Isolated culture method of adipose-derived stem cells
The method of isolating and culturing adipose-derived stem cells by pretreatment with acetic acid has solved the problem of low survival rate of adipose-derived stem cells, improved their survival rate and proliferation capacity in vivo, and enhanced their application effect in tissue repair and regeneration therapy.
Patent Information
- Application Number
- CN202411523834.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-30
- Publication Date
- 2025-01-10
AI Technical Summary
In current technologies, the survival rate of adipose-derived stem cell transplantation is low, there is a lack of effective strategies to simulate and improve the in vivo survival environment, and the issues of biocompatibility of biomaterials and stability of growth factors have not been effectively resolved.
An acetic acid-pretreated adipose-derived stem cell isolation and culture method, including digestion, filtration, centrifugation, culture, and passage, utilizes the protective effect of acetic acid under cellular stress conditions to simulate the in vivo microenvironment and improve cell adaptability.
Acetic acid pretreatment significantly improved the stress resistance and survival rate of adipose stem cells, enhancing their application potential in tissue repair and regenerative therapy. Pretreatment with acetic acid significantly promoted cell proliferation and the stability of cell migration and distribution in vivo.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of biomedicine and regenerative medicine, and in particular to a method for isolating and culturing adipose stem cells. Background Art
[0002] With the rapid development of regenerative medicine, adipose-derived stem cells (ADSCs) have become ideal candidate cells for tissue repair and regenerative therapy due to their multidirectional differentiation potential, self-renewal ability and low immunogenicity. ADSCs have shown great application potential in the treatment of soft tissue defects, cardiovascular diseases, neurodegenerative diseases, etc. However, the low survival rate of ADSCs after transplantation is the main bottleneck limiting its clinical application. At present, strategies to improve the survival rate of ADSCs in vivo mainly focus on improving the cell microenvironment, promoting angiogenesis, and reducing cell apoptosis. Commonly used methods include using biomaterials as cell carriers, applying growth factors, and optimizing transplantation techniques. Although these methods have improved the survival rate of ADSCs to a certain extent, there are still many challenges, such as the biocompatibility of biomaterials, the stability of growth factors, and the delivery efficiency.
[0003] Existing technologies have some defects in improving the survival rate of ADSCs in vivo, such as the biocompatibility and immunogenicity issues of biomaterials as cell carriers, the stability and delivery efficiency of growth factors, the lack of in-depth understanding of the in vivo survival microenvironment of ADSCs, and the lack of effective strategies to simulate and improve the in vivo survival environment of ADSCs, etc., resulting in a low survival rate of ADSCs in vivo. Summary of the invention
[0004] The present application proposes a method for isolating and culturing adipose stem cells to solve the problems of poor protection of adipose stem cells and low survival rate of adipose stem cells after transplantation in the prior art.
[0005] The present application proposes a method for isolating and culturing adipose stem cells, comprising the following steps:
[0006] S1: Aseptically obtain human adipose tissue;
[0007] S2: Cut the obtained fat tissue into 1-2 mm 3 The small pieces were placed in sterile culture bottles and cultured to obtain a uniform suspension after digestion;
[0008] S3: filtering the digested suspension through a cell filter, centrifuging the filtered liquid, and removing the supernatant to obtain a cell pellet;
[0009] S4: After the cell sediment is resuspended, it is inoculated into a culture dish and placed in an incubator for culture, and the culture medium is regularly replaced, wherein the culture medium contains acetic acid, and after a subculture operation, adipose stem cells after subculture are obtained;
[0010] S5: Detection and freezing.
[0011] Wherein, in step S2, a small piece of fat tissue is placed in a sterile culture bottle containing physiological saline or buffer, the fat tissue is digested with collagenase, and incubated at room temperature for 30-60 minutes to obtain a uniform suspension.
[0012] Wherein, the buffer in step S2 is PBS buffer.
[0013] Wherein, the rotation speed of the centrifugal treatment in step S3 is 300g, and the centrifugal time is 5-10 minutes.
[0014] Wherein, in step S4, when the cells grow to 70%-80% confluence, a subculture operation is performed until the cells grow to 80% confluence, thereby obtaining adipose stem cells after subculture.
[0015] Wherein, the acetic acid concentration contained in the culture medium in step S4 is 1-10mM.
[0016] The culture time in step S4 is 24-72 hours, and the culture dish in step S4 is pre-coated with collagen or fibrin.
[0017] Wherein, the temperature in the incubator in step S4 is 37° C., and the concentration of CO 2 gas in the incubator is 5%.
[0018] The present application also proposes the use of acetic acid in the isolation and culture of adipose stem cells, wherein the chemical formula of the acetic acid is CH3COOH.
[0019] The present application also proposes the application of adipose-derived stem cells, which are applied to tissue repair and regeneration therapy.
[0020] The present application comprises the following steps: resuspending the cell precipitate, inoculating it into a culture dish and placing it in an incubator for culture, regularly replacing the culture medium, wherein the culture medium contains acetic acid, performing a subculture operation, obtaining adipose stem cells after subculture, utilizing the protective effect of acetic acid under cell stress conditions, improving the stress resistance of adipose stem cells through pretreatment, simulating the in vivo survival microenvironment through a pretreatment strategy, improving the adaptability of adipose stem cells, thereby improving their survival rate in the survival microenvironment after transplantation, and enhancing their application potential in tissue repair and regenerative therapy. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] By reading the detailed description below with reference to the accompanying drawings, the above and other purposes, features and advantages of the exemplary embodiments of the present disclosure will become readily understood. In the accompanying drawings, several embodiments of the present disclosure are shown in an exemplary and non-limiting manner, and the same or corresponding reference numerals represent the same or corresponding parts, wherein:
[0022] Figure 1 This is a comparison chart of the proliferation of adipose stem cells pretreated with acetic acid in the examples of the present application;
[0023] Figure 2 This is a statistical comparison of fluorescence intensity of the survival of adipose stem cells pretreated with acetic acid in mice in an embodiment of the present application;
[0024] Figure 3 This is a comparison chart of the immunofluorescence display results of the survival of adipose stem cells pretreated with acetic acid in mice in another embodiment of the present application. DETAILED DESCRIPTION
[0025] The following will be combined with the drawings in the embodiments of the present disclosure to clearly and completely describe the technical solutions in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present disclosure.
[0026] The present invention first provides a method for isolating and culturing adipose stem cells, comprising the following steps:
[0027] S1: Aseptically obtain human adipose tissue;
[0028] The fat tissue obtained is usually taken from the abdomen or thigh of the human body, and the acquisition process needs to be carried out in a sterile environment to reduce the risk of infection.
[0029] S2: Cut the obtained fat tissue into 1-2 mm 3 The small pieces were placed in sterile culture bottles and cultured to obtain a uniform suspension after digestion;
[0030] The obtained fat tissue was cut into 1-2 mm 3After the small pieces are removed, they are placed in a sterile culture bottle containing physiological saline or PBS buffer. The PBS buffer, whose main components are Na2HPO4, KH2PO4, NaCl and KCl, acts as a dissolving protective agent. Based on the secondary dissociation of Na2HPO4 and KH2PO4, the pH value range of the buffer is relatively wide. The main function of NaCl and KCl is to increase the salt ion concentration in the solution. After the obtained adipose tissue is placed in a sterile culture bottle, the adipose tissue is digested with collagenase, wherein the temperature of collagenase digesting the adipose tissue is room temperature. After 30-60 minutes, the adipose tissue is digested into a uniform suspension.
[0031] S3: filtering the digested suspension through a cell filter, centrifuging the filtered liquid, and removing the supernatant to obtain a cell pellet;
[0032] The digested suspension is filtered through a cell filter, which can be a 70um cell sieve. After filtering through the cell filter, incompletely digested fat tissue pieces are removed. The filtered liquid is transferred to a centrifuge tube for centrifugation. The speed of the centrifugation is 300g, and the centrifugation time is 5-10 minutes. After centrifugation, layered supernatant and cell precipitation are obtained. The supernatant is removed from the centrifuge tube to obtain the cell precipitation.
[0033] S4: After the cell sediment is resuspended, it is inoculated into a culture dish and placed in an incubator for culture, and the culture medium is regularly replaced, wherein the culture medium contains acetic acid, and after a subculture operation, adipose stem cells after subculture are obtained;
[0034] The cell pellet is resuspended in a sterile medium, wherein the sterile medium can be a high-glucose DMEM medium, wherein the high-glucose DMEM medium contains various amino acids and glucose, and the glucose content is higher than 4500 mg / L. After resuspending, the cells are counted and adjusted to an appropriate concentration, such as 1x10 5 Up to 1x10 6 cells / ml.
[0035] The cells are seeded into a culture dish that is pre-coated with collagen or fibrin and placed in an incubator for culture. The temperature in the incubator is 37°C and the concentration of CO2 gas in the incubator is 5%. The culture medium is replaced regularly to maintain cell life.
[0036] The morphology and growth of the cells were observed under a microscope. When the cells reached 70%-80% confluence, the cells were subcultured, i.e., the cells were digested with trypsin and inoculated into a new culture dish to expand the cell amount. The isolated adipose stem cells were transferred to high-glucose DMEM medium containing 10% (m / v) fetal bovine serum and continued to be cultured until the cells reached 80% confluence.
[0037] The culture medium contains acetic acid, and the concentration of acetic acid can be 1-10 mM, preferably 5 mM, which is not limited here.
[0038] S5: Detection and freezing.
[0039] The cells are subjected to biological efficacy testing, cell differentiation ability testing, etc., and the qualified adipose stem cells are added to a freezing tube, and freezing solution is added thereto. After being mixed and shaken evenly, they are frozen in a refrigerator and transferred to liquid nitrogen for long-term storage. The detection process and the freezing process can be carried out simultaneously to improve processing efficiency.
[0040] Example 1
[0041] This example is an experimental group. In this example, the method for isolating and culturing adipose stem cells includes the following steps:
[0042] S1: Obtain human abdominal adipose tissue aseptically.
[0043] S2: Cut the obtained fat tissue into 1-2 mm 3 After being divided into small pieces, the adipose tissue is placed in a sterile culture bottle containing physiological saline, and the adipose tissue is digested with collagenase at room temperature for 60 minutes. After digestion, the adipose tissue becomes a uniform suspension.
[0044] S3: The digested suspension was filtered through a 70 um cell sieve, and the filtered liquid was transferred to a centrifuge tube for centrifugation at a speed of 300 g for 10 minutes. After centrifugation, layered supernatant and cell pellet were obtained. The supernatant was removed from the centrifuge tube to obtain the cell pellet.
[0045] S4: Resuspend the cell pellet in sterile high-glucose DMEM medium, count the cells after resuspension and adjust the cell concentration to 1x10 6 cells / ml, the cells were inoculated into a culture dish pre-coated with collagen or fibrin, and placed in an incubator for 24 hours. The temperature in the incubator was 37°C, the concentration of CO2 gas was 5%, and the culture medium was replaced regularly to maintain cell life. The morphology and growth of the cells were observed under a microscope. When the cells reached 80% confluence, the passage operation was performed, and the separated adipose stem cells were transferred to a high-glucose DMEM culture medium containing 10% (m / v) fetal bovine serum and continued to be cultured until the cells reached 80% confluence. The culture medium contained acetic acid at a concentration of 5mM.
[0046] Comparative Example
[0047] This comparative example is a control group, and the isolation and culture method of adipose stem cells is the same as that in Example 1 (experimental group), except that the sterile culture medium used in step S4 does not contain acetic acid.
[0048] The proliferation of adipose stem cells in the experimental and control groups was detected using a CCK-8 kit. Before and after pretreatment, the reagent was added and incubated for 2 hours according to the instructions of the CCK-8 kit. The absorbance (OD) value was measured using an enzyme reader, and CCK-8 detection was performed at 24 hours, 48 hours, and 72 hours, respectively.
[0049] Figure 1 This is a comparison chart of the proliferation of adipose stem cells pretreated with acetic acid in Example 1 of the present application and the situation in the comparative example, as shown in Figure 1 As shown in the figure, at 24 hours, the OD value (representing cell activity and proliferation level) of the experimental group was 0.52±0.03, while the OD value of the control group was 0.45±0.02, and the difference between the two groups did not reach statistical significance (P=0.07). As the culture time increased, the OD value of the experimental group gradually increased compared with that of the control group. At 48 hours, the OD value of the experimental group was 0.76±0.05, and the OD value of the control group was 0.63±0.04 (P<0.05). At 72 hours, the OD value of the experimental group was 0.91±0.06, and the OD value of the control group was 0.72±0.05 (P<0.01). In addition, cell proliferation was evaluated by cell counting and microscopic observation. At 72 hours, the number of cells in the experimental group was significantly higher than that in the control group, which was (2.1±0.2)×10^5 and (1.4±0.1)×10^5, respectively (P<0.01). Under the microscope, the cells in the experimental group were plump and densely grown, while the cells in the control group grew relatively slowly. The above results show that acetic acid pretreatment can significantly promote the proliferation of ADSCs.
[0050] In the present application, adipose-derived stem cells are further subjected to Dio staining, and the steps include: (1) using conventional methods to collect adipose-derived stem cells grown to 80% confluence, aspirating the culture medium, and washing the cells once with PBS; (2) adding an appropriate amount of 0.25% trypsin-EDTA solution thereto to detach the cells from the surface of the culture plate, and after the cells are completely detached, adding serum-free culture medium to neutralize trypsin, gently blowing the cell suspension, and collecting the cells in a centrifuge tube; (3) using a cell counter to count the cells, centrifuging the cells, and removing the supernatant; (4) according to the instructions of the Dio dye, using Prepare a working concentration of Dio staining solution with PBS, resuspend the cell pellet in the Dio staining solution, usually with a staining concentration of 1-10 μg / mL, and incubate the cell suspension at 37°C for 15-30 minutes; (5) After staining, add serum-free culture medium to terminate the staining process. After centrifugation, remove the staining solution and wash the cells with PBS at least twice to remove unbound dye; (6) Resuspend the washed cells in serum-free culture medium, inoculate the cells back into the culture plate or bottle, and continue culturing; (7) Observe the Dio-stained cells using a fluorescence microscope to confirm the staining of the cell membrane.
[0051] Further small animal live imaging experiments were performed on the mice. When conducting mouse live imaging experiments, (1) the Dio-stained cells were resuspended to an appropriate concentration, usually 10 6 Up to 10 7 cells / mL; (2) Anesthetize the mice properly before the experiment and ensure that the mice do not move during the injection process; (3) Select the appropriate site of the mouse for injection, usually the subcutaneous part of the back, use a syringe to draw an appropriate amount of cell suspension, and then carefully inject it into the subcutaneous part of the mouse; (4) After injection, perform in vivo imaging at different time points according to the experimental design, place the mouse on the platform of the in vivo imaging system, and adjust it to the appropriate position; (5) Select excitation and emission filters that match the fluorescence characteristics of Dio. Set the appropriate exposure time and gain to obtain clear fluorescence images; (6) Use the in vivo imaging system software to analyze the intensity and distribution of the fluorescence signal. By comparing the imaging results at different time points, evaluate the migration and survival of cells.
[0052] Figure 2 : is a statistical comparison of fluorescence intensity of the survival of adipose stem cells pretreated with acetic acid in mice in an embodiment of the present application, such as Figure 2As shown, in vivo imaging analysis showed that at 24 hours after injection, the fluorescence signal intensity of the experimental group was significantly higher than that of the control group (P<0.01), indicating that the early survival rate of acetic acid pretreated ADSCs in vivo was significantly improved. This difference was further expanded at 48 hours, and the fluorescence intensity of the experimental group continued to remain at a high level and showed a statistically significant difference compared with the control group (P<0.001), indicating that the survival advantage of acetic acid pretreated ADSCs in vivo is persistent. In addition, through the spatial distribution and morphological analysis of the fluorescence signal, we observed that the fluorescence signal of the experimental group was more concentrated at the injection site, indicating that the aggregation and distribution of acetic acid pretreated ADSCs in vivo were more stable. Combined with the in vivo imaging results, acetic acid pretreatment significantly improved the survival rate of ADSCs in nude mice, especially at 24 hours and 48 hours after transplantation.
[0053] The present application further conducted an immunofluorescence staining experiment, the experimental process of which includes: (1) after the in vivo imaging experiment, the mice were euthanized and handled in accordance with ethical regulations, and tissue samples around the injection site were collected; (2) the collected tissue samples were fixed with an appropriate fixative (such as 4% paraformaldehyde); (3) the fixed tissue samples were paraffin-embedded or frozen and sliced to prepare thin slices; (4) the tissue slices were dewaxed and hydrated, the slices were washed with PBS, and then treated with an appropriate permeabilizing agent to increase the permeability of the antibody, the slices were blocked to reduce nonspecific binding, and specific primary antibodies were added to target surface markers of ADSCs, such as CD34, CD90, etc., and incubated at 4°C overnight. After washing, fluorescently labeled secondary antibodies were added and incubated at room temperature for 1-2 hours, and nuclear staining was performed using DAPI dye for cell nucleus staining; (5) the slices were observed using a fluorescence microscope to collect Dio fluorescence signals and immunofluorescence signals; (6) the co-localization of fluorescence signals was analyzed to evaluate the distribution and survival status of Dio-stained adipose stem cells.
[0054] Figure 3 This is a comparison chart of immunofluorescence display results of the survival of adipose stem cells pretreated with acetic acid in mice in another embodiment of the present application, such as Figure 3 As shown, the immunofluorescence results showed that there were significant differences between the experimental group and the control group, and the experimental group showed a higher proportion of Dio-positive cells at both time points, indicating that more ADSCs survived after transplantation.
[0055] Specifically, on the second day of the experiment, the proportion of Dio-positive cells in the experimental group was 58.3%±3.5%, while that in the control group was 34.7%±4.1% (P<0.001). Immunofluorescence images showed that the cell morphology in the experimental group was more complete and the connections between cells were tighter, which may be related to the fact that acetic acid pretreatment enhanced the anti-apoptotic ability of cells and promoted the interaction between cells.
[0056] Combined with the results of immunofluorescence staining, acetic acid pretreatment significantly increased the survival rate of ADSCs in vivo, and this effect remained significant for at least 14 days after transplantation.
[0057] The analysis showed that acetic acid pretreatment can improve the survival rate of ADSCs in vivo by enhancing the antioxidant capacity of ADSCs, improving the anti-apoptosis ability of cells, and regulating immune response. The potential advantages of acetic acid pretreatment include: enhancing antioxidant capacity, acetic acid pretreatment can enhance the antioxidant capacity of ADSCs and reduce oxidative stress damage by activating antioxidant signaling pathways; improving anti-apoptosis ability: acetic acid can improve the anti-apoptosis ability of ADSCs by regulating the expression of apoptosis-related molecules such as Bcl-2 family proteins and Caspase family proteins; regulating immune response: acetic acid pretreatment can reduce immune attacks and improve cell survival by regulating immune-related molecules on the surface of ADSCs, such as MHC I and MHC II; promoting cell metabolism: acetic acid promotes the metabolic activity of ADSCs by participating in the TCA cycle, providing energy for cells and supporting cell survival and function.
[0058] The application of adipose-derived stem cells in the present application is to apply the adipose-derived stem cells to tissue repair and regenerative therapy. The adipose-derived stem cells are pretreated with acetic acid to improve the adaptability of the adipose-derived stem cells, thereby improving their survival rate in the survival microenvironment after transplantation and enhancing their application potential in tissue repair and regenerative therapy.
[0059] The present application utilizes the protective effect of acetic acid under cell stress conditions, improves the stress resistance of adipose stem cells through acetic acid pretreatment, simulates the in vivo survival microenvironment through pretreatment strategies, improves the adaptability of adipose stem cells, and thus improves their survival rate in the survival microenvironment after transplantation.
[0060] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.
[0061] Although the preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present application.
[0062] Finally, it should be noted that, in this application, relational terms such as first and second, etc., are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article including the elements.
[0063] Specific examples are used in this application to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core idea of this application. At the same time, for those skilled in the art, according to the idea of this application, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on this application.
Claims
1. A method for isolating and culturing adipose stem cells, characterized in that: The following steps are involved: S1: Aseptically obtain human adipose tissue; S2: Cut the obtained fat tissue into 1-2 mm 3 The small pieces were placed in sterile culture bottles and cultured to obtain a uniform suspension after digestion; S3: filtering the digested suspension through a cell filter, centrifuging the filtered liquid, and removing the supernatant to obtain a cell pellet; S4: After the cell sediment is resuspended, it is inoculated into a culture dish and placed in an incubator for culture, and the culture medium is regularly replaced, wherein the culture medium contains acetic acid, and after a subculture operation, adipose stem cells after subculture are obtained; S5: Detection and freezing.
2. The method for isolating and culturing adipose stem cells according to claim 1, characterized in that: In step S2, a small piece of fat tissue is placed in a sterile culture bottle containing physiological saline or buffer, and the fat tissue is digested with collagenase. After incubation at room temperature for 30-60 minutes, a uniform suspension is obtained.
3. The method for isolating and culturing adipose stem cells according to claim 2, characterized in that: The buffer in step S2 is PBS buffer.
4. The method for isolating and culturing adipose stem cells according to claim 1, characterized in that: The rotation speed of the centrifugal treatment in step S3 is 300g, and the centrifugal time is 5-10 minutes.
5. The method for isolating and culturing adipose stem cells according to claim 1, characterized in that: In step S4, when the cells grow to 70%-80% confluence, a subculture operation is performed until the cells grow to 80% confluence, thereby obtaining adipose stem cells after subculture.
6. The method for isolating and culturing adipose stem cells according to claim 5, characterized in that: The acetic acid concentration contained in the culture medium in step S4 is 1-10 mM.
7. The method for isolating and culturing adipose stem cells according to claim 6, characterized in that: The culture time in step S4 is 24-72 hours, and the culture dish in step S4 is pre-coated with collagen or fibrin.
8. The method for isolating and culturing adipose stem cells according to claim 7, characterized in that: The temperature of the incubator in step S4 is 37° C., and the concentration of CO 2 gas in the incubator is 5%.
9. Application of acetic acid in the isolation and culture of adipose stem cells, wherein the chemical formula of the acetic acid is CH3COOH.
10. Application of adipose-derived stem cells for tissue repair and regeneration therapy.