CN115322961B - Dissociation liquid and dissociation method for in vitro culture of myocardial cells - Google Patents
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- 238000010494 dissociation reaction Methods 0.000 title claims abstract description 96
- 230000005593 dissociations Effects 0.000 title claims abstract description 96
- 230000002107 myocardial effect Effects 0.000 title claims abstract description 41
- 239000007788 liquid Substances 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000000338 in vitro Methods 0.000 title claims abstract description 14
- 210000004027 cell Anatomy 0.000 claims abstract description 106
- 102000004142 Trypsin Human genes 0.000 claims abstract description 24
- 108090000631 Trypsin Proteins 0.000 claims abstract description 24
- 239000012588 trypsin Substances 0.000 claims abstract description 24
- 108010007093 dispase Proteins 0.000 claims abstract description 13
- LZAXPYOBKSJSEX-UHFFFAOYSA-N blebbistatin Chemical compound C1CC2(O)C(=O)C3=CC(C)=CC=C3N=C2N1C1=CC=CC=C1 LZAXPYOBKSJSEX-UHFFFAOYSA-N 0.000 claims abstract description 10
- 210000004413 cardiac myocyte Anatomy 0.000 claims description 30
- 238000010009 beating Methods 0.000 claims description 23
- 238000006386 neutralization reaction Methods 0.000 claims description 17
- 108090000790 Enzymes Proteins 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 13
- 229960003531 phenolsulfonphthalein Drugs 0.000 claims description 9
- 210000002966 serum Anatomy 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000006143 cell culture medium Substances 0.000 claims description 3
- 210000004748 cultured cell Anatomy 0.000 claims description 3
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 claims description 2
- 210000001778 pluripotent stem cell Anatomy 0.000 claims description 2
- 230000029087 digestion Effects 0.000 abstract description 20
- 230000000694 effects Effects 0.000 abstract description 14
- 210000000130 stem cell Anatomy 0.000 abstract description 9
- 239000003814 drug Substances 0.000 abstract description 2
- 230000001172 regenerating effect Effects 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 15
- 238000002474 experimental method Methods 0.000 description 11
- 230000003833 cell viability Effects 0.000 description 9
- 238000007667 floating Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- 239000003223 protective agent Substances 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 238000011160 research Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
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- 239000000203 mixture Substances 0.000 description 2
- 210000002894 multi-fate stem cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- LZAXPYOBKSJSEX-GOSISDBHSA-N (S)-blebbistatin Chemical compound O=C([C@]1(O)CC2)C3=CC(C)=CC=C3N=C1N2C1=CC=CC=C1 LZAXPYOBKSJSEX-GOSISDBHSA-N 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
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- 238000011156 evaluation Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000009067 heart development Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 210000005003 heart tissue Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 210000001087 myotubule Anatomy 0.000 description 1
- 230000003472 neutralizing 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
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0657—Cardiomyocytes; Heart cells
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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 discloses a dissociation solution and a dissociation method for in-vitro culture of myocardial cells, belonging to the field of stem cell biology and regenerative medicine. The dissociation solution for isolated culture of myocardial cells of the present invention includes Tryple, dispase II, trypsin, dnase I and blebbistatin. Wherein, tryple, dispase II, trypsin and Dnase I are used as mixed digestion liquid to play a role in digestion and dissociation. And blebbistatin as a protectant during digestion. The invention also discloses a dissociation method, and the dissociation liquid and the dissociation method can be used for rapidly and efficiently obtaining the isolated cultured myocardial cells with the activity rate higher than 85 percent and obtaining a large number of myocardial living cells.
Description
Technical Field
The invention relates to the field of stem cell biology and regenerative medicine, in particular to a dissociation solution and a dissociation method for digesting and culturing myocardial cells in vitro.
Background
The cardiomyocyte is also called myocardial fiber, belongs to involuntary muscle with transverse lines, has the capability of exciting and contracting, and the tail ends of all myocardial fiber branches can be connected with each other to form a muscle fiber net. The present myocardial cell model has wide application in heart diseases, heart development, drug effect evaluation, basic research and preclinical research of drug toxicity test and cell therapy, and the like.
The existing technology for digesting myocardial cells provides various digestion ways, different digestion ways/digestion difficulty, different cell number and different cell viability are presented, and the biggest problem is that a higher level cannot be achieved, namely, a higher cell yield and a higher cell viability are maintained.
Therefore, it is important to explore a dissociation solution and a dissociation method with high dissociation efficiency, high cell yield and high cell activity.
Disclosure of Invention
The invention aims to provide a technical scheme for effectively digesting and culturing myocardial cells in vitro, which comprises dissociation liquid and dissociation method, wherein the dissociation liquid has definite chemical composition, no animal-derived composition and stable batch. By adopting the technical scheme of the invention, the myocardial cells with high yield and high activity rate can be obtained easily, and the obtained cells can be used for subsequent reattachment culture experiments or index detection experiments.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention firstly provides a dissociation solution for in-vitro culture of myocardial cells, wherein the dissociation solution comprises Tryple, dispase II, trypsin, dnase I and blebbistatin.
The dissociation liquid of the invention is different from the traditional dissociation liquid of myocardial cells, and mainly aims at digestion of isolated cultured myocardial cells. The source of the isolated cultured cardiomyocytes comprises multipotent stem cell-derived cardiomyocytes and primary cardiomyocyte re-spread cultured cells; both human and murine sources are included. Whereas most of the traditional cardiomyocyte dissociation solution digests and dissociates heart tissue, so as to obtain cardiomyocytes.
In addition, the dissociated cardiomyocytes obtained by the invention can be used for subsequent reattachment culture experiments or index detection experiments. After re-attachment experiment and re-culture, the cells are normally attached, the floating unattached dead cells are observed to be less by naked eyes, the attached myocardial cells can recover beating 24-36 hours after attachment, and the frequency and range of the beating are unchanged from those before dissociation. It can be seen that the cells obtained by the present invention are more suitable as a cardiomyocyte model for basic research, preclinical research, etc.
In the dissociation solution, tryple, dispase II, trypsin and Dnase I are used as mixed digestion solution to play a role in digestion and dissociation. The blebbistatin is used as a protective agent in the digestion process, mainly inhibits the contraction of myocardial cells, and can play a role in protecting cells after short treatment, so that the cell viability after digestion is improved.
Further, in the dissociation solution, the concentration of the dispease II in the dissociation solution is 0.2-0.6U/mL; preferably, the concentration is 0.5U/mL.
In the dissociation solution, the concentration of the trypsin in the dissociation solution is 0.04-0.06%; preferably, the concentration is 0.05% trypsin.
In the dissociation solution, the concentration of the Dnase I in the dissociation solution is 50-60U/mL; preferably, the concentration is 50U/mL.
In the dissociation solution, the concentration of the blebbistatin in the dissociation solution is 20-30 mu mol/L; preferably, the concentration is 25. Mu. Mol/L.
In the dissociation liquid, the balance of the dissociation liquid is Tryple; preferably, the Tryple is TrypLE TM Express enzyme, phenol red free.
The invention further provides a dissociation method for culturing myocardial cells in vitro, which comprises the following steps: adding a certain volume of dissociation solution into isolated cultured myocardial cells, dissociating for a certain time, observing that more than 80% of cells are mutually separated under a microscope, then adding neutralization solution for neutralization, collecting cells, centrifuging again, discarding the supernatant, and finally adding a certain amount of myocardial cell culture medium, and uniformly mixing.
In the above dissociation method, the isolated cultured cardiomyocytes are stably beating cardiomyocytes, and the source thereof includes pluripotent stem cell-derived cardiomyocytes or primary cardiomyocyte-re-spread cultured cells.
Further, the volume of the added dissociation liquid is 40-60 mu L/cm 2 。
Further, the dissociation condition and time are CO 2 Incubator, incubate at 37℃for 5-8 min.
Further, the neutralization solution is 10% serum.
The final dissociation criteria of the dissociation method are the difficulty of eluting the cells, and the cell yield and the cell activity.
The beneficial effects of the invention are as follows:
the dissociation liquid of the invention has definite chemical components, no animal-derived components and stable batch. The dissociation liquid and the dissociation method can be used for rapidly and efficiently obtaining the isolated cultured myocardial cells with the activity rate higher than 85 percent and obtaining a large number of myocardial living cells.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the drawings.
FIG. 1 shows the comparison of the number of living cells of examples 1-3 and comparative examples 1-3.
FIG. 2 shows the comparison of the activity rates of examples 1-3 and comparative examples 1-3.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments and the accompanying drawings. Like parts in the drawings are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
The materials used in the examples described below, unless otherwise indicated, are all materials commonly used in the art and are commercially available.
The instruments, reagents, cell sources used in the examples of the present invention are as follows:
an automated cell count analyzer, countStar;
detecting fluorescent dye, countStar by AOPI activity;
TrypLE TM express enzyme (1X) -phenol red free (Thermo Fisher Scientific);
Dispase II，(Thermo Fisher Scientific)；
trypsin (Trypsin, 0.25%, EDTA-containing), (Transgen);
DnaseⅠ，(Transgen)；
(-)-Blebbistatin，(Selleck)；
multipotent stem cell-derived cardiomyocytes, (Wicell);
cardiomyocyte medium, (Stem Cell).
Example 1
A dissociation solution for in vitro culture of myocardial cells is prepared from TrypLE TM Express enzyme (1X) -phenol red-free (Thermo Fisher Scientific), dispase II, trypsin, dnase I and (-) -Blebbbistatin;
wherein in the dissociation solution, the final concentration of Dispase II is 0.2U/mL, the final concentration of Trypsin is 0.04% of Trypsin, the final concentration of Dnase I is 50U/mL, the final concentration of (-) -Blebbbistatin is 20 mu mol/L, and the balance is TrypLE TM Express enzyme (1X) -phenol red free (Thermo Fisher Scientific).
The dissociation method comprises the following steps: adding 50. Mu.L/cm to human induced multifunctional Stem cell derived cardiomyocytes 2 The dissociation liquid, CO, is exemplified by 12-well plate 2 Dissociation was carried out in an incubator at 37℃for 5-8 minutes, and it was observed under a microscope that more than 80% of the cells were separated from each other, then neutralization was carried out by adding into a neutralization solution (10% serum), gently beating, collecting the cells, re-centrifuging, discarding the supernatant, finally adding into a certain amount of cardiomyocyte medium, gently beating and mixing, then cell counting was carried out by using an automatic cell count analyzer, and cell viability data were obtained.
The specific experimental results are shown in table 1. The number of living cells was (1.26.+ -. 0.040). Times.10 6 The activity was (85.32.+ -. 0.014)%. After re-attachment experiments and re-culture, the cells are normally attached, the floating unattached dead cells are observed to be less by naked eyes, the attached myocardial cells can recover beating 24-36 hours after attachment, and the frequency and range of the beating are unchanged from those before dissociation.
TABLE 1 digestion of cells in example 1
Example 2
A dissociation solution for in vitro culture of myocardial cells is prepared from TrypLE TM Express enzyme (1X) -phenol red-free (Thermo Fisher Scientific), dispase II, trypsin, dnase I and (-) -Blebbbistatin;
wherein, in the dissociation solution, the final concentration of the dispersose II is 0.5U/mL, trypsinTrypsin with final concentration of 0.05%, dnase I with final concentration of 50U/mL, (-) -Blebbbistatin with final concentration of 25 μmol/L, balance TrypLE TM Express enzyme (1X) -phenol red free (Thermo Fisher Scientific).
The dissociation method comprises the following steps: adding 50. Mu.L/cm to human induced multifunctional Stem cell derived cardiomyocytes 2 The dissociation liquid, CO, is exemplified by 12-well plate 2 Dissociation was carried out in an incubator at 37℃for 5-8 minutes, and it was observed under a microscope that more than 80% of the cells were separated from each other, then neutralization was carried out by adding into a neutralization solution (10% serum), gently beating, collecting the cells, re-centrifuging, discarding the supernatant, finally adding into a certain amount of cardiomyocyte medium, gently beating and mixing, then cell counting was carried out by using an automatic cell count analyzer, and cell viability data were obtained.
The specific experimental results are shown in Table 2. The number of living cells was (1.28.+ -. 0.036). Times.10 6 The survival rate is (89.39+/-0.015)%, after re-attachment experiment and re-culture, the cells are normally attached, the floating unattached dead cells are less observed by naked eyes, the attached myocardial cells can recover beating 24-36 hours after attachment, and the frequency and range of the beating are unchanged from those before dissociation.
TABLE 2 digestion of cells in example 2
Example 3
A dissociation solution for in vitro culture of myocardial cells is prepared from TrypLE TM Express enzyme (1X) -phenol red-free (Thermo Fisher Scientific), dispase II, trypsin, dnase I and (-) -Blebbbistatin;
wherein in the dissociation solution, the final concentration of Dispase II is 0.6U/mL, the final concentration of Trypsin is 0.06% of Trypsin, the final concentration of Dnase I is 60U/mL, the final concentration of (-) -Blebbbistatin is 30 mu mol/L, and the balance is TrypLE TM Express enzyme (1X) -phenol red free (Thermo Fisher Scientific).
The dissociation method comprises the following steps: addition of 50 mu to human induced multifunctional Stem cell derived cardiomyocytesL/cm 2 The dissociation liquid, CO, is exemplified by 12-well plate 2 Dissociation was carried out in an incubator at 37℃for 5-8 minutes, and it was observed under a microscope that more than 80% of the cells were separated from each other, then neutralization was carried out by adding into a neutralization solution (10% serum), gently beating, collecting the cells, re-centrifuging, discarding the supernatant, finally adding into a certain amount of cardiomyocyte medium, gently beating and mixing, then cell counting was carried out by using an automatic cell count analyzer, and cell viability data were obtained.
The specific experimental results are shown in Table 3. The number of living cells was (1.20.+ -. 0.035). Times.10 6 The survival rate is (85.12+/-0.008)%, after re-attachment experiment and re-culture, the cells are normally attached, the floating unattached dead cells are less observed by naked eyes, the attached myocardial cells can recover beating 24-36 hours after attachment, and the frequency and range of the beating are unchanged from those before dissociation.
TABLE 3 digestion of cells in example 3
Comparative example 1
A dissociation solution for in vitro culture of myocardial cells is prepared from TrypLE TM Express enzyme (1X) -phenol red free (Thermo Fisher Scientific), dispase II, trypsin, and Dnase i;
wherein in the dissociation solution, the final concentration of Dispase II is 0.5U/mL, the final concentration of Trypsin is 0.05% of Trypsin, the final concentration of Dnase I is 50U/mL, and the balance is TrypLE TM Express enzyme (1X) -phenol red free (Thermo Fisher Scientific).
The dissociation method comprises the following steps: adding 50. Mu.L/cm to human induced multifunctional Stem cell derived cardiomyocytes 2 The dissociation liquid, CO, is exemplified by 12-well plate 2 Dissociating at 37deg.C for 5-8 min in incubator, observing that more than 80% of cells are separated from each other under microscope, adding neutralizing solution (10% serum) for neutralization, gently blowing, collecting cells, centrifuging, discarding supernatant, adding a certain amount of myocardial cell culture medium, gently blowing, mixing, and makingCell counts were performed with an automated cell count analyzer and cell viability data was obtained.
The specific experimental results are shown in Table 4. The number of living cells was (1.01.+ -. 1.101). Times.10 6 The survival rate is (74.70 +/-0.059)%, after re-attachment experiment and re-culture, the cells are normally attached, the floating unattached dead cells are observed with naked eyes, and the attached myocardial cells can recover beating after 48-72 hours after attachment.
TABLE 4 digestion of cells in comparative example 1
Comparative example 2
A dissociation solution for in vitro culture of myocardial cells is prepared from TrypLE TM Express enzyme (1X) -phenol-free red (Thermo Fisher Scientific) (Tryple is often selected in literature for digestion and dissociation of cardiomyocytes) and (-) -Blebbbistatin;
wherein, in the dissociation solution, the final concentration of (-) -Blebbbistatin is 25 mu mol/L, and the balance is TrypLE TM Express enzyme (1X) -phenol red free (Thermo Fisher Scientific).
The dissociation method comprises the following steps: adding 50. Mu.L/cm to human induced multifunctional Stem cell derived cardiomyocytes 2 The dissociation liquid, CO, is exemplified by 12-well plate 2 Dissociation was carried out in an incubator at 37℃for 5-8 minutes, and it was observed under a microscope that more than 80% of the cells were separated from each other, then neutralization was carried out by adding into a neutralization solution (10% serum), gently beating, collecting the cells, re-centrifuging, discarding the supernatant, finally adding into a certain amount of cardiomyocyte medium, gently beating and mixing, then cell counting was carried out by using an automatic cell count analyzer, and cell viability data were obtained.
The specific experimental results are shown in Table 5. The number of living cells was (0.97.+ -. 0.064). Times.10 6 The survival rate is (78.23 +/-0.013)%, after re-attachment experiments and re-culture, the cells are normally attached, the floating unattached dead cells are observed to be more with naked eyes, and the attached myocardial cells can resume beating after 36-48 hours after attachment.
TABLE 5 digestion of cells in comparative example 2
Comparative example 3
A dissociation solution for isolated culture of cardiomyocytes, the dissociation solution comprising Trypsin (Trypsin is often used for digestion and dissociation of cardiomyocytes in literature) and (-) -Blebbbistatin;
wherein, in the dissociation solution, the final concentration of the Trypsin is 0.25 percent, and the final concentration of the (-) -Blebbbistatin is 25 mu mol/L.
The dissociation method comprises the following steps: adding 50. Mu.L/cm to human induced multifunctional Stem cell derived cardiomyocytes 2 The dissociation liquid, CO, is exemplified by 12-well plate 2 Dissociation was carried out in an incubator at 37℃for 5-8 minutes, and it was observed under a microscope that more than 80% of the cells were separated from each other, then neutralization was carried out by adding into a neutralization solution (10% serum), gently beating, collecting the cells, re-centrifuging, discarding the supernatant, finally adding into a certain amount of cardiomyocyte medium, gently beating and mixing, then cell counting was carried out by using an automatic cell count analyzer, and cell viability data were obtained.
The specific experimental results are shown in Table 6. The number of living cells was (1.05.+ -. 0.064). Times.10 6 The survival rate is (78.23 +/-0.013)%, after re-attachment experiments and re-culture, the cells are normally attached, the floating unattached dead cells are observed to be more with naked eyes, and the attached myocardial cells can resume beating after 36-48 hours after attachment.
TABLE 6 digestion of cells in comparative example 3
Furthermore, the above experimental results are shown in comparison of fig. 1 and 2:
FIG. 1 shows a comparison of the number of living cells of examples 1-3 and comparative examples 1-3. Example 2 has the highest average number of living cells, example 3 times, followed by example 1, and the number of living cells of comparative examples 1-3 is lower than that of example. It can be seen that the number of living cells in each example of the present invention is higher than that in each comparative example, and that of living cells to which the protective agent blebbistatin was added is higher than that to which the protective agent was not added.
The comparative cases of the activity rates of examples 1 to 3 and comparative examples 1 to 3 are shown in FIG. 2. Example 2 has the highest average activity, example 3 times followed by example 1, and comparative examples 1-3 have lower activity than examples. It can be seen that the activity rate of each example of the present invention is higher than that of each comparative example, and the activity rate of the added protective agent blebbistatin is higher than that of the unprotected agent.
In conclusion, the survival rate of isolated and isolated cultured myocardial cells can be obviously improved by adding the protective agent blebbistatin, and the concentration range of the protective agent is 20-30 mu mol/L. In addition, compared with single digestive juice, tryple or Trypsin, which is commonly used for myocardial cells in the literature, the composite dissociation liquid has more stable digestion and dissociation capabilities, and can obtain myocardial cells with higher activity.
It should be understood that the foregoing examples of the present invention are provided merely for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention, and that various other changes and modifications may be made therein by one skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims.
Claims (7)
1. A dissociation solution for in vitro culture of myocardial cells, which is characterized by comprising Tryple, dispase II, trypsin, dnase I and blebbistatin; wherein,,
the concentration of the dispease II in the dissociation liquid is 0.2-0.6U/mL;
trypsin with the concentration of 0.04-0.06% in the dissociation liquid;
the concentration of the Dnase I in the dissociation liquid is 50-60U/mL;
the concentration of the blebbistatin in the dissociation liquid is 20-30 mu mol/L;
the balance of the dissociation liquid is Tryple.
2. The dissociation liquid of claim 1, wherein said Tryple is Tryple TM Express enzyme, phenol red free.
3. A method of dissociating in vitro cultured cardiomyocytes, the method comprising: adding a certain volume of dissociation solution according to claim 1 or 2 into isolated cultured myocardial cells, dissociating for a certain time, observing that more than 80% of cells are mutually separated under a microscope, adding a neutralization solution for neutralization, collecting cells, centrifuging, discarding the supernatant, and finally adding a certain amount of myocardial cell culture medium, and uniformly mixing.
4. The method of claim 3, wherein the isolated cultured cardiomyocytes are stably beating cardiomyocytes derived from a source comprising pluripotent stem cell-derived cardiomyocytes or primary cardiomyocyte-re-spread cultured cells.
5. A dissociation method as claimed in claim 3, wherein the volume of the dissociation liquid added is 40-60. Mu.L/cm 2 。
6. The method of claim 3, wherein the conditions and time of dissociation are CO 2 Incubator, incubate at 37℃for 5-8 min.
7. A method of dissociation according to claim 3, characterised in that the neutralisation solution is 10% serum.
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