CN114591883B - Preparation method of aortic cell single-cell suspension - Google Patents
Preparation method of aortic cell single-cell suspension Download PDFInfo
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- 239000006285 cell suspension Substances 0.000 title claims abstract description 61
- 238000002360 preparation method Methods 0.000 title abstract description 15
- 230000029087 digestion Effects 0.000 claims abstract description 26
- 230000002792 vascular Effects 0.000 claims abstract description 26
- 210000001519 tissue Anatomy 0.000 claims description 55
- 239000012888 bovine serum Substances 0.000 claims description 28
- 239000012634 fragment Substances 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 15
- 239000000725 suspension Substances 0.000 claims description 14
- 108010019160 Pancreatin Proteins 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 11
- 229940055695 pancreatin Drugs 0.000 claims description 11
- 102000029816 Collagenase Human genes 0.000 claims description 9
- 108060005980 Collagenase Proteins 0.000 claims description 9
- YRQNKMKHABXEJZ-UVQQGXFZSA-N chembl176323 Chemical compound C1C[C@]2(C)[C@@]3(C)CC(N=C4C[C@]5(C)CCC6[C@]7(C)CC[C@@H]([C@]7(CC[C@]6(C)[C@@]5(C)CC4=N4)C)CCCCCCCC)=C4C[C@]3(C)CCC2[C@]2(C)CC[C@H](CCCCCCCC)[C@]21C YRQNKMKHABXEJZ-UVQQGXFZSA-N 0.000 claims description 9
- 229960002424 collagenase Drugs 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 108010003272 Hyaluronate lyase Proteins 0.000 claims description 7
- 102000001974 Hyaluronidases Human genes 0.000 claims description 7
- 229960002773 hyaluronidase Drugs 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 6
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- 238000005054 agglomeration Methods 0.000 description 4
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- 208000025494 Aortic disease Diseases 0.000 description 3
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- 108010053770 Deoxyribonucleases Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
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- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 208000002251 Dissecting Aneurysm Diseases 0.000 description 1
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- 206010002895 aortic dissection Diseases 0.000 description 1
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- 238000012163 sequencing technique Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000000472 traumatic effect Effects 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention provides a preparation method of an aortic cell single-cell suspension, which is characterized in that aortic vascular tissues are treated firstly, and then secondary digestion reaction is carried out, so that single-cell suspension with high single-cell yield, a plurality of single-cell suspensions, high cell activity and strong cell functions is obtained, and the technical problem of poor cell quality obtained by low efficiency of the traditional aortic cell single-cell suspension is solved.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a preparation method of aortic cell single-cell suspension.
Background
Aortic disease is a relatively serious cardiovascular disease, and common diseases include aortic dissection, wall hematoma, atherosclerosis, aneurysm, ma Fanzeng syndrome, congenital aortic disease, traumatic disease, aortic inflammation, etc. Aortic lesions have a complex mechanism and are involved in a wide variety of cells. With the development of single cell sequencing technology, the analysis results of related participating cells have important influence on the attack of aortic diseases. However, due to the high toughness of the aortic blood vessels, the preparation of single cell suspension is difficult, and single cells with high efficiency, high activity and strong functions are difficult to obtain for the research of related pathology.
Therefore, a preparation method for obtaining single-cell suspension with high efficiency and high quality is imperative.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a preparation method of an aortic cell single-cell suspension, which aims to solve the technical problem of low cell quality obtained by low efficiency of the traditional aortic cell single-cell suspension in the related art.
The invention provides a preparation method of aortic cell single-cell suspension, which comprises the following steps:
S1, taking aortic vascular tissue, cleaning, and then aseptically culturing in a 1640 culture medium containing 8% -20% of bovine serum at a first temperature for later use;
S2, taking the aortic vascular tissue prepared in the step S1, peeling fat and connective tissue, and chopping the aortic vascular tissue into tissue fragments of 1-2 mm 3 for later use;
S3, respectively taking the tissue fragments for standby and the existing tissue digestion suspension in the step S2 according to the volume ratio of (5-8), mixing, placing in an environment of 37 ℃ for a first time of digestion reaction until no formed tissue fragments exist, filtering, centrifuging, and removing supernatant to obtain a first cell suspension;
S4, according to the volume ratio of 1: respectively taking the first cell suspension and pancreatin obtained in the step S3 according to the ratio of (1.8-3), mixing, placing in an environment of 37 ℃ for a second digestion reaction for a second period of time, filtering, centrifuging, and removing supernatant to obtain a second cell suspension;
s5, re-suspending the second cell suspension obtained in the step S4 by using a 1640 culture medium containing 10% bovine serum to obtain the target single cell suspension.
Optionally, in step S3, the tissue digestion suspension comprises collagenase I in a volume ratio (10:10:10:0:70-35:35:29:1:0): collagenase II: hyaluronidase: dnase: 1640 medium with 10% bovine serum.
Optionally, in step S1, the first temperature is 0 to 4 ℃.
Optionally, in step S1, the specific step of aseptically culturing in 1640 medium containing 8% -20% bovine serum at the first temperature includes:
Placed on ice and aseptically cultured in 1640 medium containing 10% bovine serum.
Optionally, in steps S3 and S4, the specific steps of filtering and centrifuging include:
the mixture was filtered through a 100um sieve and centrifuged at 1500rpm for 25 to 45 minutes.
Optionally, in step S4, the pancreatin is pancreatin with a mass concentration of 0.02%.
Optionally, in step S3, the first duration is 4 to 5 hours; and/or the number of the groups of groups,
In step S4, the second duration is 18 to 35 minutes.
Optionally, the bovine serum is neonatal bovine serum.
Compared with the prior art, the invention has the following beneficial effects:
In the technology of the invention, the preparation method of the single-cell suspension of the aortic cells is designed, the aortic vascular tissue is treated firstly, and then the single-cell suspension is obtained through at least secondary digestion reaction, so that the technical barriers that the aortic vascular tissue is high in toughness and the single-cell suspension of the aortic vascular cells is difficult to prepare are overcome, the single-cell suspension with high single-cell yield, multiple numbers, high cell activity and strong cell function is obtained, and the technical problem of low cell quality obtained by the traditional single-cell suspension of the aortic cells is effectively solved.
Drawings
FIG. 1 is an electron micrograph of a single cell suspension prepared by mechanically disrupting aortic vascular tissue;
FIG. 2 is data of results of a cell test on single cell suspensions prepared by mechanically disrupting aortic vascular tissue;
fig. 3 to 8 are electron microscopic images of single cell suspensions prepared by treating aortic vascular tissues with tissue digestion suspensions of different volume ratios according to the present invention.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, the technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention provides a preparation method of aortic cell single-cell suspension, which comprises the following steps:
S1, taking aortic vascular tissue, cleaning, and then aseptically culturing in a 1640 culture medium containing 8% -20% of bovine serum at a first temperature for later use;
S2, taking the aortic vascular tissue prepared in the step S1, peeling fat and connective tissue, and chopping the aortic vascular tissue into tissue fragments of 1-2 mm 3 for later use;
S3, respectively taking the tissue fragments for standby and the existing tissue digestion suspension in the step S2 according to the volume ratio of (5-8), mixing, placing in an environment of 37 ℃ for a first time of digestion reaction until no formed tissue fragments exist, filtering, centrifuging, and removing supernatant to obtain a first cell suspension;
S4, according to the volume ratio of 1: respectively taking the first cell suspension and pancreatin obtained in the step S3 according to the ratio of (1.8-3), mixing, placing in an environment of 37 ℃ for a second digestion reaction for a second period of time, filtering, centrifuging, and removing supernatant to obtain a second cell suspension;
s5, re-suspending the second cell suspension obtained in the step S4 by using a 1640 culture medium containing 10% bovine serum to obtain the target single cell suspension.
Optionally, in step S3, the tissue digestion suspension comprises collagenase I in a volume ratio (10:10:10:0:70-35:35:29:1:0): collagenase II: hyaluronidase: dnase: 1640 medium with 10% bovine serum. For example, but not limited to, in one embodiment, collagenase I: collagenase II: hyaluronidase: dnase: the volume ratio of the 1640 culture medium containing 10% of bovine serum is 25:25:30:1:9.
It will be appreciated that the above formulated tissue digestion suspension needs to be filtered through a 0.22um filter membrane to be sterilized.
For example, but not limited to, in step S3, the tissue fragments prepared in step S2 and the freshly prepared tissue digestion suspension are taken in a ratio of 1:6 by volume, mixed, and subjected to a first digestion reaction in an environment at 37℃for 4 hours. In the step S4, the first cell suspension and pancreatin obtained in the step S3 are respectively taken according to the volume ratio of 1:2, and are mixed and then placed in the environment of 37 ℃ for a second digestion reaction for 20min.
Optionally, in step S1, the first temperature is 0 to 4 ℃. For example, but not limited to, the first temperature is 4 ℃.
Optionally, in step S1, the specific step of aseptically culturing in 1640 medium containing 8% -20% bovine serum at the first temperature includes:
Placed on ice and aseptically cultured in 1640 medium containing 10% bovine serum.
Optionally, in steps S3 and S4, the specific steps of filtering and centrifuging include:
The mixture was filtered through a 100um sieve and centrifuged at 1500rpm for 25 to 45 minutes. For example, but not limited to, the centrifugation time is 30 minutes.
Optionally, in step S4, the pancreatin is pancreatin with a mass concentration of 0.02%.
Optionally, in step S3, the first duration is 4 to 5 hours. For example, but not limited to, the first duration is 4.5 hours.
Optionally, in step S4, the second duration is 18-35 minutes. For example, but not limited to, the second duration is 20 minutes.
Optionally, the bovine serum is neonatal bovine serum.
To further illustrate the excellent effects of the method for preparing aortic cell single-cell suspension provided by the invention, the following examples are selected for illustration. It should be understood that the following examples are only for illustrating the effect of the preparation method of the present invention and are not limiting the preparation method of the present invention.
The experimental methods used in the following examples were conventional methods unless otherwise specified. Materials, reagents, equipment and the like used in the examples described below are commercially available unless otherwise specified. Specific reagents used in the following example sets were as follows:
Collagenase I (sigma company, cat# 1148089, concentration 4.1 mg/mL);
Collagenase II (sigma company, cat# 1148090, concentration 2.6 mg/mL);
hyaluronidase (sigma company, cat# H1115000, concentration 1.6 mg/mL);
DNase (sigma company, cat# 10104159001, concentration 1.0 mg/mL).
Preparation of single cell suspension by mechanical disruption of aortic vascular tissue
1. Experimental operation: taking aortic vascular tissues of a person, cleaning blood and fragment tissues, immediately placing the aortic vascular tissues into a culture flask containing 1640 culture medium of 10% bovine serum for aseptic culture, and placing the culture flask on ice for later use; taking the spare aortic vascular tissue, peeling fat and connective tissue, and mechanically crushing for later use; the single cell suspension of aortic vascular tissue was resuspended in 1640 medium with 10% fcs. Performing electron microscope observation on the single cell suspension to obtain an electron microscope image shown in figure 1; the single cell suspension was tested to obtain a cell test sheet as shown in FIG. 2.
2. Analysis of results: as can be seen from FIG. 1, the single cell suspension obtained by this method has a large number of cell fragments and serious cell adhesion phenomenon. As can be seen from FIG. 2, the single cell suspension prepared by the method has a cell yield of only 16%, the average diameter of the cells is 6.21um, and the agglomeration rate is as high as 69.83%.
Example group single cell suspensions obtained after treatment of different concentrations of tissue digestion suspensions
1. Experimental operation:
S1, taking aortic vascular tissues, cleaning blood and fragment tissues, immediately placing the aortic vascular tissues into a culture flask containing 1640 culture medium of 10% bovine serum for aseptic culture, and placing the culture flask on ice for later use;
s2, taking the aortic vascular tissue prepared in the step S1, peeling fat and connective tissue, shearing the aortic vascular tissue to tissue fragments of 1-2 mm 3 by using aseptic surgical scissors, and transferring the tissue fragments into a 15ml centrifuge tube for standby;
S3, respectively taking the tissue fragments and the freshly prepared tissue digestion suspension (the components of the tissue digestion suspension and the volume of the components are shown in the table 1) which are prepared in the step S2 according to the volume ratio of 1:6, mixing, then placing the mixture in an environment of 37 ℃ for shaking to perform a first digestion reaction for 4.5 hours until no formed tissue fragments exist, filtering the mixture through a 100um screen, centrifuging the mixture at 1500rpm for 25-45 minutes, and removing supernatant to obtain a first cell suspension;
S4, according to the volume ratio of 1:2, respectively taking the first cell suspension obtained in the step S3 and pancreatin with the concentration of 0.02%, mixing, placing in an environment of 37 ℃ for a second digestion reaction for 20min, filtering by a 100um screen, centrifuging at 1500rpm for 25-45 min, and removing supernatant to obtain a second cell suspension;
S5, re-suspending the second cell suspension obtained in the step S4 by using a 1640 culture medium containing 10% FCS to obtain the target single cell suspension. Electron microscopy was performed to obtain electron microscopy images as shown in fig. 3 to 8, and data of single cell suspensions treated with the tissue digestion suspensions of different volume ratios in table 1.
TABLE 1 influence of different volume fractions of treatment of aortic vascular tissue on the prepared single cell suspensions
Note that: the volume ratios of the components of the tissue digestion suspensions in table 1 refer to collagenase I: collagenase II: hyaluronidase: dnase: the volume ratio of the 1640 culture medium containing 10% of bovine serum.
2. Analysis of results: as can be seen from fig. 3 to 8, the single cell suspension obtained by treating aortic vascular tissue by the preparation method of the single cell suspension of aortic cells proposed in the present invention has less cell debris and no obvious cell connection agglomeration phenomenon.
As can be seen from table 1, when collagenase I: collagenase II: the volume ratio of the hyaluronic acid to the hyaluronic acid is too low, and DNase: when the ratio of the 1640 culture medium containing 10% of bovine serum is too high, the cell yield of single cell suspension is low and the caking rate is high; specifically, cell yields can be as low as 45.09% and cell clumping rates can be as high as 60.12%. When collagenase I: collagenase II: the volume ratio of the hyaluronic acid to the hyaluronic acid is too high, and the DNase: when the ratio of the 1640 culture medium containing 10% of bovine serum is too low, the cell yield is improved, but the cell aggregation rate is not obvious, and the cell aggregation rate is reduced; specifically, the cell yield can reach 47.24%, and the cell agglomeration rate can reach 52.14%. In collagenase I: collagenase II: hyaluronidase: dnase: when the volume ratio of the 1640 culture medium containing 10% of bovine serum is 25:25:30:1:9, the cell yield can reach 95.78, and the cell agglomeration rate can be as low as 17.33%; and the average diameter of the cells isolated from the cell suspension was 11um.
In conclusion, the single-cell suspension prepared by the preparation method of the single-cell suspension of the aortic cells has the characteristics of high single-cell yield, multiple cell numbers, high cell activity, strong cell function and the like, effectively solves the technical problem of poor cell quality obtained by low efficiency of the traditional single-cell suspension of the aortic cells, and has great significance for clinical medicine and vascular cell research.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (4)
1. A method of preparing an aortic cell single cell suspension comprising:
S1, taking aortic vascular tissue, cleaning, and then aseptically culturing in a 1640 culture medium containing 8% -20% of bovine serum at a first temperature for later use;
S2, taking the aortic vascular tissue prepared in the step S1, peeling fat and connective tissue, and chopping the aortic vascular tissue into tissue fragments of 1-2 mm 3 for later use;
S3, respectively taking the tissue fragments for standby and the existing tissue digestion suspension in the step S2 according to the volume ratio of (5-8), mixing, placing in an environment of 37 ℃ for a first time of digestion reaction until no formed tissue fragments exist, filtering, centrifuging, and removing supernatant to obtain a first cell suspension;
S4, according to the volume ratio of 1: respectively taking the first cell suspension and pancreatin obtained in the step S3 according to the ratio of (1.8-3), mixing, placing in an environment of 37 ℃ for a second digestion reaction for a second period of time, filtering, centrifuging, and removing supernatant to obtain a second cell suspension;
s5, re-suspending the second cell suspension obtained in the step S4 by using a 1640 culture medium containing 10% bovine serum to obtain a target single cell suspension;
The tissue digestion suspension comprises collagenase I: collagenase II: hyaluronidase: dnase: the volume ratio of the 1640 culture medium containing 10% bovine serum is 25:25:30:1:9, wherein the pancreatin is pancreatin with the mass concentration of 0.02%, and the first time period is 4-5 hours; and/or, in the step S4, the second time period is 18-35 minutes; the first temperature is 0-4 ℃.
2. The method of claim 1, wherein in step S1, the step of aseptically culturing in 1640 medium containing 8% -20% bovine serum at the first temperature comprises:
Placed on ice and aseptically cultured in 1640 medium containing 10% bovine serum.
3. The method of claim 1, wherein in steps S3 and S4, the specific steps of filtering and centrifuging include:
the mixture was filtered through a 100um sieve and centrifuged at 1500rpm for 25 to 45 minutes.
4. A method of preparing an aortic cell single cell suspension as claimed in any one of claims 1 to 3 wherein the bovine serum is neonatal bovine serum.
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| Hu Desheng 等.Preparation of Single Cell Suspensions from Mouse Aorta.Bio-protocol.2016,第6卷(第11期),全文. * |
| 贾连群 等.常见心血管病的分子免疫基础与临床.北京：中国医药科技出版社,2013,第178页第2段. * |
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