Plastic reverse-age cell as well as preparation method and application thereof
By continuously extruding the cells with microfluidic chips, the high expression of Oct4 gene is induced, which solves the problems of poor cell age effect and low reprogramming efficiency in the prior art, and achieves efficient cell age and direct reprogramming effects.
Patent Information
- Application Number
- CN202411345041.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-25
- Publication Date
- 2025-01-03
AI Technical Summary
The prior art has poor effect on cell age and is inefficient in reprogramming, making it difficult to effectively restore cells to a young state.
The cells are continuously squeezed through at least two microfluidic chips, gradually reducing the size of the microchannel, inducing high expression of Oct4 gene in somatic cells, and achieving the plasticity of the cells.
The direct reprogramming efficiency was improved to more than 90% in mice age of 20 months, significantly improving the anti-age effect of cells.
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Figure CN119242571A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of medical technology, and in particular, to a plastic anti-aging cell and a preparation method and application thereof. Background Art
[0002] Reversing cell age refers to the process of restoring cells from a relatively old or aged state to a younger state. Cells that achieve anti-aging can be used in many aspects, such as cell therapy, organ welding under special environments, organ printing, etc. Some researchers use different mechanical means to improve the plasticity of somatic cells, promote gene editing efficiency or achieve aging reversal. In addition, a small number of studies have confirmed that by stacking cells and activating the cell stemness factor Oct4 to a certain extent, cell age can be reversed to a certain extent. However, the cell anti-aging effect of the above traditional technologies is poor. Summary of the invention
[0003] The present application provides a plastic anti-aging cell and a preparation method and application thereof. The preparation method has a good cell anti-aging effect and a high direct reprogramming efficiency.
[0004] This application is implemented as follows: In a first aspect, the present application provides a method for preparing plastic anti-aging cells, comprising the following steps: The cells are squeezed through at least two microfluidic chips in sequence and then cultured to obtain plastic anti-aging cells; The microchannel size of each microfluidic chip is different, and the microchannel size of the microfluidic chips passed in sequence gradually decreases; the inner diameter of the channel of the microfluidic chip gradually decreases from ≥1 / 3 of the cell diameter to ≤1 / 5 of the cell diameter.
[0005] In a possible implementation manner, the microchannel of the microfluidic chip is square.
[0006] In a possible implementation manner, the widths of two adjacent microfluidic chips for squeezing the cells differ by 1 to 3 μm.
[0007] In one possible embodiment, the cells include at least one of somatic cells and mesenchymal stem cells.
[0008] In one possible embodiment, the somatic cells include at least one of skin fibroblasts, immune cells and cardiomyocytes.
[0009] In a possible embodiment, the somatic cells include skin fibroblasts, and the skin fibroblasts are extruded through the three microfluidic chips, and the microchannel sizes of the three microfluidic chips are 7×13 μm, 5×13 μm, and 3×13 μm, respectively.
[0010] In a possible implementation, the cells are squeezed sequentially through at least two microfluidic chips so that the cells undergo 80-90% deformation.
[0011] In a possible embodiment, before injecting the cells into the microchannel of the microfluidic chip, the cells are first digested and suspended using trypsin; after the cells are squeezed using the microfluidic chip, the squeezed cells are cultured in a culture dish coated with matrix gel.
[0012] In a second aspect, the present application provides a plastic anti-aging cell, which is prepared by the preparation method of the plastic anti-aging cell of the first aspect.
[0013] In a third aspect, the present application provides a use of the plastic anti-aging cells according to the second aspect in the preparation of organ repair materials, anti-aging biomaterials, cell therapy materials, and drug screening materials.
[0014] This application has at least the following beneficial effects: In the preparation method of the plastic anti-aging cells of the present application, the high expression of the Oct4 gene in somatic cells can be induced by continuous squeezing of the cells, which has a good cell anti-aging effect. Experiments have shown that 20-month-old mice (about 80-year-old humans) can be reversed to within 3 months of age, and the direct reprogramming efficiency of the cells can be increased to more than 90%. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.
[0016] Figure 1 The relative value detection results of DNA methylation levels of age-related genes in fibroblasts before reprogramming and reversibly aged cells after reprogramming in Comparative Example 1 of the present application; Figure 2 This is a diagram showing the direct reprogramming of fibroblasts into neuronal cells in Comparative Example 1; Figure 3 This is a picture of the reversibly aged cells obtained after extrusion culture in Example 1; Figure 4 The test results of active oxygen levels of the untreated fibroblasts and the prepared plastic anti-aging cells in Example 1; Figure 5The test results of the cell autophagy level of the untreated fibroblasts and the prepared plastic anti-aging cells in Example 1; Figure 6 The test results of the autophagy levels of the untreated macrophages and the prepared plastic anti-aging cells in Example 2; Figure 7 These are the test results of the autophagy levels of the untreated cardiomyocytes and the prepared plastic anti-aging cells in Example 3. DETAILED DESCRIPTION
[0017] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0018] The following is a specific description of a plastic anti-aging cell and its preparation method and application according to an embodiment of the present application: The present application provides a method for preparing plastic anti-aging cells, comprising the following steps: The cells are squeezed through at least two microfluidic chips in sequence and then cultured to obtain plastic anti-aging cells; for example, there are two, three, four or more microfluidic chips.
[0019] The microchannel size of each microfluidic chip is different, and the microchannel size of the microfluidic chips passed in sequence gradually decreases; the inner diameter of the channel of the microfluidic chip gradually decreases from ≥1 / 3 of the cell diameter to ≤1 / 5 of the cell diameter.
[0020] The cells are pushed to move in the microchannels of the microfluidic chip by the pressure generated by the pressure pump and syringe equipped in the microfluidic chip.
[0021] The preparation method of the plastic anti-aging cells of the present application sequentially squeezes the cells through at least two microfluidic chips, and the microchannel size of the sequentially passed microfluidic chips gradually decreases, that is, the cells are subjected to multiple consecutive instantaneous nuclear deformations, and each squeezing intensity is stronger than the previous squeezing, which can induce high expression of the Oct4 gene in somatic cells, and has a good cell anti-aging effect. Experiments have shown that 48-month-old mice (about 80-year-old humans) can be reversed to within 3 months of age, and the direct reprogramming efficiency of the cells can be increased to more than 90%.
[0022] Exemplarily, the cells are squeezed through at least two microfluidic chips in sequence so that the cells are deformed by 80-90%. The inventors of the present application found in the study that if the cells are deformed by more than 90%, the cells will die, and if the deformation of the cells is less than 80%, although the anti-aging effect can be achieved, the effect is not significant. Optionally, the cells are squeezed through at least two microfluidic chips in sequence so that the cells are deformed by 80%, 83%, 85%, 88% or 90%. It should be noted that the deformation of the cells by 80-90% means that the deformation ability of the cells increases after squeezing, and they can pass through a channel of 10-20% of the original cell volume.
[0023] Optionally, in the embodiment of the present application, the cells used include at least one of somatic cells and mesenchymal stem cells. Somatic cells include at least one of skin fibroblasts, immune cells and cardiomyocytes. Optionally, immune cells include one of macrophages and T cells. The inventors of the present application have found that, taking the direct reprogramming of skin fibroblasts into neuronal cells as an example, the direct reprogramming efficiency of this method is about 3%, and a single extrusion of a microfluidic chip can promote the direct reprogramming efficiency to about 20%. The present application improves the plasticity of skin fibroblasts by performing multiple consecutive transient nuclear deformations on the cells, and each extrusion intensity is stronger than the previous extrusion, and the direct reprogramming efficiency of the cells can be increased to more than 90%. Among them, direct reprogramming efficiency = number of neuronal cells / number of cells inoculated at the beginning of the experiment.
[0024] In some embodiments, the microchannel of the microfluidic chip is square, and the width of two adjacent microfluidic chips for squeezing cells differs by 1 to 3 μm. It should be noted that the microchannel size of the microfluidic chip is different according to the diameter of the cells to be squeezed. For example, when the cells are skin fibroblasts, the skin fibroblasts are squeezed through three microfluidic chips, and the microchannel sizes of the three microfluidic chips are 7×13μm, 5×13μm, and 3×13μm, respectively.
[0025] Before injecting the cells into the microchannel of the microfluidic chip, the cells were first digested and suspended with trypsin, and after squeezing the cells with the microfluidic chip, the squeezed cells were cultured in a culture dish coated with matrix gel. After the last squeezing, the squeezed cells were cultured in a culture dish coated with matrix gel (Matrigel), and iPS culture medium was used for culture. After the remaining squeezing, the squeezed cells were cultured in a culture dish coated with matrix gel (Matrigel), and DMEM+10%FBS+1%P / S culture medium was used for culture.
[0026] In a second aspect, the present application provides a plastic anti-aging cell, which is prepared by the above-mentioned method for preparing the plastic anti-aging cell.
[0027] The plastic anti-aging cells prepared by the preparation method of plastic anti-aging cells are somatic cells with extremely high plasticity but without stemness. Such cells will not easily differentiate, and will not rapidly proliferate indefinitely to induce cancer. Based on the plastic anti-aging cells of this application, personalized cell therapy can be performed for patients, organ welding and organ printing can be achieved under special environments, and efficient multi-lineage drug screening can be achieved.
[0028] In a third aspect, the present application provides an application of the plastic anti-aging cells of the second aspect in the preparation of organ repair materials, anti-aging biomaterials, cell therapy materials, and drug screening materials.
[0029] The plastic anti-aging cells of this application can realize tissue and organ printing and welding of damaged organs. This feature is particularly important in the field of aerospace medicine. It is difficult to quickly repair the organs of astronauts in the space station after the tissues and organs are damaged, which endangers the lives of astronauts. However, a large number of highly plastic cells can be obtained in a short time through the preparation method of this application (or highly plastic cells can be prepared for astronauts in advance), and the cells come from the astronauts themselves and will not produce foreign body reactions. At this time, the required cells of damaged organs can be directly edited and injected on orbit and quickly to achieve remote organ welding and repair.
[0030] Moreover, on the one hand, the plastic anti-aging cells of the present application can be used to replace senescent cells to achieve tissue anti-aging. On the other hand, the biological products (such as EVs, supernatant products, etc.) extracted from the anti-aging cells can be injected into the blood to achieve systemic rejuvenation.
[0031] The following is a further detailed description of a plastic anti-aging cell of the present application and its preparation method and application in conjunction with the embodiments. Example
[0032] This embodiment provides a method for preparing plastic anti-aging cells, comprising the following steps: (1) After skin-derived mouse fibroblasts were digested and suspended with trypsin, the fibroblasts (diameter of about 20 μm) were squeezed through the microchannel of the first microfluidic chip, and the cross-sectional size of the microchannel of the first microfluidic chip was 7×13 μm (width*height); then they were cultured in a Matrigel-coated culture dish with DMEM+10% FBS+1% P / S culture medium for 12 hours.
[0033] (2) After the cultured fibroblasts in step (1) are digested and suspended with trypsin, the fibroblasts are squeezed through the microchannel of the second microfluidic chip, where the cross-sectional size of the microchannel of the second microfluidic chip is 5×13 μm (width×height); and then cultured in a Matrigel-coated culture dish with DMEM+10% FBS+1% P / S culture medium for 12 hours.
[0034] (3) The cultured fibroblasts in step (2) were digested and suspended with trypsin, and then squeezed through the microchannel of the third microfluidic chip, the cross-sectional size of the microchannel of the third microfluidic chip was 3×13 μm (width×height), and the deformation of the obtained cells was 85%; then they were cultured in a Matrigel-coated culture dish for 5 days, and then cultured with iPS culture medium, and the medium was changed every other day. After 5 days of culture, plastic anti-aging cells were obtained. Example
[0035] The preparation method of this embodiment is different from that of embodiment 1 in that the fibroblasts in embodiment 1 are replaced by macrophages (with a diameter of about 30 μm), and finally the cell deformation is 80%. Example
[0036] The preparation method of this embodiment is different from that of embodiment 1 in that the fibroblasts in embodiment 1 are replaced with cardiomyocytes (with a diameter of about 25 μm), and the deformation of the cells finally obtained is 82%.
[0037] Comparative Example 1 Comparative Example 1 provides a method for preparing plastic anti-aging cells, which directly reprograms skin-derived fibroblasts using CRISPR / Cas9 knock-in technology, and detects cell age using the epigenetic clock as a standard, and can reverse the age of 20-month (20M) mice (approximately 80-year-old humans) to 2-3 (3M) months old. The results are as follows Figure 1 As shown. Among them, Figure 1 The vertical axis represents the relative value of the DNA methylation level of age-related genes, where 20M corresponds to the experimental results of fibroblasts before reprogramming, and 3M represents the experimental results of plastic anti-aging cells after reprogramming. 20M represents 20 months of age, and 3M represents 3 months of age.
[0038] in addition, Figure 2 This is a diagram showing the direct reprogramming of fibroblasts into neuronal cells in Comparative Example 1. Figure 3 These are cells obtained after squeezing and culturing. The green part in the picture is a nerve cell. Figure 2 and Figure 3 , you can know Figure 3 The proportion of neurons in Figure 2This indicates that the method of Example 1 of the present application greatly improves the direct reprogramming efficiency of cells.
[0039] Test Example 1 The untreated fibroblasts and the obtained plastic anti-aging cells in Example 1 were tested for ROS (reactive oxygen species) level and cell autophagy level, and the results were as follows: Figure 4 and Figure 5 As shown, the horizontal axis 20M (month) represents 20 months of age, and 3M represents 3 months of age.
[0040] The fibroblasts not treated in Example 1 correspond to the result with the horizontal axis of 20 months (month), and the plastic anti-aging cells prepared in Example 1 correspond to the result with the horizontal axis of 3 months. Figure 4 and Figure 5 From the results of ROS (reactive oxygen species) levels and cellular autophagy levels, it can be seen that the age of the untreated fibroblasts is 20 months old, and the age of the plastic anti-aging cells prepared in Example 1 is 3 months old, which shows that the method of Example 1 can reverse the age of 20-month-old mice (about 80-year-old humans) to 3 months old.
[0041] Test Example 2 The untreated macrophages and the obtained plastic anti-aging cells of Example 2, and the untreated cardiomyocytes and the obtained plastic anti-aging cells of Example 3 were subjected to DNA methylation levels of age-related genes, and the results were as follows: Figure 6 and Figure 7 As shown. Among them, Figure 6 and Figure 7 The vertical axis represents the relative value of DNA methylation level of age-related genes, where 20M corresponds to the experimental results of macrophages or cardiomyocytes before reprogramming, and 3M represents the experimental results of plastic anti-aging cells after reprogramming. 20M represents 20 months of age, and 3M represents 3 months of age.
[0042] according to Figure 6 and Figure 7 From the results, it can be seen that the age of the untreated macrophages and cardiomyocytes is 20 months old, and the plastic anti-aging cells prepared in Example 2 and Example 3 are 3 months old, which shows that the methods of Example 2 and Example 3 can reverse the age of 20-month-old mice (about 80-year-old humans) to 3 months old.
[0043] The above description is only a specific embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A method for preparing plastic anti-aging cells, characterized in that: The following steps are involved: The cells are squeezed through at least two microfluidic chips in sequence and then cultured to obtain plastic anti-aging cells; The microchannel size of each microfluidic chip is different, and the microchannel size of the microfluidic chips passed in sequence gradually decreases; the inner diameter of the channel of the microfluidic chip gradually decreases from ≥1 / 3 of the cell diameter to ≤1 / 5 of the cell diameter.
2. The method for preparing plastic anti-aging cells according to claim 1, characterized in that: The microchannel of the microfluidic chip is square.
3. The method for preparing plastic anti-aging cells according to claim 2, characterized in that: The widths of the two adjacent microfluidic chips for squeezing the cells differ by 1 to 3 μm.
4. The method for preparing plastic anti-aging cells according to any one of claims 1 to 3, characterized in that: The cells include at least one of somatic cells and mesenchymal-like stem cells.
5. The method for preparing plastic anti-aging cells according to claim 4, characterized in that: The somatic cells include at least one of skin fibroblasts, immune cells and cardiomyocytes.
6. The method for preparing plastic anti-aging cells according to claim 4, characterized in that: The somatic cells are skin fibroblasts, which are squeezed through the three microfluidic chips, and the sizes of the microchannels of the three microfluidic chips are 7×13 μm, 5×13 μm, and 3×13 μm, respectively.
7. The method for preparing plastic anti-aging cells according to any one of claims 1 to 3, characterized in that: The cells are squeezed by at least two microfluidic chips in sequence, causing the cells to deform by 80-90%.
8. The method for preparing plastic anti-aging cells according to any one of claims 1 to 3, characterized in that: Before injecting the cells into the microchannel of the microfluidic chip, the cells are first digested and suspended by using trypsin; after the cells are squeezed by using the microfluidic chip, the squeezed cells are cultured in a culture dish coated with matrix gel.
9. A plastic anti-aging cell, which is prepared by the preparation method of the plastic anti-aging cell according to any one of claims 1 to 8.
10. Use of the plastic anti-aging cells according to claim 9 in the preparation of organ repair materials, anti-aging biomaterials, cell therapy materials, and drug screening materials.