CN117721073A - Method for constructing muscle tissue based on Faraday wave and application - Google Patents
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Abstract
The invention discloses a method for constructing muscle tissue based on Faraday waves, which comprises the following steps: adding the suspension of the assembling unit into an assembling chamber for standing; introducing Faraday waves into the assembly chamber to enable living cells in the suspension of the assembly unit to be gathered into a sound potential well of the sound field to form a pattern; solidifying the assembly unit suspension with the formed pattern to obtain a first assembly unit; adding the suspension of the assembly unit again on the basis of the first assembly unit, and repeating the steps; repeating the steps for n times to obtain an (n+1) -th assembled unit, and finally obtaining a three-dimensional micro-assembled structure; cell culture was performed. According to the invention, faraday waves are applied to the assembled unit suspension to control living cells and carry out high-flux cell unit tissues, so that muscle tissues with excellent communication and excellent rod-shaped muscle fiber bundles, no cell damage and tight cell-cell connection can be formed. The invention also provides application of the method for constructing muscle tissue based on Faraday waves.
Description
Technical Field
The invention relates to the technical field of biological manufacturing, in particular to a method for constructing muscle tissue based on Faraday waves and application thereof.
Background
In recent years, muscle transplantation or regeneration technology brings great hope for rehabilitation to patients who cause muscle tissue injury in congenital or acquired; moreover, the muscle regeneration technology is applied to the manufacture of artificial meat, and can also alleviate the problem of the rapid increase of meat consumption worldwide.
With the development of biotechnology and the advent of interdigitation of multidisciplinary technology, the use of tissue engineering and biological manufacturing techniques to construct muscle tissue in vitro has become a research hotspot in the fields of biomedical and cell culture meat (meat analogue). The technology mainly assembles living cells into tissue blocks with muscle tissue structures through biological materials, cell growth factors and nutrient substances required by cell growth, and finally cultures the tissue blocks into muscle tissues with biological functions, and can be applied to aspects of muscle transplantation, drug screening, food industry and the like, so that the recovery rate of muscle tissue wounded persons can be improved, and the utilization rate of resources can be improved.
The microstructure composition of muscle tissue is complex, which makes it very difficult to construct muscle tissue with biological functions in vitro. Biological 3D printing technology is the mainstream technical method for constructing tissue and organs in vitro due to good plasticity. The biological 3D printing technology simulates the tissue organ structure in the body through computer modeling, mixes living cells with biological ink to print out muscle tissue with similar morphology, and cultures the muscle tissue into a three-dimensional structure functional body after solidification. However, biological 3D printing technology is a muscle tissue formed with biological ink as a main carrier, so that it has the following limitations: (1) biological 3D printing techniques are difficult to achieve cell heterogeneity: the living cells are mixed in the biological ink, cannot be uniformly distributed, and cannot meet the characteristics of ectopic distribution growth of multiple types of cells in the body; (2) Because of defects of the printing process, such as extrusion printing, ink-jet printing and the like, cells can be subjected to fluid shear force, mechanical and thermal damage, collision damage and the like in the printing process, so that the survival rate of muscle stem cells is reduced, the cells are seriously damaged, and finally, the growth direction of muscle fibers is inconsistent, the mechanical property is poor, the electrical conductivity is inconsistent, and a good bionic effect cannot be achieved; such as the muscle tissue, used for cell culture of meat (meat analogue), also affects the taste of meat; (3) Biological 3D printing technology adopts single cells to construct muscle tissues, which leads to insufficient intercellular gap connection communication: single cells have larger and uneven intermediate distances in the bio-ink, and are difficult to generate tight connection among cells to carry out contact-dependent communication, so that the biological function is poor; (4) The cell content in the ink used for printing is only about 50%, and a small number of cells cannot meet the differentiation of cells to form muscle fibers; (5) The biological 3D printing does not have a muscle fiber anchor point, and muscle fibers shrink into a bulk shape when performing contraction movement, so that the long strip shape cannot be maintained.
In view of the above, there are limitations in the biological 3D printing technology in constructing muscle tissue in vitro, and new methods are urgently needed to solve the above problems.
Disclosure of Invention
In order to solve the problems, the invention provides a method for constructing muscle tissue based on Faraday waves, which applies Faraday waves in assembled unit suspension through a split tissue technology to control living cells and carry out high-flux cell unit tissue, so that muscle tissue with excellent communication and no cell damage and tight cell-to-cell connection can be formed by the rod-shaped muscle fiber bundles. The invention also provides application of the method for constructing muscle tissue based on Faraday waves.
In a first aspect, a first object of the present invention is to provide a method for constructing muscle tissue based on faraday waves, comprising the steps of:
adding the suspension of the assembling unit into an assembling chamber for standing;
introducing Faraday waves into the assembly chamber to enable living cells in the suspension of the assembly unit to be gathered into a sound potential well of the sound field to form a pattern;
solidifying the assembly unit suspension with the formed pattern to obtain a first assembly unit;
adding the suspension of the assembly unit again on the basis of the first assembly unit, and repeating the steps;
repeating the steps for n times to obtain an (n+1) -th assembled unit, and finally obtaining a three-dimensional micro-assembled structure;
cell culture was performed.
In some preferred embodiments thereof, the faraday wave is generated by a faraday wave generation apparatus comprising:
a function signal generator for generating a waveform signal including a sine signal, a cosine signal, or a multi-wavelength synthesized signal;
the power amplifier is connected with the function signal generator and is used for amplifying the amplitude of the waveform signal;
the vibration exciter is connected with the power amplifier and is used for converting the waveform signal into mechanical vibration in the vertical direction to form a sound field;
the assembly chamber is arranged above the vibration exciter.
The shape of the assembly chamber is one of a circle, a triangle, a square, a polygon or a special shape.
In some preferred embodiments, at least one of an ultraviolet light source, a heating element or a cooling element is also provided above or below the assembly chamber.
In some preferred embodiments, the faraday wave generation apparatus applies faraday waves at a loading condition of 85Hz and 100mVpp amplitude and introduces into the assembly chamber;
the assembly chamber is a square chamber, the specification of the assembly chamber is 15mm or 15mm, and the thickness of the assembly chamber is 2mm; the assembled unit suspension is a hydrogel prepolymer comprising living cells.
In some preferred embodiments, faraday waves are introduced into the assembly chamber, the living cells forming an assembly modality, the assembly modality being a bar-shaped modality formed by the tight junctions of living cells; the strip mode has directivity and is consistent in direction or neutral symmetry.
In some preferred embodiments, the method further comprises preparing the assembled cell suspension prior to adding the assembled cell suspension to the assembly chamber for standing; the assembled unit suspension comprises at least living cells, hydrogel, initiator and cell growth factor.
In some preferred embodiments, the living cells are single cells, micro-tissue masses, organoids, cell microspheres, cell-containing hydrogel microspheres, or cell-containing carrier microparticles, or a mixture of any of them.
In some preferred embodiments, the cell-containing hydrogel microspheres or cell-containing carrier microparticles are prepared by microfluidic, electrostatic spraying or inkjet printing, and curing with one or more of temperature sensitive, photosensitive or chemical reactions.
In some preferred embodiments, the hydrogel is one or a mixture of any of a photo-setting hydrogel, a temperature sensitive hydrogel, an enzyme linked hydrogel or a chemical hydrogel.
In some preferred embodiments thereof, the photocurable hydrogel comprises at least one of GelMA, HAMA, algMA, methacryloylated collagen, methacryloylated silk fibroin, methacryloylated chitosan, PEGDA, methacryloylated dextran, methacryloylated carboxymethyl chitosan, methacryloylated heparin, or methacryloylated chondroitin sulfate;
the temperature-sensitive hydrogel comprises at least one of NIPAAm, gelatin or fibrinogen;
the enzyme-linked hydrogel is fibrinogen;
the chemical hydrogel is at least one of alginic acid, modified hyaluronic acid, modified collagen, modified silk fibroin, modified chitosan or modified gelatin;
the initiator is at least one of photoinitiator or thrombin.
In a second aspect, a second object of the present invention is to apply the above method for constructing muscle tissue based on faraday waves to construct a physiological or pathological model of a tissue organ, drug screening, artificial meat manufacturing or tissue repair.
Based on the technical scheme, compared with the prior art, the invention has the following technical effects:
1. the method for constructing muscle tissue based on Faraday waves, provided by the invention, utilizes the function signal generator, the power amplifier and the vibration exciter to form Faraday waves, and applies the Faraday waves into the assembly unit suspension containing living cells, and the living cells in the assembly unit suspension are gathered in the lowest potential energy part (sound potential well) of a sound field by optimizing the assembly conditions, so that the construction of a complex and ordered cell patterning structure is realized.
2. According to the method for constructing the muscle tissue based on the Faraday wave, provided by the invention, various cells are controlled in the assembled unit suspension through the differential assembly technology, so that a muscle tissue structure which is more complex in structure and is close to the real physiology of a human body can be constructed; secondly, after a patterned structure is constructed by utilizing Faraday wave sound field, curing according to the crosslinking characteristic of the suspension of the assembly unit; finally, tension is provided by micropillars around the assembled chamber, and the solidified tissue blocks are placed into an incubator for subsequent cell culture, so as to form the muscle fibers in an extended state.
3. The method for constructing muscle tissue based on Faraday waves is applied to the construction of physiological or pathological models of tissues and organs, drug screening, artificial meat manufacturing or tissue repair. Compared with the biological 3D printing technology, the method can effectively improve the cell proliferation rate and the cell utilization rate and improve the cell survival rate and the forming stability, thereby being beneficial to forming in-vitro muscle tissues and ensuring that the intercellular gap connection communication is not blocked.
4. According to the method for constructing muscle tissue based on Faraday waves, faraday waves are introduced into the assembly cavity, so that muscle cells are gathered and connected to form a strip-shaped mode or a symmetrical mode. Compared with the non-modal muscle cell aggregation, the consistency of the growth direction of the muscle cells promotes the differentiation and maturation of the muscle fibers, accelerates the formation process from the muscle cells to the muscle fibers, ensures that the orderly muscle fibers are more attached to the in-vivo form, and can increase the success rate of muscle transplantation, the taste of artificial meat and the like.
Drawings
Fig. 1 is a flow chart of the method of constructing muscle tissue based on faraday waves of the present invention.
FIG. 2 is a graph of the internal flow velocity field distribution of the fluid at 87Hz frequency for example 1.
FIG. 3 is a graph of the internal pressure field distribution of the fluid at 87Hz for example 1.
Fig. 4 is a graph of the potential energy field distribution of the interior of the fluid at 87Hz frequency for example 1.
FIG. 5 is a diagram showing immunofluorescence of example 1 for detecting skeletal muscle-specific protein MyHC.
Fig. 6 is a graph comparing the quantitative analysis of the degree of aggregation of the patterning and non-patterning of comparative example 1.
Fig. 7 is a bright field contrast plot of comparative example 1 patterned and unpatterned.
FIG. 8 is a directional contrast plot of the growth of the patterned and unpatterned cell units of comparative example 1.
FIG. 9 is a graph of the directional contrast statistics of the growth of the patterned and unpatterned cell units of comparative example 1.
FIG. 10 is a graph showing the comparison of the expression of the differentiation marker genes of the patterned and unpatterned myocytes of comparative example 1.
Fig. 11 is a differential assembly simulation diagram of comparative example 2.
FIG. 12 is a diagram showing the differential assembly of the myocyte unit and cell-encapsulating microgel spheres of example 2.
FIG. 13 is a diagram of the differential assembly of the myocyte unit and endothelial cell unit of example 2.
FIG. 14 is a diagram showing the differential assembly of the myocyte unit and the microgel pellet of example 2.
Fig. 15 is a square chamber design of the anchored myofiber bundle of example 3.
Fig. 16 is a stretch of anchored myofibers of example 3.
Fig. 17 is a contraction view of unanchored muscle fibers of example 3.
Detailed Description
In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments that are illustrated in the appended drawings. The drawings illustrate preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
It will be understood that when an element is referred to as being "fixed to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present.
In the description of the present invention, it should be noted that, directions or positional relationships indicated by terms such as "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., are directions or positional relationships based on those shown in the drawings, or are directions or positional relationships conventionally put in use of the inventive product, are merely for convenience of describing the present invention and simplifying the description, and are not indicative or implying that the apparatus or element to be referred to must have a specific direction, be constructed and operated in a specific direction, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," "third," and the like are used merely to distinguish between descriptions and should not be construed as indicating or implying relative importance.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
A method of constructing muscle tissue based on faraday waves, comprising the steps of:
101. adding the suspension of the assembling unit into an assembling chamber for standing;
102. introducing Faraday waves into the assembly chamber to enable living cells in the suspension of the assembly unit to be gathered into a sound potential well of the sound field to form a pattern;
103. solidifying the assembly unit suspension with the formed pattern to obtain a first assembly unit;
104. adding the suspension of the assembly unit again on the basis of the first assembly unit, and repeating the steps;
105. repeating the steps for n times to obtain an (n+1) -th assembled unit, and finally obtaining a three-dimensional micro-assembled structure;
106. cell culture was performed.
In the step of introducing a faraday wave into the assembly chamber, the faraday wave is generated by a faraday wave generation apparatus comprising a function signal generator, a power amplifier and a vibration exciter.
In the faraday wave generation apparatus, a function signal generator is used to generate a waveform signal, which is an electrical signal of a specific wavelength, such as a sine signal, a cosine signal, or a multi-wavelength composite signal; the power amplifier is connected with the function signal generator and is used for amplifying the amplitude of the waveform signal and providing power for driving the vibration exciter; the vibration exciter is connected with the power amplifier and is used for converting the waveform signal into mechanical vibration in the vertical direction to form a sound field; the assembling chamber is a space for loading and solidifying the assembling unit suspension, and the assembling chamber is arranged above the vibration exciter.
Muscle tissue is composed of a plurality of muscle bundles arranged and piled up at high heights. By highly simulating the composition of the muscle tissue in the body, the bionic effect of the muscle tissue in the body can be improved. Faraday waves, in turn, are a surface standing wave generated at the gas-liquid interface, a representative one of acoustic bioaugmentation. The cells can be patterned in a rapid, non-destructive and non-contact manner outside the body by generating a faraday wave by a faraday wave generating device and applying it to an assembly chamber where the assembly cell suspension is held.
As shown in fig. 2, the high-flux cell units are assembled by faraday waves, so that a muscle tissue of a rod-shaped muscle fiber bundle can be formed, the muscle tissue is free from cell damage, and the intercellular gap junction communication is good. This embodiment uses faraday applied to the assembly chamber where the assembled unit suspension is resting for muscle assembly, a very desirable technique for constructing biomimetic muscle tissue in vitro.
The method for constructing muscle tissue based on Faraday waves of the embodiment is mainly based on assembly unit suspension of muscle cells and biological manufacturing technology of Faraday waves. Firstly, a Faraday wave generating device formed by instruments such as a function signal generator, a power amplifier, a vibration exciter and the like is utilized to form Faraday waves (standing waves), and living cells in the suspension of the assembly unit are captured (gathered) to the lowest potential energy position (sound potential well) under the combined action of the properties of an assembly medium, the geometric boundary conditions of an assembly cavity and Faraday wave driving parameters (physical fields), so that the construction of a complex and ordered cell patterning structure is realized. Secondly, after a patterned structure is constructed by utilizing Faraday wave sound field, curing according to the hydrogel crosslinking characteristic in an assembly medium; finally, tension is provided by micropillars around the assembled chamber, and the solidified tissue blocks are placed into an incubator for subsequent cell culture, so as to form the muscle fibers in an extended state.
In some embodiments, the carrier of the assembly chamber may be one of a common multi-well plate, a culture dish, a glass bottom dish, or a cell chamber, and the shape of the assembly chamber is one of a circle, a triangle, a square, a polygon, or a profile.
The assembling chamber is nested above the vibration exciter, and at least one of an ultraviolet light source, a heating piece or a refrigerating piece is arranged above or below the assembling chamber, so that the assembling unit suspension can be subjected to photosensitive or temperature-sensitive crosslinking solidification.
In some embodiments, the faraday wave generation apparatus applies a faraday wave at a loading condition of 85Hz and 100mVpp amplitude and introduces it into the assembly chamber; the assembly chamber is a square chamber, the specification of the assembly chamber is 15mm, and the thickness of the assembly chamber is 2mm; the assembled unit suspension is a hydrogel prepolymer comprising living cells.
Introducing Faraday waves into an assembly chamber, and forming an assembly mode by the living cells, wherein the assembly mode is a strip mode formed by tightly connecting the living cells; the strip mode has directivity and is direction consistency or neutral symmetry.
The microcolumns exist at the boundary of the assembly chamber, which can provide tension for muscle fibers, maintain the stretching state of the muscle fibers and be more fit with the internal muscle tissue structure.
Preferably, the method for constructing muscle tissue based on faraday waves provided in this embodiment, before adding the assembled unit suspension to the assembly chamber for standing, further includes:
100. preparing an assembly unit suspension; the assembled unit suspension comprises at least living cells, hydrogel, initiator and cell growth factor.
Wherein the living cells are one or more cells constituting muscle tissue, specifically, the living cells are one or a mixture of any of single cells, micro-tissue blocks, organoids, cell microspheres, cell-containing hydrogel microspheres or cell-containing carrier particles. The hydrogel microsphere containing cells or the carrier particle containing cells is prepared by curing one or more of temperature-sensitive, photosensitive or chemical reactions in a microfluidic, electrostatic spraying or ink-jet printing mode.
The hydrogel is biological ink, and the hydrogel is one or a mixture of any of photo-curing hydrogel, temperature-sensitive hydrogel, enzyme-linked hydrogel or chemical hydrogel, or ion-curing hydrogel.
Wherein the photocurable hydrogel comprises at least one of GelMA, HAMA, algMA, methacryloylated collagen, methacryloylated silk fibroin, methacryloylated chitosan, PEGDA, methacryloylated dextran, methacryloylated carboxymethyl chitosan, methacryloylated heparin, or methacryloylated chondroitin sulfate;
the temperature sensitive hydrogel comprises at least one of NIPAAm, gelatin, or fibrinogen;
the enzyme-linked hydrogel is fibrinogen;
the chemical hydrogel is at least one of alginic acid, modified hyaluronic acid, modified collagen, modified silk fibroin, modified chitosan or modified gelatin;
the initiator is at least one of photoinitiator or thrombin.
In 106, culturing the cells by adopting a culture medium, serum, p/s, cell growth factors and other nutrient substances required by cell growth, wherein the culture medium provides nutrition for the cells and maintains the growth; the serum contains bovine serum for culture and horse serum for promoting differentiation; p/s is used for diabody and antibacterial.
Based on the method, the embodiment forms the high-flux assembly unit in the tissue chamber, and the patterning assembly of the cell units is realized by optimizing the liquid property in the suspension of the assembly unit, the geometric boundary condition of the assembly chamber and the Faraday wave driving parameter, so that the assembly of a plurality of cells in the same plane and the same assembly medium is realized, different cell units are distributed at different positions to form the rod-shaped muscle fiber bundles of various modes, thereby being more similar to the cell structure distribution in the muscle tissue in vivo and finally being beneficial to the formation of skeletal muscle tissue in vitro.
In the process of muscle tissue assembly, living cells tend to have the lowest potential energy (sound potential well), so that the cells are distributed more tightly, the inter-cell gap connection communication is not blocked, and the cells are favorably tightly attached to form a large-bundle muscle strip in culture; in addition, the micro-column exists at the boundary of the cavity, which can provide tension for the muscle fiber, maintain the stretching state of the muscle fiber and be more fit with the internal muscle tissue structure.
According to the embodiment, the living cells in the suspension of the assembly unit are controlled and arranged in a Faraday wave sound field mode, mechanical extrusion is not needed, the cells are basically not damaged, different methods such as illumination (ultraviolet light and blue light), enzyme, temperature, chemical bonds and the like can be adopted for solidification, the damage to the cells is small, and the survival rate and the forming stability of the cells are improved.
In the first aspect, the method for constructing muscle tissue based on faraday waves provided in this embodiment can improve the cell proliferation rate. In the method, the sound wave can improve the gas mass transfer rate sound wave, provide high nutrient substances and improve the homogenization rate of metabolic substances, promote the tight connection between cells, thereby promoting the proliferation of cells, and can improve the proliferation rate of cells by 36% +/-12% compared with the biological 3D printing technology for the above reasons.
In a second aspect, the method for constructing muscle tissue based on faraday waves provided in this embodiment can improve the cell utilization rate. In the process of adopting Faraday waves to organize muscle living cells, the activity of the cells is more than 95%, the cell damage can be reduced, and the cell damage in the 3D printing technology reaches 55%, in addition, the sound waves can reduce the cell residual loss, which is lower than the cell residual in the biological 3D printing technology (the cell residual in the 3D printing technology is about 5%), and the method for constructing muscle tissue by using Faraday waves can improve the cell utilization rate by about 60% -70% based on the reasons.
In a third aspect, the method for constructing muscle tissue based on faraday waves provided in this embodiment may reduce costs. Compared with the biological 3D printing technology, the sound wave can improve the cell proliferation rate by 36% +/-12% and the cell utilization rate by about 60% -70%, so that the use rate of the culture medium and the growth factors can be reduced by 50% -60%, and the cost can be reduced by about 30-40% (the culture medium and the growth factors account for about 60% -70% of the artificial meat); by adopting the method for constructing muscle tissue based on Faraday waves, the flaky artificial meat with orderly arranged muscle fibers can be formed, and the toughness and the taste of the artificial meat are improved.
By adopting the method for constructing the muscle tissue based on Faraday waves, the construction structure is more complex and is close to the muscle tissue structure of the real physiology of the human body, and the application requirements of constructing the physiological or pathological model of the tissue organ, drug screening, artificial meat manufacturing or tissue repair can be met.
Example 1
In this example, 7 muscle fiber bundles were formed by assembling C2C12 muscle cell units in a square chamber using a method of constructing muscle tissue based on faraday waves. The method comprises the following steps:
in the Faraday wave generating device, a function signal generator is utilized to generate a sine signal, the sine signal is amplified to a required frequency value by a power amplifier, then a vibration exciter is connected, the vibration exciter converts the sine wave into vertical mechanical force to form a surface standing wave, and therefore cells are controlled in an assembled unit suspension (hydrogel medium). The signal frequency of the function signal generator is adjusted to around 87 HZ.
As shown in fig. 2, the flow velocity fields are aligned in a straight line. As shown in fig. 3, the pressure fields are uniformly arranged in stripes. As shown in fig. 4, the cells tend to move toward the lowest point of potential energy. From the algorithmic simulation, the potential to form large-scale muscle strips is expected at frequencies around 87 Hz.
Based on the Faraday wave generating device, the muscle tissue assembly is completed mainly through the following steps:
C2C12 myocyte unit (cytoball): and uniformly transferring the assembled unit suspension containing single cells into an agarose microarray hole culture dish, placing the assembled unit suspension into a cell box for culturing for 1-3 days after the cells naturally settle into micropores, and automatically aggregating the single cells in the micropores into balls to form the high-flux muscle cell unit.
The main reagent adopted is as follows:
GM growth medium: mixing DMEM basal medium, FBS and P/S in proportion;
DM differentiation medium: uniformly mixing a DMEM basic culture medium, HS and P/S according to a proportion;
GelMA hydrogel: an edible photosensitive liquid gel is prepared from GM and GelMA solids through proportional mixing, dissolving in water bath at 70 deg.C until no deposit and no dense foam are generated, and filtering and sterilizing with 0.22 microns filter screen. Before use, the solution was put into a 37 ℃ metal bath for rewarming.
And (3) a photoinitiator: after ultraviolet light irradiation, the GelMA hydrogel can be crosslinked and cured.
(1) And (3) liquid assembly environment construction:
preparing photosensitive liquid gel, mixing GelMA solid and GM according to a proportion, fully heating in a water bath kettle at 70 ℃ to dissolve the GelMA solid, taking out the mixed liquid from the water bath kettle after no obvious solid residue is observed, and filtering the mixed liquid by using a 22um filter membrane to obtain the gel. And mixing the filtered GelMA solution with a photoinitiator to form the hydrogel prepolymer. The prepared hydrogel prepolymer and cell sediment are used for resuspension to obtain an assembled unit suspension, and the concentration of the hydrogel prepolymer can be adjusted according to specific assembly requirements in the process.
(2) Assembling cells based on faraday waves:
450uL of hydrogel prepolymer containing muscle cell units is added into an assembling chamber, the assembling chamber is a square chamber with the specification of 15mm by 15mm and the thickness of 2mm, and after living cells in suspension of the assembled units naturally subside to the bottom of the assembling chamber, faraday waves are applied under the loading condition of 'frequency 85Hz and amplitude of 100 mVpp'.
After about 1min of faraday wave action, a stable corresponding pattern is evident at the bottom of the assembly chamber. The living cells form an assembly mode, and the assembly mode is a strip mode formed by closely connecting the living cells; the bar mode has directivity. Because the microcolumns exist at the boundary of the assembly chamber, tension can be provided for muscle fibers, the muscle fibers are maintained in an extended state, and the muscle fibers are more attached to the internal muscle tissue structure.
The hydrogel prepolymer in the assembly chamber was treated with a portable blue flashlight for 30s to cure the hydrogel. Subsequently, the cured hydrogel was peeled from the chamber, transferred to an orifice plate, and 2mL of GM was added, and placed in a cell incubator (37 ℃,5% CO) 2 ) Culturing in a cell incubator (37 ℃ C., 5% CO) with 2mL GM 2 ) Is cultured. The operation should be carried out in the whole process in the dark.
(3) Engineering cell meat culture:
cells were cultured, GM was added the first day of culture, and subsequently replaced with DM, and after a period of 7-14d of culture, the meat analogue was removed from the petri dish and subsequently identified, as shown in fig. 5, as positive for skeletal muscle-specific protein MyHC immunodetection.
Comparative example 1 (axial comparison of assembled and unassembled)
The directional arrangement and density packing of muscle bundles are inherent existence forms of skeletal muscle tissues, and have important significance for maintaining normal physiological structures and functions of the muscle tissues.
The unassembled and assembled musculature were set as control and experimental groups, respectively.
As can be seen from fig. 6 (a-B), patterning has a significant driving effect on the assembled units, and the bright field patterns before and after assembly are quantitatively analyzed for concentration (as shown by C in fig. 6), and the assembled units are concentrated and dispersed in specific areas, and obvious close-packed stripes appear. Further, the interior of the GelMA hydrogel was observed.
On day 0, the assembled cells in the control group exhibited random distribution within the hydrogel (fig. 7-top left) with no division of the region, whereas cells assembled using faraday waves could form interstitial regions with few cells and multi-layered stacked banding regions (fig. 6-top right), and the cells appeared to be oriented in the patterned direction. After 13 days of incubation, unordered ligation occurred in the control group (bottom left of fig. 7). Whereas the Gap region of the experimental group only had a small number of cells, the Band region appeared to have a stable directional junction (FIG. 7-bottom right).
Subsequently, hoechst/PI double staining (fig. 8 left) was performed on the pre-and post-assembly units, and quantitative analysis (fig. 8 right) showed that acoustic assembly of muscle tissue using faraday waves had no significant effect on cell activity compared to the pre-and post-assembly units. Finally, the orientation of the cells was measured and found to be non-directional in the control and significantly better in the assembled cells than in the control (fig. 9).
It can be seen that faraday wave biological assembly has no effect on cell activity, can effectively promote cell aggregation and dispersion in specific areas, and cells in the aggregation areas are arranged in a significant direction. This facilitates the bionic physiological structure of human skeletal muscle and promotes its further differentiation.
Comparative example 2 ((assembled versus unassembled Gene expression)
The effect of alignment, accumulation on C2C12 myoblast differentiation was compared, and the unassembled and assembled groups were designated as control group and experimental group, respectively, and the expression levels of myoblast differentiation marker genes Myod1, myoG, MRF4 and MyHC at days 1,7, 13 were examined using RT-qPCR method, as shown in fig. 10A-D).
The results show that: the 4 myogenic differentiation marker genes are expressed in a random control group and an assembly experimental group, and all show a certain regularity, namely, the myogenic differentiation marker genes are increased along with the increase of differentiation culture time. The 4 myogenic differentiation marker genes were found to be significantly increased in the experimental group compared to the control group in the test on day 13, and the induction of skeletal muscle formation by the arrangement and accumulation after faraday wave assembly was further verified.
Example 2 (comparison between cytogel sphere differential and xenogeneic cytosphere differential, microgel sphere differential)
This example uses a method of constructing muscle tissue based on faraday waves to differentially assemble C2C12 cell spheres (about 70-200 μm) and microgel spheres (gel spheres containing single cells) (about 500-700 μm) in a square chamber to form a sheet-like meat analogue, as shown in fig. 11, the volume of the sheet-like meat analogue formed being v=15 mm×15mm×1 mm=225 μl.
The method comprises the following steps:
in the Faraday wave generating device, a function signal generator is utilized to generate a sine signal, the sine signal is amplified to a required frequency value through a power amplifier, then a vibration exciter is connected, the vibration exciter converts the sine wave into vertical mechanical force to form a surface standing wave, and therefore cell balls and microgel balls are controlled in an assembled unit suspension (hydrogel medium). And adjusting the signal frequency of the function signal generator to be about 120HZ, and realizing real-time differential assembly of the heterogeneous cell-containing assembly unit suspension in the Faraday wave sound field by adjusting the size of the heterogeneous cell-containing sphere. During assembly, the C2C12 cell spheres (about 70-200 μm) tend to move toward the lowest point of potential energy, i.e., the node center, while the microgel spheres (gel spheres containing single cells, 500-700 μm) move toward the anti-node, forming a large piece of artificial muscle.
Based on the Faraday wave generating device, the muscle tissue assembly is completed mainly through the following steps:
hydrogel-coated single cells: gelMA is selected as hydrogel for wrapping single cells, the hydrogel is manufactured by utilizing a microfluidic water-in-oil principle, and photo-curing is performed by utilizing a photoinitiator to obtain microgel spheres, and finally the microgel spheres and the C2C12 cell spheres are mixed to form an assembled unit suspension.
The procedure is as in example 1, except that the signal frequency of the function signal generator is adjusted to 120 HZ.
Further, the present example is divided into experimental group a, experimental group B and experimental group C. Wherein,
experiment group a: differential assembly of hydrogel-coated HUVEC single-cell formed microgel spheres (about 500-700 μm) +C2C12 cell spheres (about 70-200 μm).
Experimental group B: differential assembly of HUVEC cell spheres (about 300-500 μm) +C2C12 cell spheres (about 70-200 μm).
Experiment group C: differential assembly of microgel spheres (cell-free, about 500-700 μm) +c2c12 cell spheres (about 70-150 μm).
In the test results, the experimental group a (fig. 12) is compared with the experimental group B (fig. 13), and the experimental group a can realize the 3D growth of different cells in different areas, so that the assembled muscle tissue analogue is more close to the muscle tissue structure in the organism. In the experimental group B, HUVEC cell spheres formed by self-aggregation, which are not wrapped by hydrogel, have the diameter of more than 500 mu m, and the cell spheres formed by self-aggregation are excessively large (> 200 mu m) and easily cause cell death in the spheres, so that the assembled artificial meat tissue slices die.
In the test results, the advantage of the experimental group a (fig. 12) compared with the differential assembly of the experimental group C (fig. 14) is that the slice-shaped meat analogue assembled by the heterogeneous cell ball has a more abundant taste.
Example 3 (comparison of anchored versus unanchored)
The use of microgel-encapsulated xenogeneic cells has technical advantages.
The data from the very long culture proves that the use of the anchoring structure of the chamber has technical advantages, so that the constructed muscle tissue does not shrink
Referring to fig. 15-17, the square chamber design for anchoring the muscle fiber bundle is shown in fig. 15, the muscle fiber bundle has a contraction function, and the muscle fiber is easily contracted into spheroids when the muscle fiber is lost from tensile support along with the degradation of hydrogel, such as the two ends of the unanchored muscle fiber bundle, after long-term culture, as shown in fig. 17. By anchoring the two ends of the muscle fiber bundle, the muscle fiber retraction can be effectively prevented, the culture time is increased, and the sheet-shaped muscle piece is formed, as shown in fig. 16. The bottom layer is a filter membrane structure (shown as c in fig. 15), which can be soaked in the culture medium and is favorable for the mass exchange of cells at the bottom of the gel.
A method of constructing muscle tissue based on faraday waves, comprising the steps of:
anchoring structure: the material of the anchoring structure is polyethylene glycol, gelatin, hyaluronic acid, collagen, fibronectin and other substances or mixtures.
Assembling the muscle fiber bundles: the remaining steps are as in example 1, except that the function signal generator can be tuned to any frequency to form a bar pattern.
Anchoring the muscle fiber bundles: the assembled and cured muscle fiber bundles were moved into the anchoring chamber, the collagen solution was injected from the f-hole in fig. 15 and the tendon structure as the anchored muscle fiber bundles was cured at 37 ℃ to connect with the muscle fiber bundles, and the remaining steps were shown in the examples.
Compared to the assembled group of unanchored myocyte units, the anchored myocyte units are more ductile and more biomimetic in that the myofiber bundles remain in their stretched state.
The foregoing is merely illustrative and explanatory of the invention as it is described in more detail and is not thereby to be construed as limiting the scope of the invention. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the spirit of the invention, and that these obvious alternatives fall within the scope of the invention.
Claims (12)
1. A method of constructing muscle tissue based on faraday waves, characterized by: the method comprises the following steps:
adding the suspension of the assembling unit into an assembling chamber for standing;
introducing Faraday waves into the assembly chamber to enable living cells in the suspension of the assembly unit to be gathered into a sound potential well of the sound field to form a pattern;
solidifying the assembly unit suspension with the formed pattern to obtain a first assembly unit;
adding the suspension of the assembly unit again on the basis of the first assembly unit, and repeating the steps;
repeating the steps for n times to obtain an (n+1) -th assembled unit, and finally obtaining a three-dimensional micro-assembled structure;
cell culture was performed.
2. The method of constructing muscle tissue based on faraday waves according to claim 1, wherein the faraday waves are generated by faraday wave generation apparatus comprising:
a function signal generator for generating a waveform signal including a sine signal, a cosine signal, or a multi-wavelength synthesized signal;
the power amplifier is connected with the function signal generator and is used for amplifying the amplitude of the waveform signal;
the vibration exciter is connected with the power amplifier and is used for converting the waveform signal into mechanical vibration in the vertical direction to form a sound field; the assembly chamber is arranged above the vibration exciter.
3. The method of constructing muscle tissue based on faraday waves of claim 2, wherein the shape of the assembly chamber is one of circular, triangular, square, polygonal or shaped.
4. The method of constructing muscle tissue based on faraday waves of claim 2, wherein at least one of an ultraviolet light source, a heating element or a cooling element is further provided above or below the assembly chamber.
5. The method of constructing muscle tissue based on faraday waves according to claim 2, characterized in that the faraday wave generating means applies faraday waves under loading conditions of a frequency of 85Hz and an amplitude of 100mVpp and is introduced into the assembly chamber; the assembly chamber is a square chamber, the specification of the assembly chamber is 15mm or 15mm, and the thickness of the assembly chamber is 2mm; the assembled unit suspension is a hydrogel prepolymer comprising living cells.
6. The method of constructing muscle tissue based on faraday waves of claim 5, wherein faraday waves are introduced into the assembly chamber, the living cells form an assembly modality, the assembly modality being a bar modality formed by tight junctions of living cells; the strip mode has directivity and is consistent in direction or neutral symmetry.
7. The method of constructing muscle tissue based on faraday waves of claim 1, further comprising preparing the assembled unit suspension before adding the assembled unit suspension to the assembly chamber for standing; the assembled unit suspension comprises at least living cells, hydrogel, initiator and cell growth factor.
8. The method of constructing muscle tissue based on faraday waves according to claim 7, wherein the living cells are one or a mixture of any of single cells, micro-tissue blocks, organoids, cell microspheres, cell-containing hydrogel microspheres or cell-containing carrier microparticles.
9. The method for constructing muscle tissue based on faraday waves according to claim 8, wherein the hydrogel microsphere containing cells or the carrier particle containing cells is prepared by means of micro-fluidic, electrostatic spraying or ink-jet printing and curing by one or more of temperature-sensitive, photosensitive or chemical reactions.
10. The method for constructing muscle tissue based on faraday waves according to claim 7, wherein the hydrogel is one or a mixture of any of photo-curing hydrogel, temperature sensitive hydrogel, enzyme linked hydrogel or chemical hydrogel.
11. The method of constructing muscle tissue based on faraday waves of claim 7, wherein the photo-cured hydrogel comprises at least one of GelMA, HAMA, algMA, methacryloylated collagen, methacryloylated silk fibroin, methacryloylated chitosan, PEGDA, methacryloylated dextran, methacryloylated carboxymethyl chitosan, methacryloylated heparin, or methacryloylated chondroitin sulfate;
the temperature-sensitive hydrogel comprises at least one of NIPAAm, gelatin or fibrinogen;
the enzyme-linked hydrogel is fibrinogen;
the chemical hydrogel is at least one of alginic acid, modified hyaluronic acid, modified collagen, modified silk fibroin, modified chitosan or modified gelatin;
the initiator is at least one of photoinitiator or thrombin.
12. Use of faraday-based construction of muscle tissue, characterized in that the faraday-based construction of muscle tissue according to any of claims 1-11 is applied for constructing physiological or pathological models of tissue organs, drug screening, artificial meat manufacture or tissue repair.
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