Separation method of three-dimensional cell public culture area

Abstract

The invention belongs to the field of cell culture, and discloses a method for separating a three-dimensional cell public culture area. The method is used for carrying out physical isolation arrangement on a three-dimensional cell culture area in a three-dimensional cell public culture process, and comprises the following steps: after completing culture configuration operation of three-dimensional cells in a culture dish with a gel substrate, assembling a top cover with a partition plate and the culture dish; enabling the gel substrate to be in contact with the partition plate to form a physically isolated three-dimensional cell culture area; or after the culture solution is injected from the open culture platform on the upper layer of the culture device, the liquid level of the culture solution is adjusted to be located in the isolation chamber on the lower layer of the culture device to form a physically isolated three-dimensional cell culture area. According to the method, reversible physical isolation setting is carried out in the three-dimensional cell culture process, so that the management efficiency of cell populations is improved, cross infection is prevented, the operation efficiency and reliability of high-throughput cell culture are improved, and the method is suitable for drug screening, toxicology testing, disease model research and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field related to cell culture and drug development, and more specifically, relates to a method for separating a three-dimensional cell common culture area. Background Art

[0002] In the field of biotechnology and drug development, cell culture plays a vital role as a basic technology. Especially in drug discovery and disease model research, cell culture technology provides an important platform for evaluating biological responses and drug action mechanisms. With the development of cell culture technology, from the initial monolayer cell culture to today's three-dimensional culture technology, scientists have been exploring how to more biologically simulate the natural environment of cells in the body. Three-dimensional culture technology has proven that it can more accurately simulate the microenvironment of cells in tissues than traditional two-dimensional culture, including cell-to-cell interactions, gradient distribution of nutrients, and the composition of the extracellular matrix, which are important factors affecting cell behavior and drug response.

[0003] However, with the increasing application of three-dimensional culture technology in high-throughput screening, the limitations of related technologies are becoming increasingly apparent. Traditional high-throughput cell culture methods still face many challenges, the most pressing of which is how to effectively isolate different cell populations in a shared culture environment. Although there are various attempts to solve this problem, including the direct use of partitions or microfluidic devices, they usually have the disadvantages of complex implementation, high cost, and inconvenient cell manipulation. These problems limit the application of three-dimensional culture technology in high-throughput experiments, especially when multiple cell types need to be cultured simultaneously.

[0004] In high-throughput culture experiments with multiple cell types, cross-contamination between cells becomes a significant problem, which not only affects the accuracy and reliability of experimental data, but also may lead to false positive or false negative results in the drug discovery process.

[0005] In addition, current separation methods usually cannot provide a long-term stable culture environment, and frequent replacement of culture medium can cause cell stress, affecting the cell physiological state and experimental results. These methods also usually do not support automated operations, which is particularly important in large-scale drug screening experiments. Summary of the invention

[0006] In view of the above defects or improvement needs of the prior art, the present invention provides a method for separating a three-dimensional cell common culture area, the purpose of which is to establish physical isolation through the assembly of a culture device during the operation of three-dimensional cell common culture, thereby solving the difference problem that three-dimensional cells cannot be tested under the same culture environment, and effectively avoiding the cross-contamination problem of three-dimensional cell culture.

[0007] To achieve the above object, according to one aspect of the present invention, a method for separating a three-dimensional cell common culture area is provided, wherein the method is to perform physical isolation of the three-dimensional cell culture area during the three-dimensional cell common culture process, comprising:

[0008] After completing the three-dimensional cell culture configuration operation in the culture dish with the gel substrate, assembling the top cover with the partition with the culture dish, so that the gel substrate contacts the partition to form a physically isolated three-dimensional cell culture area;

[0009] Alternatively, after injecting culture fluid from an open culture platform on an upper layer of a culture device, the liquid level of the culture fluid is adjusted to be located in an isolation chamber on a lower layer of the culture device to form a physically isolated three-dimensional cell culture area; wherein the culture device comprises an open culture platform on an upper layer and an isolation chamber on a lower layer.

[0010] As a preferred embodiment of the present invention, during the three-dimensional cell public culture, the physical isolation is set in a reversible manner so that the culture conditions of the three-dimensional cell public culture are uniformly set.

[0011] As a preferred embodiment of the present invention, after the physically isolated three-dimensional cell culture area is formed, each isolation chamber therein can be operated independently.

[0012] As a preferred embodiment of the present invention, the top cover with the partition is assembled with the culture dish, comprising:

[0013] A physically isolated three-dimensional cell culture area is formed by assembling the top cover until the gel substrate contacts the partition; wherein the contact formation comprises interfacial contact between the gel substrate and the partition or the gel substrate is interrupted by the partition.

[0014] As a preferred embodiment of the present invention, there are a plurality of partitions which are regularly arranged.

[0015] As a preferred embodiment of the present invention, a physically isolated three-dimensional cell culture area is formed in an isolation chamber located in the lower layer of the culture device by adjusting the liquid level of the culture solution; wherein the open culture platform is in a connected state, and the cover plate covering the top of the open culture platform is an open structure.

[0016] As a preferred embodiment of the present invention, a physically isolated three-dimensional cell culture area is formed in an isolation chamber located at a lower layer of the culture device by adjusting the liquid level of the culture solution; wherein the isolation chambers are arranged in an array.

[0017] As a preferred embodiment of the present invention, the method can be integrated into a cell culture system, and the cell culture system includes a control unit, a liquid flow circulation unit, a temperature control unit, a carbon dioxide control unit and a sterilization unit, and the liquid flow, temperature, carbon dioxide and sterilization operations in the cell culture system are controlled by the control unit; wherein the liquid flow circulation unit includes a liquid circulation pump for controlling the injection and outflow of the culture solution.

[0018] As a preferred embodiment of the present invention, the three-dimensional cells include primary cells, stem cells, tumor cells or organoids.

[0019] In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art:

[0020] (1) The method for separating the three-dimensional cell common culture area provided by the present invention is different from the existing method of directly using a culture dish with separated areas. During the common culture process, the common culture area is separated into several physically isolated areas through the operation of the culture device, thereby achieving efficient management of the cell population in the culture area, improving the efficiency of high-throughput cell culture and multiple control experiments, and effectively preventing cross-contamination between different cell populations, thereby ensuring the accuracy and reliability of the experimental results.

[0021] (2) The method for separating the three-dimensional cell common culture area provided by the present invention, wherein the physical isolation method of the separation is a reversible setting, so that the three-dimensional cell common culture can uniformly set the common culture conditions for common culture and can also perform specific operations on the cell culture in any chamber, thereby improving the efficiency of processing samples and the consistency of the same culture environment, while also ensuring the accuracy and repeatability under comparative experiments, and is more suitable for conducting multiple groups of control experiments.

[0022] (3) The design of the present invention is compatible with automated culture and analysis equipment, which has the potential to further reduce the workload of operators. The method of the present invention is suitable for large-scale cell culture operations and can be applied to large-scale biological production and drug development processes.

[0023] In summary, the method of the present invention improves the management efficiency of different cell populations in the public culture area by means of physical isolation, which not only improves the efficiency of high-throughput public culture of three-dimensional cells and ensures the consistency and stability of the three-dimensional public culture environment of cells, but also improves the efficiency of public culture of seed cell populations, the accuracy and repeatability of control experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of separating a common culture area by bonding a partition of a top cover to a gel substrate in a culture dish so that the two are in contact to form a physical separation.

[0025] Figure 2 It is a schematic diagram of separating the common culture area by truncating the gel according to an example of the present invention.

[0026] Figure 3 It is a schematic diagram of separating common culture areas to form physical separation by adjusting the liquid level according to an example of the present invention.

[0027] Throughout the drawings, the same reference numerals are used to denote the same elements or structures, wherein:

[0028] 1-top cover, 2-partition, 3-gel base, 4-culture dish. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] Embodiment 1: Based on a culture device comprising a culture dish 4 with a gel substrate 3 and a top cover 1 with a partition 2 covering the culture dish, a physically isolated three-dimensional cell culture area is formed by assembling the top cover after completing the three-dimensional cell culture configuration operation in the culture dish.

[0031] like Figure 1 As shown, the gel substrate 3 in the culture dish is bonded to the partition 2 of the top cover 1, so that the two are in contact to form a physical separation. In the process of contacting to form physical isolation, specifically as Figure 1 As shown, the top cover 1 is pressed down on the culture dish 4, so that the entire common culture dish 4 forms an isolated chamber. This method is suitable for cell groups that need to grow independently under the same culture conditions, and the top cover 1 can be easily removed or installed to facilitate experimental operations and the removal of cell samples.

[0032] In this embodiment, the partitions 2 of the top cover 1 are arranged regularly, and the partitions 2 are covered with a soft material. The partitions are made of a water-impermeable hard material and are covered with a soft material to prevent the mixing of liquids in different culture chambers.

[0033] In this embodiment, each partition plate 2 on the top cover 1 is not an open structure, and each independent chamber can be operated separately.

[0034] In this embodiment, the physically isolated three-dimensional cell culture area is formed by contact between the gel substrate 3 and the partition 2; wherein the contact formation includes contact between the gel substrate 3 and the partition interface or the gel substrate 3 is cut off by the partition 2. For example, Figure 2As shown, a method for implementing separation of common culture areas by cutting off the gel substrate is shown. Specifically, the method requires that the common culture substrate is a patterned hydrogel material, allowing cells to be cultured together on the gel material. When the culture reaches the processing stage, the gel can be directly cut and the separated gels can be operated separately.

[0035] Example 2: Based on a culture device including an open culture platform disposed on an isolation chamber, a physically isolated three-dimensional cell culture area is formed by controlling the height difference of the liquid surface of the culture solution injected.

[0036] like Figure 3 As shown, the implementation method of physically separating the common culture area by adjusting the liquid level of the culture solution is specifically described. The open common culture chip designed in this embodiment is a culture device including an open culture platform provided on an isolation chamber; each culture chamber requires that the chambers above are all in a connected state, and each layer of chambers must have an operating platform for adjusting the liquid level of the culture solution. By controlling the inflow and outflow of the culture solution in each chamber, a physical barrier appears between the chambers due to the lowering of the liquid level, thereby achieving separation between adjacent chambers.

[0037] like Figure 3 As shown, the multi-layer structure constitutes a public culture device, including: the uppermost layer of the public culture layer (open cover), the middle layer of the public culture layer with array-arranged holes, and the bottom plate with an array-arranged pattern. When the samples in the public culture device are cultured in public, when the liquid level of the culture liquid is in the height interval of the uppermost layer, only a single row or column can be cultured in public. At this time, different treatments can be performed on the single row or column. When the liquid level of the culture liquid drops to the height interval of the middle layer, each culture interval will be completely independent, and each independent chamber can be operated independently.

[0038] In this embodiment, it is particularly important to note that relatively independent culture chambers need to be hydrophilically modified so that by precisely controlling the amount of culture fluid flowing into and out of each chamber, a liquid level height difference can be formed between the chambers while the liquid surface is concave, thereby achieving physical separation between adjacent chambers, which can prevent both the mutual flow of liquids and the mutual migration of cells, thereby avoiding cross contamination.

[0039] In this embodiment, the common culture area is an open structure, allowing unobstructed subsequent operation and observation.

[0040] In this embodiment, the open shared culture chip includes a plurality of culture chambers of preset shapes, including but not limited to circular, rectangular, etc.

[0041] In some embodiments, the preparation of open shared culture chips includes but is not limited to the use of polymethyl methacrylate or gel. For example, to prepare an open shared culture chip: first, peel off the upper and lower layers of 1.5 mm polymethyl methacrylate for standby use; use a laser engraving machine to emboss on polymethyl methacrylate, prepare another piece of polymethyl methacrylate, peel off the film on one side, stick a special double-sided tape, and then directly use a laser engraving machine to engrave the patterned array microfluidic chip and the upper structure; peel off the double-sided tape layer of the engraved patterned array microfluidic chip and the upper structure under the action of the fixed column for bonding; put the bonded chip into a hot press, the upper and lower pressing surface temperature is 60 ° C, 20min; wait for the temperature of the hot pressed chip to return to room temperature, put it in pure water for ultrasonication for 24h, and blow it dry with a nitrogen gun; use a polypropylene glycol and ethylene oxide addition polymer, drip polypropylene glycol and ethylene oxide addition polymer on each macropore, completely cover the macropore and then suck it out, wait for it to air dry to complete the modification, put it into a transparent plastic bag and seal it for standby use. Before conducting the experiment, place the sealed chip in a UV disinfection cabinet or clean bench for 30 minutes for subsequent experiments.

[0042] In some embodiments, the methods of the present invention are applicable to cell cultures including but not limited to primary cells, stem cells, tumor cells, and organoids.

[0043] Example 3: The above-mentioned culture area separation method is further integrated with an automated system. The automated system includes an automated liquid flow circulation unit, a control unit, a temperature control unit, a carbon dioxide control unit, and a sterilization unit. The automated system in this embodiment can automatically adjust the inflow and outflow of the culture solution according to a preset program, and can even perform automated cell culture and biological tests. Through the control unit, researchers can remotely operate, set experimental parameters, monitor experimental progress, and obtain real-time data.

[0044] For example, when using the automated system integration, first (1) inoculate cells: first, digest the colorectal cancer organoids into a single cell suspension and adjust the cell concentration to 5×10 5 / ml, and 1mL of the cell suspension is inoculated into the open shared culture chip. The chip has an optimized geometric design to support the independent and uniform growth of organoids; during inoculation, the cell suspension is mixed with PBS to a total volume of 4mL, and the mixed liquid is quickly and evenly introduced into the culture channel of the microfluidic chip through the entrance of the open chamber; after a 20-minute sedimentation period, the cells begin to settle in their respective depressions in the microcavity, and then the upper PBS is slowly aspirated and replaced with a suitable organoid culture medium to supply the necessary nutrients and growth factors. (2) Docking with automated instruments: Place the inoculated chip into the automated instrument, and the inlet and outlet of the chip can be directly docked with the automated instrument; the chip is subjected to circulating flow culture through the corresponding program set inside the instrument, and the culture does not require an independent operation stage.

[0045] Specifically, for the partition that is bonded to the top cover through the gel base in the culture dish, the two are in contact to form a physical separation. A cover structure is arranged on the top of the automated equipment, and a partition corresponding to the open chip is arranged on the cover. The automated equipment controls the downward pressure, and each partition is in contact with the internal base of the corresponding chamber to form an isolation barrier.

[0046] Or, specifically, for the implementation method of physically separating the common culture area by adjusting the liquid level of the culture solution, the outflow of the culture solution from each chamber is precisely controlled from the chip outlet through the program setting in the instrument, and the liquid level height difference between the chambers is formed while the liquid surface is concave, thereby achieving physical separation between adjacent chambers. This isolation method can prevent both the flow of liquids and the migration of cells, thereby avoiding cross contamination.

[0047] Example 4: Implementation of drug screening application.

[0048] (1) Cell planting and shared environment culture: The same cell population is planted in different culture chambers preset in the open shared culture platform. Cell culture is carried out under standard conditions, so that the cell spheres can grow stably in the shared culture medium environment to a size suitable for drug treatment.

[0049] (2) Cell chamber separation: For example, when a three-dimensional cell culture area is activated to form a physical isolation by controlling the height difference of the liquid level when the culture medium is injected, or when a three-dimensional cell culture area is activated to form a physical isolation by assembling a top cover, this step may not be performed, and partitions may be directly set on the drug-sensitive plate to be processed in the next step. Each partition is in contact with the internal base of the corresponding chamber to form an isolation barrier.

[0050] (3) Drug treatment: A drug-sensitive plate is set on the top of the chip. The drug-sensitive plate has multiple drug probes. The probes on the drug-sensitive plate are pressed to make them contact with the liquid surface of each independent chamber to achieve quantitative drug delivery; or, the separated chambers are directly added with drugs independently to ensure that different chambers receive drugs of different concentrations.

[0051] (4) Real-time monitoring and analysis: Use an automated system to monitor cell growth and biological responses after drug administration. Collect data on cells in each chamber in real time through imaging technology and biomarker detection. Summarize and analyze the growth data and biological responses of cells in all chambers to quantitatively evaluate the effects of each drug. Use statistical software to compare differences between different treatment groups to screen out effective drug candidate molecules. Confirm the toxicity and efficacy of candidate drugs on target cells for subsequent experimental verification and clinical research advancement.

[0052] Example 6: Implementation of various biological tests

[0053] This example demonstrates how to use the culture zone separation method of the present invention to perform a variety of biological tests.

[0054] The method for making the open public culture chip, the method for inoculating cells and the separation strategy of this embodiment are basically the same as those of embodiment 1, wherein the separation strategy can also be manual, which will not be described in detail again. This embodiment mainly focuses on the application of the public culture chamber after separation.

[0055] It enables independent separation of each culture chamber by controlling the outflow of culture medium, so that the cells in each chamber can continue to grow under appropriate conditions after separation. At the same time, the open independent structure allows manual further multiple tests on the same batch of cultured cell spheroids.

[0056] When a physically isolated three-dimensional cell culture area is formed by assembling a top cover, there is a hollow structure between the partitions on the cover, and each partition is in contact with the inner base of the corresponding chamber, forming an isolation barrier. The hollow structure also allows further multiple tests on the same batch of cultured cell spheroids.

[0057] By using the culture area separation method of the present invention, multiple biological tests can be performed on different cell populations at the same time, such as toxicity tests, cell proliferation and migration tests, gene expression analysis, etc. This method not only significantly improves the throughput of the experiment, but also allows the acquisition of multiple data under the same experimental conditions, providing more dimensional information for complex biological systems.

[0058] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for separating a three-dimensional cell common culture area, characterized in that: The method is to perform physical isolation setting of a three-dimensional cell culture area during the three-dimensional cell public culture process, comprising: After completing the three-dimensional cell culture configuration operation in the culture dish with the gel substrate, assembling the top cover with the partition with the culture dish, so that the gel substrate contacts the partition to form a physically isolated three-dimensional cell culture area; Alternatively, after injecting culture fluid from an open culture platform on an upper layer of a culture device, the liquid level of the culture fluid is adjusted to be located in an isolation chamber on a lower layer of the culture device to form a physically isolated three-dimensional cell culture area; wherein the culture device comprises an open culture platform on an upper layer and an isolation chamber on a lower layer.

2. The method for separating a three-dimensional cell common culture area according to claim 1, characterized in that: During the three-dimensional cell public culture process, the physical isolation is set in a reversible manner, so that the culture conditions of the three-dimensional cell public culture are uniformly set.

3. The method for separating a three-dimensional cell common culture area according to claim 1, characterized in that: After the physically isolated three-dimensional cell culture area is formed, each isolation chamber therein can be operated independently.

4. The method for separating a three-dimensional cell common culture area according to claim 1, characterized in that: Assemble the top cover with the partition with the culture dish, including: A physically isolated three-dimensional cell culture area is formed by assembling the top cover until the gel substrate contacts the partition; wherein the contact formation comprises interfacial contact between the gel substrate and the partition or the gel substrate is interrupted by the partition.

5. The method for separating a three-dimensional cell common culture area according to claim 1, characterized in that: There are a plurality of partitions which are regularly arranged.

6. The method for separating a three-dimensional cell common culture area according to claim 1, characterized in that: A physically isolated three-dimensional cell culture area is formed in the isolation chamber at the lower layer of the culture device by adjusting the liquid level of the culture solution; wherein the open culture platform is in a connected state, and the cover plate covering the top of the open culture platform is an open structure.

7. The method for separating a three-dimensional cell common culture area according to claim 1, characterized in that: A physically isolated three-dimensional cell culture area is formed in an isolation chamber located at the lower layer of the culture device by adjusting the liquid level of the culture solution; wherein the isolation chambers are arranged in an array.

8. The method for separating a three-dimensional cell common culture area according to claim 1, characterized in that: The method can be integrated into a cell culture system, and the cell culture system includes a control unit, a liquid flow circulation unit, a temperature control unit, a carbon dioxide control unit and a sterilization unit, and the liquid flow, temperature, carbon dioxide and sterilization operations in the cell culture system are controlled by the control unit; wherein the liquid flow circulation unit includes a liquid circulation pump for controlling the injection and outflow of the culture solution.

9. The method for separating a three-dimensional cell common culture area according to claim 1, characterized in that: The three-dimensional cells include primary cells, stem cells, tumor cells or organoids.

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