An organism and a method for promoting its directional growth

Abstract

The present invention discloses an organism and a method for promoting its directional growth. The organism includes at least one of a single cell, a multicellular body, a tissue and an organoid. The method includes the step of placing the organism in a gradient electric field environment from strong to weak. The present invention constructs a layered material with strontium titanate as a substrate, strontium rubidium oxide as a control layer and bismuth ferrite as an internal electric domain polarization layer, and generates a surface potential through the layered material, that is, a gradient electric field to achieve in vitro guided directional growth of the organism, thereby constructing an organoid whose morphology, structure and function highly simulate the corresponding tissue in vivo.

Description

Technical Field

[0001] The invention belongs to the field of organism culture, and in particular relates to an organism and a method for promoting the directional growth thereof. Background Art

[0002] Organoids are a type of model that is highly similar to in vivo source tissues or organs based on a 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 differentiated tissues and organs in vivo. The typical method for preparing organoids is to first isolate embryonic or pluripotent stem cells, and then culture them on a supporting medium (such as Matrigel) to enable them to grow in three dimensions. The application scenarios of organoids are very wide. They can be used for disease models, such as research on developmental-related problems, genetic diseases, tumors and cancers, and to achieve more effective and realistic testing of drug efficacy and toxicity. Compared with animal models, they largely avoid inconsistent test results due to differences between animal and human cells.

[0003] However, the organoids currently constructed in vitro have significant differences in morphology and structure from real tissues and organs. This is mainly manifested in the difficulty in guiding the directional growth of cell groups within organoids in the current in vitro environment, making their functions far different from those of the corresponding tissues in the body. Summary of the Invention

[0004] To address at least some of the issues mentioned above, the present invention uses layered materials to generate biomimetic gradient electric fields, guiding the directional growth of organisms in vitro and constructing organoids whose morphology, structure, and function closely mimic those of corresponding tissues in vivo. Specifically, the present invention encompasses the following.

[0005] In a first aspect, the present invention provides a method for promoting directional growth of an organism, comprising the steps of placing the organism in an electric field environment with a spatial gradient of electric potential from high to low.

[0006] In certain embodiments, according to the method for promoting directional growth of an organism according to the present invention, the organism comprises at least one of a single cell, a multicellular body, a tissue, and an organoid.

[0007] In certain embodiments, according to the method for promoting directional growth of an organism according to the present invention, the directional growth comprises at least one of the following:

[0008] Cells divide or proliferate along the direction of the electric potential change from high to low or along the direction of the gradient electric field;

[0009] Tissues or organoids sprout or grow along the direction of the electric potential change from high to low or along the gradient electric field.

[0010] In certain embodiments, according to the method for promoting directional growth of an organism of the present invention, the gradient electric field comprises an electric field in which the magnitude of the electric potential intensity along a certain straight line direction varies with the spatial coordinates.

[0011] In certain embodiments, according to the method for promoting directional growth of organisms of the present invention, the gradient electric field is generated by an upward surface potential.

[0012] In certain embodiments, according to the method for promoting directional growth of an organism according to the present invention, the gradient electric field is generated by polarization of electric domains inside the surface material.

[0013] In certain embodiments, according to the method for promoting directional growth of an organism of the present invention, the intensity of the gradient electric field changes in a spatial gradient that decays exponentially with the distance from the surface of the material.

[0014] In certain embodiments, according to the method for promoting directional growth of organisms of the present invention, the electric potential changes in the range of 10-150 mV.

[0015] In certain embodiments, according to the method for promoting directional growth of an organism according to the present invention, wherein the organism is an organoid, the method comprises culturing cells or cell populations on the surface of a material with a positive potential, thereby causing the organoid to grow in a vertical or approximately vertical direction.

[0016] In a second aspect of the present invention, a culture system for promoting the directional growth of organisms is provided, which includes a culture dish and a culture medium, wherein at least a portion of the surface of the culture dish in contact with the culture medium has a positive potential, thereby forming a gradient electric field in a direction perpendicular or approximately perpendicular to the surface with the positive potential.

[0017] The third aspect of the present invention provides an organism prepared by the method of the present invention.

[0018] The present invention constructs a layered material with strontium titanate as a substrate, strontium rubidium oxide as a control layer, and bismuth ferrite as an internal electric domain polarization layer. The layered material generates a surface potential, that is, a gradient electric field, to achieve in vitro guided directional growth of organisms, thereby constructing organoids whose morphology, structure, and function highly simulate those of corresponding tissues in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of using bismuth ferrite biomaterial to generate surface potential to form a biomimetic gradient electric field, where: Figure 1A is a schematic diagram of the structure of a biomaterial with surface potential, BFO is bismuth ferrite (BiFeO3), LSMO is lanthanum strontium manganese oxide (LaSrMnO3), STO is strontium titanate (SrTiO3), SRO is strontium rubidium oxide, STO is the substrate, SRO and LSMO are used to control the polarization direction of BFO, BFO+ is the upward surface potential, and BFO- is the downward surface potential; Figure 1 B and C are the spatial gradient variation characteristics of the surface potential of BFO+ and BFO- materials, respectively, which decay exponentially with height.

[0020] Figure 2 The results show that the biomimetic gradient electric field promotes the directional concentration of intracellular stress. At 2 h and 6 h after inoculation, the F-actin skeleton was rearranged under the action of the gradient electric field. The F-actin skeleton at the top of the cells in the BFO+ group and at the bottom of the cells in the BFO- group showed a high abundance and formed a cross-linked reticular structure, forming directional internal stress within the cells. The stress in the cells of the BFO+ group was concentrated at the top, and the stress in the cells of the BFO- group was concentrated at the bottom. The F-actin skeleton of the cells in the control group was in a normal and relatively uniform distribution state.

[0021] Figure 3 The figure shows how the bionic gradient electric field induces directional cell division, where A, B, and C are images of the directional division of cells in each group at metaphase, anaphase, and telophase, respectively; and D is an analysis of the division of cells in each group at anaphase and telophase. In metaphase and anaphase of mitosis, the cell division direction of the BFO+ group is vertical, while that of the BFO- and STO groups is mainly horizontal. In telophase of mitosis, the two daughter cells of the BFO+ group separate vertically, while the two daughter cells of the BFO- and STO groups separate horizontally.

[0022] Figure 4 The data show that the biomimetic gradient electric field promotes the directional growth of BMSCs organoids. Among them, on the first day of inoculation, limited cell budding occurred in the BFO+ group, the cells in the BFO- group sprouted faster, and the STO group, as a control, showed a moderate sprouting speed. On the third day of inoculation, the cells in the BFO+ group no longer sprouted and only grew in the vertical direction (the cells around the organoids were the proliferation of early shed cells), the organoids in the BFO- group sprouted faster, and the organoids in the STO group had a normal sprouting morphology. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0026] method

[0027] One aspect of the present invention provides a method for promoting the directional growth of an organism in vitro, which comprises the step of placing the organism in a gradient electric field environment.

[0028] In the present invention, the organism comprises at least one of a single cell, a multicellular body, a tissue, and an organoid. A multicellular body comprises a plurality of living cells adhered to one another. In a preferred embodiment, the organism is a single cell, and specific examples of single cells include, but are not limited to, immortalized cells, primary cells, and stem cells. In another preferred embodiment, the organism is an organoid.

[0029] In the present invention, the directional growth includes at least one of the following: cells or cell colonies divide or proliferate in a direction from high to low positive potential or in the direction of the electric field; tissues or organoids grow or sprout in a direction from high to low positive potential or in the direction of the electric field. In a preferred embodiment, directional growth refers to the growth of cells, cell colonies, tissues or organoids in a direction perpendicular or approximately perpendicular to a surface with a positive potential, or the growth of cells, cell colonies, tissues or organoids in a direction away from the surface of a material with a positive potential (i.e., the direction of the electric field) and perpendicular or approximately perpendicular to the surface of a material with a positive potential.

[0030] In the present invention, the gradient electric field includes an electric field in which the magnitude of the electric potential along a certain straight line varies with the spatial coordinates. In a preferred embodiment, the gradient electric field is generated by an upward surface potential.

[0031] In a specific embodiment of the present invention, the gradient electric field is generated by a material having a surface potential, and the electric field exhibits a spatially varying gradient that decays exponentially with distance from the material surface. In a preferred embodiment, the range of distance variation is less than 1500 nm from the material surface, for example, 0-1500 nm. In a preferred embodiment, the range of the potential gradient that varies with distance is 10-150 mV.

[0032] In a preferred embodiment, the surface potential decreases from 150 mV to 17 mV with increasing height in the height range of 30 to 1415 nm.

[0033] Without being bound by any theory, any material capable of generating a gradient electric field on its surface that decreases with increasing height can be used in the present invention. In a preferred embodiment, the gradient electric field is generated by a layered material with internal domain polarization, and a surface potential that decreases with increasing height is formed above the layered material. In a more preferred embodiment, the gradient electric field is generated by a layered material with a positive surface potential, and the layered material is a layered material that generates a surface potential and has a gradient electric field that decreases from 150 mV to 17 mV with increasing height (or when away from the positive potential side of the layered material).

[0034] In a preferred embodiment, the method for promoting directional growth of an organism of the present invention comprises culturing cells or cell populations on a surface of a material with a positive potential, thereby causing organoids to grow in a vertical or substantially vertical direction, wherein the material surface comprises, in sequence along the thickness direction, an internal domain polarization layer, a control layer, and a substrate layer. In a preferred embodiment, the internal domain polarization layer is BFO, the control layer is SRO, and the substrate layer is STO.

[0035] It should be understood that the gradient electric field in the present invention is an electrostatic field, a constant electric field, generated by surface potential, with a spatial gradient and a fixed direction. Unlike a DC electric field or a capacitive electric field, the gradient electric field in the present invention does not change in frequency or direction.

[0036] Training system

[0037] One aspect of the present invention provides a culture system for promoting directional growth of organisms, comprising a culture dish and a culture medium, wherein at least a portion of the surface of the culture dish in contact with the culture medium has a surface potential, thereby forming a gradient electric field perpendicular to the surface having 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 conventional cell or organoid culture dish and is not particularly limited to this.

[0038] organism

[0039] One aspect of the present invention provides an organism produced by the methods described herein. In a preferred embodiment, the organism is an organoid. The organoids of the present invention can closely mimic their in vivo counterparts in morphology, structure, and function.

[0040] The present invention also provides materials capable of generating a gradient electric field environment, particularly materials comprising, in sequence along the thickness direction, an internal electric domain polarization layer, a control layer, and a substrate layer, for use in preparing biological organisms. In a preferred embodiment of the present invention, the internal electric domain polarization layer of the material is BFO, the control layer is SRO, and the substrate layer is STO.

[0041] Example 1

[0042] This embodiment illustrates the process of constructing a biomimetic gradient electric field using ferroelectric materials.

[0043] 1. Experimental methods

[0044] In this embodiment, BFO (BiFeO3) is deposited on 5mm×5mm strontium titanate (SrTiO3, STO) by laser pulse deposition. 1 / 3 Sr 2 / 3 MnO3), by generating different covalent mismatches and thus generating surface potentials in two directions, where BFO+ is upward and BFO- is downward, such as Figure 1 As shown in A. Kelvin probe force microscopy (KPFM) is then used to detect the surface potential intensity at different heights on the material surface.

[0045] 2. Experimental results

[0046] like Figure 1 As shown in Figure B, the surface potential of BFO+ decreases from approximately 150 mV to approximately 17 mV over a height range of 30-1415 nm; the surface potential of BFO- decreases from approximately -150 mV to approximately -17 mV over a height range of 30-1415 nm. Both surface potentials exhibit exponential decay, forming a gradient electric field.

[0047] Example 2

[0048] This embodiment shows that the gradient electric field can induce bone marrow mesenchymal stem cells to generate directional internal stress concentration.

[0049] 1. Experimental methods

[0050] Bone marrow mesenchymal stem cells in the logarithmic growth phase were seeded into culture dishes and placed in an incubator at 37°C with 5% CO2. The cells were cultured in complete BMSC medium until they reached 70%-80% confluency. The experiment was divided into three groups: BFO+, BFO-, and STO (substrate material only, without surface potential, control). Each of the three groups was plated into a 24-well plate, and 400 μL / well of the cell suspension was added. The cells were cultured for 2 and 6 hours.

[0051] 2. Detection method

[0052] After the cells were fixed with 4% paraformaldehyde, the cytoskeleton and chromatin were stained with phalloidin and DAPI, respectively. The cells were scanned and photographed using a laser confocal microscope, and three-dimensional reconstruction was performed to form 3D images and observe the cytoskeleton morphology.

[0053] 3. Experimental results

[0054] At 2 and 6 hours after inoculation, the F-actin skeleton rearranged under the action of a gradient electric field. The F-actin skeleton showed a high abundance and a cross-linked network structure at the top of the BFO+ group and at the bottom of the BFO- group, generating directional internal stress within the cells. The stress was concentrated at the top of the BFO+ group and at the bottom of the BFO- group. In the control group, the F-actin skeleton maintained a normal, relatively uniform distribution.

[0055] Example 3

[0056] This example shows that the gradient electric field can promote the directional division of bone marrow mesenchymal stem cells.

[0057] 1. Experimental methods

[0058] Bone marrow mesenchymal stem cells in the logarithmic growth phase were seeded into culture dishes and placed in an incubator at 37°C with 5% CO2. The cells were cultured in complete bone marrow mesenchymal stem cell culture medium until they reached 70%-80% confluency. The experiment was divided into three groups: BFO+, BFO-, and STO (substrate material only, without surface potential, control). All three groups were placed in a 24-well plate, and 400 μL / well of the cell suspension was added. After the cells adhered for 1 hour, matrix gel was added to maintain the 3D culture environment and culture was continued for up to 24 hours.

[0059] 2. Detection method

[0060] The cells were fixed with 4% paraformaldehyde, incubated with microtube-specific antibodies, and then labeled with fluorescent secondary antibodies. In addition, Dil and DAPI were used to stain the cell membrane and chromatin, respectively. The cells were scanned and photographed under a laser confocal microscope. The direction of cell division in mitosis was analyzed by the spindle morphology and position and the direction of chromatin separation.

[0061] 3. Experimental results

[0062] Immunofluorescence results Figure 3 As shown in the figure, the blue and white marks are chromatin, the yellow marks are microscopic proteins, and the purple marks are cell membranes. The results showed that the separation direction of cells in the BFO+ group was vertically deflected.

[0063] Example 4

[0064] This example shows that a gradient electric field can promote the directional growth of bone marrow mesenchymal stem cell organoids.

[0065] 1. Experimental methods

[0066] First, a bone marrow mesenchymal stem cell suspension was added to AggreWell and cultured for three days to generate BMSC organoids. These organoids were then seeded onto BFO+, BFO-, and STO substrates. After one day of adhesion, matrix gel was added to maintain the 3D culture environment. Culture was continued until day three. Cell membranes and nuclei were stained with DiI and DAPI.

[0067] 2. Detection method

[0068] Laser confocal microscopy was used to perform layer scanning on the Z axis and then reconstruct it into a 3D image to analyze the growth morphology of BMSCs organoids.

[0069] 3. Experimental results

[0070] like Figure 4 As shown, the biomimetic gradient electric field promotes the directional growth of BMSCs organoids. On day one, limited cell budding was observed in the BFO+ group, while cells in the BFO- group sprouted rapidly. The STO control group, however, exhibited a moderate sprouting rate. On day three, cells in the BFO+ group ceased sprouting and grew only in a vertical direction (the cells surrounding the organoids were proliferating from cells that had shed earlier). Organoids in the BFO- group sprouted more rapidly, while those in the STO group displayed normal sprouting morphology.

[0071] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified or some of the technical features thereof may be replaced with equivalents. Such modifications or replacements do not deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for promoting directional growth of an organism, characterized in that: The method comprises the steps of placing the organism in a gradient electric field environment from strong to weak and adding Matrix gel to maintain three-dimensional culture, wherein the gradient electric field is generated by a layered material with a positive surface potential, wherein the layered material has strontium titanate as a substrate, strontium rubidium oxide as a regulating layer and bismuth ferrite as an internal electric domain polarization layer, and the intensity of the gradient electric field changes in a spatial gradient with an exponential decay as the distance from the material surface changes, and the potential range of the gradient change is 10-150 mV; the organism comprises bone marrow mesenchymal stem cells or organoids formed therefrom, and the method comprises placing the organism on the surface of a material with a positive potential for adherent culture, so that the organism grows in a direction perpendicular or approximately perpendicular to the surface with a positive potential, and the directional growth comprises at least one of the following: cells divide or proliferate in a direction of a potential change from high to low or in a direction of a gradient electric field; and organoids sprout or grow in a direction of a potential change from high to low or in a direction of a gradient electric field.

2. A use of a culture system for promoting directional growth of an organism, characterized in that: The culture system comprises Matrix gel, a culture dish and a culture medium, wherein at least a portion of the surface of the culture dish in contact with the culture medium has a positive potential, thereby forming a gradient electric field in a direction perpendicular or approximately perpendicular to the surface with the positive potential, and the gradient electric field is generated by a layered material with a positive surface potential, wherein the layered material has strontium titanate as a substrate, strontium rubidium oxide as a regulating layer and bismuth ferrite as an internal electric domain polarization layer; the organism is in a gradient electric field environment from strong to weak and is maintained in a three-dimensional culture environment of Matrix gel, and the organism is cultured on the surface of the material with a positive potential, so that the organism grows in a direction perpendicular or approximately perpendicular to the surface with a positive potential, and the directional growth includes at least one of the following: cells divide or proliferate in a direction of a potential change from high to low or in a gradient electric field direction; organoids sprout or grow in a direction of a potential change from high to low or in a gradient electric field direction, and the intensity of the gradient electric field changes in a spatial gradient exponentially attenuating with the distance from the material surface, and the potential range of the gradient change is 10-150 mV; the organism includes bone marrow mesenchymal stem cells or organoids formed therefrom.

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