Spliced cell culture pore plate and compound toxicity assessment method

Abstract

The invention discloses a spliced cell culture pore plate which comprises a pore plate main body and a pore plate cover, the pore plate comprises a pore plate main body and is characterized in that a plurality of cavity placing positions are arranged on the pore plate main body, the cavity placing positions are used for placing cavity units, each cavity unit comprises a cavity body, n holes are formed in each cavity body, and n is a positive integer larger than or equal to 1. The invention also discloses a compound toxicity assessment method. According to the spliced cell culture pore plate and the compound toxicity assessment method, all the cavity units in the same pore plate main body can have the same organ, and at the moment, the toxicity of the same compound to the organ under different concentration conditions can be tested and assessed; the same pore plate main body can contain at least two kinds of organ chamber units, so that the toxicity of the same compound to different kinds of organs under the same concentration condition can be tested and evaluated; and concentrated testing and evaluation of the organoids are realized, so that the compound toxicity evaluation efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid culture, and specifically relates to a spliced ​​cell culture well plate and a compound toxicity assessment method. Background Art

[0002] At present, humans have synthesized hundreds of millions of compounds, including various drugs, pesticides and industrial chemicals, and the number is increasing every day. The damage of these synthetic compounds to human health needs to be evaluated urgently. The traditional toxicological evaluation method is to conduct animal experiments, but this method has high costs, long evaluation cycles, and there are species differences between animals and humans. Therefore, the reliability of extrapolating animal experimental results to humans is poor.

[0003] Organoid technology solves the shortcomings of animal experiments in the field of toxicological assessment of compounds. Organoids cultured using human tissues or human induced pluripotent stem cells (iPSCs) have no species differences from humans, and after the organoids are cultured to maturity, they have all types of cells in human organs and have obvious organ characteristics. Therefore, using organoids to evaluate the toxicity of compounds can highly restore the actual impact of the compounds on the human body. Because more than 10 types of organoids and embryos are used in the in vitro toxicity assessment of compounds, and they need to be tested and evaluated at the same time. However, their culture conditions and culture times are inconsistent, which makes the experimental operation complicated and difficult to integrate them into a platform for efficient evaluation. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a spliced ​​cell culture well plate and a compound toxicity assessment method, which can differentiate and culture different organoids and embryoids respectively, and centrally test and evaluate all organoids and embryoids.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention first proposes a spliced ​​cell culture well plate, including a well plate body and a well plate cover; the well plate body is provided with a plurality of chamber placement positions, the chamber placement positions are used to place chamber units, the chamber unit includes a chamber body, the chamber body is provided with n holes, and n is a positive integer greater than or equal to 1.

[0007] Furthermore, the chamber placement array is arranged on the orifice plate body.

[0008] Further, the number of holes N of the orifice plate body is:

[0009] N=n*m

[0010] Wherein: n is the number of holes provided in the chamber unit; m is the number of chamber placement positions; and * represents the product.

[0011] Furthermore, a positioning structure for positioning the placement direction of the chamber unit is provided inside the chamber unit.

[0012] Furthermore, the chamber placement position is a chamber unit placement groove arranged in the orifice plate body, and one end of the chamber unit placement groove is set as a first arc surface and the other end is set as a first plane; one end of the chamber unit is set as a second arc surface matching the first arc surface and the other end is set as a second plane matching the first plane.

[0013] Furthermore, the bottom structure of the hole adopts a porous bottom, a three-fold bottom, a flat bottom or a U-shaped bottom.

[0014] Furthermore, the porous bottom adopts micropores arranged in an array, and the pore diameter of the micropores is 20-2000um; the shape of the micropores is regular hexagon, circle or square.

[0015] Furthermore, the chamber body is integrally formed; or, in addition to the chamber body with a U-shaped bottom, the chamber body with a porous bottom, a tri-fold bottom and a flat bottom is separately arranged as a chamber body and a bottom plate located below the chamber body, a through hole is provided in the chamber body, a bottom structure is provided on the bottom plate corresponding to the through hole, and the through hole and the bottom structure are combined to form the hole.

[0016] Furthermore, the thickness of the bottom plate is 0.1-3 mm; the bottom plate and the chamber body are connected by bonding, hot pressing bonding or double-sided adhesive bonding.

[0017] Further, the orifice plate body is biocompatible and is made of one of polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cycloolefin copolymer, polymethyl methacrylate, hydrogel and transparent resin;

[0018] The well plate cover has light transmittance and biocompatibility and is made of one of glass, polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cycloolefin copolymer, polymethyl methacrylate and transparent resin;

[0019] The base plate is light-transmissive and biocompatible and is made of one of glass, polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cyclic olefin copolymer, polymethyl methacrylate, hydrogel and transparent resin.

[0020] The present invention also proposes a method for evaluating the toxicity of a compound, comprising the following steps:

[0021] Step 1: Cultivate iPSCs: Cultivate iPSCs in the first well plate until the confluence reaches the set requirement;

[0022] Step 2: Cultivating embryoid bodies: iPSCs in the first well plate are prepared into a single cell suspension, inoculated into the chamber unit at a specified density, and then cultured in an incubator to increase the diameter of the embryoid bodies;

[0023] Step 3: Embryoid differentiation: Place the same chamber unit on the same patchwork cell culture well plate and induce differentiation of the same organoid; after the organoid matures, remove the chamber unit and place the chamber units with different organoids in the same patchwork cell culture well plate;

[0024] Step 4: Compound toxicity assessment:

[0025] To evaluate the toxicity of the same compound at different concentrations to the same type of organ; or

[0026] The chamber units were split and combined so that the same spliced ​​cell culture well plate contained chamber units with at least two organoids to evaluate the toxicity of the same compound at the same concentration on different organoids.

[0027] The beneficial effects of the present invention are:

[0028] The spliced ​​cell culture well plate of the present invention has a plurality of chamber placement positions on the well plate body for placing chamber units. In this way, different organoids and embryo-like cells can be cultured in different chamber units respectively, and then the chamber units can be transferred to the chamber placement positions of the well plate body. All chamber units in the same well plate body can have the same type of organ, and the toxicity of the same compound to the organoids under different concentration conditions can be tested and evaluated. The same well plate body can also contain chamber units of at least two organoids, and the toxicity of the same compound to different organoids under the same concentration conditions can be tested and evaluated. Centralized testing and evaluation of organoids can be achieved, thereby improving the efficiency of compound toxicity evaluation.

[0029] The spliced ​​cell culture well plate and compound toxicity assessment method of the present invention also have the following beneficial effects:

[0030] (1) The toxicity evaluation of compounds using iPSC-induced organoids and embryos has a higher reference value;

[0031] (2) Various organoids and embryoids (including morula-like models, blastocyst-like models, amniotic cavity embryoids, pancreatic islet organoids, kidney organoids, cerebral cortex organoids, lung organoids, etc.) are cultured separately through different chamber units and then integrated into the spliced ​​cell culture well plate, providing a more economical and efficient platform for the toxicity evaluation of compounds;

[0032] (3) It can avoid cross contamination: Taking a 96-well plate as an example, if organoids are cultured on an ordinary 96-well plate, all 96 wells cannot be cultured with organoids, because the evaluation of a compound does not need to be repeated 96 times, and the excess organoids will be wasted; if the toxicity evaluation of multiple compounds is performed on a 96-well plate, cross contamination is likely to occur; therefore, after adopting the spliced ​​cell culture well plate of the present invention, by culturing organoids and embryos in each chamber unit separately and then centrally conducting compound toxicity testing and evaluation, not only time is saved, but also each chamber unit of the well plate is fully utilized to reduce waste.

[0033] (4) If different types of organoids are transferred from different well plates to one well plate using a pipette, there is a risk of organoid loss and cumbersome repetitive operations. The present invention directly transfers the chamber unit without the need to transfer the organoids using a pipette, which eliminates the risk of organoid loss and is quick and convenient.

[0034] (5) The spliced ​​culture well plate can be designed based on the existing standard well plate and is compatible with equipment such as enzyme-linked immunosorbent assay (ELISA). Therefore, after assembling the chamber units of various mature organoids and embryo-like cells on a well plate, it can also be directly used in equipment such as ELISA, which is convenient and quick. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0036] Figure 1 It is a schematic structural diagram of an embodiment of a spliced ​​cell culture well plate of the present invention;

[0037] Figure 2 It is a structural schematic diagram of the orifice plate body;

[0038] Figure 3 Schematic diagrams of chamber units with different bottom structures; (a) U-shaped bottom; (b) flat bottom; (c) tri-fold bottom; (d) porous bottom;

[0039] Figure 4 Top view of a chamber unit with a porous bottom; (a) micropore diameter 800um; (b) micropore diameter 400um. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0041] like Figure 1 As shown in FIG. 1 , the spliced ​​cell culture well plate of this embodiment includes a well plate body 10 and a well plate cover 20. Figure 2As shown, a plurality of chamber placement positions 11 are provided on the orifice plate body 10, and the chamber placement positions 11 are used to place the chamber unit 30. Specifically, in this embodiment, the chamber unit 30 includes a chamber body 31, and n holes 32 are provided in the chamber body 31, and n is a positive integer greater than or equal to 1. In this embodiment, the chamber placement positions 11 are arranged in an array on the orifice plate body 10. In this embodiment, the orifice plate body 10 is designed according to a standard 96-well plate, and 24 chamber placement positions 11 are provided in the orifice plate body 10, and 4 holes 32 are provided in the chamber body 31, that is, the chamber unit 30 is a quadruple chamber unit. That is, the number of holes N of the orifice plate body 10 is:

[0042] N=n*m

[0043] Wherein: n is the number of holes provided in the chamber unit; m is the number of chamber placement positions; and * represents the product.

[0044] In some other embodiments, the orifice plate 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 of holes N of the orifice plate body 10 is an integer multiple of the number of holes n of the chamber unit 30.

[0045] In order to locate the placement direction of the chamber unit 30 in the chamber placement position 11, a positioning structure for locating the placement direction of the chamber unit 30 is provided in the chamber unit placement 11 of this embodiment. Specifically, the chamber placement position 11 of this embodiment is a chamber unit placement groove provided in the orifice plate body 10, and one end of the chamber unit placement groove is provided with a first arc surface and the other end is provided with a first plane. And one end of the chamber unit 30 is provided with a second arc surface matching the first arc surface and the other end is provided with a second plane matching the first plane.

[0046] Specifically, the bottom structure of the hole 32 adopts a porous bottom, a trifold bottom, a flat bottom or a U-shaped bottom. In the chamber unit 30 placed in the orifice plate body 10, all chamber units 30 can adopt the same bottom structure or different bottom structures. Specifically, when the hole 32 adopts a porous bottom, the porous bottom adopts micropores arranged in an array, and the pore size 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.

[0047] Specifically, the chamber body 31 is integrally formed, and the chamber body 31 can be obtained by integral micro-injection molding, mechanical milling, 3D printing technology and soft lithography technology. 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 body with a porous bottom, a trifold bottom and a flat bottom can also be divided into a chamber body and a bottom plate located below the chamber body, and a through hole is provided in the chamber body, and a bottom structure is provided on the bottom plate corresponding to the through hole, and the through hole and the bottom structure are combined to form a 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 bonding or double-sided adhesive bonding. The bottom plate has light transmittance and biocompatibility, and is made of one of glass, polydimethylsiloxane (PDMS), polypropylene (PP), polystyrene (PS), polycarbonate (PC), cycloolefin copolymer (COC), polymethyl methacrylate (PMMA), hydrogel and transparent resin.

[0048] Specifically, the well plate body 10 is biocompatible and is made of one of polydimethylsiloxane (PDMS), polypropylene (PP), polystyrene (PS), polycarbonate (PC), cycloolefin copolymer (COC), polymethyl methacrylate (PMMA), hydrogel and transparent resin.

[0049] Specifically, the well plate cover 20 has light transmittance and biocompatibility, and is made of one of glass, polydimethylsiloxane (PDMS), polypropylene (PP), polystyrene (PS), polycarbonate (PC), cycloolefin copolymer (COC), polymethyl methacrylate (PMMA) and transparent resin.

[0050] This embodiment also proposes a method for evaluating the toxicity of a compound, comprising the following steps:

[0051] Step 1: Cultivate iPSCs: Cultivate iPSCs in the first well plate until the confluence reaches the set requirement;

[0052] Step 2: Cultivating embryoid bodies: iPSCs in the first well plate are prepared into a single cell suspension, inoculated into the chamber unit at a specified density, and then cultured in an incubator to increase the diameter of the embryoid bodies;

[0053] Step 3: Embryoid differentiation: Place the same chamber unit on the same patchwork cell culture well plate and induce differentiation of the same organoid; after the organoid matures, remove the chamber unit and place the chamber units with different organoids in the same patchwork cell culture well plate;

[0054] Step 4: Compound toxicity assessment:

[0055] Evaluate the toxicity of the same compound at the same concentration in different organoids; or

[0056] The chamber units were split and combined so that the same spliced ​​cell culture well plate contained chamber units with at least two organoids to evaluate the toxicity of the same compound at the same concentration on different organoids.

[0057] The following is a detailed description of the compound toxicity assessment method in conjunction with specific experiments.

[0058] 1. Experimental instruments and consumables

[0059] Spliced ​​cell culture well plates, pipettes, constant temperature carbon dioxide incubator, live cell imager, microscope, centrifuge tubes, clean bench, refrigerator, etc.

[0060] 2. Experimental reagents

[0061] Culture medium, digestion solution, anhydrous ethanol, PBS solution, pure water, matrigel, ROCK inhibitor, EDTA, hiPSC, DMSO, induction medium I (99.99 parts of mTeSRTMPlus at a concentration of 1X, 0.01 parts of CHIR99021 at a concentration of 5uM), induction medium II (99.99-99.995 parts of mTeSRTMPlus at a concentration of 1X, 0.005-0.001 parts of CHIR99021 at a concentration of 2-8uM), induction medium III (97 parts of RPMI 1640 at a concentration of 1X, 1 part of Glutamax at a concentration of 1X, 2 parts of B27 at a concentration of 1X).

[0062] 3. Experimental preparation

[0063] 3.1. Chamber unit disinfection

[0064] The method for pre-treating the chamber is: using alcohol and PBS buffer to clean the chamber in turn, and then placing the chamber under ultraviolet light for disinfection. Specifically, the chamber unit (quadruple chamber) is placed in 75% alcohol, treated for one minute, taken out and placed in PBS buffer for cleaning, shaken slightly, treated for one minute, taken out and dried, and placed under ultraviolet light for 30 minutes to complete the disinfection of the quadruple chamber.

[0065] 3.2. Preparation of reagents and prepared plates

[0066] Aliquot and dilute Matrigel, prepare ROCK inhibitor, and prepare E8 complete medium.

[0067] Thaw the matrix gel on ice at 4°C, dilute the matrix gel with KO-DMEM, add 1 mL of the matrix gel solution to each well of the first well plate (6-well plate), shake gently to ensure that the surface of the well is completely covered, and obtain a coated plate.

[0068] 4. Experimental steps

[0069] Step 1: Cultivate iPSCs: Cultivate iPSCs in the first well plate until the confluence reaches the set requirement.

[0070] Specifically, iPSCs were taken out of liquid nitrogen, thawed and transferred to a KO-DMEM centrifuge tube, centrifuged at 180 g for 5 minutes, and the supernatant was discarded. Then, E8 medium with ROCK inhibitor was added to mix the cells, and the cells were seeded into the matrix gel-coated wells of the first well plate and cultured in an incubator until the cell confluence reached 80%.

[0071] Step 2: Cultivating embryoid bodies: iPSCs in the first well plate are prepared into a single cell suspension, inoculated into the chamber unit at a specified density, and then cultured in an incubator to increase the diameter of the embryoid bodies.

[0072] Step 3: Embryoid differentiation: Place the same chamber unit on the same patchwork cell culture well plate and induce differentiation of the same organoid type; after the organoids mature, remove the chamber unit and place the chamber units with different organoids into the same patchwork cell culture well plate.

[0073] Step 4: Compound toxicity assessment:

[0074] In some of the embodiments, the toxicity of the same compound to the same organoid at different concentrations can be evaluated. Specifically, a patchwork cell culture well plate is prepared, and one well plate is used to test one compound. The chamber unit cultured with the same organoid is transferred to the same patchwork cell culture well plate to be tested, and a blank control is retained. Different concentrations of compounds are added to the remaining chambers. After a period of time, ATP detection solution is added to lyse the organoid, and the luminescence value is detected using an enzyme reader. By comparing the luminescence value results of different chambers, the toxicity of the compound to the organoid at different concentrations is evaluated.

[0075] In some other embodiments, the toxicity of the same compound at the same concentration to different organoids can also be evaluated. That is, the chamber units can be split and combined so that the same spliced ​​cell culture well plate contains chamber units with at least two organoids, and the toxicity of the same compound at the same concentration to different organoids can be evaluated. Specifically, a spliced ​​cell culture well plate is prepared, and one well plate is used to test one compound. The chamber units with different organoids cultured are transferred to the same spliced ​​cell culture well plate to be tested, and the same compound at the same concentration is added to all chambers. After a period of time, ATP detection solution is added to lyse the organoids, and the luminescence value is detected using an enzyme reader. By comparing the luminescence value results of different chambers, the toxicity of the same compound at the same concentration to different organoids is evaluated.

[0076] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A spliced ​​cell culture well plate, comprising a well plate body and a well plate cover; characterized in that: The orifice plate body is provided with a plurality of chamber placement positions, wherein the chamber placement positions are used to place chamber units, wherein the chamber units include a chamber body, wherein n holes are provided in the chamber body, and n is a positive integer greater than or equal to 1.

2. The spliced ​​cell culture well plate according to claim 1, characterized in that: The chamber placement array is arranged on the orifice plate body.

3. The spliced ​​cell culture well plate according to claim 1, characterized in that: The number of holes N of the orifice plate body is: N=n*m Wherein: n is the number of holes provided in the chamber unit; m is the number of chamber placement positions; and * represents the product.

4. The spliced ​​cell culture well plate according to claim 1, characterized in that: A positioning structure for positioning the placement direction of the chamber unit is provided inside the chamber unit.

5. The spliced ​​cell culture well plate according to claim 4, characterized in that: The chamber placement position is a chamber unit placement groove arranged in the orifice plate body, and one end of the chamber unit placement groove is set as a first arc surface and the other end is set as a first plane; one end of the chamber unit is set as a second arc surface matching the first arc surface and the other end is provided with a second plane matching the first plane.

6. The spliced ​​cell culture well plate according to claim 1, characterized in that: The bottom structure of the hole adopts a porous bottom, a three-fold bottom, a flat bottom or a U-shaped bottom.

7. The spliced ​​cell culture well plate according to claim 6, characterized in that: The porous bottom adopts micropores arranged in an array, and the pore diameter of the micropores is 20-2000um; the shape of the micropores is regular hexagon, circle or square.

8. The spliced ​​cell culture well plate according to claim 1, characterized in that: 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 tri-fold bottom and a flat bottom is separately arranged as a chamber body and a bottom plate located below the chamber body, a through hole is provided in the chamber body, a bottom structure is provided on the bottom plate corresponding to the through hole, and the through hole and the bottom structure are combined to form the hole.

9. The spliced ​​cell culture well plate according to claim 8, characterized in that: The thickness of the bottom plate is 0.1-3 mm; the bottom plate and the chamber body are connected by bonding, hot pressing bonding or double-sided adhesive bonding.

10. The spliced ​​cell culture well plate according to claim 8, characterized in that: The orifice plate body is biocompatible and is made of one of polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cycloolefin copolymer, polymethyl methacrylate, hydrogel and transparent resin; The well plate cover has light transmittance and biocompatibility and is made of one of glass, polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cycloolefin copolymer, polymethyl methacrylate and transparent resin; The base plate is light-transmissive and biocompatible and is made of one of glass, polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cyclic olefin copolymer, polymethyl methacrylate, hydrogel and transparent resin.

11. A method for evaluating the toxicity of a compound, characterized in that: The steps include: Step 1: Cultivate iPSCs: Cultivate iPSCs in the first well plate until the confluence reaches the set requirement; Step 2: Cultivating embryoid bodies: iPSCs in the first well plate are prepared into a single cell suspension, inoculated into the chamber unit at a specified density, and then cultured in an incubator to increase the diameter of the embryoid bodies; Step 3: Embryoid differentiation: transfer the chamber unit to the patchwork cell culture well plate, and perform differentiation induction of the same organoid in each patchwork cell culture well plate until the organoid matures; Step 4: Compound toxicity assessment: To evaluate the toxicity of the same compound at different concentrations to the same type of organ; or The chamber units were split and combined so that the same spliced ​​cell culture well plate contained chamber units with at least two organoids to evaluate the toxicity of the same compound at the same concentration on different organoids.

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