CN114774354B - Preparation method and application of cell spheres - Google Patents
Preparation method and application of cell spheres Download PDFInfo
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- CN114774354B CN114774354B CN202210565593.0A CN202210565593A CN114774354B CN 114774354 B CN114774354 B CN 114774354B CN 202210565593 A CN202210565593 A CN 202210565593A CN 114774354 B CN114774354 B CN 114774354B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0062—General methods for three-dimensional culture
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/37—Digestive system
- A61K35/407—Liver; Hepatocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0662—Stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/067—Hepatocytes
- C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
Abstract
The application belongs to the technical field of biology, and particularly relates to a preparation method and application of a cell sphere. The application provides a preparation method of a cell sphere, which comprises the following steps: under a magnetic environment, mixing and culturing the magnetic micropore array, the suspended cells and the culture base in a culture dish, and centrifuging after removing the magnetic environment to obtain cell spheres; the surface of the magnetic micropore array is provided with a micro blind hole. According to the method, the plurality of magnetic micropore arrays are pulled by magnetic force to capture suspended cells in the three-dimensional space of the culture medium, and the method has the advantage of high space utilization rate. Thus, the cell pellet yield of the single multi-well culture dish of the method of the present application is higher. Meanwhile, the prepared hepatocyte pellets can effectively increase the survival rate of mice with acute liver failure and promote the repair of damaged liver tissues. Therefore, the application provides a preparation method and application of the cell ball, which are used for solving the technical defects that the cell balls prepared by the prior art are often different in size or difficult to prepare in a large scale.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to a preparation method and application of a cell sphere.
Background
The cytosphere is a three-dimensional cell aggregate composed of a plurality of cells, and can better simulate the structure and function of the primary tissue compared with the traditional in-vitro two-dimensional cell culture. Cells can generally be pelleted in a short time by space constraints (hanging drops, porous dishes or microspheres) and by external forces (stirring, centrifugation or magnetic levitation), or cells can proliferate to form cell pellets over a longer period of time. Wherein, the low adsorption culture dish has no adsorption sites of cells on the surface, so that random interaction can occur among cells and form cell balls; the porous culture dish can limit the interaction between cells to a specific space range; hanging drop culture is to add a trace amount of cell suspension onto a plane, form hanging drops by inversion so that cells settle and aggregate in the hanging drops to form cell balls; microspheres prepared based on microfluidic technology can encapsulate cells in a space of a specific size to promote aggregation of cells into spheres and control their size; the stirring bioreactor can improve the probability of cell-cell contact by controlling the stirring speed so as to accelerate cell balling; cells can also realize magnetic suspension and aggregation into balls in the culture solution through endocytosis of magnetic particles and traction of an external magnetic field.
Although the existing preparation methods of the cell pellet are various, the prepared cell pellet is often different in size or difficult to prepare in large scale, so that the double requirements of the cell pellet number and quality in actual treatment cannot be met, for example: cell spheres prepared by stirring culture based on a low-adsorption culture dish or a bioreactor are often nonuniform in size and are easy to be mutually fused with each other to form large-size cell spheres with central necrosis; the method for preparing the cell ball by hanging drop culture is time-consuming and labor-consuming, and the difficulty for collecting the cell ball is high; the method for preparing the cell spheres by using the conventional two-dimensional porous culture plate needs a large space; the cell ball preparation method based on the microfluidics technology is complex to operate and has high requirements on operation experience and equipment micromachining technology. Although cells can also aggregate into spheres by endocytosis of magnetic particles and traction by external magnetic fields, studies have shown that: the degradation of the magnetic particles within the cell sphere is slow and its long-term massive accumulation can lead to cellular malfunction.
Disclosure of Invention
Aiming at the problems, the application provides a preparation method of cell spheres based on a magnetic micropore array, which is simple to operate, high in space utilization rate, easy to realize mass preparation and collection of uniform cell spheres, and capable of recycling the magnetic micropore array.
The application provides a preparation method of a cell sphere, which comprises the following steps:
under a magnetic environment, mixing and culturing the magnetic micropore array, the suspended cells and the culture in a culture dish; removing the magnetic environment, and then performing centrifugal treatment to obtain a cell ball;
the surface of the magnetic micropore array is provided with a micro blind hole.
In another embodiment, the magnetic microwell array is one or more of circular, triangular, rectangular, and polygonal in shape; the total area of the magnetic micropore array ranges from 0.001 to 1000 square centimeters; the thickness of the magnetic micropore array ranges from 0.01 to 10 millimeters.
Specifically, the overall projection area of the square micropore array is 5 mm×5 mm; the overall projected area of the circular array of microwells is 0.5 square centimeters.
In another embodiment, the magnetic microwell array is a double sided structure; the micro blind holes on the surface of the magnetic micro hole array are one or more of cones, cylinders, polygonal columns, tetrahedrons, spheres and hemispheres; the diameter range of the micro blind holes is 0.01-1 mm; the depth range of the micro blind holes is 0.01-10 mm.
Specifically, all the micro blind holes are cones with the opening diameter of 500 micrometers and the depth of 300 micrometers.
In another embodiment, the material of the magnetic micropore array comprises magnetic particles; the magnetic particles are mostly micro-nano particles with magnetism; the magnetic particles are selected from one or more compounds of iron, cobalt, nickel and manganese; the concentration range of the magnetic particles in the magnetic micropore array material is 0.001-100 mg/ml.
Specifically, the magnetic particles are ferroferric oxide nano particles.
Specifically, the concentration of the magnetic particles in the material is 2 mg/ml, 3 mg/ml, 4 mg/ml or 5 mg/ml.
In another embodiment, the method for preparing the magnetic microwell array comprises:
mixing magnetic particles and a gel material to obtain a mixture; pouring the mixture into a mould and then curing and shaping; and separating the mixture from the die to obtain the magnetic micropore array.
In another embodiment, the gel material is selected from one or more of polydimethylsiloxane, alginate hydrogel, agarose hydrogel, fibrin hydrogel, collagen hydrogel, gelatin, hyaluronic acid hydrogel or polyethylene glycol and its derivative hydrogel.
In another embodiment, the method further comprises vacuum treatment, wherein the mixture is poured into a mold, then is kept stand in a vacuum environment for 1-60 minutes, and then is cured and shaped. Preferably, the treatment is carried out in a vacuum environment for 10 to 30 minutes.
Specifically, the treatment time in the vacuum environment is 10 minutes.
Specifically, the preparation method of the magnetic micropore array comprises the following steps:
1. the magnetic particles and the gel material are uniformly mixed to obtain the magnetic gel, and the overall magnetic strength of the magnetic micropore array and the magnetic response sensitivity under magnetic driving can be correspondingly adjusted by changing the adding amount of the magnetic particles. A mold with an array of pyramids is placed over the magnetic gel prior to its gelling to compress and form an array of magnetic micro-holes with blind single-sided holes.
2. Pouring the new magnetic gel on the mould with the same cone array, then coating the mould with the magnetic micropore array with the single-sided micro blind holes on the mould with the same cone array to extrude redundant magnetic gel, and separating the mould with the cone array after the magnetic gel is glued to finish the preparation of the magnetic micropore array with the double-sided micro blind holes.
Wherein the size and number of micro-blind holes can be controlled by the mould used. The method can select natural and synthetic various gels such as ion crosslinked alginate hydrogel, thermal cured agarose hydrogel, polydimethylsiloxane elastomer, photo-cured methacrylic acid gelatin, methacrylic acid hyaluronic acid and the like. Regardless of which gel is used, it is vacuum treated for at least 10 minutes prior to gelling to remove air bubbles generated during the compression of the mold.
In another embodiment, the magnetic microwell array is present in the culture medium in an amount ranging from 1 to 100 sheets/ml.
Specifically, the number of the magnetic micropore arrays is 3 sheets/ml, 4 sheets/ml, 5 sheets/ml and 6 sheets/ml.
In another embodiment, the magnetic environment is a permanent magnet disposed on top of the culture dish to provide a constant magnetic environment.
Specifically, the permanent magnet is an N52 NdFeB permanent magnet (size: 50 mm. Times.25 mm. Times.10 mm).
In another embodiment, the cells are selected from one or more of mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, fibroblasts, cardiomyocytes, macrophages, vascular endothelial cells, islet beta cells, hepatocytes, chondrocytes, and bone cells.
Specifically, the cells are hepatocytes and mesenchymal stem cells.
In another embodiment, the suspension cells have a density of 2-10 in the medium 7 And each milliliter.
Specifically, the density of the cells was 30 k/ml, 75 k/ml, 150 k/ml and 300 k/ml; specifically, the number of cells was 30k, 75k, 150k and 300k.
The application of the hepatocyte pellets prepared by the preparation method in the preparation of medicaments for treating acute liver failure by subcutaneous implantation.
In another embodiment, the cytoball used by the cell to treat acute liver failure is a hepatocyte ball.
Specifically, hepatocytes are implanted by subcutaneous injection; specifically, the number of implanted cells is one million hepatocytes.
According to the method, the magnetic micropore array with different magnetic intensities and different numbers and sizes of micro blind holes can be prepared, and is driven to move in the culture medium in a three-dimensional space under a magnetic environment, so that suspended cells in the culture medium are actively captured, the captured cells are limited by the space of the micro blind holes, and cell balls are aggregated in the micro blind holes. Because the front surface and the back surface of the double-sided magnetic micropore array are both provided with the micro blind hole structures, the method is not limited by the orientation of the micro blind holes on the magnetic micropore array when capturing cells, the magnetic micropore array actively captures cells under the drive of external magnetic force, and the process is realized in a three-dimensional space, so that the method has the advantage of high space utilization rate. Thus, the cell pellet can be prepared in large quantities using this advantage. Moreover, the size of the prepared cell pellet can be controlled by the size of the micro blind hole on the magnetic micro hole array and the feeding ratio of the micro blind hole to cells, and the size uniformity is good. The method is simple and quick to operate, has no experience requirement on operators, can be used for preparing the cell balls by uniformly mixing the cells and the magnetic micropore array in a specific proportion and placing the magnet at the top of the culture dish, and can be used for collecting the cell balls by simple centrifugation. The hepatocyte spheroid prepared by the method can effectively improve the survival rate of mice with acute liver failure and promote the repair of damaged liver tissues.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
Fig. 1 is a process flow of preparing a double-sided magnetic micro-pore array according to embodiment 1 of the present application, wherein (1) is a step of forming magnetic gel after mixing magnetic particles and gel materials, (2) is a step of forming a single-sided magnetic micro-pore array by casting the magnetic gel on a mold with a conical array, and (3) is a step of forming a double-sided magnetic micro-pore array by stacking the back surface of the magnetic micro-pore array with single-sided micro-blind holes on a mold with the magnetic gel cast thereon;
fig. 2 is an external view of a micropore array with different shapes and an external view of a micropore array prepared by adding ferroferric oxide nano magnetic particles with different concentrations, which are provided in example 1 of the present application, wherein a first graph from left to right is a circular micropore array without magnetism, a second graph from left to right is a square micropore array added with ferroferric oxide nano magnetic particles with different concentrations, and a seventh graph from left to right is a double-sided micro blind hole microscopic graph of a magnetic micropore array prepared by adding 4 mg/ml of ferroferric oxide nano magnetic particles;
FIG. 3 is a schematic diagram of the procedure of the embodiment 2 of the present application, wherein after the magnetic microwell array, the suspended cells (single cells) and the culture are mixed and cultured in the culture dish, the plurality of magnetic microwell arrays are pulled by the magnetic force to capture the suspended cells in the three-dimensional space of the culture medium, and the suspended cells are aggregated to form cell spheres under the space limitation, wherein (1) the step of uniformly mixing the single cells with the magnetic microwell arrays in a specific ratio, (2) the step of capturing the three-dimensional cells under the magnetic force, and (3) the step of aggregating/proliferating the cells in the blind micro holes to form the cell spheres;
FIG. 4 is a graph showing a hepatocyte pellet prepared based on a magnetic microwell array under the conditions of different cell inoculum sizes and magnetic environmental driving or not under the optical microscope provided in example 2 of the present application, wherein the upper left graph is a graph showing a mixture of 150k hepatocytes and 4 magnetic microwell arrays under the non-magnetic environmental driving overnight, the upper right graph is a graph showing a mixture of 30k hepatocytes and 4 magnetic microwell arrays under the magnetic environmental driving overnight, the lower left graph is a graph showing a mixture of 150k hepatocytes and 4 magnetic microwell arrays under the magnetic environmental driving overnight, and the lower right graph is a graph showing a mixture of 300k hepatocytes and 4 magnetic microwell arrays under the magnetic environmental driving overnight;
FIG. 5 is a graph showing the distribution of scattered points of the hepatocyte spheroids, wherein the left graph is a histogram of the mean value of the hepatocyte spheroids, and the right graph is a graph showing the statistical analysis of the cross-sectional areas of the hepatocyte spheroids prepared based on the magnetic microporous arrays under the culture conditions of the four groups of cell inoculations and the driving of the magnetic environment;
fig. 6 is a diagram of a hepatocyte pellet prepared by the same hepatocyte inoculation amount and magnetic traction according to the different magnetic micropore array input amounts under the optical microscope provided in example 2 of the present application, wherein the upper left diagram is a diagram of a mixture of 150k hepatocytes and 3 magnetic micropore arrays cultured overnight under magnetic environment driving, the upper right diagram is a diagram of a mixture of 150k hepatocytes and 4 magnetic micropore arrays cultured overnight under magnetic environment driving, the lower left diagram is a diagram of a mixture of 150k hepatocytes and 5 magnetic micropore arrays cultured overnight under magnetic environment driving, and the lower right diagram is a diagram of a mixture of 150k hepatocytes and 6 magnetic micropore arrays cultured overnight under magnetic environment driving;
FIG. 7 is a graph showing statistics of hepatocyte pellets obtained by the four different magnetic microwell array inputs in FIG. 6, wherein the first graph from top to bottom shows a mean value statistical analysis based on cross-sectional area of the hepatocyte pellets obtained after 150k hepatocytes are mixed-cultured overnight with 3, 4, 5 and 6 magnetic microwell arrays, respectively, the second graph from top to bottom shows a statistical analysis based on number of hepatocyte pellets obtained after 150k hepatocytes are mixed-cultured overnight with 3, 4, 5 and 6 magnetic microwell arrays, respectively, and the third graph from top to bottom shows a scatter distribution based on cross-sectional area of the hepatocyte pellets obtained after 150k hepatocytes are mixed-cultured overnight with 3, 4, 5 and 6 magnetic microwell arrays, respectively, under magnetic environment driving;
FIG. 8 is a graph showing the fluorescent staining of live dead cells after overnight mixed culture of 30k mesenchymal stem cells and 4 magnetic microwell arrays, a graph showing the fluorescent staining of live dead cells after overnight mixed culture of 75k mesenchymal stem cells and 4 magnetic microwell arrays, and a graph showing the fluorescent staining of live dead cells after overnight mixed culture of 150k mesenchymal stem cells and 4 magnetic microwell arrays;
FIG. 9 is a graph showing survival curves of a treatment experimental group of mice treated with immunodeficiency and a control group of mice untreated by subcutaneously implanting single cells and cell spheres of the same hepatocyte number as provided in example 4 of the present application;
FIG. 10 shows the levels of aspartate aminotransferase (left panel), albumin (middle panel) and total bile acid (right panel) in serum of a treatment experimental group of immunodeficient mice and a control group of untreated immunodeficient mice, which were subcutaneously implanted with single cells and cell spheres of the same hepatocyte number, as provided in example 4 of the present application;
FIG. 11 is a graph showing in situ apoptosis staining of liver tissue TUNEL of a treatment experimental group of mice treated with immunodeficiency and a control group of mice untreated by subcutaneous implantation of single cells and cell spheres of the same hepatocyte number as provided in example 4 of the present application;
FIG. 12 is a graph showing survival curves of a treatment experimental group of immunocompetent mice subcutaneously implanted with single cells and cell spheres of the same hepatocyte number and a control group of immunocompetent mice without treatment provided in example 5 of the present application;
FIG. 13 is a photograph showing the liver of mice surviving 7 days in the experimental group of immunocompetent mice treated by subcutaneously implanting single cells and cytoballs of the same hepatocyte number and the control group of immunocompetent mice untreated, provided in example 5 of the present application, the red color range being abnormal liver tissue morphology;
fig. 14 is a liver tissue section hematoxylin/eosin composite staining chart of a treatment experimental group of immunocompetent mice and a control group of immunocompetent mice without treatment, which are subcutaneously implanted with single cells and cell spheres of the same liver cell number, provided in example 5 of the present application, wherein the white dotted line range is a tissue necrosis region, and the red dotted line range is an inflammatory cell infiltration region.
Detailed Description
The application provides a preparation method and application of a cell ball, which are used for solving the technical defect that the cell balls prepared by the prior art are often different in size or difficult to prepare on a large scale.
The following description of the technical solutions in the embodiments of the present application will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
Wherein, the raw materials or reagents used in the following examples are all commercially available or self-made.
Example 1
The embodiment of the application provides a preparation method of a magnetic micropore array, which specifically comprises the following steps:
the magnetic microwell array was prepared according to the preparation flow of the double-sided magnetic microwell array provided in fig. 1, with polydimethylsiloxane as the gel material. Wherein, the whole projection area of the square array mould with the cone is 5 mm multiplied by 5 mm; the integral projection area of the circular array die with the cone is 0.5 square centimeter; the bottom surface of the cone is circular with the diameter of 500 microns, and the height of the cone is 300 microns.
Firstly adding ferroferric oxide nano magnetic particles with different concentrations into an initiator, adding a dimethyl siloxane base solution after ultrasonic 30 minutes of resuspension, and stirring and uniformly mixing to obtain a gel precursor; standing for 5 min to remove bubbles, pouring the gel after uniform mixing into moulds with different shapes and different numbers of cone arrays, and vacuum-treating for 10 min. The mold with the cast gel precursor was pressed onto a clean plane to extrude excess gel precursor. Vacuum processing for 10 min again, transferring to an oven, heating at 80deg.C for 2 hr to solidify and shape the gel, and separating the mold to obtain the magnetic micropore array with blind holes on one side.
Next, a new magnetic gel precursor is poured on the mold with the same cone array, and the bubbles generated in the pouring process are removed by vacuum treatment for 10 minutes. And then coating the magnetic micropore array with the single-sided magnetic blind holes on the magnetic micropore array to extrude redundant magnetic gel precursors, carrying out vacuum treatment for 10 minutes, and then carrying out heat curing at 80 ℃ for 2 hours, and separating the die with the cone array after the magnetic gel is shaped to finish the preparation of the magnetic micropore array with the double-sided blind holes. Different magnetic micropore arrays containing ferroferric oxide nano magnetic particles with different concentrations and double-sided micro blind holes are prepared, the appearance of the magnetic micropore arrays is shown in fig. 2, and the rightmost diagram in fig. 2 is a double-sided micro blind hole microscopic image of the magnetic micropore array prepared by adding 4 mg/ml ferroferric oxide nano magnetic particles.
Example 2
The embodiment of the application provides a preparation method of a hepatocyte spheroid, which specifically comprises the following steps:
the embodiment of the application adopts a square magnetic micropore array which is prepared by adding 4 mg/ml ferroferric oxide nano magnetic particles, and the whole projection area of the blind holes is 5 mm multiplied by 5 mm. The magnetic microwell array prepared in the above example was sterilized by immersing in 75% alcohol for 30 minutes in advance and washed twice with physiological saline.
In this example, the size of the hepatocyte pellets is controlled by changing the cell inoculum size, and the hepatocyte pellets are divided into four groups, wherein three groups are magnetic traction experimental groups containing different numbers of hepatocytes, and one group is a control group without magnetic traction. After the magnetic micropore array, the suspended cells (single cells) and the culture are mixed and cultured in the culture dish, the magnetic force drives the (permanent magnet) to pull the plurality of magnetic micropore arrays to capture the suspended cells in the three-dimensional space of the culture medium, and the suspended cells are aggregated to form cell balls under the space limitation according to the flow chart of FIG. 3. Wherein, (1) is a step of uniformly mixing single cells and a magnetic micropore array according to a specific proportion, (2) is a step of capturing three-dimensional cells under the drive of magnetic force, and (3) is a step of aggregating/proliferating cells in blind micropores to form cell spheres.
Specifically, the magnetic micropore array and the liver cells are uniformly mixed in a 48-well plate containing a culture medium, the numbers of the liver cells in the culture medium are respectively 30k, 150k and 300k, the using amount of the culture medium in the 48-well plate is 1 milliliter, the numbers of the magnetic micropore arrays are respectively 4, and an N52 neodymium iron boron permanent magnet (the sizes: 50 mm multiplied by 25 mm multiplied by 10 mm) is placed on the top of the 48-well plate (the numbers of the liver cells are respectively 30k, 150k and 300 k) of the three experimental groups so as to form a static magnetic field to drive the magnetic micropore array to move upwards. The magnetic microwell array can contact suspended hepatocytes during migration and collect them into blind micropockets to complete capture of hepatocytes. Due to the limitation of the liquid level, the magnetic micropore array can hover in the culture solution at the gas-liquid interface. After overnight culture, hepatocytes aggregate under the spatial restriction of blind micro-pores, forming hepatocyte spheroids by interactions between hepatocytes. The separation of the hepatocyte spheroids can be achieved by a simple centrifugation method. The other group is a control group 48-well plate (the number of liver cells is 150 k), and N52-free NdFeB permanent magnet is not placed on the top of the control group 48-well plate, and then the control group is cultured overnight in the same culture environment with the other three groups 48-well plates. After overnight incubation, the hepatocyte spheroids were separated by centrifugation, the morphology of the four sets of hepatocyte spheroids was microscopic imaged and the distribution of their cross-sectional areas and the mean statistical analysis were performed, and the results are shown in fig. 4 and 5. The size of the prepared hepatocyte balls is correspondingly increased along with the increase of the cell inoculation amount through microscopic imaging, and compared with a control group without magnetic attraction, the size distribution range of the hepatocyte balls prepared by magnetic force driven magnetic micropore arrays is narrower under the 150k hepatocyte inoculation amount, so that the uniformity is better. However, a higher cell seeding amount (300 k hepatocytes) resulted in a larger size distribution of the resulting hepatocyte spheroids, and thus the average cross-sectional area thereof was not increased correspondingly.
In addition, the size and the yield of the hepatocyte balls are controlled by changing the feeding amount of the magnetic micropore arrays, and the hepatocyte balls are divided into four groups, so that the hepatocyte balls are magnetically pulled after the magnetic micropore arrays with different numbers and cells with the same inoculation amount are mixed. Specifically, the magnetic micropore array and the liver cells are uniformly mixed in a 48-pore plate containing a culture medium, the number of the liver cells in the culture medium is 150k, the using amount of the culture medium in the 48-pore plate is 1 milliliter, the number of the magnetic micropore array is divided into 3, 4, 5 and 6, and an N52 neodymium iron boron permanent magnet (the size is 50 mm multiplied by 25 mm multiplied by 10 mm) is placed on the top of the 48-pore plate to form a static magnetic field so as to drive the magnetic micropore array to move upwards. The magnetic microwell array can contact suspended hepatocytes during migration and collect them into blind micropockets to complete capture of hepatocytes. Due to the limitation of the liquid level, the magnetic micropore array can hover in the culture solution at the gas-liquid interface. After overnight culture, hepatocytes aggregate under the spatial restriction of blind micro-pores, forming hepatocyte spheroids by interactions between hepatocytes. The separation of the hepatocyte spheroids can be achieved by a simple centrifugation method. The morphology of four sets of hepatocyte spheroids was microscopically imaged and statistically analyzed for their average cross-sectional area, number of spheroids, and cross-sectional area distribution, and the results are shown in fig. 6 and 7. The increase of the input amount of the magnetic micropore array can be observed through microscopic imaging, and the size and the yield of the prepared hepatocyte balls are correspondingly increased, so that the increase of the magnetic micropore array can improve the capture success rate of cells. However, the cross-sectional area distribution of the hepatocyte spheroids also widens with increasing input of the magnetic microwell array. Compared with the number of hepatocyte balls prepared by magnetic traction of 3 magnetic micropore arrays, the 4 magnetic micropore arrays have more hepatocyte balls, and the distribution range of the cross sectional area of the 4 magnetic micropore arrays is not obviously increased. Thus, 4 magnetic microwell arrays and 150k hepatocytes were the optimal feed ratios in this example.
Example 3
The embodiment provides a preparation method of mesenchymal stem cell spheres, which specifically comprises the following steps:
the embodiment of the application adopts a square magnetic micropore array which is prepared by adding 4 mg/ml ferroferric oxide nano magnetic particles, and the whole projection area of the blind holes is 5 mm multiplied by 5 mm. The magnetic microwell array prepared in the above example was sterilized by immersing in 75% alcohol for 30 minutes in advance and washed twice with physiological saline.
In this example, the size of the mesenchymal stem cell pellets was controlled by changing the cell seeding amount, and the mesenchymal stem cells were divided into three groups, and magnetic traction was applied. Specifically, the magnetic micropore array and the mesenchymal stem cells are uniformly mixed in a 48-pore plate containing a culture medium, the number of the mesenchymal stem cells in the culture medium is 30k, 75k and 150k respectively, the using amount of the culture medium in the 48-pore plate is 1 ml, the number of the magnetic micropore array is 4, and an N52 NdFeB permanent magnet (the size is 50 mm multiplied by 25 mm multiplied by 10 mm) is placed on the top of the 48-pore plate to form a static magnetic field to drive the magnetic micropore array to move upwards. The magnetic micropore array can contact the suspended mesenchymal stem cells in the migration process and collect the mesenchymal stem cells into the blind micropores so as to complete the capture of the mesenchymal stem cells. Due to the limitation of the liquid level, the magnetic micropore array can hover in the culture solution at the gas-liquid interface. After overnight culture, mesenchymal stem cells aggregate under the limitation of the micro blind hole space, and mesenchymal stem cell spheres are formed through the interaction among the mesenchymal stem cells. The separation of the mesenchymal stem cell pellet can be achieved by a simple centrifugation method. After overnight culture, mesenchymal stem cell pellets were stained live and imaged by fluorescence microscopy, and the results are shown in fig. 8. The size of the prepared mesenchymal stem cell sphere is correspondingly increased along with the increase of the cell inoculation amount through microscopic imaging, and the cell activity is better.
Example 4
The embodiment provides a method for treating acute liver failure by using a hepatocyte spheroid, which specifically comprises the following steps:
in the examples of the present application, hepatocyte pellets were prepared by the method of example 2 for the treatment of acute liver failure. Specifically, 27 female immunodeficiency Balb/c-nude mice (18-21 g) of 6 weeks old were selected and randomly divided into 3 groups of 9. Carbon tetrachloride (CCl) 4 ) Dissolved in olive oil to give a volume concentration of 40%, and CCl of 4. Mu.l 4 The mice were intraperitoneally dosed at a dose per gram of mouse body weight to construct a model of acute liver failure. Group I is a control group, and treatment is not performed after molding. Groups II and III are experimental groups, 100 microliter of single cell suspension containing one million liver cells and hepatocyte pellets are slowly injected subcutaneously into the backs of mice in the experimental groups at a constant speed after molding for 24 hours, and the mice are placed in an SPF environment for conventional feeding for 1 week. Mice survival was observed daily and survival curves were drawn. After 1 week the experiment was ended and animals were anesthetized for analysis of material. Specifically, 0.6% pentobarbital sodium anesthetic (10 μl/g animal body weight dose) was injected intraperitoneally, 0.5 ml was collected from the infraorbital venous plexus, and serum was isolated for detection of albumin, aspartate aminotransferase and total bile acid content. Mice were sacrificed after excessive anesthesia after blood collection and liver tissue samples were collected for TUNEL in situ apoptosis staining, the results are shown in fig. 9, 10 and 11. The survival rate of the acute liver failure Balb/c-nude mice after 7 days can be obviously improved by 100% by subcutaneously implanting the liver cell pellets, compared with single cells with the same liver cell number, the liver function index value of aspartic transaminase and total bile acid in serum of the acute liver failure mice after 7 days is smaller, and the liver function index value of albumin in serum is larger. In addition, they are treated with globus hystericusThe number of apoptotic cells in liver tissue of Balb/c-nude mice after 7 days was significantly less than in the control group and the single cell treatment experimental group receiving the same number of hepatocytes.
Example 5
The embodiment provides a method for treating acute liver failure by using a hepatocyte spheroid, which specifically comprises the following steps:
in the examples of the present application, hepatocyte pellets were prepared by the method of example 2 for the treatment of acute liver failure. Specifically, 27 female immune sound ICR mice (18-21 g) of 6 weeks of age were selected and randomly divided into 3 groups of 9 animals each. Carbon tetrachloride (CCl) 4 ) Dissolved in olive oil to give a volume concentration of 40%, and CCl of 4. Mu.l 4 The mice were intraperitoneally dosed at a dose per gram of mouse body weight to construct a model of acute liver failure. Group I is a control group, and treatment is not performed after molding. Groups II and III are experimental groups, 100 microliter of single cell suspension containing one million liver cells and hepatocyte pellets are slowly injected subcutaneously into the backs of mice in the experimental groups at a constant speed after molding for 24 hours, and the mice are placed in an SPF environment for conventional feeding for 1 week. Mice survival was observed daily and survival curves were drawn. After 1 week, the experiment was ended, mice were sacrificed after excessive anesthesia for cervical dislocation, liver tissue samples were collected for paraffin embedding and slicing, and liver tissue slices were subjected to hematoxylin/eosin complex staining, and the results are shown in fig. 12, 13 and 14. Compared with single cell suspension, the survival rate of ICR mice with acute liver failure after 7 days can be obviously improved by subcutaneously implanting the hepatocyte balls with the same hepatocyte number (the cell ball treatment group vs single cell treatment group: 67% vs 22%). In addition, among the ICR mice with acute liver failure surviving 7 days later, the apparent morphology of the liver of ICR mice treated with the hepatocyte spheroids of the same hepatocyte number was more normal, and the necrotic tissue area in the liver tissue section was smaller, and the inflammatory cell infiltration degree was lighter, compared to the untreated control group and the experimental group treated with the single cell suspension.
In summary, the design of the magnetic micro-pore array with the double-sided micro-blind holes according to the embodiments of the present application enables the three-dimensional cell capturing and cell sphere forming process under the driving of magnetic force to fully contact and collect the cells no matter which side of the micro-blind holes is facing upwards. The moving speed of the magnetic micropore array in the water phase can be correspondingly adjusted by controlling the coating amount of the magnetic particles in the magnetic micropore array and the static magnetic field intensity generated by the permanent magnet. Wherein, the whole projection area of the micro blind holes of the magnetic micro hole array needs to be smaller than the cross section of the multi-hole culture dish so that all the magnetic micro hole arrays can receive cells in the upward migration process. Meanwhile, sufficient culture solution needs to be added into the 48-well plate so as to provide a migration space for the magnetic micropore array. The size of the cell sphere can be controlled by the size of the micro-blind hole of the magnetic micro-pore array and the feed ratio of the micro-blind hole to the cells. The prepared hepatocyte spheroids can effectively improve the survival rate of mice with acute liver failure and promote the repair of damaged liver tissues.
The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application and are intended to be within the scope of the present application.
Claims (8)
1. A method for preparing a cell pellet, comprising:
under a magnetic environment, mixing and culturing the magnetic micropore array, the suspended cells and the culture in a culture dish; removing the magnetic environment, and then performing centrifugal treatment to obtain a cell ball;
the surface of the magnetic micropore array is provided with a micro blind hole;
the preparation method of the magnetic micropore array comprises the following steps:
mixing magnetic particles and a gel material to obtain a mixture; pouring the mixture into a mould and then curing and shaping; separating the mixture from the mold to obtain the magnetic micropore array;
the concentration range of the magnetic particles in the magnetic micropore array material is 2-5 mg/ml;
the gel material is polydimethylsiloxane;
the diameter range of the micro blind holes is 0.01-1 mm; the depth range of the micro blind holes is 0.01-10 mm;
the density of the suspended cells in the culture medium is 30000-10 7 And each milliliter.
2. The method of claim 1, wherein the array of magnetic microwells is one or more of rectangular, circular, triangular, and polygonal in shape; the total projection area of the magnetic micropore array ranges from 0.001 to 1000 square centimeters; the thickness of the magnetic micropore array is 0.01-10 mm.
3. The method of claim 1, wherein the array of magnetic microwells is a double-sided structure; the surface blind holes of the magnetic micropore array are one or more of cones, cylinders, polygonal cylinders, tetrahedrons, spheres and hemispheres.
4. The method of claim 1, wherein the material of the magnetic microwell array comprises magnetic particles; the magnetic particles are selected from one or more compounds of iron, cobalt, nickel and manganese.
5. The method according to claim 1, further comprising vacuum treatment, wherein the mixture is placed in a vacuum environment for 10 to 60 minutes after being poured into a mold, and then cured and set.
6. The method of claim 1, wherein the magnetic microwell array is present in the medium in an amount of 1 to 100 sheets/ml.
7. The method of claim 1, wherein the cells are selected from one or more of mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, fibroblasts, cardiomyocytes, macrophages, vascular endothelial cells, islet beta cells, hepatocytes, chondrocytes, and bone cells.
8. Use of the hepatocyte pellets prepared by the preparation method according to claim 1 for the preparation of a medicament for the treatment of acute liver failure by subcutaneous implantation.
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