CN111647509A - Sitting drop type cell ball culture chip and its use method - Google Patents
Sitting drop type cell ball culture chip and its use method Download PDFInfo
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- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0062—General methods for three-dimensional culture
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- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- C12N2539/00—Supports and/or coatings for cell culture characterised by properties
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- G—PHYSICS
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- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Abstract
The sitting drop type cell ball culture chip comprises a sample adding cavity and a microcavity array, wherein the microcavity array is arranged at the bottom of the sample adding cavity, the surfaces of the sample adding cavity and the microcavity array are provided with super-hydrophobic surfaces, the microcavity array is provided with a plurality of microcavities, at least one through hole is arranged in each microcavity, and the section of each through hole is smaller than that of each microcavity. The invention also discloses a using method of the sitting-drop cell ball culture chip. According to the invention, the microcavity array is combined with the super-hydrophobic surface, and the hydrophobic effect of the super-hydrophobic surface, the surface tension of the liquid sample and the self-gravity synergistic effect are utilized, so that the automatic distribution of cell suspension, the simple and fast liquid change in the culture process and the simple and fast recovery of cultured cell spheres can be realized, and the problems of difficult culture medium update, poor air permeability, poor cell sphere uniformity, difficult in-situ observation, high cost, difficult cell sphere taking-out and the like in the prior art are effectively solved.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a sitting drop type cell ball culture chip and a using method thereof.
Background
Compared with the traditional monolayer cell culture technology, the cell spheres formed by three-dimensional cell culture can better reflect the close and rich three-dimensional relation between cells and between the cells and the external microenvironment, better simulate the inherent metabolism (such as nutrients, oxygen, metabolites and the like) and the proliferation gradient of the cells in the natural physiological environment, can track the diffusion path of the drugs, and is an ideal model for cell research. Due to its unique advantages, in recent years, three-dimensional cell sphere culture has been widely used in cell biology and medical research, and has become an indispensable powerful tool for research in drug screening, tumor therapy mechanism analysis, stem cell differentiation, regenerative medicine and other aspects. The existing methods for preparing three-dimensional cell spheres mainly comprise rotary stirring type culture, hanging drop type culture, micro-cavity type culture based on low cell adhesion surface, acoustic focusing type culture based on surface acoustic wave drive, magnetic suspension type culture, gel embedding type culture and the like. However, these methods have respective limitations, for example, the rotary stirring culture method is difficult to realize uniform culture of cell balls, the shearing force is easy to cause cell damage, and the culture process cannot be observed in real time; the hanging drop type culture has the problems that culture liquid drops are easy to fall off due to mechanical disturbance, liquid change is difficult in the culture process and the like; the micro-cavity type culture with low adhesion surface requires complex surface treatment operation, and the cell balls are positioned at a deeper part below the liquid level of the nutrient solution and are difficult to exchange with the external atmosphere; both the acoustic focusing type culture and the magnetic suspension type culture need special auxiliary devices, so that the culture system is complex and the experiment cost is high; in the process of cell culture, the exogenous matrix existing around the cells cannot accurately reflect the physiological activities of the real microenvironment of the cells, which leads to the loss of physiological signals and even obtains wrong research results. Therefore, a new device and a method for culturing three-dimensional cell spheres, which are simple and convenient to operate, low in cost, good in biocompatibility, high in stability, good in consistency, less in exogenous interference and convenient to observe in situ, are urgently needed to be developed so as to promote the development of cell biology and medicine.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a sitting-drop cell ball culture chip and a use method thereof, wherein a microcavity array is combined with a super-hydrophobic surface, and the automatic distribution of cell suspension, the simple and fast liquid change in the culture process and the simple and fast recovery of cultured cell balls can be realized through the synergistic effect of the hydrophobic effect of the super-hydrophobic surface, the surface tension of a liquid sample and the self gravity, so that the problems of difficult culture medium updating, poor air permeability, poor cell ball uniformity, difficult in-situ observation, high cost, difficult cell ball taking-out and the like in the prior art are effectively solved.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows: the utility model provides a sitting and dropping type cell ball cultivates chip, includes application of sample chamber and microcavity array, and the microcavity array sets up in application of sample chamber bottom, and application of sample chamber and microcavity array surface are provided with super hydrophobic surface, and the microcavity array is provided with a plurality of microcavities, is provided with at least one through-hole in the microcavity, and the through-hole cross-section is less than the microcavity cross-section.
Further, the microcavity array is provided with at least 4 microcavities.
Furthermore, the microcavity body is cylindrical, the diameter is larger than 2mm, and the bottom of the microcavity is arc-shaped.
Furthermore, the base material of the sitting-drop cell culture chip is one of polystyrene, polycarbonate, polymethyl methacrylate, polydimethylsiloxane, acrylonitrile-butadiene-styrene, polypropylene, polyethylene, polyvinyl chloride, polytetrafluoroethylene and epoxy resin.
Further, the microcavity array is prepared by numerical control engraving or injection molding process.
Further, the super-hydrophobic surface is prepared by the following method: and spraying or dip-coating the nano material suspension on a base material of the sitting-drop cell ball culture chip, and drying to obtain the super-hydrophobic surface.
Further, drying at 70 deg.C for at least 1 h.
Further, the nanometer material suspension is a mixed solution formed by mixing the nanometer material, glue and a dispersing agent according to the mass ratio of 1-2: 1-3: 15.
Further, the nano material is nano particles, nano fibers or nano rods, and the characteristic dimension is 5-500 nm; the glue is epoxy resin, organic silicon resin or structural adhesive; the dispersant is tetrahydrofuran, absolute ethyl alcohol, isopropanol or methane halogenated hydrocarbon.
Further, the nano material is silica nanoparticles, alumina nanoparticles, titanium dioxide nanoparticles, carbon nanofibers, polyacrylonitrile nanofibers, polyvinylidene fluoride nanofibers, carbon nanotubes or metal nanorods.
The use method of the sitting-drop cell ball culture chip sequentially comprises the following steps:
(1) cleaning and drying the sitting drop type cell ball culture chip, and then carrying out ultraviolet disinfection;
(2) adding the cell suspension into the sterilized sample adding cavity, tilting, rotating or shaking the chip, and sucking out residual liquid when the microcavity array is filled with the cell suspension;
(3) culturing the chip subjected to sample distribution in an environment with the temperature of 37 ℃ and the carbon dioxide atmosphere containing 5% and the relative humidity of more than 90% for 24-72 h, then adding a culture medium or a drug to be detected into a sample adding cavity, inclining or rotating the chip, sucking out residual liquid after full diffusion, and continuing culturing under the original culture condition; according to the requirement of cell ball culture, the liquid changing process can be repeated for many times until the end, and the cell balls are collected.
Further, when collecting the cell balls, turning the chip to transfer the liquid drops containing the cell balls in each microcavity to a plate glass or a culture dish; or directly pouring the liquid drops containing the cell balls in each microcavity into a culture dish filled with a culture medium; or the cultured cell pellet is aspirated from the single microcavity by a pipette or pipette.
Further, during ultraviolet disinfection, the radiation intensity is more than 70W/cm2Sterilizing for 15-60 min under an ultraviolet lamp.
In summary, the invention has the following advantages:
1. the sitting-drop cell ball culture chip disclosed by the invention utilizes the microcavity array to be combined with the super-hydrophobic surface, can realize automatic distribution of cell suspension, simple and quick liquid change in the culture process and simple and quick recovery of cultured cell balls through the synergistic effect of the hydrophobic effect of the super-hydrophobic surface, the surface tension of a liquid sample and self gravity, is simple and efficient to operate and low in use cost, and effectively solves the problems of difficulty in culture medium update, poor air permeability, poor cell ball uniformity, difficulty in-situ observation, high cost, difficulty in cell ball taking out and the like in the prior art.
2. The chip is provided with a through hole type micro-cavity array chip with a super-hydrophobic surface characteristic, and can realize automatic distribution of cell suspension in the super-hydrophobic micro-cavity array through the synergistic effect of the hydrophobic effect of the super-hydrophobic surface, the surface tension of a liquid sample and the self gravity of fluid, so that an independent cell suspension liquid drop is automatically formed in each micro-cavity, and the chip is applied to high-throughput culture of cell spheres. Compared with the existing hanging drop type cell ball culture device and the microcavity array type cell ball culture chip based on the arc bottom surface, the sitting drop type culture chip disclosed by the invention has better advantages in the aspects of long-term stability of culture liquid drops, simplicity and convenience in liquid sample distribution and nutrient solution updating, exchange rate of culture atmosphere, in-situ observability and the like, provides an efficient platform for culture and drug screening of three-dimensional cells, and has important application value in the aspects of cell biology and medical research.
3. The invention can simply, conveniently and quickly realize the operations of distribution of cell suspension, liquid exchange in the culture process, drug loading, recovery of cultured cell spheres and the like, has strong mechanical disturbance resistance of the culture liquid drop and good air permeability of the culture liquid drop, does not need a complex external auxiliary device, avoids the difficult problems of difficult operation, poor controllability, high material cost, difficult in-situ observation and the like of the traditional cell sphere culture method, and is expected to promote the rapid development and wide application of the three-dimensional cell culture technology in the aspects of biological basic research and medical research.
4. Because of the support of the substrate and the limitation of the microcavity, the phenomenon of drop falling caused by external disturbance can not occur in the culture process, and the culture medium has better mechanical stability; due to the super-hydrophobic microcavity design, culture liquid drops are not required to be distributed onto a culture plate one by one through a liquid transfer device, and all the culture liquid drops can be quickly distributed through the gravity action and the surface tension action by loading cell suspension once and simply inclining and rotating a chip, so that the labor intensity of experimenters is greatly reduced; suspension drop culture is difficult to change liquid in culture due to poor stability of liquid drops, and the invention utilizes super-hydrophobic microcavity design and combines with sitting drop type scheme, so that the liquid change operation can be conveniently and rapidly realized without worrying about the problem of drop falling in the liquid change process.
5. Because the bottom of the microcavity has a through hole structure, and because the microcavity is a super-hydrophobic surface, the culture liquid drop is not in complete contact with the solid surface of the microcavity, and a large amount of gas exists, the exchange between the cell ball at the bottom of the liquid drop and the external atmosphere in the culture process is facilitated, while the cell ball cultured in the microporous plate type is positioned at the closed bottom of the microcavity, and has a larger distance from the liquid surface in contact with the atmosphere, the gas exchange is difficult, and the growth of the cell ball is not facilitated; the micro-porous plate type culture usually has the culture solution communication between the micro-cavities for culturing the cell balls, and the diffusion and the interference of signal molecules exist between the micro-cavities, but the invention has the advantages that because of the design of the super-hydrophobic micro-cavities, the micro-cavities are independent, the exchange and the interference of the signal molecules do not exist in the culture process, and each micro-cavity can carry out independent drug screening or stimulation factor test, so that a single chip can realize multiple experiments, and has better flexibility and economy.
6. When the chip is used, firstly, sterilizing the sitting drop type cell ball culture chip by ultraviolet radiation; then adding a certain amount of cell suspension liquid into the chip sample adding cavity after the sterilization treatment, wherein the added cell suspension liquid can freely roll in the sample adding cavity due to the super-hydrophobic characteristic of the surface of the chip sample adding cavity, properly incline, rotate or slightly shake the chip, so that a liquid sample passes through all micro-cavities and is filled into the micro-cavities under the action of gravity, and meanwhile, the filling liquid sample part in the micro-cavities is separated from a liquid sample main body by utilizing the synergistic action of the surface tension of the liquid sample and the self gravity of the liquid sample, so that independent cell suspension liquid drops are formed in each micro-cavity; then removing redundant cell suspension in the sample adding cavity, and placing the chip which finishes liquid sample distribution in a high-humidity and set constant-temperature environment for cell ball culture; in the culture process, culture medium replacement is carried out regularly, the operation mode of liquid replacement is similar to the sample addition process, namely, a certain amount of culture medium is added into a sample addition cavity, the chip is properly inclined and rotated, so that a liquid sample passes through all micro cavities, the exchange between each cell culture liquid drop and nutrient components and metabolite molecules of a fresh culture medium is realized by utilizing the molecular diffusion effect, and then the liquid sample which is subjected to diffusion exchange in the sample addition cavity is removed; and finally collecting the cell balls after the cell balls in the micro-cavity are cultured to the expected size. The method is simple and easy to operate, can change liquid simply and quickly, is convenient for collecting the cell balls, and is convenient for popularization and use of the sitting-drop cell ball culture chip.
Drawings
FIG. 1 is a schematic diagram of a sitting-drop cell pellet culture chip;
FIG. 2 is a schematic flow chart of preparing sitting drop type cell ball culture chip by numerical control engraving technology;
FIG. 3 is a schematic view showing a sample application process of the sitting-drop cell pellet culture chip;
FIG. 4 is a schematic diagram of the liquid exchange and drug loading process of the sitting-drop cell-ball culture chip;
FIG. 5 is a schematic view showing the process of in-situ staining and observation of a cell pellet in a sitting-drop cell pellet culture chip;
FIG. 6 is a schematic diagram of a cell pellet recycling process of the sitting-drop cell pellet culture chip;
FIG. 7 is a microphotograph of human highly metastatic hepatoma carcinoma cell balls cultured and collected based on the sitting drop type cell ball culture chip of the present invention.
Detailed Description
Example 1 sitting-drop type cell ball culture chip and preparation
The utility model provides a sitting drop-type cell ball cultivates chip, includes application of sample chamber and microcavity array, and the microcavity array sets up in application of sample chamber bottom, and application of sample chamber and microcavity array surface are provided with super hydrophobic surface, and the microcavity array is provided with a plurality of microcavities, is provided with at least one through-hole in the microcavity, and the through-hole cross-section is less than the microcavity cross-section. The microcavity array is provided with at least 4 microcavities; the microcavity body is cylindrical, the diameter is larger than 2mm, and the microcavity bottom is arc.
The sitting drop type cell culture chip can be prepared by a numerical control engraving method (as shown in figure 2), and the method comprises the following specific steps:
(1) drawing a microcavity array graph to be manufactured by using AutoCAD software, then importing the design graph into numerical control software to generate a numerical control code, and setting a proper cutter rotating speed and a proper feeding speed;
(2) selecting a PMMA plate with the thickness of about 6mm as a plate, fixing the PMMA plate on a machining platform of a numerical control engraving machine, selecting a ball-end milling cutter with the diameter of 2-8 mm, adding a proper amount of water into a water tank to ensure that the plate is completely immersed, and starting a numerical control engraving program to process a microcavity array main body structure;
(3) after the microcavity array main body structure is completed, a cutter is replaced by a drill bit of 0.5-1.5 mm, and a through hole structure is manufactured in the center of the bottom of each microcavity;
(4) cutting the plate with the manufactured structure under a laser engraving machine, and removing redundant plates around the microcavity array structure; cutting a rectangular frame structure from another PMMA plate with the thickness of 6mm, wherein the size and the shape of the periphery of the frame are the same as those of the microcavity array plate, and the size of the inner frame of the frame is slightly larger than that of the microcavity array region;
(5) aligning the cut frame with the microcavity structure surface of the microcavity array plate, and bonding the cut frame and the microcavity structure surface of the microcavity array plate by using organic glass glue to ensure that the frame forms a sample adding cavity on the microcavity array plate to obtain a chip substrate;
(6) mixing the nano silicon dioxide particles, the structural adhesive and the chloroform according to the mass ratio of 1:2:15, adding the mixed solution into a liquid adding cavity of a spray pen after uniformly mixing, starting an air pump, and uniformly spraying the mixed solution on the surface of a chip;
(7) and (3) drying the sprayed chip in an oven at 70 ℃ for at least 1h to obtain the sitting-drop cell ball culture chip.
EXAMPLE 2 sample application of sitting-drop cell ball culture chip
The sitting-drop cell ball culture chip can be applied to high-throughput culture of cell balls (as shown in FIG. 3) by the following method:
(1) cleaning the sitting drop type cell ball culture chip, drying, and sterilizing under an ultraviolet lamp for 40 min;
(2) adding MHCC97-H (human highly metastatic hepatoma carcinoma cell) cell suspension into a chip sample adding cavity, and properly inclining, rotating or slightly shaking the chip to enable the cell suspension to enter each microcavity of the microcavity array and form independent liquid drops;
(3) after all the micro-cavities are filled with the cell suspension, sucking out the redundant cell suspension in the chip sample adding cavity to finish the sample adding and distribution of the chip;
(4) and (3) placing the chip after the sample adding into a culture dish, adding a moisturizing cotton ball, and then placing the chip into a carbon dioxide incubator at 37 ℃ to culture the cell balls.
EXAMPLE 3 liquid exchange and drug Loading of sitting-drop cell-ball culture chip
In the process of cell ball culture, metabolic waste generated in the growth process of cells is often removed by changing a culture solution, a fresh culture medium is supplemented at the same time, and the cells can normally grow, or medicines are added into the culture solution to study the influence of the medicines on the cell ball growth, so that medicine screening is realized.
The sitting-drop cell ball culture chip can realize the operations of liquid changing and medicine loading by the following methods (as shown in figure 4): taking out the chip to be subjected to liquid exchange after being cultured for a certain time from the culture dish, adding a fresh culture medium or a fresh culture medium dissolved with drug molecules into the sample adding cavity of the chip, covering each microcavity with the fresh culture medium or the fresh culture medium dissolved with the drug molecules, standing for 10-20 min to ensure that sufficient molecular diffusion exchange occurs between liquid drops in the microcavities and a loading culture medium, sucking out the culture medium which completes the diffusion exchange in the sample adding cavity, and placing the chip in the original culture environment for continuous culture.
EXAMPLE 4 in-situ staining and Observation of cell pellets in sitting-drop cell pellet culture chip
The sitting-drop type cell ball culture chip can realize in-situ staining and observation of cultured cell balls, and the specific operations are as follows (as shown in figure 5):
taking out the chip after culturing for a certain time from the culture environment, removing part of the supernatant of the liquid drop in the microcavity where the selected cell ball to be stained is located by a liquid transfer device, adding the prepared cell staining solution into the microcavity, and placing the chip in the original culture environment for incubation for 30 min; after the staining incubation is completed, the chip is taken out, part of supernatant of the liquid drop in the staining microcavity is removed through a pipettor, PBS buffer is added, the PBS buffer liquid changing process is repeated twice, and finally the chip is placed under a fluorescence microscope for observation.
EXAMPLE 5 Collection of cultured cell pellets in sitting-drop cell pellet culture chip
The sitting-drop cell ball culture chip can collect the cell balls cultured in the chip for subsequent analysis and application by the following method (as shown in FIG. 6):
the method comprises the following steps: taking a plate glass or a culture dish after disinfection treatment, covering one surface of the plate glass or the culture dish which is to receive the cell balls on the chip, then inverting the combination, transferring the liquid drops in each microcavity of the chip and the culture cell balls contained in the liquid drops to the plate glass or the culture dish under the action of gravity, and removing the chip to finish cell ball collection.
The second method comprises the following steps: and moving the chip to be collected for culturing the cell balls to the upper part of a culture dish containing a culture medium, and inclining the chip to ensure that the liquid drops in each microcavity of the chip and the cultured cell balls contained in the liquid drops flow into the culture dish under the action of gravity to finish the collection of the cell balls. FIG. 7 is a photomicrograph of cell spheres collected based on this method. The collected cell balls can be applied to subsequent researches such as drug screening and the like.
The third method comprises the following steps: and (3) independently sucking the cultured cell balls from the selected micro-cavities by using a pipette or a suction pipe to realize selective collection of the cell balls.
While the present invention has been described in detail with reference to the illustrated embodiments, it should not be construed as limited to the scope of the present patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.
Claims (10)
1. The utility model provides a sitting drop type cell ball cultivates chip which characterized in that, includes application of sample chamber and microcavity array, the microcavity array sets up application of sample chamber bottom, just application of sample chamber with microcavity array surface is provided with super hydrophobic surface, the microcavity array is provided with a plurality of microcavities, be provided with at least one through-hole in the microcavity, the through-hole cross-section is less than the microcavity cross-section.
2. The sitting-drop cell culture ball chip of claim 1, wherein the micro-cavity array is provided with at least 4 micro-cavities.
3. The sitting-drop cell culture chip of claim 1, wherein the microcavity is cylindrical with a diameter greater than 2mm, and the bottom of the microcavity is arc-shaped.
4. The sitting-drop cell-ball culture chip of claim 1, wherein the substrate material of the sitting-drop cell-ball culture chip is one of polystyrene, polycarbonate, polymethyl methacrylate, polydimethylsiloxane, acrylonitrile-butadiene-styrene, polypropylene, polyethylene, polyvinyl chloride, polytetrafluoroethylene and epoxy resin.
5. The sitting-drop cell culture ball chip of claim 1, wherein the microcavity array is prepared by numerical control engraving or injection molding.
6. The sitting-drop cell-ball culture chip of claim 1, wherein the super-hydrophobic surface is prepared by the following method: and spraying or dip-coating the nano material suspension on a base material of the sitting-drop cell ball culture chip, and drying to obtain the super-hydrophobic surface.
7. The sitting-drop type cell culture chip according to claim 6, wherein the nano-material suspension is a mixed solution prepared by mixing a nano-material, glue and a dispersing agent according to a mass ratio of 1-2: 1-3: 15.
8. The sitting-drop cell-ball culture chip of claim 7, wherein the nano-materials are nanoparticles, nanofibers or nanorods; the glue is epoxy resin, organic silicon resin or structural glue; the dispersing agent is tetrahydrofuran, absolute ethyl alcohol, isopropanol or methane halogenated hydrocarbon.
9. The use method of the sitting-drop cell-ball culture chip as claimed in any one of claims 1 to 8, which comprises the following steps in sequence:
(1) cleaning and drying the sitting drop type cell ball culture chip, and then carrying out ultraviolet disinfection;
(2) adding the cell suspension into the sterilized sample adding cavity, tilting, rotating or shaking the chip, and sucking out residual liquid when the microcavity array is filled with the cell suspension;
(3) culturing the chip subjected to sample distribution in an environment with the temperature of 37 ℃ and the carbon dioxide atmosphere containing 5% and the relative humidity of more than 90% for 24-72 h, then adding a culture medium or a drug to be detected into a sample adding cavity, inclining or rotating the chip, sucking out residual liquid after full diffusion, and continuing culturing under the original culture condition; according to the requirement of cell ball culture, the liquid changing process can be repeated for many times until the end, and the cell balls are collected.
10. The method of claim 9, wherein when collecting the cell balls, the chip is turned over to transfer the cell ball-containing droplets in each microcavity to a plate glass or a petri dish; or directly pouring the liquid drops containing the cell balls in each microcavity into a culture dish filled with a culture medium; or the cultured cell pellet is aspirated from the single microcavity by a pipette or pipette.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010650943.4A CN111647509A (en) | 2020-07-08 | 2020-07-08 | Sitting drop type cell ball culture chip and its use method |
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