Pet stem cell induction medium, preparation method and culture method thereof

Abstract

The present application relates to stem cell technology, in particular to a pet stem cell induction medium, a preparation method and a culture method, the medium significantly improves the proliferation efficiency and multi-directional differentiation potential of adipose-derived stem cells by optimizing the ratio of specific growth factors. The preparation method comprises the steps of preparing a basic medium, adding growth factors and sterile filtering. Experimental verification shows that compared with the control group, the adipogenic differentiation efficiency of ADSCs cultured in the medium is increased by 40%, the osteogenic differentiation ALP activity is increased by 65%, and the positive rates of surface markers CD29, CD44 and CD90 are all greater than or equal to 95%. The present application solves the problems of animal source pathogen contamination risk and low induction efficiency of traditional culture medium, and is suitable for pet medical treatment.

Description

Technical Field

[0001] This invention relates to stem cell technology, and more specifically, to a pet stem cell induction culture medium, its preparation method, and its culture method. Background Technology

[0002] Adipose-derived stem cells (ADSCs) have become core seed cells in pet regenerative medicine due to their abundant source, strong proliferative capacity, and low immunogenicity. However, current ADSC culture technologies face the following bottlenecks:

[0003] 1. Deficiencies in culture medium composition: Traditional culture media (such as DMEM+10% FBS) rely on animal serum, which poses a risk of contamination by exogenous pathogens and has poor batch-to-batch stability.

[0004] 2. Low induction efficiency: Existing induction protocols (such as dexamethasone + indomethacin) are not efficient enough in differentiating pet ADSCs, with adipogenic differentiation rates typically below 30%.

[0005] 3. Outdated passage technology: The use of bulk passage or high-density seeding results in a long cell expansion cycle (requiring 14-21 days per generation) and makes it easy to accumulate genetic variations.

[0006] 4. Insufficient function maintenance: The expression of stem cell markers (such as Oct-4 and Nanog) decreases during long-term culture, affecting the therapeutic effect.

[0007] Existing technology CN103255103A discloses a serum-free adipose-derived mesenchymal stem cell culture medium, using low-glucose DMEM as the basal medium and adding components such as basic fibroblast growth factor (bFGF), heparin, and glutamine. Its advantage lies in avoiding the use of serum and reducing the risk of exogenous contamination. However, experiments have shown that this formulation suffers from poor cell adhesion and insufficient proliferation rate, possibly related to the lack of key growth factors (such as IGF-1) and antioxidant components.

[0008] Existing technology CN118370772 discloses a culture medium containing SCF (stem cell factor), nicotinamide, and VEGF for the treatment of vascular injury with adipose-derived stem cells. Experiments show that this culture medium can enhance cell migration and miR-146b-3p expression, and delay cell senescence. However, it relies on 10% fetal bovine serum (FBS), posing a risk of batch-to-batch variation, and it does not address the issue of cell adhesion in serum-free systems.

[0009] Existing technology CN10679696A discloses a kit containing tissue preservation solution, basal culture medium, and growth factors I / II, emphasizing the efficiency of adipose-derived stem cell isolation and expansion. Its culture medium additives include fetal bovine serum albumin, ethyl methylcellulose, and xylitol, which can improve cell viability. However, the serum-free formulation is not clearly defined, and high concentrations of serum albumin may increase costs and immunogenicity risks.

[0010] The prior art CN108410808A discloses a formulation containing DMEM basal medium, human serum albumin (HSA), transferrin, and plant-extracted peptides. Experiments show that it can maintain cell morphology and activity, but it does not solve the problem of decreased expression of stem genes (such as Oct-4 and Sox-2) caused by long-term passage (such as above P10), which may be related to the lack of specific epigenetic regulatory components (such as pengaloyl glucose).

[0011] Existing technology CN117138121B discloses an induction medium using low-glucose DMEM + 10% FBS, with the addition of dexamethasone, palmitic acid, and amino acids to improve the adipogenic differentiation rate of adipose-derived stem cells. Although the adipogenic efficiency is improved, the use of FBS increases batch-to-batch inconsistency, and high concentrations of dexamethasone may inhibit cell proliferation, limiting their multi-lineage differentiation potential.

[0012] To address the aforementioned issues, some studies have attempted to improve culture medium formulations. For example, some literature suggests adding L-ascorbic acid and FGF2 to prolong cell passage times, but this does not solve the problem of animal-derived components; some literature uses serum-free formulations, but the induction differentiation efficiency is still not ideal. Therefore, there is an urgent need to develop a safe, efficient, and scalable culture medium for inducing pet ADSCs. Summary of the Invention

[0013] The present invention first provides a pet adipose stem cell induction culture medium, comprising animal-free DMEM / F12 basal medium, SCF, IGF-1, bFGF, L-ascorbic acid, transferrin, sodium selenate, N-acetylcysteine ​​(NAC) and sodium bicarbonate.

[0014] Preferably, the SCF concentration is 1-10 ng / mL.

[0015] Preferably, the IGF-1 concentration is 1-10 ng / mL.

[0016] Preferably, the concentration of bFGF is 1-10 ng / mL.

[0017] Preferably, the concentration of SCF is 2 ng / mL, the concentration of IGF-1 is 2 ng / mL, and the concentration of bFGF is 2 ng / mL.

[0018] Preferably, the concentration of L-ascorbic acid is 20-100 μg / mL, the concentration of transferrin is 1-10 μg / mL, the concentration of sodium selenate is 1-20 ng / mL, the concentration of N-acetylcysteine ​​(NAC) is 1-10 μM, and the concentration of sodium bicarbonate (NaHCO3) is 1-5 g / L.

[0019] Preferably, the concentration of L-ascorbic acid is 80 μg / mL, the concentration of transferrin is 8 μg / mL, the concentration of sodium selenate is 15 ng / mL, the concentration of N-acetylcysteine ​​(NAC) is 5 μM, and the concentration of sodium bicarbonate (NaHCO3) is 3.7 g / L.

[0020] The present invention also provides a method for preparing the pet stem cell induction culture medium as described above, the method comprising the following steps: weighing SCF, IGF-1, bFGF, L-ascorbic acid, transferrin, sodium selenate, N-acetylcysteine ​​(NAC) and sodium bicarbonate, and adding them to animal-free DMEM / F12 basal culture medium in the specified amounts.

[0021] Preferably, the SCF concentration is 1-10 ng / mL.

[0022] Preferably, the IGF-1 concentration is 1-10 ng / mL.

[0023] Preferably, the concentration of bFGF is 1-10 ng / mL.

[0024] Preferably, the concentration of SCF is 2 ng / mL, the concentration of IGF-1 is 2 ng / mL, and the concentration of bFGF is 2 ng / mL.

[0025] Preferably, the concentration of L-ascorbic acid is 20-100 μg / mL, the concentration of transferrin is 1-10 μg / mL, the concentration of sodium selenate is 1-20 ng / mL, the concentration of N-acetylcysteine ​​(NAC) is 1-10 μM, and the concentration of sodium bicarbonate (NaHCO3) is 1-5 g / L.

[0026] Preferably, the concentration of L-ascorbic acid is 80 μg / mL, the concentration of transferrin is 8 μg / mL, the concentration of sodium selenate is 15 ng / mL, the concentration of N-acetylcysteine ​​(NAC) is 5 μM, and the concentration of sodium bicarbonate (NaHCO3) is 3.7 g / L.

[0027] Finally, the present invention provides a method for culturing pet adipose stem cells, the method comprising the following steps: (1) separating and extracting pet adipose stem cells; (2) culturing pet adipose stem cells using the above-mentioned culture medium.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects:

[0029] This invention improves upon existing culture media by using active factors and optimizing the components and ratios. Experiments have demonstrated that this culture medium significantly enhances the proliferation efficiency and differentiation potential of pet ADSCs through the synergistic effect of SCF, IGF-1, and bFGF, without the risk of contamination from animal-derived components, thus providing reliable technical support for pet regenerative medicine. Detailed Implementation

[0030] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0031] Example 1. Culture medium formulation

[0032] The culture medium contains:

[0033] Basic culture medium: animal-free DMEM / F12 (1:1 mixture);

[0034] Growth factors: Recombinant human stem cell growth factor (SCF) 2 ng / mL, insulin-like growth factor-1 (IGF-1) 2 ng / mL, basic fibroblast growth factor (bFGF) 2 ng / mL;

[0035] Additives: L-ascorbic acid 80 μg / mL, transferrin 8 μg / mL, sodium selenate 15 ng / mL; Antioxidant: N-acetylcysteine ​​(NAC) 5 μM; Osmotic pressure regulator: sodium bicarbonate (NaHCO3) 3.7 g / L;

[0036] Preparation method

[0037] (1) Preparation of basic culture medium: Mix DMEM / F12 in proportion, add L-ascorbic acid, recombinant human insulin, transferrin, sodium selenate, NAC and NaHCO3, and adjust the pH to 7.3.

[0038] (2) Addition of growth factors: Add SCF, IGF-1 and bFGF according to the concentration gradient and store at 4℃ in the dark.

[0039] (3) Sterile filtration: Sterilize by filtration through a 0.22 μm filter membrane, and freeze at -20℃ after dispensing.

[0040] Culture medium 2: Compared with culture medium 1, SCF was omitted, and it only contained 3 ng / mL IGF-1 and 3 ng / mL bFGF, with the other conditions being the same.

[0041] Culture medium 3: Compared with culture medium 1, IGF-1 is omitted, and it contains only 3 ng / mL SCF and 3 ng / mL bFGF, with the other conditions being the same.

[0042] Culture medium 4: Compared with culture medium 1, bFGF is omitted, and it contains only 3 ng / mL SCF and 3 ng / mL IGF-1, with the other conditions being the same.

[0043] Culture medium 5: Compared with culture medium 1, SCF and IGF-1 are omitted, and only 6 ng / mL bFGF is contained, with the other conditions being the same.

[0044] Culture medium 6: Compared with culture medium 1, SCF and bFGF are omitted, and only 6 ng / mL IGF-1 is contained, with the other conditions being the same.

[0045] Culture medium 7: Compared with culture medium 1, IGF-1 and bFGF are omitted, and only 6 ng / mL SCF is contained, with the other conditions being the same.

[0046] Culture medium 8: Compared with culture medium 1, it does not contain SCF, IGF-1, or bFGF, but all other conditions are the same.

[0047] Example 2. Cultivation Method

[0048] (1) Under sterile conditions, subcutaneous adipose tissue of pet dogs was obtained. The adipose tissue was rinsed with physiological saline, cut into small pieces, and dispensed into 50 mL centrifuge tubes at a rate of 5 mL / tube. An equal volume of mixed solution of type I collagenase and type III collagenase (mass percentage concentrations of 0.05% and 0.05%, respectively) was added. The mixture was digested at 37°C for 60 min, and then the enzyme activity was inactivated to obtain the adipose tissue hydrolysate.

[0049] (2) Centrifuge the adipose tissue enzymatic hydrolysate from step (1) at 1500 rpm for 15 minutes and retain the adipose stem cell cluster at the bottom layer.

[0050] (3) Transfer the adipose stem cell cluster from step (2) to a new centrifuge tube, add 10 mL of phosphate buffer solution, repeatedly pipette to resuspend the cells to make a uniform single-cell suspension, wash thoroughly, let the cells pass through a filter with a diameter of 70 micrometers, collect single cells, and then centrifuge at 1500 rpm for 10 minutes, retaining the bottom adipose stem cell cluster; repeat this washing step twice to obtain adipose stem cell single-cell suspension.

[0051] (4) Add the single-cell suspension of adipose stem cells from step (3) into a culture flask containing DMEM complete medium, and place it in a 5% CO2 incubator at 37°C for culture. Change the medium every 3 days. When the cells reach 80% confluence, the cells can be passaged.

[0052] (5) After discarding the primary culture medium, the cells were dissociated using 0.25% trypsin solution for 5 minutes. Before terminating the reaction, the cell morphology was observed under an inverted microscope, and the adherent cells were completely detached by physical shaking.

[0053] (6) Add culture medium to stop digestion and pipette the cells to a new centrifuge tube. Rinse the culture flask with buffer and collect the remaining cells. Centrifuge at 400g for 3 minutes. Discard the supernatant after centrifugation, resuspend the cell pellet, and vortex to mix. Take 100μL of the cell suspension for trypan blue staining and counting. Seed the cells into T75 culture flasks according to the international cell culture standard at a passage ratio of 1:3.

[0054] Third-generation cells with uniform morphology were selected for subsequent experiments. Immunophenotyping of P3 generation cells was performed using flow cytometry to detect the fluorescence expression intensity of MSCs-specific markers such as CD29 (integrin β1), CD49 (lamin receptor), CD73 (extracellular enzyme), CD90 (Thy-1), and CD105 (endothelial glycoprotein), verifying that the cell stemness characteristics met the mesenchymal stem cell identification criteria established by ISCT.

[0055] (7) Following the steps in (6), culture the adipose stem cells to the 3rd generation and discard the supernatant; add 10 mL of fresh culture medium 1 and continue culturing at 37°C with 5% CO2 concentration. When the cell confluence reaches 80%, collect the cell supernatant. Then filter the harvested supernatant through a 0.22 μm filter.

[0056] Comparative Example 1

[0057] Except for replacing culture medium 1 with culture medium 2, the other conditions were the same as in Example 2.

[0058] Comparative Example 2

[0059] Except for replacing culture medium 1 with culture medium 3, the other conditions were the same as in Example 2.

[0060] Comparative Example 3

[0061] Except for replacing culture medium 1 with culture medium 4, the other conditions were the same as in Example 2.

[0062] Comparative Example 4

[0063] Except for replacing culture medium 1 with culture medium 5, the other conditions were the same as in Example 2.

[0064] Comparative Example 5

[0065] Except for replacing culture medium 1 with culture medium 6, the other conditions were the same as in Example 2.

[0066] Comparative Example 6

[0067] Except for replacing culture medium 1 with culture medium 7, the other conditions were the same as in Example 2.

[0068] Comparative Example 7

[0069] Except for replacing culture medium 1 with culture medium 8, the other conditions were the same as in Example 2.

[0070] Example 3. Viability of adipose-derived stem cells

[0071] Adipose-derived stem cells were expanded according to the culture protocols of Example 2 and Comparative Examples 1-7, and viable cell counts were performed using 0.4% trypan blue solution. Specific procedures included: mixing cell suspension and staining agent at a 1:1 ratio, and assessing cell viability using a hemocytometer under an inverted phase-contrast microscope. A baseline value was set based on the cell proliferation level of Comparative Example 7, and the relative proliferation fold of each experimental group relative to the control group (i.e., number of cells in the experimental group / number of cells in Comparative Example 7) was calculated. Quantitative data are expressed as mean ± standard deviation, n=3. Significant differences were verified by one-way ANOVA (P<0.05), and the results are detailed in Table 1.

[0072] Table 1 Relative proliferation rate

[0073] Group P10 Example 2 12±1.1 Comparative Example 1 8±0.7 Comparative Example 2 6±0.5 Comparative Example 3 5±0.8 Comparative Example 4 4±0.7 Comparative Example 5 3±0.6 Comparative Example 6 3±0.5

[0074] The results in Table 1 show that culture medium 1 in Example 2 increased the number of cells by 12-fold, which was highly significant compared with the control (Comparative Example 7) (p<0.05). This indicates that culture medium 1 can significantly promote the proliferation of adipose-derived stem cells.

[0075] Example 4. Verification of multi-directional differentiation ability

[0076] 1. Adipogenic differentiation:

[0077] Experimental group: Adipose-derived stem cells obtained by culturing in culture medium 1;

[0078] Control group: Adipose-derived stem cells obtained from culture medium 8 in Comparative Example 7;

[0079] Induction protocol: culture medium of Example 1 + dexamethasone (1 μM) + IBMX (0.5 mM) + insulin (10 μg / mL).

[0080] Oil Red O staining showed that the lipid content of the experimental group (68.3%) was significantly higher than that of the control group (42.1%).

[0081] 2. Osteogenic differentiation:

[0082] Induction protocol: culture medium of Example 1 + dexamethasone (0.1 μM) + sodium β-glycerophosphate (10 mM).

[0083] ALP activity assay: On day 14, the activity of the experimental group was 1.8 U / mg protein, which was 150% higher than that of the control group (0.7 U / mg).

[0084] 3. Surface marker analysis

[0085] Flow cytometry: The positive rates of CD29 (96.2%±2.1%), CD44 (94.7%±3.5%), and CD90 (93.8%±1.9%) were significantly higher than those of the control group (p<0.05).

[0086] Conclusion: This culture medium significantly enhances the proliferation efficiency and differentiation potential of pet ADSCs through the synergistic effect of SCF, IGF-1 and bFGF, without the risk of contamination from animal-derived components, providing reliable technical support for pet regenerative medicine.

[0087] It should be understood that the above descriptions are only some embodiments of the present invention. It should be noted that those skilled in the art can make other modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A pet adipose-derived stem cell induction culture medium, characterized in that, The pet adipose stem cell induction medium consists of animal-free DMEM / F12 basal medium, SCF, IGF-1, bFGF, L-ascorbic acid, transferrin, sodium selenate, N-acetylcysteine, and sodium bicarbonate. The concentration of SCF is 2 ng / mL, the concentration of IGF-1 is 2 ng / mL, and the concentration of bFGF is 2 ng / mL. The concentrations of L-ascorbic acid, transferrin, sodium selenate, N-acetylcysteine, and sodium bicarbonate were 80 μg / mL, 8 μg / mL, 15 ng / mL, 5 μM, and 3.7 g / L, respectively. The pet in question is a dog.

2. A method for preparing the pet adipose stem cell induction culture medium as described in claim 1, characterized in that, The method includes the following steps: weighing SCF, IGF-1, bFGF, L-ascorbic acid, transferrin, sodium selenate, N-acetylcysteine ​​and sodium bicarbonate, and adding them to animal-free DMEM / F12 basal medium, wherein the pet is a dog.

3. The application of the pet adipose stem cell induction culture medium as described in claim 1, characterized in that... Prepare pet adipose stem cells, wherein the pet is a dog.

4. A method for culturing pet adipose stem cells, characterized in that: The method comprises the following steps: (1) separating and extracting pet adipose stem cells; (2) culturing pet adipose stem cells using the culture medium described in claim 1, wherein the pet is a dog.