Composition for inducing autologous fibroblast reprogramming and application thereof

Abstract

The invention relates to the field of cells, in particular to a composition for inducing autologous fibroblast reprogramming and application of the composition. According to the induction sequence, the composition comprises a first-stage small molecule compound, a second-stage small molecule compound and a third-stage protein molecule, and the first-stage small molecule compound is composed of Y27632, SAG, forskolin and Rapamycin; the second-stage small molecule compound is prepared from Y27632, SAG, forskolin, Rapamycin, AXL1717, RA and TNF-alpha (Tumor Necrosis Factor-alpha); the third-stage protein molecule is bFGF (basic fibroblast growth factor). The composition disclosed by the invention can be used for reprogramming fibroblasts so as to change the omics characteristics of the fibroblasts, so that the fibroblasts have enhanced immunoregulation, tissue repair and other biological functions related to treatment, and further safe, effective and unlimited autologous cell disease treatment is realized.

Description

Technical Field

[0001] The present invention relates to the field of cells, and in particular to a composition for inducing reprogramming of autologous fibroblasts and applications thereof. Background Art

[0002] Traditional treatment concepts largely remain on the simple "disease-target-drug" model, but only a very small number of them can be truly targeted. Cell therapy provides a new hierarchical model that does not focus on removing obstacles, but on building and supporting a healthy environment. It will become the third pillar of future medicine. Although the current development trend of the cell industry is good, unstable efficacy is still the biggest reason for the intermittent results of clinical trials. Although there are many cases that demonstrate the magic of cell therapy, not every treatment is effective, nor is it effective for everyone. Among them, the phenotypic and functional heterogeneity of cell preparations themselves seriously hinders the efficiency and reproducibility of basic research and clinical applications. Secondly, even if the same cell product is used for clinical treatment, not every patient can obtain the same benefits. This is because whether it is stem cells or mature functional cells, after entering the patient's body, they will undergo immune rejection or exhaustion under the influence of the disease microenvironment. Therefore, the different immune states of the patients themselves will also have an important impact on the efficacy of cells.

[0003] Taking central nervous system diseases as an example, the abnormally activated neuroimmune microenvironment destroys the soil for neural circuit repair. Although many teams at home and abroad have developed neural progenitor cells for transplantation therapy, the transplanted stem cells are usually difficult to fully exert their functions, and the clinical effects are usually limited. If the treatment is carried out under the condition of improved soil, the survival rate of transplanted cells and the integration effect of neural circuits will be greatly improved; in addition, the immune microenvironment soil will reactivate the plasticity of the nervous system in a steady state, restore the self-regeneration of some functional cells and restore their functions. Therefore, regardless of whether functional cells are used to treat the disease, improving the pathological immune microenvironment should be the top priority.

[0004] Currently, the cell therapy products that are widely used in clinical trials include various immune cells, hematopoietic stem cells, mesenchymal stem cells (MSCs), and functional cells derived from human iPSCs (neural progenitor cells, pancreatic islet cells, hepatocytes, etc.). Due to the various types of diseases, including central nervous system diseases, functional cells used for replacement therapy are usually difficult to obtain directly and need to be differentiated from stem cells (including iPSCs). At present, stem cells such as iPSCs have made breakthroughs in the preparation of general-purpose commodities. Chemical reprogramming technology uses a combination of chemical small molecules to effectively regulate cell fate. It can reverse differentiated somatic cells into pluripotent stem cells, providing a new strategy for the preparation of universal human pluripotent stem cells. In 2022, Deng Hongkui's team reported that small molecules can effectively reprogram human somatic cells into pluripotent stem cells, and in 2025, they developed a rapid reprogramming system that can generate hCiPS cells in just 10 days. Compared with the traditional reprogramming strategy of overexpressing transcription factors, chemical reprogramming technology has outstanding advantages: chemical small molecules can regulate cell fate in a simpler, flexible and controllable manner by directly targeting signal pathways and epigenetic factors, while also effectively avoiding the potential safety risks of traditional transgenic strategies; in addition, chemical small molecules also have the advantages of easy large-scale synthesis and standardized production.

[0005] Based on theoretical and technical feasibility, a number of functional cells (i.e., seed cells) have been developed for potential clinical cell replacement therapies. However, their efficacy and stability are limited by the individual pathological immune state (the environment) of the patient. There is an urgent need for the development of cell products and preparation technologies specifically tailored to the environment. However, limited development of cell products specifically tailored to immune state is currently available. Chinese patent application number CN202010998258.0 provides a culture medium containing one or more additional growth factors: VEGF, EGF, and PDGF, for culturing mesenchymal stem cells from embryonic stem cells or induced pluripotent stem cells, which exhibit certain immunomodulatory abilities. NurOwn cells, based on the patient's own mesenchymal stem cells, combine small molecule reprogramming technology with a culture medium containing cAMP, hbFGF, PDGF-AA, and Heregulin β1. These cells retain the basic immunomodulatory abilities of MSCs while also enhancing their neurotrophic secretion, aiming to further improve the pathological microenvironment (the environment). However, in 2023, the FDA rejected NurOwn's drug application for the treatment of amyotrophic lateral sclerosis, primarily due to insufficiently stable and significant clinical efficacy. Therefore, there is a huge market application for cell preparations and preparation methods that regulate the immune microenvironment (soil) and promote the special function of tissue repair, but there is a large gap. Summary of the Invention

[0006] The purpose of the present invention is to provide a composition for inducing autologous fibroblast reprogramming, which can reprogram fibroblasts and thereby change their genomic characteristics so that they acquire enhanced biological functions related to therapy, such as immune regulation and tissue repair, thereby achieving safe, effective and unlimited autologous cell disease treatment.

[0007] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a composition for inducing fibroblast reprogramming, wherein the composition is used to reprogram fibroblasts into fibroblasts with enhanced immunoregulatory and tissue repair abilities, wherein the composition comprises, in order of induction, a first-stage small molecule compound, a second-stage small molecule compound, and a third-stage protein molecule; The first phase of small molecule compounds consists of Y27632, SAG, forskolin, and Rapamycin; The second phase of small molecule compounds consists of Y27632, SAG, forskolin, Rapamycin, AXL1717, RA, and TNF-α; The third stage protein molecule is bFGF.

[0008] In a second aspect, the present invention provides a culture system for inducing fibroblast reprogramming, which includes the composition described above. The culture system includes a first culture system, a second culture system and a third culture system. The first culture system contains the first-stage small molecule compound, the second culture system contains the second-stage small molecule compound, and the third culture system contains the third-stage protein molecule.

[0009] Furthermore, in the first culture system, the second culture system and the third culture system, the Y27632 concentration is 2.5-5 μM; the SAG concentration is 0.25-0.5 μM; the forskolin concentration is 5-10 μM; the Rapamycin concentration is 0.1-0.2 μM; the TNF-α concentration is 10-20 ng / ml; the AXL1717 concentration is 1-2 μM; the RA concentration is 1-2 μM; and the bFGF concentration is 10-20 ng / mL.

[0010] In a third aspect, the present invention provides a culture medium for inducing fibroblast reprogramming, which includes the culture system.

[0011] In a fourth aspect, the present invention provides a kit for inducing fibroblast reprogramming, which comprises the culture system.

[0012] In a fifth aspect, the present invention provides use of the composition, the culture system, the culture medium or the kit in inducing the preparation of reprogrammed fibroblasts with enhanced immunoregulatory and tissue repair capabilities.

[0013] The composition, kit or culture medium can be used to induce the preparation of reprogrammed fibroblasts in vitro with enhanced immunoregulatory and tissue repair capabilities. The small molecule compound can be used for basic research, preclinical research, clinical treatment and the production of cell product-related products.

[0014] In a sixth aspect, the present invention provides a method for inducing fibroblast reprogramming, which is performed using the aforementioned culture system, and the method comprises: The fibroblasts are cultured in the first culture system for 4-6 days, then transferred to the second culture system for 3-5 days, and then transferred to the third culture system for 3 days, and then replaced with a conventional mesenchymal stem cell culture medium for maintenance culture and passage expansion.

[0015] Specifically, cultured autologous primary fibroblasts are co-cultured with a ROCK inhibitor (Y27632), a Smoothened (Smo) receptor agonist (SAG), a cAMP agonist (Forskolin), and Rapamycin (as an AMPK activator and mTOR inhibitor). After 4-6 days of induction culture, the autologous primary fibroblasts enter a pre-induction state, stop proliferating and undergo deformation, while significantly increasing the level of autophagy and reducing the accumulation of toxic products. Furthermore, the pre-induction autologous primary fibroblasts were co-cultured with an IGF-1R inhibitor (AXL1717), a RAR agonist (RA), a ROCK inhibitor (Y27632), a Smoothened (Smo) receptor agonist (SAG), a cAMP agonist (Forskolin), Rapamycin (an AMPK activator and mTOR inhibitor), and TNF-α (an NF-κB activator, a TGF-β agonist, a JAK-STAT agonist, and an mTORC1 activator) to activate immune factor responsiveness and trophic factor secretion pathways in the pre-induction autologous primary fibroblasts, thereby generating the reprogrammed fibroblasts. In the third stage, the reprogrammed fibroblasts were co-cultured with the fibroblast growth factor (bFGF) to restore their proliferative capacity, allowing for production and expansion without compromising the enhanced functional phenotype achieved through induced reprogramming.

[0016] It should be noted that the small molecule compounds and protein molecules mentioned above are only examples of signal pathway regulators. In fact, other small molecule compounds with the same function can also play a similar role and achieve the same effect.

[0017] Furthermore, the autologous fibroblasts are derived from fibroblasts behind the ears, limbs, abdomen, and buttocks of mammals. Preferably, the autologous fibroblasts are human behind-the-ear fibroblasts, without limitation to age, gender, or disease.

[0018] In a seventh aspect, the present invention provides a fibroblast with enhanced immunoregulatory and tissue repair abilities, which is prepared by the method.

[0019] In an eighth aspect, the present invention provides the use of the fibroblasts in the preparation of disease treatment drugs, and the reprogrammed fibroblasts obtained by using the small molecule compound combination provided by the present invention have enhanced immunomodulatory ability and tissue repair ability, and have limited stemness characteristics, are stable in passage, and can be expanded at least 5 times. Its process is induced reprogramming according to an orderly combination, and the process operation can be standardized and scaled. The present invention can obtain reprogrammed fibroblasts of autologous origin, can efficiently regulate the immune state and immune microenvironment, provide nutritional support, promote tissue repair, and can develop clinical treatment strategies for related diseases that can benefit from immunomodulation, including but not limited to diabetes, Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, frailty, etc.

[0020] Compared with the prior art, the present invention has the following technical effects: By utilizing the composition provided by the present invention and inducing and culturing autologous fibroblasts according to the process steps, reprogrammed fibroblasts that are stably passaged and amplified in large quantities can be obtained, which have enhanced immunomodulatory ability and the ability to promote tissue repair. Potential routes of administration, including but not limited to intravenous drip, nasal administration, subcutaneous injection, intramuscular injection, etc., can be used for basic medical research, preclinical research, clinical treatment and cell product-related product production. It fills the gap in reprogrammed autologous cell products that target and regulate the immune microenvironment to promote tissue repair, and can also be used as an adjuvant therapy for other functional cells. After regulating the body's soil homeostasis, it is beneficial to the efficacy of functional cell transplantation therapy. This technology and the reprogrammed fibroblasts obtained by this technology have good and unique industrial prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure shows the cell morphology of human postauricular fibroblasts induced to transdifferentiate into reprogrammed fibroblasts by the composition of the present invention.

[0022] Figure 2 Figure 2 shows the peripheral immunosuppressive capacity of reprogrammed fibroblasts.

[0023] Figure 3 A diagram showing the central immunosuppressive capacity of reprogrammed fibroblasts.

[0024] Figure 4 A diagram showing the therapeutic outcomes of transplanting reprogrammed fibroblasts into inflammatory model mice and the differences in administration routes.

[0025] Figure 5 Figure 2 shows the results of in vivo tumorigenicity (A) and tumor-promoting ability (B) detection of reprogrammed fibroblasts.

[0026] Figure 6 A diagram showing the therapeutic results of transplanting reprogrammed fibroblasts into ALS model mice. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0028] The small molecule compounds used in the following examples are: Y27632: CAS No.:146986-50-7.

[0029] SAG:CAS No.: 912545-86-9.

[0030] Forskolin: CAS No.:66575-29-9.

[0031] Rapamycin: CAS No.: 53123-88-9.

[0032] AXL1717: CAS No.:477-47-4.

[0033] RA:CAS No. : 302-79-4.

[0034] TNF-α: CAS No.: 144796-71-4.

[0035] bFGF: Protein ID: P09038.

[0036] Example 1 1. Skin tissue sample collection, transportation and storage 1.1. Sample Collection: Collect and record the basic information and medical history of volunteers or patients, conduct pathogen screening (including HIV, syphilis, hepatitis C, and hepatitis B), sign an informed consent form, strictly follow process control principles to prevent contamination and cross-contamination, and have the attending physician sample the skin tissue (approximately 1 cm2 spindle-shaped) behind the ear in the operating room of a second-level or higher hospital.

[0037] 1.2. Place the surgically excised skin tissue in sample collection and preservation solution (DMEM + 0.08 mg / mL gentamicin sulfate) for temporary storage. Seal the tissue with parafilm and ziplock bag. Place the bag in a low-temperature transport box at 2-8°C and transport it to the GMP laboratory for primary isolation as quickly as possible, within 4 hours.

[0038] 2. Skin tissue sample reception 2.1. Verify the basic information and medical history of the volunteer or patient, and confirm that all pathogen screening results are negative.

[0039] 2.2. Confirm that the entire tissue transportation process complies with low-temperature transportation conditions of 2-8°C.

[0040] 2.3. Confirm that the tissue transport container is leak-free and free of contamination.

[0041] 3. Isolation of autologous fibroblasts 3.1. Prepare surgical instruments for primary cell isolation (tissue forceps and microscissors) and skin tissue washing buffer (sodium chloride injection + 0.08 mg / mL gentamicin sulfate), and prepare complete fibroblast culture medium 1 (DMEM + 5% UltraGRO™ - Advanced GMP grade).

[0042] 3.2. Transfer the skin tissue to a cell culture dish containing washing buffer and take photos to record the original state of the skin tissue sample.

[0043] 3.3. Place the skin tissue under a stereo microscope, fix the skin tissue with tissue forceps, use microscissors to scrape off as much blood and impurities as possible on the surface of the tissue, then cut off the fat and skin layer, leaving the dermis.

[0044] 3.4. Wash the dermis tissue 5 times and transfer the dermis tissue to a clean culture dish lid.

[0045] 3.5. Use microscissors to cut the dermis into pieces of 1 mm 3 Use forceps to evenly distribute the tissue blocks on a 6 cm 2 Place the cell culture dish in a cell culture incubator to dry for 30 minutes.

[0046] 3.6. After drying is complete, slowly add 3 mL of complete medium 1 and gently shake the culture dish until the culture medium covers all tissue pieces. Place the culture dish in a cell culture incubator and continue culturing.

[0047] 4. Primary cell culture medium replacement 4.1. On the second day of primary culture preparation, gently remove the culture dish and check the adhesion of the tissue block. Observe under a microscope to preliminarily determine whether there is bacterial contamination.

[0048] 4.2. Collect the cell culture supernatant from the culture dish and perform sterility and endotoxin tests; 4.3. Replace the culture medium with 3 mL of fresh complete medium 1, and replace the culture medium with fresh complete medium 1 every 3 days thereafter.

[0049] 4.4. Around day 13-18, observe the fibroblast crawling out of the tissue block and the cell density under a microscope. When the cell confluence reaches about 80%, the cells can be digested and passaged.

[0050] 5. Subculture and expansion of primary cells 5.1. When the primary fibroblasts adjacent to the tissue block reach approximately 80% confluence, remove the culture dish, aspirate the cell culture supernatant, add 3 mL of sodium chloride injection, gently shake the dish to wash away the remaining cell culture medium, aspirate the supernatant, and add 1 mL of Gibco TrypLE™ Express. Place the dish in the incubator for digestion for 3-5 minutes.

[0051] 5.2. Observe the cell digestion process under a microscope. When the cells gradually shrink from a long spindle shape, become round, and become single cells, add an equal volume of complete medium 1 to terminate the digestion. Use a pipette to collect the cell suspension into a 15 mL centrifuge tube.

[0052] 5.3. Pass the collected cell suspension through a 70 μm cell sieve to remove residual tissue fragments and cell clusters. Centrifuge at 400 g for 3 minutes, discard the supernatant, resuspend the cell pellet in 1 mL of complete medium 1, and take 20 μL of the cell suspension for cell counting.

[0053] 5.4. Based on autologous products, the number of primary fibroblasts harvested from different skin tissue sources will vary. Based on the counting results, the number of primary fibroblasts is calculated based on 1×10 4 cells / cm 2 Seeding density: Inoculate cells into cell culture flasks of appropriate surface area, mark the cells as P0, and culture them in a cell culture incubator. The volume of complete medium 1 corresponding to culture flasks of different surface areas is: T25cm 2 -5mL, T75cm 2 -15mL.

[0054] 5.5. After 72 hours of culture, when the confluence of the skin fibroblasts reaches 80-90%, discard the cell culture supernatant, add 5-8 mL of sodium chloride injection, and gently shake the culture flask to wash away the residual cell culture medium.

[0055] 5.6. Aspirate the sodium chloride injection solution and add the corresponding volume of Gibco TrypLE™ Express. Place the tube in the incubator for digestion for 3-5 minutes. The volume of Gibco TrypLE™ Express corresponding to the surface area of ​​the culture flask with different surface areas is: T25cm 2 -1mL, T75cm 2 -3mL, T175cm 2 -4mL, T225cm 2 -5mL.

[0056] 5.7. Observe the cell digestion process under a microscope. When the cells gradually shrink from a long spindle shape, become round, and become single cells, add an equal volume of complete medium 1 to terminate the digestion. Use a pipette to collect the cell suspension into a 50 mL centrifuge tube.

[0057] 5.8. Rinse the remaining suspended cells in the cell culture flask with 8 mL of sodium chloride injection and collect them into the above centrifuge tube; centrifuge the collected cell suspension at 400g for 3 minutes; discard the supernatant, resuspend the cell pellet in 5 mL of complete culture medium, and take 20 μL of the cell suspension for cell counting.

[0058] 5.9, according to 1×10 4 cells / cm 2 Seeding density: Inoculate cells into cell culture flasks of appropriate surface area, label the cells as P1, and culture them in a cell culture incubator. The volume of complete medium 1 corresponding to culture flasks of different surface areas is: T25cm 2 -5mL, T75cm 2 -15mL.

[0059] 5.10. Follow the same procedure to expand to P5.

[0060] 6. Reprogramming 6.1. Take photos to record cell morphology. Dilute the small molecule combination from the first stage, Y27632 (5 μM), Forskolin (10 μM), SAG (0.5 μM), and Rapamycin (0.1 μM), into 50 mL of complete fibroblast medium 1 (DMEM + 5% UltraGRO™-Advanced GMP grade), and label it as solution 1. Discard the cell culture supernatant in the culture flask and add 12.5 mL of the prepared solution 1 to a T75 culture flask. Incubate the cells at 37°C with 5% CO. 2 The cells were cultured statically in a cell culture incubator, and the day was recorded as day 0.

[0061] 6.2. On the third day, replace the cells in the above culture flasks with new solution No. 1, add 12.5 mL to each culture flask, and place the cells at 37°C, 5% CO2 Culture the cells statically in a cell culture incubator.

[0062] 6.3. On day 5, take photos to record cell morphology and dilute the second stage small molecule combination, Y27632 (5 μM), Forskolin (10 μM), SAG (0.5 μM), Rapamycin (0.1 μM), AXL1717 (1 μM), Retinoic Acid (1 μM), and TNF-α (10 ng / mL), into 25 mL of fibroblast complete medium 1, labeled as medium 2. Replace the cells in the above culture flasks with new medium 2, add 12.5 mL to each culture flask, and incubate the cells at 37°C with 5% CO. 2 Culture the cells statically in a cell culture incubator.

[0063] 6.4. On day 8, take photos to record cell morphology and dilute the third stage small molecule (bFGF, 10 ng / mL) into 30 mL of fibroblast complete medium 1, labeled as liquid 3. Rinse the cells in the above culture flasks with 5 mL of fresh complete medium 1 and replace with new liquid 3. Add 15 mL to each culture flask and incubate the cells at 37°C, 5% CO 2 Culture the cells statically in a cell culture incubator.

[0064] 6.5. On day 11, take photos to record cell morphology. At this point, the induction process is complete and the cell supernatant is collected for microbial testing (bacteria, fungi, mycoplasma, and endotoxin).

[0065] 6.6. The cells can then be stably passaged and maintained, expanded, and cultured using conventional mesenchymal stem cell culture medium. The successfully induced reprogrammed fibroblasts are designated ARC001.

[0066] The cell morphology of human postauricular fibroblasts induced by small molecule compounds to transdifferentiate into reprogrammed fibroblasts Figure 1 As shown by Figure 1 It can be seen that ARC001 has a spindle-shaped morphology and grows adherently, which is consistent with the basic characteristics of mesenchymal stromal cells.

[0067] Example 2 Carboxyfluorescein diacetate succinimidyl ester (CFSE) staining is a novel dye that can fluorescently label living cells. It easily penetrates cell membranes and covalently binds to intracellular proteins within living cells, releasing green fluorescence upon hydrolysis. Fluorescent CFSE covalently binds to amino groups within the cell, coupling to proteins. As cell division progresses, it is evenly distributed among daughter cells, reducing the fluorescence intensity of these cells by half. Fluorescence intensity decreases with each daughter cell division. Flow cytometry can detect peaks of varying fluorescence intensity, allowing for monitoring cell division and proliferation. By staining PBMC with CFSE and marking them with fluorescence, the CFSE fluorescence value on each cell will decrease after PBMC activation and expansion, that is, the lower the fluorescence value, the higher the level of activation and expansion; then, by co-culturing with the test cells, including fibroblasts (FIB) before induction or ARC001 cells after induction and reprogramming, the changes in fluorescence values ​​are detected by flow cytometry to calculate the regulatory ability of the test cells on the activated and expanded PBMCs, which represents the level of their regulatory effect on peripheral immunity.

[0068] 1. Isolation of mononuclear cells (PBMCs) 1.1. Collect 5 mL of sodium heparin-anticoagulant blood, gently mix by inverting it upside down, and pipette the blood sample into a 15 mL centrifuge tube. (Avoid aspirating blood clots during this step; use a pipette to aspirate, as this will contaminate the small-scale gun.)

[0069] 1.2. Use normal saline to dilute, the dilution multiple should be greater than one.

[0070] 1.3. Slowly add the anticoagulant diluent to the upper layer of the prepared equal volume of Lympholyte®-H separation solution (anticoagulant: saline: separation solution = 1:1:1), making sure to maintain a clear boundary (Note: Handle the centrifuge tube with care to prevent the anticoagulant diluent from entering the lower layer of separation solution).

[0071] 1.4. Centrifuge at room temperature with the brake off and speed at 800 g for 20 min.

[0072] 1.5. After centrifugation, remove the tube and gently place it aside. The liquid in the centrifuge tube will separate into three layers: the upper layer is a clear, translucent liquid primarily composed of plasma; the middle layer is a white, membrane-like layer composed of mononuclear cells; and the lower layer is a red precipitate composed of platelets, granulocytes, and red blood cells. Remove the upper layer and collect the cells in the middle buffy coat layer into a 15 mL centrifuge tube (approximately 2 mL). Add the cells to a previously prepared 15 mL centrifuge tube containing 8 mL of normal saline and centrifuge for 8 minutes at 300 g.

[0073] 1.6 After centrifugation, remove the supernatant, add 10 mL of normal saline to resuspend the cell pellet, and centrifuge at 300 g for 8 min to wash the pellet a second time.

[0074] 1.7 After centrifugation, remove the supernatant and add 1 mL of 1X ACK working solution to resuspend the cells by pipetting. Let the cells stand at room temperature for 3 minutes to break up the red blood cells. Add 10 times the volume of normal saline to stop the suspension. Invert the tube to mix thoroughly and centrifuge at 300 g for 8 minutes.

[0075] 1.8. Centrifuge and remove the supernatant to obtain mononuclear cells (PBMCs), add 1640 complete medium, mix well, count, and adjust the concentration to 1×10 6 / mL, inoculated into 24-well plates for suspension culture.

[0076] 2. PBMC activation and expansion 2.1. Vortex the CD3 / CD28 magnetic bead working solution to mix thoroughly. Then, take out a certain volume of magnetic beads and add them to a 1.5 mL EP tube for washing. 25 μL of magnetic beads is required for every 1×106 cells.

[0077] 2.2. Add an equal volume of magnetic bead washing buffer and vortex for 5 seconds. Place on a magnetic rack and let it stand for 1 minute. Then remove the supernatant, add an equal volume of 1640 complete medium to resuspend, and add to PBMCs cultured in a 24-well plate.

[0078] 2.3. Add 50 ng / mL IL2 factor for stimulation.

[0079] 2.4. Co-stimulation for 3-5 days.

[0080] 3. Co-culture of ARC001 and PBMC and flow cytometry detection 3.1. Day 1: 10 μg / mL mitomycin C was added to αMEM blank medium and cultured for 2 h. The cells were then washed three times with saline. ARC001 cells were trypsinized for 1-3 minutes and terminated with complete medium containing serum replacement. The cells were then centrifuged at 500 g for 5 minutes to wash the cells and adjust the cell density to 3 × 10 4 / mL, inoculated into 24-well plates, 1 mL per well, and cultured overnight.

[0081] 3.2, Day 0: Collect the prepared PBMCs, centrifuge at 300 g for 8 minutes, wash twice, and resuspend in 1× PBS containing 1 uM CFSE at a density of 5×10 6 / mL, incubate at room temperature in the dark for 20 minutes; dilute fivefold with αMEM complete medium, let stand at 37°C for 5 minutes to stop labeling; then centrifuge at 300g for 8 minutes, wash three times, and resuspend in αMEM complete medium; inoculate at a ratio of 1:5, with 1.5×10 per well of a 24-well plate. 5 / well, and added to the ARC001 cell culture wells to be tested for co-culture.

[0082] 3.3. Day 2: After 2 days of co-culture, collect the suspended cells, sieve them (300-mesh filter), and analyze them on a flow cytometer. Detect the FITC channel. Define the analysis interval based on the negative and positive proliferation groups. PBMC proliferation inhibition rate (%) = 1 - the proliferation rate of the test cells in co-culture / the proliferation rate of PBMC cultured alone.

[0083] The results are as follows Figure 2 As shown, the PBMC proliferation inhibition rate was calculated and paired t-test was performed on fibroblasts (FIB) from the same donor and fibroblasts (ARC001) reprogrammed by this technology. Figure 2 It can be seen that after reprogramming, the PBMC proliferation inhibition rate (%) increased significantly. p = 0.0427, p The value is less than 0.05, which is statistically significant, that is, after reprogramming with this technology, ARC001 has acquired enhanced peripheral immunosuppressive function.

[0084] Example 3 Microglia are resident immune effector cells within the central nervous system (CNS) and are involved in the development and progression of a range of CNS degenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Microglial activation, proliferation, and neuroinflammation are key features of neuropathology, accompanied by an increase in glial number and phenotypic changes, a phenomenon known as reactive gliosis. Inhibiting microglial proliferation and activation in disease states can reduce microglial-mediated chronic inflammation, thereby suppressing the imbalance of CNS immune homeostasis and restoring the neuronal supportive microenvironment. BV2 cells are a mouse-derived microglial cell line immortalized by retroviral transfection with v-raf / v-myc. They can spontaneously activate and proliferate in vitro. BV2 cells exhibit many morphological, characteristic, and functional characteristics of microglia, such as phagocytic capacity and reactive proliferation upon activation. BV2 cells are a commonly used in vitro model in neuroscience research, simulating neurological diseases and studying related disease mechanisms. Co-culture of BV2 cells with test cells or their secretory supernatants can be used to determine whether the test drug has a feedback mechanism on BV2 cells, promoting or inhibiting their proliferation, thereby verifying its mechanism of action, which activates or inhibits central immune responses. CCK8, short for Cell Counting Kit-8, is a reagent used for simple and accurate cell proliferation and toxicity analysis. This reagent contains the water-soluble tetrazolium salt WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonate)-2H-tetrazolium monosodium salt). Under the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS), it is reduced by cellular dehydrogenases to a highly water-soluble yellow formazan dye. The amount of formazan generated is proportional to the number and activity of viable cells and can be measured using a microplate reader. This property allows for cell proliferation and toxicity analysis.

[0085] To eliminate the influence of co-cultured test cells on formazan production, the secretory supernatant of test cells, such as ARC001 and pre-induced fibroblasts (FIB), was selected as the co-culture test substance to analyze whether the test cells have a paracrine mechanism to regulate central immune cells. After co-culture, the absorbance at 450 nm was measured using a microplate reader to reflect the proliferation activity of BV2 cells, and the BV2 inhibition rate was calculated according to the formula.

[0086] 1. Processing and culture of ARC001 cell samples 1.1. ARC001 cells from different donors were cultured in αMEM containing 5% serum replacement in a 5% CO atmosphere. 2 , subcultured at 37°C; ARC001 cells in the logarithmic growth phase were adjusted to a density of 1×10 5 12 mL of cell suspension was inoculated into T75 and cultured in an incubator for 72 h. The supernatant was collected and aliquoted into 10 mL tubes and stored at -80°C for future use.

[0087] 2. BV2 cell culture and experimental grouping 2.1. Remove the BV2 cells stored in the cryovial from liquid nitrogen and quickly place them in a 37°C water bath. Shake continuously to thaw evenly. Place in a 4°C centrifuge and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant in the cryovial and add 1 mL of cell culture medium to the cryovial. Slowly pipette and mix. Transfer 1 mL of cell suspension to a cell culture flask and add 5-6 mL of culture medium. Blow the cell suspension evenly to distribute the cells evenly in the flask. Place the flask in a cell incubator at 37°C and 5% CO. 2 After culturing for 12-24 hours, observe the cell status using a cell microscope and replace with new culture medium to continue culturing for 24 hours;

[0088] 2.2. BV2 microglia were cultured in 1640 complete medium with 5% CO 2 , cultured at 37°C.

[0089] 2.3. Cell Passaging: Discard the original culture medium, add PBS, rinse the bottom of the cell flask, discard the PBS, repeat this step three times, add an appropriate amount of PBS and trypsin, and return the cells to the cell incubator for digestion for 1 min. When the cells become rounded and no longer adhere to or float in the PBS under a microscope, add an appropriate amount of culture medium to stop digestion. BV2 cells are then stirred evenly, discarding 2 / 3 of the cell suspension. Add an appropriate amount of culture medium, mix thoroughly, and return the cells to the cell incubator. Allow BV2 cells to adhere evenly to the cell flask for 2-3 days, covering the bottom of the flask.

[0090] 2.4. BV2 cells in the logarithmic growth phase were taken and adjusted to a density of 7.5×10 4Cells were plated at 400 μL / mL and inoculated into 96-well cell culture plates, 100 μL per well, and cultured in a cell culture incubator for 24 hours. The experiment was divided into a control group, a test article experimental group-ARC001 supernatant group, a test article control group-FIB supernatant group, and an immunosuppression-positive group (umbilical cord mesenchymal stem cell supernatant). Six wells were set up for technical replicates in each group. Six additional wells were set up as blank wells without BV2 cells, and 100 μL of 1640 complete medium was added. After 24 hours of culture, the supernatant was discarded, and 100 μL of αMEM complete medium was added to the blank wells, 100 μL of αMEM complete medium was added to the control wells, and 100 μL of different conditioned media, including the test article experimental group-ARC001 supernatant, the test article control group-FIB supernatant, and the umbilical cord mesenchymal stem cell supernatant, was added to each well in a 5% CO incubator. 2 The cells were cultured in a 37°C cell culture incubator for 24 h before subsequent experiments.

[0091] 3. Inhibition rate determination 3.1. After BV2 microglial cells were cultured in the new culture medium for 24 h, 10 μL of CCK8 solution was added to each well, and the cells were cultured in the dark for another 1 h. The absorbance of each well was measured at 450 nm on an automatic microplate reader and corrected. The cell inhibition rate of each group was calculated as follows: cell inhibition rate = [(A value of the control group - A value of the blank well) - (A value of the intervention group - A value of the blank well)] / (A value of the control group - A value of the blank well) × 100%.

[0092] The results are as follows Figure 3 As shown, the inhibition rate of BV2 microglia proliferation was calculated and paired t-test was performed on fibroblasts (FIB) from the same donor and fibroblasts (ARC001) reprogrammed by this technology. Figure 3 It can be seen that after reprogramming, the PBMC proliferation inhibition rate (%) increased significantly. p = 0.0126, p The value is less than 0.05, which is statistically significant, that is, after reprogramming with this technology, ARC001 has acquired enhanced central immune suppressive function.

[0093] Example 4 The body's response to infection / stress is based on physiological and pathological conditions. Under these conditions, cross-talk between the three immune systems (circulatory immunity, central immunity, and neural function) is crucial. Endotoxin injection can be used to induce immune activation, mimicking the natural immune / stress response to infection. Lipopolysaccharide (LPS), part of the outer membrane of Gram-negative bacteria, acts as an endotoxin and activates pathogenic molecular patterns (PAMPs) that activate the immune system. Intraperitoneal injection of LPS binds to and activates immune cells, triggering an inflammatory storm in the peripheral circulation through a cellular cascade. This inflammatory response is then transmitted to the central nervous system through signal transduction pathways, the lung-brain axis, and the vagal axis, activating central inflammation. Repeated intraperitoneal injections of LPS induce peripheral immune tolerance or paralysis in mice, as well as immune tolerance in the brain, activating chronic inflammation and evoking behavioral phenotypes of depression or anxiety. Chronic central inflammation is considered a common pathological feature of most central nervous system (CNS) diseases. Restoring immune homeostasis, whether in the periphery or the CNS, is crucial for disease progression. By establishing a mouse model with four consecutive LPS injections, we simulated perturbations in both peripheral and central immune homeostasis and evaluated ARC001's ability to modulate immune function and promote (neural) tissue repair in vivo.

[0094] 1. Preparation method and injection frequency of modeling reagents All preparations must be performed aseptically in a biosafety cabinet. The reagent bottles used must be sterilized or sterile containers must be used. There is no need to avoid light during the preparation process (see Table 1 for details).

[0095] The prepared modeling preparation is labeled with the concentration.

[0096] Table 1 Preparation method of modeling preparation Grouping and modeling arrangements are shown in Table 2: Table 2 Grouping and modeling arrangements Note: N: number of animals; ip: intraperitoneal injection.

[0097] 2. Dosage, method and frequency of administration The dosage, administration method and dosage of each group are shown in Table 3: Table 3 Dosage, administration method and dosage of each group All doses / concentrations shown in the table are theoretical doses / concentrations and all animals were male.

[0098] In this trial, the dosing frequency was once a day. The first dosing day was defined as Day 1 (Day 1 / D1), the day before the first dosing was defined as (Day -1 / D-1), and so on.

[0099] 3. Open-field behavioral evaluation The open field consisted of a black acrylic panel with a white background, measuring 40 x 40 x 40 mm. A camera was mounted on a bracket above the open field box and connected to a computer via USB or video cable. Before testing, the box was clean and odorless, wiped with 70% ethanol and sterile tissue. One hour before testing, the designated mouse was allowed to acclimate to the experimental environment. The experimental mouse was removed from its cage and placed in the center of the open field box. The experimenter then left the open field box to avoid disturbing the mouse. After recording for 10 minutes, the video was removed from the open field box and returned to its cage. Before the next animal was tested, the entire open field box was cleaned with 70% ethanol and sterile tissue. The saved video was analyzed for total distance traveled using Tracking Master V3.0.

[0100] The results are as follows Figure 4 As shown by Figure 4 It can be seen that the total distance of movement (Ambulatory Distance) in open field behavior was significantly decreased in the LPS control group compared with the blank control group. p = 0.0009, indicating that the model was successfully established; the total movement distance of the ARC001 intravenous group (ARC001-iv) administered via the tail vein increased significantly, and the disease-related phenotype was restored, and there was no significant difference from the blank control group ( p = 0.1403); no significant therapeutic effect was observed in the ARC001 nasal group (ARC001-nasal) administered through the nasal cavity; however, the combined tail vein and nasal administration (ARC001-combination) maximized the therapeutic effect, and compared with ARC001-iv, the total distance of exercise was increased more. p = 0.0061, p The value is less than 0.05, which is statistically significant, that is, ARC001 can treat behavioral damage related to the inflammatory storm caused by LPS, suggesting its enhanced immunosuppressive function and tissue repair function; and the combined intravenous and nasal administration routes can provide more significant therapeutic effects.

[0101] Example 5 1. Hep G2 cell culture 1.1. For the first passaging, a 1:2 passaging ratio is recommended. Discard the old culture medium and add 1-2 ml of 0.25% trypsin to the culture flask. Place the flask in a 37°C incubator for digestion for 5-10 minutes. Observe the cell digestion process under a microscope. Ensure that the cell digestion is complete until the cell clumps fall off the bottom of the culture flask. Quickly return the flask to the operating table and add 3-6 ml of complete culture medium to terminate the digestion. Gently pipette the cells to disperse the culture medium into small cell clumps. Aspirate the culture medium into a centrifuge tube and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend in 1-3 ml of complete culture medium. Transfer the resuspended cell suspension to two new T25 culture flasks and add 8-10 ml of new complete culture medium per flask. The cells should be fully grown in 3-4 days.

[0102] 1.2. Complete culture medium preparation: DMEM medium, 90%; high-quality fetal bovine serum, 10%; complete cell culture medium 92% + DMSO 8%.

[0103] 2. Tumor Cell Preparation 2.1. Place the Matrigel on ice and thaw overnight at 4°C. Use a pre-cooled pipette or pipette tip to mix the Matrigel until uniform.

[0104] 2.2. Prepare Hep-G2 cells in logarithmic phase growth with a cell density of approximately 80-90%. Replace the culture medium with fresh medium the night before cell collection. Digest the cells with trypsin. When the cells become round but do not detach from the culture dish, remove the trypsin and add serum-free culture medium to make a cell suspension. Centrifuge the collected cell suspension at 300×g for 5 minutes and wash twice with PBS. Resuspend the cells in PBS to a final concentration of 8×10 7 cells / mL. Keep cells on ice until use.

[0105] 2.3. Mix the cell suspension and matrigel at a ratio of 1:1 at 4°C to a final concentration of 0.5×10 7 -5×10 7 cells / mL.

[0106] 3. Tumor formation experiment 3.1 Use an alcohol cotton ball to disinfect the area to be injected (skin on the axillary side of the upper limb).

[0107] 3.2 Use a 1 mL syringe without a needle to draw the cell suspension and Matrigel mixture into the syringe and then attach the needle.

[0108] 3.3 Grasp and secure the nude mouse with your left hand and inject subcutaneously into the left axilla. Insert the needle slightly deeper, approximately 1 cm, to minimize spillage of the cell suspension from the needle. The inoculation volume is typically 100 µL per mouse.

[0109] 3.4 The nude mice were returned to the cage and continued to be raised. A more obvious tumor mass was observed in about 2-4 weeks. According to the experimental design, the tumor volume did not exceed 1000 mm. 3 The nude mice were euthanized before surgery, the tumors were removed, and photos were taken and the tumor volume was recorded using a vernier caliper.

[0110] 3.5 The injection conditions for ARC001 products are the same as above.

[0111] 4. Tumor promotion experiment 4.1. Place the matrix gel on ice and melt it at 4°C overnight. Use a pre-cooled pipette or pipette tip to mix the matrix gel until it is uniform. Prepare Hep-G2 cells in logarithmic phase with a cell density of about 80-90%, and replace the culture medium the night before collecting the cells. Digest the cells with trypsin. When the cells become round but do not detach from the culture dish, remove the trypsin and add serum-free culture medium to make a cell suspension. Centrifuge the collected cell suspension at 300×g for 5 minutes and wash twice with PBS. Resuspend the cells in PBS to a final concentration of 8×10 7 cells / mL. Keep cells on ice until use.

[0112] 4.2. Mix the cell suspension and matrigel at a ratio of 1:1 at 4°C to a final concentration of 0.5×10 7 -5×10 7 cells / mL.

[0113] 4.3 Use an alcohol cotton ball to disinfect the area to be injected (skin on the axillary side of the upper limb).

[0114] 4.4 Use a 1 mL syringe without a needle to draw the cell suspension and Matrigel mixture into the syringe and then attach the needle.

[0115] 4.5 Grasp and secure the nude mouse with your left hand and inject subcutaneously into the left axilla. Insert the needle slightly deeper, approximately 1 cm, to minimize spillage of the cell suspension from the needle. The inoculation volume is typically 100 µL per mouse.

[0116] 4.6 Return the nude mice to their cages for continued feeding. Smaller tumor masses will be observed in about 1-2 weeks. Take photos and record the baseline tumor volume using a vernier caliper.

[0117] 4.7 ARC001 test product samples were injected into the mouse vein via tail vein injection, with a volume of 100µL / 25g / mouse and a 1×10 ARC001 cell count. 6 , which is the highest safe tolerable dose in mice.

[0118] 4.8 Return the nude mice to their cages and continue to observe for about 1-2 weeks. 3The nude mice were euthanized before the experiment, the tumors were removed, photos were taken, and the tumor volume was recorded with a vernier caliper. Finally, the difference between the tumor volume and the baseline volume was calculated.

[0119] The results are as follows Figure 5 As shown by Figure 5 A shows that HepG2 cells can form tumors after injection, but no mice injected with ARC001 develop tumors, which means that ARC001 cannot be observed to have tumorigenicity. Figure 5 As shown in Figure B, HepG2 tumor-bearing mice were divided into two comparable subgroups: one group received tail vein injection of ARC001, and the other group received intravenous injection of vehicle as a control (CTR). The results showed no difference between the groups, indicating that ARC001 did not promote tumor growth. These results suggest that after a series of small molecule reprogramming, ARC001 has no risk of tumorigenesis or tumor promotion.

[0120] Example 6 Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by degeneration of nerve cells in the brain and spinal cord. Approximately 5,000 people are diagnosed with ALS each year, with an average life expectancy of 3-5 years after diagnosis. The main pathological features of ALS patients are progressive death of motor neurons, abnormal activation of central glial immune cells, and imbalance in peripheral immune homeostasis. ALS poses a serious threat to human health, but effective treatment strategies are currently lacking. ARC001 is an autologous fibroblast cell induced through small molecule reprogramming technology. By regulating the immune state, central immune inflammation, and neurotrophic microenvironment, it can be clinically developed for the treatment of amyotrophic lateral sclerosis (ALS).

[0121] SOD1 G93A is currently the most commonly used mouse model for ALS preclinical research. It exhibits classic ALS-like clinical phenotypes and is widely used in ALS research and therapeutic strategy development. The GPT-B6-hSOD1 G93A mouse model, with an older age of onset, is closer to the onset of human ALS than other SOD1 disease models, such as Jax-hSOD1 G93A (G1H), with a median age of onset of 5.5 months, with a margin of error of 1.5 months. Therefore, this study used GPT-B6-hSOD1 G93A mice (hereafter referred to as SOD1 mice) for in vivo efficacy testing.

[0122] 1. Before the first dose, all animals were acclimated for at least 5 days in the animal room of this research institution. Due to the limited number of off-the-shelf model mice purchased for the pre-experiment and limited gender, the groups were grouped to ensure comparability in body weight and human SOD1 G93A copy number. Mice were 22 weeks (5.5 months) old when dosing began.

[0123] ALS treatment groups and dosing regimens are shown in Table 4: Table 4 ALS treatment groups and dosing regimens 2. Treatment and medication 2.1. Cell preparations were transported on wet ice to the animal room for animal administration (aseptic operation).

[0124] 2.2. Before administration, mix thoroughly by pipetting up and down. Draw out / absorb cells with a syringe / pipette before administration. Mix thoroughly every 3-5 minutes. Keep the preparation on wet ice during administration.

[0125] 2.3. Secure the mouse in an appropriate position. Use a pipette (20 μL) to draw up the drug solution. Place a drop of the solution into one nostril, allowing the animal to inhale it during breathing. Once the animal has inhaled the previous drop, continue with the next drop. This is repeated 3-5 times, alternating between nostrils. After each nostril instillation, slightly tilt the mouse's head upward for at least 60 seconds to prevent the drug from leaking out.

[0126] 2.4. Perform nasal instillation first, then complete tail vein injection within 10 minutes.

[0127] 2.5. Administer the drug once a week for a total of 6 times (D1, D7, D14, D21, D28, and D35).

[0128] 3. Limb grip strength test 3.1. All surviving animals were tested for limb grip strength before each treatment (after disease scoring and body weight measurement) from before the first treatment to one week after the last treatment (Day 1, 7, 14, 21, 28, 35, and 42).

[0129] 3.2. Weigh each test mouse before testing and bring the animal into the testing room at least one hour before testing to allow them to acclimate. Gently remove the animal from the test cage and gently grasp its tail. Allow the animal to grasp the digital dynamometer while gently pulling parallel to the bar with its tail. Record the grip dynamometer reading at the point where the mouse exerts maximum force. Repeat the measurement three times to ensure accurate results. Clean the instrument with alcohol after each test.

[0130] 3.3. The maximum force applied (in Newtons (N)) was divided by the body weight to obtain force / body weight (N / g).

[0131] The results are as follows Figure 6 As shown by Figure 6It can be seen that there was no significant difference between the ARC001 treatment group and the placebo control group before treatment (D1); after treatment, the slope of grip strength decline in the ARC001 treatment group was significantly smaller, and the grip strength of the ARC001 treatment group was higher than that of the control group. Repeated measures analysis of variance p = 0.0106, p The value was less than 0.05, which was statistically significant, suggesting that ARC001 could promote muscle strength recovery and delay disease progression in ALS mice.

[0132] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0133] Although the preferred embodiments of the present invention 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 present invention.

Claims

1. A composition for inducing fibroblast reprogramming, characterized in that: The composition is used to reprogram fibroblasts into fibroblasts with enhanced immunomodulatory and tissue repair capabilities, and includes, in order of induction, a first-stage small molecule compound, a second-stage small molecule compound, and a third-stage protein molecule; The first phase of small molecule compounds consists of Y27632, SAG, forskolin, and Rapamycin; The second phase of small molecule compounds consists of Y27632, SAG, forskolin, Rapamycin, AXL1717, RA, and TNF-α; The third stage protein molecule is bFGF.

2. A culture system for inducing fibroblast reprogramming, characterized in that: It includes the composition according to claim 1, wherein the culture system includes a first culture system, a second culture system and a third culture system, the first culture system contains the first-stage small molecule compound, the second culture system contains the second-stage small molecule compound, and the third culture system contains the third-stage protein molecule.

3. A culture system for inducing fibroblast reprogramming according to claim 2, characterized in that: In the first culture system, the second culture system and the third culture system, the Y27632 concentration is 2.5-5 μM; the SAG concentration is 0.25-0.5 μM; the forskolin concentration is 5-10 μM; the Rapamycin concentration is 0.1-0.2 μM; the TNF-α concentration is 10-20 ng / ml; the AXL1717 concentration is 1-2 μM; the RA concentration is 1-2 μM; and the bFGF concentration is 10-20 ng / mL.

4. A culture medium for inducing fibroblast reprogramming, characterized in that It comprises the culture system according to claim 3.

5. A kit for inducing fibroblast reprogramming, characterized in that: It comprises the culture system according to claim 3.

6. Use of the composition of claim 1, the culture system of claim 3, the culture medium of claim 4, or the kit of claim 5 in inducing the preparation of reprogrammed fibroblasts with enhanced immunoregulatory and tissue repair abilities.

7. A method for inducing fibroblast reprogramming, characterized in that: Utilizing the culture system of claim 3, the method comprises: The fibroblasts are cultured in the first culture system for 4-6 days, then transferred to the second culture system for 3-5 days, and then transferred to the third culture system for 3 days, and then replaced with a conventional mesenchymal stem cell culture medium for maintenance culture and passage expansion.

8. The method according to claim 7, characterized in that The fibroblasts are derived from the fibroblasts behind the ears, limbs, abdomen and buttocks of mammals.

9. A fibroblast with enhanced immunomodulatory and tissue repair abilities, characterized in that: Prepared by the method according to any one of claims 7 to 8.

10. Use of the fibroblasts according to claim 9 in preparing a drug for treating a disease, characterized in that: Such diseases include Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, and frailty.

Images