Method for establishing a goat bone marrow mesenchymal stem cell line

Abstract

The application discloses a method for establishing a goat bone marrow mesenchymal stem cell line and belongs to the technical field of stem cell culture. The application adopts DMEM / F12 to rinse goat bone marrow to obtain a rinsing product; the rinsing product and complete culture medium are mixed and blown to be a single cell suspension, and then inoculation culture is carried out; after the inoculation culture is completed, subculture is carried out; the subculture method is as follows: when the cell fusion degree reaches 80%, the culture medium is discarded, rinsing, digestion and digestion termination are carried out to obtain a digested cell suspension; the digested cell suspension is blown, centrifuged, precipitated and resuspended, and then inoculated into a new culture bottle; after one hour, the non-adherent cell suspension is transferred to a new bottle, and the adherent BMSCs are reserved for continuous culture, so that the subculture is completed; the main cell bank is established by subculture to the third generation; the aging phenomenon that the proliferation speed of BMSCs is slowed down and the differentiation ability is decreased is effectively delayed, and the differentiation potential stability of the BMSCs is ensured.

Description

Technical Field

[0001] This invention belongs to the field of stem cell culture technology, specifically relating to a method for establishing a goat bone marrow mesenchymal stem cell line. Background Technology

[0002] Bone marrow mesenchymal stem cells (BMSCs) are a type of pluripotent stem cells found in the bone marrow stroma. They possess self-renewal capacity and the potential to differentiate into multiple lineages, including osteoblasts, chondrocytes, adipocytes, and myocytes, under specific induction conditions. Goat BMSCs, due to their differentiation orientation closely related to meat quality traits (such as intramuscular fat content and muscle development), have become an important in vitro model for studying the molecular mechanisms of adipogenesis and myogenic differentiation, as well as for applications in livestock breeding.

[0003] The isolation and purification of BMSCs is the first step in conducting related research. Currently, common isolation techniques mainly include the following categories: Traditional separation methods include whole bone marrow adherence, density gradient centrifugation, and bone tissue block digestion. Among these, density gradient centrifugation is time-consuming (usually exceeding 3 hours), during which cells easily lose their essential microenvironment, leading to slow subsequent proliferation and accelerated cell aging. Bone tissue digestion relies heavily on precise control of enzymatic digestion conditions (such as enzyme concentration, reaction time, and temperature), which are demanding. Improper digestion can easily damage cell membrane integrity and even induce apoptosis.

[0004] Emerging sorting strategies include magnetic immunobead sorting (MACS) and flow cytometry sorting (FACS). These technologies sort cells based on cell surface-specific markers, yielding cell populations with high purity. However, they generally suffer from drawbacks such as high cost, complex operation, and heavy reliance on specialized equipment, making them difficult to widely implement in routine laboratories or large-scale applications.

[0005] In contrast, the whole bone marrow adherence method leverages the adhesion and growth characteristics of BMSCs on plastic culture dishes. It involves directly seeding and culturing a single-cell suspension of bone marrow cells, utilizing differential adhesion to achieve initial screening and enrichment of target cells. This method is simple and quick, avoids physical damage from repeated centrifugation, reduces the risk of contamination, and is relatively low-cost. More importantly, this method maximizes the preservation of inherent endogenous growth factors in bone marrow fluid, which helps cells adhere rapidly, shortens the latency period, and increases the initial proliferation rate. After appropriate passage and expansion, cell purity can reach over 95%.

[0006] After obtaining BMSCs, maintaining their stability in quantity and functional characteristics during in vitro expansion is crucial, which depends on optimizing the culture system. Generally, α-MEM or DMEM / F12, suitable basal media for mesenchymal stem cell growth, are preferred, supplemented with 10% fetal bovine serum to provide necessary growth factors and nutrients, and 1% penicillin antibiotics are added to prevent microbial contamination. Cell seeding density must be controlled within an appropriate range to avoid apoptosis due to too low a density or contact inhibition due to too high a density. Simultaneously, passage should be performed promptly when cell confluence reaches approximately 80% (usually after 72-96 hours of culture). Prolonged culture or excessive passages can easily induce replicative senescence, manifested as telomere shortening, decreased proliferative capacity, and reduced multi-lineage differentiation potential.

[0007] In summary, although existing technologies provide a certain pathway for obtaining goat BMSCs, they still have many limitations in terms of isolation efficiency, cost control, ease of operation, and protection of cell viability and functional integrity. Therefore, developing a more efficient, economical, mild, and easily standardized goat BMSCs isolation and culture protocol is of significant practical importance and technical necessity for stably obtaining high-quality cell resources, further elucidating the cellular and molecular mechanisms affecting goat meat production performance, and promoting its practical application in molecular breeding. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a method for establishing a goat bone marrow mesenchymal stem cell line. This invention effectively delays the aging phenomenon of BMSCs, which leads to a slowdown in proliferation rate and a decline in differentiation capacity, and ensures the stability of the differentiation potential of BMSCs.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for establishing a goat bone marrow mesenchymal stem cell line, comprising the following steps: S1. Goat bone marrow was rinsed with DMEM / F12 to obtain the rinsed material; S2. Mix the rinse material and complete culture medium and pipette to form a single-cell suspension, then inoculate and culture. S3. After the inoculation culture is completed, perform subculture. The passage culture method is as follows: When the cell confluence reaches 80%, discard the culture medium, rinse, digest, and terminate the digestion to obtain a digested cell suspension; After the digested cell suspension is pipetted, centrifuged, and resuspended by precipitation, it is seeded into a new culture flask. One hour after seeding, the non-adherent cell suspension is transferred to a new flask, and the adherent BMSCs are retained for further culture to complete the passage culture. S4. After passage to the 3rd generation, establish a master cell bank and preserve the cells by cryopreservation using a cryopreservation solution containing 90% DMEM / F12 medium and 10% DMSO.

[0010] The method for establishing the master cell bank is as follows: When BMSCs (P3) form a dense monolayer on the culture dish, the cells can be detached from the surface by trypsin digestion, resuspended in DMEM / F12 medium containing 10% FBS, centrifuged for 5 minutes to collect the cell pellet, and then resuspended in cryopreservation buffer and aliquoted into cryovials. The cells are then transferred to liquid nitrogen for long-term storage using a programmed cooling method.

[0011] Preferably, the goat bone marrow in S1 is obtained from the femur of a goat.

[0012] Preferably, the rinsing in step S1 includes blowing, washing, and centrifugation; the number of rinsing cycles is 2-4.

[0013] Furthermore, the DMEM / F12 is commercially available.

[0014] Preferably, the density of the single-cell suspension in S2 is 1×10⁻⁶. 6 cells / mL.

[0015] Preferably, the inoculation and culture temperature in S2 is 37°C and 5% CO2; the culture process is as follows: when the cells are cultured for 6 hours, the complete culture medium is partially replaced; when the cells are cultured for 24 hours, the complete culture medium is completely replaced; thereafter, the medium is completely replaced once every 3 days.

[0016] Furthermore, the complete culture medium consists of: DMEM / F12 medium, 10% fetal bovine serum (FBS), and 1% penicillin and streptomycin.

[0017] Preferably, the digestion step in S3 is as follows: digestion with 0.25% trypsin-EDTA at 37°C for 2 minutes.

[0018] Preferably, the digestion termination step in S3 is as follows: after digestion, discard the trypsin and immediately add complete culture medium to terminate digestion.

[0019] Preferably, the centrifugation parameters in S3 are 1300 rpm for 5 minutes.

[0020] Preferably, the inoculation density in S3 is 1×10⁻⁶. 4 cells / cm 2 .

[0021] It contains at least the following beneficial technical effects: This invention provides a highly efficient and rapid method for the efficient enrichment of bone marrow mesenchymal stem cells (BMSCs) through whole bone marrow adherence. It involves direct inoculation of bone marrow single-cell suspensions, utilizing the natural adhesion properties of BMSCs for selective enrichment. This method eliminates complex steps, significantly shortens separation time, and reduces the cost of expensive reagents and equipment. It preserves endogenous growth factors (such as SDF-1 and PDGF) in bone marrow plasma, promoting early cell adhesion and proliferation. It avoids repeated centrifugation that could damage cells, reducing contamination. Cell passage can increase BMSC purity to over 95%. A complete separation strategy and culture conditions ensure the quality of BMSCs, effectively delaying the aging process of slowed proliferation and decreased differentiation capacity, and guaranteeing the stability of BMSC differentiation potential. Attached Figure Description

[0022] Figure 1 These are micrographs from different periods.

[0023] Figure 2 This is a cell immunofluorescence identification image.

[0024] Figure 3 This is a diagram illustrating the adipogenic differentiation of BMSCs.

[0025] Figure 4 This is a diagram illustrating the myogenic differentiation of BMSCs. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0032] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0033] Reagents or raw materials: 0.25% trypsin-EDTA (Gibco); fetal bovine serum (Gibco); DMEM / F12 (Gibco); penicillin / streptomycin solution (Gibco); oleic acid (Sigma); dexamethasone (Sigma); IBMX (Sigma); insulin (Beyotime); 5-Aza (Sigam); DMSO (Solepro); horse serum (Biosharp).

[0034] Example 1 (1) Seven-day-old goats were euthanized and sterilely cleaned, and their cells were separated in a sterile environment.

[0035] (2) Scrape and disinfect the femur of the goat, remove excess muscle tissue, puncture the femur with a bone marrow aspiration needle, extract bone marrow, add an equal volume of DMEM / F12 and centrifuge briefly to remove the upper layer of oil, then add DMEM / F12 again, blow, wash and centrifuge, and rinse a total of 3 times.

[0036] (3) After rinsing, the rinsed material is added to complete culture medium and pipetted into a single-cell suspension at a ratio of 2×10⁻⁶. 5 Cells were seeded at a density of cells / cm² into 25 cm² cartridge bottles and incubated at 37°C with 5% CO₂. After 6 hours of culture, the cartridge bottles were gently shaken to partially change the complete culture medium. After 24 hours of culture, the bottles were rinsed and the medium was completely changed, and thereafter, the medium was completely changed every 3 days.

[0037] When the primary cells reached 80% confluence, discard the culture medium, wash twice with PBS, add 0.25% trypsin-EDTA, and digest at 37°C for 2 minutes (monitor cell retraction under a microscope). Discard the trypsin and immediately add complete culture medium to terminate the digestion. Collect the cell suspension by pipetting, centrifuge at 1300 rpm for 5 minutes, resuspend the pellet, and subculture at a 1:4 ratio into new culture flasks (density 1×10⁶ cells / year). 4 cells / cm 2 To improve purity, a differential adhesion method was used: 1 hour after inoculation, the non-adherent cell suspension was transferred to a new bottle, while the adherent BMSCs were cultured to complete one passage.

[0038] (4) Passage culture to 3 generations to establish master cell bank The cells were passaged continuously to the P3 generation to establish a master cell bank. The cryopreservation solution was 90% DMEM / F12 + 10% DMSO. The temperature was programmed (4℃, 30min → -20℃, 2h → -80℃, overnight). Finally, the cells were transferred to liquid nitrogen for long-term storage.

[0039] Experimental Example Bone marrow mesenchymal stem cell identification: Morphological evaluation of third-generation BMSCs (spindle-shaped proportion >90%), immunofluorescence identification of surface markers of bone marrow mesenchymal stem cells, multiple differentiation potential tests of BMSCs, and multi-faceted cell identification.

[0040] 1. Morphological assessment of BMSCs: Cell status was observed under an inverted phase-contrast microscope, and morphological changes were recorded after primary seeding and passage. Figure 1 BMSCs in P0 (100×); BMSCs in P1 (100×); BMSCs in P3 (100×); BMSCs in P7 (100×); BMSCs in P11 (100×); BMSCs in P13 (100×); BMSCs in P27 (100×); BMSCs in P31 (100×).

[0041] The cells had been continuously passaged to passage 31 (P31). Primary cell culture: adherent cells were mostly typical spindle-shaped, with some irregular polygonal or short spindle-shaped cells; the cytoplasm was clear, with a small number of round hematopoietic cells suspended in suspension. Immunofluorescence identification of surface markers of bone marrow mesenchymal stem cells: P5 passage BMSCs were seeded in 96-well plates (density 1×10⁻⁶). 4 cells / cm 2After 24 h, fix with 4% paraformaldehyde for 30 min, wash three times with PBS for 10 min each time, and block with blocking buffer (2% BSA, 2% horse serum, 0.2 mol / L glycine and 0.2% Triton X-100) for 2 h. Primary antibody incubation: overnight at 4°C (CD29 [1:200, Bioss bs-0486R], CD90 [1:500, Bioss bs-0778R], CD34 [1:300, Bioss bs-0646R]). Secondary antibody incubation: 2 h at room temperature in the dark (FITC Conjugated AffiniPure [1:1000, BOSTER BM2012], Hoechst counterstaining nuclei [1:100, Beyotime C1028]).

[0042] 2. Imaging Analysis: Images were acquired using a fluorescence microscope (Olympus IX-73), and fluorescence intensity was quantified using ImageJ. Immunofluorescence staining confirmed that the cultured bone marrow mesenchymal stem cells (BMSCs) met the minimum international ISCT standards for BMSCs: positive expression rates of CD29 (integrin β1) and CD90 (Thy-1 antigen) were both ≥95%, and the positive expression rate of CD34 (a hematopoietic stem cell marker) was ≤2% (see [link to relevant documentation]). Figure 2 ).

[0043] 3. Adipogenic differentiation of BMSCs: When bone marrow mesenchymal stem cells in the culture dish adhered and grew to 85% confluence, they were passaged and seeded into P5 generation cells into 6-well or 48-well plates. When the cells grew to 80% confluence, the medium was replaced with adipogenic induction medium (DMEM / F12 basal medium + 10% fetal bovine serum + 1 μM dexamethasone + 0.5 mM IBMX + 50 nM insulin + 50 μM oleic acid) to induce adipogenic differentiation of BMSCs. The medium was changed every 2 days and induced for 15 days. Cells were then harvested according to the specific experimental protocol for subsequent experiments (Oil Red O staining and semi-quantitative and real-time quantitative PCR).

[0044] The results showed that dynamic monitoring of lipid droplet formation using Oil Red O staining revealed that as the adipogenic induction time increased (0-15 days), the cellular lipid content increased in stages—in the early stage (1-9 days), large lipid droplets were predominantly distributed locally, and the Oil Red O-positive areas were relatively concentrated. Figure 3 In the middle stage (A); towards the later stage (10-15 days), large lipid droplets gradually break down into uniformly distributed clusters of small lipid droplets ( Figure 3 (A). Semi-quantitative analysis using Oil Red O staining showed that with increasing induction time, the cellular lipid content and lipid droplet accumulation significantly increased in the early stage, while the lipid droplet number density increased but the total lipid content decreased during the transition between the early and late stages. Figure 3(B). Dynamic detection of key transcription factors in adipogenic differentiation showed that C / EBPα, as a core transcription factor in adipogenic differentiation, had its mRNA expression level significantly upregulated throughout the entire BMSC differentiation process (0-15 days) compared to adipogenic differentiation day 0. Figure 3 C; C / EBPβ mRNA expression was significantly upregulated in the early differentiation phase (days 0-7) compared to day 0 of adipogenic differentiation, driving the initiation of the early adipogenic program ( Figure 3 (D); while PPARγ exhibits dynamic oscillations in the early stages, including brief downregulation phases (such as induction 2d and 5d), reflecting the complexity of its regulation ( Figure 3 In the later stages of cell differentiation (10-15 days), the mature adipocyte phenotype is established, leading to a decrease in the absolute dependence on transcription factors C / EBPβ and PPARγ.

[0045] 4. Myogenic differentiation of BMSCs: P5 generation cells were seeded into 6-well or 24-well plates. The next day, the medium was replaced with myogenic induction medium (DMEM / F12 basal medium + 10% horse serum + 10 μM 5-Aza). After 48 hours of induction, the medium was replaced with complete medium every 2 days. After 16, 28 and 39 days of induction, the cells were harvested according to the specific experimental protocol for subsequent experiments (morphological assessment, cell immunofluorescence detection and real-time quantitative PCR).

[0046] The results showed that BMSCs underwent directed aggregation at 16 days of myogenic differentiation, forming myogenic colonies. Immunofluorescence staining for Desmin was positive, revealing a small number of cell fusions. Figure 4 In the middle A and E); increased myogenic colonies and cell fusion in myoblasts at 28 days ( Figure 4 In the middle B and E regions, myogenic colonies and cell fusion were further increased at 39 days of myogenesis, and typical myotube structures were visible under bright field microscopy with an inverted microscope. At the same time, the mRNA expression levels of myogenic marker genes MYOG, MYOD1, MYF5 and DESM were all significantly upregulated. Figure 4 (CF).

[0047] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for establishing a goat bone marrow mesenchymal stem cell line, characterized in that, Includes the following steps: S1. Goat bone marrow was rinsed with DMEM / F12 to obtain the rinsed material; S2. Mix the rinse material and complete culture medium and pipette to form a single-cell suspension, then inoculate and culture. S3. After the inoculation culture is completed, perform subculture. The passage culture method is as follows: When the cell confluence reaches 80%, discard the culture medium, rinse, digest, and terminate the digestion to obtain a digested cell suspension; After the digested cell suspension is pipetted, centrifuged, and resuspended by precipitation, it is seeded into a new culture flask. One hour after seeding, the non-adherent cell suspension is transferred to a new flask, and the adherent BMSCs are retained for further culture to complete the passage culture. S4. After passage to the 3rd generation, establish a master cell bank and preserve the cells by cryopreservation using a cryopreservation solution containing 90% DMEM / F12 medium and 10% DMSO.

2. The method for establishing according to claim 1, characterized in that, The goat bone marrow in S1 is collected from the femur of a goat.

3. The method for establishing according to claim 1, characterized in that, The rinsing process in S1 includes blowing, washing, and centrifugation; the number of rinsing cycles is 2-4.

4. The method for establishing according to claim 1, characterized in that, The density of the single-cell suspension in S2 is 1×10⁻⁶. 6 cells / mL.

5. The method for establishing according to claim 1, characterized in that, The inoculation and culture temperature in S2 is 37℃ and 5% CO2. The culture process is as follows: when the cells are cultured for 6 hours, the complete culture medium is partially replaced; when the cells are cultured for 24 hours, the complete culture medium is completely replaced; thereafter, the medium is completely replaced once every 3 days.

6. The method for establishing according to claim 1, characterized in that, The digestion step in S3 is as follows: digestion with 0.25% trypsin-EDTA at 37°C for 2 minutes.

7. The method for establishing according to claim 1, characterized in that, The step to terminate digestion in S3 is as follows: after digestion, discard the trypsin and immediately add complete culture medium to terminate digestion.

8. The method for establishing according to claim 1, characterized in that, The centrifugation parameters in S3 are 1300 rpm for 5 minutes.

9. The method for establishing according to claim 1, characterized in that, The inoculation density in S3 is 1×10⁻⁶. 4 cells / cm 2 .

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