1.5 mg / mL Type II collagenase 2mg / mL Type III collagenase 1.5 mg / mL Type IV collagenase 1.5 mg / mL Hyaluronidase 1.5 mg / mL elastase 1.5 mg / mL neutral protease 1.5 mg / mL Deoxyribonuclease 0.1 mg / mL fetal bovine serum 5voL% Calcium chloride 0.5 mg / mL

[0140] Test Example 1

[0141] The number of viable cells, cell viability, and cohesion rate of the ligament tissue cell suspensions prepared in the examples and comparative examples were tested, and the test results are shown in Tables 2 and 3.

[0142] Table 2

[0143] Cell viability % viable cell count (cells / mL) Total number of live cells (cells) Example 1 92.23 3.58E+05 3.58E+05 Example 2 90.6 8.38E+05 1.68E+05 Example 3 84.64 7.22E+05 3.53E+05 Comparative Example 1 82.32 7.00E+04 7.00E+04 Comparative Example 2 91.04 3.48E+04 3.48E+04 Comparative Example 3 84.06 1.15E+05 1.03E+05 Comparative Example 4 62.84 2.43E+04 2.43E+04 Comparative Example 5 45.80 1.29E+04 1.29E+04 Comparative Example 6 88.07 1.56E+05 1.56E+05

[0144] Test Example 2

[0145] Apoptosis tests were performed on the ligament tissue cell suspensions prepared in the examples and comparative examples. The results are shown in Table 3 and... Figure 1 As shown.

[0146] Table 3

[0147] Instance number Q1-LR (%) Q1-UR(%) Q1-UR+Q1-LR(%) Example 1 15.91 5.92 21.83 Comparative Example 3 23.48 5.09 28.57

[0148] Q1-LR represents the early apoptosis rate, Q1-UR represents the late apoptosis rate, and Q1-UR+Q1-LR represents the total apoptosis rate.

[0149] As shown in Table 2, comparing Example 1 with Comparative Examples 2 and 3, the cell viability and number of live cells obtained by preparing ligament tissue cell suspension using the method of the present invention are significantly higher than those obtained by the literature method and the cell suspension prepared using digestive solution I. Comparing Example 1 with Comparative Examples 1 and 6, the optimal treatment time with trypsin solution is about 10 minutes. Failure to perform trypsin treatment or excessively long trypsin treatment time will reduce the quality, cell viability, and number of live cells in the obtained cell suspension. Comparing Example 2, Example 3 with Comparative Examples 4 and 5, the method of the present invention can still obtain high-quality cell suspension after ligament tissue is preserved for 24 hours and 48 hours, while the effect of the literature method is very poor.

[0150] From Table 3 and Figure 1 It can be seen that the total apoptosis rate of the cell suspension prepared by the method of the present invention is lower than that of the methods of the prior art; in particular, the early apoptosis rate is much lower than that of the methods of the prior art, and the late apoptosis rate is slightly higher than that of the methods of the prior art.

[0151] from Figures 2 to 5 As can be seen, the cell suspension prepared by the method of the present invention has high viability and fewer impurities and cell clumps.

[0152] The results above show that the ligament tissue cell suspension prepared using the technical solution provided by this invention has a higher cell viability and a greater number of viable cells. The method for preparing the ligament tissue cell suspension provided by this invention is faster and more efficient, and has significant application value.

[0153] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various specific technical features in any suitable manner. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. However, these simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A digestive fluid for obtaining ligament cells, characterized in that, The digestive fluid contains type I collagenase, type II collagenase, type III collagenase, type IV collagenase, hyaluronidase, elastase, neutral protease, and deoxyribonuclease. Relative to 100 parts by weight of the type I collagenase, the content of the type II collagenase is 50-200 parts by weight, the content of the type III collagenase is 50-200 parts by weight, the content of the type IV collagenase is 50-200 parts by weight, the content of the hyaluronidase is 50-200 parts by weight, the content of the elastase is 50-200 parts by weight, the content of the neutral protease is 50-200 parts by weight, and the content of the deoxyribonuclease is 2.5-15 parts by weight.

2. The digestive fluid according to claim 1, characterized in that, The digestion solution also contains DMEM culture medium and FBS; Preferably, in the digestive fluid, the concentration of type I collagenase is 1-2 mg / mL, the concentration of type II collagenase is 1-2 mg / mL, the concentration of type III collagenase is 1-2 mg / mL, the concentration of type IV collagenase is 1-2 mg / mL, the concentration of hyaluronidase is 1-2 mg / mL, the concentration of elastase is 1-2 mg / mL, the concentration of neutral protease is 1-2 mg / mL, and the concentration of deoxyribonuclease is 0.05-0.15 mg / mL.

3. The application of the digestive solution for obtaining ligament cells as described in claim 1 or 2 in single-cell analysis.

4. A method for preparing a suspension of ligament tissue cells, characterized in that, The method includes: (1) The ligament tissue was pretreated with pancreatic enzyme solution to obtain intermediate I; (2) The intermediate I is digested with digestion liquid to obtain material I. The material I is then filtered and centrifuged in sequence to obtain the first cell precipitate. (3) The first cell precipitate is subjected to a first resuspension treatment with the resuspension solution to obtain material II. Material II is subjected to red blood cell lysis treatment and second centrifugation treatment in sequence to obtain the second cell precipitate. (4) The second cell precipitate is subjected to a second resuspension treatment with the resuspension to obtain a ligament tissue cell suspension; In step (2), the digestive fluid is the digestive fluid for obtaining ligament cells as described in claim 1 or 2.

5. The method according to claim 4, characterized in that, In step (1), the amount of pancreatic enzyme solution used is 3-10 uL relative to 1 mg of the ligament tissue.

6. The method according to claim 4 or 5, characterized in that, In step (2), the amount of digestion solution used is 10-50 μL relative to 1 mg of intermediate I on a dry basis. 。 7. The method according to any one of claims 4-6, characterized in that, The resuspension solution is PBS buffer with a volume content of 4-6 vol% FBS.

8. The method according to any one of claims 4-7, characterized in that, In step (1), the pretreatment conditions include: temperature of 36-38℃, time of 8-12min, and rotation speed of 70-90rpm.

9. The method according to any one of claims 4-8, characterized in that, In step (2), the digestion conditions include: a temperature of 36-38°C, a time of 40-50 min, and a rotation speed of 60-100 rpm.

10. A ligament tissue cell suspension prepared by the method according to any one of claims 4-9.

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