CN109609439B - Method for isolating single cells from abnormal tissue - Google Patents
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- CN109609439B CN109609439B CN201811594140.0A CN201811594140A CN109609439B CN 109609439 B CN109609439 B CN 109609439B CN 201811594140 A CN201811594140 A CN 201811594140A CN 109609439 B CN109609439 B CN 109609439B
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Abstract
The invention relates to the field of single cell separation, and discloses a method for separating single cells from abnormal tissues. The method comprises the following steps: and mixing the abnormal tissue sample with a digestive fluid for digestion, wherein the digestive fluid contains VIII type collagenase, neutral protease, trypsin inhibitor and deoxyribonuclease, and the ratio of the enzyme activity units of the VIII type collagenase, the neutral protease, the trypsin inhibitor and the deoxyribonuclease is 100-312.5:1-5:5000-25000: 1. The invention can realize the extraction of the single cells with high yield and high survival rate under the condition of short total digestion time, particularly the pancreatic tissue, and solves the problem of low yield and survival rate of the single cells of the conventional pancreatic tissue.
Description
Technical Field
The invention relates to the field of single cell separation, in particular to a method for separating single cells from abnormal tissues.
Background
The pancreas is an organ in the human body that has both endocrine and exocrine functions. Histologically contains a number of cellular components including islet cells, acinar cells, ductal cells, and stromal cells. Pancreatic juice secreted by the pancreas contains various digestive enzymes such as trypsin, pancreatic lipase and pancreatic amylase. With the recent rise of single cell analysis techniques, it is clear that the importance of the entire tissue analysis, in which different components are mixed and interfere with each other, have been advanced to the study of individual cells constituting a tissue or organ. In the field of pancreatic research, pancreatic single cell research is often stopped at the first step, namely, the acquisition of pancreatic tissue single cells for analysis, because pancreatic organs are generally soft and fragile, histologically complex, and self-digestion easily occurs due to potent pancreatic enzymes.
The single cell separating method for tissue and organ of human body or animal model includes the initial capillary suction, flow sorting and microfluid technology, and many single cell separating technologies obtain more or less satisfactory results in different tissues, but are not satisfactory in pancreatic tissues. According to the literature, most researchers currently adopt enzyme digestion dissociation methods mainly comprising type II collagenase when isolating mouse pancreatic cancer single cells, but all researchers of pancreatic single cells have shown that great challenges are met when isolating pancreatic single cells, and the single cell yield is not high or the cell viability is low, so that it is of great importance to develop a preparation or method of pancreatic single cells capable of being effectively isolated.
Disclosure of Invention
The invention aims to overcome the problems of low single cell yield and low cell survival rate in the prior art and provide a novel method for separating single cells from abnormal tissues.
In order to achieve the above object, the present invention provides a method for isolating a single cell from an abnormal tissue, the method comprising: and mixing the abnormal tissue sample with a digestive fluid for digestion, wherein the digestive fluid contains VIII type collagenase, neutral protease, trypsin inhibitor and deoxyribonuclease, and the ratio of the enzyme activity units of the VIII type collagenase, the neutral protease, the trypsin inhibitor and the deoxyribonuclease is 100-312.5:1-5:5000-25000: 1.
Through the technical scheme, the method can realize the extraction of the single cells with high yield and high survival rate under the condition of short total digestion time, particularly solves the problem of low yield and survival rate of the single cells of the conventional pancreatic tissue based on the separation of the single cells of the pancreatic tissue, points out the direction for the separation of the single cells of the pancreatic tissue, and is favorable for promoting the further research on the pancreatic tissue.
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FIG. 1 is a graph showing the results of isolating single cells from pancreatic tissue using a method according to an embodiment of the present invention;
FIGS. 2 to 5 are graphs showing the results of isolating single cells from pancreatic tissue using different reference methods, respectively.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In the present invention, the term "enzyme activity unit" used in the case where no description is made to the contrary, refers to the amount of enzyme content, which is the amount of enzyme required to convert 1. mu. mol of a substrate or 1. mu. mol of a group of interest in the substrate in 1 minute under a specific condition, and is referred to as one enzyme activity unit (IU, also called U).
Wherein, the enzyme activity unit of the VIII type collagenase is defined as: the amount of enzyme that hydrolyzes collagen within 5 hours under the conditions of pH 7.5 and 37 ℃ to produce L-leucine equivalent to 1. mu. mol is one enzyme activity unit.
The definition of the enzyme activity unit of the neutral protease is: the amount of enzyme that hydrolyzes casein at pH 7.5 and 37 ℃ per minute to produce Folin positive amino acids or peptides corresponding to 1. mu. mol of tyrosine was one enzyme activity unit.
The definition of the enzyme activity unit of trypsin inhibitor is: the activity of inhibiting one trypsin enzyme activity unit is called as one inhibitor enzyme activity unit (U), and under the conditions of pH 7.5 and 25 ℃, 1 mu mol of N-benzoyl-L-arginine ethyl ester (BAEE) is hydrolyzed per second to form one trypsin enzyme activity unit.
The definition of the enzyme activity unit of dnase is: the amount of enzyme required to completely degrade 1. mu.g of pBR322 plasmid DNA in 10 minutes at pH 8.0 and 37 ℃ was one unit of enzyme activity.
The method for separating single cells from abnormal tissues comprises the following steps: the abnormal tissue sample is digested by mixing with a digestive fluid containing collagenase type VIII, neutral protease, trypsin inhibitor and DNase.
In the method, the ratio of the activity units of the collagenase type VIII and the DNase is 100-312.5:1, such as 100:1, 120:1, 130:1, 150:1, 180:1, 200:1, 220:1, 240:1, 260:1, 280:1, 300:1, 310:1, 312:1 and 312.5:1, or any value in the above ranges.
In the method, the ratio of the enzyme activity units of the neutral protease and the DNase is 1-5:1, such as 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1 or any value in the above values.
In the method, the ratio of the enzyme activity units of the trypsin inhibitor and the DNase is 5000-25000:1, such as 5000:1, 8000:1, 9000:1, 10000:1, 12000:1, 15000:1, 18000:1, 20000:1, 22000:1, 24000:1, 25000:1 or any value in between.
Collagenase is the only protease that degrades native collagen fibers with a triple supercoiled structure, which are found extensively in connective tissue. The collagenase is mainly prepared by fermentation culture, extraction and refining of Clostridium histolyticum, and can also be extracted from pig pancreas. In the present invention, the collagenase is collagenase of type VIII, and may be Cat: C2139 from Sigma, for example.
In the present invention, there is no particular requirement for the specific types of neutral protease and DNase, and various options are available as are common in the art.
Preferably, the neutral protease is Dispase II. For example, Roche Cat: 4942078001.
Preferably, the dnase is dnase i. For example, NEB's Cat: M0303S.
In the present invention, the trypsin inhibitor can inhibit trypsin and chymotrypsin, prevent activation of other active proproteinases in the pancreas and self-activation of trypsinogen, and can be selected from trypsin inhibitors conventionally used in the art. For example, Cat from Sigma: T6522.
In the present invention, the digestion solution contains a solvent. The solvent may be present in an amount of 0.1-12.5mL, preferably 0.2-3mL, such as 0.2mL, 0.4mL, 0.5mL, 0.8mL, 1mL, 1.2mL, 1.3mL, 1.5mL, 2mL, 2.5mL or any value in between, per mg of collagenase type VIII.
In the present invention, the solvent may be various common reagents for preserving or dissolving the enzyme preparation, and preferably, the solvent is a phosphate buffer containing 4-10 vol% fetal bovine serum.
In the present invention, the amount of the digestion solution used is not particularly limited as long as the sample can be immersed. Preferably 1cm with respect to the volume3The amount of the digestion solution is 20-45mL, such as 20mL, 25mL, 26mL, 27mL, 28mL, 30mL, 32mL, 35mL, 40mL, 45mL, or any value therebetween.
In the present invention, the volume of the abnormal tissue sample is preferably 1-2cm × 1-2cm × 0.5-2cm, such as 1.5cm × 1.5cm × 0.5cm, for better digestion.
In the present invention, the digestion may be performed under conventional conditions. Preferably, the conditions for digestion include a temperature of 35-38 ℃, such as 35 ℃, 36 ℃, 36.5 ℃, 36.8 ℃, 37 ℃, 37.2 ℃, 37.5 ℃, 38 ℃ or any value in between.
Preferably, the digestion conditions further comprise a time of 5-80min, such as 5min, 10min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, 65min, 70min, 75min, 80min or any value therebetween.
The digestion may be performed on a shaker, preferably, the conditions of the digestion further comprise a shaking rate of 40-60 rpm/min.
In the present invention, to further accelerate digestion of the cohesive mass of the sample, the sample may be sheared and/or blown through with a pipette (e.g., a sterile buchner pipette). Or digesting for a period of time, filtering by a cell sieve, and supplementing the undigested sample with digestive juice to continue digestion. That is, the digestion may be in the form of: mixing the sample with the first part of digestive juice, shearing the sample, mixing the sheared sample with the second part of digestive juice, blowing the sample, sieving the sample, supplementing the digestive juice to the undigested sample, continuing digestion, sieving the sample with the cell sieve, and combining filtrates of the two cell sieves. Relative volume of 1cm3The amount of the first portion of the digestion solution of the abnormal tissue sample of (1) may be 400-1000. mu.L. Relative volume of 1cm3The amount of the second portion of the digestion solution may be 10-20 mL. Relative volume of 1cm3The amount of the supplemented digestive juice can be 10-20 mL.
In the present invention, in order to further promote the digestion, the method further comprises: the abnormal tissue samples were removed of fiber, fat and necrotic tissue in a tissue preservation solution prior to digestion.
The amount of the tissue preservation solution used is not particularly limited as long as the sample can be immersed therein. The tissue preservation solution may be of conventional choice and may, for example, be a serum-free cell cryopreservation medium containing fetal bovine serum and a protease inhibitor.
In the present invention, the method further comprises: and contacting the digested sample with a red blood cell lysate to lyse red blood cells, and washing to obtain separated single cells. The digested sample is cell suspension, and is centrifuged to obtain precipitate, the precipitate and red blood cellsCell lysate contact to remove red blood cells. The amount of the erythrocyte lysate may be conventionally selected, for example, 1cm relative to the volume3The amount of the erythrocyte lysate used in the abnormal tissue sample of (1) may be 3 to 8 ml. The washing solution used for washing may also be a conventional one, and may be, for example, a phosphate buffer containing 0.02 to 0.08% by weight of bovine serum albumin. The amount of the washing liquid to be used is not particularly limited, and is, for example, 1cm relative to the volume3The washing solution may be used in an amount of 3 to 8 ml.
According to a particular embodiment of the invention, the method comprises the following steps:
1) in the tissue preservation solution, fibers, fat, necrotic tissues and the like on an abnormal tissue sample (particularly a pancreatic tumor sample) are trimmed by using sterile ophthalmic scissors;
2) washing with sterile PBS;
3) the sample was placed in a sterile centrifuge tube and the first portion of the digest (1 cm relative to volume) was added3The amount of the first part of the digestive juice may be 400-;
4) tissue is cut into pieces (about 2X 1 mm) using sterile ophthalmic scissors3) Operating on ice, wherein the shearing time is not more than 5 minutes;
5) transferring the tissue debris into a sterile tube, adding a second portion of the digestion solution, and placing on a shaker for digestion (1 cm relative to volume)3The amount of the second portion of the digestive fluid may be 10-20 mL);
6) blowing and uniformly mixing the adhered and conglobated tissue fragments by using a sterile Brinell suction pipe;
7) observing the state of the tissue fragments after continuously digesting, and repeating the steps 6) -7) if the tissue fragments are still adhered and agglomerated, and continuously digesting if the tissue fragments are scattered, wherein the total digestion time is not more than 30 min;
8) after digestion, blowing the tissue fragments by using a sterile Brinell suction tube again, standing, collecting supernatant, passing through a cell sieve to obtain filtrate, and placing the filtrate on ice;
9) undigested tissue was supplemented with digestive juice (1 cm relative to volume)3Of (2) isNormal tissue sample, the dosage of the added digestive juice can be 10-20mL), continue digesting, using sterile Brinell pipette to blow tissue debris, and passing through a cell sieve together with the digestive juice and the tissue debris to obtain filtrate;
10) combining the filtrates obtained in the two steps, and centrifuging;
11) discarding the supernatant, adding erythrocyte lysate, gently blowing, resuspending the precipitate, preventing photodisruption at room temperature, and removing flocculent insoluble substance by cell sieve;
12) transferring the heavy suspension to a sterile centrifuge tube for centrifugation;
13) discarding the supernatant, adding the first part of washing solution, gently blowing to beat the resuspended precipitate, if flocculent insoluble matters exist, removing the flocculent precipitate through a cell sieve again, transferring the resuspended solution to a new sterile centrifuge tube, and continuously adding the second part of washing solution (the volume ratio of the first part of washing solution to the second part of washing solution can be 1: 4-5);
14) centrifuging;
15) repeating step 13);
16) and (4) centrifuging.
According to another particular embodiment of the invention, the method comprises the following steps:
1) in the tissue preservation solution, fibers, fat, necrotic tissues and the like on an abnormal tissue sample (particularly pancreas at the part where the tumor is located) are sheared off by using sterile ophthalmic scissors;
2) washing with sterile PBS;
3) the sample was placed in a sterile centrifuge tube and the first portion of the digest (1 cm relative to volume) was added3The amount of the first part of the digestive juice may be 400-;
4) tissue is cut into pieces (about 2X 1 mm) using sterile ophthalmic scissors3) Operating on ice, wherein the shearing time is not more than 5 minutes;
5) the tissue debris was transferred to a sterile petri dish and a second portion of the digestion solution (1 cm relative to volume) was added3The amount of the second portion of the digestive fluid may be 5-20 mL);
6) blowing with sterile Buchner suction pipe, collecting supernatant, sieving with cell sieve, and collecting filtrateThe undigested tissue debris is supplemented with digestive juice and continued to be digested in a sterile culture dish until the tissue digestion is complete or satisfactory normal tissue single cells (relative to a volume of 1 cm)3The dosage of the digestive juice supplemented each time can be 5-20 mL);
7) centrifuging the filtrate obtained in step 6);
8) discarding the supernatant, adding lysate, gently blowing, resuspending the precipitate, preventing photodisruption at room temperature, and removing flocculent insoluble substance by cell sieve;
9) transferring the heavy suspension to a sterile centrifuge tube for centrifugation;
10) discarding the supernatant, adding the first part of washing solution, gently blowing to beat the resuspended precipitate, if flocculent insoluble matters exist, removing the flocculent precipitate through a cell sieve again, transferring the resuspended solution to a new sterile centrifuge tube, and continuously adding the second part of washing solution (the volume ratio of the first part of washing solution to the second part of washing solution can be 1: 4-5);
11) centrifuging;
12) repeating step 10);
13) and (4) centrifuging.
In the present invention, the method may further comprise culturing the single cells obtained by digestion with a cell culture medium. The cell culture medium may be a conventional medium for expanding cells, or may be a three-dimensional medium containing growth factors to grow the obtained single cells into a three-dimensional organoid model.
In the present invention, the abnormal tissue sample may be a tissue sample which is abnormal due to a disease or the like, and may be a pancreatic tissue sample, for example, a tumor tissue derived from pancreas. The method of the invention is particularly suitable for isolating single cells from a pancreatic sample, and therefore, according to a preferred embodiment of the invention, the non-normal tissue sample is a pancreatic tumor sample (pancreatic tumor tissue) and/or the pancreas (pancreatic tissue) at the site of the tumor.
The present invention will be described in detail below by way of examples.
TABLE 1
TABLE 2
Example 1
This example is intended to illustrate the method of isolating single cells from normal pancreatic tissue (pancreas at the site of tumor) according to the present invention, wherein the tissue preservation solution, the digestive juice, the lysis solution and the washing solution used are shown in table 1, the enzyme activity unit values and the concentrations of each component per liter of the digestive juice are shown in table 2, and the digestive juice is prepared by the following method: mixing the VIII type collagenase, Dispase II and trypsin inhibitor, dissolving the mixed powder by using a solvent, adding DNaseI, and mixing uniformly.
The samples were divided into five groups for testing (see table 3), and the specific procedures were as follows:
1) in the tissue preservation solution, the specimen was trimmed with sterile ophthalmic scissors to remove fibers, fat, necrotic tissue, etc. (normal pancreatic tissue piece size about 1.5X 0.5 cm)3Yellow in color, soft in the middle, without black coke necrosis and too much blood on the surface, clear pancreatic lobule partition can be seen in the section, the section is from a pancreatic tumor patient, the section is obtained by cutting, and the patient signs an informed consent).
2) Sterile PBS was washed 3 times.
3) The sample was placed in a 5ml sterile EP tube (eppendorf) and about 500. mu.l of the digest was added.
4) Tissue is cut into pieces (about 2X 1 mm) using sterile ophthalmic scissors3) The operation is carried out on ice, the shearing time does not exceed 5 minutes.
5) The tissue debris was transferred to a 10cm sterile petri dish, 10ml of the digestion solution was added, and the mixture was digested in an incubator at 37 ℃.
6) The cells were pipetted and mixed with a sterile Buchner pipette every 5min, and the dissociation of the cells was observed under 10X 20 and 10X 40 times microscope. Suspending single cells in the digestive juice can be seen under a microscope at the first 10min, collecting the digestive juice and the tissue debris into a sterile 50ml centrifuge tube, gently blowing and beating the centrifuge tube, standing the centrifuge tube, collecting the supernatant, and screening the supernatant through a 40 mu m cell screen (cellstrainer), and taking the filtrate. Adding 10ml of undigested tissue debris into a 10cm sterile culture dish, continuing digestion until the tissue digestion is complete or satisfactory pancreatic normal tissue single cells are obtained (the total duration is 50min), and discarding the filtrate collected in the first 10min, wherein the single cells are difficult to separate due to the fact that the filtrate contains more impurities. And (4) respectively reserving the filtrates obtained by digestion every 10min later, respectively carrying out the following operation steps, and finally selecting the single cell suspension sample with the optimal activity rate and cell number.
7) 800g of the filtrate obtained in step 6) were centrifuged for 4min (using a horizontal rotor).
8) Discarding the supernatant, adding 5ml of lysate, gently blowing, resuspending the precipitate, and avoiding photodisruption at room temperature for 5min (if flocculent insoluble matter exists, removing by a 40 μm cell sieve again).
9) The resuspension solution was transferred to a sterile 15ml centrifuge tube for centrifugation, centrifuge force: 800g, time: and 4 min.
10) The supernatant was discarded, the resuspended pellet was gently flushed with 1ml of wash solution (again through a 40 μm cell sieve if there was any flocculent insoluble material), the resuspended solution was transferred to a fresh sterile 15ml centrifuge tube and 5ml of wash solution was added.
11) Centrifuge at 600g for 5 min.
12) Repeat step 10).
13) Centrifuge at 400g for 6 min.
14) The supernatant was discarded, an appropriate amount of wash was resuspended (pellet covered at the bottom of 15ml centrifuge tube, resuspended with 100. mu.l wash), an equal volume of cells was mixed with trypan blue, the morphology, viability, impurities, etc. were examined, and the cell viability (the percentage of viable cells per ml product to the total number of cells) and concentration (total number of cells per ml product, including dead cells) were measured with a cell counter (life cell counter), the results are shown in FIGS. 1-5 and Table 3.
In FIGS. 1 to 5, the upper graph shows the results of observation of the digestion effect under a normal light microscope (40X) after digestion, and the lower graph shows the results of observation of the cell viability under a normal light microscope (40X) after further dilution.
TABLE 3
As can be seen from FIG. 1 and Table 3, the method of the present invention can effectively separate single cells from normal tissues, and the single cells are basically free from aggregation, and the cell survival rate is above 85%.
As can be seen from FIG. 2, FIG. 3 and Table 3, if the ratio between the components in the digestion solution is not within the range of the present invention, aggregation of cells occurs after digestion and the cell viability is low.
As can be seen from fig. 4 and table 3, using collagenase type VIII enables significantly more single cells to be obtained and the cell viability rate to be higher than other types of collagenase.
As can be seen from FIG. 5 and Table 3, only the combination of collagenase type VIII, neutral protease, trypsin inhibitor and DNase can effectively increase the number of single cells (less aggregation) and the cell viability, and the four components are one of the four components, so that the four components can synergistically play a role.
Example 2
This example is intended to illustrate the method for isolating single cells from a tumor tissue derived from pancreas according to the present invention, wherein the tissue preservation solution, the digestive juice, the lysate and the washing solution used are shown in Table 1, and the enzyme activity unit value and the concentration of each component in each liter of the digestive juice (the preparation method is the same as in example 1) are shown in I-1 of Table 2.
1) Pancreatic tumor samples (1.5X 0.5 cm) were trimmed away in tissue preservation solution using sterile ophthalmic scissors3Surgically excised, with the patient signed an informed consent) of fiber, fat, and necrotic tissue, etc.
2) Sterile PBS was washed 3 times.
3) The sample was placed in a 5mL sterile EP tube (eppendorf) and about 500. mu.l of the digest was added.
4) Tissue is cut into pieces (about 2X 1 mm) using sterile ophthalmic scissors3) The operation is carried out on ice, the shearing time does not exceed 5 minutes.
5) The tissue debris was transferred to a 50ml sterile tube, 15ml of digestion solution was added, and the tube was digested at 37 ℃ on a 50rmp/min shaker.
6) After 5min, the clumped tissue pieces were blown up and mixed well using a sterile buchner pipette.
7) And (4) observing the state of the tissue debris after continuously digesting for 5min, repeating the steps 6) -7) if the tissue debris is still adhered and agglomerated, and continuously digesting for 20min if the tissue debris is scattered, wherein the total digestion time is not more than 30 min.
8) After digestion, the tissue debris was again blown with a sterile buchner pipette and allowed to stand for 3min, the supernatant was collected and passed through a 40 μm cell sieve (cell filter) and the filtrate was placed on ice.
9) Adding 15ml of digestive juice into undigested tissue, continuously digesting for 20min, blowing tissue debris by using a sterile Brinell pipette, and sieving the tissue debris together with the digestive juice and the tissue debris by using a 40-micron cell sieve to obtain filtrate.
10) The filtrates obtained in two times were combined and centrifuged at 800g for 4min (using a horizontal rotor).
11) Discarding the supernatant, adding 5ml of lysate, gently blowing, resuspending the precipitate, and avoiding photodisruption at room temperature for 5min (if flocculent insoluble matter exists, removing by a 40 μm cell sieve again).
12) The resuspension solution was transferred to a sterile 15ml centrifuge tube for centrifugation, centrifuge force: 800g, time: and 4 min.
13) The supernatant was discarded, the resuspended pellet was gently flushed with 1ml of wash solution (again through a 40 μm cell sieve if there was any flocculent insoluble material), the resuspended solution was transferred to a fresh sterile 15ml centrifuge tube and 5ml of wash solution was added.
14) Centrifuge at 600g for 5 min.
15) Repeat step 13).
16) Centrifuge at 400g for 6 min.
17) Abandoning the supernatant, resuspending the precipitate with a proper amount of lotion (covering the precipitate at the bottom of a 15ml centrifuge tube, resuspending with 100 mul of lotion), taking equal volume of cells, mixing with trypan blue, examining the cell morphology, the cell viability, the impurity condition and the like by a microscope, detecting the cell viability and the cell concentration by a cell counter (life cell counter), and obtaining a result similar to that of the embodiment 1, wherein the method can effectively separate single cells from tumor tissues, basically has no aggregation, and the cell viability is more than 80%; if the ratio of each component in the digestive juice is not in the range of the invention, the cells after digestion agglomerate, and the cell survival rate is lower; and using collagenase of type VIII enables to obtain significantly more single cells and higher cell viability rates than other types of collagenase. In addition, under the condition of lacking one component, the number of single cells and the cell survival rate are not ideal, and further, the four components are lack of one component and cannot act synergistically.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. A method for isolating a single cell from an abnormal tissue, the method comprising: mixing an abnormal tissue sample with a digestive fluid for digestion, wherein the digestive fluid contains VIII type collagenase, neutral protease, trypsin inhibitor and deoxyribonuclease, and the ratio of enzyme activity units of the VIII type collagenase, the neutral protease, the trypsin inhibitor and the deoxyribonuclease is 100-312.5:1-5:5000-25000: 1;
wherein the abnormal tissue includes at least one of pancreatic tumor tissue and pancreatic tissue at a site of the tumor.
2. The method of claim 1, wherein the neutral protease is dispaseII;
and/or, the dnase is dnase i.
3. The method according to claim 1 or 2, wherein the amount of solvent in the digest is 0.1-12.5mL per mg of collagenase type VIII.
4. The method of claim 3, wherein the solvent is a phosphate buffer containing 4-10 vol% fetal bovine serum.
5. The method of claim 1, wherein the relative volume is 1cm3The dosage of the digestive juice is 20-45 mL.
6. The method of claim 1 or 5, wherein the volume of the abnormal tissue sample is 1-2cm x 0.5-2 cm.
7. The method of claim 1, wherein the conditions of digestion comprise: the temperature is 35-38 deg.C, the time is 5-80min, and the vibration speed is 40-60 rpm/min.
8. The method of claim 1, wherein the method further comprises: the abnormal tissue samples were removed of fiber, fat and necrotic tissue in a tissue preservation solution prior to digestion.
9. The method of claim 8, wherein the tissue preservation fluid is serum-free cell cryopreservation media comprising fetal bovine serum and a protease inhibitor.
10. The method of claim 1, 8 or 9, wherein the method further comprises: and contacting the digested sample with a red blood cell lysate to lyse red blood cells, and washing to obtain separated single cells.
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