Method for regulating and controlling chiral growth of cells

Abstract

The invention provides a cell chiral growth regulation and control method, and relates to the technical field of cell chiral culture. The regulation and control method provided by the invention comprises the following steps: carrying out full-exposure pre-shaping on a photo-crosslinking substrate, then inoculating cells to be subjected to chiral culture, carrying out adherent culture, then carrying out patterning exposure and continuous culture, and differentiating to obtain a cell chiral array; the photo-crosslinking substrate is provided with photo-crosslinking gel and a matched photoinitiator. In-situ regulation and control on cell chirality can be realized on the basis of the cell chirality growth direction through a double-exposure method, so that cell chirality arrays with different chirality orientations can be obtained after differentiation, and the structure and function of the heart and basic unit myocardial cells thereof can be known and simulated; the method is of great significance to cardiovascular disease research and drug screening.

Description

Technical Field

[0001] This invention relates to the field of cell chiral culture technology, and more particularly to a method for regulating cell chiral growth. Background Technology

[0002] Chiral growth of cells refers to the asymmetry exhibited by cells during growth and development, similar to that of chiral molecules. This asymmetry manifests in various aspects, including cell morphology, structure, physiological function, and the distribution of intracellular molecules. As the core organ of the human body, the heart's function depends on its unique anatomical structure. During embryonic development, chiral structures are gradually established through complex morphogenesis events ("C-loops," chiral arrangement of cardiomyocytes), ultimately resulting in the spatial asymmetry of the left and right ventricles. This allows the heart to efficiently distinguish between oxygenated and deoxygenated blood and propel blood circulation in a directional manner through coordinated myocardial contraction.

[0003] Studies have shown that the chiral arrangement of cardiomyocytes is the basis of cardiac contractile function. However, abnormal development of cardiac chirality or pathological remodeling (myocardial fibrosis) can lead to the loss of ventricular wall mechanical coordination, which can easily cause heart failure or arrhythmia. At the same time, once the development of the cardiac chiral pattern is incorrect, it is difficult to cooperate correctly with other organs. However, mature cardiomyocytes have almost no regenerative capacity, and functional repair after cardiac injury faces severe challenges. Therefore, constructing biomimetic cardiac models or functional myocardial tissues in vitro to simulate the chiral structure of the heart is of great significance for cardiovascular disease research and drug screening.

[0004] Existing technologies for achieving chiral regulation of cardiomyocytes mainly include drugs, electrical stimulation, construction of mechanical microenvironments, gene editing, and the application of biomaterials. Among these, constructing mechanical microenvironments can achieve synchronous regulation of the orientation and differentiation of myogenic progenitor cells, thus becoming a powerful means to improve the chiral regulation of cardiomyocytes. However, this method has poor flexibility in cell chiral culture and is difficult to achieve real-time in-situ regulation of cell orientation during the culture process. Therefore, there is an urgent need to provide a solution to improve the above problems. Summary of the Invention

[0005] The purpose of this invention is to provide a method for regulating cell chiral growth. By using a double-exposure method, in-situ regulation of cell chirality can be achieved based on the direction of cell chiral growth, thereby obtaining cell chiral arrays with different chiral orientations after differentiation.

[0006] The present invention provides a method for regulating chiral cell growth, comprising: pre-shaping a photocrosslinked substrate by full exposure, inoculating cells to be cultured chirally, performing adherent culture, patterning exposure, and continuous culture and differentiation to obtain a chiral cell array; wherein the photocrosslinked substrate has a photocrosslinked gel and a photoinitiator.

[0007] The chiral growth regulation method provided by this invention can adjust the local cross-linking degree of the photocrosslinked substrate through patterned exposure, thereby forming a topological microenvironment with undulating surfaces on the substrate surface. Furthermore, by performing patterned exposure after adherent culture, it is possible to regulate the chiral orientation at different stages of cell chiral growth, based on the direction of cell chiral growth. In addition, by adjusting the exposure shape of the patterned exposure, in-situ flexible regulation of cell chirality can be achieved, which is helpful in promoting the establishment of in vitro cardiac models and is of great significance for elucidating the pathogenesis of cardiovascular diseases and drug screening.

[0008] Optionally, the mass fraction of the photocrosslinking gel in the photocrosslinking substrate is 5%-15%.

[0009] Optionally, the mass fraction of the photoinitiator in the photocrosslinked substrate is 0.1%-1%.

[0010] Optionally, the photoinitiator includes Irgacure 2959.

[0011] Optionally, and / or, the photocrosslinking gel comprises methacrylic anhydride gelatin.

[0012] Optionally, the photocrosslinked substrate further comprises a biological buffer, and the biological buffer includes a phosphate buffer.

[0013] Optionally, the photocrosslinked substrate is fully exposed to light with a wavelength of 400nm-435nm; and / or, the photocrosslinked substrate is fully exposed for 3s-7s for pre-shaping.

[0014] Optionally, when inoculating cells to be chiralized, the process includes: mixing the cells to be chiralized with the cell culture medium before inoculation.

[0015] Optionally, after inoculating the cells to be chiralized, cell culture medium is added for cell culture.

[0016] Optionally, during the adherent culture, the initial cell density is (0.5-2)×10⁻⁶. 6 per mL.

[0017] Optionally, the cells to be chiralized include muscle cells; wherein the muscle cells include C2C12 cells.

[0018] Optionally, after patterning exposure and continuous culture until the cell density reaches 75%-85%, the cell differentiation medium is replaced to obtain a chiral cell array.

[0019] Optionally, the cells can be adhered to the culture vessel until the cell density reaches 50%-65% before patterning and exposure.

[0020] Optionally, after adhering culture, patterned exposure can be performed under light with a wavelength of 400nm-435nm.

[0021] Optionally, after adhering to the culture wall, patterning exposure can be performed for 8-12 seconds.

[0022] Optionally, patterned exposure is performed using a mask with a light-transmitting area and a light-blocking area, wherein the shape of the light-transmitting area is related to the morphology of cell chiral growth. Attached Figure Description

[0023] Figure 1 A schematic diagram illustrating the morphological changes of a photocrosslinked substrate during exposure, as provided by the present invention, regarding a method for regulating chiral cell growth.

[0024] Figure 2 This is a schematic flowchart of a method for regulating chiral cell growth provided in Embodiment 1 of the present invention;

[0025] Figure 3 This is a flowchart illustrating a method for regulating chiral cell growth provided in Embodiment 2 of the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0027] This invention provides a method for regulating chiral cell growth, comprising: pre-shaping a photocrosslinked substrate by full exposure, inoculating cells to be cultured chirally, performing adherent culture, patterning exposure, and continuous culture and differentiation to obtain a chiral cell array.

[0028] In fact, see Figure 1 By fully exposing the photocrosslinked substrate, a homogeneous substrate material with low crosslinking degree can be formed. Then, by secondary patterning exposure, the crosslinking degree of local areas of the substrate can be controlled. Furthermore, the substrate's water absorption and swelling effect can form an uneven pattern structure, thereby regulating the chiral growth of cells.

[0029] Specifically, the photocrosslinking substrate contains a photocrosslinking gel and a photoinitiator that works in conjunction with the photocrosslinking gel, and the photocrosslinking gel is crosslinked by initiating the crosslinking in an exposure environment. In some embodiments, the mass fraction of the photocrosslinking gel in the photocrosslinking substrate is 5%-15%, and the mass fraction of the photoinitiator is 0.1%-1%. In practice, the amount of photoinitiator in the photocrosslinking substrate is related to the photocrosslinking gel, and the photoinitiator is necessary to promote the crosslinking of the photocrosslinking gel.

[0030] Specifically, the photocrosslinking gel in the photocrosslinking substrate may include methacrylic anhydride gelatin, and the photoinitiator may include Irgacure 2959. In fact, the photocrosslinking substrate also contains a biological buffer, and the biological buffer used includes phosphate buffer. Furthermore, the photocrosslinking substrate can be pre-shaped by full exposure to 400nm-435nm (UV) light for 3s-7s.

[0031] In some embodiments, when inoculating cells to be chiralized, the cells can be pre-mixed with the cell culture medium before inoculation, or the cells can be pre-inoculated into a pre-shaped photocrosslinked substrate before adding cell culture medium for cell culture. In practice, the initial cell density during adherent culture after inoculation is (0.5-2) × 10⁻⁶. 6 per mL.

[0032] In some embodiments, the cells to be cultured for chiral formation include myocytes, specifically C2C12 cells. In practice, after adherent culture to a cell density of 50%-65%, patterning exposure is performed under light at a wavelength of 400nm-435nm for 8-12 seconds. Culture is then continued until the cell density reaches 75%-85%, at which point the cell differentiation medium is replaced to obtain a chiral cell array. Specifically, during patterning exposure, a mask with both transparent and opaque regions is used, and the shape of the transparent region is related to the morphology of chiral cell growth.

[0033] Specifically, this invention uses a fully exposed, pre-shaped, photocrosslinked substrate as a cell culture medium. Cells to be cultured chirally are seeded onto the substrate for adherent culture, and after adhering to a certain extent, they undergo patterned secondary exposure before differentiation culture to obtain a cell array with chiral orientation. In fact, by adjusting the timing of patterned exposure, the chiral orientation at different stages can be regulated based on the cell's own chiral growth direction. Furthermore, using patterned exposures of different shapes allows for in-situ, flexible control of cell chirality.

[0034] Example 1

[0035] See Figure 2 This embodiment 1 provides a method for regulating cell chiral growth, including:

[0036] S1. In a 37°C water bath and in the dark, methacrylic anhydride gelatin (GelMA) and Irgacure 2959 were stirred and dissolved in phosphate buffer to prepare a photocrosslinked substrate; wherein the substrate contained 10wt% GelMA and 0.5wt% Irgacure 2959.

[0037] S2. The photocrosslinked substrate is dropped onto a glass slide, and the thickness of the substrate is fixed to 100μm using a 100μm feeler gauge and a coverslip. Then, the substrate is fully exposed to ultraviolet light for 5s to pre-shape the substrate.

[0038] S3. Place the pre-shaped substrate into a 35mm culture dish containing PBS solution. After sterilizing in a biosafety cabinet for 30 minutes, wash three times with PBS buffer. Centrifuge the collected myoblast C2C12 cells at 1×10⁻⁶ ppm. 6 The cells were dispersed at a density of 1 / mL in C2C12 medium;

[0039] S4. Take 2m C2C12 cell dispersion and drop it onto the cleaned substrate. After culturing in a cell culture incubator for 2 hours, change the cell culture medium and culture in a cell culture incubator. When the density of C2C12 cells reaches 60% using an inverted fluorescence microscope, perform a second exposure for 10 seconds under a strip mask.

[0040] S5. After the substrate that has been exposed twice and inoculated with cells is washed three times with PBS buffer, new cell culture medium is injected and cultured in a cell culture incubator for 2 days. Then the cell culture medium is replaced with cell differentiation medium. The differentiation medium is replaced every other day. After 5 days of culture, a mature skeletal muscle myotube array with chiral orientation and contractile function is obtained.

[0041] Example 2

[0042] See Figure 3 This embodiment 2 provides a method for regulating cell chiral growth. The difference from embodiment 1 is that in step S4, when the density of C2C12 cells reaches 60%, a second exposure is performed under a ring mask for 10 seconds; in step S5, a ring myotube array with chiral orientation and contractile function, similar to a sphincter, is obtained.

[0043] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A method for regulating chiral cell growth, characterized in that, include: After the photocrosslinked substrate is fully exposed and pre-shaped, cells to be cultured chirally are inoculated, adhered to the substrate, patterned and exposed, and then continuously cultured and differentiated to obtain a chiral cell array; the photocrosslinked substrate has a photocrosslinked gel and a photoinitiator.

2. The control method according to claim 1, characterized in that: The photocrosslinking gel in the photocrosslinking substrate has a mass fraction of 5%-15%; and / or, the photoinitiator in the photocrosslinking substrate has a mass fraction of 0.1%-1%; and / or, the photoinitiator includes Irgacure 2959; and / or, the photocrosslinking gel includes methacrylic anhydride gelatin.

3. The control method according to claim 1, characterized in that: The photocrosslinked substrate also has a biological buffer solution, and the biological buffer solution includes a phosphate buffer solution; and / or, the photocrosslinked substrate is fully exposed to light with a wavelength of 400nm-435nm; and / or, the photocrosslinked substrate is fully exposed to light for 3s-7s for pre-shaping.

4. The control method according to claim 1, characterized in that, When inoculating cells intended for chiral culture, the following methods are included: pre-mixing the cells intended for chiral culture with cell culture medium before inoculation; or, adding cell culture medium after inoculation for cell culture; wherein, during the adherent culture, the initial cell density is (0.5-2)×10⁻⁶. 6 per mL.

5. The control method according to claim 1 or 4, characterized in that: The cells to be chiralized include muscle cells; wherein the muscle cells include C2C12 cells.

6. The control method according to claim 4, characterized in that, After patterning and exposure and continuous culturing until the cell density reaches 75%-85%, the cell differentiation medium is replaced to obtain a chiral cell array.

7. The control method according to claim 1, characterized in that: Patterning exposure is performed after adherent culture to a cell density of 50%-65%; and / or, patterning exposure is performed under light with a wavelength of 400nm-435nm after adherent culture; and / or, patterning exposure is performed for 8s-12s after adherent culture.

8. The control method according to claim 1, characterized in that: Patterned exposure is performed using a mask with a light-transmitting area and a light-blocking area, wherein the shape of the light-transmitting area is related to the morphology of cell chiral growth.

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