Anti-tumor drug screening device based on hanging drop method three-dimensional cell culture
By designing a three-dimensional cell culture device for hanging drop method containing permeable membranes and labeling grooves, the problem that two-dimensional cell culture cannot reflect tumor spatial heterogeneity and cumbersome operation of hanging drop method is solved, independent hanging drop culture, simplified dosing operations and accurate drug classification are achieved, and the accuracy of anti-tumor drug screening is improved.
Patent Information
- Application Number
- CN202421363595.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-15
- Publication Date
- 2025-05-30
- Estimated Expiration
- 2034-06-15
AI Technical Summary
The existing two-dimensional monolayer cell culture methods cannot effectively reflect the spatial heterogeneity of tumors, resulting in a large gap between the screening results of anti-tumor drugs and the in vivo conditions. At the same time, the three-dimensional cell culture of hanging drops method has problems such as hanging drop mixing, cumbersome dosing operations and difficulty in labeling, which affects the accuracy of the screening results.
An anti-tumor drug screening device based on three-dimensional cell culture of the hanging drop method is designed, and a support mechanism and a culture screening mechanism are adopted, including a first through tube and a second through tube, drug dosing is performed through a permeable membrane, and drug classification is performed using marking grooves and marking paper.
The independent culture of adjacent suspended droplets is achieved, the dosing operation is simplified, the loss of suspended droplets is avoided, and the drug classification is carried out through the labeling tank, which improves the accuracy of the screening results.
Smart Images
Figure CN222923154U_ABST
Abstract
Description
Technical Field
[0001] The utility model relates to the technical field of anti-tumor drug screening, and more specifically, to an anti-tumor drug screening device based on three-dimensional cell culture by the hanging drop method. Background Art
[0002] In the research of anti-tumor drugs, tumor cells cultured in vitro are often used for preliminary screening of the anti-tumor activity of the drug to be tested. In the past, the culture of tumor cells mostly adopted the traditional two-dimensional monolayer culture, that is, cells adhered to the bottom of a glass or plastic culture dish to form a flat monolayer of cells, which were exposed to oxygen, nutrients, and drugs without restriction, lacking cell-cell and cell-extracellular matrix interactions and unable to reflect the spatial heterogeneity of tumors. Therefore, the tumor cells cultured in two-dimensional monolayers are very different from the tumor cells growing in vivo, with significant differences in gene expression patterns, metabolic patterns, etc. Using tumor cells cultured in two-dimensional monolayers for anti-tumor drug screening, there is a large gap between the obtained results and the results of animal models and clinical trials.
[0003] Different from two-dimensional culture, three-dimensional culture forms a three-dimensional cell mass, which well maintains the interaction between cells and between cells and the extracellular matrix, and to a certain extent reproduces the microenvironment of tumors in vivo, and can better reflect the characteristics of tumor cells, thus becoming an ideal model for anti-tumor drug screening.
[0004] Three-dimensional cell culture by the hanging drop method is a culture method in which a small amount of tumor cell suspension is inverted and suspended on a specific plane, and the surface tension of the microdroplets is used to make the tumor cells aggregate and grow into three-dimensional cell spheres. Currently, the method for most laboratories to perform three-dimensional cell culture by the hanging drop method is to drop 30-50 microliters of cell suspension on the lid of a culture dish at a certain interval, and then turn the lid of the culture dish over and buckle it on the culture dish for culture. Such a culture method has three disadvantages:
[0005] 1. The distance between the hanging drops is relatively close and there is no physical barrier. When moving the culture dish, with a slight shake, the hanging drops will be mixed together, making it impossible to perform normal screening subsequently.
[0006] 2. When adding drugs, it is necessary to turn the lid of the culture dish over, add a certain volume of liquid medicine to the hanging drops, and then buckle the lid back onto the bottom of the dish. This not only makes the operation cumbersome, but also easily destroys the morphology of the hanging drops during the liquid addition process.
[0007] 3. Each hanging drop cannot be marked correspondingly, and it is very easy to confuse the groups during subsequent detection.
[0008] All of these seriously affect the accuracy of drug screening results. Content of the Utility Model
[0009] To make up for the above deficiencies, the present utility model provides an anti-tumor drug screening device based on three-dimensional cell culture by the hanging drop method.
[0010] The present utility model is implemented as follows:
[0011] An anti-tumor drug screening device based on three-dimensional cell culture by the hanging drop method, comprising a support mechanism and a culture and screening mechanism. The support mechanism includes a culture dish and a culture dish cover covering the top of the culture dish. The culture and screening mechanism includes a plurality of first through pipes and second through pipes. A plurality of slots are equidistantly formed in the top of the culture dish cover, and the first through pipes are installed at the bottoms of the slots, and the second through pipes are installed at the tops of the slots. A permeable membrane is adhered inside the slots. Marking slots are formed in the top of the culture dish cover below each slot.
[0012] The beneficial effect of adopting the above further solution is that when the culture dish cover is turned over so that the bottom of the first through pipe faces upward, the user can drop 30-50 microliters of tumor cell suspension into each first through pipe. Then, the culture dish cover is covered on the culture dish, and the tumor cells in the first through pipe grow in a hanging drop three-dimensionally. Generally, three-dimensional cell spheres can be formed in 3-5 days. Since each hanging drop is separated by the first through pipe, the adjacent hanging drops will not be mixed together. After the three-dimensional cell spheres are formed, the drug to be tested is added from the second through pipe and penetrates into the first through pipe through the permeable membrane. The final concentration of the drug in the first through pipe can be calculated according to the total amount of the liquid in the two through pipes. The marking slots are used to store marking papers, and the marking papers are marked in the order of I, II, and III, and each marking paper corresponds to the through pipe above it respectively. In this way, the classification of the drug to be tested is completed to achieve the purpose of facilitating screening.
[0013] Further, the capacity of the first through pipe is 30-50 microliters.
[0014] The beneficial effect of adopting the above further solution is that a first through pipe with a suitable capacity is selected according to the situation, and the capacity selection range of the first through pipe is between 30 and 50 microliters. During the process of moving and placing the culture dish, the adjacent hanging drops will not converge with each other due to a small displacement.
[0015] Further, the permeable membrane is a polycarbonate membrane densely covered with small holes with a pore diameter of 0.6 microns.
[0016] The beneficial effect of adopting the above further solution is that when adding the drug, the drug can be directly dropped into the second through pipe. At this time, the drug drop will penetrate through the permeable membrane and immerse into the hanging drop below, acting on the three-dimensional cell sphere. In this way, there is no need to open the culture dish cover to add the drug, avoiding cumbersome operations and losses of hanging drops.
[0017] Further, a first connecting block is installed on one side of the culture dish cover. A groove is formed on one side of the first connecting block, and a rotating shaft is installed in the groove. A Z-shaped block is sleeved on the outer side of the rotating shaft, and a baffle is connected to one side of the Z-shaped block.
[0018] Further, a metal sheet is installed on one side of the baffle, a second connecting block is installed on one side of the culture dish cover, and a magnet is embedded in the top of the second connecting block. The magnet and the metal sheet are adsorbed to each other.
[0019] The beneficial effect of adopting the above further scheme is that since the baffle has the ability to flip, when adding medicine, the baffle can be turned over, and when there is no need to add medicine, the baffle can be closed, so as to cover a number of second through pipes, avoiding the evaporation of the medicine. At the same time, through the use of the metal sheet and the magnet, the closed baffle can be fixed.
[0020] Further, the number of the second through pipes is the same as that of the first through pipes.
[0021] Further, both the culture dish and the culture dish cover are made of transparent plastic.
[0022] The beneficial effect of adopting the above further scheme is that the culture dish and the culture dish cover made of transparent plastic are conducive to the user observing the medicine inside.
[0023] The beneficial effect of the present utility model is as follows: An anti-tumor drug screening device based on the hanging drop method three-dimensional cell culture obtained by the above design of the present utility model. When the culture dish cover is turned over so that the bottom of the first through pipe faces upward, the user can drop the tumor cell suspension into each first through pipe for hanging drop culture. Since each cell hanging drop is restricted in the first through pipe, adjacent hanging drops will not be mixed together. By using the second through pipe, the drug to be tested can be directly dropped into the second through pipe from above and penetrate into the first through pipe through the membrane, so that there is no need to turn over the culture dish cover to add medicine. First, the operation is simplified. Second, it is conducive to sequential drug addition. Third, it avoids the loss of cell hanging drops caused by turning over the culture dish cover. And the marking groove is used to store the marking paper. There are marks in the order of I, II, and III on the marking paper, and each piece of marking paper corresponds to the first through pipe above it respectively, thus completing the classification of the drug to be tested and facilitating the subsequent screening. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present utility model, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present utility model, so they should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.
[0025] Figure 1 Schematic three - dimensional structure diagram of an anti - tumor drug screening device based on hanging - drop three - dimensional cell culture provided by the present utility model;
[0026] Figure 2 is Figure 1 Enlarged structure diagram of structure A in
[0027] Figure 3 Exploded structure diagram of an anti - tumor drug screening device based on hanging - drop three - dimensional cell culture provided by the present utility model;
[0028] Figure 4 Exploded structure diagram of the culture dish cover of an anti - tumor drug screening device based on hanging - drop three - dimensional cell culture provided by the present utility model.
[0029] In the figure: 100, support mechanism; 1001, culture dish; 1002, culture dish cover; 1003, baffle; 1004, metal sheet; 1005, first connection block; 1006, second connection block; 1007, Z - shaped block; 1008, magnet; 200, culture and screening mechanism; 2001, slotted opening; 2002, permeable membrane; 2003, marking groove; 2004, first through - tube; 2005, second through - tube. Specific implementation manners
[0030] To make the objectives, technical solutions and advantages of the embodiments of the present utility model clearer, the technical solutions in the embodiments of the present utility model will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present utility model. Obviously, the described embodiments are some, but not all, of the embodiments of the present utility model. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present utility model without making creative efforts fall within the scope of protection of the present utility model.
[0031] Therefore, the following detailed description of the embodiments of the present utility model provided in the drawings is not intended to limit the scope of the present utility model claimed, but merely represents selected embodiments of the present utility model. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present utility model without making creative efforts fall within the scope of protection of the present utility model.
[0032] Embodiment 1 of an anti - tumor drug screening device based on hanging - drop three - dimensional cell culture of the present utility model
[0033] The present utility model provides the following technical solutions: As Figures 1 to 4, including a support mechanism 100 and a culture and screening mechanism 200. The support mechanism 100 includes a culture dish 1001 and a culture dish cover 1002 covering the top of the culture dish 1001. The culture and screening mechanism 200 includes a number of first through-tubes 2004 and second through-tubes 2005. A number of slots 2001 are equidistantly opened at the top of the culture dish cover 1002, and the first through-tubes 2004 are installed at the bottom of the slots 2001, and the second through-tubes 2005 are installed at the top of the slots 2001. A permeable membrane 2002 is adhered inside the slots 2001. Marking slots 2003 are opened at the top of the culture dish cover 1002 below each slot 2001. Flip the culture dish cover so that the bottom of the first through-tube faces upward. The user drops 30 - 50 microliters of tumor cell suspension into each first through-tube, and then covers the culture dish with the culture dish cover. After 3 - 5 days, the tumor cells grow into three-dimensional cell spheres under hanging drop conditions. Add the drug to be tested into the second through-tube 2005, and place a marked label paper in the marking slot. After the drug acts for 3 - 5 days, use the adenosine triphosphate detection method (ATP method) to detect whether the added drug inhibits cell growth.
[0034] Embodiment 2 of an anti-tumor drug screening device based on three-dimensional cell culture by hanging drop method of the present utility model
[0035] Refer to Figures 1 to 4 , specifically, the permeable membrane 2002 is a polycarbonate membrane densely covered with small holes with a pore diameter of 0.6 microns. If the sequential action of two drugs needs to be detected, one day or two days after the first drug has been added, the second drug can be directly added into the second through-tube 2005. The drug will also penetrate through the permeable membrane 2002 and immerse into the first through-tube, acting on the three-dimensional tumor cell spheres. In this way, there is no need to open the culture dish cover 1002 to add the drug for the second time.
[0036] Embodiment 3 of an anti-tumor drug screening device based on three-dimensional cell culture by hanging drop method of the present utility model
[0037] Refer to Figures 1 to 4, Specifically, a second connecting block 1006 is installed on one side of the culture dish cover 1002. A groove is formed on one side of the second connecting block 1006, and a rotating shaft is installed in the groove. A Z-shaped block 1007 is sleeved outside the rotating shaft, and a baffle 1003 is connected to one side of the Z-shaped block 1007. A metal sheet 1004 is installed on one side of the baffle 1003. A first connecting block 1005 is installed on one side of the culture dish cover 1002, and a magnet 1008 is embedded in the top of the first connecting block 1005. The magnet 1008 and the metal sheet 1004 adsorb each other. Since the baffle 1003 has the ability to flip, when adding medicine, the baffle 1003 can be turned over. When medicine addition is not required, the baffle 1003 can be closed, thereby covering a plurality of slots 2002 to avoid evaporation of the medicine. At the same time, by using the metal sheet 1004 and the magnet 1008, the closed baffle 1003 can be fixed.
[0038] Specifically, the working principle of a kind of anti-tumor drug screening device based on the hanging drop method for three-dimensional cell culture: When in use, first add PPS in the culture dish to maintain the saturated humidity inside. Then turn over the culture dish cover 1002 so that the bottom of the first through pipe 2004 faces upward. The user can add the tumor cell suspension into each first through pipe 2004. After that, cover the culture dish cover 1002 on the culture dish 1001, and then close the baffle 1003 to cover a plurality of slots 2002 to avoid evaporation of the liquid. The tumor cells can grow into three-dimensional cell spheres after 3 - 5 days under the hanging drop culture condition. At this time, the baffle 1003 can be turned over, and the drug to be tested can be directly dropped into the second through pipe 2004. At this time, the drug drop will penetrate through the permeable membrane 2002 and immerse into the first through pipe to act on the tumor cell spheres. Finally, close the baffle 1003 again. Finally, when recovering the drug, the drug can be sucked out from the second through pipe 2005 through a suction device.
[0039] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, various changes and modifications can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. An anti-tumor drug screening device based on three-dimensional cell culture using the hanging drop method, characterized in that: The invention comprises a supporting mechanism (100) and a culture screening mechanism (200), wherein the supporting mechanism (100) comprises a culture dish (1001) and a culture dish cover (1002) covering the top of the culture dish (1001), and the culture screening mechanism (200) comprises a plurality of first through tubes (2004) and a second through tube (2005), wherein the top of the culture dish cover (1002) is provided with a plurality of slots (2001) at equal distances, and the first through tubes (2004) are installed at the bottom of the slots (2001), and the second through tubes (2005) are installed at the top of the slots (2001), and a transparent membrane (2002) is adhered inside the slots (2001), and a marking slot (2003) is provided at the top of the culture dish cover (1002) below each slot (2001).
2. The anti-tumor drug screening device based on hanging drop three-dimensional cell culture according to claim 1, characterized in that: The capacity of the first through tube (2004) is between 30 and 50 microliters.
3. The anti-tumor drug screening device based on hanging drop three-dimensional cell culture according to claim 1, characterized in that: The permeable membrane (2002) is a polycarbonate membrane densely covered with pores with a pore size of 0.6 micrometers.
4. The anti-tumor drug screening device based on hanging drop three-dimensional cell culture according to claim 1, characterized in that: A first connecting block (1006) is installed on one side of the culture dish cover (1002), a groove is provided on one side of the first connecting block (1006), a rotating shaft is installed in the groove, a Z-shaped block (1007) is sleeved on the outer side of the rotating shaft, and a baffle (1003) is connected to one side of the Z-shaped block (1007).
5. The anti-tumor drug screening device based on hanging drop three-dimensional cell culture according to claim 4, characterized in that: A metal sheet (1004) is installed on one side of the baffle (1003), a second connection block (1005) is installed on one side of the culture dish cover (1002), and a magnet (1008) is embedded on the top of the second connection block (1005), and the magnet (1008) and the metal sheet (1004) are attracted to each other.
6. The anti-tumor drug screening device based on hanging drop three-dimensional cell culture according to claim 1, characterized in that: The number of the slots (2001) and the number of the first through pipes (2004) are the same.
7. The anti-tumor drug screening device based on hanging drop three-dimensional cell culture according to claim 1, characterized in that: The culture dish (1001) and the culture dish cover (1002) are both made of transparent plastic.