CN118325828A - Hydrogel for artificial heart in-vitro drug screening model and application - Google Patents
Hydrogel for artificial heart in-vitro drug screening model and application Download PDFInfo
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- CN118325828A CN118325828A CN202410521926.9A CN202410521926A CN118325828A CN 118325828 A CN118325828 A CN 118325828A CN 202410521926 A CN202410521926 A CN 202410521926A CN 118325828 A CN118325828 A CN 118325828A
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- 238000000338 in vitro Methods 0.000 title claims abstract description 41
- 238000007877 drug screening Methods 0.000 title claims abstract description 18
- 239000000017 hydrogel Substances 0.000 title claims abstract description 18
- 238000007639 printing Methods 0.000 claims abstract description 18
- 239000011159 matrix material Substances 0.000 claims abstract description 17
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 14
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 14
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 14
- 239000000661 sodium alginate Substances 0.000 claims abstract description 14
- 230000002107 myocardial effect Effects 0.000 claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 8
- 210000004413 cardiac myocyte Anatomy 0.000 claims description 7
- 238000001125 extrusion Methods 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 4
- 238000010146 3D printing Methods 0.000 claims description 3
- 238000004132 cross linking Methods 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 2
- 238000005057 refrigeration Methods 0.000 claims description 2
- 239000000843 powder Substances 0.000 abstract description 14
- 239000003814 drug Substances 0.000 abstract description 6
- 210000005003 heart tissue Anatomy 0.000 abstract description 6
- 241000282414 Homo sapiens Species 0.000 abstract description 5
- 238000010009 beating Methods 0.000 abstract description 5
- 239000012620 biological material Substances 0.000 abstract description 5
- 230000004069 differentiation Effects 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- 230000004217 heart function Effects 0.000 abstract description 2
- 238000013537 high throughput screening Methods 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract description 2
- 238000010923 batch production Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 7
- 238000010171 animal model Methods 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 208000019622 heart disease Diseases 0.000 description 2
- 239000002547 new drug Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000004088 simulation Methods 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 102000010825 Actinin Human genes 0.000 description 1
- 108010063503 Actinin Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- BQRGNLJZBFXNCZ-UHFFFAOYSA-N calcein am Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O)=C(OC(C)=O)C=C1OC1=C2C=C(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(=O)C)C(OC(C)=O)=C1 BQRGNLJZBFXNCZ-UHFFFAOYSA-N 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
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- 210000002064 heart cell Anatomy 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 210000004165 myocardium Anatomy 0.000 description 1
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- 238000010186 staining Methods 0.000 description 1
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- Materials For Medical Uses (AREA)
Abstract
The invention discloses a hydrogel for an artificial heart in-vitro drug screening model and application thereof, wherein the hydrogel comprises: heart acellular matrix, gelMA and sodium alginate; wherein, the mass concentration of the heart acellular matrix is 5% -10%; the mass concentration of GelMA is 6-10%; the mass concentration of the sodium alginate is 0.5-1.0%. According to the invention, the heart tissue of the mouse is separated, and then the acellular matrix is separated, and the matrix is mixed with GelMA powder and sodium alginate powder to obtain the biological material of the artificial heart, so that an environment required by growth and differentiation is provided for myocardial cells; meanwhile, the artificial heart is prepared by taking hydrogel as a biological material, and the 3D biological printing technology is used for in-vitro large-scale batch production, so that high-throughput screening of medicines is realized, the artificial heart in-vitro shows the mature heart function phenotype of myocardial beating and the like, is fit with the real heart of a human, and provides a good model for subsequent medicine screening.
Description
Technical Field
The invention relates to the technical field of artificial heart model construction, in particular to hydrogel for an artificial heart in-vitro drug screening model and application thereof.
Background
Heart related diseases have become one of the major causes of threats to human health. In the development process of the traditional new medicine, an animal model plays a key role, and the curative effect and side effect of the medicine can be predicted to a certain extent. However, as large quantities of new drugs are put into clinical trials, the shortcomings of animal models are also becoming more pronounced. As a drug screening means, animal models cannot achieve complete reconstruction of human pathophysiological structures, and suitable animal models are not found for many heart diseases. In addition, animal models have long experimental periods and heavy economic burden, which is another obstacle to development of new drugs. Therefore, in vitro drug screening models based on human or animal cells have been attracting attention from students at home and abroad in recent years.
For in vitro drug screening models, the common 2D cultured cells have serious dedifferentiation phenomenon, and the specific functions of primary myocardial cells are difficult to be preserved. The 3D culture can provide a good growth environment for the myocardial cells, and each function of the primary myocardial cells is reserved to the greatest extent. In addition, 3D bioprinting can further accurately control the distribution of myocardial cells and biological materials in space, and provides possibility for reconstruction of complex heart structures. Since cardiomyocytes are extremely demanding in terms of growth environment, most biological materials have difficulty providing cardiomyocytes with the environment required for growth and differentiation. Therefore, there is a need for a model that can simulate the physiological and pathological characteristics of the heart in vitro to solve the problem of drug screening in vitro for heart diseases.
Therefore, how to provide a material for in vitro simulation of the heart and to construct an in vitro simulation heart model is a problem to be solved by the person skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a hydrogel for an artificial heart in-vitro drug screening model and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a hydrogel for an artificial heart in vitro drug screening model, comprising: heart acellular matrix, gelMA and sodium alginate; wherein, the mass concentration of the heart acellular matrix is 5% -10%; the mass concentration of GelMA is 6-10%; the mass concentration of the sodium alginate is 0.5-1.0%.
After the heart acellular matrix is separated, the heart acellular matrix is mixed with GelMA powder and sodium alginate powder to be dissolved in PBS, and the final mass concentration of the GelMA powder, the sodium alginate powder and the sodium alginate powder is preferably 5%, 8% and 0.5%.
As the invention concept same as the technical scheme, the invention also claims the application of the hydrogel in constructing an artificial heart in-vitro drug screening model.
As the invention concept same as the technical scheme, the invention also claims an artificial heart in-vitro drug screening model, which comprises the following raw materials: hydrogel and neonatal mouse cardiomyocytes.
As the same inventive concept as the above technical solution, the present invention also claims a 3D printing method for constructing an artificial heart in vitro drug screening model, the process comprising:
1) The mixed decellularized matrix hydrogel is mixed with neonatal mouse cardiomyocytes (5X 10 6/ml) at 37 ℃ to prepare the bio-ink required for printing;
2) The hydrogel is placed at 4 ℃ for refrigeration for 8-10min, the diameter of a proper extrusion nozzle is selected, the temperature of a printing nozzle is adjusted to 10-15 ℃, the temperature of a printing platform is adjusted to 5-10 ℃, the bio-ink is printed in a 6cm sterile culture dish by using the extrusion speed of 2-4mm 3/s, and then the printed heart print is placed in CaCl 2 solution with the mass concentration of 3% for crosslinking, and then the heart print is cultivated for a long time.
Preferably, in step 1), the final concentration of cardiomyocytes is 5X 10 6/mL.
Preferably, in step 2), 405nm ultraviolet light is used to cure the print body during printing.
Compared with the prior art, the invention separates the mouse heart tissue, and further divides the acellular matrix, the matrix is mixed with GelMA powder and sodium alginate powder to obtain the biological material of the artificial heart, and provides the environment for the growth and differentiation of myocardial cells; furthermore, the decellularized matrix of the heart provides necessary environment for proliferation and differentiation of myocardial cells, and ensures that the heart printing body has mature function. GelMA and sodium alginate have good printing and curing characteristics, and can be rapidly cured through ultraviolet irradiation and double-crosslinking means of Ca +, so that the artificial heart tissue is constructed.
The artificial heart prepared by the invention can be produced in large scale in vitro by a 3D biological printing technology, realizes high throughput screening of medicines, shows mature heart function phenotypes such as myocardial beating in vitro, is fit with the real heart of human beings, and provides a good model for subsequent medicine screening.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of in vitro heart model construction;
FIG. 2 is a drawing of an artificial heart cultured in vitro;
FIG. 3 is a photograph of an artificial heart under a microscope for 3, 7, and 14 days of in vitro culture;
FIG. 4 is a graph showing cell viability of artificial hearts cultured in vitro for 3, 7, and 14 days;
FIG. 5 is a graph showing myocardial beating curves of artificial hearts cultured in vitro for 3, 7, and 14 days;
FIG. 6 is a graph showing the beating image of an artificial heart cultured in vitro for 3 and 5 days;
FIG. 7 is a graph showing immunofluorescent staining of alpha-actin (alpha-actin) at 3, 7, and 14 days of in vitro culture of artificial hearts.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The heart tissue of the neonatal C57 mice was surgically isolated and surface blood stained with PBS after perfusion. The heart tissue was then incubated in PBS containing 1% Sodium Dodecyl Sulfate (SDS) and 1% Triton X-100 for 72 hours for decellularization. The decellularized heart tissue was lyophilized, the lyophilized powder was dissolved in PBS containing 3% acetic acid (Merck Millipore, USA) and 1mg/ml pepsin (SIGMA ALDRICH, USA), shaken at 37℃for 72 hours, and finally the pH of the dissolved heart decellularized matrix was adjusted to neutral using NaOH. Mixing the powder with GelMA powder and sodium alginate powder, and dissolving in PBS, wherein the final mass concentrations of the powder, the sodium alginate powder and the PBS are 5%, 8% and 4%, respectively. After the acellular matrix hydrogel was prepared, it was mixed with heart cells and then chilled at 4℃for 20 minutes, and the printability was measured. Then selecting proper extrusion nozzle diameter, regulating the temperature of the printing nozzle to 10-15 ℃ and regulating the temperature of the printing platform to 5-10 ℃. The cell-containing bio-ink was printed in a 6cm sterile petri dish using an extrusion speed of 2-4mm3/s, and the print was cured using 405nm uv light during printing. The printed heart prints were placed in 3% CaCl 2 solution to rapidly crosslink them, which were then cultured in vitro for long periods of time. See fig. 1 and 2.
Example 2
Imaging the printed artificial heart under a microscope when the artificial heart is cultured in vitro for 3,7 and 14 days, and observing that myocardial cells begin to aggregate under the microscope after a period of in vitro culture, so that a more obvious connecting structure between the cardiac muscles appears. See fig. 3.
Example 3
The printed artificial hearts are respectively added with Calcein-AM (2 μl) when cultured in vitro for 3, 7 and 14 days, and incubated at 37deg.C for 20-25 min in the absence of light. The supernatant was aspirated and medium with PI dye (2. Mu.l) was added again and stained at room temperature in the dark for 5min. The staining solution was removed and rinsed three times for five minutes with PBS. The staining results were quantified as shown in fig. 4. When cultured in vitro for 3, 7 and 14 days, most of myocardial cells in the artificial heart survive, and the ratio of the viable cells is higher than 95%
Example 4
The print artificial heart cultured in vitro processes and quantifies the microscopic imaging pictures in 3, 7 and 14 days of contraction and relaxation, and the imaging pictures are treated and quantified by using imageJ. See fig. 5.
Example 5
Printed artificial hearts cultured in vitro were imaged under a microscope at 3, 5 days of systolic and diastolic conditions, and the data were analyzed using imageJ. With the extension of in vitro culture time, the beating area of the heart printing body is increased, and the printing body functions are gradually matured. See fig. 6.
Example 6
Print artificial hearts cultured in vitro were fixed with 4% paraformaldehyde at days 3, 7, and 14, washed 3 times with PBS for 5min each. Blocking treatment was performed for 30min with a blocking solution containing 3% fetal bovine serum. An a-actinin primary antibody solution was added to the dish and incubated at room temperature for 1 hour. The PBS was rinsed 3 times for 5min each. The secondary antibody solution containing DAPI is added and incubated for 1h at room temperature in a dark place. The PBS was rinsed 3 times for 5min each. The expression of α -actinin was observed under a microscope, and after a period of in vitro culture, the apparent sarcomere structure was visible under the microscope, indicating that the printed body function was mature. See fig. 7.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. A hydrogel for an artificial heart in vitro drug screening model, comprising: heart acellular matrix, gelMA and sodium alginate; wherein, the mass concentration of the heart acellular matrix is 5% -10%; the mass concentration of GelMA is 6-10%; the mass concentration of the sodium alginate is 0.5-1.0%.
2. Use of the hydrogel of claim 1 in constructing an artificial heart in vitro drug screening model.
3. An artificial heart in-vitro drug screening model is characterized in that the raw materials comprise: hydrogel and neonatal mouse cardiomyocytes.
4. The 3D printing method for constructing the artificial heart in-vitro drug screening model is characterized by comprising the following steps of:
1) Mixing the mixed acellular matrix hydrogel with neonatal mouse myocardial cells at 37 ℃ to prepare the bio-ink required by printing;
2) The hydrogel is placed at 4 ℃ for refrigeration for 8-10min, the diameter of a proper extrusion nozzle is selected, the temperature of a printing nozzle is adjusted to 10-15 ℃, the temperature of a printing platform is adjusted to 5-10 ℃, the bio-ink is printed in a 6cm sterile culture dish by using the extrusion speed of 2-4mm 3/s, and then the printed heart print is placed in CaCl 2 solution with the mass concentration of 3% for crosslinking, and then the heart print is cultivated for a long time.
5. The method according to claim 4, wherein in step 1), the final concentration of the cardiomyocytes is 5 x 10 6/mL.
6. The 3D printing method for constructing an artificial heart in vitro drug screening model according to claim 5, wherein in the step 2), 405nm ultraviolet light is used for curing the printing body during the printing process.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410521926.9A CN118325828A (en) | 2024-04-28 | 2024-04-28 | Hydrogel for artificial heart in-vitro drug screening model and application |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202410521926.9A CN118325828A (en) | 2024-04-28 | 2024-04-28 | Hydrogel for artificial heart in-vitro drug screening model and application |
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| Publication Number | Publication Date |
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| CN118325828A true CN118325828A (en) | 2024-07-12 |
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| CN202410521926.9A Pending CN118325828A (en) | 2024-04-28 | 2024-04-28 | Hydrogel for artificial heart in-vitro drug screening model and application |
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| CN (1) | CN118325828A (en) |
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- 2024-04-28 CN CN202410521926.9A patent/CN118325828A/en active Pending
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