CN111647509A - Sitting drop type cell ball culture chip and its use method - Google Patents

Abstract

The present invention provides a sitting drop cell spheroid culture chip and a method for using the same. The sitting drop cell spheroid culture chip comprises a sample feeding cavity and a microcavity array, wherein the microcavity array is arranged at the bottom of the sample feeding cavity, and the The sample loading cavity and the microcavity array are provided with superhydrophobic surfaces, the microcavity array is provided with several microcavities, and at least one through hole is set in the microcavity, and the cross section of the through hole is smaller than that of the microcavity. section. The present invention also includes a method for using the sitting drop cell spheroid culture chip. The invention utilizes the micro-cavity array combined with the super-hydrophobic surface, and can realize the automatic distribution of the cell suspension, the simple and rapid change of the medium in the culture process, and the replacement of the cultured cell spheroid through the hydrophobic effect of the super-hydrophobic surface, the surface tension of the liquid sample and the self-gravity synergy. It is simple and quick to recover, and effectively solves the problems in the prior art, such as difficulty in medium renewal, poor air permeability, poor uniformity of cell spheroids, difficulty in in-situ observation, high cost, and difficulty in taking out cell spheroids.

Description

A sitting drop cell spheroid culture chip and method of using the same

technical field

The invention belongs to the technical field of cell biology, and particularly relates to a sitting drop cell spheroid culture chip and a method for using the same.

Background technique

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 connection between cells and between cells and the external microenvironment, and better simulate the natural physiological environment. Cell intrinsic metabolism (such as nutrients, oxygen, metabolites, etc.) and proliferative gradients, which can track the diffusion path of drugs, are ideal models for cell research. Due to its unique advantages, in recent years, 3D cell spheroid culture has been widely used in cell biology and medical research, and has become an indispensable and powerful tool for drug screening, tumor treatment mechanism analysis, stem cell differentiation and regenerative medicine. At present, the methods used to prepare 3D cell spheroids mainly include rotary stirring culture, hanging drop culture, microcavity culture based on low cell adhesion surface, acoustic focusing culture driven by surface acoustic wave, magnetic suspension culture, gel Embedded culture, etc. However, these methods all have their own limitations. For example, the rotary stirring culture method is difficult to achieve uniform culture of cell spheroids, and the shear force can easily cause cell damage, so the culture process cannot be observed in real time; Disturbance and shedding, difficulty in changing the medium during the culture process, etc.; low-adhesion surface microcavity culture requires tedious surface treatment operations, and the cell sphere is located deep below the nutrient solution level, making it difficult to exchange with the outside atmosphere; acoustic focusing type In addition to the magnetic suspension culture, special auxiliary devices are required, resulting in a complex culture system and high experimental cost; while the gel-embedded culture makes the existence of exogenous matrix around the cells in the process of cell culture, which cannot accurately reflect the physiological activities of the real microenvironment of the cells. , which will lead to the lack of physiological signals or even to obtain erroneous research results. Therefore, it is urgent to develop a new device and method for culturing 3D cell spheroids with simple operation, low cost, good biocompatibility, high stability, good consistency, less external interference and convenient in situ observation, so as to promote cell biology and medical development.

SUMMARY OF THE INVENTION

In view of the above problems existing in the prior art, the present invention provides a sitting drop cell spheroid culture chip and a method for using the same, which utilizes a microcavity array combined with a superhydrophobic surface, through the superhydrophobic surface hydrophobic effect, liquid sample surface tension and self- The synergistic effect of gravity can realize the automatic distribution of cell suspension, the simple and fast liquid exchange in the culture process, and the simple and fast recovery of the cultured cell spheroids, which effectively solves the difficulty of medium renewal, poor air permeability, and poor cell spheroid uniformity in the prior art. Difficult to observe in situ, high cost, and difficult to remove cell spheroids.

In order to achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is: to provide a sitting drop cell spheroid culture chip, which includes a sample adding cavity and a microcavity array, and the microcavity array is arranged at the bottom of the sample adding cavity, and the adding The surfaces of the sample cavity and the microcavity array are provided with superhydrophobic surfaces, the microcavity array is provided with several microcavities, and at least one through hole is arranged in the microcavity, and the cross section of the through hole is smaller than the cross section of the microcavity.

Further, the microcavity array is provided with at least 4 microcavities.

Further, the microcavity cavity is cylindrical, the diameter is greater than 2 mm, and the bottom of the microcavity is arc-shaped.

Further, the base material of the sitting drop cell spheroid culture chip is polystyrene, polycarbonate, polymethyl methacrylate, polydimethylsiloxane, acrylonitrile-butadiene-styrene, polypropylene, One of polyethylene, polyvinyl chloride, teflon and epoxy resin.

Further, the microcavity array is prepared by numerical control engraving or injection molding process.

Further, the superhydrophobic surface is prepared by the following method: spraying or dipping the nanomaterial suspension on the substrate of the sitting drop cell spheroid culture chip, and drying to obtain the superhydrophobic surface.

Further, dry at 70°C for at least 1 h.

Further, the nanomaterial suspension is a mixed solution obtained by mixing nanomaterials, glue and dispersant in a mass ratio of 1-2:1-3:15.

Further, the nanomaterial is nanoparticle, nanofiber or nanorod, and the characteristic scale is 5-500 nm; the glue is epoxy resin, silicone resin or structural glue; the dispersant is tetrahydrofuran, absolute ethanol, isopropanol or methane halogenated hydrocarbon.

Further, the nanomaterials are silica nanoparticles, alumina nanoparticles, titania nanoparticles, carbon nanofibers, polyacrylonitrile nanofibers, polyvinylidene fluoride nanofibers, carbon nanotubes or metal nanorods.

The using method of the above-mentioned sitting drop cell spheroid culture chip includes the following steps in turn:

(1) Wash and dry the sitting drop cell spheroid culture chip, and then sterilize with UV light;

(2) Add the cell suspension into the sterilized sample loading chamber, tilt, rotate or shake the chip, and when the cell suspension fills the microcavity array, suck out the remaining liquid;

(3) Incubate the chip with the completed sample distribution at 37°C in an environment containing 5% carbon dioxide and a relative humidity greater than 90% for 24 to 72 hours, then add culture medium or the drug to be tested in the sample loading chamber, tilt or rotate After the chip is fully diffused, the remaining liquid is sucked out, and the culture is continued under the original culture conditions; according to the needs of cell spheroid culture, the process of changing the medium can be repeated many times until the end, and the cell spheroids are collected.

Further, when collecting cell spheroids, flip the chip to transfer the droplets of cell spheroids in each microchamber to a flat glass or a petri dish; or pour the droplets of cell spheroids in each microchamber directly into a culture dish filled with culture medium. ; or by pipetting or pipetting the cultured spheroid from a single microchamber.

Further, during ultraviolet disinfection, disinfection is performed under an ultraviolet lamp with a radiation intensity greater than 70W/cm ² for 15 to 60 minutes.

To sum up, the present invention has the following advantages:

1. The sitting drop cell spheroid culture chip of the present invention utilizes a micro-cavity array combined with a super-hydrophobic surface, and can realize the automatic distribution and culture process of cell suspension through the hydrophobic effect of the super-hydrophobic surface, the synergistic effect of the surface tension of the liquid sample and its own gravity. The simple and fast medium exchange and the convenient and fast recovery of the cultured cell spheroids, the operation is simple and efficient, and the use cost is low. And the cell spheroid is not easy to take out and so on.

2. The chip has a through-hole microcavity array chip with superhydrophobic surface characteristics. The chip can realize the superhydrophobicity of the cell suspension through the synergistic effect of the hydrophobic effect of the superhydrophobic surface, the surface tension of the liquid sample and the gravity of the fluid itself. The automatic distribution in the cavity array enables the automatic formation of an independent droplet of cell suspension in each microchamber for high-throughput culture of cell spheroids. Compared with the existing hanging drop cell spheroid culture device and the microcavity array cell spheroid culture chip based on the curved bottom surface, the sitting drop culture chip disclosed in the present invention has the advantages of long-term stability of the culture droplets, liquid sample distribution and improvement The simplicity of nutrient solution renewal, the exchange rate of the culture atmosphere, and the in situ observability reflect better advantages, providing an efficient platform for 3D cell culture and drug screening. It has important application value.

3. The present invention can easily and quickly realize operations such as cell suspension distribution, medium exchange during culture, drug loading, and recovery of cultured cell spheroids, and the culture droplets have strong resistance to mechanical disturbances, good air permeability of culture droplets, and do not require complicated equipment. The external auxiliary device avoids the difficult operation, poor controllability, high cost of consumables, and difficult in situ observation of the current cell spheroid culture method, and is expected to promote the rapid development and wide application of 3D cell culture technology in basic biological research and medical research. .

4. Because of the support of the substrate and the limitation of the microcavity, the droplet will not fall off due to external disturbance during the culture process, and it has better mechanical stability; due to the design of the superhydrophobic microcavity, there is no need to pass a pipette Distribute the culture droplets to the culture plate one by one, and only need to load the cell suspension once and simply tilt and rotate the chip to achieve rapid distribution of all culture droplets through the action of gravity and surface tension, which greatly reduces the labor intensity of the experimenter; Due to the poor stability of droplets in the hanging drop culture, it is often difficult to change the liquid during the culture. However, the present invention utilizes the superhydrophobic microcavity design, combined with the sitting drop solution, which can conveniently and quickly realize the liquid change operation without worrying about changing the liquid. The problem of droplet shedding during the process.

5. Due to the through-hole structure at the bottom of the microcavity, and because the microcavity is a super-hydrophobic surface, the culture droplet and the solid surface of the microcavity are not in complete contact, and there is a large amount of gas, which is beneficial to the liquid at the bottom of the droplet during the culture process. The cell spheroid exchanges with the outside atmosphere, while the cell spheroid cultured in the microplate type is located at the closed bottom of the microcavity, and has a large distance from the liquid surface in contact with the atmosphere. The gas exchange is difficult, which is not conducive to the growth of the cell spheroid. Usually, the culture microcavities of the cell spheroids are connected by the culture medium, and there is diffusion and interference of signal molecules between them. However, in the present invention, due to the design of the superhydrophobic microcavities, each culture microcavity is independent, and there is no signal during the culture process. Molecular exchange and interference, each microcavity can perform independent drug screening or stimulation factor tests, so a single chip can realize multiple experiments, with better flexibility and economy.

6. When the chip is in use, first sterilize the sitting drop cell spheroid culture chip by ultraviolet radiation; then add a certain amount of cell suspension into the sterilized chip sample chamber, due to the surface of the chip sample chamber. Super-hydrophobic properties, the added cell suspension can roll freely in the sample chamber, properly tilt, turn or shake the chip slightly, so that the liquid sample passes through all the micro-chambers and is filled into the micro-chambers under the action of gravity. The synergistic effect of the sample surface tension and its own gravity separates the filled liquid sample portion in the microchamber from the liquid sample body, thereby forming an independent droplet of cell suspension in each microchamber; then remove the excess in the sample loading chamber Cell suspension, and the chip after the liquid sample distribution is placed in a high humidity and set constant temperature environment for cell spheroid culture; during the culture process, the medium is replaced regularly. Add a certain amount of culture medium to the sample loading chamber, tilt and rotate the chip appropriately, make the liquid sample pass through all the microcavities, and use molecular diffusion to realize the exchange of nutrients and metabolite molecules between each cell culture droplet and the fresh culture medium, and then Remove the liquid sample that has completed the diffusion exchange in the sample loading chamber; after the cell spheroids in the micro-chamber are cultured to the expected size, the cell spheroids are finally collected. The method is simple and easy to operate, can easily and quickly perform medium exchange, and is convenient to collect the cell spheroids, which is convenient for the popularization and application of the sitting drop cell spheroid culture chip.

Description of drawings

Fig. 1 is a schematic diagram of the structure of a sitting drop cell spheroid culture chip;

Fig. 2 is a schematic diagram of the process flow of preparing a sitting drop cell spheroid culture chip by CNC engraving technology;

Figure 3 is a schematic diagram of the sample adding process of the sitting drop cell spheroid culture chip;

Fig. 4 is a schematic diagram of the liquid exchange and drug loading process of the sitting drop cell spheroid culture chip;

Figure 5 is a schematic diagram of the in situ staining and observation process of cell spheroids in a sitting drop cell spheroid culture chip;

FIG. 6 is a schematic diagram of the recovery process of the cell spheroids of the sitting drop cell spheroid culture chip;

Fig. 7 is a micrograph of human highly metastatic hepatoma cell spheroids cultured and collected based on the sitting drop cell spheroid culture chip of the present invention.

Detailed ways

Example 1 Sitting drop cell spheroid culture chip and preparation

A sitting drop cell spheroid culture chip includes a sample loading cavity and a microcavity array, the microcavity array is arranged at the bottom of the sample loading cavity, the surfaces of the sample loading cavity and the microcavity array are provided with superhydrophobic surfaces, and the microcavity array is provided with several The micro-cavity is provided with at least one through hole, and the cross-section of the through-hole is smaller than the cross-section of the micro-cavity. The microcavity array is provided with at least 4 microcavities; the microcavity cavity is cylindrical, the diameter is greater than 2 mm, and the bottom of the microcavity is arc-shaped.

The preparation of the sitting drop cell spheroid culture chip can be made by the numerical control engraving method (as shown in Figure 2), and the specific steps are as follows:

(1) Use AutoCAD software to draw the micro-cavity array graphic to be produced, then import the design graphic into the numerical control software, generate numerical control code, and set the appropriate tool speed and feed speed;

(2) Choose a PMMA board with a thickness of about 6mm as a plate, fix it on the CNC engraving machine processing platform, choose a ball end milling cutter with a diameter of 2-8mm, and then add an appropriate amount of water to the water tank to ensure that the plate is completely immersed , start the CNC engraving program to process the main structure of the microcavity array;

(3) After completing the main structure of the microcavity array, replace the tool with a 0.5-1.5mm drill bit, and make a through-hole structure in the center of the bottom of each microcavity;

(4) Place the fabricated plate under the laser engraving machine for cutting, and remove the excess plates around the microcavity array structure; take another PMMA plate with a thickness of 6mm, and cut a rectangular frame structure, the size, shape and micro The cavity array plate is the same, and its inner frame is slightly larger than the microcavity array area;

(5) Align the cut frame with the microcavity structure surface of the microcavity array plate, and bond the two with plexiglass glue, so that the frame forms a sample loading cavity on the microcavity array plate to obtain a chip substrate;

(6) Mix the nano-silica particles, structural adhesive and chloroform in a mass ratio of 1:2:15. After mixing evenly, add the mixture into the liquid-filling chamber of the airbrush, turn on the air pump, and spray the mixture evenly on the chip surface;

(7) Place the sprayed chip in a 70° C. oven to dry for at least 1 hour to obtain a sitting drop cell spheroid culture chip.

Example 2 Loading of Sitting Drop Cell Spheroid Culture Chip

The sitting drop cell spheroid culture chip can be applied to the high-throughput culture of cell spheroids by the following methods (as shown in Figure 3):

(1) the obtained sitting drop cell spheroid culture chip is cleaned, dried, and placed under an ultraviolet lamp for sterilization for 40min;

(2) Add the MHCC97-H (human highly metastatic hepatoma cell) cell suspension into the chip loading chamber, properly tilt, turn or shake the chip slightly, so that the cell suspension enters each microchamber of the microchamber array, and Form independent droplets;

(3) After all the microcavities are filled with the cell suspension, suck out the excess cell suspension in the chip loading chamber to complete the chip loading and distribution;

(4) Put the chip after adding the sample into a petri dish, add a moisturizing cotton ball, and then put it into a 37° C. carbon dioxide incubator to culture the cell ball.

Example 3 Liquid exchange and drug loading of the sitting drop cell spheroid culture chip

In the process of cell spheroid culture, it is often necessary to remove the metabolic waste produced by the cells during the growth process by changing the medium, and at the same time replenish fresh medium to maintain the normal growth of the cells, or add drugs to the culture medium to study the effect of drugs on the cell spheroids. Growth effects to enable drug screening.

The sitting drop cell spheroid culture chip can realize the liquid change and drug loading operations by the following methods (as shown in Figure 4): take out the chip to be changed after cultured for a certain period of time from the petri dish, add fresh medium or dissolved drugs The fresh medium of molecules is added to the chip loading chamber, so that the fresh medium or the fresh medium dissolved with drug molecules covers each micro-chamber, and let stand for 10-20 minutes, so that the droplets in the micro-chamber and the loading medium are between the micro-chambers. When sufficient molecular diffusion exchange occurs, the medium in the sample loading chamber that has completed the diffusion exchange is aspirated, and the chip is placed in the original culture environment to continue culturing.

Example 4 In situ staining and observation of cell spheroids in a sitting drop cell spheroid culture chip

The sitting drop cell spheroid culture chip can realize in situ staining and observation of cultured cell spheroids. The specific operations are as follows (as shown in Figure 5):

After culturing for a certain period of time, the chip is taken out from the culture environment, and the supernatant liquid of the droplet in the microchamber where the selected to-be-stained cell spheroid is located is removed by a pipette, and then the prepared cell staining solution is added to the microchamber. The chip was placed in the original culture environment and incubated for 30 min; after the staining incubation was completed, the chip was taken out, and part of the supernatant of the droplets in the staining microchamber was removed by a pipette, and PBS buffer was added. The PBS buffer exchange process was repeated twice, and finally the chip was placed under a fluorescence microscope for observation.

Example 5 Collection of cultured cell spheroids in a sitting drop cell spheroid culture chip

The sitting drop cell spheroid culture chip can collect the cell spheroids cultured in the chip by the following methods for subsequent analysis and application (as shown in Figure 6):

Method 1: Take a sterilized flat glass or petri dish, cover the side of the cell sphere to be received on the chip, and then turn the assembly upside down. Transfer to a glass plate or petri dish and remove the chip to complete the spheroid collection.

Method 2: Move the chip to be used for the collection of cultured cell spheroids above a culture dish containing culture medium, and tilt the chip so that the droplets in each microcavity of the chip and the cultured cell spheroids contained in it flow into the culture dish under the action of gravity. , to complete the spheroid collection. Figure 7 is a photomicrograph of spheroids collected based on this method. The collected spheroids can be used in subsequent studies such as drug screening.

Method 3: Using a pipette or a pipette, individually suck the cultured cell spheroids from the selected microcavity to achieve selective collection of the cell spheroids.

Although the specific embodiments of the present invention have been described in detail with reference to the accompanying drawings, they should not be construed as limiting the protection scope of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative efforts still belong to the protection scope of this patent.

Claims (10)

1. A sitting drop cell spheroid culture chip, characterized in that it comprises a sample-adding cavity and a micro-cavity array, wherein the micro-cavity array is arranged at the bottom of the sample-adding cavity, and the sample-adding cavity and the micro-cavity are The surface of the array is provided with a superhydrophobic surface, the microcavity array is provided with several microcavities, and at least one through hole is arranged in the microcavity, and the cross section of the through hole is smaller than the cross section of the microcavity. 2 . The sitting drop cell spheroid culture chip according to claim 1 , wherein the microcavity array is provided with at least 4 microcavities. 3 . 3 . The sitting drop cell spheroid culture chip according to claim 1 , wherein the microcavity cavity is cylindrical with a diameter greater than 2 mm, and the bottom of the microcavity is arc-shaped. 4 . 4. The sitting drop cell spheroid culture chip according to claim 1, wherein the base material of the sitting drop cell spheroid culture chip is polystyrene, polycarbonate, polymethyl methacrylate, One of polydimethylsiloxane, acrylonitrile-butadiene-styrene, polypropylene, polyethylene, polyvinyl chloride, polytetrafluoroethylene and epoxy resin. 5 . The sitting drop cell spheroid culture chip according to claim 1 , wherein the microcavity array is prepared by numerical control engraving or injection molding process. 6 . 6. The sitting drop cell spheroid culture chip according to claim 1, wherein the superhydrophobic surface is prepared by the following method: spraying or dipping the nanomaterial suspension on the sitting drop cell spheroid culture chip The substrate is dried to obtain a superhydrophobic surface. 7 . The sitting drop cell spheroid culture chip according to claim 6 , wherein the nanomaterial suspension is formed by mixing nanomaterials, glue and dispersant in a mass ratio of 1～2:1～3:15. 8 . of the mixture. 8. The sitting drop cell spheroid culture chip of claim 7, wherein the nanomaterial is nanoparticle, nanofiber or nanorod; and the glue is epoxy resin, silicone resin or structural glue; The dispersant is tetrahydrofuran, absolute ethanol, isopropanol or methane halogenated hydrocarbon. 9. The method for using the sitting drop cell spheroid culture chip according to any one of claims 1 to 8, characterized in that it comprises the following steps in sequence: (1) Wash and dry the sitting drop cell spheroid culture chip, and then sterilize with UV light; (2) Add the cell suspension into the sterilized sample loading chamber, tilt, rotate or shake the chip, and when the cell suspension fills the microcavity array, suck out the remaining liquid; (3) Incubate the chip with the completed sample distribution at 37°C in an environment containing 5% carbon dioxide and a relative humidity greater than 90% for 24 to 72 hours, then add culture medium or the drug to be tested in the sample loading chamber, tilt or rotate After the chip is fully diffused, the remaining liquid is sucked out, and the culture is continued under the original culture conditions; according to the needs of cell spheroid culture, the process of changing the medium can be repeated many times until the end, and the cell spheroids are collected. 10. The method for using a sitting drop cell spheroid culture chip according to claim 9, wherein when collecting the cell spheroids, the chip is turned over to transfer the droplets of the cell spheroids in each microcavity to a flat glass or a petri dish; Or pour the droplets of cell spheroids in each microchamber directly into a culture dish filled with culture medium; or aspirate the cultured cell spheroids from a single microchamber through a pipette or pipette.

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