CN114181801A - Method for constructing high-throughput cell mass model by droplet microfluidic gravity positioning - Google Patents
Method for constructing high-throughput cell mass model by droplet microfluidic gravity positioning Download PDFInfo
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Abstract
The invention discloses a method for constructing a high-throughput cell cluster model by droplet microfluidic gravity positioning. Standing in a cell culture box, forming cell accumulation bodies by means of gravity sedimentation, and adhering cell masses after culture. The time for the initial formation of the cell mass is only 12 hours. The gel has good biocompatibility, forms physical gel below the gel-sol transition temperature, and is in a liquid state when the temperature is raised to the gel-sol transition temperature. The cell mass culture is carried out at a temperature not higher than the gel-sol transition temperature, and the cell mass is collected at a temperature not lower than the gel-sol transition temperature. The cell clusters constructed by the invention have the advantages of high flux, short time consumption, good consistency, simple and convenient recovery and reduction of pollution caused by contact with air in a closed chip environment. The cell mass model construction method can be really applied to the fields of drug activity and substance toxicity screening and the like.
Description
Technical Field
The invention relates to a biological model construction method, in particular to a construction method of a quantitative micro cell mass which can be suitable for high-throughput drug and substance safety tests.
Background
The biological model is a basic model necessary for testing the toxicity and the effectiveness of the medicine of the substance. The biological models traditionally used are mainly animal models and artificially cultured cell models. The animal model and the human have obvious biological difference, so the test result of the animal model is difficult to be directly suitable for the human, even different experimental effects can be obtained, and the animal model is difficult to replace the human experiment. Compared with animals, the artificially cultured cell model is closer to a human body, but the artificial culture is usually carried out in a two-dimensional state, many cells in the human body grow in a three-dimensional and dynamic environment, and the extracellular matrix, the mass transfer process and the metabolic cycle of the cells are obviously different from those of the artificially cultured cells, so that the two-dimensional cell model is also greatly different from the human body, and the test result is obviously different from the actual result. Therefore, the development of a novel cell culture system, the simulation of extracellular matrix and environment, the simulation of physiological mass transfer process and the construction of a novel cell model with certain human tissue functions have important value.
Cell clusters are a biological model formed by many cells combined together and having certain biological functions. The cells in the cell mass are organically combined under the three-dimensional condition, and the function of the human cell mass can be better simulated, so that the cell mass has the advantages over the traditional biological model in drug screening and toxicity testing. The micro cell mass usually has a diameter of hundreds of microns, can simulate the process of diffusion exchange between human tissues and the outside, does not need to construct an additional vascular system to maintain the state of the cell mass, and therefore has higher manufacturing and using convenience. The construction of the micro cell mass by the quantitative technology also provides a biological model with better consistency for high-throughput drug screening and toxicity testing, is more suitable for automatic production compared with the traditional model, greatly reduces the production cost of the biological model, improves the quality and the biological safety, and becomes a key object for competitive research and development in the field of home and abroad biological medicines. However, the existing technology still has certain limitations on the sterile operation, the automation degree, the flux and the consistency of the constructed model of the cell mass construction. The droplet microfluidic technology is a key technology for automatically realizing high-flux uniform droplet manufacturing by utilizing the fluid characteristics of a microfluidic chip. The application of the droplet microfluidic technology to the preparation of high-throughput cell clusters has realized a great breakthrough. However, the prior art mainly depends on cell division and growth in the liquid drop and finally grows into a cell mass, so that the cell mass is constructed. Because the micro-droplets have a certain upper limit on the cell density, it usually takes several days for the cells to grow into a dense cell mass, which is several times longer than the conventional method for constructing a cell mass by stacking and adhering cells, and the latter usually only needs 12 hours to realize the preliminary molding of the cell mass. However, conventional macroscopic cell mass construction methods generally lack consistency and throughput, which in turn limits their application in drug and toxicity screening. Microarray chips partially address the issues of consistency and throughput, but on top of this, operations involving cell pellet retrieval during cell pellet construction typically need to be performed manually and may involve extensive air exposure, risking sterile handling. Furthermore, cell clumps in an array typically need to be cultured in a solution in microwells for recovery, which limits the use of extracellular matrix-like materials in micro-organ construction processes. Therefore, how to construct a miniature cell mass with high consistency and efficiency in a quantitative way becomes the key point for truly applying the cell mass to screening of pharmaceutical activity and substance toxicity.
Disclosure of Invention
In order to solve the problem that the traditional droplet microfluidics in the prior art needs several days for constructing cell clusters, the invention aims to overcome the defects in the prior art, and provides a method for constructing a high-throughput cell cluster model by droplet microfluidics gravity positioning, which can construct a high-consistency minicell cluster in a high-throughput manner within 12 hours. The invention combines the advantages of high flux and consistency of droplet microfluidics, has the advantages of rapidness and high efficiency of traditional cell cluster construction, and can be used for preparing micro cell clusters with the number of more than thousands and the size of tens of micrometers to hundreds of micrometers in a short time. The cell clusters prepared by the invention are in a gel environment, so that the subsequent operation is convenient, and the direct contact among the cell clusters is avoided.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for constructing a high-throughput cell mass model by droplet microfluidic gravity positioning comprises the following steps:
a. microfluidic preparation of cell microdroplets:
building a constant-temperature micro-fluidic device, preparing a mixed solution of gel and cells as a water phase, and preparing oil containing a surfactant as an oil phase, so that the micro-fluidic chip generates uniform multi-cell micro-droplets;
b. synchronous cell gravity sedimentation:
collecting the multicellular micro-droplets, and depositing and adhering the cells by means of gravity to construct a cell aggregation group;
c. cell mass culture:
culturing the cell aggregate in a gel maintaining the cell activity in an environment at a gel-sol transition temperature;
d. shaped cell pellet model recovery
And (3) heating to the gel-sol transition temperature or higher, destroying a gel system containing the cell aggregation group, releasing the cell clusters, and recovering the cell cluster model. The formed cell mass can be used for subsequent detection.
Preferably, in the step a, the microfluidic chip consists of a water phase inlet, an oil phase inlet and an oil phase outlet, and the oil phase is used as an entrained flow to form high-flux uniform-size droplets, wherein the droplet size is 50-500 microns.
Preferably, in the step a, a thermostatic micro-fluidic device is adopted to control the temperature of the aqueous phase solution entering the micro-fluidic chip and the chip for forming the liquid drop, so as to maintain the gel precursor in a flowable state.
Preferably, in the step a, the microfluidic chip is kept at a constant temperature of 25-45 ℃ to form stable and uniform multicellular micro-droplets.
Preferably, in the step a, the gel is at least one of natural gel, synthetic gel and composite gel; or the gel adopts at least one of gels which have reversible temperature-sensitive phase change behavior in physiological solution, form physical gel below the gel-sol transition temperature, are in liquid state above the gel-sol transition temperature and can maintain cell activity.
Preferably, in the step a, the mass concentration of the gel in the mixed solution of the water phase is 3-20%, and the cell concentration is 1 × 105-1×108Each per milliliter.
Preferably, in the step b, when the cell aggregation group is constructed by depositing and adhering cells by gravity sedimentation, the collected container of the multicellular micro-droplets is placed in an incubator for standing, and the cells are deposited and accumulated by gravity sedimentation, and then the cells are adhered to form the cell aggregation group. The collection vessel is preferably sealed with an oil phase having a specific gravity less than water to maintain the droplet size.
Preferably, in the step b, the cell micro-droplets are collected, and an oil phase with a specific gravity lighter than water is added on the collection container for sealing, so as to maintain the size of the droplets unchanged; then placing the mixture into an incubator for standing, and enabling the cells to gather at the bottom of the liquid drop by means of gravity sedimentation to form a dense cell aggregation group.
Preferably, in the step c, when the cell mass is cultured, the gel material can form a physical gel below the gel-sol transition temperature, and the cell aggregate mass formed by sedimentation is placed in an environment below the gel-sol transition temperature to form a physical gel; then replacing oil phase with cell culture solution, and placing the cell aggregation mass back to the environment below the gel-sol transition temperature for continuous culture.
Preferably, in the step c, the settled cell micro-droplets are placed in an environment below the gel-sol transition temperature, and waiting for gelling to form cell gel spheres; then transferring the cell gel balls into a cell culture solution, and culturing at the temperature below the gel-sol transition temperature to ensure that the cells of the cell mass are attached and tightly connected with each other, thereby gradually forming the cell mass.
Preferably, in said step d, since the gel material is capable of being transformed from the physical gel into a solution state above the gel-sol transition temperature, the physical gel is dissolved, wherein the encapsulated cell mass is released.
Compared with the prior art, the invention has the following obvious and prominent substantive characteristics and remarkable advantages:
1. the invention combines the droplet microfluidic technology with gravity sedimentation, realizes the preparation of the rapid, high-flux and uniform cold cell mass microspheres, and realizes the regulation and control of the size of the cell mass microspheres. The microfluidic droplet technology obtains more accurate control on the cell mass cell number in each droplet by generating the cell-containing droplets with ultra-narrow size distribution, thereby having high uniformity of cell mass while having high flux; the final size of the cell mass can be effectively regulated by regulating the density of the cells in the liquid drops, so that the technology is endowed with the size flexibility of cell mass production;
2. the invention adopts gravity sedimentation, thus overcoming the problem that the traditional droplet microfluidics construction cell cluster needs to wait for the dispersed cells loaded in the droplets to gradually grow and fuse, and can form a uniform cell cluster through multi-day culture: the cells in the liquid drop can form an aggregation state within ten minutes through gravity sedimentation, and a sticky cell mass can be formed through culture for no more than 12 hours, so that the cell mass preparation efficiency is greatly improved; compared with a macroscopic system, the micro-droplets have smaller settling distance, so that cell aggregates can be formed in shorter time to occupy efficiency advantage;
3. the physical gel component is added in the cell environment, and a gel bracket can be formed by short-time cooling after the cells are settled, so that the gel bracket is closer to the extracellular matrix in the human body, and the physiological state can be better simulated; after the cell mass grows up, the gel is also very beneficial to the recovery of the cell mass, and avoids the damage to the cell mass caused by the interaction between different cell masses and environmental factors in the recovery process; from the aspect of operation, the preparation process of the cell mass is based on the operation of a microfluidic chip and a test tube, can be completely automated, does not have the operation of large-area exposure to the air, and greatly reduces the risk of cell pollution; therefore, the method is more suitable for biological medicine screening and toxicity testing occasions with strict safety and reliability requirements.
Drawings
FIG. 1 is a schematic diagram of the basic structure of a chip for preparing uniform cell droplets in high throughput by droplet microfluidics according to a preferred embodiment of the present invention and a Fenix test diagram. Wherein, fig. 1(a) is a basic structure schematic diagram of the chip, and 1, 2 and 3 are respectively a water phase inlet, an outlet and an oil phase inlet; FIG. 1(b) is a photograph of a cell-containing droplet in real form; FIG. 1(c) is a statistical chart of the sizes of generated droplets. Cell density 1.5X 107piece/mL, oil phase flow rate of 300 u L/h, aqueous phase flow rate of 100 u L/h, scale for 100 u m.
FIG. 2 is a graph of the analysis of cell aggregation assisted by gravity sedimentation according to an embodiment of the present invention. Wherein, FIG. 2(a) is a timing diagram of cell sedimentation in a droplet, which is a screen shot of an actual video; FIG. 2(b) is a graph of sedimentation distance of cells in droplets versus time, comparing the sedimentation process of cells in a macroscopic solution; FIG. 2(c) is a confocal image of unsettled cell gel and GelMA gel of cells settled for 12 hours. Green are live cells and red are dead cells. Cell density 1.5X 107piece/mL, oil phase flow rate of 300 u L/h, aqueous phase flow rate of 100 u L/h, scale for 100 u m.
FIG. 3 is a confocal characterization diagram of single cell gel pellet after 12 hours of culturing of secondary sedimentation cells according to the embodiment of the present invention, wherein FIG. 3(a) is a light field, green fluorescence, red fluorescence, three-in-one diagram, and 3D fluorescence mapping diagram. Large field of view cell pellet gel pellet pictures; FIG. 3(b) is a statistical chart of the number of cells in the gel. Cell density 1.5X 107piece/mL, oil phase flow rate of 300 u L/h, aqueous phase flow rate of 100 u L/h, scale for 100 u m. The average number of cells in the gel was 10-25.
FIG. 4 shows the encapsulation of gel beads at different cell concentrations according to example two of the present invention. In FIG. 4(a), green is live cells and red is dead cells in the confocal images. FIG. 4(b) is a graph showing the relationship between the number of encapsulated cells and the cell concentration. The flow rate of the oil phase was 300. mu.L/h, and the flow rate of the water phase was 100. mu.L/h, as measured on a scale of 100. mu.m.
Detailed Description
The above-described scheme is further illustrated below with reference to specific embodiments, which are detailed below:
the first embodiment is as follows:
in this embodiment, a method for constructing a high-throughput cell mass model by droplet microfluidic gravity positioning includes the following steps:
a. microfluidic preparation of cell microdroplets:
preparing a mixed solution of gel and cells as a water phase, wherein the gel is natural gel, synthetic gel or composite gel and comprises all gels which have reversible temperature-sensitive phase change behavior in physiological solution, form physical gel below the gel-sol transition temperature, are in a liquid state above the gel-sol transition temperature and can maintain the cell activity; preparing oil containing surfactant as oil phase; the mass concentration of the gel in the water-phase mixed solution is 3-20%, and the cell concentration is 1 multiplied by 105-1×108Per milliliter, and generating uniform micro liquid drops containing cells by a liquid drop micro flow control chip;
b. synchronous cell gravity sedimentation:
collecting cell micro-droplets, and adding an oil phase with a specific gravity lighter than water on a collection container for sealing to maintain the size of the droplets unchanged; then placing the mixture into an incubator for standing, and enabling the cells to gather at the bottom of the liquid drop by means of gravity sedimentation to form a dense cell aggregation group;
c. cell mass culture:
placing the settled cell micro-droplets in an environment below the gel-sol transition temperature, waiting for the cell micro-droplets to gel, and forming cell gel spheres; then transferring the cell gel spheres into a cell culture solution for culture at a temperature below the gel-sol transition temperature, so that cell groups are attached to each other and tightly connected to form cell groups gradually;
d. shaped cell pellet model recovery
And (3) heating to the gel-sol transition temperature or higher, destroying a gel system containing the cell aggregation group, releasing the cell clusters, and recovering the cell cluster model. The formed cell mass can be used for subsequent detection.
The embodiment is based on a method for constructing a cell mass model by microfluidics and gravity, firstly, a multi-cell micro-droplet is prepared at high flux by utilizing a droplet microfluidics method, the size of a cell mass is regulated and controlled by adjusting the cell concentration, the cell mass is kept stand in a cell culture box, a cell accumulation body is formed by gravity sedimentation, cells are further adhered into the cell mass after being cultured, the time for primarily forming the cell mass only needs 12 hours, the gel has good biocompatibility, a physical gel is formed below the gel-sol transition temperature, the temperature is raised to the gel-sol transition temperature and is in a liquid state, therefore, the cell mass culture can be carried out below the gel-sol transition temperature, and the cell mass is recovered above the gel-sol transition temperature; the cell clusters constructed in the embodiment have the advantages of high flux, short time consumption, good consistency, simple and convenient recovery and reduction of pollution caused by contact with air in a closed chip environment. The cell mass model construction method of the embodiment can be really applied to the fields of drug activity and substance toxicity screening and the like.
Example two:
this embodiment is substantially the same as the first embodiment, and is characterized in that:
a method for constructing a cell cluster in high flux, which adopts the microfluidic chip to generate gel spheres to wrap cells, comprises the following steps:
as shown in figure 1, the micro-fluidic method is used to form uniform cell micro-droplets, wherein the gel is natural gel, synthetic gel or composite gel, and includes all gels which have reversible temperature-sensitive phase change behavior in physiological solution, form physical gel below the gel-sol transition temperature, are in liquid state above the gel-sol transition temperature, and can maintain cell activity. Cells in exponential growth phase were first lysed and resuspended in fresh medium, the cell concentration was adjusted to 1X 108Using culture solution with adjusted cell concentration to prepare 10% methacrylic acid anhydrified gelatin serving as an aqueous phase, HFE7500 containing 2% EA surfactant produced by RAN Biotechnology, US serving as an oil phase, enabling the aqueous phase to flow at 300 mu L/h and the oil phase to flow at the flow rate of 100 mu L/h, and enabling the chip to be kept at the constant temperature of 25-45 ℃ to form stable and uniform liquid drops. Collecting cell micro-droplets, and adding oil with a specific gravity lower than that of water into the collecting containerThe rows are sealed to stabilize the droplets. Then the mixture is placed into an incubator and stands for 3 hours, and the cells are gathered at the bottom of the liquid drop by gravity sedimentation to form a dense cell aggregation group. Placing the settled cell micro-droplets in a temperature environment for forming physical gel, and waiting for the cell micro-droplets to form gel to form cell gel spheres; and then transferring the cell gel spheres into a cell culture solution, culturing at 4 ℃ to ensure that cells in the gel spheres are attached to each other and tightly bonded, thereby gradually forming cell masses, and continuously culturing for 12 hours to obtain the cell masses, wherein the cell masses are shown in figure 2. And heating the gel ball coated with the cell mass to 37 ℃ to recover the cell mass.
FIG. 3 is a confocal characterization diagram of a single cell mass gel sphere after 12 hours of culture of settled cells in the present embodiment, wherein FIG. 3(a) is a diagram of bright field, green fluorescence, red fluorescence and triad. Large field of view cell pellet gel pellet pictures; FIG. 3(b) is a statistical chart of the number of cells in the gel. Cell density 1.5X 107piece/mL, oil phase flow rate of 300 u L/h, aqueous phase flow rate of 100 u L/h, scale for 100 u m. The average number of cells in the gel was 10-25.
FIG. 4 shows the encapsulation of gel beads at different cell concentrations in this example. In FIG. 4(a), green is live cells and red is dead cells in the confocal images. FIG. 4(b) is a graph showing the relationship between the number of encapsulated cells and the cell concentration. The flow rate of the oil phase was 300. mu.L/h, and the flow rate of the water phase was 100. mu.L/h, as measured on a scale of 100. mu.m.
In the embodiment, the droplet microfluidic technology is combined with gravity sedimentation, so that the rapid, high-flux and uniform preparation of the cold cell mass microspheres is realized, and the size of the cell mass microspheres is regulated. The microfluidic droplet technology obtains relatively accurate control of the number of cells in cell clusters in each droplet by generating cell-containing droplets with ultra-narrow size distribution, thereby having high uniformity of cell clusters while having high flux. The final size of the cell mass can be effectively regulated by regulating the density of the cells in the liquid drops, thereby endowing the technology with the flexibility of producing the size of the cell mass. Because the gravity sedimentation is adopted, the problem that the traditional liquid drop microfluidic construction of cell clusters requires waiting for the dispersed cells loaded in the liquid drops to gradually grow and fuse, and a uniform cell cluster can be formed through multi-day culture is solved: the cells in the liquid drop can form an aggregation state within ten minutes through gravity sedimentation, and a sticky cell mass can be formed through culture for no more than 12 hours, so that the cell mass preparation efficiency is greatly improved; compared with a macroscopic system, the micro-droplets have smaller settling distance, so that cell aggregates can be formed in shorter time to occupy efficiency advantage.
Example three:
this embodiment is substantially the same as the above embodiment, and is characterized in that:
a method for constructing a cell cluster in high flux, which adopts the microfluidic chip to generate gel spheres to wrap cells, comprises the following steps:
as shown in figure 1, the micro-fluidic method is used to form uniform cell micro-droplets, wherein the gel is natural gel, synthetic gel or composite gel, and includes all gels which have reversible temperature-sensitive phase change behavior in physiological solution, form physical gel below the gel-sol transition temperature, are in liquid state above the gel-sol transition temperature, and can maintain cell activity. Cells in exponential growth phase were first lysed and resuspended in fresh medium, the cell concentration was adjusted to 5X 106Using culture solution with adjusted cell concentration to prepare 10% methacrylic acid anhydrified gelatin serving as an aqueous phase, HFE7500 containing 2% EA surfactant produced by RAN Biotechnology, US serving as an oil phase, enabling the aqueous phase to flow at 300 mu L/h and the oil phase to flow at the flow rate of 100 mu L/h, and enabling the chip to be kept at the constant temperature of 25-45 ℃ to form stable and uniform liquid drops. The cell microdroplets were collected and sealed by adding an oil phase lighter in weight than water to the collection container to stabilize the droplets. Then the mixture is placed into an incubator and stands for 3 hours, and the cells are gathered at the bottom of the liquid drop by gravity sedimentation to form a dense cell aggregation group. Placing the settled cell micro-droplets in a temperature environment for forming physical gel, and waiting for the cell micro-droplets to form gel to form cell gel spheres; and then transferring the cell gel spheres into a cell culture solution, culturing at 4 ℃ to ensure that cells in the gel spheres are attached to each other and tightly bonded, thereby gradually forming cell masses, and continuously culturing for 12 hours to obtain the cell masses. Heating the gel ball coated with cell mass to 37 deg.C to recover cellsAnd (4) clustering. The method combines the advantages of high flux and consistency of droplet microfluidics, has the advantages of rapidness and high efficiency of traditional cell mass construction, and can be used for preparing micro cell masses with the number of more than thousand and the size of tens of micrometers to hundreds of micrometers in a short time. The cell clusters prepared by the method are in a gel environment, so that the subsequent operation is facilitated, and the direct contact among the cell clusters is avoided.
Example four:
this embodiment is substantially the same as the above embodiment, and is characterized in that:
a method for constructing a cell cluster in high flux, which adopts the microfluidic chip to generate gel spheres to wrap cells, comprises the following steps:
as shown in figure 1, the micro-fluidic method is used to form uniform cell micro-droplets, wherein the gel is natural gel, synthetic gel or composite gel, and includes all gels which have reversible temperature-sensitive phase change behavior in physiological solution, form physical gel below the gel-sol transition temperature, are in liquid state above the gel-sol transition temperature, and can maintain cell activity. Cells in exponential growth phase were first lysed and resuspended in fresh medium, the cell concentration was adjusted to 1X 105The cell concentration of the cells was adjusted to 5% by mass of methacrylic anhydrified gelatin as an aqueous phase, HFE7500 containing 0.1% EA surfactant produced by RAN Biotechnology, US as an oil phase, the flow rate of the aqueous phase was 300. mu.L/h, and the flow rate of the oil phase was 100. mu.L/h, and the chip was thermostatted at 25 to 45 ℃ to form stable and uniform droplets. The cell microdroplets were collected and sealed by adding an oil phase lighter in weight than water to the collection container to stabilize the droplets. Then the mixture is placed into an incubator and stands for 1 hour, and the cells are gathered at the bottom of the liquid drop by gravity sedimentation to form a dense cell aggregation group. Placing the settled cell micro-droplets in a temperature environment for forming physical gel, and waiting for the cell micro-droplets to form gel to form cell gel spheres; and then transferring the cell gel spheres into a cell culture solution, culturing at 20 ℃ to ensure that cells in the gel spheres are attached to each other and tightly bonded, thereby gradually forming cell masses, and continuously culturing for 12 hours to obtain the cell masses. To be coated with cell pelletsThe gel ball is heated to 30 ℃ to recover the cell mass. The method combines the advantages of high flux and consistency of droplet microfluidics, has the advantages of rapidness and high efficiency of traditional cell mass construction, and can be used for preparing micro cell masses with the number of more than thousand and the size of tens of micrometers to hundreds of micrometers in a short time. The cell clusters prepared by the method are in a gel environment, so that the subsequent operation is facilitated, and the direct contact among the cell clusters is avoided.
Example five:
this embodiment is substantially the same as the above embodiment, and is characterized in that:
a method for constructing a cell cluster in high flux, which adopts the microfluidic chip to generate gel spheres to wrap cells, comprises the following steps:
as shown in figure 1, the micro-fluidic method is used to form uniform cell micro-droplets, wherein the gel is natural gel, synthetic gel or composite gel, and includes all gels which have reversible temperature-sensitive phase change behavior in physiological solution, form physical gel below the gel-sol transition temperature, are in liquid state above the gel-sol transition temperature, and can maintain cell activity. Cells in exponential growth phase were first lysed and resuspended in fresh medium, the cell concentration was adjusted to 1X 107Using culture solution with adjusted cell concentration to prepare 20% methacrylic acid anhydrified gelatin serving as an aqueous phase, HFE7500 containing 1% EA surfactant produced by RAN Biotechnology, US serving as an oil phase, enabling the aqueous phase to flow at 200 mu L/h and enabling the flow rate of the oil phase to be 200 mu L/h, and enabling the chip to be kept at a constant temperature of 25-45 ℃ to form stable and uniform liquid drops. The cell microdroplets were collected and sealed by adding an oil phase lighter in weight than water to the collection container to stabilize the droplets. Then the mixture is placed into an incubator and stands for 10 minutes, and the cells are gathered at the bottom of the liquid drop by gravity sedimentation to form a dense cell aggregation group. Placing the settled cell micro-droplets in a temperature environment for forming physical gel, and waiting for the cell micro-droplets to form gel to form cell gel spheres; then transferring the cell gel spheres into a cell culture solution and culturing at 15 ℃ to ensure that the cells in the gel spheres are attached to each other and tightly bonded, thereby gradually forming cell masses, and continuously culturing for 12 hours to obtain the cell gel spheresObtaining cell mass. And heating the gel ball coated with the cell mass to 37 ℃ to recover the cell mass. The method combines the advantages of high flux and consistency of droplet microfluidics, has the advantages of rapidness and high efficiency of traditional cell mass construction, and can be used for preparing micro cell masses with the number of more than thousand and the size of tens of micrometers to hundreds of micrometers in a short time. The cell clusters prepared by the method are in a gel environment, so that the subsequent operation is facilitated, and the direct contact among the cell clusters is avoided.
Example six:
this embodiment is substantially the same as the above embodiment, and is characterized in that:
a method for constructing a cell cluster in high flux, which adopts the microfluidic chip to generate gel spheres to wrap cells, comprises the following steps:
as shown in figure 1, the micro-fluidic method is used to form uniform cell micro-droplets, wherein the gel is natural gel, synthetic gel or composite gel, and includes all gels which have reversible temperature-sensitive phase change behavior in physiological solution, form physical gel below the gel-sol transition temperature, are in liquid state above the gel-sol transition temperature, and can maintain cell activity. Cells in exponential growth phase were first lysed and resuspended in fresh medium, cell concentration was adjusted to 2X 106Using culture solution with adjusted cell concentration to prepare 10% methacrylic acid anhydrified gelatin serving as an aqueous phase, HFE7500 containing 0.2% EA surfactant produced by RAN Biotechnology, US serving as an oil phase, wherein the flow rate of the aqueous phase is 500 mu L/h and the flow rate of the oil phase is 50 mu L/h, and keeping the chip at a constant temperature of 25-45 ℃ to form stable and uniform liquid drops. The cell microdroplets were collected and sealed by adding an oil phase lighter in weight than water to the collection container to stabilize the droplets. Then the mixture is placed into an incubator and stands for 1 hour, and the cells are gathered at the bottom of the liquid drop by gravity sedimentation to form a dense cell aggregation group. Placing the settled cell micro-droplets in a temperature environment for forming physical gel, and waiting for the cell micro-droplets to form gel to form cell gel spheres; then transferring the cell gel spheres into a cell culture solution and culturing at 10 ℃ to ensure that the cells in the gel spheres are attached to each other and tightly bonded, thereby gradually formingAnd (5) continuously culturing the cell mass for 12 hours to obtain the cell mass. And heating the gel ball coated with the cell mass to 37 ℃ to recover the cell mass. The method combines the advantages of high flux and consistency of droplet microfluidics, has the advantages of rapidness and high efficiency of traditional cell mass construction, and can be used for preparing micro cell masses with the number of more than thousand and the size of tens of micrometers to hundreds of micrometers in a short time. The cell clusters prepared by the method are in a gel environment, so that the subsequent operation is facilitated, and the direct contact among the cell clusters is avoided.
Example seven:
this embodiment is substantially the same as the above embodiment, and is characterized in that:
a method for constructing a cell cluster in high flux, which adopts the microfluidic chip to generate gel spheres to wrap cells, comprises the following steps:
as shown in figure 1, the micro-fluidic method is used to form uniform cell micro-droplets, wherein the gel is natural gel, synthetic gel or composite gel, and includes all gels which have reversible temperature-sensitive phase change behavior in physiological solution, form physical gel below the gel-sol transition temperature, are in liquid state above the gel-sol transition temperature, and can maintain cell activity. Cells in exponential growth phase were first lysed and resuspended in fresh medium, the cell concentration was adjusted to 1.5X 107Using culture solution with adjusted cell concentration to prepare 10% methacrylic acid anhydrified gelatin serving as an aqueous phase, HFE7500 containing 1% EA surfactant produced by RAN Biotechnology, US serving as an oil phase, enabling the aqueous phase to flow at 300 mu L/h and the oil phase to flow at the flow rate of 100 mu L/h, and enabling the chip to be kept at the constant temperature of 25-45 ℃ to form stable and uniform liquid drops. The cell microdroplets were collected and sealed by adding an oil phase lighter in weight than water to the collection container to stabilize the droplets. Then the mixture is placed into an incubator and stands for 0.5 hour, and the cells are gathered at the bottom of the liquid drop by gravity sedimentation to form a dense cell aggregation group. Placing the settled cell micro-droplets in a temperature environment for forming physical gel, and waiting for the cell micro-droplets to form gel to form cell gel spheres; then transferring the cell gel spheres into a cell culture solution and culturing at 20 ℃ to ensure that the intercellular phase in the gel spheresAdhering tightly to form cell mass gradually, and culturing for 12 hr to obtain cell mass. And heating the gel ball coated with the cell mass to 32 ℃ to recover the cell mass. The method combines the advantages of high flux and consistency of droplet microfluidics, has the advantages of rapidness and high efficiency of traditional cell mass construction, and can be used for preparing micro cell masses with the number of more than thousand and the size of tens of micrometers to hundreds of micrometers in a short time. The cell clusters prepared by the method are in a gel environment, so that the subsequent operation is facilitated, and the direct contact among the cell clusters is avoided.
Example eight:
this embodiment is substantially the same as the above embodiment, and is characterized in that:
a method for constructing a cell cluster in high flux, which adopts the microfluidic chip to generate gel spheres to wrap cells, comprises the following steps:
as shown in figure 1, the micro-fluidic method is used to form uniform cell micro-droplets, wherein the gel is natural gel, synthetic gel or composite gel, and includes all gels which have reversible temperature-sensitive phase change behavior in physiological solution, form physical gel below the gel-sol transition temperature, are in liquid state above the gel-sol transition temperature, and can maintain cell activity. Cells in exponential growth phase were first lysed and resuspended in fresh medium, the cell concentration was adjusted to 1.5X 107Using culture solution with adjusted cell concentration to prepare 10% methacrylic acid anhydrified gelatin serving as an aqueous phase, HFE7500 containing 0.5% EA surfactant produced by RAN Biotechnology, US serving as an oil phase, wherein the flow rate of the aqueous phase is 100 mu L/h, and the flow rate of the oil phase is 500 mu L/h, and keeping the chip at a constant temperature of 25-45 ℃ to form stable and uniform liquid drops. The cell microdroplets were collected and sealed by adding an oil phase lighter in weight than water to the collection container to stabilize the droplets. Then the mixture is placed into an incubator and stands for 3 hours, and the cells are gathered at the bottom of the liquid drop by gravity sedimentation to form a dense cell aggregation group. Placing the settled cell micro-droplets in a temperature environment for forming physical gel, and waiting for the cell micro-droplets to form gel to form cell gel spheres; the cell gel beads were then transferred to cell culture media at 20 deg.CCulturing to make the cells in the gel ball adhere tightly to form cell mass gradually, and culturing for 12 hr to obtain cell mass. And heating the gel ball coated with the cell mass to 35 ℃ to recover the cell mass.
According to the embodiment of the invention, the physical gel component is added in the cell environment, and the gel scaffold can be formed through short-time cooling after the cells are settled, so that the gel scaffold is closer to the extracellular matrix in the human body, and the physiological state can be better simulated. In the above embodiment, after the cell mass grows, the gel is also very beneficial to the recovery of the cell mass, and avoids the mutual action among different cell masses and the damage of environmental factors to the cell mass in the recovery process. From the aspect of operation, the preparation process of the cell mass is based on the operation of a microfluidic chip and a test tube, can be completely automated, does not have the operation of large-area exposure in the air, and greatly reduces the risk of cell pollution. Therefore, the method of the embodiment is more suitable for biological medicine screening and toxicity testing occasions with strict safety and reliability requirements.
The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made according to the purpose of the invention, and any changes, modifications, substitutions, combinations or simplifications made according to the spirit and principle of the technical solution of the present invention should be replaced with equivalents as long as the object of the present invention is met, and the technical principle and the inventive concept of the present invention are not departed from the scope of the present invention.
Claims (10)
1. A method for constructing a high-throughput cell mass model by droplet microfluidic gravity positioning is characterized by comprising the following steps:
a. microfluidic preparation of cell microdroplets:
building a constant-temperature micro-fluidic device, preparing a mixed solution of gel and cells as a water phase, and preparing oil containing a surfactant as an oil phase, so that the micro-fluidic chip generates uniform multi-cell micro-droplets;
b. synchronous cell gravity sedimentation:
collecting the multicellular micro-droplets, and depositing and adhering the cells by means of gravity to construct a cell aggregation group;
c. cell mass culture:
culturing the cell aggregate in a gel maintaining the cell activity in an environment at a gel-sol transition temperature;
d. shaped cell pellet model recovery
And (3) heating to the gel-sol transition temperature or higher, destroying a gel system containing the cell aggregation group, releasing the cell clusters, and recovering the cell cluster model.
2. The method for constructing the high-throughput cell mass model by the microfluidic gravity positioning of the liquid drops according to claim 1, is characterized in that: in the step a, the microfluidic chip consists of a water phase inlet, an oil phase inlet and an oil phase outlet, and the oil phase is used as an entrained flow to form high-flux uniform-size droplets, wherein the droplet size is 50-500 microns.
3. The method for constructing the high-throughput cell mass model by the microfluidic gravity positioning of the liquid drops according to claim 1, is characterized in that: in the step a, a constant-temperature micro-fluidic device is adopted to control the temperature of the aqueous phase solution entering the micro-fluidic chip and the chip forming the liquid drop, and the gel precursor is maintained in a flowable state.
4. The method for constructing the high-throughput cell mass model by the microfluidic gravity positioning of the liquid drops according to claim 1, is characterized in that: in the step a, the micro-fluidic chip is kept at a constant temperature of 25-45 ℃ to form stable and uniform multi-cell micro-droplets.
5. The method for constructing the high-throughput cell mass model by the microfluidic gravity positioning of the liquid drops according to claim 1, is characterized in that: in the step a, the gel is at least one of natural gel, synthetic gel and composite gel; or the gel adopts at least one of gels which have reversible temperature-sensitive phase change behavior in physiological solution, form physical gel below the gel-sol transition temperature, are in liquid state above the gel-sol transition temperature and can maintain cell activity.
6. The method for constructing the high-throughput cell mass model by the microfluidic gravity positioning of the liquid drops according to claim 1, is characterized in that: in the step a, the mass concentration of the gel in the mixed solution of the water phase is 3-20%, and the cell concentration is 1 x 105-1×108Each per milliliter.
7. The method for constructing the high-throughput cell mass model by the microfluidic gravity positioning of the liquid drops according to claim 1, is characterized in that: in the step b, when the cells are settled and adhered by gravity to construct the cell aggregation group, the collected container of the multicellular micro-droplets is placed in an incubator to be stood, the cells are settled and accumulated by gravity, and then the cells are adhered to form the cell aggregation group.
8. The method for constructing the high-throughput cell mass model by the microfluidic gravity positioning of the liquid drops according to claim 1, is characterized in that: in the step b, collecting cell micro-droplets, and adding an oil phase with a specific gravity lighter than that of water on a collection container for sealing so as to maintain the size of the droplets unchanged; then placing the mixture into an incubator for standing, and enabling the cells to gather at the bottom of the liquid drop by means of gravity sedimentation to form a dense cell aggregation group.
9. The method for constructing the high-throughput cell mass model by the microfluidic gravity positioning of the liquid drops according to claim 1, is characterized in that: in the step c, when the cell mass is cultured, the cell aggregate formed by sedimentation is placed in an environment of a gel-sol transition temperature or lower to form a physical gel; then replacing oil phase with cell culture solution, and placing the cell aggregation mass back to the environment below the gel-sol transition temperature for continuous culture.
10. The method for constructing the high-throughput cell mass model by the microfluidic gravity positioning of the liquid drops according to claim 1, is characterized in that: in the step c, the settled cell micro-droplets are placed in an environment below the gel-sol transition temperature to wait for gelling, so as to form cell gel spheres; then transferring the cell gel balls into a cell culture solution, and culturing at the temperature below the gel-sol transition temperature to ensure that the cells of the cell mass are attached and tightly connected with each other, thereby gradually forming the cell mass.
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