A microfluidic organoid continuous perfusion culture device and method based on low-frequency sound waves

Abstract

This invention relates to the field of microfluidic cell culture and biomedical engineering, and discloses a microfluidic organoid continuous perfusion culture device and method based on low-frequency acoustic waves. The device includes a microfluidic chip, fluid drive and acoustic drive components; the chip is composed of a channel layer, a thin film layer, and a rigid substrate layer stacked together. The channel layer has microfluidic channels and liquid storage grooves, and the thin film layer has through-culture wells. The two are vertically aligned and sealed at the bottom by the substrate layer, forming a stepped chamber with liquid storage / bubble trapping at the top and culture at the bottom. Acoustic wave elements are located on the lower surface of the substrate layer and correspond to the culture wells, coupling low-frequency acoustic waves to the culture area, causing dispersed cells to rapidly accumulate into a central cell cluster. The method includes pre-filling with liquid, sample injection, positioning, acoustic enrichment, static stabilization, and continuous perfusion culture. This invention can reduce bubble interference, improve cluster density and shear resistance, and is suitable for tumor organoid construction, drug screening, and personalized medicine research.

Description

Technical Field

[0001] This invention relates to the fields of microfluidics, acoustic manipulation, three-dimensional cell culture and biomedical engineering, and specifically to a microfluidic organoid continuous perfusion culture device and method based on low-frequency sound waves. Background Technology

[0002] Organoids are three-dimensional cellular structures formed in vitro from stem cells, tumor cells, or primary cells. They can mimic the structural features, cell interactions, and functional responses of tissues and organs to a certain extent, and have important application value in disease modeling, drug screening, precision medicine, and regenerative medicine research.

[0003] Microfluidic chips can regulate fluid flow rate, nutrient supply, metabolite removal, and drug concentration gradients within a microscale space, providing a stable, low-consumption, and image-enabled in vitro culture platform for organoid culture. However, existing microfluidic organoid culture devices still suffer from problems such as air bubbles easily entering the culture zone, slow cell aggregation due to natural sedimentation, insufficient density and uniformity of the aggregates, and easy drift or disintegration of cell aggregates under continuous perfusion.

[0004] Acoustic manipulation enables non-contact manipulation of particles and cells through acoustic radiation or acoustic flow. However, existing acoustic fluid control schemes are mostly used for short-term sorting, capture, or mixing, and rarely integrate acoustic enrichment, bubble suppression structures, and continuous perfusion culture processes into a device suitable for long-term organoid culture. Therefore, it is necessary to provide a microfluidic organoid continuous culture device and method that takes into account rapid aggregation, bubble suppression, low-shear perfusion, and in-situ observation. Summary of the Invention

[0005] The purpose of this invention is to provide a microfluidic organoid continuous perfusion culture device and method based on low-frequency sound waves, so as to solve the problems of low cell aggregation efficiency, large bubble interference, poor continuous perfusion stability and inconvenience of in situ observation in the prior art.

[0006] To achieve the above objectives, the device provided by this invention includes a microfluidic chip assembly, a fluid drive assembly, and an acoustic drive assembly. The microfluidic chip assembly includes a channel layer, a thin film layer, and a rigid substrate layer. Microfluidic channels and liquid storage grooves are provided in the channel layer, and through-culture wells are provided in the thin film layer. The liquid storage grooves correspond vertically to the culture wells, and the bottom of the culture wells is sealed by the rigid substrate layer, thereby forming a stepped culture chamber with an upper liquid storage / bubble trapping space and a lower culture space. An acoustic wave generating element is disposed on the lower surface of the rigid substrate layer and corresponds to the culture well, enabling low-frequency acoustic waves to couple to the culture well region through the rigid substrate layer.

[0007] The method provided by this invention includes prefilling with a solution, sample introduction, positioning, acoustic enrichment, static stabilization, and continuous perfusion culture steps. After cells enter the culture wells, they rapidly accumulate in the central region of the culture wells under the action of low-frequency sound waves to form a central cell cluster; subsequently, the sound field is turned off and the cells are statically stabilized to enhance cell-cell adhesion; finally, they are cultured under low-shear continuous perfusion conditions to form three-dimensional organoids.

[0008] Compared with the prior art, the present invention has at least the following beneficial effects: First, the stepped culture chamber achieves bubble retention and culture medium buffering through the upper expansion space, significantly reducing the probability of bubbles entering the lower culture area; Second, low-frequency acoustic enrichment can promote the formation of dense cell clusters in a short time, improving clustering efficiency and consistency; Third, the static stabilization step can improve the shear resistance of the clusters before continuous perfusion, reducing the risk of disintegration and drift caused by subsequent perfusion; Fourth, the device has a simple structure, is easy to integrate with an injection pump, imaging module and signal generator, and can be used for tumor organoid construction, drug screening and personalized medicine research. Attached Figure Description

[0009] Figure 1 This is an exploded structural diagram of the overall culture device of the present invention.

[0010] Figure 2 This is a side view of the assembly structure of the overall culture device of the present invention.

[0011] Figure 3 This is a layered exploded view of the microfluidic chip assembly of the present invention.

[0012] Figure 4 This is a schematic diagram of the planar structure of the flow channel layer.

[0013] Figure 5 This is a schematic diagram of the longitudinal section of the stepped culture chamber and a schematic diagram of the bubble retention path.

[0014] Figure 6 This is a schematic diagram showing the coaxial coupling relationship between the acoustic wave generating element, the rigid substrate, and the culture well.

[0015] Figure 7 The impedance-frequency spectrum of the PZT4 piezoelectric ring is shown.

[0016] Figure 8 Comparison of enrichment effects of microspheres of different sizes under different driving frequencies.

[0017] Figure 9 A comparison chart showing the enrichment effect under different driving voltages.

[0018] Figure 10 This is a comparison chart of chip temperatures under different driving voltages.

[0019] Figure 11 A comparison diagram showing the enrichment adaptability of microspheres of different concentrations and sizes.

[0020] Figure 12 This is a comparison chart of the stability of microspheres under continuous infusion conditions.

[0021] Figure 13 Images showing the dynamic aggregation process of lung cancer cells in culture wells.

[0022] Figure 14 This is a comparison of cell cluster stability under continuous perfusion conditions.

[0023] Figure 15 This is a comparison of cell activity before and after enrichment.

[0024] Figure 16 This is a comparison of organoid morphology and live / dead staining results after 5 days of continuous culture.

[0025] Explanation of reference numerals in the attached figures 1-Injection pump; 2-Defoamer; 3-Liquid delivery tube; 4-Chip fixing fixture; 5-Microfluidic chip; 51-Flow channel layer; 511-Microfluidic channel; 512-Reservoir groove; 52-Thin film layer; 521-Cultivation well; 53-Rigid substrate layer; 54-Acoustic wave generating element; 6-Optical imaging module; 7-Reflector; 8-Outer shell base; 9-Waste liquid cylinder; 10-Signal generator; 11-Signal amplifier. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the following embodiments. The parameters, materials and steps in the embodiments are used to illustrate the technical principles and feasibility of the present invention. Without departing from the essence of the present invention, those skilled in the art can make various substitutions, equivalent changes or combinations thereof.

[0027] Example 1: Overall Device Structure. The microfluidic organoid continuous perfusion culture device of this example is as follows: Figures 1 to 2 As shown, the system includes a syringe pump 1, a defoamer 2, a fluid delivery tube 3, a chip holder 4, a microfluidic chip 5, an optical imaging module 6, a reflector 7, a housing base 8, a waste liquid tank 9, a signal generator 10, and a signal amplifier 11. The syringe pump 1 provides a controllable flow rate, the defoamer 2 reduces the air bubble load in the input fluid, the microfluidic chip 5 is mounted within the chip holder 4, and the optical imaging module 6, in conjunction with the reflector 7, enables in-situ observation of the bottom of the culture well region. An acoustic wave generator 54 is connected to the signal generator 10 and the signal amplifier 11 via wires to receive AC drive signals.

[0028] Example 2: Microfluidic chip structure. For example... Figures 3 to 6As shown, the microfluidic chip 5 adopts a four-layer structure, consisting of a flow channel layer 51, a thin film layer 52, a rigid substrate layer 53, and an acoustic wave generating element 54 from top to bottom. Microfluidic channels 511 are provided within the flow channel layer 51, and liquid storage grooves 512 are provided at positions corresponding to the culture sites. Culture pores 521 are provided on the thin film layer 52, corresponding vertically to the liquid storage grooves 512, and the bottom is sealed by the rigid substrate layer 53, thus forming a stepped culture chamber. Preferably, the lateral dimension of the liquid storage groove 512 is not less than the lateral dimension of the culture pore 521, so as to form an expansion space in the upper part for liquid storage and bubble trapping; the culture pores 521 are located in the lower part, serving as the main space for cell sedimentation, enrichment, and development.

[0029] Preferably, the height of the microfluidic channel is 0.1 mm-1.0 mm, and the width of the microfluidic channel is 0.5 mm-10 mm; the equivalent diameter of the culture well is 0.5 mm-8 mm, and the thickness of the film layer is 0.1 mm-2.0 mm. The equivalent lateral dimension D1 of the liquid storage groove and the equivalent lateral dimension D2 of the culture well satisfy D1 / D2=α, and 1.0≤α≤3.0.

[0030] Example 3: Acoustic Mechanism and Theoretical Explanation. In this invention, the low-frequency sound waves generated by the sound wave generating element 54 are coupled into the culture well region through the rigid substrate layer 53, forming an acoustic radiation force around the cells. For a cell or particle with radius a, the dominant acoustic radiation force it experiences in a one-dimensional standing wave field can be simplified as follows: F_rad = 4πa^3kE_acΦ sin(2kx) Where k is the wavenumber, E_ac is the average energy density of the acoustic field, Φ is the acoustic contrast factor, and x is the position of the particle relative to the acoustic pressure node. When the acoustic contrast factor between the cell and the culture medium is positive, the cells tend to aggregate towards the acoustic pressure node, thus forming a dense cell cluster in the central region of the culture well. Meanwhile, the resistance of the cells in the fluid can be approximated as: F_d = 6πμau Where μ is the dynamic viscosity of the culture medium, and u is the velocity of the cell relative to the fluid. When the acoustic radiation force is greater than the viscous resistance, cells are more likely to detach from their random dispersion state and migrate to a stable aggregation location. Therefore, this invention, by constructing a stable low-frequency acoustic field in the culture well region, facilitates rapid cell enrichment in a short time.

[0031] The wavelength of sound waves in a liquid medium can be estimated using the following formula: λ = c / f Where c is the velocity of sound in the liquid phase, and f is the driving frequency. Assuming the velocity of sound in the culture medium is approximately 1500 m / s, the theoretical wavelength is approximately 15.15 mm when f is 99 kHz; and approximately 14.15 mm when f is 106 kHz. Since the actual system consists of a piezoelectric element, a rigid substrate, a PDMS structure, and liquid coupling, the optimal operating frequency does not necessarily coincide perfectly with the electrical resonant frequency of the piezoelectric element. Therefore, it is necessary to combine impedance testing and enrichment experiments to determine the optimal operating point.

[0032] Example 4: Fluid and Shear Conditions Explanation. The rapid injection and low-speed positioning steps of this invention correspond to two stages: a higher flow rate and a lower flow rate, respectively. For an approximately rectangular microchannel with width b and height h, the wall shear stress level can be estimated by the following formula: τ ≈ 6μQ / (bh^2) Where Q is the volumetric flow rate and μ is the dynamic viscosity of the culture medium. Based on the preferred dimensions disclosed in this invention (b≈4 mm, h≈0.2 mm, μ≈1.0×10⁻³ Pa·s), the wall shear stress level is approximately 3.13×10⁻² Pa when Q is 50 μL / min; and approximately 6.25×10⁻⁴ Pa when Q is reduced to 1 μL / min. These results indicate that the shear stress during the low-speed positioning and continuous perfusion stages is significantly lower than that during the rapid injection stage, which helps reduce the disturbance to the formed cell clusters and theoretically supports the process design of "acoustic enrichment followed by static stabilization and then continuous perfusion."

[0033] Example 5: Chip Fabrication and Assembly. Preferably, both the flow channel layer 51 and the thin film layer 52 are made of polydimethylsiloxane, and the rigid substrate layer 53 is made of an ultrathin coverslip to balance biocompatibility, light transmittance, and sound transmission efficiency. The acoustic wave generating element 54 can be a PZT4 piezoelectric ceramic ring, which is bonded to the lower surface of the rigid substrate layer 53 with epoxy resin adhesive. Before assembly, each layer of the chip can be cleaned, subjected to plasma surface treatment, and aligned and bonded to form a sealed integral structure.

[0034] Example 6: Organoid Culture Process. Step S1, Pre-filling: Inject 50 μL of culture medium through the chip inlet using a pipette to wet the channels and remove initial air. Step S2, Rapid Injection: Inject 100 μL of cell suspension into the chip using a syringe pump at a flow rate of 50 μL / min. Step S3, Low-speed Positioning: Reduce the flow rate to 1 μL / min to allow cells to slowly enter the culture wells and settle to the lower culture space. Step S4, Acoustic Enrichment: Use an external signal generator to drive a PZT4 piezoelectric ring, applying a 99 kHz, 40 Vpp AC signal for 2 min to rapidly enrich the dispersed cells in the culture wells, forming a central cell cluster. Step S5, Stabilization: Turn off the acoustic field and stop perfusion, allowing the cells to stabilize for 3 h to enhance cell-cell adhesion, cluster integrity, and shear resistance. Step S6, continuous perfusion culture: Place the device in a cell culture environment and continuously perfuse fresh culture medium at a flow rate of 1 μL / min, so that the enriched cell clusters gradually develop into three-dimensional organoids.

[0035] Example 7: Parameter optimization and experimental observation results. For example... Figures 7 to 12 As shown, based on existing experimental results, the impedance-frequency spectrum of the PZT4 piezoelectric ring indicates that its electrical resonance peak is located at approximately 106 kHz. When enriching microspheres at three frequencies—90 kHz, 99 kHz, and 106 kHz—the aggregates formed at 99 kHz were the densest and had the clearest outlines, indicating a shift between the optimal operating point of the chip-liquid coupling system and the electrical resonance peak of the bare piezoelectric element. Further comparison of four voltages (16 Vpp, 32 Vpp, 40 Vpp, and 56 Vpp) at 99 kHz shows that 40 Vpp balances high enrichment efficiency with low temperature rise risk. For microsphere or cell concentrations in the range of 1000-20000 cells / mL, this invention exhibits good aggregation adaptability.

[0036] Example 8: Validation of stability and activity during continuous perfusion. Figures 13 to 15 As shown, after acoustic enrichment of lung cancer cells at 99 kHz, 40 Vpp, and 2 min, the cells rapidly formed central clumps and exhibited higher structural stability after a subsequent 3 h resting period. Under continuous perfusion conditions of 1 μL / min, the microspheres formed by acoustic enrichment did not show significant disintegration or drift within 30 min. Immediate live-cell staining after enrichment revealed strong green fluorescence, indicating that the acoustic manipulation did not cause significant cell damage.

[0037] Example 9: Long-term organoid culture. For example... Figure 16As shown, after 5 days of continuous perfusion culture, the acoustic enrichment group formed more dense and clearly defined three-dimensional organoid masses compared to the non-enriched group. The live and dead staining results showed a higher proportion of live cells, proving that the device and method described in this invention are beneficial for obtaining highly active organoid models.

Claims

1. A microfluidic organoid continuous perfusion culture device based on low-frequency acoustic waves, characterized in that, The device includes a microfluidic chip assembly, a fluid drive assembly connected to the microfluidic chip assembly, and an acoustic drive assembly acoustically coupled to the microfluidic chip assembly. The microfluidic chip assembly includes a channel layer, a thin film layer, and a rigid substrate layer stacked sequentially from top to bottom. The channel layer has microfluidic channels and liquid reservoirs corresponding to culture sites. The thin film layer has through-hole culture pores. The liquid reservoirs correspond vertically to the culture pores, and the bottom of the culture pores is sealed by the rigid substrate layer, so that the liquid reservoirs and the culture pores together form a stepped culture chamber. The stepped culture chamber includes a liquid reservoir / bubble trapping space at the top and a culture space at the bottom. The acoustic drive assembly includes a sound wave generating element disposed on the lower surface of the rigid substrate layer. The sound wave generating element corresponds vertically to the culture pores to couple low-frequency sound waves through the rigid substrate layer into the culture pores, so that the dispersed cells entering the culture pores are enriched into clusters for continuous perfusion culture.

2. The apparatus according to claim 1, characterized in that, The equivalent lateral dimension D1 of the liquid storage groove and the equivalent lateral dimension D2 of the culture well satisfy D1 / D2=α, and 1.0≤α≤3.0; preferably, 1.05≤α≤2.0; the volume V1 of the upper liquid storage / bubble interception space and the volume V2 of the lower culture space satisfy 0.5≤V1 / V2≤10, so that the bubbles entering the stepped culture chamber are preferentially retained in the upper liquid storage / bubble interception space.

3. The apparatus according to claim 1, characterized in that, The acoustic wave generating element is a piezoelectric ceramic sheet, a piezoelectric ceramic ring, or a piezoelectric composite vibrator; when the acoustic wave generating element is a piezoelectric ceramic ring, the central hole axis of the piezoelectric ceramic ring is coaxial or nearly coaxial with the central axis of the culture well.

4. The apparatus according to claim 1, characterized in that, The operating frequency f of the sound wave generating element is 20kHz-500kHz, preferably 70kHz-150kHz, and more preferably 90kHz-110kHz; when the electrical resonant frequency of the sound wave generating element is fr, the operating frequency f satisfies f=γfr, where 0.80≤γ≤1.10, preferably 0.90≤γ≤1.

00.

5. The apparatus according to claim 1, characterized in that, The microfluidic chip assembly has two or more culture units spaced apart from each other. Each culture unit includes a liquid storage groove, a culture well, and an acoustic wave generating element corresponding to the culture well. Multiple culture units can be arranged in series along the same microfluidic channel or connected to independent microfluidic channels respectively.

6. The apparatus according to claim 1, characterized in that, The flow channel layer and / or the thin film layer are made of an elastic polymer material, including polydimethylsiloxane; the rigid substrate layer is a glass substrate, cover glass, quartz sheet or other transparent biocompatible rigid substrate; the flow channel layer, thin film layer and rigid substrate layer are sealed and connected by plasma treatment, adhesive bonding, hot pressing or mechanical clamping.

7. The apparatus according to claim 1, characterized in that, The fluid drive assembly includes an injection pump, a peristaltic pump, or a pressure-driven pump; the device also includes a defoamer disposed on the fluid input side, a waste liquid collection unit disposed on the fluid output side, a chip fixing fixture for fixing the microfluidic chip assembly, a signal generator and / or power amplifier electrically connected to the acoustic wave generating element, and an optical imaging module for observing the culture well region.

8. A method for continuous perfusion culture of organoids using the apparatus according to any one of claims 1 to 7, characterized in that, Includes the following steps: S1, Introduce culture medium into the microfluidic chip assembly, expel initial air from the microfluidic channels, and wet the channels; S2, Introduce a cell suspension containing dispersed cells into the microfluidic chip assembly; S3, Reduce the flow rate to allow cells in the cell suspension to enter and settle into the culture wells; S4, Apply an AC drive signal to the acoustic wave generator to couple low-frequency acoustic waves to the culture well region, causing the dispersed cells to enrich into central cell clusters within the culture wells; S5, Turn off the low-frequency acoustic waves and stop or reduce perfusion, allowing the central cell clusters to stabilize within the culture wells; S6, Supply culture medium to the microfluidic chip assembly at a continuous perfusion flow rate, allowing the central cell clusters to develop into three-dimensional organoids within the culture wells.

9. The method according to claim 8, characterized in that, In step S4, the AC drive signal frequency is 70 kHz-150 kHz, the voltage is 5 Vpp-80 Vpp, and the action time is 0.5 min-10 min; preferably, the AC drive signal frequency is 99 kHz, the voltage is 40 Vpp, and the action time is 2 min; in step S5, the settling time is 0.5 h-6 h, preferably 3 h; in step S6, the continuous perfusion flow rate is 0.1 μL / min-10 μL / min, preferably 1 μL / min.

10. The method according to claim 8, characterized in that, In step S6, the wall shear stress level τp during the continuous perfusion stage satisfies τp≈6μQp / (bh²), where μ is the dynamic viscosity of the culture medium, Qp is the continuous perfusion flow rate, b is the microfluidic channel width, and h is the microfluidic channel height; and τp is less than 1 / 10 of the wall shear stress level τs during the rapid injection stage in step S2; in step S4, the acoustic radiation force Frad and viscous resistance Fd experienced by the cells satisfy Frad / Fd≥1, where Frad=4πa³kEacΦsin(2kx), Fd=6πμau.

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