Cell cloning passage device and passage method

Abstract

The invention provides a cell cloning passage device. The cell cloning passage device comprises a passage assembly and a fixing assembly, the passage assembly comprises a plurality of cutting tool bits, a mounting part and an operating handle, the plurality of cutting tool bits are suitable for cutting cell clones cultured in the culture appliance, the mounting part is suitable for mounting the plurality of cutting tool bits, and the operating handle is mounted at one end, far away from the plurality of cutting tool bits, of the mounting part and is suitable for holding and driving the plurality of cutting tool bits to cut the cell clones; and the fixing assembly is suitable for accommodating and fixing the culture appliance. On the other hand, the invention also provides a passage method using the cell cloning passage device.

Description

Technical Field

[0001] The present disclosure relates to the technical field of cell culture devices for cell biology experiments, and more specifically, to a cell cloning and cell culture device and a cell culture method. Background Art

[0002] Cell culture devices play a vital role in many cell biology experiments. They not only support the smooth progress of experiments, but also ensure the acquisition of high-quality cell samples. Especially when dealing with cells that grow in clones, daily subculture and maintenance culture place extremely strict technical requirements on the experimental operators, and require them to have rich experience in order to make accurate judgments during the subculture process. If the experimenter fails to meet high standards in the subculture operation, the cloned cells are very likely to differentiate or deteriorate, which will affect the subsequent experiments.

[0003] At present, conventional cell passaging methods are mainly divided into two types: chemical digestion and physical cutting. However, the chemical digestion method often causes great damage to cells and is difficult to operate; while the passaging tools used in the physical cutting method have problems such as non-reusability, high cost, complex operation and high cell loss rate.

[0004] In view of this, there is an urgent need to develop a device specifically for cell cloning and subculturing, which should have the characteristics of saving costs, simplifying operating procedures, and lowering technical barriers. It should also be able to solve the various shortcomings of existing methods while ensuring the acquisition of high-quality cells. Summary of the invention

[0005] In view of this, the present disclosure provides a cell cloning and passage device, which can simply and conveniently realize physical cutting of cell clones during the process of cell cloning and passage, and obtain high-quality passage cells.

[0006] One aspect of the present disclosure provides a cell cloning passage device, including a passage component and a fixing component, the passage component including: a plurality of cutting heads, suitable for cutting cell clones cultured in a culture vessel; a mounting portion, suitable for mounting the plurality of cutting heads; and an operating handle, mounted on one end of the mounting portion away from the plurality of cutting heads, suitable for holding and driving the plurality of cutting heads to cut the cell clones; the fixing component is suitable for accommodating and fixing the culture vessel.

[0007] According to an embodiment of the present disclosure, the above-mentioned culture vessel includes a culture dish; the above-mentioned mounting portion includes a first square mounting mold, and the plurality of the above-mentioned cutting heads are installed at one end of the above-mentioned first mounting mold at intervals in the width direction of the above-mentioned first mounting mold to cut the above-mentioned cell clones in the culture dish.

[0008] According to an embodiment of the present disclosure, the above-mentioned fixing assembly includes: a fixing box, which is configured to be opened at the top to form a accommodating cavity for accommodating the above-mentioned culture dish, and the two facing side walls of the above-mentioned accommodating cavity are symmetrically provided with spaced first limiting grooves; and a baffle, which is installed between the two symmetrical first limiting grooves to limit the movement of the above-mentioned culture dish.

[0009] According to an embodiment of the present disclosure, the above-mentioned culture vessel includes a culture plate; the above-mentioned mounting portion includes a second cylindrical mounting mold, and the plurality of the above-mentioned cutting heads are arranged in sequence in rows and / or columns at the bottom end of the above-mentioned second mounting mold to cut the above-mentioned cell clones in the culture holes on the above-mentioned culture plate.

[0010] According to an embodiment of the present disclosure, the above-mentioned fixing assembly includes: a fixing box, which is constructed with a top opening to form a accommodating cavity for accommodating the above-mentioned culture plate, and the two facing side walls of the above-mentioned accommodating cavity are symmetrically provided with spaced-apart second limiting grooves; and a clamping rod, which is suitable for cooperating with the two symmetrical second limiting grooves on the side walls of the accommodating cavity to limit the movement of the above-mentioned culture plate.

[0011] According to an embodiment of the present disclosure, the mounting portion further comprises: a mounting sleeve, a bottom end of which is provided with a mounting cavity for accommodating the second mounting mold; and a connecting rod mounted on a top end of the mounting sleeve.

[0012] According to an embodiment of the present disclosure, the above-mentioned cell cloning passage device further includes a connecting elbow, which is configured in an arc shape and is suitable for connecting the above-mentioned connecting rod and the above-mentioned operating handle.

[0013] According to an embodiment of the present disclosure, at least two first limiting ridges extending in the circumferential direction and arranged at intervals are arranged on the inner side wall of the above-mentioned installation cavity, and a limiting groove is formed between each of the above-mentioned first limiting ridges and the upper wall of the above-mentioned installation cavity; at least two second limiting ridges cooperating with the above-mentioned limiting grooves are arranged at intervals on the outer side surface of the above-mentioned second installation mold, and each of the above-mentioned second limiting ridges slides into the above-mentioned limiting groove between the two adjacent above-mentioned first limiting ridges, so that the above-mentioned second installation mold is maintained in the above-mentioned installation cavity.

[0014] According to an embodiment of the present disclosure, the distance between the top ends of two adjacent cutting blades is 50-500 μm.

[0015] Another aspect of the present disclosure provides a cell clone passaging method using the above-mentioned cell clone passaging device, comprising using a culture vessel to culture cells to obtain cell clones; installing the above-mentioned culture vessel with a growth density of the above-mentioned cell clones reaching 70-80% in a fixed component and using a passaging component to cut the above-mentioned cell clones to obtain sheet cell clones; extracting the above-mentioned sheet cell clones and centrifuging them to obtain passaging cells.

[0016] According to an embodiment of the present disclosure, a cell cloning passaging device is provided, which is composed of a passaging component and a fixing component. The passaging component includes a plurality of cutting heads for accurately cutting cell clones in a culture vessel; a mounting portion is used to fix these cutting heads; and an operating handle is located at the other end of the mounting portion, which is convenient for holding and driving the cutting heads to cut cell clones. The fixing component is used to accommodate and stabilize the culture vessel. In this way, the operational process of cell passaging is simplified, the technical threshold is lowered, and at the same time, the damage and loss of cell clones are reduced, ensuring the acquisition of high-quality passaging cells, and effectively solving the problems of high cost, complex operation and high cell loss rate existing in existing passaging methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and other objects, features and advantages of the present disclosure will become more apparent through the following description of the embodiments of the present disclosure with reference to the accompanying drawings, in which:

[0018] Figure 1 A perspective view schematically shows a cell cloning subculture component of a cell cloning subculture device according to an embodiment of the present disclosure;

[0019] Figure 2 Schematically shows Figure 1 A front view of the passaging assembly is shown;

[0020] Figure 3 Schematically shows Figure 1 A side view of the passaging assembly is shown;

[0021] Figure 4 Schematically shows Figure 1 Bottom view of the passaging assembly shown;

[0022] Figure 5 A perspective view schematically shows a fixing component of a cell cloning passaging device according to an embodiment of the present disclosure;

[0023] Figure 6 The schematic diagram schematically shows the use process of the cell cloning and subculture device according to the embodiment of the present disclosure;

[0024] Figure 7 A perspective view schematically shows a cell cloning subculture component of a cell cloning subculture device according to another embodiment of the present disclosure;

[0025] Figure 8 A schematic diagram of the use process of the cell cloning and subculture device according to another embodiment of the present disclosure is schematically shown;

[0026] Fig. 9 Schematically shows Figure 7 A perspective view of the mounting portion of the passaging assembly shown;

[0027] Fig.10 Schematically shows Figure 8 A cross-sectional view of the mounting portion shown;

[0028] Fig.11 Schematically shows an exploded view of a mounting portion according to another embodiment of the present disclosure;

[0029] Fig.12 A flow chart of a method for passage according to an embodiment of the present disclosure is schematically shown;

[0030] Fig.13 A schematic diagram schematically shows a cutting process of a passaging component according to an embodiment of the present disclosure;

[0031] Fig.14 The schematic diagram of extracting cell clones after cutting according to the embodiment of the present disclosure is schematically shown;

[0032] Fig.15 Schematically showing the state of cell clones before and after cutting under a microscope according to an embodiment of the present disclosure;

[0033] Fig.16 The diagram schematically shows the state of the passaged cells under a microscope according to an embodiment of the present disclosure.

[0034] In the drawings, the meanings of the reference numerals are as follows:

[0035] 1. Passage components;

[0036] 11. Cutting head;

[0037] 12. Installation department;

[0038] 121, first installation mold;

[0039] 122, second installation mold;

[0040] 1221, second limiting convex strip;

[0041] 123. Installation sleeve;

[0042] 1231, installation cavity;

[0043] 1232, first limiting convex strip;

[0044] 124. Connecting rod;

[0045] 13. Operating handle;

[0046] 131, connecting part;

[0047] 14. Connect elbow;

[0048] 2. Fix the components;

[0049] 21. Fixed box;

[0050] 211, first limiting groove;

[0051] 212, second limiting groove;

[0052] 22. Baffle;

[0053] 23. Card stick;

[0054] 3. Pasteur pipette; and

[0055] 4. Centrifuge tube. DETAILED DESCRIPTION

[0056] Hereinafter, embodiments of the present disclosure will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present disclosure. In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. However, it is apparent that one or more embodiments may also be implemented without these specific details. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present disclosure.

[0057] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. The terms "include", "comprising", etc. used herein indicate the existence of the features, steps, operations and / or mechanisms, but do not exclude the existence or addition of one or more other features, steps, operations or mechanisms.

[0058] All terms (including technical and scientific terms) used herein have the meanings commonly understood by those skilled in the art unless otherwise defined. It should be noted that the terms used herein should be interpreted as having a meaning consistent with the context of this specification and should not be interpreted in an idealized or overly rigid manner.

[0059] When using expressions such as "at least one of A, B, and C, etc.", they should generally be interpreted according to the meaning of the expression commonly understood by those skilled in the art (for example, "a system having at least one of A, B, and C" should include but is not limited to a system having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, C, etc.).

[0060] An embodiment of the present disclosure provides a cell cloning passaging device, comprising a passaging component 1 and a fixing component 2, wherein the passaging component 1 comprises a plurality of cutting heads 11 suitable for cutting cell clones cultured in a culture vessel, and a mounting portion 12 suitable for mounting the plurality of cutting heads 11, and an operating handle 13 mounted on an end of the mounting portion 12 away from the plurality of cutting heads 11, wherein the operating handle 13 is suitable for an operator to hold and operate to drive the plurality of cutting heads 11 to cut the cell clones; the fixing component 2 is suitable for accommodating and fixing the culture vessel.

[0061] According to the above-mentioned setting method, the cell cloning passage device includes a passage component 1 and a fixing component 2. The fixing component 2 is used to fix the culture vessel and the multiple cutting heads 2 of the passage component 1 are used to cut the cell clones cultured in the culture vessel, which simplifies the process of obtaining passage cells by physically cutting cell clones in the cell passage operation process, reduces the operation difficulty and technical threshold, and reduces the damage and loss of cell clones, ensuring the acquisition of high-quality passage cells, effectively solving the problems of high cost, complex operation and high cell loss rate in the existing passage methods.

[0062] In an illustrative embodiment, the cutting blade head is made of a hard material, and the hard material includes plastic, plastic, resin, metal alloy or ceramic material.

[0063] In detail, such material selection ensures that the cutting head 11 has sufficient hardness and durability to effectively cut the cell clones in the culture dish. At the same time, the preparation material can be selected according to the specific application requirements of the cutting head. For example, if the cutting head needs to be sterilized frequently, metal alloys or ceramic materials may be better choices in order to withstand the effects of high temperatures and chemical disinfectants. The price of plastic or plastics focuses more on cost factors.

[0064] Figure 1 A perspective view schematically shows a cell cloning subculture component of a cell cloning subculture device according to an embodiment of the present disclosure; Figure 2 Schematically shows Figure 1 A front view of the passaging assembly is shown; Figure 3 Schematically shows Figure 1 A side view of the passaging assembly is shown; Figure 4 Schematically shows Figure 1 Bottom view of the passaging assembly is shown.

[0065] In an illustrative embodiment, Figures 1 to 4As shown, the culture apparatus includes a culture dish; the transfer component 1 includes a brush-like transfer tool, the mounting portion 12 of the brush-like transfer tool includes a first mounting mold 121 configured as a square, and a plurality of cutting heads 11 are installed at one end of the first mounting mold 121 at intervals in the width direction of the square first mounting mold 121 for cutting the cultured cell clones in the culture dish.

[0066] According to the above-mentioned configuration, such a layout enables the cutting head 11 to effectively and precisely cut the cell clones growing in the culture dish, thereby realizing the cell clone subculture operation, making the cell subculture process simpler and more efficient, and helping to reduce damage to the cells, so that the obtained subculture cells remain in a high-quality state.

[0067] In the above exemplary embodiments, Figures 1 to 4 As shown, the cutting head 11 is constructed as a cone with a height of 2-10 mm, a radius r of 0.1-0.3 mm at the bottom, preferably 0.2 mm, and a spacing of 50-500 μm between the tip blades of two adjacent cutting heads 11, preferably 250 μm.

[0068] In detail, the first mounting mold 121 has a width of 3-4 cm, a length of 3-4 cm, and a height of 1.5-2 cm.

[0069] It should be noted that the size of a common culture dish used for cell culture is 6 cm-15 cm, and the width of the first mounting mold 121 enables the multiple cutting heads 11 mounted thereon to adapt to culture dishes of various sizes, thereby increasing the adaptability of the transfer component.

[0070] In an illustrative embodiment, the first mounting mold 121 is made of a high molecular polymer, and the high molecular polymer includes silicone, soft polyvinyl chloride or thermoplastic elastomer.

[0071] In detail, the high molecular polymer can provide properties such as heat resistance, flexibility, chemical resistance, biocompatibility, cost-effectiveness, plasticity and durability to meet different application requirements and facilitate consideration of the processing performance and cost factors of the first mounting mold 121.

[0072] In an illustrative embodiment, the operating handle 13 of the brush-shaped passaging tool has a length of 5-20 cm and a width of 1-2 cm; preferably, the length of the operating handle 13 is 10 cm.

[0073] In an illustrative embodiment, the operating handle 13 is made of a hard material, such as polypropylene, polyethylene, polycarbonate, polyethylene terephthalate, polymethyl methacrylate, polylactic acid, epoxy resin, etc., to ensure the firmness and durability of the operating handle 13 .

[0074] In an illustrative embodiment, Figures 1 to 4 As shown, the operating handle 13 includes a connecting portion 131, which is disposed at one end of the operating handle 13 facing the first mounting mold 121 and is configured as an expansion structure gradually extending toward the first mounting mold 121, and the side surface of the connecting portion 131 forms a smooth arc surface.

[0075] According to the above arrangement, the connection portion 131 gradually extends in cross section from the operating handle 13 toward the first installation mold to increase the contact area with the first installation mold 121, thereby increasing the stability of the connection.

[0076] Figure 5 The figure schematically shows a perspective view of a fixing component of a cell cloning passaging device according to an embodiment of the present disclosure.

[0077] In an illustrative embodiment, Figure 5 As shown, the fixing assembly 2 includes a fixing box 21 and a baffle 22. The fixing box 21 is configured to have an opening at the top to form a receiving cavity for receiving the culture dish, and the two facing side walls of the receiving cavity are symmetrically provided with spaced first limiting grooves 211; the baffle 22 is installed between the two symmetrical first limiting grooves 211 to limit the movement of the culture dish.

[0078] According to the above-mentioned setting method, a receiving cavity with a top opening is effectively formed on the fixing box 21 to stabilize the culture dish. The first limiting grooves 211 on the two side walls cooperate with the baffle 22 to achieve precise restriction of the movement of the culture dish, thereby improving the stability of the culture dish placement and the safety of experimental operations.

[0079] In an illustrative embodiment, Figure 5 As shown, each first limiting groove 211 extends on the side wall of the accommodating cavity in the height direction or vertical direction of the fixing box 21, and extends to the top of the side wall to form a notch, so that the baffle 22 can be inserted through two symmetrical notches on the two side walls to limit the movement of the culture dish placed in the accommodating cavity.

[0080] In an illustrative embodiment, the length of the baffle 22 is greater than the width of the accommodating cavity of the fixing box 22 and is less than or equal to the distance between the two symmetrical first limiting grooves 211 , so that the baffle 22 can be stuck between the two first limiting grooves 211 .

[0081] Figure 6 The figure schematically shows the use process of the cell cloning passaging device according to the embodiment of the present disclosure.

[0082] In an illustrative embodiment, Figure 6As shown, first take out the fixing box 21 of the fixing assembly 2, carefully place the culture dish into the accommodating cavity, and push it until the edge abuts against the side wall of the accommodating cavity, then insert the baffle 22 into the two symmetrical first limiting grooves 211 that are just located at the tangent horizontal line of the culture dish or closest to the outer edge of the culture dish, and form a match with the side wall of the accommodating cavity to complete the fixation of the culture dish.

[0083] Figure 7 The figure schematically shows a perspective view of a cell cloning passaging component of a cell cloning passaging device according to another embodiment of the present disclosure.

[0084] In an exemplary alternative embodiment, Figure 7 As shown, the culture apparatus includes a culture plate; the transfer component 1 includes a nail-head cake-shaped transfer tool, the mounting portion 12 of the nail-head cake-shaped transfer tool includes a cylindrical second mounting mold 122, and a plurality of cutting heads 11 are arranged in sequence in rows and / or columns at the bottom end of the second mounting mold 122 to cut cell clones in culture holes on the culture plate.

[0085] In detail, the culture plate includes 4-well plate, 6-well plate, 8-well plate and other suitable non-conventional well plates, etc. The smallest well plate can be used for 24-well plate. The size of each well of the culture plate is 35 mm x 10 mm, which is suitable for culturing multiple samples at the same time.

[0086] According to the above-mentioned setting mode, such a layout enables the cutting head 11 to effectively and accurately cut the cell clones growing in the wells on the culture plate, thereby realizing the cell clone subculture operation, making the cell subculture process simpler and more efficient, and helping to reduce damage to the cells, so that the obtained subculture cells remain in a high-quality state.

[0087] In the above-mentioned exemplary embodiment, the cutting head 11 is embedded in the bottom end of the second mounting mold and is configured as a cone with a height of 1-5 mm, preferably 2 mm. The spacing between the tip blades of two adjacent cutting heads 11 in the row or column direction is 50-500 μm, preferably 200 μm.

[0088] In detail, the diameter 26 of the second mounting mold 122 is 10-50 mm, preferably 26 mm, so that it can be applied to culture plates of different specifications and sizes to improve applicability.

[0089] In an illustrative embodiment, the second mounting mold 122 is made of a high molecular polymer, and the high molecular polymer includes silicone, soft polyvinyl chloride or thermoplastic elastomer.

[0090] In an exemplary alternative embodiment, Figure 5As shown, the fixing assembly 2 includes a fixing box 21 and a clamping rod 23. The fixing box 21 is constructed with an opening at the top to form a receiving cavity for receiving the culture plate. Second limiting grooves 212 are symmetrically arranged with intervals on two facing side walls of the receiving cavity. The clamping rod 23 is suitable for cooperating with two symmetrical second limiting grooves 212 on the side walls of the receiving cavity to limit the movement of the culture plate.

[0091] According to the above-mentioned setting method, a accommodating cavity with a top opening is effectively formed on the fixing box 21 to stabilize the culture plate, and the cooperation between the second limiting grooves 212 on the two side walls and the clamping rod 23 realizes the precise restriction of the movement of the culture plate, thereby improving the stability of the culture dish placement and the safety of the experimental operation.

[0092] In an illustrative embodiment, Figure 5 As shown, each second limiting groove 212 extends from the top of the side wall of the fixed box 21 and passes through the outer wall and the inner wall to form a groove for accommodating the card rod 23, so that the card rod 23 can be placed in two symmetrical grooves on the two side walls, thereby limiting the movement of the culture plate placed in the accommodating cavity.

[0093] In an illustrative embodiment, the length of the clamping rod 23 is greater than the distance between the two symmetrical second limiting grooves 212 , so that the clamping rod 23 can be placed on the two second limiting grooves 212 .

[0094] Figure 8 The figure schematically shows the use process of the cell cloning passaging device according to another embodiment of the present disclosure.

[0095] In an illustrative embodiment, Figure 8 As shown, first take out the fixing box 21 of the fixing assembly 2, carefully place the culture plate into the accommodating cavity, and push it until the edge abuts against the side wall of the accommodating cavity, then place the clamping rod 23 into two symmetrical second limiting grooves 212 that are just flush with the side edge of the culture dish or closest to the outer edge of the culture plate to cooperate with the side wall of the accommodating cavity to complete the fixation of the culture plate.

[0096] Fig. 9 Schematically shows Figure 7 A perspective view of the mounting portion of the passaging assembly shown; Fig.10 Schematically shows Figure 8 A cross-sectional view of the mounting portion is shown.

[0097] In an illustrative embodiment, Figures 9 and 10 As shown, the mounting portion 12 further includes a mounting sleeve 123 and a connecting rod 124 . The bottom end of the mounting sleeve 123 is provided with a mounting cavity 1231 for accommodating the second mounting mold 122 ; the connecting rod 124 is mounted on the top end of the mounting sleeve 123 .

[0098] According to the above arrangement, the bottom end of the mounting sleeve 123 is provided with a mounting cavity 1231 specifically used to accommodate the second mounting mold 122, which provides a stable fixed position for the second mounting mold 122. The connecting rod 124 is installed on the top of the mounting sleeve 123. Such a configuration helps to connect the mounting part 12 with other components, thereby achieving stable installation and precise adjustment of the entire device. The assembly accuracy between the various structures of the generation component is improved, and the structural stability and operational flexibility of the entire component are enhanced.

[0099] In an illustrative embodiment, the total height of the mounting sleeve 123 and the connecting rod 124 is 10-40 mm, preferably 20 mm.

[0100] In an illustrative embodiment, Figure 7 As shown, the cell cloning passage device further comprises a connecting elbow 14 which is configured in an arc shape and is suitable for connecting the connecting rod 124 and the operating handle 13 .

[0101] In an exemplary embodiment, the turning angle or central angle of the connecting elbow 14 is 60-80 degrees, preferably 75 degrees. Such an angle selection can provide a moderate bending radius, facilitate the operator to perform manual operation, and improve the accuracy of operation.

[0102] In an illustrative embodiment, Figure 7 As shown, the connecting elbow 14 is threadedly connected to the connecting rod 124 and the operating handle 13 respectively.

[0103] In an illustrative embodiment, the length of the operating handle 13 is 100-400 mm, and preferably 150 mm.

[0104] In an illustrative embodiment, the operating handle 13 and the connecting elbow 14 are made of a polymer material, and the polymer material includes polypropylene, polyethylene, polycarbonate, polyethylene terephthalate, polymethyl methacrylate, polylactic acid, epoxy resin, thermoplastic polyurethane and the like.

[0105] In an illustrative embodiment, Fig.10 As shown, at least two first limiting ridges 1232 extending in the circumferential direction and spaced apart are provided on the inner side wall of the installation cavity 1231, and a limiting groove is formed between each first limiting ridge 1232 and the upper wall of the installation cavity 1231; at least two second limiting ridges 1221 cooperating with the limiting groove are spaced apart on the outer surface of the second installation mold 122, and each second limiting ridge slides into the limiting groove between two adjacent first limiting ridges, so that the second installation mold 122 is kept in the installation cavity 1231.

[0106] Fig.11An exploded view of a mounting portion according to another embodiment of the present disclosure is schematically shown.

[0107] In an exemplary alternative embodiment, Fig.11 As shown, two layers of limiting units distributed along the axial direction are arranged on the inner wall of the installation cavity 1231, and each layer of limiting units includes at least two first limiting ridges 1232 extending circumferentially and arranged at intervals on the inner wall of the installation cavity 1231, and two first limiting ridges 1232 adjacent to each other in the axial direction form a limiting groove; at least two second limiting ridges 1221 cooperating with the limiting grooves are arranged at intervals on the outer surface of the second installation mold 122, and each second limiting ridge slides into the limiting groove between two adjacent first limiting ridges, so that the second installation mold 122 is maintained in the installation cavity 1231.

[0108] According to the above-mentioned setting method, the positioning accuracy and stability of the second mounting mold 122 in the mounting cavity 1231 are improved, the accidental movement of the second mounting mold 122 during operation is prevented, the safety and reliability of the entire transfer tool are improved, and the rapid installation and disassembly of the mold is facilitated.

[0109] In an illustrative embodiment, Figure 5 As shown, the fixed box 21 is a transparent cuboid with a length of 20 cm, a width of 8.5 cm, and a height of 3 cm. The bottom and surrounding edges of the fixed box 21 are provided with marking scales with a minimum scale of 0.1 cm and a maximum scale of 0.5 cm.

[0110] In an illustrative embodiment, Figure 5 As shown, a plurality of first limiting slots 211 and a plurality of second limiting slots 212 are alternately arranged on the side wall of the accommodating cavity of the fixing box 21 .

[0111] In detail, the first limiting slot 211 has a depth of 1.5 cm and a width of 0.1 cm; the second limiting slot 212 has a depth and a width of 0.1 cm.

[0112] Furthermore, the baffle 22 has a width of 2 cm, a height of 0.1 cm, and a length that matches the spacing between the two symmetrical first limit slots 211. The clamping rod 23 has a length of 12 cm and a diameter of 0.1 cm.

[0113] In an illustrative embodiment, a plurality of first limit slots 211 and a plurality of second limit slots 212 are regularly and alternately distributed on two side arms extending in the length direction of the fixed box 21, wherein a distance between two adjacent first limit slots 211 is 4 cm, and a distance between two adjacent second limit slots 212 is 1 cm.

[0114] In an illustrative embodiment, the fixing box 21, the baffle 22 and the clamping rod 23 are all made of a polymer material, and the polymer material includes polypropylene, polyethylene, polycarbonate, polyethylene terephthalate, polymethyl methacrylate, polylactic acid, epoxy resin and other materials.

[0115] According to the above arrangement, the firmness, durability and chemical stability of the fixed component are ensured, and it is also convenient to carry out large-scale production through processes such as injection molding, thereby reducing costs. In addition, these materials generally have good biocompatibility and are suitable for use in biological experimental environments.

[0116] Fig.12 The figure schematically shows a flow chart of a method for cell culture according to an embodiment of the present disclosure. Fig.13 The figure schematically shows a cutting process of a passaging assembly according to an embodiment of the present disclosure. Fig.14 The schematic diagram of extracting cell clones after cutting according to an embodiment of the present disclosure is schematically shown.

[0117] The embodiments of the present disclosure also provide a cell cloning and cell subculture method using the above-mentioned cell cloning and cell subculture device, such as Fig.12 As shown, including operations S100~S300:

[0118] In operation S100 , cells are cultured using a culture vessel to obtain cell clones.

[0119] In an embodiment of the present disclosure, the culture vessel includes a culture dish and a culture plate.

[0120] In detail, pluripotent stem cells are cultured using a culture dish or a culture plate to obtain pluripotent stem cell clones.

[0121] In operation S200, a culture vessel with a cell clone growth density of 70-80% is installed in a fixing component and the cell clone is cut using a subculture component to obtain a sheet-like cell clone.

[0122] Specifically, the culture vessel with a growth density of 70-80% is taken out of the incubator, the culture medium in the culture vessel is removed, and cell culture buffer that can cover the bottom of the culture vessel is added according to the size of the culture vessel. The cell culture buffer is gently shaken and removed to wash the cell clones attached to the bottom of the culture vessel, remove metabolic waste and impurities, and provide a clean and healthy cell environment for subsequent cell passaging or experimental operations.

[0123] Then, the fixing assembly was taken out from the biosafety cabinet, a cell culture buffer of 1 / 4 of the holding volume was added to the culture vessel, and the culture vessel was mounted on the fixing assembly.

[0124] Finally, take out the passaging kit in the biosafety cabinet and use the passaging kit to cut the cell clones. Fig.13 As shown, first fix a direction and scratch the cells along the teeth for many times without repetition. The force should be such that scratches can be seen under the microscope but the clones do not fall off in strips. You can try many times until you find the right force. After completing the scratch in one direction, rotate the fixed box 90 degrees and complete the scratch again as described above. If more cell clones are needed, you can rotate along the inner wall of the culture vessel to create scratches. After the cutting is completed, the clones that have been cut can be seen under the microscope. The size of the clones is about 50-500um, and they are in the form of flakes that are easy to fall off.

[0125] In an embodiment of the present disclosure, the cell culture buffer includes Gibco DPBS (Cat. No. 14190144), which is a calcium-free and magnesium-free Dulbecco's phosphate buffered saline, mainly used for various applications of cell culture.

[0126] It should be noted that Dulbecco's Phosphate-Buffered Saline (DPBS) is a commonly used cell culture buffer, which is mainly used for cell washing, cell separation, tissue culture and as a diluent for cell culture fluid.

[0127] In an illustrative embodiment, a brush-shaped passaging tool is used to cut the cell clones in the culture dish, and a nail-head pie-shaped passaging tool is used to cut the cell clones in the culture plate.

[0128] In operation S300, sheet-like cell clones are extracted and centrifuged to obtain passage cells.

[0129] Detailed, such as Fig.14 As shown, a Pasteur pipette 3 is used to obtain a cell suspension, including using the Pasteur pipette 3 to absorb 1-2 mL of cell culture buffer in the culture vessel. To ensure that the cells are evenly suspended, the Pasteur pipette 3 is used to gently blow on the bottom of the dish 2-5 times with moderate force, and the cells on the bottom of the dish are observed with the naked eye to ensure that there are no obvious cell clone clumps and the cells are evenly dispersed in the buffer.

[0130] Then, the obtained cell suspension is transferred to a pre-cooled centrifuge tube 4 to avoid bubble generation and cell damage. The centrifuge tube 4 is placed in a centrifuge, the centrifugal force is set to 200g, and centrifuged for 1 minute at a low temperature of 4 degrees Celsius to precipitate cells and remove impurities in the supernatant, providing a pure cell sample for subsequent cell separation, washing or passage operations.

[0131] Finally, use a Pasteur pipette 3 or a pipette to gently aspirate the supernatant on the upper layer to avoid disturbing the cells precipitated at the bottom of the tube. According to the cell type and the required subculture ratio, add an appropriate amount of fresh culture medium to the centrifuge tube 4, gently blow or shake the centrifuge tube 4 to evenly suspend the cells in the culture medium to complete the subculture.

[0132] In an illustrative embodiment, the passaging ratio is 1:4 to 1:6.

[0133] In an embodiment of the present disclosure, after the passaging is completed, the passaging device is rinsed in sterile distilled water for 2-3 times, then sterilized by ultraviolet light in a biosafety cabinet, and stored for multiple uses. The purpose of rinsing with sterile distilled water is to allow the remaining cells to absorb water and burst, and to remove cell debris for the next use.

[0134] Fig.15 Schematically showing the state of cell clones before and after cutting under a microscope according to an embodiment of the present disclosure; Fig.16 The diagram schematically shows the state of the passaged cells under a microscope according to an embodiment of the present disclosure.

[0135] According to the embodiments of the present disclosure, Fig.15 As shown, Fig.15 On the left is a photo taken under a microscope before the cell clones were cut using a passaging tool. The picture shows that the cells are currently in good condition and suitable for passaging. Fig.15 The right side shows the cell clones that have been cut. The cell clones are about 50-500um in size and are in the form of flakes that are easy to fall off. Then take a Pasteur pipette, such as Fig.14 As shown, aspirate 1-2 mL of DPBS in the dish and pipette the bottom of the dish 2-5 times until no obvious clones are visible to the naked eye. Then transfer the cell suspension to a centrifuge tube and centrifuge at 200 g, 4 degrees, for 1 minute.

[0136] According to the embodiments of the present disclosure, Fig.16 As shown, Fig.16 The left side shows the adhesion state of pluripotent stem cells on the first day after passage. As can be seen from the figure, the cells are in good condition, with no signs of differentiation and no high mortality rate observed, which indicates that the cells can quickly adapt to the new growth environment after passage. Fig.16 The right side shows the cell clones after six days of culture. The cell clones are rounded and in very good overall condition. This shows that the method of the present invention can obtain high-quality cells, which not only have a high cell survival rate but also maintain good growth characteristics.

[0137] It will be appreciated by those skilled in the art that the features described in the various embodiments of the present disclosure may be combined and / or combined in a variety of ways, even if such combinations or combinations are not explicitly described in the present disclosure. In particular, without departing from the spirit and teachings of the present disclosure, the features described in the various embodiments of the present disclosure may be combined and / or combined in a variety of ways. All of these combinations and / or combinations fall within the scope of the present disclosure.

[0138] The embodiments of the present disclosure are described above. However, these embodiments are only for illustrative purposes and are not intended to limit the scope of the present disclosure. Although the embodiments are described above, this does not mean that the measures in the various embodiments cannot be used in combination to advantage. Without departing from the scope of the present disclosure, those skilled in the art may make a variety of substitutions and modifications, which should all fall within the scope of the present disclosure.

Claims

1. A cell cloning and subculturing device, characterized in that: include: A passaging component (1) comprising: A plurality of cutting blades (11) adapted to cut cell clones cultured in a culture vessel; A mounting portion (12) adapted to mount a plurality of the cutting blade heads (11); and an operating handle (13), mounted on an end of the mounting portion (12) away from the plurality of cutting blades (11), and suitable for holding and driving the plurality of cutting blades (11) to cut the cell clones; and The fixing component (2) is suitable for accommodating and fixing the culture device.

2. The cell cloning and subculturing device according to claim 1, characterized in that: The culture vessel comprises a culture dish; The mounting portion (12) comprises a first mounting mold (121) in a square shape, and a plurality of cutting blades (11) are installed at intervals on one end of the first mounting mold (121) in a width direction of the first mounting mold (121) to cut the cell clones in the culture dish.

3. The cell cloning and subculturing device according to claim 2, characterized in that: The fixing component (2) comprises: A fixing box (21) is configured to have an open top to form a receiving cavity for receiving the culture dish, wherein two facing side walls of the receiving cavity are symmetrically provided with first limiting grooves (211) distributed at intervals; and The baffle (22) is installed between the two symmetrical first limiting grooves (211) to limit the movement of the culture dish.

4. The cell cloning and subculturing device according to claim 1, characterized in that: The culture vessel comprises a culture plate; The mounting portion (12) comprises a second mounting mold (122) in a cylindrical shape, and a plurality of the cutting blade heads (11) are sequentially arranged in rows and / or columns at the bottom end of the second mounting mold (122) to cut the cell clones in the culture wells on the culture plate.

5. The cell cloning and subculturing device according to claim 4, characterized in that: The fixing component (2) comprises: The fixing box (21) is configured to have an opening at the top to form a receiving cavity for receiving the culture plate, and second limiting grooves (212) are symmetrically arranged at intervals on two facing side walls of the receiving cavity; and The clamping rod (23) is adapted to cooperate with two symmetrical second limiting grooves (212) on the side wall of the accommodating chamber to limit the movement of the culture plate.

6. The cell cloning and subculturing device according to claim 4, characterized in that: The mounting portion (12) further comprises: An installation sleeve (123), the bottom end of which is provided with an installation cavity (1231) for accommodating the second installation mold (122); and A connecting rod (124) is mounted on the top end of the mounting sleeve (123).

7. The cell cloning and subculturing device according to claim 6, characterized in that: It also includes a connecting elbow (14) which is configured in an arc shape and is suitable for connecting the connecting rod (124) and the operating handle (13).

8. The cell cloning and subculturing device according to claim 6, characterized in that: At least two first limiting convex strips (1232) extending in the circumferential direction and arranged at intervals are arranged on the inner side wall of the installation cavity (1231), and a limiting groove is formed between each of the first limiting convex strips (1232) and the upper wall of the installation cavity (1231); At least two second limiting convex strips (1221) cooperating with the limiting groove are arranged at intervals on the outer surface of the second installation mold (122), and each of the second limiting convex strips slides into the limiting groove between two adjacent first limiting convex strips, so that the second installation mold (122) is maintained in the installation cavity (1231).

9. The cell cloning and subculturing device according to claim 1, characterized in that: The distance between the top ends of two adjacent cutting blade heads (11) is 50-500 μm.

10. A cell cloning and cell subculturing method using the cell cloning and cell subculturing device according to any one of claims 1 to 9, characterized in that: include: Using culture vessels to culture cells to obtain cell clones; The culture vessel with a cell clone growth density of 70-80% is installed in the fixed component and the cell clone is cut using the subculture component to obtain a sheet cell clone; The sheet-like cell clones are extracted and centrifuged to obtain passage cells.

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