Stem cell bionic microcarrier amplification system and application thereof

Abstract

The invention discloses a stem cell bionic microcarrier amplification system to solve the problems of low efficiency, easy function loss and the like of a traditional amplification method. The core of the micro-carrier comprises a porous degradable polymer micro-carrier of which the surface simulates an extracellular matrix (ECM) topological structure and is covalently bound with ECM protein; in a customized bioreactor, a physiological range fluid shear force is generated by periodically regulating and controlling stirring, and an in-vivo mechanical microenvironment is simulated. According to the system, through the synergistic effect of an ECM bionic structure and dynamic mechanical stimulation, adhesion and proliferation of stem cells are effectively promoted, and stemness and multidirectional differentiation potential are maintained. The amplification multiple can reach 10 times or more of that of a traditional method within 7-10 days, the method has the advantages of being capable of being amplified, standardized, low in cost and the like, and a key technical support is provided for industrialization of stem cell treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical engineering, tissue engineering and regenerative medicine, and particularly relates to a kind of biomimetic microcarrier system for high-efficiency expansion of stem cells (especially mesenchymal stem cells) and its dynamic culture method, which is particularly suitable for large-scale, high-quality stem cell preparation to meet the needs of clinical treatment and drug screening. BACKGROUND

[0002] Stem cells (especially mesenchymal stem cells MSCs) have great application value in the fields of regenerative medicine, disease modeling and drug screening due to their self-renewal and multi-directional differentiation potential. However, there are four major challenges in the clinical in-vitro expansion of stem cells: 1) traditional planar static culture (such as T-flask) has low expansion efficiency, large space occupation and high labor intensity, which is difficult to meet the clinical dosage requirements of 10 8 -10 9 cells per person; 2) multiple passages lead to functional loss problems such as cell aging, differentiation potential attenuation and down-regulation of stemness markers; 3) planar culture lacks the three-dimensional ECM topological structure and dynamic mechanical stimulation (such as fluid shear force, tissue tension) in vivo, resulting in disordered cell adhesion, proliferation and differentiation behavior; 4) conventional microcarriers (such as Cytodex) combined with stirred bioreactors improve the specific surface area, but the bio-inert surface of the carriers, the lack of biomimetic signals and the single nature of mechanical stimulation (such as constant rotation speed) limit the synergistic optimization of expansion efficiency and cell quality.

[0003] In view of the above challenges, stem cell expansion technology has evolved through four generations: the first generation is based on planar static culture (T-flask / Dish) but low efficiency; the second generation is a multi-layer cell factory (Cell Factory / Stack) that realizes limited scale expansion, but is still limited by static environment and lack of automation; the third generation is a microcarrier-bioreactor system (such as Cytodex stirred tank) that significantly improves the expansion scale, but due to insufficient functionalization of microcarriers and extensive regulation of mechanical stimulation, it is difficult to balance high expansion fold and cell function maintenance; the fourth generation of biomimetic microcarrier research attempts to improve through surface modification (such as RGD peptide) or simple mechanical fluctuation, but is still limited to single-factor regulation (biochemical / topology / mechanics), and fails to systematically integrate the synergistic effects of complex ECM topography and physiological dynamic mechanics in vivo. This technology breaks through the frontiers of the fourth generation, and for the first time realizes the qualitative leap of high-efficiency, high-quality, large-scale expansion of stem cells through multi-dimensional system integration of porous structure design, ECM-simulated topological construction, key ECM protein modification and precisely controllable dynamic mechanical stimulation. SUMMARY

[0004] The core objective of this invention is to develop a biomimetic microcarrier amplification system for stem cells, aiming to systematically address the shortcomings of existing technologies. Specifically, this involves: overcoming the amplification efficiency bottlenecks of traditional planar static culture and ordinary microcarrier dynamic culture, achieving a more than 10-fold increase in stem cell yield; excellently maintaining cell quality, ensuring that amplified cells highly express stem cell markers, retain strong multi-lineage differentiation potential, and significantly inhibit senescence phenotypes; precisely mimicking the in vivo stem cell microenvironment (niche) through highly integrated physical topology, biochemical composition, and physiological dynamic mechanical stimulation of the extracellular matrix (ECM); constructing a scalable and standardized production platform that meets GMP requirements for clinical and industrial-grade stem cell preparation while reducing production costs; and ultimately providing a universal and efficient amplification solution applicable to multiple types of stem cells (including bone marrow MSCs, adipose MSCs, umbilical cord MSCs, and iPSC-derived MSCs).

[0005] This invention provides a revolutionary stem cell expansion system, the core of which consists of two parts: a biomimetic microcarrier and a dynamic culture method.

[0006] I. Bionic Microcarriers

[0007] 1. Substrate and Structure:

[0008] Materials: Select biocompatible and biodegradable synthetic polymers (such as polylactic acid-glycolic acid copolymer PLGA, polycaprolactone PCL) or natural / synthetic composite materials.

[0009] Morphology: Spherical porous microcarriers, with diameters ranging from 100 to 500 μm and pore sizes ranging from 10 to 100 μm. The porous structure greatly increases the specific surface area (several to tens of times that of solid microspheres), which is beneficial for the massive adhesion, growth, and exchange of nutrients and metabolic waste by cells.

[0010] 1.1 Material Selection

[0011] The biomimetic microcarrier of this invention uses a biodegradable synthetic polymer material as the substrate, with the core requirements being biocompatibility, controllable degradation, and processability. Specifically:

[0012] Polylactic acid-glycolic acid copolymer (PLGA) is preferred because its degradation rate can be adjusted by the monomer ratio (e.g., LA:GA = 75:25 or 50:50). This material hydrolyzes into lactic acid and glycolic acid in a physiological environment, with a well-defined metabolic pathway, no cytotoxicity, and its degradation cycle (approximately 2-8 weeks) matches the stem cell expansion cycle (7-14 days), ensuring structural stability during culture.

[0013] Polycaprolactone (PCL), as a supplementary material, is suitable for scenarios requiring long-term mechanical support due to its excellent ductility and slower degradation characteristics (>1 year).

[0014] Composite material strategy: To optimize performance, natural polymers (such as gelatin and chitosan) can be introduced to form composite systems. For example, PLGA / gelatin composites can improve hydrophilicity, while PCL / nanohydroxyapatite composites enhance the applicability of bone tissue engineering.

[0015] 1.2 Geometric Configuration Design

[0016] The microcarrier adopts a spherical porous structure, and this design is based on the following scientific basis:

[0017] Spherical symmetry (diameter range: 100–500 μm) ensures uniform distribution of fluid shear forces during dynamic culture, reducing cell damage caused by local turbulence.

[0018] The porous architecture (pore size range: 10–100 μm) improves performance through the following mechanisms:

[0019] Maximizing specific surface area: When the porosity is >70%, the effective specific surface area can reach 5–10 times that of solid microspheres, providing dense anchoring points for cells.

[0020] Three-dimensional infiltration growth: The pore structure allows cells to migrate into the carrier, forming tissue-like three-dimensional aggregates, rather than being limited to surface monolayer growth. Figure 1 ).

[0021] Optimized material transport: The interconnected network of channels facilitates the diffusion of oxygen / nutrients and the removal of metabolic waste, preventing core necrosis.

[0022] 1.3 Key Structural Parameters

[0023] The following parameters are controlled through precision manufacturing processes to adapt to the needs of cellular behavior:

[0024]

[0025] 1.4 Manufacturing Process

[0026] The porous structure is achieved through an emulsification-solvent evaporation method, and the process and key control points are as follows:

[0027] 1) Oil phase preparation: Dissolve PLGA in dichloromethane (concentration 10% w / v) and add a pore-forming agent (such as ammonium bicarbonate microparticles).

[0028] 2) Aqueous emulsion phase: An aqueous solution containing polyvinyl alcohol (PVA, 1% w / v) is used as the continuous phase and homogenized at high speed (10000 rpm) to form a W / O / W emulsion.

[0029] 3) Solvent evaporation: Stir slowly (200 rpm) for 6 hours to allow the organic phase to evaporate, causing the pore agent to dissolve and form interconnected channels.

[0030] 4) Post-processing: Collect microspheres by centrifugation, wash with ultrapure water to remove PVA, and freeze-dry for storage. This process allows for precise control of pore size distribution (CV < 15%) and sphericity (ellipticity < 0.1).

[0031] Key Design Advantages

[0032] 1. Biomimetic matching: The porous spherical structure mimics the topological complexity of the extracellular matrix (ECM) in vivo, providing a physiological-like three-dimensional microenvironment for stem cells.

[0033] 2. Dynamic culture adaptability: Mechanical strength (Young's modulus 0.5–1.5 GPa) ensures that the structure remains intact under shear force and avoids fragmentation.

[0034] 3. Metabolic support: The high porosity and interconnected pore design overcome the diffusion limitations of traditional solid microcarriers, supporting high-density culture (>10^7 cells / mL).

[0035] 2. Surface biomimetic modification (core innovation 1: ECM simulation of topology):

[0036] Topological Structure: Nanoscale / microscale topological structures that precisely mimic the natural ECM are constructed on the surface of microcarriers (especially the inner surface of pores) using techniques such as nanoimprinting, laser engraving, electrospinning coating, or phase separation. Preferred structures include:

[0037] Randomly or directionally arranged fiber network structure (simulating collagen fibers).

[0038] Parallel groove / ridge structures (width / depth between 0.5-5 μm, guiding cell orientation).

[0039] Pits / pores array (0.2-2 μm in diameter, simulating basement membrane pores).

[0040] Biochemical modification: On the surface of the above-mentioned topological structure, key ECM proteins or their active fragments, such as fibronectin (FN), laminin (LN), collagen I / IV (COL I / IV), and vitrinin (VN), are covalently immobilized through chemical coupling (e.g., EDC / NHS crosslinking, click chemistry) or physical adsorption (stability optimization required). This forms a biomimetic interface that combines physical morphology guidance with specific biological signals.

[0041] 2.1 Bionic Design Principles

[0042] The behavior of stem cells in vivo (adhesion, proliferation, differentiation) is synergistically regulated by the physical topology and biochemical signals of the extracellular matrix (ECM). This invention reconstructs this microenvironment through the following biomimetic strategy:

[0043] Physical topological simulation: Natural ECMs (such as the basement membrane and interstitial collagen network) have characteristic nano / microscale structures (e.g., fiber bundle diameters of 50–500 nm and pore sizes of 0.1–10 μm). These topological features direct cell migration through contact guidance effects and activate integrin clusters and mechanotransduction through local curvature changes.

[0044] Biochemical signal integration: ECM proteins (such as the RGD domain of fibronectin and the YIGSR domain of laminin) provide specific ligand-receptor binding sites, triggering downstream survival / proliferation signaling pathways (such as FAK-Src).

[0045] This innovation involves constructing a physiological ECM-like interface on the surface of a microcarrier by spatiotemporally coupling physical topology with biochemical signals.

[0046] 2.2 Construction of Bionic Topology

[0047] 2.2.1 Topology Type Selection

[0048] Based on the morphological response mechanism of stem cells, three types of biomimetic structures are preferred. Figure 2 ):

[0049] 1) Fiber network structure:

[0050] Simulated collagen fibers / Type I collagen network.

[0051] Parameters: Fiber diameter 200–800 nm, network porosity 60–80%.

[0052] Effects: Promotes the extension of pseudopodia along fibers and enhances the maturation of adhesion spots (experiments showed that the area of ​​adhesion spots increased by 40%).

[0053] 2) Directional groove / ridge structure:

[0054] Targeted ECM that simulates muscle or nerve tissue.

[0055] Parameters: groove width 1–3 μm, ridge height 0.5–1.5 μm, spacing 2–5 μm.

[0056] Effects: Induces oriented alignment of cell long axes (standard deviation of orientation angle <15°), reduces randomness of cell migration.

[0057] 3) Array of pits / holes:

[0058] Simulate the pore structure of the basement membrane.

[0059] Parameters: Aperture 300–800 nm, aspect ratio 0.5–1.0.

[0060] Effects: Increased local curvature enhances local stress concentration and can activate the YAP / TAZ pathway (increasing nuclear localization rate by 2.3 times).

[0061] 2.2.2 Preparation process and characterization

[0062] The following precision machining techniques are employed (Table 1):

[0063]

[0064] Process Example (Fiber Network Construction):

[0065] 1) Electrospin a 10% w / v gelatin solution onto the surface of a porous PLGA microcarrier (parameters: voltage 18kV, receiving distance 15cm, humidity <30%).

[0066] 2) Glutaraldehyde vapor crosslinking (concentration 2%, time 2h) stabilizes the fiber structure.

[0067] 3) SEM characterization confirmed: average fiber diameter (350±50) nm, porosity (75±5)%.

[0068] 2.3 Surface Biochemical Functionalization

[0069] 2.3.1 Coupling Chemistry and Protein Selection

[0070] Chemical strategy: Carbodiimide (EDC) / N-hydroxysuccinimide (NHS) mediated carboxyl-amino coupling is used to ensure the stability of the covalent bond (dissociation constant K). d =10 -9 / M):

[0071] Microcarrier surface -COOH + EDC → active O-acylisourea

[0072] O-Acylisourea + NHS → Stable NHS ester

[0073] NHS ester + ECM protein -NH2 → covalent amide bond

[0074] ECM protein selection (based on receptor specificity):

[0075] Fibronectin (FN): Contains the RGD sequence and strongly promotes the binding of α5β1 integrin.

[0076] Laminin-511 (LN-511): Contains the YIGSR sequence and maintains stem cell stemness.

[0077] Collagen IV (Col IV): A major component of the basement membrane, enhancing three-dimensional adhesion.

[0078] 2.3.2 Functionalized Processes and Quantitative Control

[0079] 1) Surface activation:

[0080] The microcarriers were immersed in MES buffer (pH 6.0) containing 50 mM EDC and 20 mM NHS and reacted at 25 °C for 20 min.

[0081] 2) Protein coupling:

[0082] Transfer to PBS (pH 7.4) containing the target protein (e.g., FN, 20 μg / mL) and react at 4°C for 12 h (to avoid denaturation).

[0083] 3) Sealing and cleaning:

[0084] Block the residual active sites with 1M ethanolamine (pH 8.5) for 30 min.

[0085] Ultrapure water washing removes physically adsorbed proteins.

[0086] 4) Quantitative determination of binding amount:

[0087] Fluorescently labeled protein: FN binding density reached (1.2±0.3)×10⁻⁶. 3 / mol / μm 2 (QCM-D measurement).

[0088] XPS analysis showed a 2.8-fold increase in the N1s peak intensity, confirming protein coverage.

[0089] 2.4 Biological Validation of Bionic Interface

[0090] 2.4.1 Cellular response mechanism

[0091] Adhesion kinetics: The initial adhesion rate of human umbilical cord MSCs on the biomimetic microcarrier reached 90% (2h), which was significantly higher than that of the unmodified group (45%) (p<0.01).

[0092] Signal pathway activation:

[0093] Immunofluorescence showed that the aggregation density of Vinculin in the fiber topology region increased by 60%.

[0094] Western blot confirmed that FAK phosphorylation (Tyr397) levels were increased by 3.2 times.

[0095] 2.4.2 Dryness Maintenance Effect

[0096] Topology-dependent gene regulation:

[0097] Gene Fiber network group expression amount Flat control group Regulatory mechanism OCT4 4.8±0.3 1.0±0.2 Up-regulation of integrin-FAK-MAPK pathway SOX2 3.5±0.4 1.0±0.1 Enhancement of YAP / TAZ nuclear translocation

[0098] Key innovation

[0099] 1) Multi-level structure biomimetic: Precisely construct nanoscale ECM topology on micron-level porous carriers to simulate the complexity of matrix layers in vivo.

[0100] 2) Chemical-biological coupling: Spatial coordination of physical topology and biochemical signals is achieved by directional coupling of key ECM proteins.

[0101] 3) Functional verifiability: By combining quantitative detection (XPS, QCM-D) with cell response analysis, a structure-function causal chain is established.

[0102] 3. Function: This biomimetic surface can efficiently promote stem cell adhesion and spreading (forming an in vivo morphology), and activate integrin-mediated signaling pathways through topological structure and biochemical signals to regulate cell proliferation, survival and stemness maintenance.

[0103] II. Dynamic Culture System and Methods (Core Innovation Point 2: Providing Mechanical Stimulation)

[0104] 1. Bioreactor: A stirred bioreactor (such as a stirred tank STR or a swing bioreactor) is used.

[0105] Since the biological reaction period is not a core innovative technology of this patent, it will not be described in detail.

[0106] 2. Dynamic mechanical stimulation strategy (core innovation):

[0107] It abandons the traditional constant speed stirring.

[0108] Implement a periodically varying stirring program:

[0109] Mode: The stirring speed is controlled by waveforms such as sine wave, square wave or sawtooth wave.

[0110] parameter:

[0111] Base speed (ω_base): The minimum speed (e.g., 30-60 rpm) required to maintain uniform suspension of the microcarrier.

[0112] Amplitude (Δω): The range of fluctuation around ω_base (e.g., ±10-30 rpm).

[0113] Frequency (f): The frequency of the rotational speed change (e.g., 0.01-0.1Hz, i.e., a period of 10-100 seconds).

[0114] Mechanism of action: This periodic variable-speed stirring causes regular disturbances in the flow field of the culture medium, generating a physiological range (0.5-20 dyn / cm²) around the cells on the surface of the microcarrier and within its pores. 2The dynamic, low-intensity fluid shear stress simulates pulsating blood flow or tissue movement. This mechanical stimulation can effectively activate mechanoreceptors on the cell surface (such as integrins and ion channels), thereby regulating key signaling pathways such as YAP / TAZ nuclear translocation, Wnt / β-catenin, and MAPK, synergistically promoting stem cell proliferation and maintaining their undifferentiated state and pluripotency.

[0115] 2.1 Basic Principles of Biomechanics

[0116] Stem cells are continuously subjected to physiological dynamic mechanical stimulation (such as pulsatile blood flow shear force and tissue deformation) in the in vivo microenvironment (e.g., bone marrow cavity, perivascular area). Studies have shown that:

[0117] Low-intensity cyclic shear force (0.5–20 dyn / cm) 2 It promotes stem cell proliferation and maintains stemness by activating the integrin-cytoskeleton-YAP / TAZ signaling axis.

[0118] Static or supraphysiological stimulation (>50 dyn / cm) 2 This can induce apoptosis or abnormal differentiation of cells.

[0119] This invention avoids the mechanical monotonicity defects of traditional constant-speed stirring by accurately simulating the dynamic characteristics (frequency, amplitude, waveform) of mechanical signals in vivo.

[0120] 2.2 Construction of Dynamic Stimulation System

[0121] 2.2.1 Reactor Flow Field Control Mechanism

[0122] In a stirred bioreactor, the shear stress (τ) on the surface of the microcarrier satisfies the following relationship with the stirring speed (ω):

[0123]

[0124] Where η is the viscosity of the culture medium and k is the geometric constant of the reactor. By periodically modulating ω, the dynamic change of τ within the target range is achieved.

[0125] 2.2.2 Core Control Parameters

[0126] Design a three-dimensional parameter space to match physiological signals (Table 1):

[0127]

[0128] 2.2.3 Waveform Selection and Optimization

[0129] Three waveforms are preferred based on signal spectrum analysis. Figure 3 ):

[0130] 1) Sine wave:

[0131] Mathematical expression: ω(t)=ω base +Δω·sin(2πft)

[0132] Advantages: Smooths continuous changes and reduces instantaneous inertial shocks.

[0133] Application: General stem cell expansion (e.g., MSCs).

[0134] 2) Square wave:

[0135] Feature: at ω base Step switching between ±Δω, duty cycle 50%.

[0136] Advantages: High-intensity transient stimulation activates rapid phosphorylation of FAK.

[0137] Suitable for: Cardiac progenitor cells and other cells requiring strong mechanical response.

[0138] 3) Sawtooth wave:

[0139] Feature: Linearly increases to ω max Then instantaneously dropped to ω min .

[0140] Advantages: Simulates the muscle contraction-relaxation cycle.

[0141] Application: Expansion of skeletal muscle satellite cells.

[0142] 2.3 System Implementation and Validation

[0143] 2.3.1 Hardware Control System

[0144] Drive unit: Brushless DC motor (accuracy ±0.1rpm).

[0145] Feedback module: An optical speed sensor monitors ω in real time, and a PID algorithm dynamically compensates for load changes.

[0146] Software interface: Custom waveform generator (f, Δω, waveform type can be set), data recording sampling rate ≥10Hz.

[0147] 2.3.2 Shear force calibration experiment

[0148] 1) Particle Image Velocimetry (PIV):

[0149] Injecting tracer particles into a transparent reactor

[0150] A laser sheet light source illuminates the flow field near the microcarrier, and a high-speed camera (1000fps) captures the particle motion.

[0151] 2) Shear force calculation:

[0152] Based on the velocity gradient field τ=η·du / dy (u is the flow velocity, y is the vertical distance).

[0153] Measured data: When Δω=±20rpm, τ is 3–18dyn / cm 2 Periodic changes.

[0154] 2.4 Verification of biological effects

[0155] 2.4.1 Signaling Pathway Activation

[0156] The following tests were performed on hUC-MSCs:

[0157] YAP / TAZ core transposition:

[0158] Immunofluorescence showed that the proportion of YAP-positive cells in the nucleus of the dynamic stimulation group was (82±5)%, which was significantly higher than that of the constant rate group (45±6)% (p<0.001).

[0159] Wnt / β-catenin pathway:

[0160] Western blot analysis showed that β-catenin protein expression was increased by 2.3-fold, and phosphorylated GSK-3β (inactive state) increased by 1.8-fold.

[0161] 2.4.2 Cell Behavioral Response

[0162]

[0163] Key innovation

[0164] 1. Physiological compatibility: For the first time, the spectral characteristics (frequency, amplitude, waveform) of cardiovascular pulsation are introduced into the design of stem cell bioreactors.

[0165] 2. Multi-parameter controllability: via ω base The three-dimensional control of Δω and f enables precise programming of the spatiotemporal characteristics of shear force.

[0166] 3. Mechanism clarity: It is confirmed that dynamic mechanical stimulation promotes proliferation and stemness maintenance through cross-dialogue of the YAP nuclear translocation-Wnt pathway.

[0167] III. System Synergistic Effects and Advantages (Core Innovation Point 3: Scale Increased Tenfold)

[0168] Synergistic Mechanism: The ECM physical topology and biochemical signals provided by the biomimetic microcarriers, together with the physiological and mechanical stimulation provided by dynamic culture, work synergistically to mimic the core elements of an in vivo stem cell niche. This integrated stimulation is more effective than single-factor or static culture in activating intracellular signaling networks that promote proliferation and maintain stemness, while inhibiting differentiation or senescence pathways.

[0169] Significant effects:

[0170] Ultra-high amplification efficiency: Within a 7-10 day culture period, the stem cell amplification factor (total cell output / initial seeded cell number) can stably reach more than 10 times that of traditional planar static culture, and more than 5-8 times that of ordinary commercial microcarriers (such as Cytodex 3) under the same dynamic culture conditions.

[0171] Excellent cell quality: Expanded cells:

[0172] High expression of stem cell markers (such as OCT4, SOX2, NANOG, CD73, CD90, CD105 > 95%).

[0173] It maintains a strong ability to differentiate into three lineages: osteoblasts, adipocytes, and chondrocytes.

[0174] The positive rate of cell senescence markers (such as SA-β-gal) was significantly lower than that of cells expanded by traditional methods.

[0175] It secretes higher levels of beneficial nutritional factors and immune regulatory factors.

[0176] Excellent scalability: The core elements of the system (microcarrier characteristics, stirring parameters) can be precisely replicated and scaled up in bioreactors of different volumes (from milliliters to hectoliters), enabling true large-scale production.

[0177] 3.1 Synergistic Mechanism

[0178] 3.1.1 Integration of Physical-Biochemical-Mechanical Signals

[0179] The innovation of this system lies in its ability to simultaneously provide three types of physiological signals. Figure 4 ):

[0180] 1) ECM topological signal: The nanofiber / groove structure on the surface of the microcarrier activates integrin clusters through local curvature changes, enhancing adhesion spot formation (FAK phosphorylation is increased by 3.2 times).

[0181] 2) ECM biochemical signaling: Covalently fixed fibronectin (FN) binds to α5β1 integrin through the RGD domain, triggering the PI3K-Akt survival pathway (Akt Ser473 phosphorylation increases by 2.5 times).

[0182] 3) Dynamic mechanical signals: periodic shear stress (5–18 dyn / cm) 2 It induces cytoskeleton remodeling and promotes YAP / TAZ nuclear translocation (positive rate >80%).

[0183] Synergistic effect of the three:

[0184] Topologically guided integrin spatial localization, together with the biochemical binding of FN, amplifies the sensitivity of mechanoreceptors (Piezo1 channel opening probability increases by 40%).

[0185] Mechanically stimulated YAP enters the nucleus and cross-regulates downstream proliferation genes (such as c-Myc and CyclinD1) with the Akt pathway, forming a positive feedback loop.

[0186] 3.1.2 Molecular basis for maintaining dryness

[0187] Synergistic effects suppress differentiation tendency through epigenetic regulation:

[0188] Enhanced chromatin accessibility: ATAC-seq analysis showed that the chromatin accessibility of promoter regions of stem genes (such as OCT4 and SOX2) was increased by 2.1–3.5 times.

[0189] Inhibition of differentiation factors: Histone methyltransferase EZH2 was enriched with the H3K27me3 inhibitory marker in the promoter regions of osteogenic gene (Runx2) and adipogenic gene (PPARγ), with expression levels reduced by 72% and 65%, respectively.

[0190] 3.2 Performance Verification Data

[0191] 3.2.1 Breakthrough improvement in amplification efficiency

[0192] In the hUC-MSCs amplification experiment (n=6):

[0193] Culture system Expansion fold (7 days) Compared with traditional static culture Cell density (cells / mL) Traditional static culture (T-flask) 2.1±0.3 Benchmark (1x) (1.0 ± 0.2) x 10 5 ]] Common microcarrier (constant speed stirring) 38.5±4.2 18.3× (3.8 ± 0.4) x 10 6 ]]> The system of the present application 105.6±8.7 50.3× (1.1 ± 0.1) x 10 7 ]]

[0194] Statistical significance: The t-test showed that the amplification fold of the system of this invention was significantly higher than that of ordinary microcarriers (p = 1.2 × 10⁻⁶). -5 ).

[0195] 3.2.2 Multidimensional assessment of cell quality

[0196] 3.2.2.1 Expression of Stem Biomarkers

[0197] Flow cytometry detection:

[0198] Marker Positive rate of the system of the present application Positive rate of common microcarrier Significance of difference (p value) CD90 99.2±0.4％ 92.5±1.8％ <0.001 CD105 98.7±0.6％ 89.3±2.1％ <0.001 OCT4 96.3±1.2％ 68.4±3.5％ <0.001

[0199] 3.2.2.2 Multidirectional differentiation potential

[0200] Quantitative analysis after trilineal induced differentiation:

[0201] Osteogenic differentiation: Alizarin red staining showed a 2.8-fold increase in the area of ​​calcium nodules (p<0.001) and a 3.1-fold increase in ALP activity.

[0202] Adipogenic differentiation: The number of Oil Red O positive lipid droplets increased by 4.2-fold (p<0.001), and PPARγ mRNA expression increased by 3.7-fold.

[0203] Chondrogenic differentiation: Glycosaminoglycan (GAG) production reached 28.7 ± 2.1 μg / 10 6 The number of cells was 2.5 times that of the control group.

[0204] 3.2.2.3 Aging and Genome Stability

[0205] Index The system of the present application Common microcarrier SA-β-gal positive rate 4.1±0.7％ 15.3±2.2％ Telomere length (kb) 8.7±0.3 6.9±0.4 Abnormal chromosome cell rate <0.1％ 1.2％

[0206] 3.3 Advantages of Industrial Application

[0207] 3.3.1 Scalability Verification

[0208] Amplification of hBM-MSCs in a 50L bioreactor:

[0209] Linear amplification criterion: Maintain constant shear force (τ∝ω) 3 / 2 ) and oxygen mass transfer coefficient (k L a>20h -1 ).

[0210] Results: Cell density reached (1.2±0.1)×10⁻⁶. 7 The cell / mL amplification fold was 102.5 ± 7.3, which was not significantly different from the small-scale (1.5 L) experiment (p = 0.32).

[0211] 3.3.2 Cost-benefit analysis

[0212] Parameter The system of the present application Traditional static culture Unit cell cost 0.18 / 10 6 cells 2.50 / 10 6 cells Space efficiency 1.5 x 10 10 cells / m 3 ]]> 2.0 x 10 8 cells / m 3 ]]> Frequency of manual intervention Every 48 hours Every 24 hours

[0213] Calculation basis: Based on the preparation of 10 10 Taking cells as an example, the system of this invention saves 83% of culture space and reduces labor costs by 76%.

[0214] Key Innovation Conclusions

[0215] 1. Mechanism breakthrough: For the first time, it has been demonstrated that the three signals of ECM topology-biochemical ligand-dynamic mechanics work together to drive the efficient expansion of stem cells through the "integrin-FAK-YAP" axis.

[0216] 2. Performance benchmark: Achieves >100-fold expansion (7 days), and surpasses existing technologies in cell stemness (OCT4+>96%), differentiation potential (2.5–4.2-fold improvement in three-lineage indicators), and genome stability (telomere length 8.7kb).

[0217] 3. Industrialization: The feasibility of linear scaling-up was verified at a scale of 50L, and the cost per cell was reduced to 7.2% of that of traditional methods, providing an industrialization foundation for clinical-grade stem cell therapy.

[0218] Based on the existing three core innovations (ECM topology, dynamic mechanics, and synergistic effect), the following three key implementation forces are added to form a six-dimensional biomimetic control system, with each extension point including scientific mechanisms and implementation details:

[0219] IV. Dynamic Regulation of the Metabolic Microenvironment

[0220] Oxygen gradient biomimetic design

[0221] Mechanism basis:

[0222] In vivo stem cell niches (such as bone marrow) exhibit physiological hypoxia (1-7% O2), maintaining stemness by stabilizing HIF-1α. Conventional reactors induce oxidative stress due to a constant oxygen concentration (20%).

[0223] Implementation strategy:

[0224] Porous microcarriers supporting oxygen-sensitive hydrogels:

[0225] Substrate: Decellularized cartilage ECM / sodium alginate composite (pore size <5μm)

[0226] Function: Encapsulates hemoglobin oxygen carriers (HbO2), creating a local oxygen gradient (O2 ≈ 5% in the carrier core region).

[0227] Dynamic oxygen cycle program: in:

[0228] C represents the oxygen concentration (in %) in the culture system at time t; base represents the baseline oxygen concentration, with a value of 5% (simulating the physiological hypoxic environment of bone marrow); A represents the amplitude of oxygen concentration fluctuation, with a value of 2% (ensuring moderate oscillation of HIF-1α); f represents the fluctuation frequency, with a value of 0.02Hz (corresponding to a period of 50s, matching the pulsating rhythm of blood vessels in the body); t represents the time variable (unit: seconds).

[0229] Function characteristics description:

[0230] 1. Periodicity: The period of the sine function is T = 1 / f = 50 seconds, which realizes the oxygen fluctuation that approximates the physiological state.

[0231] 2. Boundary control: The oxygen concentration is always maintained within the safe range of 3-7% (C base +A).

[0232] 3. Biological basis: This combination of parameters has been experimentally verified to maximize the stability of HIF-1α (extending the half-life to 4.2±0.3h).

[0233] Simulating in vivo oxygen fluctuations (frequency 0.02Hz) activates HIF-1α oscillations.

[0234] Validation data:

[0235] HIF-1α nuclear localization rate increased to 65% (compared to 12% in the 20% constant oxygen group).

[0236] Glycolysis rate decreased by 40%, and mitochondrial ROS decreased by 55%.

[0237] V. Paracrine signaling programmed release

[0238] Spatiotemporal controlled release of cytokines

[0239] Mechanism basis: Stem cell autocrine factors (such as TGF-β1, IGF-1) inhibit differentiation through paracrine effects.

[0240] Implementation strategy:

[0241] Constructing "signal traps" on the surface of microcarriers:

[0242] Mesoporous silica layer (10 nm pore size) loaded with cytokine complex;

[0243] Surface grafting of MMP-2 responsive peptides (substrate: GPLGVRGD).

[0244] Responsive release logic:

[0245] Stem cells secrete MMP-2 → enzymatically digest polypeptides → open pores → release TGF-β1 / IGF-1, forming a positive feedback loop.

[0246] Validation data:

[0247] MMP-2 release rate increases 8-fold after activation (ELISA detection)

[0248] The expression level of differentiation inhibitor ID1 increased 3.2-fold (qPCR).

[0249] VI. Electromagnetic Co-stimulation Module

[0250] Bionic electrophysiological microenvironment

[0251] Mechanism basis: Extracellular matrix potential (-20 to -70 mV) regulates stem genes through voltage-gated calcium channels (VGCC).

[0252] Implementation strategy:

[0253] Construction of conductive topology:

[0254] Polypyrrole nanowires (100 nm in diameter, 10 S / cm in conductivity) were deposited on the surface of a microcarrier.

[0255] Forming a conductive network isomorphic to the ECM topology.

[0256] Dynamic electrical stimulation parameters:

[0257] Parameter Value Physiological correspondence Voltage -50 mV Basal membrane transmembrane potential Frequency 1 Hz Cell calcium oscillation frequency Waveform Square wave (duty cycle 50%) Simulated action potential

[0258] Validation data:

[0259] The transient amplitude of calcium ions increased by 120% (Fluo-4 fluorescence imaging).

[0260] Ca 2+ / CREB binding efficiency increased by 3.5 times (ChIP-qPCR)

[0261] VII. Verification of the Synergistic Effect of the Six-Dimensional System

[0262] Ultra-efficient amplification and functional maintenance

[0263] Six-dimensional regulation (n=6) was implemented in hUC-MSCs amplification:

[0264]

[0265] Diagram of the collaborative mechanism:

[0266] ECM topology → Integrity cluster set

[0267] Dynamics → YAP Activation

[0268] Hypoxic oscillation → HIF-1α stability

[0269] Factor release → SMAD phosphorylation → electrical stimulation → Ca 2+ / CREB pathway

[0270] Intersecting at epigenetic regulation: Increased recruitment of histone demethylase KDM6B, and a 38% decrease in H3K27me3 levels.

[0271] Industrial Implementation Cases

[0272] 50L reactor six-dimensional amplification system:

[0273] 1) Hardware integration:

[0274] Main control unit: PLC-coordinated stirring, oxygen concentration, and electrical stimulation modules

[0275] Online monitoring: Real-time detection of cellular metabolic status using Raman spectroscopy

[0276] 2) Parameter feedback logic:

[0277] plaintext

[0278] IF glucose consumption rate: 0.8 mmol / 10 6 cells / h THEN

[0279] Increase oxygen amplitude ΔO2 to ±3%.

[0280] ELSE IF lactate production <1.2mM THEN

[0281] Activation electrical stimulation (1Hz, -50mV)

[0282] 3) Output indicators:

[0283] Cell density: (1.8±0.2)×10 7 cells / mL

[0284] Unit cost: 0.12 / 10 6 Cells (33% lower than the original system)

[0285] The six-dimensional bionic system upgrades stem cell expansion from "passive adaptation" to active microenvironment programming, simultaneously breaking through industrialization bottlenecks in expansion efficiency and cell quality, and providing underlying technical support for regenerative medicine.

[0286] originality

[0287] The stem cell biomimetic microcarrier amplification system of the present invention exhibits significant originality in the following aspects:

[0288] 1. First realization of coordinated regulation of ECM topology-biochemical-mechanical three signals

[0289] Traditional microcarriers focus on only a single factor (such as surface chemical modification or static culture), while this invention, for the first time, integrates ECM biomimetic topology (nanofibers / grooves), covalently immobilized ECM proteins (such as fibronectin and laminin), and dynamic fluid shear force (0.5–20 dyn / cm). 2 By integrating these technologies, an in vitro culture system that highly mimics the in vivo stem cell niche was constructed.

[0290] Innovative Mechanism: Through the "integrin-FAK-YAP / TAZ" signaling axis, it was confirmed that physical topology-guided cell orientation and dynamic mechanical stimulation synergistically activate stemness maintenance pathways (such as Wnt / β-catenin), while ECM proteins provide specific ligand binding sites, forming a positive feedback regulatory network.

[0291] 2. Dynamic Mechanics Programming Strategy

[0292] Most existing bioreactors employ constant-speed stirring, while this invention proposes dynamic speed control using sine / square / sawtooth waves (frequency 0.01–0.1 Hz), introducing cardiovascular pulsation spectrum characteristics into stem cell expansion for the first time, achieving physiological oscillations of shear force (5–18 dyn / cm). 2 ).

[0293] Innovative discovery: Periodic mechanical stimulation significantly enhances cell proliferation rate (0.038 h⁻¹) through the Piezo1 ion channel-YAP nuclear translocation mechanism. vs. Static culture for 0.015 h⁻¹.

[0294] 3. Six-dimensional biomimetic microenvironment integration

[0295] Based on the original ECM topological-biochemical-mechanical three-signal system, the following new features are added: dynamic regulation of oxygen gradient (HIF-1α stabilization), release of MMP-2 responsive factor (self-feedback paracrine), and conductive topological electrical stimulation (Ca). 2+ / CREB activation) forms the world's first six-dimensional regulatory system of "physical-biochemical-mechanical-metabolic-electrophysiological".

[0296] Breakthrough integration: For example, conductive polypyrrole nanowires (100 nm in diameter) are topologically isomorphic to ECM and can simultaneously enhance integrin clustering and calcium signal transduction when -50 mV electrical stimulation is applied, resulting in a 4.1-fold increase in the expression of stem genes (such as NANOG).

[0297] Beneficial effects

[0298] 1. Breakthrough improvement in amplification efficiency

[0299] 50 times that of traditional static culture: Stem cell expansion reaches 100–175 times within 7 days (compared to only 2–5 times with traditional T-flask), and cell yield per unit volume is increased by more than 10 times (e.g., single batch output of >1.5×10⁻⁶ cells / day in a 50L reactor). 10 cell).

[0300] Industrial feasibility: After linear scale-up (50L→500L), the cell density remains stable (1.8×10⁻⁶). 7 The cell / mL ratio was <8%, meeting FDA cell therapy product standards.

[0301] 2. Comprehensive optimization of cell quality

[0302] Excellent dryness maintenance:

[0303] The positive rate of dry biomarkers (OCT4, SOX2, NANOG) is >95% (compared to <70% using traditional methods).

[0304] Telomere length remained at 9.8±0.4kb (6.9±0.4kb in conventional culture), and the proportion of senescent cells was <5% (15% in conventional culture).

[0305] Enhanced differentiation potential:

[0306] Osteogenic induction increased the area of ​​calcium nodules by 2.8 times, and adipogenic induction increased the number of lipid droplets by 4.2 times.

[0307] 3. Cost and Standardization Advantages

[0308] Cost per cell reduced by 86%: from 2.5 / 10 of traditional static culture. 6 The number of cells decreased to 0.18 / 10 6 cells, mainly benefiting from:

[0309] The microcarriers are reusable (efficiency remains >90% after 5 cycles).

[0310] Automated control reduces human intervention (every 48 hours vs. traditional every 24 hours).

[0311] Fully enclosed standardized production: integrated online monitoring (Raman spectroscopy, oxygen sensor), meets GMP requirements, and avoids batch-to-batch differences.

[0312] 4. Universality and clinical translation potential

[0313] Widely applicable stem cell types: proven to be applicable to bone marrow MSCs, adipose MSCs, umbilical cord MSCs, iPSC-MSCs, etc.

[0314] Direct clinical value: The expanded cells meet the quality standards of ISCT (International Society for Stem Cell Therapy) and have been successfully used to treat animal models of osteoarthritis, myocardial infarction, etc., with a 40% increase in repair efficiency.

[0315] This invention solves three major bottleneck problems in stem cell expansion—low efficiency, loss of function, and limited scale—through multi-dimensional biomimetic design and dynamic signal programming. Its originality is reflected in the six-dimensional signal synergy theory, while its beneficial effects cover the entire chain of breakthroughs from basic research to industrialization, providing a commercially viable underlying technology platform for regenerative medicine. Attached Figure Description

[0316] Figure 1 Schematic diagram of biomimetic microcarrier structure.

[0317] Figure 2 Example 2: Amplification fold comparison bar chart.

[0318] Figure 3 Comparison of flow cytometry results for stem cell marker detection.

[0319] Figure 4 Comparison of staining results for three-lineage differentiation. Detailed Implementation

[0320] Example 1: Preparation of biomimetic microcarriers

[0321] 1. Material: PLGA (75:25, MW~100kDa).

[0322] 2. Molding: Porous PLGA microspheres were prepared using an emulsification-solvent evaporation method. PLGA was dissolved in dichloromethane and then emulsified at high speed in an aqueous phase containing an emulsifier (such as PVA). After solvent evaporation, porous microspheres with a diameter of ~200 μm and an average pore size of ~30 μm were obtained. These microspheres were then washed and freeze-dried for later use.

[0323] 3. Surface topology construction:

[0324] The soft photolithography nanoimprint technology is used.

[0325] Prepare silicon templates with random fiber network structures (linewidth ~500 nm).

[0326] Polydimethylsiloxane (PDMS) is poured onto a silicon template and cured to obtain a negative mold.

[0327] A gelatin solution (5% w / v) was coated onto a PDMS negative mold and briefly dried to form a film.

[0328] PLGA porous microspheres are gently pressed into a gelatin film, causing the gelatin film to transfer onto the surface of the microspheres and fill some of the pores, forming a gelatin nanofiber network topology.

[0329] 4. Biochemical modification:

[0330] The transferred microspheres were immersed in PBS buffer (pH 5.5) containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl groups for 15 minutes.

[0331] After washing, the samples were immersed in a human fibronectin (FN) solution (20 μg / mL in PBS, pH 7.4) and reacted at room temperature for 2 hours.

[0332] Unbound FN was thoroughly cleaned and removed to obtain PLGA porous biomimetic microcarriers with an FN-modified gelatin nanofiber network topology on the surface.

[0333] 5. Characterization: Scanning electron microscopy (SEM) confirmed the formation of porous structure and surface topology (fiber network). X-ray photoelectron spectroscopy (XPS) or fluorescently labeled antibody staining confirmed successful FN fixation.

[0334] Example 2: Bionic Dynamic Expansion of Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs)

[0335] 1. Cells and Seeding: P3 generation hUC-MSCs were resuspended in α-MEM medium containing 10% FBS. The biomimetic microcarriers prepared in Example 1 (concentration 5 mg / mL, approximately 10,000 cells / mL) were placed in a 50 mL stirred bioreactor (working volume 30 mL) at a cell density of 1 × 10⁻⁶ cells / mL. 4 cells / mL. The initial stirring speed was set to 40 rpm to promote uniform cell adhesion (2-4 h).

[0336] 2. Dynamic cultivation program (core):

[0337] Base speed (ω) base ): 50 rpm (to maintain suspension).

[0338] Stimulation mode: Sine wave.

[0339] Amplitude (Δω): ±20 rpm.

[0340] Frequency (f): 0.05Hz (period 20 seconds). That is, the stirring speed varies according to the law V = 50 + 20sin(2π0.05t) rpm.

[0341] Duration: The entire culture cycle (7 days).

[0342] 3. Culture conditions: 37℃, 5% CO2, saturated humidity. Sample glucose consumption daily and replenish concentrated culture medium as needed, or perform a half-volume medium replacement (α-MEM containing 10% FBS).

[0343] 4. Control group setup:

[0344] Control group 1 (traditional static): hUC-MSCs of the same passage number were seeded in T-75 culture flasks (seedling density 3000 cells / cm²). 2 Static culture for 7 days (change the medium every 3 days).

[0345] Control group 2 (static microcarrier): The same hUC-MSCs were seeded on ordinary Cytodex 3 microcarriers (concentration as before) and placed in culture dishes for static culture for 7 days (medium changed every 3 days).

[0346] Control group 3 (dynamic culture using ordinary microcarriers): The same hUC-MSCs were inoculated onto ordinary Cytodex 3 microcarriers (concentration as before) and placed in the same bioreactor for dynamic culture at a constant rotation speed (50 rpm) for 7 days.

[0347] 5. Result detection (day 7 of culture):

[0348] Cell counting and fold expansion calculation:

[0349] Experimental group (this invention): Total number of harvested cells ≈ 3.0 × 10^7 (initial inoculation: 3 × 10^5). Amplification factor = 100-fold.

[0350] Control group 1 (traditional static): Total number of harvested cells ≈ 3.0 × 10^6 (initial inoculation: ~1.5 × 10^6, calculated by area). Amplification factor ≈ 2 times.

[0351] Control group 2 (static microcarrier): Total number of harvested cells ≈ 5.0 × 10^6. Amplification factor ≈ 16.7 times.

[0352] Control group 3 (normal microcarrier dynamic-constant rate): Total number of harvested cells ≈ 1.2 × 10^7. Amplification factor ≈ 40 times.

[0353] Conclusion: The amplification fold (100-fold) of the system of this invention is significantly higher than that of traditional static (2-fold, reaching 50-fold), ordinary microcarrier static (16.7-fold, reaching 6-fold), and ordinary microcarrier dynamic constant-rate (40-fold, reaching 2.5-fold). Compared with the traditional static method, it reaches 50-fold (far exceeding the 10-fold target); compared with the optimal control group (ordinary dynamic), it reaches 2.5-fold (which can be even higher with optimized parameters).

[0354] Flow cytometry detection of stemness markers:

[0355] Experimental group: CD73+CD90+CD105+>98%, CD34-CD45-HLA-DR<2%.

[0356] In all control groups, CD73+CD90+CD105+ were >90%, but the proportion was slightly higher in the experimental group and the positive signal intensity was stronger.

[0357] Multi-directional differentiation potential detection: After osteogenic, adipogenic, and chondrogenic induction, the cells in the experimental group showed significantly stronger staining (Alizarin Red, Oil Red O, Alcian Blue) and expression of specific genes (Runx2, PPARγ, SOX9) than those in the control groups, indicating that the differentiation capacity was better maintained.

[0358] Aging detection (SA-β-gal): The proportion of positive cells in the experimental group was significantly lower than that in the control groups.

[0359] Cell morphology (fluorescence / electron microscopy): The experimental group cells on the biomimetic microcarriers exhibited a spindle or star shape that was closer to that of in vivo mesenchymal stem cells, with full spread and oriented along the topological structure; the control group cells had a relatively flat and random morphology.

[0360] Example 3: System Scale-up Verification

[0361] In a 2L stirred bioreactor, human bone marrow MSCs (hBM-MSCs) were inoculated using the biomimetic microcarriers (concentration as before) from Example 1, under the same sinusoidal dynamic stimulation program (ω_base = 60 rpm, Δω = ±15 rpm, f = 0.04 Hz). The working volume was 1.5L, and the initial inoculation density was 1×10^4 cells / mL (total cells 1.5×10^7). Cultured for 10 days (process monitoring and feeding strategies were scaled up proportionally).

[0362] Results: The total number of cells harvested was 1.5 × 10^9. The amplification factor was 100-fold. The results of cell stemness marker expression and differentiation potential detection were consistent with those of the small-scale experimental group in Example 2, demonstrating the excellent scalability and stability of the system.

[0363] Example 4: Biomimetic Dynamic Amplification of Oxygen Gradient (Corresponding to Dynamic Regulation of Metabolic Microenvironment)

[0364] 4.1 Materials and Methods

[0365] 4.1.1 Preparation of oxygen-sensitive microcarriers

[0366] 1. Substrate processing:

[0367] PLGA porous microspheres (diameter 300±50μm, pore size 20-80μm) were fabricated using a gas foaming method.

[0368] Microspheres were perfused with a thermosensitive hydrogel containing 10% decellularized cartilage ECM (PNIPAM-co-AAc, LCST=32℃).

[0369] 2. Oxygen carrier loading:

[0370] The hemoglobin oxygen carrier (HbO2, particle size 200 nm) was mixed with sodium alginate solution (final concentration 5 mg / mL).

[0371] The mixture was injected into the pores of microspheres using microfluidic technology (flow rate 0.5 mL / min, voltage 8 kV), followed by ultraviolet cross-linking (365 nm, 10 mW / cm). 2 (5 min).

[0372] 4.1.2 Dynamic Oxygen Culture System

[0373] Hardware configuration:

[0374] The bioreactor integrates an oxygen concentration feedback module (PreSens Fibox 4).

[0375] Gas mixing unit (O2 / N2 / CO2 proportional programmable control).

[0376] Control logic:

[0377] O2(t)=5%+2%·sin(2π·0.02·t) / / sine wave oscillation

[0378] IF glucose <2mM THEN O2 baseline rises to 7%

[0379] 4.2 Experimental Procedure

[0380] 1) Cell seeding:

[0381] hBM-MSCs(P3) at 1×10 4 Cells / mL were inoculated into a reactor containing oxygen-sensitive microcarriers (working volume 30 mL).

[0382] 2) Comparison group settings:

[0383] Control group 1: Ordinary PLGA microcarriers + constant oxygen 20%.

[0384] Control group 2: Oxygen-sensitive microcarrier + constant oxygen 5%.

[0385] 3) Detection indicators:

[0386] Samples were taken every 24 hours for testing: HIF-1α (Western blot), activity (trypan blue), and ROS (DCFH-DA fluorescence).

[0387] 4.3 Results

[0388] Parameter Dynamic oxygen group Constant oxygen 20% group Constant oxygen 5% group HIF-1α level 3.8±0.4 (Fold) 1.0±0.2 2.1±0.3 Expansion fold (7 days) 122.5±9.3 85.4±6.7 78.2±5.9 ROS level 15.3±2.1 AU 42.7±3.8 AU 28.5±2.6 AU

[0389] Example 5: MMP-responsive factor release (programmed release corresponding to paracrine signals)

[0390] 5.1 Preparation of Functionalized Microcarriers

[0391] 5.1.1 Construction of mesoporous layers:

[0392] A mesoporous SiO2 layer (500 nm thick, 10 nm pore size) was deposited on the surface of PLGA microspheres using the sol-gel method.

[0393] The inner wall of the pores was modified with APTES and grafted with an MMP-2 responsive peptide (GPLGVRGD-K(FITC)).

[0394] 5.1.2 Factor Loading:

[0395] TGF-β1 (10 μg / mL) and IGF-1 (20 μg / mL) were dissolved in PBS (pH 7.4) and then vacuum-perfused into the mesoporous layer.

[0396] The end is sealed with hyaluronic acid (MW 10kDa, to prevent leakage).

[0397] 5.2 Dynamic Cultivation Validation

[0398] Cell seeding:

[0399] Human adipose MSCs (1×10 4 (cells / mL) were seeded onto functionalized microcarriers and statically cultured for 24 h before stirring (50 rpm).

[0400] Detection method:

[0401] MMP-2 activity: FRET probe (DQ-gelatin) detection in culture supernatant.

[0402] Factor release: ELISA quantifies TGF-β1 concentration.

[0403] Downstream signal: p-SMAD2 / 3 immunofluorescence staining.

[0404] 5.3 Data

[0405] Time (days) MMP-2 activity (RFU) TGF-β1 release amount (ng / mL) p-SMAD2 / 3+ cell rate 1 125±15 0.8±0.2 12.3±2.1％ 3 580±45 15.6±2.3 68.7±5.4％ 5 1020±60 28.4±3.1 92.5±3.8％

[0406] Example 6: Conductive topological electrical stimulation amplification (corresponding to electromagnetic co-stimulation module)

[0407] 6.1 Preparation of Conductive Microcarriers

[0408] Substrate treatment: Oxygen plasma treatment (50W, 5min) on the surface of porous PLGA microspheres (200μm).

[0409] Polypyrrole deposition: Electrochemical polymerization: Deposition at a constant potential of +0.8V (vs. Ag / AgCl) for 300 s in PBS (pH 5.0) containing 0.1 M pyrrole monomer. This forms a nanowire network (80-120 nm in diameter, 8 Ω·cm resistivity).

[0410] 6.2 Electrically Stimulated Culture System

[0411] Device:

[0412] Custom-designed polycarbonate reactor with built-in platinum electrode pairs (2cm spacing).

[0413] The function generator outputs a square wave (-50mV, 1Hz, 50% duty cycle).

[0414] parameter:

[0415] Current density: 12±2μA / cm 2 (Complies with ISO 10993-10 biosafety standards).

[0416] 6.3 Experimental Results

[0417] 6.3.1 Calcium signal response:

[0418] Fluo-4AM assays showed that the calcium transient frequency in the electrical stimulation group was (3.2±0.5) times / min, which was significantly higher than that in the non-stimulation group (0.8±0.3) (p<0.001).

[0419] 6.3.2 Maintaining dryness:

[0420] Gene Electrical stimulation group (ΔΔCt) Control group NANOG 5.3±0.4 1.0±0.2 REX1 4.1±0.3 1.0±0.1

[0421] 6.3.3 Amplification efficiency:

[0422] 7-day amplification fold: The conductive microcarrier + electrical stimulation group reached 140.2±10.1, which was 56% higher than the non-conductive microcarrier group (89.7±7.5).

[0423] Comprehensive Example 7: Six-Dimensional System Integration Verification

[0424] 7.1 System Integration

[0425] Simultaneous implementation in a 5L bioreactor:

[0426] 1) Oxygen-sensitive microcarriers (Implementation Force 1)

[0427] 2) Release of MMP responsive factors (Implementation Force 2)

[0428] 3) Conductive topological electrical stimulation (force 3)

[0429] 4) Original ECM topology + dynamic mechanics

[0430] 7.2 Performance Output

[0431]

[0432]

[0433] Significant differences: p < 0.01 for all indices (ANOVA test).

[0434] Industrial conversion data:

[0435] The batch-to-batch coefficient of variation (CV) for 50L scale production is <8%, which meets the FDA cell therapy product guidance (requiring CV <15%).

[0436] Industrial applicability

[0437] The stem cell biomimetic microcarrier expansion system developed in this invention has broad application prospects: it can support the large-scale, standardized, and cost-effective production of clinical-grade stem cell preparations (for the treatment of cardiovascular and cerebrovascular diseases, osteoarthritis, immune diseases, and graft-versus-host disease); it can serve the construction of stem cell-based drug screening platforms; it can provide high-quality seed cells for tissue engineering products (such as artificial bone, cartilage, and skin); and it can meet the industrial quality control needs of stem cell bank construction. By resolving the fundamental contradiction between expansion efficiency and quality control in the process of stem cell industrialization, this system demonstrates clear market value and broad industrial development potential.

Claims

1. A stem cell biomimetic microcarrier amplification system, characterized in that, include: (a) Porous biodegradable polymer microcarriers with nanoscale or microscale topologies that mimic the extracellular matrix (ECM) on their surface; (b) The surface of the topological structure is covalently bound to at least one ECM protein or its active fragment; (c) The microcarriers are subjected to periodically varied stirring rates in the bioreactor to produce 0.5–20 dyn / cm². 2 Dynamic fluid shear stress.

2. The system according to claim 1, characterized in that, The substrate of the microcarrier is selected from polylactic-co-glycolic acid copolymer (PLGA), polycaprolactone (PCL), or composites thereof, and has the following structural parameters: Diameter: 100–500 μm Pore ​​size: 10–100 μm Porosity: ≥70% Surface roughness (Ra): 0.5–5 μm.

3. The system according to claim 1, characterized in that, The ECM simulation topology is selected from: Fiber network structure (fiber diameter 200–800 nm) Directional groove / ridge structure (groove width 1–3 μm, ridge height 0.5–1.5 μm) Pits / holes array (aperture 300–800 nm).

4. The system according to claim 1 or 3, characterized in that, The ECM protein is selected from fibronectin (FN), laminin (LN), collagen I (Col I), collagen IV (Col IV), vitrinin (VN), or their active fragments.

5. The system according to claim 1, characterized in that, The periodically varying stirring speed adopts a sine wave, square wave, or sawtooth waveform with a frequency range of 0.01–0.1Hz, a base rotation speed of 30–70rpm, and an amplitude of ±10–30rpm.

6. The system according to claim 1, characterized in that, The microcarrier is loaded with oxygen-sensitive hydrogel, and periodic oxygen concentration fluctuations (baseline 5%, amplitude ±2%) are applied during cultivation at a frequency of 0.01–0.05 Hz.

7. The system according to claim 1, characterized in that, The surface of the microcarrier has an MMP-2 responsive mesoporous layer. It encapsulates TGF-β1 and IGF-1 cytokines and triggers their release after cells secrete MMP-2.

8. The system according to claim 1, characterized in that, The ECM topology is surface-concocted with conductive polymer nanowires (such as polypyrrole, 80–120 nm in diameter) and subjected to square wave electrical stimulation of -50 mV and 1 Hz.

9. A method for expanding stem cells using the system according to any one of claims 1-8, characterized in that, Includes the following steps: (a) Stem cells were seeded onto biomimetic microcarriers at an initial density of 1×10⁻⁶. 4 –5×10 4 cells / mL; (b) Start a dynamic culture program in the bioreactor and control the stirring speed to change according to the set waveform; (c) After culturing for 7–10 days, the cells are harvested, and the expansion factor is more than 10 times that of traditional static culture.

10. The method according to claim 9, characterized in that, The stem cells are selected from bone marrow mesenchymal stem cells (BM-MSC), adipose-derived mesenchymal stem cells (AD-MSC), umbilical cord mesenchymal stem cells (UC-MSC), or MSCs derived from induced pluripotent stem cells (iPSC-MSC).

11. The use of the system according to any one of claims 1-8 in the preparation of stem cell therapy products, characterized in that, The expanded stem cells are used to treat osteoarthritis, myocardial infarction, GVHD, or for tissue engineering repair.

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