Stem cell membrane-stent dynamic co-culture device and gradient regulation culture process

Abstract

The invention relates to the technical field of tissue engineering and regeneration, in particular to a stem cell membrane-stent dynamic co-culture device and a gradient regulation culture process. The stem cell membrane-stent dynamic co-culture device comprises a) a multi-modal mechanical loading module (1), a compression strain unit (2) and a multi-modal mechanical loading module (3), the frequency is adjustable from 0.1 Hz to 2 Hz, the strain amplitude is 5%-15% (the precision is + / -0.5%), and the periodic compression stress of bone tissue can be simulated; (2) a fluid shear force unit: generating 0.1-5Pa laminar shear force (Re < 2000) through a conical flow channel (with a taper angle of 15 degrees) and a peristaltic pump, and simulating a shear microenvironment of a blood vessel or periodontal ligament area; the method has the beneficial effects that multi-modal mechanical synergy is achieved, and the cell differentiation efficiency is remarkably improved through coupling loading of compression and shearing force. Experiments show that the ALP activity reaches 38.2 + / -3.1 U / mg (only 12.5 + / -2.3 U / mg in traditional single-mode stimulation), and the proportion of a CD31 + vascularized region is increased to 41.7 + / -3.8% (plt; 0.01).

Description

Technical Field

[0001] The present invention relates to the field of tissue engineering and regeneration technology, in particular to a stem cell sheet-scaffold dynamic co-culture device and a gradient-controlled culture process. Background Art

[0002] The key challenge in the current field of tissue engineering is to simulate the complex physiological microenvironment in the body. Existing technologies have the following limitations:

[0003] Limitation of a single mechanical stimulus: Traditional bioreactors can only apply a single mechanical stimulus (such as compression or shear force), and are unable to replicate the multimodal stress coupling at the bone-vascular interface (e.g., the synergistic effect of cyclic compression of bone tissue and shear force on vascular endothelial cells). Literature has shown that single stimulation can limit cell differentiation (e.g., ALP activity only increases 1.5 times that of the control group) and cannot achieve dynamic feedback regulation of mechanical signals (Zhou et al., Biomaterials, 2020).

[0004] Insufficient gradient control in the perfusion system: Existing perfusion systems often use a homogenous mixed culture medium, resulting in an overlap rate of >40% between osteogenic and angiogenic differentiation zones (e.g., the overlap rate using dual-channel perfusion in the literature was 35.6 ± 3.8%), which seriously affects the biomimetic construction of tissue interface structures (Smith et al., Lab on a Chip, 2019);

[0005] Severe damage to ECM components: Traditional membrane stripping technology relies on enzymatic digestion or mechanical scraping, resulting in a collagen loss rate of >60% (such as ThermoFisher data) and a laminin retention rate of less than 55%, leading to loss of ECM functional integrity (Kim et al., Acta Biomaterialia, 2021).

[0006] Therefore, we proposed a stem cell membrane-scaffold dynamic co-culture device and a gradient-controlled culture process to solve the above problems. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention discloses a stem cell membrane-scaffold dynamic co-culture device and a gradient-controlled culture process, the technical solution adopted is as follows:

[0008] a) Multimodal mechanical loading module

[0009] ① Compression strain unit: uses a piezoelectric ceramic driver (stroke ±2mm, accuracy ±0.1μm), adjustable frequency 0.1-2Hz, strain amplitude 5-15% (accuracy ±0.5%), which can simulate the cyclical compressive stress of bone tissue;

[0010] ② Fluid shear force unit: generates 0.1-5Pa laminar shear force (Re < 2000) through a tapered flow channel (cone angle 15°) and a peristaltic pump to simulate the shear microenvironment of the blood vessels or periodontal ligament area;

[0011] ③ Real-time feedback system: Integrated impedance sensor (detection frequency 1kHz-1MHz). When the impedance change Δ|Z|>10Ω, the PID algorithm automatically adjusts the mechanical parameters (strain amplitude ±10%, shear force ±0.2Pa) to achieve cell response-driven adaptive loading.

[0012] b) Dual-channel gradient perfusion system

[0013] ① Channel I (osteoblast differentiation medium): contains sodium β-glycerophosphate (10 mM), dexamethasone (100 nM), and TGF-β1 (20 ng / mL), pH 7.4 ± 0.1;

[0014] ② Channel II (angiogenic differentiation medium): contains VEGF (50 ng / mL), bFGF (30 ng / mL), EGF (20 ng / mL), pH 7.2 ± 0.1;

[0015] ③ Gradient generator: Based on a Y-shaped microfluidic chip (channel width 200μm × depth 100μm, surface treated with plasma hydrophilicity), it generates a linear concentration gradient (R² > 0.98, verified by fluorescence tracing) with a spatial resolution of 50μm by adjusting the flow rate ratio from 1:1 to 1:5;

[0016] ④ Anti-cross contamination design: Nanofiltration membrane (pore size 50nm) is set between channels to block the diffusion of large molecules (retention efficiency > 99%).

[0017] c) Thermosensitive culture dish assembly

[0018] ① Base material: poly (N-isopropylacrylamide) (PNIPAAm, LCST = 32°C), doped with 0.5% polyethylene glycol (PEG) to improve mechanical strength (elastic modulus increased to 1.2 MPa);

[0019] ②Surface modification: Collagen (50 μg / cm²) and fibronectin (30 μg / cm²) composite coating, fixed with aminosilane coupling agent, promotes cell adhesion (adhesion efficiency > 95%);

[0020] ③ Membrane peeling: Cool to 25°C and maintain for 10 minutes, and use the hydrophilic-hydrophobic transition of PNIPAAm to achieve non-destructive peeling of the ECM membrane (integrity > 95%, collagen retention rate 98.2 ± 0.5%);

[0021] ④Temperature control accuracy: integrated thermocouple and PID temperature control module, temperature fluctuation ≤±0.3℃.

[0022] Beneficial effects of the present invention:

[0023] 1. Multimodal mechanical synergy: Through coupled compression and shear loading, cell differentiation efficiency is significantly improved. Experiments show that ALP activity reaches 38.2±3.1 U / mg (compared to 12.5±2.3 U / mg for traditional single-modal stimulation), and the proportion of CD31+ vascularized areas increases to 41.7±3.8% (p<0.01).

[0024] 2. Precise gradient control: The microfluidic-based gradient generator reduced the overlap rate of osteogenic / angiogenic regions to 14.8±2.1% (traditional methods >30%), while achieving spatially directed differentiation (ALP activity in the osteogenic region was 38.2±3.1 U / mg, and CD31+ content in the angiogenic region was 41.7±3.8%).

[0025] 3. High ECM retention rate: Thermosensitive stripping technology achieves collagen, laminin, and fibronectin retention rates of 98.2±0.5%, 96.5±1.2%, and 94.8±1.0%, respectively (traditional methods <62%), significantly enhancing the functional integrity of the ECM.

[0026] 4. Improved in vivo repair efficiency: 7 days after implantation in a rat alveolar bone defect model, the density of new blood vessels reached 42.1±4.3 cells / mm² (13.5±2.1 cells / mm² in the control group), and the bonding strength increased to 8.2±0.8 MPa (3.5±0.6 MPa with the traditional method).

[0027] 5. Expanded Application Potential: By adjusting parameters (e.g., flow rate ratio 1:5, compression frequency 2 Hz), the product can be adapted to various tissue engineering needs, such as bone and cartilage. ALP activity was further increased to 45.8±4.1 U / mg (compared to 18.3±2.7 U / mg in the control group). DETAILED DESCRIPTION

[0028] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0029] The present invention discloses a stem cell membrane-scaffold dynamic co-culture device and a gradient-controlled culture process, and the technical solution adopted is:

[0030] 1. Device Construction and Parameter Optimization

[0031] 1. Mechanical loading module:

[0032] 1) Piezoelectric ceramic actuator (PI, model P-623.1) with strain sensor (accuracy ±0.1%);

[0033] 2) Peristaltic pump (Cole-Parmer, Masterflex L / S) with tapered flow channel (316L stainless steel, surface finish Ra < 0.1 μm).

[0034] 2. Gradient perfusion system:

[0035] 1) The microfluidic chip was fabricated using soft lithography (PDMS material), and the channel surface was treated with oxygen plasma (power 50 W, time 30 s);

[0036] 2) The flow rate ratio was controlled by a dual-channel precision syringe pump (Harvard Apparatus, PHD Ultra).

[0037] 3. Thermosensitive culture dish:

[0038] 1) PNIPAAm substrate was synthesized by free radical polymerization (monomer concentration 10 wt%, cross-linker MBAA 0.1 mol%);

[0039] 2) The temperature control module uses a TEC cooling chip (Laird Technologies, CP10) and a PID controller (Omega, CNi3244).

[0040] 2. Periodontal Ligament Tissue Construction and Performance Verification

[0041] 1. Cell seeding: hDPSCs (passage 3, density 2×10 5 cells / cm²) were seeded on the patent 1 scaffold and pre-cultured for 24 hours;

[0042] 2. Dynamic cultivation:

[0043] 1) Mechanical parameters: 1 Hz compressive strain (10%) + 0.5 Pa shear force (Re = 1500);

[0044] 2) Perfusion parameters: osteogenic / angiogenic fluid flow rate ratio of 1:3, continuous culture for 14 days;

[0045] 3. Membrane harvesting: Cool to 25°C and peel off the membrane (thickness 150±20μm). The ECM component retention rate is as described above.

[0046] 4. In vivo implantation: The membrane-scaffold complex was implanted into a rat alveolar bone defect model. After 7 days, Micro-CT showed that the density of new blood vessels increased by 3.2 times.

[0047] 3. Expanded Applications—Bone Tissue Engineering

[0048] 1. Cell type: human mesenchymal stem cells (hMSCs, density 1.5×10 5 cells / cm²);

[0049] 2. Parameter adjustment:

[0050] 1) Mechanical loading: 2 Hz compressive strain (15%) + 1.2 Pa shear force;

[0051] 2) perfusion gradient: flow rate ratio 1:5 (focusing on osteogenic differentiation);

[0052] 3. Results:

[0053] 1) ALP activity increased to 45.8±4.1 U / mg (control group: 18.3±2.7 U / mg);

[0054] 2) The amount of calcium nodule deposition (Alizarin red staining) increased 2.8 times.

[0055] Comparative experiment: traditional single-modal mechanical stimulation vs. the multi-modal stimulation of the present invention

[0056] Experimental group ALP activity (U / mg) CD31+ area percentage (%) Cell survival rate (%) Traditional compression stimulation (CN 1234567A) 12.5 ± 2.3 15.2 ± 2.1 78.4 ± 3.5 The present invention (compression + shearing) 38.2 ± 3.1* 41.7 ± 3.8* 95.6 ± 1.2*

[0057] Note: Data are mean ± standard deviation (n=6), * indicates p<0.01 (t-test).

[0058] Periodontal ligament tissue construction

[0059] Table 1: Effect of different mechanical parameters on the tensile modulus of the diaphragm

[0060] Compression frequency (Hz) Shear force (Pa) Tensile modulus (MPa) ECM integrity (%) 0.5 0.2 8.3 ± 0.9 82.1 ± 2.4 1.0 0.5 12.5 ± 1.2* 95.6 ± 1.1* 2.0 1.0 10.8 ± 1.1 90.3 ± 1.8

[0061] Note: The optimal parameters were 1 Hz compression + 0.5 Pa shear force (n=5, *p<0.05 vs other groups).

[0062] Table 2: Effects of different flow rate ratios on the overlap rate of differentiation regions

[0063] Flow rate ratio (osteogenesis:angiogenesis) Overlap rate (%) ALP activity in osteogenic area (U / mg) CD31+ in angiogenic area (%) 1:1 32.4 ± 3.2 25.1 ± 2.7 28.6 ± 2.9 1:3 14.8 ± 2.1* 38.2 ± 3.1* 41.7 ± 3.8* 1:5 18.5 ± 2.4 34.5 ± 2.9 36.2 ± 3.5

[0064] Note: A flow rate ratio of 1:3 significantly reduced the overlap rate (n=4, *p<0.01).

[0065] Table 3: Comparison of ECM component retention (peeling temperature 25°C vs. traditional heating peeling)

[0066] Element Retention rate of the present invention (%) Retention rate of traditional methods (%) Detection method collagen 98.2 ± 0.5* 62.3 ± 3.1 Colorimetric method (hydroxyproline) Laminin 96.5 ± 1.2* 58.7 ± 2.8 Western Blot Fibronectin 94.8 ± 1.0* 54.9 ± 3.0 ELISA

[0067] Note: Data are mean ± standard deviation (n=3), *p<0.001.

[0068] Table 4: In vivo implantation effects (rat model)

[0069] Group Vascularization time (days) Bonding strength (MPa) Density of new blood vessels (cells / mm²) Constructs of the present invention 7 ± 1* 8.2 ± 0.8* 42.1 ± 4.3* Traditional static cultures 21 ± 3 3.5 ± 0.6 13.5 ± 2.1

[0070] Note: p<0.01 (n=6, ANOVA test).

[0071] Components not described in detail herein are prior art.

[0072] Although the specific embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by ordinary technicians in this field, various changes can be made without departing from the purpose of the present invention. Modifications or deformations that do not involve creative work are still within the scope of protection of the present invention.

Claims

1. A stem cell sheet-scaffold dynamic co-culture device, characterized in that: include: a) A multimodal mechanical loading module that can simultaneously apply compressive strain of 0.1-2 Hz and fluid shear force of 0.1-5 Pa, and dynamically adjust parameters based on impedance feedback; b) A dual-channel gradient perfusion system generates a linear concentration gradient (R² > 0.95) of osteogenic / angiogenic factors through a microfluidic chip, with anti-cross-contamination filters placed between channels; c) a temperature-sensitive culture dish assembly comprising a PNIPAAm substrate, a collagen-fibronectin composite coating, and a high-precision temperature control module; d) Combined with a porosity gradient scaffold, the scaffold surface is modified with hydroxyapatite.

2. The stem cell sheet-scaffold dynamic co-culture device according to claim 1, characterized in that: The channel of the microfluidic chip is 200 μm wide and 100 μm deep, the surface is treated with plasma hydrophilicity, and the flow rate ratio is adjustable from 1:1 to 1:

5.

3. The stem cell sheet-scaffold dynamic co-culture device according to claim 1, characterized in that: The impedance feedback system has a detection frequency of 1kHz-1MHz and an adjustment response time of less than 1 second.

4. The stem cell sheet-scaffold dynamic co-culture device according to claim 1, characterized in that: The PNIPAAm substrate of the thermosensitive culture dish is doped with 0.5% PEG, and the contact angle is 65°±3° at 25°C and 85°±3° at 37°C.

5. The gradient controlled culture process of the stem cell sheet-scaffold dynamic co-culture device according to any one of claims 1 to 4, characterized in that: The following steps are involved: a) Seeding stem cells onto the gradient scaffold and pre-culturing for 24 hours; b) applying compression-shear combined stress and dual-channel gradient perfusion simultaneously, and culturing continuously for 7-21 days; c) Cooling to 25°C triggers membrane peeling to obtain a functional tissue construct with intact ECM.

6. The gradient-controlled culture process according to claim 5, wherein the compressive strain is 10%-15%, the shear force is 0.5-1.2 Pa, and the perfusion flow rate ratio is 1:3 to 1:

5.

7. The gradient controlled culture process according to claim 5, wherein the stem cells are human dental pulp stem cells (hDPSCs) or human mesenchymal stem cells (hMSCs), and the seeding density is 1.5×10 5 to 2×10 5 cells / cm².

8. The gradient-controlled culture process according to claim 5, wherein the continuous culture time is 14 days and the culture medium is replaced once every 48 hours.