Culture medium and culture method for canine mesenchymal stem cell culture

Abstract

The present application relates to a culture medium and a culture method for canine mesenchymal stem cell culture, wherein the culture medium is based on DMEM, and is added with exosome-free fetal bovine serum, 0.5 ng / mL glutamic acid and 0.2 ng / mL cobalt chloride, both of which synergistically promote the rapid proliferation of canine mesenchymal stem cells (entering the logarithmic growth phase 2 days in advance, and the population doubling time is shortened by about 33%), and through the dual mechanism of promoting cell proliferation and promoting unit cell exosome secretion, the exosome yield in the culture supernatant is increased by about 1.1 times; at the same time, the use of exosome-free serum eliminates the interference of serum exosomes, ensuring the purity and batch stability of the exosomes; the matching precipitation extraction process does not require ultracentrifugation, and is simple to operate and low in cost. The present application effectively solves the technical problem that it is difficult to obtain canine mesenchymal stem cells and their exosomes in large quantities in the process of veterinary pharmaceutical production, and lays a foundation for the large-scale and industrialized application thereof.

Description

Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to a method for the large-scale preparation of mesenchymal stem cells and their exosomes. Background Technology

[0003] In recent years, with the development of regenerative medicine and tissue engineering, the application of stem cells in veterinary clinical practice has become a research hotspot. Stem cells (SCs) are known in the medical field as "universal cells" because, under different culture conditions, SCs can differentiate into myoepithelial cells, osteocytes, chondrocytes, dopaminergic neurons, and acetylcholinergic neurons, among others. Mesenchymal stem cells (MSCs) are a type of adult stem cell with multi-lineage differentiation potential. MSCs originate from mesodermal mesenchyme and are mostly found in organ stroma and connective tissue, with the highest content in animal bone marrow. Currently, the most researched MSCs are mainly derived from bone marrow, umbilical cord, peripheral blood, teeth, and adipose tissue. MSCs have advantages such as convenient sourcing, rapid expansion, and maintaining multi-lineage differentiation potential during expansion, while differentiating into osteoblasts, chondrocytes, and adipocytes under specific conditions. Mesenchymal stem cells, with their outstanding multi-lineage differentiation potential, immunomodulatory properties and low immunogenicity, have become a key biological resource for veterinary clinical treatment of bone and joint diseases, nerve damage and metabolic diseases. Their derived exosomes represent a new frontier in cell-free therapy.

[0004] With the booming pet economy, pet owners are paying more attention to their pets' health. In recent years, there has been an increase in canine dermatitis, muscle, bone, and articular cartilage diseases, but existing treatments are not ideal, necessitating more effective methods. Canine mesenchymal stem cells (cMSCs) have shown great therapeutic potential in the veterinary field. MSCs possess multi-lineage differentiation potential, low immunogenicity, and strong proliferative capacity, which has accelerated the research and application of cMSCs in tissue engineering. However, given the surge in demand, how to obtain large quantities of canine mesenchymal stem cells and reduce rejection reactions during their use have become hot research topics.

[0005] Exosomes are single-membrane vesicles secreted by cells into the extracellular space, typically ranging in diameter from 30 to 150 nm. Various cell types can secrete EXOs under any conditions. Exosomes carry complex substances, including cell-type specific proteins, nucleic acids, and lipids, and possess unique biological activities. In recent years, exosomes have been extensively studied as important intercellular communication carriers, influencing both neighboring cells and other parts of the body. Mesenchymal stem cell exosomes (MSCs-EXOs) have shown excellent repair effects on organ damage and play a significant role in tissue injury treatment. Exosomes contain various bioactive substances, including microRNA, mRNA, non-coding RNA, membrane proteins and cytoplasm, lipids, cytokines, and growth factors, playing important roles in immune responses, immune regulation, inflammatory responses, and stem cell phenotypic transformation.

[0006] Compared to MSCs, nanoscale MSCs-EXOs have the advantages of smaller particle size, targeted binding, stable preservation, safe and efficient immune response, and clinical applicability. Therefore, MSCs-EXOs are expected to become a new drug delivery route and gene therapy vector with broad clinical application prospects. The function of exosomes depends on the source cell type and cell culture state, and obtaining large quantities of exosomes is a prerequisite for successful pharmaceutical manufacturing and large-scale commercial application. Summary of the Invention

[0007] The present invention aims to solve the technical problem of difficulty in obtaining large quantities of mesenchymal stem cells and their exosomes in the process of veterinary drug manufacturing, and provides a mesenchymal stem cell culture medium that facilitates the rapid proliferation of mesenchymal stem cells and promotes the production of exosomes by mesenchymal stem cells.

[0008] To solve the above technical problems, the present invention adopts the following technical solution: This invention claims protection for a culture medium for canine mesenchymal stem cell culture, characterized in that the culture medium is based on DMEM medium, with the addition of exosome-free serum, and further with the addition of glutamate and / or cobalt chloride.

[0009] Preferably, the culture medium is based on DMEM medium, with the addition of 10% by volume of exosome-free fetal bovine serum, 0.5 ng / mL of glutamate and / or 0.2 ng / mL of cobalt chloride.

[0010] Preferably, the culture medium is based on DMEM medium, with the addition of 10% by volume of exosome-free fetal bovine serum, 0.5 ng / mL of glutamate and 0.2 ng / mL of cobalt chloride.

[0011] Preferably, the exosome-free fetal bovine serum is prepared by the following method: the fetal bovine serum is subjected to ultracentrifugation at 120,000g for 4 hours, and the precipitate is discarded to obtain the exosome-free fetal bovine serum.

[0012] Preferably, the canine mesenchymal stem cells are canine bone marrow mesenchymal stem cells.

[0013] The present invention also claims protection for a method for culturing canine bone marrow mesenchymal stem cells, characterized in that the culture medium described above for culturing canine mesenchymal stem cells is used for culturing.

[0014] This invention also claims protection for a method for preparing exosomes from canine bone marrow mesenchymal stem cells, characterized in that exosomes are extracted from a culture of canine bone marrow mesenchymal stem cells obtained by the above method.

[0015] Preferably, the exosome extraction method includes the following steps: (1) Centrifuge the collected canine bone marrow mesenchymal stem cell culture supernatant at 4°C and 3000g for 20 min to remove residual dead cells and cell debris. (2) Mix the sample with the exosome precipitation reagent EPS-C at a ratio of 2:1, and mix the sample and EPS-C by inverting and let stand overnight at 2~8℃; (3) Remove the centrifuge tube for observation and mark the precipitate location. Place the centrifuge tube in a centrifuge and centrifuge at 4°C, 10000g, for 30 minutes to precipitate the exosomes; (4) Discard the supernatant, collect the precipitate, centrifuge at 10000g for 5min, carefully aspirate the remaining supernatant with a 200μL pipette tip, centrifuge at 10000g for 5min, and carefully aspirate the remaining supernatant with a 10μL pipette tip, and remove the supernatant as cleanly as possible. (5) Add 30 μL of ERS-C solution to the precipitate, gently pipette and resuspend the precipitate to obtain exosomes. If there is too much exosome precipitate, increase the amount of resuspension solution until the precipitate is completely dissolved.

[0016] Based on the above technical solution, the present invention has the following beneficial effects: The culture medium of this invention is based on DMEM, supplemented with exosome-free fetal bovine serum and 0.5 ng / mL glutamate and 0.2 ng / mL cobalt chloride. These two components synergistically promote the rapid proliferation of canine mesenchymal stem cells (entering the logarithmic growth phase 2 days earlier, shortening the population doubling time by approximately 33%). Through a dual mechanism of promoting cell proliferation and promoting exosome secretion per unit cell, the exosome yield in the culture supernatant is increased by approximately 1.1 times. Simultaneously, the use of exosome-free serum eliminates interference from serum exosomes, ensuring the purity and batch stability of exosomes. The accompanying precipitation extraction process eliminates the need for ultracentrifugation, making it simple to operate and cost-effective. This invention effectively solves the technical problem of obtaining large quantities of canine mesenchymal stem cells and their exosomes in veterinary drug manufacturing processes, laying the foundation for their large-scale, industrial application. Detailed Implementation

[0017] The following specific embodiments are used to further illustrate the present invention, so that those skilled in the art can more clearly understand the technical solution of the present invention, and are not intended to limit the present invention.

[0018] 1.1 Experimental animals: 1-year-old male Chinese rural dogs, provided by Shaanxi Novelihua Pet Drug Evaluation Center.

[0019] 1.2 Isolation and primary culture of canine bone marrow mesenchymal stem cells: The surgery was performed under strict aseptic conditions. The experimental dogs were given general anesthesia with Serta-50, secured on the operating table, and their skin was prepared. Routine disinfection and draping were performed. A 20-22 gauge bone marrow aspiration needle was inserted into the greater trochanter of the femur in the hind limb. A 20 mL syringe containing 0.6 mL of heparin sodium (3000 U / mL) was connected, and 6 mL of bone marrow was extracted. This was slowly stacked along the centrifuge tube wall into two pre-prepared centrifuge tubes containing 3 mL of lymphocyte separation medium. The tubes were centrifuged at 3000 rpm for 30 min. The milky white, cloudy mononuclear cell layer from the intermediate interface was slowly aspirated, washed twice with PBS, and completely cultured in DMEM medium containing 10% fetal bovine serum at a growth rate of 1×10⁻⁶. 8 Cells were seeded at a density of 1 / L in culture flasks and cultured in a CO2 cell culture incubator at 37°C with 0.05% CO2 and saturated humidity. After 48 hours, half the medium was replaced. After 72 hours, non-adherent cells were discarded, and the cells were washed twice with PBS, followed by a full medium replacement. The medium was then changed every 3 days thereafter. Primary culture was terminated after 10 days when the adherent cells reached approximately 80%–90% confluence.

[0020] After isolating primary cells, cell adhesion can be observed under a microscope, with the cells exhibiting an irregular, short spindle shape. After one week of culture, the cells display an elongated spindle shape and adhere to the culture surface.

[0021] 1.3 Passaging of canine bone marrow mesenchymal stem cells: When the cell density reaches approximately 80%, perform passage. First, wash the cell culture supernatant, then wash the cells three times with PBS. Digest the cells with 1.0-2.0 mL of 0.25% trypsin and 1 mmol EDTA at 37°C for 2 min. When the cells begin to shrink and the intercellular spaces begin to increase, stop the digestion with 1.5-2 mL of DMEM medium containing 10% fetal bovine serum, and add 3-5 mL of PBS. Gently pipette the cells to detach them from the culture flask wall, transfer them to a centrifuge tube, centrifuge at 1200 rpm for 3 min, discard the supernatant, add DMEM medium containing 10% fetal bovine serum to the pellet, gently mix, and seed them 1:3 into appropriate culture dishes and continue culturing in an incubator.

[0022] 1.4 Cell detection For passaged cells, flow cytometry antigen detection showed that the cells were positive for CD44, CD73, CD105, CD90 and CD166, and negative for CD34 and CD45.

[0023] When canine bone marrow mesenchymal stem cells are passaged to the 4th generation, and the cell density reaches 70%, the culture is replaced with exosome-free serum to eliminate interference from serum exosomes. When the cell growth density reaches 85%–90%, the culture supernatant is collected for exosome extraction and identification.

[0024] (1) Centrifuge the collected canine bone marrow mesenchymal stem cell culture supernatant at 4°C and 3000g for 20 min to remove residual dead cells and cell debris. (2) Mix the sample with the exosome precipitation reagent EPS-C at a ratio of 2:1, and mix the sample and EPS-C by inverting and let stand overnight at 2~8℃; (3) Remove the centrifuge tube for observation and mark the precipitate location. Place the centrifuge tube in a centrifuge and centrifuge at 4°C, 10000g, for 30 minutes to precipitate the exosomes; (4) Discard the supernatant, collect the precipitate, centrifuge at 10000g for 5min, carefully aspirate the remaining supernatant with a 200μL pipette tip, centrifuge at 10000g for 5min, and carefully aspirate the remaining supernatant with a 10μL pipette tip, and remove the supernatant as cleanly as possible. (5) Add 30 μL of ERS-C solution to the precipitate, gently pipette and resuspend the precipitate to obtain exosomes. If there is too much exosome precipitate, increase the amount of resuspension solution until the precipitate is completely dissolved.

[0025] 20 μL of exosome sample was dissolved in 1 mL of PBS and placed in a cuvette. The cuvette was then placed in a nanoparticle size analyzer for particle size measurement. Nanoparticle size analysis revealed that the extracted exosomes had a particle size between 55 and 125 nm.

[0026] Exosome protein extraction: Exosomes from canine mesenchymal stem cells were lysed on ice using RIPA lysis buffer. 1% protease inhibitor was added according to the RIPA volume, and the mixture was directly pipetted. The mixture was vortexed once every 10 minutes on ice, for a total of 3 vortexes. After centrifugation at 12000 rpm for 10 minutes at 4°C, the supernatant was collected, and protein concentration was measured using a BCA kit. Western blot analysis confirmed the expression of the exosome marker protein TSG101.

[0027] Fetal bovine serum was centrifuged at 120,000g for 4 hours, and the precipitate was discarded to obtain exosome-free serum.

[0028] Based on previous research, this invention has discovered that adjustments to various components and culture medium composition have a significant impact on the growth rate of mesenchymal stem cells and the yield of exosomes. Therefore, this invention sets up a normal control group (containing only 10% exosome-free serum and DMEM basal medium), a glutamate group (glutamate was added to the normal group to a concentration of 0.5 ng / mL), a cobalt chloride group (cobalt chloride was added to the normal group to a concentration of 0.2 ng / mL), and a glutamate + cobalt chloride group (glutamate was added to the normal group to a concentration of 0.5 ng / mL and cobalt chloride to a concentration of 0.2 ng / mL).

[0029] Single-cell suspensions were prepared from P5 generation canine bone marrow mesenchymal stem cells, and the cell concentration was adjusted to 1×10⁻⁶. 6 Cells / L were seeded into 24-well culture plates, 200 μL per well, and cultured in the normal control group, glutamate group, cobalt chloride group, and glutamate + cobalt chloride group culture medium prepared in 3.2. The medium was changed every 3 days.

[0030] Starting from day 2, the absorbance (OD490nm) of 6-well cells in each group was measured every 24 hours using the MTT assay. The average value was taken as the cell count for that day, and the following formula was used: PDT=(t-t0)×lg2 / (lgNt-lgN0); The population doubling time (PDT) during the logarithmic growth phase was calculated. One-way ANOVA was performed using SPSS software, and a 2×2 factorial analysis was used to examine the interaction between glutamate and cobalt chloride. P < 0.05 was considered statistically significant. Cell growth parameters for each group are shown in Table 1.

[0031] Table 1 Comparison of growth indices of canine bone marrow mesenchymal stem cells in different groups (X±s, n=6) normal control group 4 38.5±2.1 0.86±0.07 Glutamate group 3 33.2±1.8* 1.02±0.06* cobalt chloride group 3 32.1±1.6* 1.05±0.08* Glutamic acid + cobalt chloride group 2 25.8±1.4*# 1.31±0.09*# Note: * indicates P < 0.05 compared with the normal control group; # indicates P < 0.05 compared with the glutamate group and the cobalt chloride group.

[0032] As shown in Table 1, cells in the glutamate and cobalt chloride groups entered the logarithmic growth phase earlier than the normal control group, with a significantly shorter population doubling time (P < 0.05). Cells in the glutamate + cobalt chloride group entered the logarithmic growth phase on day 2 of culture, exhibiting the shortest population doubling time and the highest cell count on day 9, all significantly superior to the normal control group and the two groups receiving the single addition (P < 0.05). This indicates a significant synergistic effect between glutamate and cobalt chloride in promoting cell proliferation. These results suggest that both glutamate and cobalt chloride alone can promote the proliferation of canine bone marrow mesenchymal stem cells, but their combined addition has a synergistic effect, enabling cells to enter the logarithmic growth phase earlier and significantly increasing cell yield.

[0033] 3.4 Determination of exosome content in canine bone marrow mesenchymal stem cell culture supernatant P5 generation canine bone marrow mesenchymal stem cells were obtained at a concentration of 1×10⁻⁶. 6 Cells were seeded at a density of 2 mL / L in 6-well plates. Once cell confluence reached 70%, the original culture medium was discarded, and the cells were washed twice with PBS. The medium was then replaced with the prepared 3.2 medium for the normal control group, glutamate group, cobalt chloride group, and glutamate + cobalt chloride group, with 6 replicates per group. After 48 hours of culture, 5 mL of culture supernatant was collected from each group. Simultaneously, adherent cells were digested with 0.25% trypsin and counted to correct for the effect of cell number differences on exosome production.

[0034] Exosomes were separated from the supernatant of each group according to the method in 2.1. After resuspending the precipitate in 30 μL of ERS-C solution, the exosome protein concentration was determined using a BCA protein quantification kit. A standard curve was plotted using bovine serum albumin (BSA) as a standard. The BCA working solution (solution A:solution B = 50:1) was mixed with the sample at a volume ratio of 8:1 and incubated at 37℃ for 30 min. The absorbance at 562 nm was measured. The sample protein concentration was calculated based on the standard curve and converted into the exosome protein content (μg / mL) per unit volume of culture supernatant and per 10 6 Exosomal protein production per cell (μg / 10) 6 The exosome content was measured using a nanoparticle size analyzer to determine the particle concentration (particles / mL) of each group. Statistical methods were the same as in 3.3.

[0035] Table 2. Results of exosome content determination in culture supernatant of each group (x±s, n=6) normal control group 10.2±1.3 5.1±0.6 Glutamate group 13.8±1.5* 6.9±0.7* cobalt chloride group 14.6±1.4* 7.3±0.6* Glutamic acid + cobalt chloride group 21.5±1.8*# 10.7±0.9*# Note: * indicates P < 0.05 compared with the normal control group; # indicates P < 0.05 compared with the glutamate group and the cobalt chloride group.

[0036] Based on the results in Table 2, compared with the normal control group, the exosome protein content in the culture supernatant of both the glutamate group and the cobalt chloride group was significantly increased (P < 0.05). The glutamate + cobalt chloride group had the highest exosome content, significantly higher than the normal control group and the two groups that were added alone (P < 0.05). The exosome protein yield per unit cell number showed the same trend, and factorial analysis showed a significant interaction between glutamate and cobalt chloride (P < 0.05), indicating that the increase was not solely due to accelerated cell proliferation, but that the combined addition also had a synergistic promoting effect on exosome secretion in individual cells. The above results indicate that the culture medium of the present invention significantly increases the exosome yield of canine bone marrow mesenchymal stem cells through a dual mechanism of promoting cell proliferation and promoting exosome secretion per unit cell.

Claims

1. A culture medium for culturing canine mesenchymal stem cells, characterized in that, The culture medium is based on DMEM medium, with the addition of exosome-free serum, and further supplemented with glutamate and / or cobalt chloride.

2. The culture medium for canine mesenchymal stem cell culture according to claim 1, characterized in that, The culture medium is based on DMEM medium, supplemented with 10% by volume of exosome-free fetal bovine serum, 0.5 ng / mL of glutamate and / or 0.2 ng / mL of cobalt chloride.

3. The culture medium for canine mesenchymal stem cell culture according to claim 2, characterized in that, The culture medium is based on DMEM medium, supplemented with 10% by volume of exosome-free fetal bovine serum, 0.5 ng / mL of glutamate and 0.2 ng / mL of cobalt chloride.

4. The culture medium for canine mesenchymal stem cell culture according to any one of claims 1-3, characterized in that, The exosome-free fetal bovine serum was prepared by the following method: the fetal bovine serum was subjected to ultracentrifugation at 120,000g for 4 hours, and the precipitate was discarded to obtain the exosome-free fetal bovine serum.

5. The culture medium for canine mesenchymal stem cell culture according to any one of claims 1-3, characterized in that, The canine mesenchymal stem cells mentioned are canine bone marrow mesenchymal stem cells.

6. A method for culturing canine bone marrow mesenchymal stem cells, characterized in that, The cells were cultured using the culture medium for canine mesenchymal stem cell culture as described in any one of claims 1-3.

7. A method for preparing canine bone marrow mesenchymal stem cell exosomes, characterized in that, After obtaining a canine bone marrow mesenchymal stem cell culture using the method described in claim 6, exosomes are extracted.

8. The method for preparing canine bone marrow mesenchymal stem cell exosomes according to claim 7, characterized in that, The exosome extraction method includes the following steps: (1) Centrifuge the collected canine bone marrow mesenchymal stem cell culture supernatant at 4°C and 3000g for 20 min to remove residual dead cells and cell debris. (2) Mix the sample with the exosome precipitation reagent EPS-C at a ratio of 2:1, and mix the sample and EPS-C by inverting and let stand overnight at 2~8℃; (3) Remove the centrifuge tube for observation and mark the precipitate location. Place the centrifuge tube in a centrifuge and centrifuge at 4°C, 10000g, for 30 minutes to precipitate the exosomes; (4) Discard the supernatant, collect the precipitate, centrifuge at 10000g for 5min, carefully aspirate the remaining supernatant with a 200μL pipette tip, centrifuge at 10000g for 5min, and carefully aspirate the remaining supernatant with a 10μL pipette tip, and remove the supernatant as cleanly as possible. (5) Add 30 μL of ERS-C solution to the precipitate, gently pipette and resuspend the precipitate to obtain exosomes. If there is too much exosome precipitate, increase the amount of resuspension solution until the precipitate is completely dissolved.