Microfluidic device for simulating red blood cell damage in autologous blood transfusion and method for manufacturing the same

Abstract

This invention relates to the field of biomedical materials technology, and more particularly to a microfluidic device and its preparation method for simulating the damage to red blood cells during autologous blood transfusion. The technical solution includes a polymer matrix, a filter membrane, a fluid inlet and a fluid outlet interface, and a negative pressure generating device. At least one microfluidic channel and a hollow chamber for accommodating the filter membrane are formed within the polymer matrix. This invention, by employing a track-etched membrane with pore size matched to that of a clinical filter membrane and combining it with a controllable negative pressure system, can highly simulate the mechanical damage to red blood cells during critical transfusion stages. Based on microfluidic technology, the required sample volume is reduced from hundreds of milliliters to just a few milliliters, significantly reducing research costs and sample acquisition difficulties. The device is replicated using PDMS and features modular connections, making preparation and operation simple. Furthermore, the mechanical conditions can be precisely controlled by adjusting the pump speed, achieving controllability and standardization of the experimental process.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a microfluidic device that simulates the damage caused by autologous blood transfusion of red blood cells and its preparation method. Background Technology

[0002] Intraoperative Autologous Blood Transfusion (IABT) is a blood preservation technique that involves recovering blood lost during surgical procedures using negative pressure suction, followed by anticoagulation, filtration, centrifugation, and washing, before reinfusing it into the patient's own blood. This technique effectively reduces or eliminates the need for allogeneic blood transfusions, lowers the risks of transfusion-related infectious disease transmission, immune reactions, and blood shortages, and has become an important component of modern perioperative blood management.

[0003] Throughout the autologous blood recovery and reinfusion process, red blood cells are continuously exposed to various non-physiological mechanical forces, including negative pressure suction, tubing transport, filtration shock, and centrifugal shearing. These mechanical stresses can lead to damage to the red blood cell membrane structure, decreased deformability, increased fragility, and even hemolysis, collectively known as "red blood cell washing injury." This type of injury not only reduces the survival rate and oxygen-carrying capacity of the reinfused red blood cells, but the released hemoglobin and intracellular substances may also trigger inflammatory responses, affecting patient prognosis. Therefore, in-depth investigation into the specific mechanisms of red blood cell damage during autologous blood reinfusion is of significant clinical and research value for optimizing recovery equipment parameters, improving processing procedures, and ultimately enhancing the safety and effectiveness of autologous blood reinfusion.

[0004] Currently, research on erythrocyte injury related to autologous blood transfusion mainly relies on two approaches: direct clinical observation, i.e., analyzing patient blood samples processed by commercial autologous blood recovery machines; and in vitro simulations using commercially available recovery equipment in the laboratory. Both methods have significant limitations. Clinical research is constrained by multiple confounding factors such as individual patient differences, surgical type, blood loss, and concomitant medications, making it difficult to isolate, quantify, and reproducibly examine single mechanical force factors, and it also faces high ethical and accessibility requirements. On the other hand, direct in vitro experiments using large-scale clinical equipment typically require hundreds of milliliters of blood, resulting in large sample volumes, high costs, complex operations, and fixed equipment parameters that are difficult to adjust flexibly to simulate different stress conditions or study the effects of specific processes.

[0005] Microfluidics has become a powerful tool for in vitro cell mechanics research due to its ability to precisely control fluid flow on a small scale, provide repeatable shear stress environments, and perform high-throughput experiments with only trace samples. Polydimethylsiloxane (PDMS) is a commonly used material for constructing microfluidic chips due to its good biocompatibility, light transmittance, and ease of microfabrication. Track-etched films are polymeric films with precise, uniform, and through-cylindrical channels whose pore size can be controlled at the micrometer to nanometer scale, serving as ideal physical filtration barrier models.

[0006] Although microfluidic technology has significant advantages in simulating biomechanical environments, existing technologies lack an integrated, miniaturized, and parameter-controllable in vitro model that can specifically simulate the key mechanical stresses (especially the negative pressure suction filtration stage) throughout the entire clinical autologous blood transfusion process. Therefore, this application proposes a microfluidic device and its preparation method for simulating red blood cell damage during autologous blood transfusion. Summary of the Invention

[0007] The purpose of this invention is to address the lack of an integrated, miniaturized, and parameter-controllable in vitro model that can specifically simulate the key mechanical stresses (especially the negative pressure suction and filtration stage) in the entire process of clinical autologous blood transfusion. This invention proposes a microfluidic device for simulating red blood cell damage during autologous blood transfusion and its preparation method.

[0008] In a first aspect, this application provides a microfluidic device for simulating damage caused by autologous blood transfusion of red blood cells, comprising: A polymer matrix having at least one microfluidic channel and a hollow chamber for accommodating a filter membrane formed therein; A filter membrane is disposed in the hollow cavity. The filter membrane is a track-etched membrane with uniform cylindrical channels, used to simulate the filtration function of a clinical autologous blood recovery device. The fluid input interface and the fluid output interface are respectively connected to the microfluidic channel; A negative pressure generating device is connected to the fluid output interface via a connecting pipeline to provide a controllable negative pressure attraction, causing the red blood cell suspension to flow sequentially through the fluid input interface, the microfluidic channel, the filter membrane, and the fluid output interface, thereby simulating the mechanical filtration damage experienced by red blood cells during autologous blood reinfusion.

[0009] Optionally, the polymer matrix is ​​made of polydimethylsiloxane (PDMS), which is bonded to a first PDMS component and a second PDMS component, and the hollow cavity and the microfluidic channel are formed within the first PDMS component and / or the second PDMS component.

[0010] Optionally, the filter membrane is sandwiched between the first PDMS component and the second PDMS component, and is bonded and fixed to both by an adhesive, so that the filter membrane covers and separates the hollow cavity.

[0011] Optionally, the first PDMS component and the second PDMS component have different thicknesses. The PDMS component directly bonded to the filter membrane is the first thickness component, and a second thickness component is further bonded to its outer side by plasma bonding. The second thickness component is provided with a communication channel for the fluid input interface or the fluid output interface.

[0012] Optionally, the fluid input interface and fluid output interface are connected to an external container and the negative pressure generating device via polytetrafluoroethylene (PTFE) tubing; the negative pressure generating device is a microfluidic injection pump, on which a syringe is mounted, and the negative pressure attraction is generated by controlling the suction motion of the syringe.

[0013] Secondly, this application provides a method for preparing a microfluidic device that simulates autologous blood transfusion red blood cell injury, applied to the microfluidic device for simulating autologous blood transfusion red blood cell injury described in the first aspect, comprising the following steps: S1. Prepare polymer components, provide a mold, inject liquid polymer precursor into the mold, and demold after solidification to obtain at least two polymer components with microfluidic channels and hollow structures inside; S2, an integrated filter membrane, wherein the track-etched filter membrane is fixed to the hollow structural region of one of the polymer components; S3. Bonding assembly: Align and bond another polymer component with the polymer component to which the filter membrane is fixed, so that the filter membrane is sealed and held between the two, forming a microfluidic chip with a built-in filter membrane. The microfluidic channel forms a continuous flow path after bonding. S4. System connection: Connect the inlet and outlet of the microfluidic chip to the red blood cell suspension supply source and the negative pressure generating device respectively through connecting pipes to form a complete simulation device.

[0014] Optionally, in step S1, the liquid polymer precursor is a mixture of PDMS component A and component B in a mixing ratio of 10:1. After vacuum degassing, it is cured at 50℃~80℃ for 2 to 8 hours. By controlling the volume of the mixture injected into the mold, PDMS components with different thicknesses can be prepared.

[0015] Optionally, in step S2, the filter membrane is cut into a circle with a diameter larger than the opening diameter of the hollow structure on the polymer component; the filter membrane is then bonded to the opening using AB glue.

[0016] Optionally, in step S3, the bonding method is irreversible bonding after plasma treatment; for the assembly between PDMS components that are not directly bonded to the filter membrane, plasma bonding is used.

[0017] Optionally, in step S4, the negative pressure generating device is a microfluidic injection pump, and the connecting pipeline is a polytetrafluoroethylene (PTFE) flexible tube; during operation, the aspiration program of the microfluidic injection pump is started, and the aspiration flow rate is controlled within the range of 1~50 mL / min to simulate the negative pressure conditions in the clinical autologous blood reinfusion process.

[0018] Compared with the prior art, this application includes at least one of the following beneficial technical effects: This device integrates a track-etched membrane that matches the pore size of the filter membrane in clinical autologous blood recovery devices, and utilizes a microfluidic injection pump to provide controllable negative pressure attraction. It can accurately simulate the key mechanical filtration damage that red blood cells undergo during clinical autologous blood reinfusion in vitro, providing a clinically relevant model for mechanism research.

[0019] Based on microfluidic technology, this device achieves miniaturization of the experimental system, reducing the amount of red blood cell sample required for a single simulation from hundreds of milliliters in clinical practice to just a few milliliters, significantly reducing blood sample consumption and experimental costs.

[0020] The core components of the device are manufactured using mature PDMS replication molding technology and connected to standardized components (such as injection pumps and tubing), making the fabrication method simple and assembly convenient. Furthermore, by adjusting parameters such as the flow rate of the microfluidic injection pump, key mechanical conditions such as negative pressure can be easily controlled, enabling controllable simulation of different degrees of damage.

[0021] Based on a precisely shaped microfluidic chip and a programmable injection pump, this device can provide a highly consistent hydrodynamic environment, ensuring the repeatability and standardization of experimental conditions, which is beneficial for the comparison and analysis of experimental results from different batches.

[0022] In summary, this invention, by employing a track-etched membrane with pore size matched to clinical filtration membranes and combining it with a controllable negative pressure system, can simulate the mechanical damage suffered by red blood cells during critical reinfusion processes with high fidelity. Based on microfluidic technology, the required amount of experimental samples is reduced from hundreds of milliliters to just a few milliliters, significantly reducing research costs and sample acquisition difficulties. The device is made using PDMS replication molding and modular connection, making preparation and operation simple. Furthermore, the mechanical conditions can be precisely controlled by adjusting the pump speed, achieving controllability and standardization of the experimental process. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of a microfluidic chip composed of PDMS and track-etched PET film.

[0024] Figure 2 This is a schematic diagram of a microfluidic device simulating autologous blood reinfusion, consisting of a microfluidic chip, an injection pump, and a Teflon tube.

[0025] Figure 3 This image shows a comparison of the morphology of red blood cells in the radial artery with those recovered by a clinical autologous blood transfusion machine.

[0026] Figure 4 The morphology of red blood cells in the radial artery is compared with that of red blood cells after being transfused via a microfluidic device that simulates autologous blood in vitro.

[0027] Figure 5 for Figure 3 and Figure 4 Statistical results for the two scenarios are shown in the graph. Detailed Implementation

[0028] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0029] Example 1 This embodiment provides a microfluidic device for simulating the damage caused by autologous blood transfusion of red blood cells, such as... Figure 1 and Figure 2 As shown, the device mainly consists of three parts: a microfluidic chip, connecting pipes, and a drive system.

[0030] The microfluidic chip has a stacked structure, with its core consisting of polydimethylsiloxane (PDMS) and a track-etched filter membrane. Specifically, a 1.5cm diameter circular track-etched PET membrane is bonded between two 5mm thick PDMS cubes using AB glue, forming an intermediate layer structure of "5mm PDMS-membrane-5mm PDMS". At both ends of this intermediate layer structure, a 2mm thick PDMS cube is bonded to each end using plasma bonding, ultimately forming a complete chip body of "2mm PDMS-5mm PDMS-membrane-5mm PDMS-2mm PDMS". The 5mm thick PDMS cube has a 1cm diameter circular hollow cavity at its center to accommodate the filter membrane and form a filter chamber; the 2mm thick PDMS cube has a 1mm diameter circular channel at its center as a fluid inlet / outlet.

[0031] The connecting tubing is a 1mm inner diameter polytetrafluoroethylene (Teflon) flexible tube. One end of the flexible tube is inserted into the 1mm circular hole in the center of a 2mm thick PDMS block to achieve a sealed connection with the microfluidic chip.

[0032] The drive system mainly consists of a microfluidic injection pump and a syringe. One end of a PTFE tubing is connected to a 10ml syringe, which is fixedly mounted on the microfluidic injection pump. The microfluidic injection pump precisely controls the push-pull motion of the syringe, thereby generating controllable negative or positive pressure within the system.

[0033] Example 2 This embodiment provides a method for preparing a microfluidic device as described in Embodiment 1, including the following steps: Preparation of PDMS preforms: PDMS prepolymer (component A) and curing agent (component B) were mixed evenly at a mass ratio of 10:1. After vacuum degassing to remove air bubbles, the mixture was poured into a flat mold. By controlling the volume of the mixture poured into the mold, PDMS boards with thicknesses of 5 mm and 2 mm were prepared. The cured PDMS boards were then placed in a 60℃ oven for further curing for 5 hours. Subsequently, both thicknesses of PDMS boards were cut into cubes with a side length of 3 cm.

[0034] Processing the fluid channel: For a 5mm thick PDMS block, cut or carve a 1cm diameter through hole with its geometric center as the center to form a filter chamber. For a 2mm thick PDMS block, similarly, use a 1mm diameter punch to create a through hole with its center as the center to form a fluid connection channel.

[0035] Membrane integration and chip assembly: The track-etched PET film was cut into circles with a diameter of 1.5 cm.

[0036] AB glue is applied to the inner 2mm edge of a circular filter membrane with a diameter of 1.5cm. The center of the filter membrane is aligned with the center of a 5mm thick PDMS block with a pre-processed 1cm circular hole. The membrane is gently pressed to completely cover and adhere to the upper surface of the block, forming a double-layer "PDMS-membrane" structure. Another 5mm thick PDMS block with a pre-processed 1cm circular hole is used as the upper layer and aligned with the PDMS portion of the "PDMS-membrane" double-layer structure. After treating the PDMS surfaces to be bonded using a plasma treatment machine, the two are tightly bonded together to complete irreversible plasma bonding, thus sandwiching the filter membrane between the two PDMS layers, resulting in a "PDMS-membrane-PDMS" intermediate sandwich structure.

[0037] Two 2mm thick PDMS cubes with 1mm holes pre-drilled were aligned with the two sides of the aforementioned intermediate sandwich structure. All PDMS surfaces to be bonded were treated using a plasma treatment machine. Then, the 2mm thick PDMS cubes were tightly bonded to the 5mm thick PDMS cube of the intermediate sandwich structure, completing irreversible plasma bonding. This resulted in a microfluidic chip with a structure of "2mm PDMS-5mm PDMS-film-5mm PDMS-2mm PDMS".

[0038] System Connection: Insert two appropriately sized PTFE tubing pieces, each with an inner diameter of 1mm, into the 1mm circular holes on the 2mm thick PDMS blocks at the top and bottom of the microfluidic chip, ensuring a tight, leak-proof connection. Connect the other end of one of the tubing pieces to a 10ml syringe, and then mount and secure the syringe to the microfluidic injection pump.

[0039] Example 3 This embodiment demonstrates an experiment simulating autologous blood transfusion red blood cell damage in vitro using the device prepared in Example 2.

[0040] Sample preparation: Fresh anticoagulated whole blood was collected from healthy volunteers and concentrated red blood cells were obtained by centrifugation and washing. A small amount of concentrated red blood cells was taken and diluted with physiological saline or cell culture medium to prepare red blood cell dilution solution, which was then placed in a 15ml centrifuge tube.

[0041] Device connection: Insert the PTFE tubing from the end of the microfluidic chip that is not connected to the syringe (the inlet end) into the bottom of the 15ml centrifuge tube containing the red blood cell diluent. Ensure that the syringe, microfluidic chip, and sample tube are connected through the tubing.

[0042] Simulated negative pressure suction filtration: Start the microfluidic syringe pump and set it to "suction" mode. Set the pump's suction flow rate to 25 mL / min (this parameter can be adjusted according to simulation requirements). The syringe pump drives the syringe piston backward, generating negative pressure throughout the flow path system. Under this negative pressure suction, the red blood cell diluent in the centrifuge tube flows sequentially through the inlet tubing, the track etching filter membrane in the microfluidic chip (simulating clinical filtration), and is finally collected into the syringe.

[0043] Subsequent processing simulation: To more completely simulate the autologous blood reinfusion process, the syringe containing the collected red blood cell suspension can be removed from the pump, and its contents transferred to a centrifuge tube for centrifugation and washing, simulating the washing step. Afterward, the washed red blood cells can be re-drawn into another syringe, and the "ejection" mode of a microfluidic injection pump can be used to pump the red blood cells into a simulated blood storage bag at a certain flow rate.

[0044] Damage assessment: such as Figure 3 Untreated raw red blood cells (as a control) were used to treat the cells. Figure 3 (Left) Red blood cells processed by a clinical autologous blood recovery machine (e.g.) Figure 3 (right), and red blood cells processed by the microfluidic device of this embodiment (such as...) Figure 4 Smears and microscopic observations were performed to compare morphological changes. For example... Figure 5 The statistical results show that the red blood cells flowing through this microfluidic device underwent morphological damage similar to that of clinical blood recovery machines (such as an increase in acanthocytosis), proving that the device can effectively simulate and be used to study red blood cell washing damage during autologous blood transfusion.

[0045] This invention, for the first time, uses a track-etched membrane with precisely controllable pore size as a biomimetic filtration unit, integrating it with a PDMS microfluidic chip through a standardized structure to construct an integrated, miniaturized in vitro model with precisely controllable negative pressure parameters. Its innovative effects are specifically reflected in: the device successfully achieves a high degree of realism in simulating key filtration processes in clinical autologous blood transfusion, allowing red blood cells to undergo mechanical stress with properties highly consistent with those of the clinical process in micro-samples (only a few milliliters). This induces typical red blood cell morphological damage in a controllable and repeatable experiment, providing a dedicated experimental platform with high realism, low cost, and high throughput potential for in-depth research into damage mechanisms.

[0046] The above specific embodiments are merely several optional embodiments of the present invention. Based on the technical solutions of the present invention and the relevant teachings of the above embodiments, those skilled in the art can make various alternative improvements and combinations to the above specific embodiments.

Claims

1. A microfluidic device for simulating autologous blood transfusion injury of red blood cells, characterized in that, include: A polymer matrix having at least one microfluidic channel and a hollow chamber for accommodating a filter membrane formed therein; A filter membrane is disposed in the hollow cavity. The filter membrane is a track-etched membrane with uniform cylindrical channels, used to simulate the filtration function of a clinical autologous blood recovery device. The fluid input interface and the fluid output interface are respectively connected to the microfluidic channel; A negative pressure generating device is connected to the fluid output interface via a connecting pipeline to provide a controllable negative pressure attraction, causing the red blood cell suspension to flow sequentially through the fluid input interface, the microfluidic channel, the filter membrane, and the fluid output interface, thereby simulating the mechanical filtration damage experienced by red blood cells during autologous blood reinfusion.

2. The microfluidic device for simulating autologous blood transfusion red blood cell damage according to claim 1, characterized in that, The polymer matrix is ​​made of polydimethylsiloxane, which is represented by PDMS. It is formed by bonding a first PDMS component and a second PDMS component. The hollow cavity and the microfluidic channel are formed within the first PDMS component and / or the second PDMS component.

3. The microfluidic device for simulating autologous blood transfusion red blood cell damage according to claim 2, characterized in that, The filter membrane is sandwiched between the first PDMS component and the second PDMS component, and is bonded and fixed to both by an adhesive, so that the filter membrane covers and separates the hollow cavity.

4. The microfluidic device for simulating autologous blood transfusion red blood cell damage according to claim 3, characterized in that, The first PDMS component and the second PDMS component have different thicknesses. The PDMS component directly bonded to the filter membrane is the first thickness component, and a second thickness component is further bonded to its outer side by plasma bonding. The second thickness component is provided with a connecting channel for the fluid input interface or the fluid output interface.

5. The microfluidic device for simulating autologous blood transfusion red blood cell damage according to claim 1, characterized in that, The fluid input interface and fluid output interface are connected to the external container and the negative pressure generating device via polytetrafluoroethylene hoses; the negative pressure generating device is a microfluidic injection pump, on which a syringe is installed, and the negative pressure attraction is generated by controlling the suction movement of the syringe.

6. A method for preparing a microfluidic device simulating autologous blood transfusion red blood cell injury, applicable to the microfluidic device simulating autologous blood transfusion red blood cell injury as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Prepare polymer components, provide a mold, inject liquid polymer precursor into the mold, and demold after solidification to obtain at least two polymer components with microfluidic channels and hollow structures inside; S2, an integrated filter membrane, wherein the track-etched filter membrane is fixed to the hollow structural region of one of the polymer components; S3. Bonding assembly: Align and bond another polymer component with the polymer component to which the filter membrane is fixed, so that the filter membrane is sealed and held between the two, forming a microfluidic chip with a built-in filter membrane. The microfluidic channel forms a continuous flow path after bonding. S4. System connection: Connect the inlet and outlet of the microfluidic chip to the red blood cell suspension supply source and the negative pressure generating device respectively through connecting pipes to form a complete simulation device.

7. The method for preparing the microfluidic device for simulating autologous blood transfusion erythrocyte injury according to claim 6, characterized in that, In step S1, the liquid polymer precursor is a mixture of PDMS components A and B in a ratio of 10:

1. After vacuum degassing, it is cured at 50°C to 80°C for 2 to 8 hours. By controlling the volume of the mixture injected into the mold, PDMS components with different thicknesses are prepared.

8. The method for preparing the microfluidic device for simulating autologous blood transfusion erythrocyte injury according to claim 7, characterized in that, In step S2, the filter membrane is cut into a circle with a diameter larger than the opening diameter of the hollow structure on the polymer component; the filter membrane is then bonded to the opening using AB glue.

9. The method for preparing the microfluidic device for simulating autologous blood transfusion erythrocyte injury according to claim 6, characterized in that, In step S3, the bonding method is irreversible bonding after plasma treatment; for the assembly between PDMS components that are not directly bonded to the filter membrane, plasma bonding is used.

10. The method for preparing the microfluidic device for simulating autologous blood transfusion erythrocyte injury according to claim 6, characterized in that, In step S4, the negative pressure generating device is a microfluidic injection pump, and the connecting pipeline is a polytetrafluoroethylene (PTFE) tubing. During operation, the aspiration program of the microfluidic injection pump is started, and the aspiration flow rate is controlled within the range of 1~50 mL / min to simulate the negative pressure conditions in the clinical autologous blood reinfusion process.

Images