Spliced cell culture pore plate and chamber unit
By using modular cell culture plates and chamber units, the problem of inconsistent culture conditions in traditional organoids has been solved, realizing a highly efficient, economical, and cross-contamination-free platform for compound toxicity assessment, suitable for centralized testing of organoids and embryos.
Patent Information
- Application Number
- CN202422587921.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-25
- Publication Date
- 2025-11-21
- Estimated Expiration
- 2034-10-25
AI Technical Summary
Traditional animal experiments for assessing compound toxicity are costly, time-consuming, and have poor reliability in extrapolating results. Inconsistent organoid culture conditions lead to complex experimental procedures that are difficult to integrate for efficient assessment.
A modular cell culture plate and chamber unit is designed, which sets multiple chamber placement positions on the plate body to culture different organoids and embryooids respectively, and performs compound toxicity testing and evaluation on the same platform.
It has created an efficient, economical, and cross-contamination-free platform for compound toxicity assessment, reducing the complexity of experimental operations and resource waste, and is compatible with existing detection equipment.
Smart Images
Figure CN223576507U_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The utility model belongs to organoid culture technical field, and specifically for a splicing type cell culture well plate and chamber unit. BACKGROUND
[0002] At present, human has synthesized hundreds of millions of compounds, including various drugs, pesticides and industrial chemicals, and is increasing in the middle every day. The harm of these synthetic compounds to human health needs to be evaluated. The traditional toxicology evaluation method is to carry out animal experiments, but this method has high cost, long evaluation period, and species difference between animals and human body. Therefore, the reliability of the animal experiment results extrapolated to the human body is poor.
[0003] Organoid technology solves the shortcomings of animal experiments in the field of compound toxicology evaluation. The organoids cultured from human tissues or human-induced pluripotent stem cells (iPSC) have no species difference with the human body, and after the organoids are cultured to maturity, they have all kinds of cells of the human body and obvious organ characteristics. Therefore, using organoids to evaluate the toxicity of compounds can highly reduce the actual impact of compounds on the human body. Since up to 10 or more organoids and embryoids are used for compound toxicity evaluation in vitro, and testing and evaluation need to be performed at the same time. However, their culture conditions and culture time are inconsistent, leading to complex experimental operation and difficulty in integrating them into a platform for efficient evaluation. SUMMARY
[0004] Therefore, the utility model aims at providing a splicing type cell culture well plate and chamber unit, which can culture different organoids and embryoids respectively by using the chamber unit, and test and evaluate different organoids and embryoids by using the cell culture well plate.
[0005] To achieve the above-mentioned purpose, the utility model provides the following technical scheme:
[0006] The utility model discloses a chamber unit first, including chamber body, be equipped with n holes in the chamber body, and n is greater than or equal to 1 positive integer.
[0007] Further, the bottom structure of the hole adopts multi-hole bottom, three-fold bottom, flat bottom or U-shaped bottom.
[0008] Further, the multi-hole bottom adopts arrayed micropores, the pore size of the micropore is 20-2000um, and the shape of the micropore is regular hexagon, circle or square.
[0009] Further, the chamber body is integrally formed; or, in addition to the chamber body with a U-shaped bottom, the chamber body with a multi-hole bottom, a three-fold bottom and a flat bottom is separately provided as a chamber main body and a bottom plate located below the chamber main body, a through hole is arranged in the chamber main body, a bottom structure corresponding to the through hole is arranged on the bottom plate, and the through hole and the bottom structure are combined to form the hole.
[0010] Further, the thickness of the bottom plate is 0.1-3mm; the bottom plate and the chamber main body are connected by bonding, hot pressing adhesion or double-sided adhesive bonding.
[0011] Further, the bottom plate has light transmission and biocompatibility and is made of one of glass, polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cyclic olefin copolymer, polymethyl methacrylate, hydrogel and transparent resin.
[0012] The utility model also proposes a spliced cell culture hole plate, including hole plate main part and hole plate cover, a plurality of chamber unit placement positions are equipped on the hole plate main part, and the chamber unit placement position is used for placing the chamber unit.
[0013] Further, the chamber unit placement positions are arranged in an array on the hole plate main body.
[0014] Further, the number N of holes of the hole plate main body is:
[0015] N=n*m
[0016] Wherein: n is the number of holes arranged in the chamber unit; m is the number of chamber unit placement positions; * indicates the product.
[0017] Further, the chamber unit placement position is provided with a positioning structure for positioning the placement direction of the chamber unit.
[0018] Further, the chamber unit placement position is a chamber placement groove arranged in the hole plate main body, one end of the opposite two ends of the chamber placement groove is provided as a first circular arc surface, and the other end is provided as a first plane; one end of the two ends of the chamber unit is provided as a second circular arc surface matched with the first circular arc surface, and the other end is provided with a second plane matched with the first plane.
[0019] Further, the hole plate main body has biocompatibility and is made of one of polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cyclic olefin copolymer, polymethyl methacrylate, hydrogel and transparent resin.
[0020] The hole plate cover has light transmission and biocompatibility and is made of one of glass, polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cyclic olefin copolymer, polymethyl methacrylate and transparent resin.
[0021] The utility model discloses the beneficial effect lies in:
[0022] The utility model discloses a spliced cell culture well plate, a plurality of chamber placement positions are arranged on the well plate main body to place chamber units, so that different organoids and embryoid bodies can be cultured in different chamber units respectively, and then the chamber units are transferred to the chamber placement positions of the well plate main body, so that all the chamber units in the same well plate main body have the same organoid, and at this time, the toxicity of the same compound on the organoid under different concentration conditions can be tested and evaluated, or the chamber units containing at least two organoids in the same well plate main body can be used to test and evaluate the toxicity of the same compound on different organoids under the same concentration condition, and the centralized testing and evaluation of the organoids are realized, thereby improving the compound toxicity evaluation efficiency.
[0023] The utility model discloses a spliced cell culture well plate still has following beneficial effect:
[0024] (1) after various organoids and embryoid bodies (including organoid models, cystic embryo models, amniotic cavity embryo bodies, pancreatic islet organoids, kidney organoids, cerebral cortex organoids and lung organoids) are cultured in different chamber units respectively and then integrated into the spliced cell culture well plate, a more economical and efficient platform is provided for compound toxicity evaluation.
[0025] (2) cross contamination can be avoided: taking a 96-well plate as an example, if organoids are cultured on a common 96-well plate, the 96-well plate cannot be cultured with organoids, because 96 times of repeated evaluation of a compound is not needed, and the extra organoids will be wasted, if the toxicity of multiple compounds is evaluated on a 96-well plate, cross contamination is likely to occur, therefore, after the spliced cell culture well plate is used, the organoids and embryoid bodies are cultured in the chamber units respectively, and then the compound toxicity is tested and evaluated, not only time is saved, but also each chamber unit of the well plate is fully utilized, and waste is reduced.
[0026] (3) if different organoids are transferred from different well plates to one well plate by using a pipette, organoid loss and tedious repeated operation need to be faced, the utility model directly transfers the chamber units, and does not need to transfer the organoids by using the pipette, so that there is no risk of organoid loss, and the operation is quick and convenient.
[0027] (4) the spliced culture well plate can be designed based on the existing standard well plate, and can be compatible with enzyme-linked immunoassay equipment and other equipment, therefore, after the chamber units of the mature organoids and embryoid bodies are assembled on one well plate, the chamber units can be directly used for enzyme-linked immunoassay equipment and other equipment, which is convenient and quick. BRIEF DESCRIPTION OF DRAWINGS
[0028] In order to make the purpose, technical scheme and beneficial effects of the utility model clearer, the utility model provides the following drawings for description.
[0029] Figure 1 It is a structure schematic view of the embodiment of the spliced cell culture well plate of the utility model.
[0030] Figure 2 It is a structure schematic view of the well body.
[0031] Figure 3 It is a structure schematic view of the chamber unit with different bottom structures; (a) U-shaped bottom; (b) flat bottom; (c) three-fold bottom; (d) multi-hole bottom.
[0032] Figure 4 It is a top view of the chamber unit with multi-hole bottom; (a) micropore aperture 800um; (b) micropore aperture 400um. DETAILED DESCRIPTION
[0033] The utility model will be further explained in combination with the drawings and specific embodiments, so that the person skilled in the art can better understand the utility model and can be implemented, but the embodiment is not as the limitation of the utility model.
[0034] As Figure 1 shown, the spliced cell culture well plate of the embodiment includes a well body 10 and a well cover 20. As Figure 2 shown, the well body 10 is provided with a plurality of chamber placing positions 11, and the chamber placing position 11 is used to place a chamber unit 30. Specifically, in the embodiment, the chamber unit 30 includes a chamber body 31, the chamber body 31 is provided with n holes 32, and n is a positive integer greater than or equal to 1. In the embodiment, the chamber placing positions 11 are arranged on the well body 10. In the embodiment, the well body 10 is designed according to a standard 96-well plate, the well body 10 is provided with 24 chamber placing positions 11, and the chamber body 31 is provided with 4 holes 32, that is, the chamber unit 30 is a four-unit chamber unit. That is, the number N of holes of the well body 10 is:
[0035] N=n*m
[0036] Wherein: n is the number of holes provided in the chamber unit; m is the number of chamber placing positions; * represents the product.
[0037] In other embodiments, the well body 10 can also be designed according to a standard 24-well plate, a 48-well plate and a 384-well plate. The number of holes 32 provided in each chamber body 31 is 1-24. Of course, the number N of holes of the well body 10 is an integer multiple of the number n of holes of the chamber unit 30.
[0038] In order to position the placement direction of the chamber unit 30 in the chamber placement site 11, the chamber unit placement 11 of the embodiment is provided with a positioning structure for positioning the placement direction of the chamber unit 30. Specifically, the chamber placement site 11 of the embodiment is a chamber unit placement groove provided in the well plate body 10, and one end of the chamber unit placement groove is provided as a first circular arc surface, and the other end is provided as a first flat surface. One end of the chamber unit 30 is provided as a second circular arc surface matched with the first circular arc surface, and the other end is provided with a second flat surface matched with the first flat surface.
[0039] Specifically, the bottom structure of the hole 32 adopts a multi-hole bottom, a three-fold bottom, a flat bottom, or a U-shaped bottom. Among the chamber units 30 placed in the well plate body 10, all the chamber units 30 can adopt the same bottom structure, or different bottom structures. Specifically, when the hole 32 adopts a multi-hole bottom, the multi-hole bottom adopts an array of micropores, and the pore diameter of the micropores is 20-2000um, preferably 400um and 800um. The shape of the micropores is a regular hexagon, a circle, or a square, preferably a regular hexagon.
[0040] Specifically, the chamber body 31 is integrally formed and can be obtained by integral micro-injection molding, mechanical milling, 3D printing technology, soft lithography technology, etc. Of course, the chamber body 31 can also be made in parts. Specifically, in addition to the chamber body with a U-shaped bottom, the chamber bodies with a multi-hole bottom, a three-fold bottom, and a flat bottom can also be provided in parts as a chamber body and a bottom plate located below the chamber body. The chamber body is provided with a through hole, and the bottom plate is provided with a bottom structure corresponding to the through hole. The through hole and the bottom structure combine to form the hole 32. Specifically, the thickness of the bottom plate is 0.1-3mm, preferably 1mm. The bottom plate and the chamber body can be connected by bonding, hot pressing, or double-sided adhesive bonding. The bottom plate has light transmission and biocompatibility and is made of one of glass, polydimethylsiloxane (PDMS), polypropylene (PP), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), polymethyl methacrylate (PMMA), hydrogel, and transparent resin.
[0041] Specifically, the well plate body 10 has biocompatibility and is made of one of polydimethylsiloxane (PDMS), polypropylene (PP), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), polymethyl methacrylate (PMMA), hydrogel, and transparent resin.
[0042] Specifically, the well plate cover 20 has light transmission and biocompatibility and is made of one of glass, polydimethylsiloxane (PDMS), polypropylene (PP), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), polymethyl methacrylate (PMMA), and transparent resin.
[0043] The following detailed description of the specific embodiment of the compound toxicity evaluation method using the spliced cell culture plate will be described in detail.
[0044] A compound toxicity evaluation method, comprising the following steps:
[0045] Step 1: Culturing iPSCs: iPSCs are cultured in a first plate until the confluence reaches the set requirement;
[0046] Step 2: Culturing embryoid bodies: iPSCs in the first plate are prepared into a single cell suspension, inoculated into the chamber unit at the specified density, and then placed in an incubator for culture to increase the diameter of the embryoid bodies;
[0047] Step 3: Differentiation of embryoid bodies: the same chamber unit is placed on the same spliced cell culture plate, and the same type of organ differentiation induction is performed; after the organ matures, the chamber unit is removed, and the chamber units with different organs are placed in the same spliced cell culture plate;
[0048] Step 4: Compound toxicity evaluation:
[0049] To evaluate the toxicity of the same compound at the same concentration on different organs; or,
[0050] The chamber unit is split and combined so that the same spliced cell culture plate contains chamber units with at least two types of organs, and the toxicity of the same compound at the same concentration on different organs is evaluated.
[0051] The following detailed description of the specific experiment will be described in detail.
[0052] 1. Experimental instruments and consumables
[0053] Spliced cell culture plate, pipettor, constant temperature carbon dioxide incubator, live cell imaging instrument, microscope, centrifuge tube, clean bench, refrigerator, etc.
[0054] 2. Experimental reagents
[0055] Culture medium, digestive juice, anhydrous alcohol, PBS solution, pure water, Matrigel, ROCK inhibitor, EDTA, hiPSC, DMSO, induction medium I (mTeSR™ Plus 99.99 parts with a concentration of 1X, CHIR99021 0.01 part with a concentration of 5uM), induction medium II (mTeSR™ Plus 99.99-99.995 parts with a concentration of 1X, CHIR99021 0.005-0.001 parts with a concentration of 2-8uM), induction medium III (RPMI 1640 97 parts with a concentration of 1X, Glutamax 1 part with a concentration of 1X, B27 2 parts with a concentration of 1X).
[0056] 3. Experimental preparation
[0057] 3.1. Chamber unit disinfection
[0058] The method for pre-treating the chamber is to clean the chamber with alcohol and PBS buffer in turn, and then disinfect the chamber under ultraviolet light. Specifically, the chamber unit (quadruple chamber) is placed in 75% concentration alcohol, treated for one minute, then taken out and washed in PBS buffer, slightly shaken, treated for one minute, then taken out and spun dry, and placed under ultraviolet light for 30 minutes to complete the disinfection of the quadruple chamber.
[0059] 3.2. Preparation of reagents and preparation of coated plates
[0060] The Matrigel is dispensed and diluted, the ROCK inhibitor is prepared, and the E8 complete medium is prepared.
[0061] The Matrigel is thawed on ice at 4℃, diluted with KO-DMEM, 1mL of Matrigel solution is added to each well of the first well plate (6-well plate), and the surface of the well is covered with slight shaking to obtain a coated plate.
[0062] 4. Experimental steps
[0063] Step one: culture iPSC: culture iPSC in the first well plate until the confluence reaches the set requirement.
[0064] Specifically, the iPSC is taken out from liquid nitrogen and transferred to a centrifuge tube containing KO-DMEM after thawing, centrifuged at 180g for 5 minutes, and the supernatant is discarded. Then the cells are mixed with E8 medium containing ROCK inhibitor and planted in the Matrigel coated wells of the first well plate, and placed in an incubator for culture until the cell confluence reaches 80%.
[0065] Step two: cultivate embryoid bodies: prepare the iPSC in the first well plate into a single cell suspension, inoculate the chamber unit at the specified density, and place it in an incubator for culture to increase the diameter of the embryoid bodies.
[0066] Step three: embryoid differentiation: placing the same chamber units on the same assembled cell culture plate and conducting the same kind of organ differentiation induction; after the organ matures, the chamber units with different organs are placed in the same assembled cell culture plate.
[0067] Step four: compound toxicity evaluation:
[0068] In some embodiments, the toxicity of the same compound at different concentrations on the same kind of organ can be evaluated. Specifically, the assembled cell culture plate is prepared, and one plate tests one compound. The chamber units cultured with the same organ are transferred to the same assembled cell culture plate to be tested, a blank control is reserved, different concentrations of the compound are added to the remaining chambers, after a period of time, the organ is lysed by adding ATP detection solution, and the luminescence value is detected by using an enzyme marker. By comparing the luminescence value results of different chambers, the toxicity of the compound on the organ at different concentrations is evaluated.
[0069] In some other embodiments, the toxicity of the same compound at the same concentration on different organs can also be evaluated. That is, the chamber units can be split and combined so that the same assembled cell culture plate contains chamber units with at least two kinds of organs, and the toxicity of the same compound at the same concentration on different organs is evaluated. Specifically, the assembled cell culture plate is prepared, and one plate tests one compound. The chamber units cultured with different organs are transferred to the same assembled cell culture plate to be tested, the same kind of compound at the same concentration is added to all chambers, after a period of time, the organ is lysed by adding ATP detection solution, and the luminescence value is detected by using an enzyme marker. By comparing the luminescence value results of different chambers, the toxicity of the same compound at the same concentration on different organs is evaluated.
[0070] The above embodiments are only preferred embodiments for fully illustrating the present application, and the protection scope of the present application is not limited thereto. The equivalent substitutions or transformations made by the person skilled in the art on the basis of the present application are all within the protection scope of the present application. The protection scope of the present application is subject to the claims.
Claims
1. A chamber unit, characterized in that: The device includes a chamber body, which has n holes, where n is a positive integer greater than or equal to 1; the bottom structure of the holes adopts a porous bottom, a three-fold bottom, a flat bottom, or a U-shaped bottom; the chamber body is integrally formed; or, except for the chamber body with a U-shaped bottom, the chamber body with a porous bottom, a three-fold bottom, or a flat bottom is divided into a chamber body and a bottom plate located below the chamber body, the chamber body has through holes, and the bottom plate has bottom structures corresponding to the through holes, the through holes and the bottom structures are combined to form the holes.
2. The chamber unit according to claim 1, characterized in that: The porous bottom employs an array of micropores, the pore diameter of which is 20-2000 μm; the shape of the micropores is a regular hexagon, a circle, or a square.
3. The chamber unit according to claim 1, characterized in that: The thickness of the base plate is 0.1-3mm; the base plate is connected to the main body of the chamber by bonding, hot pressing, or double-sided adhesive.
4. The chamber unit according to claim 1, characterized in that: The base plate is transparent and biocompatible, and is made of one of the following: glass, polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cyclic olefin copolymer, polymethyl methacrylate, hydrogel, and transparent resin.
5. A modular cell culture plate, comprising a plate body and a plate cap; characterized in that: The orifice plate body is provided with multiple chamber unit placement positions, which are used to place chamber units; each chamber unit placement position is a chamber placement groove provided in the orifice plate body, and the chamber unit placement position is provided with a positioning structure for positioning the placement direction of the chamber unit.
6. The modular cell culture plate according to claim 5, characterized in that: The chamber unit placement array is arranged on the orifice plate body.
7. The modular cell culture plate according to claim 5, characterized in that: The number of holes N in the main body of the perforated plate is: N=n*m Where: n is the number of holes in the chamber unit; m is the number of chamber unit placement positions; * indicates product.
8. The modular cell culture plate according to claim 5, characterized in that: Of the two opposite ends of the cavity placement slot, one end is designated as a first arc surface and the other end as a first plane; of the two ends of the cavity unit, one end is designated as a second arc surface that mates with the first arc surface, and the other end is designated as a second plane that mates with the first plane.
9. The modular cell culture plate according to claim 5, characterized in that: The main body of the perforated plate is biocompatible and is made of one of polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cyclic olefin copolymer, polymethyl methacrylate, hydrogel and transparent resin. The perforated plate cover is transparent and biocompatible, and is made of one of glass, polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cyclic olefin copolymer, polymethyl methacrylate and transparent resin.