CN112481190B - Complex enzyme digestive juice and preparation method and application thereof - Google Patents
Complex enzyme digestive juice and preparation method and application thereof Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention provides a compound enzyme digestive juice, a preparation method and application thereof. The digestive juice comprises the following components in final concentration: 200U/mL of tetrasaccharide 100-type collagenase, 1-10U/mL of type II dispase, 50-200U/mL of deoxyribonuclease I, 0.1-0.5mM of PMSF, 10-20 wt% of glycol, 5-10 wt% of polysucrose and sterile water as a solvent; the pH of the digestive juice is 7.4-8.0. The preparation method comprises the following steps: preparing materials according to the final concentration of each component of the digestive juice; firstly, dissolving collagenase type IV, dispase type II, deoxyribonuclease I, PMSF and polysucrose in sterile water, and then adding ethylene glycol and uniformly mixing; adjusting pH to 7.4-8.0, and filtering for sterilization. The compound enzyme digestive juice and the preparation method thereof are applied to organoid technology. The digestive juice of the invention can obtain complete organoid and disperse cell mass with uniform size, and has short digestion time, convenient operation and stable effect.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a compound enzyme digestive juice, and a preparation method and application thereof.
Background
Organoids (Organoids) are organ-specific collections of cells derived from stem cells or precursor cells. Unlike a single cell group cultured in a two-dimensional environment in a conventional way, organoid culture is a plurality of cell groups contained in a specific organ in a three-dimensional environment, the culture system of organoid culture is more similar to an in-vivo microenvironment, and the organoid culture is a good in-vitro disease model, and even the organoid is an optimal in-vitro drug screening model.
Since organoid culture is based on matrigel droplets, all that needs to be done first is to completely separate the organoids from the gel if they are to be collected for sub-cryopreservation or other downstream applications. The conventional separation method is to use pancreatin for digestion, however, since the organoid is in micron level and has single composition, the organoid is fragile, and the conditions of morphological disruption and cell activity reduction are easy to occur in the digestion process of the existing pancreatin digestion system, thereby influencing the subsequent experiments.
Disclosure of Invention
Aiming at the problems in the existing organoid digestion process, the invention aims to provide a compound enzyme digestion solution and a preparation method and application thereof. The technical scheme of the invention is as follows:
in a first aspect, the invention provides a complex enzyme digestion solution, which comprises the following components in final concentration: 200U/mL of tetrasaccharide 100-type collagenase, 1-10U/mL of type II dispase, 5-20U/mL of deoxyribonuclease, 0.1-0.5mM of PMSF, 10-20 wt% of glycol, 5-10 wt% of polysucrose and sterile water as a solvent; the pH of the digestive juice is 7.4-8.0.
Further, the digestive fluid further comprises a metal salt, and the final concentration of the metal salt in the digestive fluid is 0.5-20 mM.
Optionally, the metal salt is at least one of nitrate and chloride salts of sodium, zinc, manganese, calcium, magnesium, and iron.
Preferably, the metal salt is CaCl2And MgCl2The final concentrations of the two in the digestive liquid are 1-10mM and 1-2mM respectively.
Further, the digestive juice also comprises 1 wt% -5 wt% of glycerol.
Further, the digestive juice also comprises a pH regulator.
Preferably, the pH adjusting agent is NaOH.
In a second aspect, the invention provides a preparation method of the complex enzyme digestion solution, which comprises the following steps:
preparing materials according to the final concentration of each component of the digestive juice;
firstly, dissolving collagenase type IV, dispase type II, deoxyribonuclease I, PMSF and polysucrose in sterile water, and then adding ethylene glycol and uniformly mixing;
adjusting pH to 7.4-8.0, and filtering for sterilization.
Further, when the digestive juice contains a metal salt, a process of dissolving the metal salt in the sterile water is also included.
Further, when the digestive juice contains glycerin, a process of adding glycerin to the solution mixed with ethylene glycol is also included.
In a third aspect, the invention provides the complex enzyme digestive juice and the application of the preparation method thereof in organoid technology.
In a fourth aspect, the present invention provides a method of digesting organoids for pathological or passaging studies, comprising:
removing supernatant from organoid culture liquid, adding above digestive juice, and mixing;
standing at 2-8 deg.C for digestion for 10-30 min, and blowing and dispersing every 10 min;
adding EDTA solution to stop digestion, centrifuging and discarding supernatant to obtain digested organoids.
In a fifth aspect, the present invention provides a method for organoid digestion for in vitro drug screening or gene sequencing, comprising:
removing supernatant from organoid culture liquid, adding above digestive juice, and mixing;
standing at room temperature for digestion for 5-15min, and blowing and dispersing every 3 min;
adding EDTA solution to stop digestion, centrifuging and discarding supernatant to obtain cell mass with uniform size.
Further, the concentration of the EDTA solution is 0.1-1 mg/mL.
Preferably, the concentration of the EDTA solution is 0.5 mg/mL.
Further, the speed of the centrifugation process is: 1000-1500 rpm for 3-6 min.
Compared with the traditional digestive juice, the digestive juice of the invention has the following advantages:
1. the digestive juice can obtain complete organoids and cell clusters with uniform dispersion, the traditional digestive juice can only obtain the cell clusters, and the digestive juice has short digestion time, convenient operation and stable effect.
2. When the digestive juice is digested at 2-8 ℃, the damage to the organoid in the digestion process can be effectively reduced, and the complete morphological structure and function of the organoid are maintained.
3. The digestive juice can more mildly digest organoids when being digested at room temperature, the cell masses are more uniform in size, the cell death rate is low, the survival rate is high, and more cells can be recovered.
Drawings
FIG. 1 is a structural morphology diagram of a digested organoid obtained in example 12 of the present invention.
FIG. 2 is a structural morphology diagram of a digested organoid obtained in example 13 of the present invention.
FIG. 3 is a structural morphology map of a digested organoid obtained in example 14 of the present invention.
FIG. 4 is a structural morphology map of a digested organoid obtained in example 15 of the present invention.
FIG. 5 is a structural morphology map of a digested organoid obtained in example 16 of the present invention.
FIG. 6 is a structural morphology map of a digested organoid obtained in example 17 of the present invention.
FIG. 7 is a structural morphology map of a digested organoid obtained in example 18 of the present invention.
FIG. 8 is a structural morphology map of a digested organoid obtained in example 19 of the present invention.
FIG. 9 is a structural morphology map of a digested organoid obtained in example 20 of the present invention.
FIG. 10 is a structural morphology map of a digested organoid obtained in example 21 of the present invention.
FIG. 11 is a structural morphology map of a digested organoid obtained in example 22 of the present invention.
FIG. 12 is a structural morphology map of a digested organoid obtained in comparative example 1 of the present invention.
FIG. 13 is a structural morphology map of a digested organoid obtained in comparative example 2 of the present invention.
FIG. 14 is a structural morphology map of a digested organoid obtained in comparative example 3 of the present invention.
FIG. 15 is a structural morphology map of a digested organoid obtained in comparative example 4 of the present invention.
Detailed Description
In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
Example 1
The embodiment provides a compound enzyme digestion solution, which comprises the following components in final concentration: 200U/mL of collagenase type IV, 2U/mL of type II dispase, 20U/mL of DNase, 0.1mM of PMSF, 10 wt% of ethylene glycol, 10 wt% of polysucrose and sterile water as a solvent; the pH of the digest was 7.4. The preparation method of the digestive juice comprises the following steps: preparing materials according to the final concentration of each component of the digestive juice; firstly, dissolving collagenase type IV, dispase type II, deoxyribonuclease I, PMSF and polysucrose in sterile water, and then adding ethylene glycol and uniformly mixing; adjusting pH to 7.4, filtering, sterilizing, and storing the digestive juice in a refrigerator at-20 deg.C.
Example 2
The embodiment provides a complex enzyme digestion solution, which is different from the embodiment 1 in that the complex enzyme digestion solution further comprises: CaCl2 2mM，MgCl21 mM. The preparation method of the digestive juice comprises the following steps: preparing materials according to the final concentration of each component of the digestive juice; firstly collagenase type IV, dispase type II, DNAse I, PMSF and CaCl2、MgCl2Dissolving the polysucrose in sterile water, and then adding ethylene glycol and mixing uniformly; adjusting pH to 7.4, filtering, sterilizing, and storing the digestive juice in a refrigerator at-20 deg.C.
Example 3
The embodiment provides a complex enzyme digestion solution, which is different from the embodiment 1 in that the complex enzyme digestion solution further comprises: 1 wt% of glycerin. The preparation method of the digestive juice comprises the following steps: preparing materials according to the final concentration of each component of the digestive juice; firstly, dissolving collagenase type IV, dispase type II, deoxyribonuclease I, PMSF and polysucrose in sterile water, and then adding glycol and glycerol to mix uniformly; adjusting pH to 7.4, filtering, sterilizing, and storing the digestive juice in a refrigerator at-20 deg.C.
Example 4
This example provides a complex enzyme digest, and1 is distinguished by further comprising: CaCl2 2mM，MgCl21mM, glycerol 1 wt%. The preparation method of the digestive juice comprises the following steps: preparing materials according to the final concentration of each component of the digestive juice; firstly collagenase type IV, dispase type II, DNAse I, PMSF and CaCl2、MgCl2Dissolving the polysucrose in sterile water, and then adding the ethylene glycol and the glycerol to be uniformly mixed; adjusting pH to 7.4, filtering, sterilizing, and storing the digestive juice in a refrigerator at-20 deg.C.
Example 5
The embodiment provides a compound enzyme digestion solution, which comprises the following components in final concentration: collagenase type IV 100U/mL, type II dispase 1U/mL, deoxyribonuclease 5U/mL, PMSF 0.5mM, CaCl2 10mM，MgCl22mM, 20 wt% of ethylene glycol, 8 wt% of polysucrose, 5 wt% of glycerol and sterile water as a solvent; the pH of the digest was 8.0. The preparation method is the same as example 3.
Example 6
The embodiment provides a compound enzyme digestion solution, which comprises the following components in final concentration: 150U/mL collagenase type IV, 10U/mL type II dispase, 10U/mL DNase, 0.3mM PMSF, CaCl2 5mM，MgCl21.5mM, 15 wt% of glycol, 5 wt% of polysucrose, 3 wt% of glycerol and sterile water as a solvent; the pH of the digest was 7.6. The preparation method is the same as example 3.
Example 7
This example differs from example 4 in that: by KNO3Replacement of CaCl by 3mM22mM and MgCl2 1mM。
Example 8
This example differs from example 4 in that: the metal salt is CaCl2 3mM。
Example 9
This example differs from example 4 in that: the metal salt is MgCl2 3mM。
Example 10
This example differs from example 5 in that: by FeCl312mM instead of CaCl210mM and MgCl2 2mM。
Example 11
This example differs from example 6 in that: by MnCl26.5mM instead of CaCl25mM and MgCl2 1.5mM。
Example 12
The present embodiment provides a method for digesting human lung cancer organoids for in vitro drug screening studies, which uses the digestive juice of embodiment 1, and comprises the following steps:
(1) carefully sucking out supernatant in a human lung cancer organoid culture dish, adding 2-3mL of digestive juice, standing for 1 minute, and blowing off glue drops;
(2) standing at room temperature for digestion for 5-15min, and gently blowing and dispersing every 3 min;
(3) adding 3ml of 0.5mg/ml EDTA solution to stop digestion, centrifuging and removing supernatant to obtain digested cell mass, wherein the structural morphology of the cell mass is shown in figure 1.
In FIG. 1, it is shown that a cell mass was obtained, but the digestion process was not very uniform, the size difference was more significant, and there was also a case where the single cell was over-digested.
Example 13
The present example provides a method for digesting human gastric carcinoma-like organs for in vitro drug screening research, which employs the digestive juice of example 2, the process is the same as example 12, and the structural morphology is shown in fig. 2.
In FIG. 2, it is shown that the cell mass is obtained, but there is some over-digestion of the cell mass, and the dead cells resulting from over-digestion release the glial DNA, forming flocs, indicating that the digestive enzymes exert the best activity.
Example 14
The present embodiment provides a method for digesting a human liver cancer organoid for in vitro drug screening research, which employs the digestive juice of embodiment 3, the process is the same as embodiment 12, and the structural morphology is shown in fig. 3.
In FIG. 3, it is shown that the cell mass is obtained, but the digestion process of the cell mass is not very uniform and the size difference is more obvious.
Example 15
The present embodiment provides a method for digesting a human lung cancer organoid for in vitro drug screening research, which employs the digestive juice of embodiment 4, the process is the same as embodiment 12, and the structural morphology is shown in fig. 4.
In fig. 4 it is shown that an intact cell pellet is obtained and that the cell pellet is digested uniformly, with a relatively uniform size and good organoid activity.
Example 16
This example provides a method for digesting mouse colon organoids for pathological study, using the digestive juice of example 5, the process is similar to example 12, and differs from example 12 in that: standing at 2-8 deg.C for digestion for 10-30 min, and gently blowing and dispersing every 10 min, wherein the structure appearance is shown in FIG. 5.
In fig. 5 it is shown that intact organoids were obtained with small size differences and good organoid activity.
Example 17
The present example provides a method for digesting mouse intestinal organoids for pathological study, which uses the digestive juice of example 6, the process is the same as example 16, and the structural morphology is shown in fig. 6.
In figure 6 it is shown that intact organoids were obtained with small size differences and good activity.
Example 18
This example provides a method for digesting mouse large intestine organoids for pathological study, which uses the digestive juice of example 7 in the same manner as example 16, and the structural morphology is shown in fig. 7.
In figure 7 it is shown that intact organoids were obtained with less variation in organoid size and better viability.
Example 19
The present example provides a method for digesting mouse liver organoids for pathological study, which uses the digestive juice of example 8, the process is the same as example 16, and the structural morphology is shown in fig. 8.
In figure 8 it is shown that intact organoids were obtained with less variation in organoid size and better viability.
Example 20
The present example provides a method for digesting mouse liver organoids for pathological study, which uses the digestive juice of example 9 in the same manner as example 16, and the structural morphology thereof is shown in fig. 9.
In figure 9 it is shown that intact organoids were obtained with small size differences and moderate activity.
Example 21
The present example provides a method for digesting mouse lung bud organoids for pathological study, which uses the digestive juice of example 10, the process is the same as example 16, and the structural morphology is shown in fig. 10.
In figure 10 it is shown that intact organoids were obtained with less variation in organoid size and better viability.
Example 22
The present example provides a method for digesting mouse lung bud organoids for pathological study, which uses the digestive juice of example 11 in the same manner as example 16, and the structural morphology thereof is shown in fig. 11.
In figure 11 it is shown that intact organoids were obtained with small size differences and moderate activity.
The digestants of examples 1-3 lack glycerol and/or metal salts, the digestants of examples 4-11 contain glycerol and metal salts, and the metal salts used in examples 4-6 are magnesium chloride and calcium chloride. From the results of examples 12 to 22, it can be seen that, although organoids can be obtained in the absence of glycerol and metal salts, partial organoid over-digestion is highly likely to occur, and the obtained organoids have large and uneven size differences. This shows that the addition of metal salts and glycerol to the digestive juice can increase the activity of the digestive juice sufficiently, and in particular, the metal salts with calcium chloride and magnesium chloride have the best effect, which can not only protect the organoids from over-digestion, but also obtain the complete organoids and also obtain the cell clusters with dispersed and uniform sizes.
Comparative example 1
This comparative example differs from example 15 in that the digestive juices do not contain collagenase type IV.
In FIG. 12, it is shown that intact cell masses were obtained, but the size difference was large, the activity was good, the metal salts in the digestive juice components gave the best activity of the enzyme, and the glycerol components also protected the cell masses from over-digestion, but lacking the tetrasaccharidase, the intact cell masses could be obtained, and it was difficult to obtain cell masses of uniform size dispersion, indicating that the tetrasaccharidase in the digestive juice was essential.
Comparative example 2
This comparative example differs from example 15 in that the digestive juice does not contain type II dispase.
In FIG. 13, it is shown that intact cell clusters are obtained, but the size difference is significant, the activity is good, the metal salt in the digestive juice component allows the enzyme to exert the best activity, and the glycerol component also well protects the cell clusters from over-digestion, but the two-type dispase is absent, the intact cell clusters can be obtained, the cell clusters aggregate, and the cell clusters with uniform size are difficult to obtain, which indicates that the two-type dispase in the digestive juice is indispensable.
Comparative example 3
This comparative example differs from example 15 in that the digestion solution does not contain DNase I.
In FIG. 14, it is shown that intact cell clusters are obtained, but the size difference is obvious, the activity is good, the metal salt in the digestive juice component allows the enzyme to exert the best activity, and the glycerol component also well protects the cell clusters from over-digestion, but in the absence of DNase I, more intact cell clusters can be obtained, but the cell clusters with uniform size are difficult to obtain, which indicates that DNase I in the digestive juice is indispensable.
Comparative example 4
This comparative example differs from example 15 in that glycerol was replaced by ethylene glycol in the digestive juice.
FIG. 15 shows that the cell mass is over-digested, most of the cell mass is digested into single cells, the activity is poor, the metal salt in the digestive juice component enables the enzyme to exert the best activity, but the lack of glycerol protects the cell mass from over-digestion, the cell mass with uniform size can be obtained, and the complete cell mass is difficult to obtain, which indicates that the glycerol in the digestive juice is indispensable.
In conclusion, the digestive juice can obtain complete organoids and cell clusters with uniform dispersion, the traditional digestive juice can only obtain the cell clusters, and the digestive juice has short digestion time, convenient operation and stable effect.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (4)
1. A compound enzyme digestive juice for organoids is characterized in that: consists of the following components in final concentration: 200U/mL of tetrasaccharide 100-type collagenase, 1-10U/mL of type II dispase, 5-20U/mL of deoxyribonuclease I, 0.1-0.5mM of PMSF, 10-20 wt% of glycol, 5-10 wt% of polysucrose, 0.5-20mM of metal salt, 1-5 wt% of glycerol and sterile water as a solvent; the digestive juice also comprises a pH regulator, and the pH of the digestive juice is 7.4-8.0; the metal salt is CaCl21-10mM and MgCl21-2 mM; or the metal salt is KNO33 mM; or the metal salt is CaCl23 mM; or the metal salt is MgCl23 mM; or FeCl312 mM; or the metal salt is MnCl2 6.5mM。
2. The method for preparing the compound enzyme digestive juice for organoids as claimed in claim 1, which is characterized in that: the method comprises the following steps:
preparing materials according to the final concentration of each component of the digestive juice;
firstly, dissolving collagenase type IV, dispase type II, deoxyribonuclease I, PMSF, polysucrose and metal salt in sterile water, and then adding glycol and glycerol to mix evenly;
adding pH regulator to adjust pH to 7.4-8.0, and filtering for sterilization.
3. A method of digesting organoids for pathological or passaging studies, comprising: the method comprises the following steps:
removing supernatant from organoid culture medium, adding digestive juice of claim 1, and mixing;
standing at 2-8 deg.C for digestion for 10-30 min, and blowing and dispersing every 10 min;
adding EDTA solution to stop digestion, centrifuging and discarding supernatant to obtain digested organoids.
4. A method of organoid digestion for in vitro drug screening or gene sequencing, characterized by: the method comprises the following steps:
removing supernatant from organoid culture medium, adding digestive juice of claim 1, and mixing;
standing at room temperature for digestion for 5-15min, and blowing and dispersing every 3 min;
adding EDTA solution to stop digestion, centrifuging and discarding supernatant to obtain cell mass with uniform size.
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