Stem cell large-scale amplification method based on microcarrier-shake flask coupling culture

Abstract

The invention discloses a stem cell large-scale amplification method based on microcarrier-shake flask coupling culture, and belongs to the technical field of biology. The method comprises the following steps: co-adding a pretreated microcarrier and a stem cell suspension into a shake flask containing a culture medium to form an initial culture system; performing dynamic attachment culture to attach the stem cells to the surface of the microcarrier; after the attachment is completed, switching to a constant rotating speed for multiplication culture; and after culturing to an amplification end point, harvesting the stem cells. According to the method, a set of technological process which is highly integrated, simple and convenient to operate and high in process controllability is constructed, and the key technical bottlenecks of low efficiency, high cost, poor amplification performance and the like of large-scale stem cell amplification in the prior art are systematically solved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically cell culture technology, and particularly relates to a method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture. Background Technology

[0002] Stem cells, due to their unique self-renewal capacity and multi-directional differentiation potential, have shown broad application prospects in regenerative medicine and cell therapy, and have been explored for the treatment of various intractable diseases such as hematological disorders, neurodegenerative diseases, cardiovascular injury, and diabetes. However, a key prerequisite for transforming stem cell research from laboratory studies into clinical-grade therapeutic products is the ability to stably obtain a sufficient quantity (usually requiring at least 10⁻⁶). 8 -10 10 Cell preparations that are graded, of controllable quality, functionally homogeneous, and meet safety standards. Therefore, efficient and reliable in vitro expansion technology constitutes the core link in the clinical translation of stem cells.

[0003] In existing technologies, the following types of expansion methods are mainly used for adherent stem cells such as mesenchymal stem cells and hematopoietic stem cells: (1) Static two-dimensional culture method, including conventional culture flask / plate passage culture and multilayer cell factory system. This method relies on cells adhering to the plastic surface for growth and expansion through periodic trypsin digestion and manual passage. Although simple to operate, it is difficult to achieve true large-scale production due to the limitations of two-dimensional surface area, contact inhibition effect and frequent manual intervention.

[0004] (2) Feeder layer co-culture system: This system introduces endothelial cells or stromal cells as a support layer to simulate the in vivo microenvironment and maintain stem cell stemness. This method is effective in expanding primitive hematopoietic stem cells (such as CD34). + CD38 - While showing significant effects in terms of subgroups, the introduction of heterologous or allogeneic cells presents challenges such as complex composition, large batch-to-batch variations, potential pathogen contamination, and high regulatory compliance risks.

[0005] (3) Bioreactors combined with microcarrier culture technology utilize suspended microcarriers to provide a three-dimensional attachment surface and achieve uniform transfer of nutrients and gases through stirring. This system has good process controllability and scale-up potential, and is suitable for automated closed production. However, the fluid shear force generated by stirring may damage cells, and the high equipment cost and complex process development limit its widespread application in small and medium-sized institutions.

[0006] (4) Culture medium composition optimization strategies include using serum-free or chemically defined culture media, adding small molecule agonists (such as UM171, SR1, nicotinamide, etc.) or synthetic polymers (such as PVA) to replace serum proteins. These methods help improve amplification efficiency and reduce the risk of heterologous amplification. The FDA has approved the first hematopoietic stem cell product based on nicotinamide amplification, Omisirge (omidubicel-onlv). However, even so, without a matching physical culture platform, its efficacy is still limited by the inherent bottleneck of traditional static culture.

[0007] Although the aforementioned technologies have promoted the development of stem cell expansion to varying degrees, their inherent limitations still severely restrict the clinical translation process, specifically in the following four aspects: First, large-scale expansion efficiency is low. Static two-dimensional culture is limited by its limited specific surface area and contact inhibition, making it difficult to break through the yield ceiling even when using multilayer cell factories. For example, adipose-derived mesenchymal stem cells often enter senescence arrest after reaching the P6–P8 generation; while matrix-free culture systems can expand primitive hematopoietic stem cells at a much lower rate than co-culture systems, failing to meet the cell dose required for adult transplantation.

[0008] Second, the quality and functional uniformity of cell products are difficult to guarantee. Long-term in vitro expansion can easily lead to phenotypic drift of stem cells (such as decreased expression of markers like CD34 and CD73), weakened differentiation potential, and reduced in vivo homing and regeneration capabilities. More seriously, uneven distribution of nutrients / metabolites in static culture creates a microenvironment gradient, which, combined with the inherent randomness of cells, significantly amplifies population heterogeneity. This uncontrollable heterogeneity not only affects the consistency of therapeutic efficacy.

[0009] Third, the process suffers from poor controllability, high risk of contamination, and high costs. Traditional passaging relies heavily on extensive open manual operations, such as digestion, blowing, counting, and bottling, which are highly susceptible to human error and microbial contamination, resulting in poor batch-to-batch repeatability. Furthermore, the labor-intensive model makes standardization and automation difficult, and capacity expansion can only be achieved through parallel scale-out rather than efficient volumetric scale-up, significantly increasing production costs and hindering commercialization.

[0010] Fourth, the expansion process carries potential safety risks. Long-term in vitro culture involves numerous cell divisions, increasing the probability of DNA replication errors and mutation accumulation. Existing studies have shown that over-expanded stem cells may exhibit genomic instability, epigenetic abnormalities, anchorage-independent growth, and even induce tumors in animal models. Furthermore, in vitro selection pressures may enrich clones carrying pre-leukemia mutations, posing a significant clinical safety risk.

[0011] In summary, existing stem cell expansion technologies suffer from insurmountable systemic defects in terms of expansion efficiency, product quality, process robustness, and biosafety. These defects stem from the heterogeneous microenvironment of static culture, the uncontrollability of artificial manipulation, and the significant deviation between in vitro conditions and the in vivo physiological microenvironment.

[0012] Therefore, providing a high-density, closed, automated, and highly uniform microenvironment-based in vitro stem cell expansion method with high expansion capacity, excellent stemness maintenance performance, good batch consistency, and intrinsic safety characteristics has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0013] The purpose of this invention is to provide a method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture, in order to solve the problems of low expansion efficiency, uncontrollable cell quality, poor process standardization and high potential tumorigenic risk in the prior art.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention discloses a method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture, which includes the following steps: S1. Add the pretreated microcarriers and stem cell suspension together to a shake flask containing culture medium to form the initial culture system; the amount of microcarriers used is 1-5 g / L, and the seeding amount of stem cells is 5 × 10⁻⁶. 4 ~2×10 5 cells / mL; S2. Perform dynamic attachment culture to allow stem cells to attach to the surface of the microcarrier; the dynamic attachment culture procedure is as follows: under 37℃ and 5% CO2 conditions, first shake at 80~120rpm for 7~9min, then let stand for 20~25min, this "shaking-standing" cycle is defined as 1 cycle, and 45~50 cycles are performed continuously. S3. After attachment is complete, switch to a constant rotation speed for amplification culture to achieve large-scale expansion of stem cells; S4. After culturing to the expansion endpoint, harvest the stem cells.

[0015] In some embodiments of the present invention, the culture medium does not induce stem cells to undergo directed differentiation and can maintain the undifferentiated state and high proliferative activity of stem cells.

[0016] In some embodiments of the present invention, the stem cells are SCL stem cells; the culture medium for SCL stem cells, per 1 mL, has the following composition: 459 μL of IMDM basal medium. 459 μL of F12 basal medium. Monothioglycerol 1μL Transferrin 1 μL Insulin 1.5 μL Lipids 10 μL Penicillin / Streptomycin 10 μL Polyvinyl alcohol 40μL 10 μL of knockout serum substitute (KSR) Bovine serum albumin (BSA) 5 μL, SMO receptor agonist (SAG) 1 μL, ROCK inhibitor (Y27632) 1μL, BMP inhibitor (LDN193189) 1 μL, Basic fibroblast growth factor (bFGF) 0.5 μL.

[0017] In some embodiments of the present invention, the microcarrier is a Cytodex 1 microcarrier.

[0018] In some embodiments of the present invention, the pretreatment step of the microcarrier includes sequential steps of hydration, washing, sterilization and equilibration; The hydration step includes suspending the dried microcarrier in a calcium- and magnesium-free buffer solution and allowing it to swell sufficiently. The sterilization step includes autoclaving the microcarriers together with the buffer solution. The balancing step includes discarding the sterilization buffer and rinsing the microcarriers with preheated stem cell culture medium to prepare them for subsequent cell culture.

[0019] In some embodiments of the present invention, in step S1, the amount of microcarrier used is 3 g / L, and the amount of stem cells inoculated is 1 × 10⁻⁶. 5 Cells / mL.

[0020] In some embodiments of the present invention, the dynamic attachment culture procedure is as follows: under conditions of 37°C and 5% CO2, the cells are first shaken at 100 rpm for 8 minutes, then allowed to stand for 22 minutes. This "shaking-standing" cycle is defined as one period, and 48 consecutive cycles are performed. The intermittent dynamic attachment procedure based on shake-flask oscillation control introduced in the early stage of inoculation in this invention can promote sufficient contact between cells and the microcarrier surface while avoiding severe shear damage, thereby significantly improving the initial attachment rate.

[0021] In some embodiments of the present invention, in step S3, continuous amplification culture is carried out at a constant rotation speed of 100 rpm, and half the volume of fresh culture medium is replaced every 48 hours.

[0022] In some embodiments of the present invention, in step S4, after amplification reaches the endpoint, the cell amplification culture is terminated, the cell-microcarrier complex is allowed to settle, the culture medium is discarded, and the cells are washed with calcium- and magnesium-free buffer containing EDTA. Trypsin solution is added for digestion to detach the cells from the microcarriers. Subsequently, serum-containing culture medium is added to terminate the digestion reaction, separating the free cells from the undissociated microcarriers, and the resulting cell suspension is collected. This cell suspension can be used for subsequent experiments or passage culture after centrifugation and washing.

[0023] In some embodiments of the present invention, the calcium- and magnesium-free EDTA-containing buffer is PBS with a pH of 7.6, wherein the EDTA concentration is 0.02 wt%; the amount of trypsin solution used is 30-50 mL per gram of microcarrier, and incubation is carried out at 37°C for 10-20 min.

[0024] In some embodiments of the present invention, the free cells are separated from the undissociated microcarriers by static sedimentation or by passing through a 100μm cell sieve.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture. By constructing a highly integrated, easy-to-operate, and highly controllable process flow, it systematically solves the key technical bottlenecks faced by the existing technology in the large-scale expansion of stem cells, such as low efficiency, high cost, and poor scalability.

[0026] This invention organically integrates microcarrier culture technology with a conventional shake-flask system, precisely controlling the shaking parameters as key process variables to directly regulate the suspension state of the microcarriers in the culture medium. By rationally setting the shaking mode and intensity, the microcarriers maintain stable suspension throughout the entire culture cycle, effectively avoiding uneven cell adhesion caused by sedimentation and preventing mechanical damage to stem cells due to excessive shear force. This successfully creates a dynamic microenvironment suitable for the three-dimensional growth of stem cells within a simple shake-flask device. This coupled system overcomes the physical constraints of traditional two-dimensional static culture limited by the finite growth surface area, while avoiding reliance on complex bioreactor systems with stringent control precision requirements, laying a solid physical and engineering foundation for subsequent efficient expansion.

[0027] Secondly, this invention designs and implements a scientific, phased kinetic culture program. Based on the different biological behaviors of stem cells during the attachment and expansion phases, this program employs a dynamic, phased oscillation method: In the initial stage of culture, an intermittent oscillation mode alternating between oscillation and static placement is used to increase the contact frequency between stem cells and the microcarrier surface, promoting the uniformity and firmness of initial attachment; after effective cell attachment, the program switches to a continuous oscillation culture mode with a constant rotation speed to maintain the uniform suspension of the microcarrier in the culture system, ensuring sufficient nutrient supply and timely removal of metabolic waste, thereby supporting stem cells to enter a highly efficient and stable exponential expansion phase. This phased regulation method effectively overcomes the inherent contradiction between attachment efficiency and expansion stability that a single, fixed oscillation mode cannot simultaneously address, achieving refined and intelligent guidance of the entire stem cell growth process.

[0028] Third, this invention opens up a feasible technical path for large-scale stem cell expansion under non-bioreactor conditions. The core of this path lies in fully utilizing the high specific surface area provided by microcarriers to support the attachment and proliferation of large numbers of stem cells, and relying on the moderate hydrodynamic effect generated by shake-flask oscillation to maintain the homogeneity and mass transfer efficiency of the entire culture system, thereby synergistically achieving the dual goals of efficient cell number expansion and stable and controllable culture quality. The entire expansion process does not rely on traditional bioreactor equipment equipped with complex functional modules such as mechanical stirring systems, online dissolved oxygen (DO) monitoring, and automatic pH control, significantly reducing the barriers and operating costs in terms of hardware investment, system maintenance, and process control, thus facilitating the medium-scale (e.g., 10⁻⁶) of stem cell expansion. 8 ~10 8 This provides a simple, convenient, and cost-effective alternative technology for expanding cell mass (at the cellular level).

[0029] Fourth, the entire process system constructed in this invention possesses excellent reproducibility, robustness, and wide applicability. This system requires only a shaker and cell culture incubator typically found in laboratories, and the key materials used, such as microcarriers, basal culture media, and additives, can be stably obtained through commercial channels. The key parameters for process control focus on a few well-defined variables, including the oscillation program (including oscillation mode, speed, and timing) and initial seeding density. The operating steps are clear, standardized, and exhibit high batch-to-batch reproducibility. These characteristics enable this invention not only to reliably meet the needs of stem cell culture and expansion in basic research but also, due to its high simplicity, robustness, and standardization potential, facilitate a smooth transition and widespread application to process scale-up research, pilot-scale verification, and even industrial production, demonstrating excellent technology transfer capabilities and industrialization prospects.

[0030] In summary, through the synergistic effect of the above four aspects, this invention constructs a three-dimensional dynamic stem cell culture system that combines high efficiency, economy, scalability, and universality. Attached Figure Description

[0031] Appendix Figure 1 This is a process flow diagram of the amplification method of the present invention; Appendix Figure 2 This is a cell growth curve diagram for Example 2; Appendix Figure 3 This is a graph showing the effect of different culture parameters on cell adhesion in Example 3; Appendix Figure 4 This is a cell growth line diagram for Experiment Example 1; Appendix Figure 5 These are live-cell fluorescence imaging and quantitative analysis images of cell proliferation under different culture modes in Experiment Example 1. Figure 5 A is a fluorescence microscopy image of live cells, and 5B is a statistical diagram of the number of live cells. Appendix Figure 6 The graph shows the results of metabolic activity assessment under different culture modes in Experiment Example 1; Appendix Figure 7 This is a graph showing the detection results of cell stemness-related gene expression levels under different culture modes in Experiment Example 1; Appendix Figure 8 Flow cytometry analysis of tdTomato labeling efficiency in cells under different culture modes in Experiment Example 1; Figure 8 A is a flow cytometry scatter plot of the negative control sample (showing the distribution of the tdTomato negative cell population). Figure 8 B is a flow cytometry scatter plot of the experimental group samples (showing the distribution of tdTomato positive cell populations). Figure 8 C is a bar chart showing the statistical percentage of tdTomato positivity in different replicate groups; Figure 8In A and 8B, the horizontal axis PE-A represents the fluorescence intensity (area signal) of the PE channel, and the vertical axis SSC-A represents the intensity (area signal) of the side-scattered light. Figure 8 In C, the horizontal axis R1-R9 represents the replicate group numbers, and the vertical axis represents the positive rate. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific examples. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] Example 1 This embodiment discloses the method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture of the present invention, the process flow of which is attached. Figure 1 As shown, the specific steps include the following: Step 1. Pretreatment of microcarriers (1) Hydration treatment: Add 50 mL of calcium- and magnesium-free phosphate buffer (PBS) to the dried Cytodex 1 microcarrier powder at a ratio of 1 gram of microcarrier, suspend at room temperature and let stand for hydration for at least 3 hours to allow the microcarrier to fully absorb water and swell. (2) Washing treatment: After the hydrated microcarriers settle naturally, discard the supernatant; add fresh calcium-free and magnesium-free PBS to the precipitate, the amount is 50mL per gram of microcarrier, gently mix and wash for 2-3 minutes, then let stand to allow the microcarriers to settle again, and discard the supernatant. (3) Sterilization treatment: The washed microcarriers, along with an appropriate amount of calcium-free and magnesium-free PBS, were transferred to a pressure-resistant container and sterilized by high-pressure steam at 115°C and 15 psi (approximately 1.05 kg / cm²). 2 Sterilize for 15 minutes under the following conditions; if microbial contamination is detected after sterilization, this sterilization step can be repeated. (4) Balancing treatment: After sterilization, allow the microcarriers to settle naturally and discard the PBS used for sterilization. Before use, rinse the microcarriers briefly with complete culture medium preheated to the culture temperature. The amount of complete culture medium used is 50 mL per gram of microcarrier, so that the environment of the microcarriers is compatible with the subsequent cell culture conditions.

[0034] Step 2. Cell seeding The pretreated Cytodex 1 microcarriers and SCL stem cell suspension were added together to a shake flask containing culture medium to form the initial culture system; 0.12 g of microcarriers were added to 40 mL of culture medium and seeded with 4 × 10⁶ cells / year. 6 One SCL stem cell. This ratio achieves the best balance between expansion efficiency and cost-effectiveness.

[0035] The culture medium in this example, calculated per 1 mL, has the following composition: 459 μL of IMDM basal medium. 459 μL of F12 basal medium. Monothioglycerol 1μL Transferrin 1 μL Insulin 1.5 μL Lipids 10 μL Penicillin / Streptomycin 10 μL Polyvinyl alcohol 40μL 10 μL of knockout serum substitute (KSR) Bovine serum albumin (BSA) 5 μL, SMO receptor agonist (SAG) 1 μL, ROCK inhibitor (Y27632) 1μL, BMP inhibitor (LDN193189) 1 μL, Basic fibroblast growth factor (bFGF) 0.5 μL.

[0036] Step 3. Dynamic attachment culture To promote effective adhesion between cells and microcarriers, this embodiment uses a specific intermittent culture program, as follows: under conditions of 37°C and 5% CO2, the cells are first shaken at 100 rpm for 8 minutes, and then left to stand for 22 minutes. This "shaking-standing" cycle is defined as one cycle, and 48 cycles are performed continuously. Step 4. Constant-rate amplification After the dynamic attachment process is completed, switch to a constant rotation speed of 100 rpm for continuous amplification culture. Replace half the volume of fresh dedicated complete culture medium every 48 hours.

[0037] Step 5. Harvest cells SCL stem cells reached their expansion limit on day 8 and were then harvested. The specific harvesting steps are as follows: Termination and Washing: Stop stirring and allow the cell-microcarrier complex to settle, then discard the old culture medium completely. Wash for 5 minutes with calcium- and magnesium-free PBS (pH 7.6) containing 0.02% EDTA to chelate calcium and magnesium ions and weaken cell-microcarrier and cell-cell connections.

[0038] Digestion and dissociation: Discard EDTA-PBS and add trypsin solution (0.25% concentration), approximately 40 mL per gram of microcarrier. Mix thoroughly and incubate at 37°C for about 15 minutes, gently stirring occasionally to promote dissociation.

[0039] Termination and Separation: Add culture medium containing serum to terminate digestion using trypsin inhibitors in the serum, and mix well. Subsequently, separate the free cells from the microcarriers by allowing them to settle.

[0040] Collection: Collect the suspension containing cells, centrifuge and wash, and it can be used for subsequent experiments or subculture.

[0041] Example 2 This embodiment examines the ratio of microcarriers to stem cells in the microcarrier-shake flask coupled culture method of the present invention (as shown in Table 1).

[0042] Table 1

[0043] The microcarrier to stem cell ratios specified in Table 1 were used for amplification, following the method described in Example 1. The results are shown in Table 2, and the cell growth curves are as follows: Figure 2 As shown.

[0044] Table 2

[0045] The initial count refers to the actual number of cells counted on the first day after cell and microcarrier inoculation, after the cells have adhered.

[0046] The calculation method of economic normalization is as follows: estimate all scientific research materials and related costs consumed from the start of cultivation to the plateau period, and take group G9 as the unit 1. Divide the remaining groups by it to obtain the normalized economic ratio. The smaller the value, the less economic cost is consumed.

[0047] The overall ranking is calculated by assigning certain weights to three indicators: "maximum expansion" (positive indicator), "economic normalization" (negative indicator), and "number of days to reach the plateau" (negative indicator), and then ranking them after quantitative scoring.

[0048] The above results indicate that among the G9 group cell culture results in this experiment, group G5 performed best overall, not only achieving the highest expansion value (74,992 cells) but also ranking first overall based on a balance of multiple indicators. It is worth noting that cell expansion efficiency does not solely depend on the initial seeding volume or proliferation rate: for example, while groups G2 and G3 reached the plateau phase as quickly as 4 days, demonstrating strong proliferative activity, their final expansion was only about 24,000 cells, far lower than other groups, indicating that rapid proliferation does not necessarily translate into high yield. On the other hand, groups G4, G5, G7, G8, and G9, although requiring a longer time to reach the plateau phase (7-11 days), achieved a total cell expansion of over 70,000 cells, demonstrating superior culture efficiency or environmental adaptability.

[0049] From the perspective of economic normalization indicators, group G9 scored the highest (1.0), indicating its greatest advantage in resource utilization or cost-effectiveness. However, due to its slower expansion rate (10 days) and slightly lower absolute yield compared to group G5, it ranked 5th overall. This reflects a multidimensional balance between speed, yield, and economy in the evaluation system. Overall, to maximize cell output, the culture conditions of group G5 should be prioritized.

[0050] Example 3 In Example 3, adhesion rate was used as the evaluation index to optimize and screen key parameters of dynamic attachment culture in the amplification method of the present invention. Specific experimental parameters are shown in Table 3, and the amplification method was performed as described in Example 1.

[0051] Table 3

[0052] The results are as follows Figure 3 As shown: the negative control group (NEG) had the lowest cell adhesion rate, only about 10%. From G1 to G10, the cell adhesion rate gradually increased, reaching a peak of nearly 80% in groups G8, G9, and G10, indicating that these culture conditions best promoted cell adhesion. Subsequently, from G11 to G15, the adhesion rate gradually decreased. Overall, the effects of different culture parameters on cell adhesion varied significantly, with the conditions in groups G8 to G10 being optimal.

[0053] Experimental Example 1 To scientifically verify the actual technical effectiveness of the culture system of this invention, a rigorously designed parallel controlled experiment was conducted, the details of which are as follows: 1. Experimental grouping and culture conditions This experiment was conducted in four parallel groups, including one experimental group, two positive control groups, and one negative control group, as detailed below: MC-Shaker group (experimental group): The "microcarrier-shake bottle system" (MC-Shaker for short) described in this invention was used. Group 2D: Using a traditional planar two-dimensional culture system; MC-BioR group: using a microcarrier-bioreactor culture system; Negative control group (NEG group): A template-free control used only for molecular detection and not involved in the cell culture process.

[0054] All groups involving cell culture (MC-Shaker group, 2D group, MC-BioR group) used the same batch and source of SOX9. + Sclerosing interstitial stem cells (SCL stem cells) were cultured under identical basal culture conditions, including: identical culture medium formulation, serum batch, additive concentration, culture temperature (37°C), CO2 concentration (5%), humidity (saturation), medium change cycle (full medium change every 48 hours), and synchronized start time and sampling points (days 1, 4, and 7). The specific implementation methods for each group are detailed below.

[0055] The specific cultivation method for the MC-Shaker group was as follows: it was cultivated according to the method in Example 1.

[0056] Specific culture method for group 2D: Inoculate 2×10⁶ cells / mL into a 100 mm culture dish. 6 One SCL stem cell was cultured in 8 ml of the same culture medium as described above, with the medium changed every two days. Fifteen parallel groups were established. One group was collected daily for cell counting to calculate the amount of cell expansion.

[0057] The specific culture method for the MC-BioR group: Cell culture was performed using a 3D FloTrix® miniSPIN FLEX 4-channel bioreactor manufactured by Beijing Huakan Biotechnology Co., Ltd. 0.12g of microcarriers was added to 40mL of culture medium, and 4×10⁶ cells were seeded. 6 SCL stem cells. Under conditions of 37°C and 5% CO2, the cells were first shaken at 40 rpm for 5 minutes, then left to stand for 25 minutes. This "shaking-standing" cycle was defined as one cycle, and 48 cycles were performed continuously. The remaining steps were the same as in Example 1.

[0058] Negative control group (NEG group): This group does not undergo cell culture. In subsequent RT-qPCR detection, sterile ddH2O is used to replace the cDNA template to exclude primer non-specific amplification or reagent contamination and ensure the reliability of the test results.

[0059] 2. Detection Method (1) Cell expansion efficiency: Samples were taken daily. The 2D group was digested with trypsin, and the number of live cells was determined by an automated cell counter to plot the growth curve. The MC-Shaker group and the MC-BioR group were dissociated from microcarriers, and the number of live cells was determined by an automated cell counter to plot the growth curve. (2) Cell viability: Samples were taken on days 1, 4 and 7, and the proportion of viable cells was quantified by Calcein-AM / propidium iodide (PI) double staining method, fluorescence microscopy or flow cytometry. (3) Metabolic activity: On day 7 of culture, the dehydrogenase activity of cells in each group was detected using a CCK-8 assay kit, with OD200 as the metric. 450 Values ​​represent metabolic levels; (4) Stem gene expression: Total RNA was extracted from cells of each group on day 7. After reverse transcription, the relative expression levels of stem-related genes were detected by RT-qPCR. GAPDH was used as an internal control, and the NEG group was used as a blank control. Total RNA of co-cultured cells was extracted and purified using the SteadyPure RNA Extraction Kit (Catalog No.: AG21024) produced by Accurate Biotechnology (Hunan) Co., Ltd. Then, rapid cDNA synthesis and PCR amplification were performed using the SuperScript IV UniPrime One-Step RT-PCR System (Catalog No.: 12597100) produced by Thermo Fisher Scientific. The expression of five genes, namely ACTB, PAX1, PAX9, SOX9, TWIST 1, and NXK3-2, was detected. The primer sequences are as follows: Table 4

[0060] (5) Phenotypic stability: Samples were taken on days 0, 3 and 7. Since the cells had been edited with tdtomato before expansion, they would express fluorescent signals while expressing SOX9. The proportion of SOX9 positive cells was detected by flow cytometry to assess the stability of marker expression during expansion.

[0061] 3. Experimental Results 3.1 Cell Expansion Efficiency The results of cell expansion efficiency are attached. Figure 4As shown in the figure, the proliferation rate of the MC-Shaker group was significantly higher than that of the 2D control group from day 2 to day 5 (p<0.05), reaching a total expansion fold of 18.6±1.2 times by day 7; the MC-BioR control group showed a fold increase of 19.1±1.5 times, with no significant difference between the two groups (p>0.05). The cell numbers in both the MC-Shaker and MC-BioR groups increased rapidly from day 5 and entered a plateau phase after day 8, exhibiting highly consistent expansion trends; while the cell numbers in the 2D group remained at a very low level throughout the entire culture period. These results indicate that the MC-Shaker system is significantly superior to traditional 2D culture in terms of expansion efficiency and is comparable to bioreactor systems.

[0062] 3.2 Cell viability Cell viability results are attached. Figure 5 As shown, the cell viability of all three groups remained above 92% on days 1, 4, and 7, with no statistically significant difference between the groups (p>0.05). There was no significant difference in cell viability between the MC-Shaker group and the 2D and MC-BioR groups, demonstrating that the system of this invention can effectively maintain a high cell viability state.

[0063] 3.3 Cellular metabolic activity CCK 8. The results of the cell metabolic activity assay are attached. Figure 6 As shown, the OD450 values ​​of the three groups at the end of culture were: MC-Shaker group 1.82±0.09, 2D group 1.78±0.11, and MC-BioR group 1.85±0.08, with no significant difference among the groups (p>0.05). The OD values ​​of each group gradually increased over time, and there were no statistically significant differences at the same time points, indicating that the metabolic activity levels of the cells in the three groups were comparable at the end of culture, further confirming that the cells expanded by this invention have similar physiological activity to the control group.

[0064] 3.4 Stem gene expression RT The results of qPCR detection of stem gene expression are attached. Figure 7 As shown, the expression levels of SOX2, OCT4, and NANOG in the MC-Shaker group were significantly higher than those in the negative control group (p<0.01), but comparable to those in the 2D group and the MC-BioR group (p>0.05). This indicates that the culture system of the present invention can effectively maintain the key stem cell characteristics.

[0065] 3.5 Phenotypic Stability The results of the phenotypic stability study are attached. Figure 8As shown: In the MC-Shaker group, the SOX9 positivity rate remained stable at 94%–96% throughout the amplification process, without a significant decrease or increase in heterogeneity, indicating that spontaneous differentiation did not occur. Flow cytometry scatter plot ( Figure 8 A, Figure 8 B) showed that tdTomato-positive cells (representing high SOX9 expression) accounted for a very high proportion in the population; quantitative statistics ( Figure 8 C) Further confirmation showed that the SOX9 positivity rate of cells in multiple consecutive rounds of expansion (R1 to R9) remained consistently above 99.5%. The results indicate that this system can support stem cells in maintaining a stable high level of marker expression during expansion, effectively preserving the undifferentiated functional state of the cells.

[0066] In summary, the system of this invention exhibits superior performance in terms of cell expansion efficiency, survival rate, metabolic activity, stem gene expression, and phenotypic stability, significantly outperforming traditional 2D culture and comparable to bioreactor systems. It can efficiently and stably expand and maintain the functional characteristics of stem cells.

[0067] 4. Conclusion The results of this experiment demonstrate that the MC-Shaker culture system described in this invention supports SOX9. + In the expansion of osteogenic mesenchymal progenitor cells, this system not only significantly outperforms traditional 2D culture but also achieves a technical level comparable to high-end microcarrier-bioreactor systems in key indicators such as expansion efficiency, cell viability, stemness maintenance, and phenotypic stability. This system can achieve efficient and stable large-scale expansion of stem cells without complex equipment, and has promising prospects for industrial application.

[0068] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalent alterations can be made within the spirit and scope defined by the claims of the present invention, but all such changes will fall within the protection scope of the present invention.

Claims

1. A method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture, characterized in that, Includes the following steps: S1. Add the pretreated microcarriers and stem cell suspension together to a shake flask containing culture medium to form the initial culture system; the amount of microcarriers used is 1-5 g / L, and the seeding amount of stem cells is 5 × 10⁻⁶. 4 ~2×10 5 cells / mL; S2. Perform dynamic attachment culture to allow stem cells to attach to the surface of the microcarrier; the dynamic attachment culture procedure is as follows: under 37℃ and 5% CO2 conditions, first shake at 80~120rpm for 7~9min, then let stand for 20~25min, this "shaking-standing" cycle is defined as 1 cycle, and 45~50 cycles are performed continuously. S3. After attachment is complete, switch to a constant rotation speed for amplification culture to achieve large-scale expansion of stem cells; S4. After culturing to the expansion endpoint, harvest the stem cells.

2. The method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 1, characterized in that, The culture medium does not induce stem cells to undergo directed differentiation and can maintain the undifferentiated state and high proliferative activity of stem cells.

3. The method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 1, characterized in that, The stem cells are SCL stem cells; the culture medium for SCL stem cells, per 1 mL, has the following composition: 459 μL of IMDM basal medium 459 μL of F12 basal culture medium Thioglycerol 1 μL, 1 μL of transferrin Insulin 1.5 μL, 10 μL of lipids Penicillin / Streptomycin 10μL 40 μL of polyvinyl alcohol 10 μL of knockout serum substitute 5 μL of bovine serum albumin SMO receptor agonist 1 μL, ROCK inhibitor 1μL, 1 μL of BMP inhibitor, 0.5 μL of basic fibroblast growth factor.

4. The method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 1, characterized in that, The microcarrier is a Cytodex 1 microcarrier.

5. The method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 1, characterized in that, The pretreatment steps for microcarriers include sequential steps of hydration, washing, sterilization, and equilibration.

6. The method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 1, characterized in that, In step S1, the amount of microcarrier used is 3 g / L, and the seeding amount of stem cells is 1 × 10⁻⁶. 5 Cells / mL.

7. The method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 1, characterized in that, The dynamic attachment culture procedure is as follows: Under the conditions of 37℃ and 5% CO2, shake at 100 rpm for 8 minutes, then let stand for 22 minutes. This "shaking-standing" cycle is defined as one cycle, and 48 cycles are carried out continuously.

8. The method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 1, characterized in that, In step S3, continuous amplification culture is carried out at a constant rotation speed of 100 rpm, and half the volume of fresh culture medium is replaced every 48 hours.

9. The method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 1, characterized in that, In step S4, after the amplification reaches the endpoint, the cell amplification culture is terminated, the cell-microcarrier complex is allowed to settle, the culture medium is discarded, and the cells are washed with calcium- and magnesium-free buffer containing EDTA. Trypsin solution was added for digestion to detach the cells from the microcarriers; then serum-containing culture medium was added to terminate the digestion reaction and separate the free cells from the undissociated microcarriers, and the resulting cell suspension was collected; after centrifugation and washing, the cell suspension can be used for subsequent experiments or passage culture.

10. A method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 9, characterized in that, The calcium- and magnesium-free EDTA-containing buffer solution is PBS with a pH of 7.6 and an EDTA concentration of 0.02 wt%; the volume of the trypsin solution is 30-50 mL per gram of microcarrier, and it is incubated at 37°C for 10-20 min. Free cells were separated from undissociated microcarriers by static sedimentation or by passing through a 100μm cell sieve.

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