CN102174501A - Preparation method of three-dimensional histioid cardiac muscular tissue for studying simulated microgravity effect - Google Patents
Preparation method of three-dimensional histioid cardiac muscular tissue for studying simulated microgravity effect Download PDFInfo
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Abstract
The invention relates to a preparation method of a three-dimensional histioid cardiac muscular tissue for studying a simulated microgravity effect, in particular relating to a preparation method of a three-dimensional histioid cardiac muscular tissue microballoon, aiming at solving the problems that according to the current cell three-dimensional culture method for studying the simulated microgravity effect, the cytodex microcarrier material is toxic and is not conducive to generation of cardiocytes, a great number of cells are needed for culture, cell harvesting is difficult, the cells are prone to interference of shearing force when being cultured in a klinostat and the continual contraction time of the cardiocytes is short. The method comprises the following steps: preparing a mixed solution; preparing cardiocytes; preparing a cell suspension, adding the cell suspension to a divalent positive ion solution to obtain microballoons; culturing the mircoballons in a culture medium to form the three-dimensional histioid cardiac muscular tissue. The three-dimensional histioid cardiac muscular tissue prepared by the method prepared by the invention can be cultured on a long-term basis, needs no passage and can maintain contraction of the cardiac muscular tissue for a long time. The method is applied to the research fields of space biology, aerospace medicine and tissue engineering.
Description
Technical field
The present invention relates to the preparation method of a kind of three-dimensional class cardiac muscular tissue.
Background technology
Outer space environment has features such as microgravity, very low temperature, high vacuum and severe radiation, and wherein, microgravity has a significant effect to human body physiological function.The aerospace medicine investigator has carried out medical observation and the research of a large amount of microgravitys to the function of human body influence both at home and abroad, find that long-time space flight can cause that pathological change to a certain degree appears in physiological function, comprises that mainly the pathologic of cardiovascular systems, musculature, immunity system and Skeletal system changes.Because cardiovascular systems is being kept the normal activities of human body, therefore study the influence of microgravity to cardiovascular system of human body, inquire into its mechanism and formulate effective safeguard procedures, for guarantee the spacefarer when the space flight health and effectively work is significant.
Myocardial cell's model of research microgravity effect mainly is by culturing cell in gyrator at present, thereby reaches the deterministic simulation microgravity environment.Myocardial cell's three-dimensional culture method is to be used to study simulated microgravity effect cell culture processes the most widely at present, makes the myocardial cell be grown in the surface of cytodex microcarrier.There are many deficiencies in this method: (1) cytodex microcarrier material has toxicity, can have a negative impact by pair cell; The cell concentration that needs when (2) cultivating is big, collects the cell difficulty; When (3) in gyrator, cultivating, be subject to the interference of the shearing force of factor generations such as bubble; (4) myocardial cell's lasting systolic time is short.
Summary of the invention
The present invention will solve the cell three-dimensional culture method cytodex microcarrier material that is used for studying the simulated microgravity effect at present to have toxicity and the myocardial cell is had a negative impact, the cell concentration that needs during cultivation is big, collect the cell difficulty, cell is subject to the interference of shearing force when cultivating in gyrator, the problem that myocardial cell's lasting systolic time is short provides a kind of preparation method who is used to study the three-dimensional class cardiac muscular tissue microballoon of simulated microgravity effect.
A kind of preparation method who is used to study the three-dimensional class cardiac muscular tissue of simulated microgravity effect of the present invention, carry out according to the following steps: one, be 0.05%～3% sodium alginate soln with mass concentration be that 10%～50% type i collagen protein solution mixes, obtain mixing solutions mass concentration; Two, will the be born Wistar rat suckling mouse heart of back 1～3d places the PBS solution of 0～4 ℃ 0.1mol/L, with the PBS solution cleaning heart of 0～4 ℃ 0.1mol/L 3～5 times, again heart tissue is cut into 1～3mm then
3The tissue block of size, PBS solution with 0～4 ℃ 0.1mol/L cleans 3～5 times again, then tissue block is digested 10～30min with Digestive system, the DMEM substratum that adds 10 times of volumes of tissue block again, abandon supernatant after centrifugal, in precipitation, add the DMEM substratum then and make cell suspension A, cell suspension A is inoculated in the culturing bottle, adherent 90～the 120min of differential obtains the myocardial cell of purifying; Three, the myocardial cell with purifying mixes with the mixing solutions that step 1 makes, and making cell density is 0.5 * 10
6～5 * 10
6Myocardial cell's suspension of individual/mL adopts syringe needle that myocardial cell's suspension is dropwise joined in the positive solion of divalence of 25～100mmol/L, leaves standstill 5～20min, obtains wrapping up myocardial cell's microballoon; Four, to add mass concentration be after leaving standstill 5～20min in 0.1%～3% the sodium alginate soln to the microballoon that will wrap up the myocardial cell, put into mass concentration again and be 0.5～2% Poly-L-Lysine Solution and leave standstill 5～20min, to be put in the DMEM substratum through the parcel myocardial cell's of leaving standstill microballoon then and carry out 1～2 week of dimensional culture, promptly form three-dimensional class cardiac muscular tissue with the function that continues to beat; Wherein the quality of type i collagen protein solution is 0.1%～50% of a sodium alginate soln quality in the step 1; Digestive system is made up of pancreatin and collagenase II in the step 2, and the mass concentration of pancreatin is 0.02～0.2% in the Digestive system, and the mass concentration of collagenase II is 0.01～0.1%; The volume of Digestive system is 9～12 times of tissue block volume in the step 2; All contain mass concentration in the DMEM substratum in step 2 and the step 4 and be 10%～15% foetal calf serum; The cell density of cell suspension A in the step 2 is 0.5 * 10
6～5 * 10
6Individual/mL; Divalent cation is calcium ion, barium ion or strontium ion in the divalent cation solution in the step 3; The volume of sodium alginate soln is 9～12 times of microsphere volume in the step 4, and the volume of Poly-L-Lysine Solution is 9～12 times of microsphere volume.
The present invention adopts tissue engineering technique to have the class cardiac muscular tissue of myocardial structural and function in external structure, such cardiac muscular tissue has the characteristics very close with real heart tissue and is convenient to accuracy controlling, utilize such class cardiac muscular tissue to replace the myocardial cell to be used to study the myocardial structural under the simulated weightlessness conditions and the The Molecular Biology Mechanism of changing function, build a three-dimension cardiac muscle cell cultures platform that is applicable to space life science research and space environment risk assessment.
Method of the present invention has the following advantages:
1, myocardial cell's less shearing force that is subjected in microballoon is disturbed;
2, the myocardial cell forms globoferous cell group in microballoon, on biology performance with more approaching in body tissue;
3, microballoon has certain porosity, and the myocardial cell can accept nutritive substance and metabolic waste can be discharged again in microballoon;
4, but myocardial cell's long-term cultivation in microballoon need not to go down to posterity, and is more suitable for the space and carries;
5, use the three-dimensional class cardiac muscular tissue of the inventive method preparation can keep myocardial cell's contractile function for a long time, the time length is 6 months; And the lasting systolic time of present two-dimentional cultural method myocardial cell is 130 days, and it is 2 months that present three-dimensional culture method myocardial cell continues systolic time.
Description of drawings
Fig. 1 is the photo of the parcel myocardial cell's that step 3 obtains in the embodiment 24 microballoon; Fig. 2 amplifies 40 times photo for the parcel myocardial cell's that step 3 in the embodiment 24 obtains microballoon at microscopically; Fig. 3 is that the microballoon after DAPI dyeing amplifies 40 times photo in the embodiment 24 at microscopically; Fig. 4 is the photo of the microballoon of the three-dimensional class of 2 months parcel of cultivation cardiac muscular tissue in the embodiment 24; Fig. 5 is that the microballoon of cultivating 2 months the three-dimensional class of parcel cardiac muscular tissue in the embodiment 24 amplifies 400 times photo at microscopically; Fig. 6 is that 500 times stereoscan photograph amplifies in the three-dimensional class cardiac muscular tissue of cultivating in the embodiment 24 2 months; Fig. 7 is that 4000 times stereoscan photograph amplifies in the three-dimensional class cardiac muscular tissue of cultivating in the embodiment 24 2 months; Fig. 8 for three-dimensional class cardiac muscular tissue in the embodiment 24 at beat the synchronously detection figure of frequency of different incubation times.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment is a kind of to be used to study the preparation method of the three-dimensional class cardiac muscular tissue of simulated microgravity effect, carry out according to the following steps: one, be 0.05%～3% sodium alginate soln with mass concentration be that 10%～50% type i collagen protein solution mixes, obtain mixing solutions mass concentration; Two, will the be born Wistar rat suckling mouse heart of back 1～3d places the PBS solution of 0～4 ℃ 0.1mol/L, with the PBS solution cleaning heart of 0～4 ℃ 0.1mol/L 3～5 times, again heart tissue is cut into 1～3mm then
3The tissue block of size, PBS solution with 0～4 ℃ 0.1mol/L cleans 3～5 times again, then tissue block is digested 10～30min with Digestive system, the DMEM substratum that adds 10 times of volumes of tissue block again, abandon supernatant after centrifugal, in precipitation, add the DMEM substratum then and make cell suspension A, cell suspension A is inoculated in the culturing bottle, adherent 90～the 120min of differential obtains the myocardial cell of purifying; Three, the myocardial cell with purifying mixes with the mixing solutions that step 1 makes, and making cell density is 0.5 * 10
6～5 * 10
6Myocardial cell's suspension of individual/mL adopts syringe needle that myocardial cell's suspension is dropwise joined in the positive solion of divalence of 25～100mmol/L, leaves standstill 5～20min, obtains wrapping up myocardial cell's microballoon; Four, to add mass concentration be after leaving standstill 5～20min in 0.1%～3% the sodium alginate soln to the microballoon that will wrap up the myocardial cell, put into mass concentration again and be 0.5～2% Poly-L-Lysine Solution and leave standstill 5～20min, to be put in the DMEM substratum through the parcel myocardial cell's of leaving standstill microballoon then and carry out 1～2 week of dimensional culture, promptly form three-dimensional class cardiac muscular tissue with the function that continues to beat; Wherein the quality of type i collagen protein solution is 0.1%～50% of a sodium alginate soln quality in the step 1; Digestive system is made up of pancreatin and collagenase II in the step 2, and the mass concentration of pancreatin is 0.02～0.2% in the Digestive system, and the mass concentration of collagenase II is 0.01～0.1%; The volume of Digestive system is 9～12 times of tissue block volume in the step 2; All contain mass concentration in the DMEM substratum in step 2 and the step 4 and be 10%～15% foetal calf serum; The cell density of cell suspension A in the step 2 is 0.5 * 10
6～5 * 10
6Individual/mL; Divalent cation is calcium ion, barium ion or strontium ion in the divalent cation solution in the step 3; The volume of sodium alginate soln is 9～12 times of microsphere volume in the step 4, and the volume of Poly-L-Lysine Solution is 9～12 times of microsphere volume.
Type i collagen albumen in the present embodiment step 1 is rat type i collagen albumen, for purchase obtains.
The method of present embodiment has the following advantages: myocardial cell's less shearing force that is subjected in microballoon is disturbed; The myocardial cell forms globoferous cell group in microballoon, on biology performance with more approaching in body tissue; Microballoon has certain porosity, and the myocardial cell can accept nutritive substance and metabolic waste can be discharged again in microballoon; But the myocardial cell is long-term cultivation in microballoon, need not to go down to posterity, and is more suitable for the space and carries; Use the three-dimensional class cardiac muscular tissue of the inventive method preparation can keep myocardial cell's contractile function for a long time, the time length is 6 months; And the lasting systolic time of present two-dimentional cultural method myocardial cell is 130 days, and it is 2 months that present three-dimensional culture method myocardial cell continues systolic time.
Embodiment two: what present embodiment and embodiment one were different is: the mass concentration of sodium alginate soln is 0.5%～2% in the step 1.Other is identical with embodiment one.
Embodiment three: what present embodiment was different with embodiment one or two is: the mass concentration of sodium alginate soln is 1% in the step 1.Other is identical with embodiment one or two.
Embodiment four: what present embodiment was different with one of embodiment one to three is: the mass concentration of type i collagen protein solution is 20%～40% in the step 1.Other is identical with one of embodiment one to three.
Embodiment five: what present embodiment was different with one of embodiment one to three is: the mass concentration of type i collagen protein solution is 30% in the step 1.Other is identical with one of embodiment one to three.
Embodiment six: what present embodiment was different with one of embodiment one to five is: the quality of type i collagen protein solution is 5%～40% of a sodium alginate soln quality in the step 1.Other is identical with one of embodiment one to five.
Embodiment seven: what present embodiment was different with one of embodiment one to five is: the quality of type i collagen protein solution is 10%～30% of a sodium alginate soln quality in the step 1.Other is identical with one of embodiment one to five.
Embodiment eight: what present embodiment was different with one of embodiment one to five is: the quality of type i collagen protein solution is 20% of a sodium alginate soln quality in the step 1.Other is identical with one of embodiment one to five.
Embodiment nine: what present embodiment was different with one of embodiment one to eight is: the mass concentration of pancreatin is 0.05～0.15% in the Digestive system of step 2.Other is identical with one of embodiment one to eight.
Embodiment ten: what present embodiment was different with one of embodiment one to eight is: the mass concentration of pancreatin is 0.1% in the Digestive system of step 2.Other is identical with one of embodiment one to eight.
Embodiment 11: what present embodiment was different with one of embodiment one to ten is: the mass concentration of collagenase II is 0.05% in the Digestive system of step 2.Other is identical with one of embodiment one to ten.
Embodiment 12: what present embodiment was different with one of embodiment one to 11 is: the volume of Digestive system is 10 times of tissue block volume in the step 2.Other is identical with one of embodiment one to 11.
Embodiment 13: what present embodiment was different with one of embodiment one to 12 is: in the step 2 tissue block is digested 20min with Digestive system.Other is identical with one of embodiment one to 12.
Embodiment 14: what present embodiment was different with one of embodiment one to 13 is: the adherent 100min of differential in the step 2.Other is identical with one of embodiment one to 13.
Embodiment 15: what present embodiment was different with one of embodiment one to 14 is: the cell density of the cell suspension A in the step 2 is 1 * 10
6～3 * 10
6Individual/mL.Other is identical with one of embodiment one to 14.
Embodiment 16: what present embodiment was different with one of embodiment one to 14 is: the cell density of the cell suspension A in the step 2 is 2 * 10
6Individual/mL.Other is identical with one of embodiment one to 14.
Embodiment 17: what present embodiment was different with one of embodiment one to 16 is: making cell density in the step 3 is 1 * 10
6～3 * 10
6Myocardial cell's suspension of individual/mL.Other is identical with one of embodiment one to 16.
Embodiment 18: what present embodiment was different with one of embodiment one to 16 is: making cell density in the step 3 is 2 * 10
6Myocardial cell's suspension of individual/mL.Other is identical with one of embodiment one to 16.
Embodiment 19: what present embodiment was different with one of embodiment one to 18 is: in the step 3 myocardial cell's suspension is dropwise joined in the positive solion of divalence of 40～80mmol/L.Other is identical with one of embodiment one to 18.
Embodiment 20: what present embodiment was different with one of embodiment one to 18 is: in the step 3 myocardial cell's suspension is dropwise joined in the positive solion of divalence of 60mmol/L.Other is identical with one of embodiment one to 18.
Embodiment 21: what present embodiment was different with one of embodiment one to 20 is: the mass concentration of sodium alginate soln is 0.5%～2% in the step 4.Other is identical with one of embodiment one to 20.
Embodiment 22: what present embodiment was different with one of embodiment one to 20 is: the mass concentration of sodium alginate soln is 1% in the step 4.Other is identical with one of embodiment one to 20.
Embodiment 23: what present embodiment was different with one of embodiment one to 22 is: the mass concentration of Poly-L-Lysine Solution is 1% in the step 4.Other is identical with one of embodiment one to 22.
Embodiment 24: present embodiment is a kind of to be used to study the preparation method of the three-dimensional class cardiac muscular tissue of simulated microgravity effect, carry out according to the following steps: one, be 2% sodium alginate soln with mass concentration be that 30% type i collagen protein solution mixes, obtain mixing solutions mass concentration; Two, will the be born Wistar rat suckling mouse heart of back 3d places the PBS solution of 4 ℃ 0.1mol/L, with the PBS solution cleaning heart of 4 ℃ 0.1mol/L 5 times, again heart tissue is cut into 1～3mm then
3The tissue block of size, PBS solution with 4 ℃ 0.1mol/L cleans 3 times again, then tissue block is digested 30min with Digestive system, the DMEM substratum that adds 10 times of volumes of tissue block again, abandon supernatant after centrifugal, in precipitation, add the DMEM substratum then and make cell suspension A, cell suspension A is inoculated in the culturing bottle, the adherent 120min of differential obtains the myocardial cell of purifying; Three, the myocardial cell with purifying mixes with the mixing solutions that step 1 makes, and making cell density is 2 * 10
6Myocardial cell's suspension of individual/mL adopts syringe needle that myocardial cell's suspension is dropwise joined in the positive solion of divalence of 100mmol/L, leaves standstill 5min, obtains wrapping up myocardial cell's microballoon; Four, to add mass concentration be after leaving standstill 10min in 1% the sodium alginate soln to the microballoon that will wrap up the myocardial cell, the Poly-L-Lysine Solution of putting into mass concentration 0.5% again leaves standstill 10min, to be put in the DMEM substratum through the parcel myocardial cell's of leaving standstill microballoon and carry out 2 weeks of dimensional culture, promptly form three-dimensional class cardiac muscular tissue with the function that continues to beat; Wherein the quality of type i collagen protein solution is 20% of a sodium alginate soln quality in the step 1; Digestive system is made up of pancreatin and collagenase II in the step 2, and the mass concentration of pancreatin is 0.02% in the Digestive system, and the mass concentration of collagenase II is 0.01%; The volume of Digestive system is 10 times of tissue block volume in the step 2; All contain mass concentration in the DMEM substratum in step 2 and the step 4 and be 15% foetal calf serum; The cell density of cell suspension A in the step 2 is 2 * 10
6Individual/mL; Divalent cation is calcium ion, barium ion or strontium ion in the divalent cation solution in the step 3; The volume of sodium alginate soln is 10 times of microsphere volume in the step 4, and the volume of Poly-L-Lysine Solution is 10 times of microsphere volume.
The parcel myocardial cell's that the present embodiment step 3 makes microballoon as illustrated in fig. 1 and 2, Fig. 1 is parcel myocardial cell's the photo of microballoon, Fig. 2 be that microballoon amplifies 40 times photo at microscopically, can find out that the size of microballoon is even, diameter is about 2.3mm.Microballoon is carried out DAPI dyeing, and as shown in Figure 3, Fig. 3 is that the microballoon after DAPI dyeing amplifies 40 times photo at microscopically, can see that the myocardial cell is evenly distributed in microballoon.
The three-dimensional class cardiac muscular tissue of this enforcement preparation is continued to be cultured to 2 months, form macroscopic three-dimensional class cardiac muscular tissue, as shown in Figure 4 and Figure 5, Fig. 4 is the photo of the microballoon of the three-dimensional class of 2 months parcel of cultivation cardiac muscular tissue, and Fig. 5 is the photo of the microballoon of the three-dimensional class of 2 months parcel of cultivation cardiac muscular tissue 400 times of microscopically amplifications.
The three-dimensional class cardiac muscular tissue of cultivating 2 months is discharged from microballoon, carry out scanning electron microscopic observation, Fig. 6 amplifies 500 times stereoscan photograph for the three-dimensional class cardiac muscular tissue of cultivating 2 months, Fig. 7 amplifies 4000 times stereoscan photograph for the three-dimensional class cardiac muscular tissue of cultivating 2 months, can see that class cardiac muscular tissue has the cellularstructure of multilayer densification.
The three-dimensional class cardiac muscular tissue of this enforcement preparation is continued to cultivate, observe its situation of beating, Fig. 8 for three-dimensional class cardiac muscular tissue at beat the synchronously detection figure of frequency of different incubation times, show among the figure that the class myocardial cell has formed the cell-cell communication connection under three-dimensional state, sustainable 2 months of its time of beating, the frequency dimension of beating is held in about 20～30 times.
Claims (10)
1. preparation method who is used to study the three-dimensional class cardiac muscular tissue of simulated microgravity effect, it is characterized in that being used to studying the preparation method of the three-dimensional class cardiac muscular tissue of simulated microgravity effect, carry out according to the following steps: one, be 0.05%～3% sodium alginate soln with mass concentration be that 10%～50% type i collagen protein solution mixes, obtain mixing solutions mass concentration; Two, will the be born Wistar rat suckling mouse heart of back 1～3d places the PBS solution of 0～4 ℃ 0.1mol/L, with the PBS solution cleaning heart of 0～4 ℃ 0.1mol/L 3～5 times, again heart tissue is cut into 1～3mm then
3The tissue block of size, PBS solution with 0～4 ℃ 0.1mol/L cleans 3～5 times again, then tissue block is digested 10～30min with Digestive system, the DMEM substratum that adds 10 times of volumes of tissue block again, abandon supernatant after centrifugal, in precipitation, add the DMEM substratum then and make cell suspension A, cell suspension A is inoculated in the culturing bottle, adherent 90～the 120min of differential obtains the myocardial cell of purifying; Three, the myocardial cell with purifying mixes with the mixing solutions that step 1 makes, and making cell density is 0.5 * 10
6～5 * 10
6Myocardial cell's suspension of individual/mL adopts syringe needle that myocardial cell's suspension is dropwise joined in the positive solion of divalence of 25～100mmol/L, leaves standstill 5～20min, obtains wrapping up myocardial cell's microballoon; Four, to add mass concentration be after leaving standstill 5～20min in 0.1%～3% the sodium alginate soln to the microballoon that will wrap up the myocardial cell, put into mass concentration again and be 0.5～2% Poly-L-Lysine Solution and leave standstill 5～20min, to be put in the DMEM substratum through the parcel myocardial cell's of leaving standstill microballoon then and carry out 1～2 week of dimensional culture, promptly form three-dimensional class cardiac muscular tissue with the function that continues to beat; Wherein the quality of type i collagen protein solution is 0.1%～50% of a sodium alginate soln quality in the step 1; Digestive system is made up of pancreatin and collagenase II in the step 2, and the mass concentration of pancreatin is 0.02～0.2% in the Digestive system, and the mass concentration of collagenase II is 0.01～0.1%; The volume of Digestive system is 9～12 times of tissue block volume in the step 2; All contain mass concentration in the DMEM substratum in step 2 and the step 4 and be 10%～15% foetal calf serum; The cell density of cell suspension A in the step 2 is 0.5 * 10
6～5 * 10
6Individual/mL; Divalent cation is calcium ion, barium ion or strontium ion in the divalent cation solution in the step 3; The volume of sodium alginate soln is 9～12 times of microsphere volume in the step 4, and the volume of Poly-L-Lysine Solution is 9～12 times of microsphere volume.
2. a kind of preparation method who is used to study the three-dimensional class cardiac muscular tissue of simulated microgravity effect according to claim 1, the mass concentration that it is characterized in that sodium alginate soln in the step 1 is 0.5%～2%.
3. a kind of preparation method who is used to study the three-dimensional class cardiac muscular tissue of simulated microgravity effect according to claim 1 and 2, the mass concentration that it is characterized in that the type i collagen protein solution is 20%～40%.
4. a kind of preparation method who is used to study the three-dimensional class cardiac muscular tissue of simulated microgravity effect according to claim 3, the quality that it is characterized in that type i collagen protein solution in the step 1 is 5%～40% of a sodium alginate soln quality.
5. a kind of preparation method who is used to study the three-dimensional class cardiac muscular tissue of simulated microgravity effect according to claim 4 is characterized in that the mass concentration of pancreatin in the Digestive system of step 2 is 0.05～0.15%.
6. a kind of preparation method who is used to study the three-dimensional class cardiac muscular tissue of simulated microgravity effect according to claim 5 is characterized in that the mass concentration of collagenase II in the Digestive system of step 2 is 0.05%.
7. a kind of preparation method who is used to study the three-dimensional class cardiac muscular tissue of simulated microgravity effect according to claim 6, it is characterized in that making in the step 3 cell density is 1 * 10
6～3 * 10
6Myocardial cell's suspension of individual/mL.
8. a kind of preparation method who is used to study the three-dimensional class cardiac muscular tissue of simulated microgravity effect according to claim 7 is characterized in that in the step 3 myocardial cell's suspension dropwise being joined in the positive solion of divalence of 40～80mmol/L.
9. a kind of preparation method who is used to study the three-dimensional class cardiac muscular tissue of simulated microgravity effect according to claim 8, the mass concentration that it is characterized in that sodium alginate soln in the step 4 is 0.5%～2%.
10. a kind of preparation method who is used to study the three-dimensional class cardiac muscular tissue of simulated microgravity effect according to claim 9, the mass concentration that it is characterized in that Poly-L-Lysine Solution in the step 4 is 1%.
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| CN104087550A (en) * | 2014-07-10 | 2014-10-08 | 上海益诺思生物技术有限公司 | Culture method of rat myocardial cell |
| CN104087550B (en) * | 2014-07-10 | 2017-01-04 | 上海益诺思生物技术有限公司 | A kind of cultural method of rat myocardial cell |
| CN111621493A (en) * | 2020-06-10 | 2020-09-04 | 航天中心医院 | Cell-cleavable microcapsule, preparation method thereof and cell culture method |
| CN111621493B (en) * | 2020-06-10 | 2022-02-15 | 航天中心医院 | Cell-cleavable microcapsule, preparation method thereof and cell culture method |
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