CN114774354A - Preparation method and application of cell ball - Google Patents
Preparation method and application of cell ball Download PDFInfo
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- CN114774354A CN114774354A CN202210565593.0A CN202210565593A CN114774354A CN 114774354 A CN114774354 A CN 114774354A CN 202210565593 A CN202210565593 A CN 202210565593A CN 114774354 A CN114774354 A CN 114774354A
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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
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- 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
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- 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
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- 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
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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
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- 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
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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
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- C—CHEMISTRY; METALLURGY
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Abstract
The application belongs to the technical field of biology, and particularly relates to a preparation method and application of a cell ball. The application provides a preparation method of a cell ball, which comprises the following steps: in a magnetic environment, performing mixed culture on the magnetic micropore array, the suspended cells and the culture medium in a culture dish, removing the magnetic environment, and then performing centrifugal treatment to prepare cell spheres; and micro blind holes are formed in the surface of the magnetic micropore array. The method captures the suspended cells in the three-dimensional space of the culture medium by using the magnetic force to pull the magnetic micropore arrays, and has the advantage of high space utilization rate. Thus, the yield of cell pellets from a single multi-well culture dish is higher for the methods of the present application. Meanwhile, the prepared hepatocyte cell balls 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 spheres, which are used for solving the technical defects that the cell spheres prepared by the prior art are often different in size or difficult to prepare on 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 cell sphere is a three-dimensional cell aggregate consisting of a plurality of cells, and can better simulate the structure and function of a primary tissue compared with the traditional in-vitro two-dimensional cell culture. Cells can generally aggregate into spheres within a short time by space limitation (hanging drops, multi-well culture dishes or microspheres) and with the aid of external forces (stirring, centrifugation or magnetic suspension), or form cell spheres by cell proliferation over a longer period of time. Wherein, the surface of the low-adsorption culture dish does not have adsorption sites of cells, so random interaction can occur among the cells and cell spheres are formed; the multi-hole culture dish can limit the interaction among cells in a certain specific space range; the hanging drop culture is to add a trace amount of cell suspension onto a plane, form a hanging drop through inversion, enable cells to settle and aggregate in the hanging drop to form a cell ball; the microspheres prepared based on the microfluid technology can encapsulate cells in a space with a specific size so as to promote the cells to aggregate into spheres and control the size of the spheres; the stirring bioreactor can improve the probability of intercellular contact by controlling the stirring speed so as to accelerate the balling of cells; the cells can also realize magnetic suspension and aggregation into spheres in the culture solution through the endocytosis of the magnetic particles and the traction of an external magnetic field.
Although the existing preparation methods of the cell balls are various, the prepared cell balls are often different in size or difficult to prepare on a large scale, so that the dual requirements on the number and quality of the cell balls in actual treatment cannot be met, such as: cell balls prepared by stirring culture based on a low-adsorption culture dish or a bioreactor are often nonuniform in size and are easy to fuse with each other to form large-size cell balls with central necrosis; the method for preparing the cell spheres by hanging drop culture is time-consuming and labor-consuming, and the difficulty for collecting the cell spheres is higher; the conventional method for preparing the cell spheres by using the two-dimensional porous culture plate needs a larger space; the cell ball preparation method based on the microfluidic technology is complex to operate, and has higher requirements on operation experience and equipment micromachining technology. Although cells can also aggregate into spheres by endocytosis of magnetic particles and traction of external magnetic field, studies have shown that: the degradation of magnetic particles in the cell sphere is slow and their massive accumulation over a long period of time can lead to cell malfunction.
Disclosure of Invention
In order to solve the problems, the application provides a cell ball preparation method based on a magnetic micropore array, which is simple to operate, high in space utilization rate, easy to realize large-scale preparation and collection of uniform cell balls, and the magnetic micropore array can be recycled.
The application provides a preparation method of a cell ball, which comprises the following steps:
in a magnetic environment, performing mixed culture on the magnetic micropore array, the suspension state cells and the culture medium in a culture dish; removing the magnetic environment, and then carrying out centrifugal treatment to prepare cell balls;
and a micro blind hole is formed in the surface of the magnetic micropore array.
In another embodiment, the magnetic microwell array is one or more of circular, triangular, rectangular, and polygonal in shape; the total area range of the magnetic micropore array is 0.001-1000 square centimeters; the thickness range of the magnetic micropore array is 0.01-10 mm.
Specifically, the whole projection area of the square micropore array is 5 mm × 5 mm; the whole projection area of the circular micropore array is 0.5 square centimeter.
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 blind micro-vias are cones with openings of 500 microns in diameter and 300 microns in depth.
In another embodiment, the material of the magnetic micro-pore array comprises magnetic particles; the magnetic particles are mostly magnetic micro-nano particles; 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 nanoparticles.
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 the magnetic particles and the gel material to obtain a mixture; pouring the mixture into a mold and then carrying out curing and shaping treatment; and separating the mixture from the mold 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 hydrogel of polyethylene glycol and derivatives thereof.
In another embodiment, the method further comprises vacuum treatment, the mixture is poured into a mold and then stands still for 1-60 minutes in a vacuum environment, and then curing and shaping treatment is carried out. Preferably, the vacuum environment is treated for 10 to 30 minutes.
Specifically, the processing time in the vacuum environment is 10 minutes.
Specifically, the preparation method of the magnetic micropore array comprises the following steps:
1. and (3) uniformly mixing the magnetic particles with the gel material to obtain magnetic gel, and correspondingly adjusting the overall magnetic strength of the magnetic micropore array and the magnetic response sensitivity thereof under magnetic drive by changing the adding amount of the magnetic particles. And placing a mold with a cone array on the magnetic gel before the magnetic gel is formed into glue to press and form a magnetic micropore array with single-sided blind micropores.
2. And pouring the new magnetic gel on the mould with the same cone array, covering the mould with the magnetic micropore array with the single-sided micro blind hole to extrude redundant magnetic gel, and separating the mould with the cone array after the magnetic gel is gelatinized to finish the preparation of the magnetic micropore array with the double-sided micro blind hole.
Wherein the size and the number of the micro blind holes can be controlled by the used mould. The method can select various natural and synthetic gels such as ion-crosslinked alginate hydrogel, thermosetting agarose hydrogel and polydimethylsiloxane elastomer, photocuring methacrylic gelatin, methacrylic hyaluronic acid and the like. Whichever gel was used, it was subjected to vacuum treatment for at least 10 minutes prior to gelling to remove air bubbles generated during the mold pressing.
In another embodiment, the number of the magnetic microwell array in the culture medium is in the range of 1-100 pieces/ml.
Specifically, the number of the magnetic micropore arrays is 3/ml, 4/ml, 5/ml and 6/ml.
In another embodiment, the magnetic environment is a permanent magnet placed on top of the culture dish to provide a constant magnetic environment.
Specifically, the permanent magnet is an N52 neodymium iron boron permanent magnet (size: 50 mm × 25 mm × 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 osteocytes.
Specifically, the cells are liver cells and mesenchymal stem cells.
In another embodiment, the density of the suspended cells in the culture medium is 2-107One/ml.
Specifically, the density of the cells is 30 k/ml, 75 k/ml, 150 k/ml and 300 k/ml; specifically, the number of the cells was 30k, 75k, 150k and 300 k.
The application of the hepatocyte balls prepared by the preparation method disclosed by the application in the medicine for treating acute liver failure by percutaneous implantation.
In another embodiment, the cytosphere used for treating acute liver failure is a hepatocyte cytosphere.
Specifically, the hepatocytes are implanted by subcutaneous injection; specifically, the number of implanted cells is one million hepatocytes.
According to the method, the magnetic micropore arrays with different magnetic strengths, different numbers and sizes of blind micro holes can be prepared, and the magnetic micropore arrays are driven to move in a three-dimensional space culture medium 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 blind micro holes, and are gathered to form cell spheres in the blind micro holes. Because the front side and the back side 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 in the magnetic micropore array when the cells are captured, the magnetic micropore array actively captures the cells under the driving of external magnetic force, the process is realized in a three-dimensional space, and the advantage of high space utilization rate is achieved. Thus, the cell pellet can be mass-produced by utilizing this advantage. Moreover, the size of the prepared cell ball can be controlled by the size of the blind micro-hole on the magnetic micro-hole array and the charging ratio of the blind micro-hole to the cell, and the uniformity of the size is good. The method is simple and quick to operate, has no empirical requirement on an operator, can complete the preparation of the cell balls only 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 complete the collection of the cell balls only by simple centrifugation. The hepatocyte cell ball 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 flow chart of a preparation process of a double-sided magnetic micro-pore array provided in example 1 of the present application, wherein (1) is a step of mixing magnetic particles and a gel material to form a magnetic gel, (2) is a step of casting the magnetic gel into a mold with a tapered array to form a single-sided magnetic micro-pore array, and (3) is a step of stacking the back of the magnetic micro-pore array with single-sided micro blind holes on the mold cast with the magnetic gel and pressing to form the double-sided magnetic micro-pore array;
fig. 2 is an appearance diagram of a microporous array with different shapes and an appearance diagram of a microporous array prepared by adding ferroferric oxide nano-magnetic particles with different concentrations provided in example 1 of the present application, wherein a first diagram from left to right is a circular microporous array without magnetism, a second diagram from left to right to a sixth diagram are a square microporous array added with ferroferric oxide nano-magnetic particles with different concentrations, and a seventh diagram from left to right is a double-sided micro blind hole micrograph of a magnetic microporous array prepared by adding ferroferric oxide nano-magnetic particles with 4 mg/ml;
fig. 3 is an operation flow of capturing suspended cells in three-dimensional space of a culture medium by pulling a plurality of magnetic microwell arrays under magnetic force drive (permanent magnets) after mixing and culturing the magnetic microwell arrays, the suspended cells (single cells) and the culture medium in a culture dish in a magnetic environment, and the suspended cells are aggregated to form cytospheres under the spatial limitation, provided by example 2 of the present application, wherein (1) is a step of uniformly mixing the single cells and the magnetic microwell arrays in a specific ratio, (2) is a step of capturing the three-dimensional cells under magnetic force drive, and (3) is a step of aggregating/proliferating the cells in the blind microwells to form cytospheres;
fig. 4 is a top left view of a hepatocyte pellet prepared based on magnetic micropore arrays under the optical microscope and under the culture conditions of different cell inoculation amounts and driven by a nonmagnetic environment according to embodiment 2 of the present application, wherein the top left view is a micrograph of 150k hepatocytes mixed with 4 magnetic micropore arrays for overnight culture under the drive of the nonmagnetic environment, the top right view is a micrograph of 30k hepatocytes mixed with 4 magnetic micropore arrays for overnight culture under the drive of the magnetic environment, the bottom left view is a micrograph of 150k hepatocytes mixed with 4 magnetic micropore arrays for overnight culture under the drive of the magnetic environment, and the bottom right view is a micrograph of 300k hepatocytes mixed with 4 magnetic micropore arrays for overnight culture under the drive of the magnetic environment;
FIG. 5 is a cross-sectional area-based statistical analysis of hepatocyte spheroids prepared based on magnetic microwell arrays under culture conditions driven by four groups of cell inoculum sizes and a non-magnetic environment in FIG. 4, wherein the left graph is a scatter plot of hepatocyte spheroids and the right graph is a mean histogram of hepatocyte spheroids;
fig. 6 is a hepatocyte pellet prepared by the optical microscope under the same hepatocyte inoculation amount and magnetic pulling according to different magnetic micropore arrays input amount, where the upper left is a micrograph of 150k hepatocytes after mixed culture with 3 magnetic micropore arrays overnight under the driving of the magnetic environment, the upper right is a micrograph of 150k hepatocytes after mixed culture with 4 magnetic micropore arrays overnight under the driving of the magnetic environment, the lower left is a micrograph of 150k hepatocytes after mixed culture with 5 magnetic micropore arrays overnight under the driving of the magnetic environment, and the lower right is a micrograph of 150k hepatocytes after mixed culture with 6 magnetic micropore arrays overnight under the driving of the magnetic environment;
fig. 7 is statistical data of hepatocyte spheroids prepared by four different sets of magnetic micropore arrays in fig. 6, wherein the first graph from top to bottom is a mean statistical analysis of hepatocyte spheroids prepared by mixing and culturing 150k hepatocytes with 3, 4, 5, and 6 magnetic micropore arrays respectively overnight under the driving of a magnetic environment, the second graph from top to bottom is a statistical analysis of hepatocyte spheroids prepared by mixing and culturing 150k hepatocytes with 3, 4, 5, and 6 magnetic micropore arrays respectively overnight under the driving of a magnetic environment, and the third graph from top to bottom is a scattered point distribution of hepatocyte spheroids prepared by mixing and culturing 150k hepatocytes with 3, 4, 5, and 6 magnetic micropore arrays respectively overnight under the driving of a magnetic environment;
fig. 8 is a fluorescence staining diagram of live and dead cells obtained after the overnight mixed culture of the different mesenchymal stem cell inoculation amounts and 4 magnetic micropore arrays under the fluorescence microscope provided in embodiment 3 of the present application, wherein the left diagram is a fluorescence staining diagram of live and dead cells obtained after the overnight mixed culture of 30k mesenchymal stem cells and 4 magnetic micropore arrays, the middle diagram is a fluorescence staining diagram of live and dead cells obtained after the overnight mixed culture of 75k mesenchymal stem cells and 4 magnetic micropore arrays, and the right diagram is a fluorescence staining diagram of live and dead cells obtained after the overnight mixed culture of 150k mesenchymal stem cells and 4 magnetic micropore arrays;
FIG. 9 is a graph showing the survival curves of the experimental treatment group of immunodeficient mice implanted subcutaneously with the same number of hepatocytes as single cells and cytospheres and the control group of untreated immunodeficient mice as provided in example 4 of the present application;
FIG. 10 shows the serum levels of aspartate aminotransferase (left panel), albumin (middle panel) and total bile acid (right panel) in the experimental group of immunodeficient mice implanted subcutaneously with the same number of hepatocytes as single cells and cell spheres and the control group of untreated immunodeficient mice provided in example 4 of the present application;
FIG. 11 is a graph showing TUNEL in situ apoptosis staining of liver tissue of an experimental group of immunodeficient mice subcutaneously implanted with the same number of hepatocytes as single cells and cell balls and a control group of untreated immunodeficient mice as provided in example 4 of the present application;
FIG. 12 is a graph showing the survival curves of the experimental group of immune-competent mice treated with the same number of hepatocytes and single cells and cell balls implanted subcutaneously and the control group of immune-competent mice without treatment provided in example 5 of the present application;
FIG. 13 is a general image of the liver of mice surviving from 7 days after the treatment of the experimental group of immune healthy mice implanted with the same number of single cells and cell balls of hepatocytes subcutaneously and the control group of immune healthy mice without treatment provided in example 5 of the present application, and the red color range is abnormal liver tissue morphology;
fig. 14 is a hematoxylin/eosin complex staining diagram of liver tissue sections of an experiment group of treatment of immune healthy mice implanted with the same number of single cells and cytospheres subcutaneously and a control group of immune healthy mice without treatment 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 defects that the cell balls prepared by the prior art are often different in size or difficult to prepare on a large scale.
The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
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 procedure for preparing the double-sided magnetic microwell array provided in fig. 1, using polydimethylsiloxane as the gel material. Wherein, the whole projection area of the square array mould with the round cone is 5 mm multiplied by 5 mm; the integral projection area of the die with the cone circular array is 0.5 square centimeter; the base of the cone is a circle with a diameter of 500 microns and the height of the cone is 300 microns.
Adding ferroferric oxide nano magnetic particles with different concentrations into an initiator, carrying out ultrasonic treatment for 30 minutes, carrying out heavy suspension, adding a base solution of dimethyl siloxane, and uniformly stirring to obtain a gel precursor; standing for 5 minutes to remove air bubbles, pouring the uniformly mixed gel into molds with different shapes and different numbers of cone arrays, and carrying out vacuum treatment for 10 minutes. The mold with the gel precursor poured is pressed onto a clean plane to extrude excess gel precursor. And (3) carrying out vacuum treatment again for 10 minutes, transferring the mixture into an oven, heating the mixture for 2 hours at the temperature of 80 ℃ to solidify and form gel, and separating a mould to obtain the magnetic micropore array with the single-sided micro blind holes.
Next, a new precursor of the magnetic gel was cast on the mold with the same cone array and vacuumed for 10 minutes to remove air bubbles generated during the casting process. Then covering the magnetic micropore array with the single-sided magnetic micropore array to extrude redundant magnetic gel precursor, carrying out vacuum treatment for 10 minutes, then carrying out heat curing at 80 ℃ for 2 hours, and separating the mold with the cone array after the magnetic gel is shaped, thus completing the preparation of the magnetic micropore array with the double-sided magnetic micropore array. Magnetic micropore arrays with different shapes and containing ferroferric oxide nano magnetic particles with different concentrations and double-sided micro blind holes are prepared, the appearance picture of the magnetic micropore arrays is shown in figure 2, and the rightmost picture in figure 2 is a double-sided micro blind hole micrograph of the magnetic micropore array prepared by adding the ferroferric oxide nano magnetic particles with 4 mg/ml.
Example 2
The embodiment of the application provides a preparation method of a hepatocyte cell pellet, which specifically comprises the following steps:
in the embodiment of the application, a square magnetic micropore array prepared by adding 4 mg/ml ferroferric oxide nano magnetic particles is adopted, and the integral projection area of the blind micro-holes is 5 mm multiplied by 5 mm. The magnetic micro-well array prepared in the above example was previously sterilized by soaking in 75% alcohol for 30 minutes and washed twice with physiological saline.
In this example, the sizes of the spheroids of the hepatocytes are first controlled by changing the cell seeding amount, and the spheroids 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 suspension state cells (single cells) and the culture medium are mixed and cultured in a culture dish according to the flow of figure 3, a plurality of magnetic micropore arrays are drawn by magnetic force (permanent magnets) to capture the suspension state cells in the three-dimensional space of the culture medium, and the suspension state cells are aggregated to form cell spheres under the limitation of space. The method comprises the following steps of (1) uniformly mixing single cells and a magnetic micropore array in a specific ratio, (2) capturing three-dimensional cells under the drive of magnetic force, and (3) forming cell spheres by aggregating/proliferating the cells in the blind micropores.
Specifically, the magnetic micropore arrays and the hepatocytes are uniformly mixed in a 48-pore plate containing a culture medium, the number of the hepatocytes in the culture medium is respectively 30k, 150k and 300k, the dosage of the culture medium in the 48-pore plate is 1 ml, the number of the magnetic micropore arrays is 4, and a piece of 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 each of the 48-pore plate (the number of the hepatocytes is respectively 30k, 150k and 300k) of three experimental groups to form a static magnetic field to drive the magnetic micropore arrays to move upwards. The magnetic micropore array can contact suspended liver cells during migration and collect the suspended liver cells into the blind micropores to complete the capture of the liver 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, the hepatocytes will aggregate under the restriction of the blind microperforated space, forming hepatocyte spheroids through the interaction between hepatocytes. The separation of hepatocyte spheroids can be achieved by simple centrifugation methods. The other set of control 48-well plates (150 k hepatocytes) were incubated overnight in the same culture environment without NdFeB permanent magnets of N52 on top of the plates. After overnight culture, the hepatocyte spheroids were separated by centrifugation, and the morphology of the four groups of hepatocyte spheroids was microscopically imaged and the distribution of their cross-sectional areas and the mean value were statistically analyzed, the results are shown in fig. 4 and 5. The size of the prepared hepatocyte spheroids can be correspondingly increased along with the increase of the cell inoculation amount through microscopic imaging, and the size distribution range of the hepatocyte spheroids prepared by the magnetic force driven magnetic micropore array is narrower and shows better uniformity compared with a control group without magnetic attraction under the 150k hepatocyte inoculation amount. However, the higher cell inoculum size (300k hepatocytes) resulted in a larger size distribution of the hepatocyte spheroids produced, and thus the average cross-sectional area was not increased accordingly.
In addition, the size and yield of hepatocyte balls are controlled by changing the feeding amount of the magnetic micropore arrays, the hepatocyte balls are divided into four groups, and the four groups are magnetically drawn after the magnetic micropore arrays with different numbers and cells with the same inoculation amount are mixed. Specifically, the magnetic micropore arrays and the hepatocytes are uniformly mixed in a 48-pore plate containing a culture medium, the number of the hepatocytes in the culture medium is 150k, the dosage of the culture medium in the 48-pore plate is 1 ml, the number of the magnetic micropore arrays is divided into 3, 4, 5 and 6, and a piece of neodymium iron boron permanent magnet (the size: 50 mm × 25 mm × 10 mm) of N52 is placed on the top of each 48-pore plate to form a static magnetic field to drive the magnetic micropore arrays to move upwards. The magnetic micropore array can contact suspended liver cells during migration and collect the suspended liver cells into the blind micropores to complete the capture of the liver 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, the hepatocytes will aggregate under the restriction of the blind microperforated space, forming hepatocyte spheroids through the interaction between hepatocytes. The separation of hepatocyte spheroids can be achieved by simple centrifugation methods. The morphology of four groups of hepatocyte spheroids was microscopically imaged and the average cross-sectional area, the number of spheroids and the cross-sectional area distribution were statistically analyzed, and the results are shown in fig. 6 and 7. The size and the yield of the prepared hepatocyte spheres are correspondingly increased along with the increase of the input amount of the magnetic micropore array through microscopic imaging, which shows 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 spheres also becomes wider as the input amount of the magnetic micropore array increases. Compared with the 3 magnetic micropore arrays, the number of the hepatocyte balls prepared by the 4 magnetic micropore arrays through magnetic traction is more, and the cross-sectional area distribution range is not obviously increased. Therefore, 4 magnetic microwell arrays and 150k hepatocytes were the optimal dosing ratios in this example.
Example 3
The embodiment provides a preparation method of a mesenchymal stem cell ball, which specifically comprises the following steps:
in the embodiment of the application, a square magnetic micropore array prepared by adding 4 mg/ml ferroferric oxide nano magnetic particles is adopted, and the integral projection area of the blind micro-holes is 5 mm multiplied by 5 mm. The magnetic micro-well array prepared in the above example was previously sterilized by soaking in 75% alcohol for 30 minutes and washed twice with physiological saline.
In the embodiment, the size of the mesenchymal stem cell spheroids is controlled by changing the cell inoculation amount, the mesenchymal stem cells are divided into three groups in total, and magnetic traction is applied to the three groups. Specifically, the magnetic micropore arrays 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 respectively 30k, 75k and 150k, the using amount of the culture medium in the 48-pore plate is 1 ml, the number of the magnetic micropore arrays is 4, and a piece of NdFeB permanent magnet (the size is 50 mm multiplied by 25 mm multiplied by 10 mm) of N52 is placed on the top of each 48-pore plate to form a static magnetic field to drive the magnetic micropore arrays to move upwards. The magnetic micropore array can contact and collect the suspended mesenchymal stem cells into the blind micropores during the migration process 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, the mesenchymal stem cells can gather under the restriction of the blind micro-hole space, and mesenchymal stem cell balls are formed through the interaction among the mesenchymal stem cells. The separation of the mesenchymal stem cell ball can be realized by a simple centrifugation method. After overnight culture, the mesenchymal stem cell pellet was subjected to live-dead staining and imaged by a fluorescence microscope, and the result is shown in fig. 8. The size of the prepared mesenchymal stem cell ball 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 cell, which specifically comprises the following steps:
in the examples of the present application, the hepatocyte spheroids prepared by the method in example 2 are used for treating acute liver failure. Specifically, 27 female immunodeficiency Balb/c-nude mice (18-21 g) with the age of 6 weeks were selected and randomly divided into 3 groups of 9 mice each. Mixing carbon tetrachloride (CCl)4) Dissolved in olive oil to a concentration of 40% by volume, at 4. mu.l CCl4The dosage of each gram of the body weight of the mouse is administrated to the abdominal cavity of the mouse to construct an acute liver failure model. Group I was a control group and was not treated after molding. And the group II and the group III are experimental groups, 100 microliters of single cell suspension containing one million liver cells and liver cell balls are respectively injected into the backs of the mice in the experimental groups at a constant speed and slowly after 24 hours of modeling, and the mice are placed in an SPF environment for conventional feeding for 1 week. Mice were observed daily for survival and survival curves were plotted. The experiment was terminated after 1 week and the animals were anesthetized for sampling analysis. Specifically, 0.6% sodium pentobarbital anesthetic (10 microliter/gram animal body weight dose) is injected into the abdominal cavity, 0.5 milliliter of blood is collected through the infraorbital venous plexus, and serum is separated for useDetecting the contents of albumin, aspartate aminotransferase and total bile acid. After blood collection, the mice were sacrificed by cervical dislocation after excessive anesthesia, and liver tissue samples were collected for TUNEL in situ apoptosis staining, and 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 implanting the hepatocyte balls under the skin, and compared with single cells with the same number of hepatocytes, the indexes of aspartate aminotransferase and total bile acid liver injury in the serum of the acute liver failure mice living after 7 days are smaller, and the index of the liver function of albumin in the serum is larger. In addition, the number of apoptotic cells in the liver tissue of 7 days after the treatment of acute hepatic failure Balb/c-nude mice by the hepatocyte spheroids is obviously less than that of a control group and a single cell treatment experimental group receiving the same number of hepatocytes.
Example 5
The embodiment provides a method for treating acute liver failure by a hepatocyte spheroid, which specifically comprises the following steps:
in the examples of the present application, the hepatocyte spheroids prepared by the method in example 2 are used for treating acute liver failure. Specifically, 6-week-old females were selected to immunize 27 healthy ICR mice (18-21 g) and randomly divided into 3 groups of 9 mice each. Mixing carbon tetrachloride (CCl)4) Dissolved in olive oil to a concentration of 40% by volume, in 4 microlitres of CCl4The dosage of each gram of the body weight of the mouse is administrated to the abdominal cavity of the mouse to construct an acute liver failure model. Group I was a control group and was not treated after molding. And the group II and the group III are experimental groups, 100 microliters of single cell suspension containing one million liver cells and liver cell balls are respectively injected into the backs of the mice in the experimental groups at a constant speed and slowly after 24 hours of modeling, and the mice are placed in an SPF environment for conventional feeding for 1 week. Mice survival was observed daily and survival curves were plotted. After 1 week, the experiment was terminated, the mice were sacrificed by cervical dislocation after excessive anesthesia, liver tissue samples were collected for paraffin tissue embedding and sectioning, and liver tissue sections 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 the ICR mice with acute liver failure in 7 days can be obviously improved by implanting the hepatocyte balls with the same hepatocyte number subcutaneously (a cell ball treatment group vs single cell treatment group: 67% vs 22%). In addition to this, the present invention is,in 7-day-later-surviving acute liver failure ICR mice, compared with untreated control groups and experimental groups treated by single cell suspension, ICR mice treated by hepatocyte spheroids with the same hepatocyte number have more normal liver appearance forms, smaller necrotic tissue areas in liver tissue slices and lighter inflammatory cell infiltration degree.
In summary, the magnetic micro-pore array with double-sided micro-blind holes according to the embodiments of the present application is designed such that cells can be fully contacted and collected no matter which side of the micro-blind holes faces upwards in the three-dimensional cell capture and cell sphere formation process under magnetic force driving. The movement rate of the magnetic micropore array in the water phase can be correspondingly adjusted by controlling the loading amount of the magnetic particles in the magnetic micropore array and the static magnetic field intensity generated by the permanent magnet. The overall projected area of the blind micro-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. At the same time, the 48-well plate requires sufficient media to provide room for the magnetic microwell array to migrate. The size of the cell ball can be controlled by the size of the blind micro-hole of the magnetic micro-hole array and the feeding ratio of the blind micro-hole to the cell. 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 only a preferred embodiment of the present application and it should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.
Claims (10)
1. A method for preparing a cell pellet, comprising:
in a magnetic environment, performing mixed culture on the magnetic micropore array, the suspension state cells and the culture medium in a culture dish; removing the magnetic environment, and then carrying out centrifugal treatment to prepare cell balls;
and a micro blind hole is formed in the surface of the magnetic micropore array.
2. The method of claim 1, wherein the magnetic microwell array has a shape of one or more of a rectangle, a circle, a triangle, and a polygon; the total projection area range of the magnetic micropore array is 0.001-1000 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 hole of the magnetic micropore array is in one or more of a cone, a cylinder, a polygonal column, a tetrahedron, a sphere and a hemisphere; 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.
4. The method according to claim 1, wherein the material of the magnetic micro-pore array comprises magnetic particles; 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.
5. The method of claim 1, wherein the method of preparing the magnetic microwell array comprises:
mixing the magnetic particles and the gel material to obtain a mixture; pouring the mixture into a mold and then carrying out curing and shaping treatment; and separating the mixture from the mold to obtain the magnetic micropore array.
6. The method according to claim 5, wherein 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 hydrogel and derivatives thereof.
7. The preparation method according to claim 5, further comprising a 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 subjected to a curing and shaping treatment.
8. The method according to claim 1, wherein the number of the magnetic micro-well array in the culture medium is 1-100 pieces/ml, and the density of the suspension cells in the culture medium is 2-107One/ml.
9. 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 osteocytes.
10. The application of the hepatocyte cell balls prepared by the preparation method of claim 1 in the percutaneous implantation of drugs for treating acute liver failure.
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| 张瑞;韩宝三;吴薇;吴旭波;彭承宏;: "微载体及其在肝细胞培养中的作用与应用", 中国组织工程研究与临床康复, no. 32 * |
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