Organism and method for promoting directional growth thereof

Abstract

Disclosed in the present invention are an organism and a method for promoting the directional growth thereof. The organism comprises at least one of a single cell, a multicellular body, a tissue and an organoid. The method comprises the step of subjecting the organism to a strong-to-weak gradient electric field environment. In the present invention, a layered material is constructed using strontium titanate as a substrate, strontium ruthenate as a regulating layer, and bismuth ferrite as an internal electric domain polarization layer. A surface potential, i.e., a gradient electric field, is generated by the layered material so as to guide the directional growth of the organism in vitro, thereby constructing an organoid that highly mimics the morphology, structure and function of a corresponding tissue in vivo.

Description

An organism and a method for promoting directional growth thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to the patent application with the application number CN202411358347.3, the title of which is “An organism and a method for promoting directional growth thereof”, filed on September 27, 2024, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application belongs to the field of organism culture, in particular relates to an organism and a method for promoting directional growth thereof. BACKGROUND

[0004] Organoids are a model that is highly similar to in vivo derived tissues or organs based on 3D in vitro cell culture system. These 3D in vitro culture systems can replicate the complex spatial morphology of differentiated tissues and can achieve similar physiological responses to in vivo differentiated tissues and organs. The typical method of organoid preparation first separates embryonic or pluripotent stem cells, and then cultures them on a support medium (such as Matrigel) to enable three-dimensional growth. The application scenarios of organoids are very wide, which can be used for disease models, such as the study of development-related problems, genetic diseases, tumor cancers, and the like, and can achieve more effective and more realistic detection of drug efficacy and toxicity, which greatly avoids the inconsistency of detection results caused by the differences between animal and human cells compared to animal models.

[0005] However, the organoids constructed in vitro at present have significant differences in morphology and structure from real tissues and organs, mainly manifested in that it is difficult to guide the directional growth of cell populations in the organoids in the current in vitro environment, so that the function thereof is far different from the corresponding tissues in vivo. SUMMARY

[0006] To solve at least part of the problems in the prior art, the present application generates a biomimetic gradient electric field through a layered material, realizes the directional growth of an organism in vitro, and constructs an organoid that highly simulates the morphology, structure and function of the corresponding tissue in vivo. Specifically, the present application includes the following contents.

[0007] In a first aspect, the present application provides a method for promoting directional growth of an organism, comprising the step of placing the organism in a spatial gradient electric field with a high-to-low electric potential.

[0008] In some embodiments, the method for promoting directional growth of an organism according to the present application, wherein the organism comprises at least one of a single cell, a multicellular body, a tissue and an organoid.

[0009] In some embodiments, the method for promoting directional growth of an organism according to the present application, wherein the directional growth comprises at least one of the following:

[0010] cells divide or proliferate along the direction of high to low electric potential change or along the direction of the gradient electric field;

[0011] tissues or organoids sprout or grow along the direction of high to low electric potential change or along the direction of the gradient electric field.

[0012] In some embodiments, the method for promoting directional growth of an organism according to the present application, wherein the gradient electric field comprises an electric field whose electric potential strength varies with spatial coordinates along a certain straight line direction.

[0013] In some embodiments, the method for promoting directional growth of an organism according to the present application, wherein the gradient electric field is generated by an upward surface electric potential.

[0014] In some embodiments, the method for promoting directional growth of an organism according to the present application, wherein the gradient electric field is generated by internal domain polarization of a surface material.

[0015] In some embodiments, the method for promoting directional growth of an organism according to the present application, wherein the gradient electric field has a spatial gradient variation whose strength exponentially decays with the distance from the surface of the material.

[0016] In some embodiments, the method for promoting directional growth of an organism according to the present application, wherein the electric potential variation ranges from 10 to 150 mV.

[0017] In some embodiments, the method for promoting directional growth of an organism according to the present application, wherein the organism is an organoid, and the method comprises adherent culture of cells or cell populations on a surface of a material with positive electric potential, so as to make the organoid directionally grow in a vertical or substantially vertical direction.

[0018] In a second aspect, the present application provides a culture system for promoting directional growth of an organism, comprising a culture dish and a culture medium, wherein at least part of the surface of the culture dish in contact with the culture medium has a positive electric potential, so as to form a gradient electric field in a vertical or substantially vertical direction along the surface with positive electric potential.

[0019] In a third aspect, the present application provides an organism prepared by the method according to the present application.

[0020] The application constructs a layered material with strontium titanate as a substrate, strontium rubidate as a control layer and bismuth ferrite as an internal domain polarization layer, generates a surface potential, that is, a gradient electric field, through the layered material, realizes in-vitro guiding of directional growth of organisms, and thus constructs an organoid highly simulating the morphology, structure and function of corresponding tissues in vivo. BRIEF DESCRIPTION OF DRAWINGS

[0021] FIG. 1 is a schematic diagram of generating a surface potential by using bismuth ferrite biomaterials to form a biomimetic gradient electric field, wherein FIG. 1A is a structural schematic diagram of biomaterials with a surface potential, BFO is bismuth ferrite (BiFeO3), LSMO is lanthanum strontium manganese oxide (LaSrMnO3), STO is strontium titanate (SrTiO3), SRO is strontium rubidate, STO is a substrate, SRO and LSMO are respectively used for controlling the polarization direction of BFO, BFO+ is a surface potential upward, and BFO- is a surface potential downward; FIG. 1B and FIG. 1C are respectively the spatial gradient variation characteristics of the surface potential of BFO+ and BFO- materials exponentially decaying with the change of height.

[0022] FIG. 2 shows the phenomenon of directional concentration of intracellular stress promoted by the biomimetic gradient electric field, wherein at 2h and 6h after inoculation, the phenomenon of rearrangement of F-actin skeleton under the action of the gradient electric field appears, the F-actin skeleton at the top of the BFO+ group and at the bottom of the BFO- group appears in a crosslinked form with high abundance and a reticular structure, and directional intracellular stress is formed, wherein the intracellular stress of the BFO+ group is concentrated at the top, the intracellular stress of the BFO- group is concentrated at the bottom, and the F-actin skeleton of the control group is in a normal relatively uniform distribution state.

[0023] FIG. 3 shows the case of directional division of cells promoted by the biomimetic gradient electric field, wherein A, B and C are respectively images of directional division of cells in metaphase, anaphase and telophase of mitosis, and D is analysis of the division of cells in anaphase and telophase of mitosis; in metaphase and anaphase of mitosis, the division direction of the BFO+ group is vertical, and the division direction of the BFO- group and the STO group is mainly horizontal; in telophase of mitosis, two daughter cells of the BFO+ group are separated vertically, and two daughter cells of the BFO- group and the STO group are separated horizontally.

[0024] FIG. 4 shows the case of directional growth of BMSCs organoids promoted by the biomimetic gradient electric field, wherein at 1 day after inoculation, the BFO+ group appears limited cell budding, the BFO- group has a faster cell budding speed, and the STO group as a control shows a moderate budding speed; at 3 days after inoculation, the cells of the BFO+ group no longer bud and only grow in the vertical direction (the cells around the organoids are early shed cells proliferated), the BFO- group accelerates the budding of the organoids, and the STO group is in a normal budding form. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be non-limiting examples of the present application, and are understood to be a further description of certain aspects, features and embodiments of the present application.

[0026] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise stated, the inclusion of either extreme or any other value within the ranges is expressly submitted to be included within the scope of the application. The disclosure of any range of values is a disclosure of each value and each smaller range within the range. The disclosure of any range of values is also a disclosure of each value and of each end point of the range.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the patents, patent applications, publications, and descriptions are cited.

[0028] Method

[0029] In one aspect of the present application, there is provided a method for promoting directional growth of an organism in vitro, comprising the step of subjecting the organism to a gradient electric field environment.

[0030] In the present application, the organism comprises at least one of a single cell, a multicellular body, a tissue, and an organoid. In one preferred embodiment, the organism is a single cell, and specific examples of the single cell include, but are not limited to, immortalized cells, primary cells, stem cells, and the like. In another preferred embodiment, the organism is an organoid.

[0031] In the present application, the directional growth comprises at least one of the following: a cell or a cell population splits or proliferates in a direction from high to low of a positive electric potential or in a direction of an electric field; a tissue or an organoid grows or sprouts in a direction from high to low of a positive electric potential or in a direction of an electric field. In one preferred embodiment, the directional growth refers to growth of a cell, a cell population, a tissue, or an organoid in a direction perpendicular or substantially perpendicular to a surface having a positive electric potential, or growth of a cell, a cell population, a tissue, or an organoid in a direction away from a surface of a material having a positive electric potential (i.e., a direction of an electric field) and in a direction perpendicular or substantially perpendicular to the surface of the material having a positive electric potential.

[0032] In the present application, the gradient electric field includes an electric field whose potential magnitude along a certain linear direction varies with spatial coordinates. In a preferred embodiment, the gradient electric field is generated by a surface potential in an upward direction.

[0033] In a specific embodiment of the present application, the gradient electric field is generated by a material with a surface potential and a spatial gradient that exponentially decays with the distance from the surface of the material. In a preferred embodiment, the distance variation refers to a distance from the surface of the material less than 1500 nm, for example, 0-1500 nm. In a preferred embodiment, the potential gradient varies from 10-150 mV with the above-mentioned distance variation.

[0034] In a preferred embodiment, the surface potential decreases from 150 mV to 17 mV with an increase in height in the range of 30-1415 nm.

[0035] Without being bound by any theory, any material that can generate a gradient electric field with a gradient decrease with an increase in height on its surface can be used in the present application. In a preferred embodiment, the gradient electric field is generated by a layered material with internal domain polarization, above which a surface potential with a gradient decrease with an increase in height is formed. In a more preferred embodiment, the gradient electric field is generated by a layered material with a positive surface potential, which generates a surface potential and has a gradient decrease from 150 mV to 17 mV with an increase in height (or away from the positive potential side of the layered material).

[0036] In a preferred embodiment, the method for promoting directional growth of organisms of the present application includes adherent culture of cells or cell populations on a material surface with a positive potential, so that the organoids grow directionally in a vertical or substantially vertical direction, wherein the material surface sequentially includes an internal domain polarization layer, a regulation layer and a substrate layer in the thickness direction. In a preferred embodiment, the internal domain polarization layer is BFO, the regulation layer is SRO, and the substrate layer is STO.

[0037] It should be understood that the gradient electric field in the present application is an electrostatic field, a constant electric field, generated by a surface potential, with a spatial gradient and a fixed direction, which is different from a direct current electric field or a capacitive electric field in that the gradient electric field of the present application has no frequency or direction variation.

[0038] Culture system

[0039] In one aspect of the present application, a culture system for promoting directional growth of organisms is provided, which comprises a culture dish and a culture medium, wherein at least part of the surface of the culture dish in contact with the culture medium has a surface potential, thereby forming a gradient electric field in the direction perpendicular to the surface with the surface potential. The specific composition of the culture medium is not particularly limited and can be adjusted according to the type of cells or organoids to be cultured. The culture dish can be a commonly used cell or organoid culture dish, which is not particularly limited.

[0040] Organisms

[0041] In one aspect of the present application, an organism prepared by the method described in the present application is provided. In a preferred embodiment, the organism is an organoid. The organoid of the present application can highly simulate the corresponding organ in vivo in morphology, structure and function.

[0042] The present application also provides a material capable of generating a gradient electric field environment, in particular a material comprising an internal domain polarization layer, a regulation layer and a substrate layer in sequence along the thickness direction, for use in preparing organisms. In a preferred embodiment of the application, the internal domain polarization layer of the material is BFO, the regulation layer is SRO, and the substrate layer is STO.

[0043] Example 1

[0044] This example shows the process of constructing a biomimetic gradient electric field using ferroelectric-like materials.

[0045] 1. Experimental method

[0046] In this example, BFO (BiFeO3) was deposited on a 5mm x 5mm sized strontium titanate (SrTiO3, STO) using a laser pulse deposition method. Among them, SRO (SrRuO3) and LSMO (La 1 / 3 Sr 2 / 3 MnO3) were used to generate different covalent mismatches and thus generate surface potentials in two directions, with BFO+ upward and BFO- downward, as shown in Figure 1A. Then the surface potential intensity of different height surfaces of the material was detected using Kelvin probe force microscopy (KPFM).

[0047] 2. Experimental results

[0048] As shown in Figure 1B, the surface potential of BFO+ decreases from about 150 mV to about 17 mV in the height range of 30-1415 nm; the surface potential of BFO- decreases from about -150 mV to about -17 mV in the height range of 30-1415 nm. Both surface potentials show exponential decay characteristics, forming a gradient electric field.

[0049] Example 2

[0050] This example shows that the gradient electric field can promote the internal stress directional concentration of bone marrow mesenchymal stem cells.

[0051] 1. Experimental method

[0052] Bone marrow mesenchymal stem cells in the logarithmic growth phase were inoculated into culture dishes and placed in a 37°C, 5% CO2 incubator. The cells were cultured using bone marrow mesenchymal stem cell complete medium to a confluence of 70%-80%. The experiment was divided into the following three groups: BFO+ group, BFO- group and STO group (only substrate material, no surface potential, control group). The three groups of materials were placed in a 24-well plate, and then 400 μL / well of cell suspension was added. After 1 h of cell adhesion, Matrix gel was added to maintain a 3D culture environment, and the cells were cultured for 24 h.

[0053] 2. Detection method

[0054] After the cells were fixed with 4% paraformaldehyde, the cytoskeleton and chromatin were stained with phalloidin and DAPI, respectively. Laser confocal microscopy was used to scan and take pictures, and 3D images were formed by three-dimensional reconstruction to observe the morphology of the cytoskeleton.

[0055] 3. Experimental results

[0056] At 2 h and 6 h after inoculation, the F-actin skeleton showed rearrangement under the action of the gradient electric field. In the BFO+ group, the F-actin skeleton at the top of the cell showed a high abundance and reticular structure of cross-linking morphology, forming an internal stress in the cell. In the BFO- group, the F-actin skeleton at the bottom of the cell showed a high abundance and reticular structure of cross-linking morphology, forming an internal stress in the cell. In the control group, the F-actin skeleton was in a normal, relatively uniform distribution state.

[0057] Example 3

[0058] This example shows that the gradient electric field can promote the directional division of bone marrow mesenchymal stem cells.

[0059] 1. Experimental method

[0060] Bone marrow mesenchymal stem cells in the logarithmic growth phase were inoculated into culture dishes and placed in a 37°C, 5% CO2 incubator. The cells were cultured using bone marrow mesenchymal stem cell complete medium to a confluence of 70%-80%. The experiment was divided into the following three groups: BFO+ group, BFO- group and STO group (only substrate material, no surface potential, control group). The three groups of materials were placed in a 24-well plate, and then 400 μL / well of cell suspension was added. After 1 h of cell adhesion, Matrix gel was added to maintain a 3D culture environment, and the cells were cultured for 24 h.

[0061] 2. Detection method

[0062] After the cells were fixed with 4% paraformaldehyde, microtube specific antibody was used for incubation, and then labeled by fluorescent secondary antibody. In addition, Dil and DAPI were used to stain cell membrane and chromatin, respectively. Laser confocal microscope was used for scanning and shooting. The division direction of cells in mitosis was analyzed by spindle morphology and position, and chromatin separation direction.

[0063] 3. Experimental results

[0064] The immunofluorescence results are shown in FIG. 3, in which blue-white color marks chromatin, yellow color marks microtubule, and purple color marks cell membrane. The results show that the separation direction of cells in the BFO+ group is vertically deflected.

[0065] Example 4

[0066] This example shows that the gradient electric field can promote the directional growth of bone marrow mesenchymal stem cell organoids.

[0067] 1. Experimental method

[0068] First, the bone marrow mesenchymal stem cell suspension was added to AggreWell, and cultured for 3 days to prepare BMSCs organoids. Then, the organoids were inoculated on BFO+, BFO-, and STO groups, respectively. After adhesion for 1 day, Matrix gel was added to maintain the 3D culture environment. The culture was continued to the third day. Dil and DAPI were used to stain cell membrane and cell nucleus.

[0069] 2. Detection method

[0070] Laser confocal microscope was used to scan on Z axis, and then reconstruct 3D image to analyze the growth morphology of BMSCs organoids.

[0071] 3. Experimental results

[0072] As shown in FIG. 4, the biomimetic gradient electric field promotes the directional growth of BMSCs organoids. At 1 day after inoculation, limited cell budding occurs in the BFO+ group, the cell budding speed is faster in the BFO- group, and the STO group as a control shows moderate budding speed. At 3 days after inoculation, the cells in the BFO+ group no longer bud, but grow only in the vertical direction (the cells around the organoids are early shed cells proliferated), the BFO- group organoids accelerate budding, and the STO group organoids show normal budding morphology.

[0073] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; and although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacements for some of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for promoting the directed growth of an organism, characterized by, comprising subjecting the organism to a gradient electric field environment ranging from strong to weak.

2. The method for facilitating directed growth of an organism of claim 1, wherein, The organism comprises at least one of a single cell, a multicellular organism, a tissue, and an organoid.

3. The method for facilitating directed growth of an organism of claim 1, wherein, The directed growth comprises at least one of: cell division or proliferation along a direction of change in electric potential or a direction of a gradient electric field from high to low; tissue or organoid sprouting or growth along a direction of change in electric potential or a direction of a gradient electric field from high to low.

4. The method for facilitating directed growth of an organism of claim 1, wherein, The gradient electric field comprises an electric field whose magnitude of electric potential along a certain linear direction varies with spatial coordinates.

5. The method for facilitating directed growth of an organism of claim 1, wherein, The gradient electric field is generated by a surface electric potential that is upward.

6. The method for facilitating directed growth of an organism of claim 1, wherein, The gradient electric field is generated by internal domain polarization of a surface material.

7. The method for facilitating directed growth of an organism of claim 1, wherein, The gradient electric field has a spatial gradient variation in intensity that exponentially decays with distance from a surface of the material, wherein the surface electric potential varies in a range of 10-150 mV.

8. The method for facilitating directed growth of an organism of claim 1, wherein, The organism is an organoid, and the method comprises subjecting a cell or a population of cells to an adherent culture on a surface of a material having a positive electric potential, thereby causing the organoid to grow in a direction that is perpendicular or substantially perpendicular.

9. A culture system for promoting the directional growth of organisms, characterized in that, comprising a culture dish and a culture medium, wherein at least a portion of a surface of the culture dish in contact with the culture medium has a positive electric potential, thereby forming a gradient electric field in a direction that is perpendicular or substantially perpendicular to the surface having the positive electric potential.

10. An organism, characterized in that, prepared by the method of any one of claims 1-8.

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