PDMS (Polydimethylsiloxane) micro-fluidic chip for long-term culture of living cells and preparation method of PDMS micro-fluidic chip

Abstract

The invention discloses a PDMS (polydimethylsiloxane) micro-fluidic chip for long-term culture of living cells and a preparation method thereof, and relates to the technical field of micro-fluidic cell culture.The PDMS micro-fluidic chip comprises a PDMS main body, and at least one culture flow channel is arranged in the PDMS main body; the culture flow channel is provided with a fluid inlet and a fluid outlet; according to the PDMS micro-fluidic chip for long-term culture of living cells and the preparation method of the PDMS micro-fluidic chip, the mechanical microenvironment of an in-vivo tissue vasculature is effectively simulated, and meanwhile, the design of an open pool with a one-way valve at the tail end is combined, so that automatic removal of bubbles, stable maintenance of system pressure and low-damage convenient recovery of cell samples are realized; therefore, a plurality of key steps of cell capture, long-term culture, sample acquisition and the like are integrated into a simplified and reliable operation process, and the defects of complicated operation, high dependency on auxiliary equipment and insufficient microenvironment uniformity are overcome.

Description

Technical Field

[0001] This invention relates to the field of microfluidic cell culture technology, specifically to a PDMS microfluidic chip for long-term culture of live cells and its preparation method. Background Technology

[0002] In vitro cell culture is a key technology in biomedical research, drug screening, and toxicity testing. Traditional two-dimensional cell culture relies primarily on culture dishes or plates. While this method is simple to operate, it struggles to simulate the three-dimensional microenvironment in vivo and the interactions between cells and the matrix, leading to significant differences in physiological responses compared to real tissues. Furthermore, two-dimensional culture typically requires large cell volumes and culture reagents, resulting in high costs and making long-term, dynamic in-situ monitoring difficult. In recent years, microfluidic technology has provided a new platform for cell culture. Through micrometer-scale fluid control and microstructure design, microfluidic chips can construct three-dimensional culture environments that more closely resemble physiological conditions and achieve precise control of culture parameters. In particular, polydimethylsiloxane (PDMS) material, due to its excellent biocompatibility, permeability, and optical transparency, is widely used in constructing cell culture chips.

[0003] However, existing microfluidic chips for long-term live cell culture still have some limitations. For example, patent publication CN108485972B describes a microfluidic chip and its method of use for cell and tissue culture and real-time monitoring. This chip achieves dielectrophoretic capture and electrical impedance spectroscopy monitoring through integrated microelectrodes, and facilitates cell aggregation and release by switching between vertical and horizontal chip placement. While this chip has certain advantages in cell manipulation and real-time detection, its structure relies on specific orientation and gravity assistance, making the operation process relatively complex. Furthermore, during long-term culture, cell retention and nutrient exchange efficiency are still limited by the microfluidic channel structure. In addition, there is still room for improvement in the scalability of culture units, adaptability to multi-condition parallel culture, and convenient acquisition of cell samples later. Therefore, there is still a need to develop a PDMS microfluidic chip with a simpler structure, suitable for long-term stable live cell culture, supporting multi-channel parallel experiments, and facilitating sample recovery to meet the needs of high-throughput drug screening, tissue engineering, and cell behavior research. Summary of the Invention

[0004] The purpose of this invention is to provide a PDMS microfluidic chip for long-term culture of live cells and its preparation method, so as to solve the problems mentioned in the background art.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a PDMS microfluidic chip for long-term culture of live cells, comprising:

[0006] A PDMS body, wherein at least one culture channel is provided within the PDMS body;

[0007] The culture channel has a fluid inlet and a fluid outlet;

[0008] The culture channel includes at least one main chamber connected in series. When there are two or more main chambers, adjacent main chambers are connected by a first connecting channel.

[0009] The culture channel also includes an open pool located downstream of the last main chamber and connected to the fluid outlet.

[0010] Furthermore, the outline of the main chamber is elliptical;

[0011] The length of the first connecting channel is less than the major axis dimension of the main chamber;

[0012] The cross-sectional area of ​​the first connecting channel is smaller than that of the main chamber; microchannels may be added between the main chamber and the fluid inlet, the main chamber and the fluid outlet or open pool, and the cross-sectional area of ​​the microchannels is smaller than that of the first connecting channel, for precise control of the culture medium flow rate.

[0013] Furthermore, at least one satellite retention chamber is connected to the side wall of at least one of the main chambers;

[0014] The satellite retention chamber is connected to the main chamber via a second connecting channel;

[0015] The cross-sectional area of ​​the second connection channel is smaller than that of the first connection channel.

[0016] Furthermore, the open pool is a hemispherical chamber with a volume larger than that of the main chamber;

[0017] The top of the open pool has an opening that connects to the outside atmosphere;

[0018] A one-way valve structure is provided at the connection between the open pool and the last main chamber;

[0019] The one-way valve structure is integrally formed from PDMS material, which can remain open or slightly open when the fluid flows in the culture channel in the forward direction, and increase the degree of opening when negative pressure is applied from the open pool side.

[0020] Furthermore, the PDMS body has a bonding surface; the area of ​​the PDMS body corresponding to the main chamber is a hollow structure, and a quartz glass substrate is bonded to the bonding surface. The quartz glass substrate covers the hollow area of ​​the main chamber, and the area corresponding to the main chamber is a light-transmitting area, which can reduce the interference of PDMS material on optical detection signals such as Raman spectroscopy.

[0021] On the bonding surface, the surface hydrophilicity corresponding to the main chamber region is stronger than the surface hydrophilicity corresponding to the first connection channel region;

[0022] The surface hydrophilicity is achieved through differentiated surface treatment processes.

[0023] Furthermore, the differentiated surface treatment process includes:

[0024] The entire bonding surface is subjected to oxygen plasma treatment under first conditions;

[0025] Subsequently, the region corresponding to the main chamber is subjected to oxygen plasma treatment under the second condition;

[0026] Wherein, the processing power of the second condition is greater than the processing power of the first condition, and / or the processing time of the second condition is longer than the processing time of the first condition.

[0027] Furthermore, following the differentiated surface treatment, at least a portion of the bonding surface is coated with a polylysine coating.

[0028] Furthermore, a self-sealing structure is provided at the fluid inlet;

[0029] The self-sealing structure can be punctured by a syringe needle and form a sealed connection.

[0030] A method for fabricating a PDMS microfluidic chip for long-term live cell culture, comprising the following steps:

[0031] S1: Provide a mold with microstructure protrusions, the microstructure protrusions corresponding to the negative structure of the culture channel and open cell;

[0032] S2: Pour the PDMS prepolymer onto the mold, and after curing, demold to obtain a PDMS layer with a microchannel structure;

[0033] S3: Perform surface modification treatment on the bonding surface of the PDMS layer;

[0034] S4: The surface-modified PDMS layer is bonded to a quartz glass substrate to form a closed microchannel. The quartz glass substrate precisely corresponds to the hollowed-out area of ​​the main cavity of the PDMS layer, which is used to adapt to optical inspection scenarios.

[0035] Furthermore, the surface modification treatment of the bonding surface of the PDMS layer includes:

[0036] First, the entire bonding surface is subjected to a first-stage oxygen plasma treatment with the first power and the first time.

[0037] Then, the region corresponding to the non-main chamber on the bonding surface is covered with a mask, and the exposed region corresponding to the main chamber is subjected to a second-stage oxygen plasma treatment with a second power and a second time, wherein the second power is greater than the first power and / or the second time is longer than the first time.

[0038] Next, the entire PDMS layer was immersed in a poly-L-lysine solution, incubated, and then dried. Poly-L-lysine modification can be achieved in three ways: Method 1: Immerse the entire PDMS layer in a poly-L-lysine solution, incubate at 20-37°C for 30 minutes to 2 hours, and then dry. Method 2: Immerse the bonding surface of a quartz slide in a poly-L-lysine solution with a concentration of 0.01 mg / mL-0.1 mg / mL (pH 7.4-8.5), incubate at 37°C for 1 hour, dry, and then bond it to the surface-modified PDMS layer. Method 3: After the PDMS layer and quartz slide have bonded to form a closed microchannel, inject the above-mentioned poly-L-lysine solution into the chip, incubate for 1 hour, aspirate the solution, and place the chip in a fume hood to air dry for later use. In all three methods, the incubation target is the bonding surface of PDMS and quartz slide, and all methods can achieve the formation of a bioactive coating to meet the requirements of cell adhesion.

[0039] This invention provides a PDMS microfluidic chip for long-term live cell culture and its preparation method. It has the following beneficial effects:

[0040] This PDMS microfluidic chip for long-term live cell culture and its fabrication method spontaneously generate a spatially heterogeneous fluid shear force distribution and mass exchange gradient within the chip by utilizing the fluid's own dynamic behavior, without relying on external complex force fields or physical flipping operations. This structure effectively simulates the mechanical microenvironment of the in vivo tissue vascular system. Combined with an open pool design with a one-way valve at the end, it achieves automatic bubble removal, stable system pressure maintenance, and low-damage, convenient recovery of cell samples. This integrates multiple key steps such as cell capture, long-term culture, and sample acquisition into a simplified and reliable operating procedure, overcoming the shortcomings of cumbersome operation, high dependence on auxiliary equipment, and insufficient microenvironment uniformity.

[0041] This PDMS microfluidic chip for long-term live cell culture and its fabrication method utilize gradient surface engineering combining mask-assisted partitioned oxygen plasma treatment and poly-L-lysine modification to construct a spatially selective hydrophilic and bioactive interface on a PDMS substrate. This surface characteristic not only guides cells to preferentially attach to the target culture area, improving cell utilization and distribution control, but also effectively enhances the wetting and retention of the culture medium within the chip, mitigating local evaporation and providing a chemically stable, osmotically balanced microenvironment for long-term cell viability and function. The chip's structural design supports parallel and array-based expansion, with each culture unit being independent and its microenvironment controllable, providing an efficient and stable platform for high-throughput drug screening and multi-condition controlled cell biology research. Attached Figure Description

[0042] Figure 1 This is a data flow diagram of a PDMS microfluidic chip for long-term culture of live cells and its preparation method according to the present invention;

[0043] Figure 2 This image shows a comparison of the surface treatment effects of a PDMS microfluidic chip for long-term culture of live cells and its preparation method, as described in this invention. Detailed Implementation

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Please see Figure 1 and Figure 2 This invention provides a technical solution: a PDMS microfluidic chip for long-term culture of live cells, comprising:

[0046] The PDMS body contains at least one culture channel.

[0047] The culture channel has a fluid inlet and a fluid outlet;

[0048] The culture channel includes at least one main chamber connected in series. When there are two or more main chambers, adjacent main chambers are connected by a first connecting channel.

[0049] The culture channel also includes an open pool located downstream of the last main chamber and connected to the fluid outlet.

[0050] It should be further explained that a PDMS microfluidic chip for long-term live cell culture has at least one culture channel within its PDMS body. This culture channel has a fluid inlet and a fluid outlet. The culture channel includes at least one main chamber connected in series. When there are two or more main chambers, adjacent main chambers are connected by a first connecting channel. The culture channel also includes an open pool located downstream of the last main chamber and connected to the fluid outlet. During implementation, the number of main chambers can be flexibly adjusted according to experimental needs. When only a single culture microenvironment is required, one main chamber can be set up, and the first connecting channel between the main chambers can be omitted, directly connecting the fluid inlet, main chamber, and fluid outlet through the microfluidic channel. When multi-condition parallel experiments or the construction of gradient mechanical microenvironments are required, three or more main chambers can be set up, connected in series by the first connecting channel to form differentiated culture regions, balancing the needs of single-system and high-throughput experiments.

[0051] In practice, the main chamber is designed with an elliptical outline. The length of the first connecting channel is smaller than the major axis of the main chamber, and its cross-sectional area is also significantly smaller than that of the main chamber. This periodic expansion and contraction flow channel structure, under low flow rate perfusion, such as 1 to 5 microliters per minute, can form a low-speed vortex in the central region and an effective material exchange in the near-wall region in each main chamber. This allows for the spontaneous formation of a progressive fluid shear force distribution without the need for an external mixing device, providing cells with heterogeneous stimulation that mimics the mechanical microenvironment at the branching points of blood vessels in vivo.

[0052] The open chamber is designed as a hemispherical chamber with a volume larger than the main chamber. Its top has an opening that communicates with the atmosphere. This opening serves as the system's atmospheric pressure point, effectively eliminating pressure fluctuations caused by thermal expansion and contraction of the fluid or trace evaporation, ensuring long-term stability of the perfusion flow rate. The connection between the open chamber and the final main chamber is integrally formed with a thin-walled structure made of PDMS material. This structure remains open under normal perfusion pressure within the flow channel, allowing culture medium to flow into the open chamber and exit from the outlet. When a slight negative pressure is applied from the opening of the open chamber using a pipette, the thin-walled structure deforms, increasing the degree of opening. This allows cells or tissue pellets cultured in the chamber to be completely and minimally damaged, and can be aspirated and recovered. This design replaces the existing cell release methods that rely on physical flipping of the chip or complex external force, simplifying the operation and avoiding sample damage.

[0053] The surface hydrophilicity of the bonding surface of the PDMS chip is differentiated. Specifically, the surface hydrophilicity corresponding to the main chamber region is significantly stronger than that corresponding to the connecting channel region through longer treatment time or higher power oxygen plasma treatment. This gradient hydrophilicity makes it easier for cell suspension to selectively wet and remain in the main chamber region during perfusion, improving cell seeding efficiency. At the same time, the strongly hydrophilic chamber surface is conducive to maintaining a stable liquid film during long-term culture and reducing the evaporation rate of the culture medium.

[0054] The entire chip organically combines a series vortex chamber structure, an open cell at the end with atmospheric pressure, and a gradient hydrophilic surface to construct a biomimetic culture microenvironment that can autonomously maintain a stable chemical and mechanical gradient and is easy to operate. This effectively solves the problems of cumbersome operation, uneven microenvironment, and difficult sample recovery in existing closed microfluidic chips during long-term culture.

[0055] The main chamber has an elliptical outline;

[0056] The length of the first connecting channel is less than the major axis dimension of the main chamber;

[0057] The cross-sectional area of ​​the first connecting channel is smaller than that of the main chamber. To further optimize the fluid shear force distribution and culture medium flow rate within the main chamber, microchannels can be added between the main chamber and the fluid inlet, and between the main chamber and the open pool. These microchannels have a smaller cross-sectional area than the first connecting channel, enabling precise control of the flow rate of the culture medium into and out of the main chamber, ensuring the stability of the culture microenvironment. In practical applications, the microchannel between the main chamber and the fluid inlet acts as a buffer, preventing the culture medium from directly impacting the main chamber and causing cell detachment. The microchannel between the main chamber and the open pool slows down the rate at which fluid enters the open pool, ensuring sufficient material exchange within the main chamber without affecting the pressure balance function of the open pool.

[0058] It should be further explained that the main chamber is designed with an elliptical outline, with its major axis dimension ranging from three to eight millimeters, and the minor axis dimension adjusted accordingly to maintain a suitable aspect ratio. The length of the first connecting channel is set to be significantly shorter than the major axis dimension of the main chamber, typically not exceeding half of the major axis dimension, in order to achieve a compact connection between the chambers. At the same time, the cross-sectional area of ​​the first connecting channel is significantly smaller than that of the main chamber, for example, the channel width is 100 to 300 micrometers and the height is 50 to 150 micrometers, while the cross-sectional area of ​​the main chamber is increased by radially expanding its elliptical outline.

[0059] The periodic alternation of elliptical chambers and short, narrow connecting channels guides fluid dynamics during continuous perfusion at low flow rates by alternating changes in the cross-sectional area of ​​the channels: within each elliptical main chamber, the fluid expands, the velocity decreases, and a local vortex region is formed, generating lower fluid shear forces, which is conducive to stable cell adhesion and aggregation; at the narrow first connecting channel, the fluid cross-section contracts, leading to an increase in velocity and generating higher fluid shear forces, promoting efficient exchange of nutrients and metabolic waste.

[0060] By precisely controlling the size ratio of the chambers and channels, this structure can spontaneously form a spatially heterogeneous fluid shear force distribution that resets from low to high in a series of tandem chambers. This simulates the complex mechanical microenvironment at the branching points of the vascular system in vivo, thereby optimizing the chemical gradient and mechanical stimulation in the culture microenvironment without relying on external active mixing or complex flow field control devices. This effectively overcomes the defects of uneven fluid distribution and insufficient material exchange caused by existing straight channel or single-chamber designs, providing a dynamic substrate that is closer to physiological conditions for long-term culture of live cells.

[0061] At least one satellite retention chamber is connected to the side wall of at least one main chamber;

[0062] The satellite retention chamber is connected to the main chamber via a second connecting channel;

[0063] The cross-sectional area of ​​the second connecting channel is smaller than that of the first connecting channel.

[0064] It should be further explained that the satellite retention cavity has an approximately circular or slightly elliptical structure, and is connected and led out from the side wall of the main chamber through a second connecting channel. The cross-sectional area of ​​the second connecting channel is further reduced, with its width set between fifty and one hundred micrometers and its height between thirty and eighty micrometers, making its flow cross-sectional area significantly smaller than that of the first connecting channel. The diameter or main dimensions of this satellite retention cavity are typically designed to be one-third to one-half the minor axis dimension of the main chamber.

[0065] During fluid perfusion, the flow resistance generated by the narrow size of the second connecting channel and capillary action significantly reduce the fluid velocity entering the satellite retention chamber, creating a near-static microenvironment where fluid shear force approaches zero. This sheltered chamber provides cells with a region to avoid the periodic shear forces within the main chamber, facilitating initial cell attachment, natural extracellular matrix deposition, and the stable formation of tight junctions or spherical clusters. This spontaneously constructs a composite microenvironment within a single culture unit, characterized by both heterogeneous active culture and static shelter zones. Through physical separation and flow resistance design, this structure simulates the material exchange and functional partitioning between the main flow channels and collateral nutrient sinuses in biological tissues. It effectively solves the problems of uneven cell distribution, difficulty in forming stable three-dimensional structures, and dead zones due to insufficient nutrient exchange at the edges caused by a single flow field in existing microfluidic chips. This provides a crucial structural basis for observing cell behavior under differentiated mechanical conditions, studying cell-matrix interactions, and achieving long-term three-dimensional culture that more closely resembles in vivo tissues.

[0066] The open pool is a hemispherical chamber with a volume larger than that of the main chamber;

[0067] The top of the open pool has an opening that connects to the outside atmosphere;

[0068] A one-way valve structure is installed at the connection between the open pool and the last main chamber;

[0069] The one-way valve structure is integrally molded from PDMS material, which can remain open or slightly open when the fluid flows in the culture channel in the forward direction, and increase the degree of opening when negative pressure is applied from the open pool side.

[0070] It should be further explained that the open pool is a hemispherical chamber with a diameter between two and four millimeters and a volume larger than that of any main chamber. The top of the open pool has a circular opening with a diameter of 0.5 to 1 millimeter, which is always connected to the outside atmosphere and serves as the atmospheric pressure point of the entire flow channel system.

[0071] At the connection between the open pool and the last main chamber, a thin-walled flexible structure made of PDMS material is integrally molded by a mold, which constitutes a one-way valve; the thickness of the thin-walled structure is between 20 micrometers and 100 micrometers, and its shape is an arc or cantilever beam protruding into the open pool.

[0072] When the culture medium flows in from the fluid inlet and through this connection point under the drive of the injection pump, the fluid pressure causes the thin-walled structure to undergo micro-elastic deformation and remain open or slightly open, ensuring that the fluid flows smoothly into the open cell and is discharged from the fluid outlet. At the same time, this open state allows the pressure fluctuations inside the chip caused by temperature changes or liquid evaporation to be eliminated by balancing with the atmosphere through the opening of the open cell.

[0073] When it is necessary to recover cultured cells or tissues, the pipette tip is aligned with the top opening of the open cell and a slight negative pressure is applied for aspiration. This negative pressure causes the thin-walled structure to deform more towards the main chamber, significantly increasing the gap between it and the opposite sidewall, forming a low-resistance recovery channel that leads directly from the main chamber to the open cell. At this time, the cells or tissue spheres located in the main chamber can be completely and smoothly drawn into the pipette through this increased gap under the negative pressure, completing the non-destructive sample recovery. This integrated one-way valve structure replaces the sample release methods in traditional chips that rely on physical flipping, gravity detachment, or additional complex mechanical structures. It integrates pressure balancing, air bubble removal, and controllable sample recovery functions into a single open cell structure, which not only simplifies the operation steps but, more importantly, avoids the physical damage that may be caused to fragile cells and tissues by flipping or mechanical intervention. It achieves a seamless connection from long-term culture to sample acquisition, solving the prominent contradiction between operational convenience and sample integrity in existing closed microfluidic chips.

[0074] The PDMS body has a bonding surface; to meet the optical detection requirements such as Raman acquisition during cell culture, the main chamber area of ​​the PDMS body is designed as a hollow structure, and the bonding surface is made of quartz glass slide as substrate. The quartz glass slide is directly used as the bottom bearing surface of the main chamber. By utilizing the low optical interference characteristics of quartz material, the Raman signal of the PDMS matrix is ​​avoided from blocking the cell detection signal, thus ensuring the accuracy of optical detection.

[0075] On the bonding surface, the surface hydrophilicity corresponding to the main chamber region is stronger than the surface hydrophilicity corresponding to the first connecting channel region;

[0076] Surface hydrophilicity is achieved through differentiated surface treatment processes.

[0077] It should be further explained that the differentiated control of the surface hydrophilicity of the PDMS bonding surface is achieved through regional and conditional surface treatment processes. The surface corresponding to the main chamber region on the bonding surface exhibits significantly enhanced hydrophilicity after stronger or longer treatment, manifested as a reduced contact angle with water; while the surface corresponding to the first connection channel region maintains relatively weak hydrophilicity. This differentiated characteristic is achieved through the following method: First, before bonding the PDMS layer to the substrate, the entire bonding surface undergoes a first-stage oxygen plasma treatment, for example, treatment at a power of 30 watts for 30 seconds, which imparts basic hydrophilicity to the entire surface.

[0078] Subsequently, a physical mask with precisely corresponding cutouts corresponding to the shape and position of all main chambers is used to cover the bonding surface, while other areas such as the connecting channels are concealed and protected. Next, the exposed main chamber areas undergo a second stage of oxygen plasma treatment, for example, at a power of 60 watts for three minutes. This stronger treatment condition causes more complete oxidation of the PDMS material on the surface of the main chamber areas, forming a denser and more durable hydrophilic layer. After treatment, the mask is removed, revealing a hydrophilic gradient distribution on the bonding surface: the main chamber areas are superhydrophilic with extremely small contact angles; the connecting channel areas are moderately hydrophilic.

[0079] This differentiated hydrophilic surface, during cell seeding, guides the cell suspension to preferentially and stably wet and retain in the target culture zone, i.e., the main chamber, improving cell utilization and controllable distribution. In long-term perfusion culture, the strongly hydrophilic surface of the main chamber better maintains hydration, effectively slowing down the local evaporation rate of the culture medium in this area, thus providing a more stable osmotic pressure and chemical microenvironment for the cells. This surface engineering strategy overcomes the inherent hydrophobicity of PDMS materials and the non-specific adsorption problems within the flow channels that may result from uniform modification. Through spatially selective surface property modulation, it achieves active guidance of cell adhesion behavior and microenvironment stability.

[0080] Differentiated surface treatment processes include:

[0081] The bonding surface is subjected to oxygen plasma treatment under the first condition.

[0082] Then, the region corresponding to the main chamber is subjected to oxygen plasma treatment under the second condition;

[0083] Wherein, the processing power of the second condition is greater than that of the first condition, and / or the processing time of the second condition is longer than that of the first condition.

[0084] It should be further explained that when implementing the differentiated surface treatment process, the PDMS layer that has completed microstructure forming but has not yet bonded is first placed in the chamber of the oxygen plasma treatment equipment with the bonding surface with the microchannel structure facing upwards; oxygen plasma treatment under the first condition is carried out, with the treatment power ranging from 20 watts to 40 watts and the treatment time ranging from 20 seconds to 60 seconds. This step enables the PDMS bonding surface to obtain basic and uniform hydrophilic modification.

[0085] Subsequently, a pre-prepared mask is tightly bonded to the bonding surface of the PDMS layer. The mask is made of polyimide or metal sheet and has precisely machined cutout patterns that are completely consistent with the shape, size and position of all main chambers in the chip design. The mask portions corresponding to other areas such as connection channels and open cells are solid covers. After the mask is placed and fixed, the PDMS layer together with the mask is placed back into the oxygen plasma processing equipment for the second condition processing. The processing power of the second condition is significantly higher than that of the first condition, ranging from 50 watts to 80 watts, and the processing time is also significantly longer than that of the first condition, ranging from two minutes to five minutes. In this stage, the high-energy plasma can only act on the PDMS surface below through the cutout areas on the mask, that is, the area corresponding to the main chambers, while other covered areas are protected. After these two stages of differentiated treatment, the surface properties of the PDMS bonding surface undergo spatially selective changes: the region corresponding to the main chamber, due to the higher energy and longer oxidation time, has broken siloxane chains on its surface and formed more abundant silanol groups, resulting in greatly enhanced hydrophilicity, manifested as a significant reduction in the water droplet contact angle; while the covered connecting channels and other regions retain only the moderate hydrophilicity imparted by the first stage of treatment.

[0086] This process, through precise control of mask patterns and plasma treatment parameters, directly constructs regionalized surfaces with varying degrees of hydrophilicity on a single PDMS substrate. This non-uniform surface energy distribution actively guides the spreading and wetting behavior of liquids within the flow channels, making cell suspensions more likely to reside in the strongly hydrophilic main chamber. Simultaneously, it reduces non-specific adsorption of cells and proteins in non-target areas. Furthermore, the strongly hydrophilic main chamber surface energy more effectively binds water molecules to form a stable hydration layer, thereby inhibiting excessive evaporation of local liquids during long-term dynamic perfusion culture and maintaining the chemical stability and osmotic pressure balance of the culture microenvironment. This mask-assisted, zoned, and intensity-differentiated plasma treatment method is key to achieving spatial programming of the surface properties of PDMS microfluidic chips. It overcomes the contradiction that traditional monolithic treatment cannot simultaneously achieve selective cell adhesion and long-term culture stability.

[0087] Following the differentiated surface treatment, a polylysine coating is also applied to at least a portion of the bonding surface. It should be further explained that the poly-L-lysine coating modification of the PDMS layer bonding surface after the differential oxygen plasma treatment is carried out through the following specific steps: Prepare a poly-L-lysine aqueous solution with a concentration of 0.01 mg to 0.1 mg per milliliter, and adjust the pH of the solution to between 7.4 and 8.5 using phosphate buffer; completely immerse the PDMS layer, which has undergone differential plasma treatment and had its mask removed, in the poly-L-lysine solution, and incubate it at a temperature of 20°C to 37°C for 30 minutes to 2 hours. During this process, positively charged poly-L-lysine molecules adsorb onto the negatively charged, oxygen plasma-activated PDMS surface through electrostatic interactions; after incubation, remove the PDMS layer from the solution and gently rinse it three to five times with sterile deionized water or phosphate buffer to remove loosely adsorbed poly-L-lysine molecules; subsequently, place the PDMS layer in a clean environment and air-dry it at room temperature or in an incubator at 37°C to form a stable poly-L-lysine modified layer.

[0088] In particular, due to the pre-differentiated treatment of the bonding surface, the stronger hydrophilicity and richer surface polar groups in the main chamber region result in more dense and robust adsorption of poly-L-lysine in this region, while the adsorption layer in areas such as connecting channels is relatively thin, which further enhances the regional heterogeneity of surface properties. This poly-L-lysine coating provides a biomimetic attachment interface rich in positive charge for subsequent inoculation of mammalian cells, significantly promoting initial cell adhesion, spreading, and subsequent proliferation activities. Compared with single plasma treatment, this composite surface treatment strategy combines the advantages of both physical and biochemical modification. It not only guides cell sap distribution using hydrophilic gradients but also actively regulates cell behavior through the spatially differentiated distribution of bioactive molecules, thereby producing a synergistic enhancement effect in cell inoculation efficiency, attachment uniformity, and maintenance of cell viability during long-term culture.

[0089] A self-sealing structure is provided at the fluid inlet;

[0090] The self-sealing structure can be punctured by a syringe needle and form a sealed connection. It should be further explained that the self-sealing structure at the fluid inlet is achieved as follows: During the molding process of the PDMS body, a solid cylindrical boss or thickened area of ​​PDMS material is designed and machined at the location corresponding to the fluid inlet. This area is integrally molded with the PDMS body, with a diameter between 1.5 mm and 3 mm and a height between 0.5 mm and 2 mm. It is made by mixing and curing the same prepolymer and curing agent in the same ratio as the PDMS body, thus ensuring the consistency of the material's elasticity and self-healing properties.

[0091] When connecting an external fluid drive device, such as a syringe pump, the operator uses a standard-sized syringe needle, vertically aligning it with the center of the protrusion, and applies appropriate axial pressure to insert it. The PDMS material, in a highly elastic state, is stretched by the needle and tightly wraps around the outer wall of the needle, forming the first circumferential seal. Due to the elastic memory and self-recovering properties of PDMS, the material continuously applies a clamping force to the outer wall of the needle throughout the entire process of needle insertion and connection, effectively preventing fluid leakage along the needle wall.

[0092] When the experiment is finished and disconnection is required, the needle is pulled out axially. The puncture channel left on the PDMS material will quickly close under the elastic restoring force of the material itself, achieving self-healing and preventing leakage of culture medium or intrusion of contaminants. This self-sealing structure eliminates the complex external interfaces commonly found in traditional microfluidic chips, such as Luer connectors, silicone tube sleeves, or adhesive flanges, making the connection between the chip and the fluid system extremely fast and simple. This significantly reduces the risk of leakage, air bubble introduction, or contamination due to improper interface assembly. At the same time, the one-piece molded PDMS boss structure avoids potential failure hazards at the interface of different materials, improving the connection reliability during long-term perfusion. This design fully aligns with the chip's overall design philosophy of pursuing ease of operation and stable reliability, enabling users to easily replace culture medium, add drugs, or perform continuous perfusion.

[0093] A method for fabricating a PDMS microfluidic chip for long-term live cell culture, comprising the following steps:

[0094] S1: Provide a mold with microstructure protrusions, the microstructure protrusions corresponding to the negative structure of the culture channel and open cell;

[0095] S2: Pour the PDMS prepolymer onto the mold, and after curing, demold to obtain a PDMS layer with a microchannel structure;

[0096] S3: Perform surface modification treatment on the bonding surface of the PDMS layer;

[0097] S4: The surface-modified PDMS layer is bonded to a quartz glass substrate to form a closed microchannel. The cutout area of ​​the quartz glass substrate and the main chamber of the PDMS layer are precisely aligned to adapt to optical inspection scenarios. During the molding of the PDMS layer, the cutout processing of the main chamber area is completed simultaneously to ensure that its size is precisely matched with the light-transmitting area of ​​the quartz glass.

[0098] Further explanation is needed regarding the specific implementation of the preparation method: First, a mold with microstructured protrusions on its surface is provided. This mold is processed onto a silicon substrate using photolithography. The negative photoresist used is SU-83050, and the exposure dose is controlled between 300 and 400 millijoules per square centimeter. After development, a negative groove structure corresponding to the culture channels and open cell structure in the target chip is formed. Subsequently, polydimethylsiloxane (PDMS) prepolymer and its curing agent are thoroughly mixed at a weight ratio of 10:1 and degassed. The mixture is then poured onto the surface of the mold and heated in an oven at 80 degrees Celsius. After curing for two hours, the PDMS layer is peeled off from the mold to obtain a PDMS layer with a complete microfluidic structure. Fluid inlet holes and open cell top openings are then machined at predetermined locations using a punch. Next, the bonding surfaces of the PDMS layer are subjected to surface modification treatment. This treatment involves placing the PDMS layer in an oxygen plasma device and treating it with 30 watts of power for 30 seconds to achieve overall basic activation. Then, a precision metal mask is used to tightly cover the bonding surfaces, exposing only the areas corresponding to all main chamber shapes. The exposed areas are then treated again with 60 watts of power for 3 minutes to complete the differentiated hydrophilic modification.

[0099] Subsequently, the plasma-treated PDMS layer was immersed in a polylysine phosphate buffer solution with a concentration of 0.1 mg / mL and incubated at 37°C for one hour. After removal, it was rinsed with deionized water and dried to form a bioactive coating. Finally, the surface-modified PDMS layer was aligned and bonded to a cleaned quartz glass substrate and placed together in an oxygen plasma device for a short treatment at low power for ten seconds. Immediately, uniform pressure was applied to bring the two into close contact. Irreversible Si-O-Si covalent bonds were formed between the silanol groups formed on the treated surface to achieve permanent bonding, thereby completing the fabrication of a closed microchannel chip with a gradient hydrophilicity and bioactive coating on the inner surface.

[0100] This preparation method integrates mold forming, material curing, partitioned surface activation, biomolecular modification and low-temperature bonding process system, and in particular introduces a mask-assisted partitioned plasma treatment step to ensure precise differential control of the surface properties of the core functional area and non-core flow channel area of ​​the chip.

[0101] Surface modification treatment of the bonding surface of the PDMS layer includes:

[0102] First, the entire bonding surface is subjected to a first-stage oxygen plasma treatment with the first power and the first time.

[0103] Then, the region corresponding to the non-main chamber on the bonding surface is covered with a mask, and the exposed region corresponding to the main chamber is subjected to a second-stage oxygen plasma treatment with a second power and a second time, wherein the second power is greater than the first power and / or the second time is longer than the first time.

[0104] Next, the entire PDMS layer is immersed in a poly-L-lysine solution, incubated, and then dried. In this embodiment, method two can be used for modification: the bonding surface of a quartz slide is immersed in a poly-L-lysine solution (0.1 mg / mL, pH 7.4), incubated at 37°C for 1 hour, then removed, rinsed three times with sterile deionized water, dried at room temperature, and then aligned and bonded with the PDMS layer treated with oxygen plasma. If method three is used, after bonding, poly-L-lysine solution is injected through the fluid inlet to fill the microchannel and main chamber. After incubation for 1 hour, the solution is aspirated with a syringe and air-dried in a fume hood for later use.

[0105] It should be further explained that the surface modification treatment in the preparation method is implemented as follows: First, the PDMS layer that has completed microstructure forming and perforation, with the bonding surface with the microfluidic structure facing upward, is placed in the chamber of the oxygen plasma treatment equipment; the first stage of oxygen plasma treatment is carried out, with the treatment power set between 20 watts and 40 watts and the treatment time controlled between 20 seconds and 60 seconds. This step aims to make the entire bonding surface basically and uniformly activated, and initially form a hydrophilic surface. Subsequently, a pre-processed mask is tightly bonded to the PDMS bonding surface. This mask is made of a metal sheet with a thickness of 50 to 100 micrometers, such as stainless steel or polyimide film. Its surface is processed by laser cutting or photolithography to create an array of vias that perfectly match the shape, size, and planar position of all main chambers in the chip design. The areas on the mask corresponding to the connection channels, open cells, and chip edges are solid, hole-free covers. The PDMS layer with the mask bonded is then placed in an oxygen plasma treatment device for a second stage of processing. The processing power in this stage is increased to between 50 and 80 watts, and the processing time is extended to two to five minutes. During this process, the highly reactive oxygen plasma can only act on the exposed PDMS surface area below through the vias in the mask, that is, precisely corresponding to the inner surface of each main chamber in the future chip. The remaining areas covered by the solid mask are protected, avoiding further deep oxidation.

[0106] After these two stages of differentiated treatment, the surface chemical properties of the PDMS bonding surface exhibit precise spatial heterogeneity: the main chamber region, having received higher energy doses and longer plasma irradiation, shows more complete cleavage of its surface siloxane network and a higher concentration of generated silanol groups, thus exhibiting extremely strong hydrophilicity; while the covered region retains the moderate hydrophilicity formed in the first stage treatment. Subsequently, the PDMS layer was immersed in a polylysine solution prepared with phosphate buffer at a concentration of 0.01 mg to 0.1 mg / mL and a pH of 7.4 to 8.5, and incubated at 20°C to 37°C for 30 minutes to 2 hours. After incubation, the layer was removed and rinsed three to five times with sterile phosphate buffer or deionized water to remove any loosely adsorbed polylysine molecules, and then air-dried in a clean environment or gently blow-dried at 37°C.

[0107] Through the aforementioned process, not only was a physical hydrophilic gradient formed by plasma treatment constructed on the PDMS surface, but a denser and more stable poly-lysine bioactive molecular layer was also superimposed on the strongly hydrophilic main chamber region. This enabled synergistic spatial programming of surface physicochemical properties and biological functions on a single substrate. This refined and zoned surface engineering method is a key and reproducible manufacturing step in realizing the core functions of the chip, namely guiding selective cell attachment, enhancing long-term culture stability, and optimizing the microenvironment.

[0108] By employing a multi-level series chamber structure resembling a "skewer of candied hawthorns" combined with a satellite retention chamber, a spatially heterogeneous fluid shear force distribution and mass exchange gradient are spontaneously generated within the chip, utilizing the inherent dynamics of fluids, without relying on complex external force fields or physical flipping operations. This structure effectively simulates the mechanical microenvironment of the in vivo tissue vascular system. Combined with an open pool design featuring a one-way valve at the end, it achieves automatic bubble removal, stable system pressure maintenance, and low-damage, convenient recovery of cell samples. This integrates multiple key steps, including cell capture, long-term culture, and sample acquisition, into a simplified and reliable operating procedure, overcoming the shortcomings of cumbersome operation, high dependence on auxiliary equipment, and insufficient microenvironment uniformity.

[0109] A spatially selective hydrophilic and bioactive interface was constructed on a PDMS substrate through gradient surface engineering combining mask-assisted partitioned oxygen plasma treatment with poly-L-lysine modification. This surface property not only guides cells to preferentially attach to the target culture area, improving cell utilization and distribution control, but also effectively enhances the wetting and retention of the culture medium within the chip, mitigating local evaporation and providing a chemically stable, osmotically balanced microenvironment for long-term cell viability and function. The chip structure design supports parallel and array-based expansion, with each culture unit being independent and its microenvironment controllable, providing an efficient and stable platform for high-throughput drug screening and multi-condition controlled cell biology research.

[0110] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0111] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A PDMS microfluidic chip for long-term culture of live cells, characterized in that, include: A PDMS body, wherein at least one culture channel is provided within the PDMS body; The culture channel has a fluid inlet and a fluid outlet; The culture channel includes at least one main chamber connected in series. When there are two or more main chambers, adjacent main chambers are connected by a first connecting channel. The culture channel also includes an open pool located downstream of the last main chamber and connected to the fluid outlet.

2. The PDMS microfluidic chip for long-term live cell culture according to claim 1, characterized in that: The main chamber has an elliptical outline; The length of the first connecting channel is less than the major axis dimension of the main chamber; The cross-sectional area of ​​the first connecting channel is smaller than the cross-sectional area of ​​the main chamber.

3. A PDMS microfluidic chip for long-term live cell culture according to claim 2, characterized in that: At least one of the main chambers has a side wall connected to at least one satellite retention chamber; The satellite retention chamber is connected to the main chamber via a second connecting channel; The cross-sectional area of ​​the second connection channel is smaller than that of the first connection channel.

4. A PDMS microfluidic chip for long-term live cell culture according to claim 3, characterized in that: The open pool is a hemispherical chamber with a volume larger than that of the main chamber; The top of the open pool has an opening that connects to the outside atmosphere; A one-way valve structure is provided at the connection between the open pool and the last main chamber; The one-way valve structure is integrally formed from PDMS material, which can remain open or slightly open when the fluid flows in the culture channel in the forward direction, and increase the degree of opening when negative pressure is applied from the open pool side.

5. A PDMS microfluidic chip for long-term live cell culture according to claim 4, characterized in that: The PDMS body has a bonding surface; the area of ​​the PDMS body corresponding to the main chamber is a hollow structure, a quartz glass substrate is bonded to the bonding surface, the quartz glass substrate covers the hollow area of ​​the main chamber, and the area corresponding to the main chamber is a light-transmitting area. On the bonding surface, the surface hydrophilicity corresponding to the main chamber region is stronger than the surface hydrophilicity corresponding to the first connection channel region; The surface hydrophilicity is achieved through differentiated surface treatment processes.

6. A PDMS microfluidic chip for long-term live cell culture according to claim 5, characterized in that: The differentiated surface treatment processes include: The entire bonding surface is subjected to oxygen plasma treatment under first conditions; Subsequently, the region corresponding to the main chamber is subjected to oxygen plasma treatment under the second condition; Wherein, the processing power of the second condition is greater than the processing power of the first condition, and / or the processing time of the second condition is longer than the processing time of the first condition.

7. A PDMS microfluidic chip for long-term live cell culture according to claim 6, characterized in that: Following the differentiated surface treatment, a polylysine coating is also applied to at least a portion of the bonding surface.

8. A PDMS microfluidic chip for long-term live cell culture according to claim 7, characterized in that: The fluid inlet is equipped with a self-sealing structure; The self-sealing structure can be punctured by a syringe needle and form a sealed connection.

9. A method for fabricating a PDMS microfluidic chip for long-term live cell culture, comprising the method for fabricating a PDMS microfluidic chip for long-term live cell culture as described in any one of claims 1 to 8, characterized in that, Includes the following steps: S1: Provide a mold with microstructure protrusions, the microstructure protrusions corresponding to the negative structure of the culture channel and open cell; S2: Pour the PDMS prepolymer onto the mold, and after curing, demold to obtain a PDMS layer with a microchannel structure; S3: Perform surface modification treatment on the bonding surface of the PDMS layer; S4: The surface-modified PDMS layer is bonded to a quartz glass substrate to form a closed microchannel.

10. The method for fabricating a PDMS microfluidic chip for long-term culture of live cells according to claim 9, characterized in that: Surface modification treatment of the bonding surface of the PDMS layer includes: First, the entire bonding surface is subjected to a first-stage oxygen plasma treatment with the first power and the first time. Then, the region corresponding to the non-main chamber on the bonding surface is covered with a mask, and the exposed region corresponding to the main chamber is subjected to a second-stage oxygen plasma treatment with a second power and a second time, wherein the second power is greater than the first power and / or the second time is longer than the first time. Then, the entire PDMS layer was immersed in a poly-L-lysine solution, incubated, and then removed and dried.

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