Microfluidic channel for extruding cells, microfluidic chip and application thereof

Abstract

This invention relates to the field of biomedical technology, specifically to a microfluidic channel, a microfluidic chip, and their applications for squeezing cells. The microfluidic channel is composed of several continuously arranged cell squeezing units. Each cell squeezing unit consists of a main channel and a squeezing region. The main channel is composed of connecting sections and curved sections. The squeezing region includes a first squeezing section, a squeezing slit, and a second squeezing section. The width of the first squeezing section gradually decreases from the main channel, reaching its minimum at the squeezing slit, and then gradually increases towards the second squeezing section until it matches the width of the main channel. The squeezing slit is rectangular, with a width of 4-10 μm and a length of 20-60 μm. The microfluidic channel provided by this invention applies relatively uniform and dispersed mechanical stress to the cell membrane, reducing cell breakage and mortality rates, and allowing cells to restore their integrity through the cell membrane's self-repair mechanism after squeezing.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a microfluidic channel for squeezing cells, a microfluidic chip, and their applications. Background Technology

[0002] Neutrophils are the most abundant type of white blood cell in human blood and serve as the body's first line of defense against infection, recruited from the circulatory system to various sites of inflammation, including cancer. In particular, when the level of interleukin-8 (IL-8) in a localized area reaches a certain level, it promotes neutrophil infiltration. Induced by IL-8, neutrophils can cross vascular endothelial cells (HUVECs) and the blood-brain barrier to reach the site of inflammation or tumor. Furthermore, neutrophils have been shown to sense the receptor for advanced glycation end products (RAGE) on tumor cells, meaning that neutrophils can specifically recognize and target tumor cells for elimination without causing significant damage to healthy cells. Therefore, neutrophils can be chosen as a cellular carrier for targeted drug delivery to glioblastoma (GBM). However, neutrophils typically cannot proliferate and have a short survival time (approximately 2-3 days), making it difficult to achieve rapid drug loading using conventional methods, which greatly limits their application in cell therapy.

[0003] Traditional methods of drug delivery to cells typically involve co-incubating cells and drugs in PBS or culture medium. To achieve the desired drug loading, incubation times usually range from 24 to 48 hours. For example, Chinese invention patent CN118460467A discloses a method for preparing drug-loaded neutrophils. This method involves co-incubating neutrophils with the drug to be loaded in a buffer solution containing nicotinamide, Q-VD-Oph, and vitamin C to form a neutrophil-based drug delivery formulation. This preparation method requires a co-incubation time of up to 30 minutes, and this incubation time is based on the use of doxorubicin hydrochloride liposomes as the target drug, the original drug Doxil, which is very expensive. For neutrophils, which have a very short in vitro lifespan, using this method to prepare neutrophil-based drug delivery formulations for cell therapy presents significant limitations.

[0004] Chinese invention patent CN115254214A discloses a microfluidic channel, a microfluidic chip, and a method for delivering biochemical molecules. This method uses a microfluidic chip to compress cells, delivering extracellular biochemical molecules into the cells, achieving a cell viability rate as high as 80%. However, the chip in this method uses sharp, point-like compression, which exerts significant local mechanical stress on the cell membrane, resulting in considerable cell damage. Furthermore, the drug transmembrane delivery efficiency is not high, with the highest delivery efficiency not exceeding 60%. Summary of the Invention

[0005] To address the above problems, this invention proposes a microfluidic channel for squeezing cells, a microfluidic chip, and their applications.

[0006] This invention provides a microfluidic channel for squeezing cells. The microfluidic channel is composed of several cell squeezing units arranged continuously. Each cell squeezing unit consists of a main channel and a squeezing zone. The main channel is composed of a connecting section and a curved section. The connecting section and the curved section have the same width. One end of the curved section is connected to the connecting section, and the other end is connected to the squeezing zone. The squeezing zone includes a first squeezing section, a squeezing slit, and a second squeezing section. The curved section is connected to the first squeezing section. One end of the squeezing slit is connected to the first squeezing section, and the other end is connected to the second squeezing section. The width of the first squeezing section gradually decreases from the main channel to its minimum at the squeezing slit, and then gradually increases at the second squeezing section until it matches the width of the main channel. One end of the second squeezing section is connected to the squeezing slit, and the other end is connected to the main channel connecting section of the next squeezing unit. The squeezing slit is rectangular, with a width of 4-10 μm and a length of 20-60 μm.

[0007] Furthermore, the curved section is a serpentine channel formed by several semi-circular channels arranged alternately and connected vertically.

[0008] Furthermore, the width of the extrusion section one and extrusion section two gradually decreases in a stepped manner, divided into five levels. The first level is connected to the main channel and has the same width as the main channel. The fifth level has an opening at one end that is connected to the extrusion slit. From the first level to the fifth level, the width gradually decreases.

[0009] Furthermore, the microfluidic channel is composed of 10 cell compression units arranged in a continuous manner.

[0010] Furthermore, the width of the main channel is 100μm.

[0011] Furthermore, the semi-circular channel has a circular radius of 200 μm and a channel width of 100 μm.

[0012] Furthermore, the first stage width of the extrusion section one and the extrusion section two is 100μm; the second stage width is 80μm and the length is 50μm; the third stage width is 60μm and the length is 50μm; the fourth stage width is 40μm and the length is 50μm; and the fifth stage width is 20μm and the length is 50μm.

[0013] Furthermore, the microfluidic chip consists of an inlet, a first transition region, a microfluidic channel array, a second transition region, and an outlet; the microfluidic channel array is formed by several microfluidic channels arranged in parallel.

[0014] The microfluidic channel for squeezing cells of the present invention is used in the preparation of cell drug delivery carriers.

[0015] The microfluidic chip for squeezing cells of the present invention is used in the preparation of cell drug delivery carriers.

[0016] The beneficial effects of this invention are as follows:

[0017] 1. The extrusion slit of the microfluidic channel provided by the present invention is a key extrusion area with a rectangular structure. The mechanical stress applied to the cell membrane by this structure is relatively uniform and dispersed, which can reduce the cell breakage rate and mortality rate, and enable the cell to restore its integrity through the cell membrane self-repair mechanism after being extruded.

[0018] 2. Each microfluidic channel of this invention contains an array of continuously periodic cell squeezing units. Each cell squeezing unit consists of a main channel and squeezing zones. The main channel is composed of connecting sections and curved sections. This structure is based on the cell membrane stress relaxation theory and aims to subject cells to a cyclical mechanical stimulation of "squeezing-elastic recovery-re-squeezing-re-elastic recovery" as they continuously flow through the cell squeezing zones. This periodic mechanical stimulation can induce dynamic reconstruction of the cell membrane lipid bilayer, promoting the gradient distribution of transmembrane potential, thereby significantly improving the transmembrane delivery efficiency of exogenous macromolecules. The curved sections of the main channel are composed of serpentine channels, and the cell squeezing zones are connected by a complex structure with gradually contracting structures. The presence of serpentine channels allows cells to pass through the center of the main channel, reducing chip clogging. A gradually contracting flow channel configuration is used before and after the cell squeezing zones, decreasing in width from 100 μm in the main channel to the minimum squeezing size. This structure provides moderate mechanical stimulation while avoiding cell damage caused by sudden shear stress changes. Microfluidic chips prepared using the microfluidic channels of this invention can achieve a maximum delivery efficiency of 95.8% for cell delivery.

[0019] 3. The microfluidic chip of this invention employs a progressively contracting flow channel configuration before and after the cell extrusion section, gradually decreasing from a main channel width of 100 μm to the minimum extrusion size. This structure provides moderate mechanical stimulation while avoiding cell damage caused by sudden shear stress changes. Using the microfluidic chip of this invention for cell delivery, the viability of collected intact cells can reach 99%.

[0020] 4. This invention enables the rapid preparation of a neutrophil-based drug delivery carrier. Albumin-based paclitaxel (PTX-ALB) was selected as the drug to be loaded, and the prepared drug-loaded neutrophils were used for the treatment of glioblastoma (GBM) at the cellular level. The preparation of drug-loaded cells using this invention takes only 8 minutes, compared to the 30 minutes required by conventional co-incubation techniques, reducing the preparation time by approximately 3.75 times.

[0021] 5. The present invention has the advantage of being able to deliver virtually any macromolecule into virtually any cell type in a high-throughput and rapid manner. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of a microfluidic chip structure;

[0023] Figure 2 for Figure 1 A magnified structural diagram of the location marked A in the middle;

[0024] Figure 3 for Figure 2 A magnified structural diagram of the location marked B in the middle;

[0025] Figure 4 The following is a flow cytometry plot of delivery efficiency under different slit sizes and flow rates in Example 2;

[0026] Figure 5 The bar chart shows the delivery efficiency under different slit sizes and flow rates in Example 2;

[0027] Figure 6 The bar chart shows the loading efficiency under different slit sizes and flow rates in Example 2;

[0028] Figure 7 This is a schematic diagram of the process of HL-60 cells undergoing DMSO-induced differentiation and mechanical deformation to deliver drugs in Example 3;

[0029] Figure 8 This is a flow cytometry diagram showing the expression level of CD11b in HL-60 cells induced by DMSO after differentiation in Example 3.

[0030] Figure 9 Flow cytometry plot of neutrophil-like cells loaded with FITC-BSA in Example 3;

[0031] Figure 10 This is a graph showing the ELISA quantitative results of neutrophil-like cells loaded with PTX-ALB in Example 4.

[0032] Figure 11 This is a schematic diagram of the process for co-culturing PTX-ALB-loaded neutrophil-like cells with U87-eGFP glioma cells in Example 5.

[0033] Figure 12 This is a bar chart showing the relative viability of U87-eGFP cells under different co-culture conditions in Example 5;

[0034] Figure 13 Fluorescence images of U87-eGFP cells under different treatment conditions in Example 5. Detailed Implementation

[0035] The present invention will be further described below with reference to the embodiments.

[0036] 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.

[0037] Example 1: Preparation of a microfluidic chip for squeezing cells:

[0038] 1.1 Design of Microfluidic Chips:

[0039] like Figures 1 to 3 As shown, the microfluidic chip consists of an inlet 1, a first transition zone 2, a microfluidic channel array 3, a second transition zone 4, and an outlet 5. The microfluidic channel array 3 consists of 12 parallel microfluidic channels. Each microfluidic channel consists of 10 continuously arranged cell extrusion units. Each cell extrusion unit consists of a main channel and an extrusion zone 8. The main channel consists of a connecting section 6 and a curved section 7. The connecting section 6 and the curved section 7 have the same width. One end of the curved section 7 is connected to the connecting section 6, and the other end is connected to the extrusion zone 8. The extrusion zone includes an extrusion section 9, an extrusion slit 11, and an extrusion section 10. The curved section 7 is connected to the extrusion section 9. One end of the extrusion slit 11 is connected to the extrusion section 9, and the other end is connected to the extrusion section 10. The width of the extrusion section 9 gradually decreases from the main channel, reaches its minimum at the extrusion slit 11, and then gradually increases at the extrusion section 10 until it matches the main channel. One end of the extrusion section 10 is connected to the extrusion slit 11, and the other end is connected to the main channel connecting section of the next extrusion unit.

[0040] Specifically, the curved segment 7 is a serpentine channel formed by several semi-circular channels arranged alternately vertically. The serpentine channel uses alternating semicircles with a radius of curvature of 200 μm, with a main channel width of 100 μm. The compression slits 11 in the cell compression section are composed of rectangles 30 μm long, designed in four groups with slit widths of 4 μm, 6 μm, 8 μm, and 10 μm respectively. The chip height is 11 μm. Compression slits 11 are critical locations where cells are compressed. The rectangular structure applies relatively uniform and dispersed mechanical stress to the cell membrane, reducing cell breakage and mortality rates, allowing cells to recover their integrity through the cell membrane's self-repair mechanism after compression.

[0041] Specifically, the width of the extrusion section 9 and extrusion section 10 gradually decreases in a stepped manner, divided into five levels. The first level is connected to the main channel and has the same width as the main channel. The fifth level 15 has an opening at one end connected to the extrusion slit 11. From the first level to the fifth level, the width gradually decreases. The width of the first level of extrusion section 9 and extrusion section 10 is 100μm; the width of the second level 12 is 80μm and the length is 50μm; the width of the third level 13 is 60μm and the length is 50μm; the width of the fourth level 14 is 40μm and the length is 50μm; and the width of the fifth level 15 is 20μm and the length is 50μm.

[0042] 1.2 Fabrication of Microfluidic Chips: The chip template is fabricated using soft photolithography. A 4-inch silicon wafer is selected, and SM8-2015 photoresist is uniformly spin-coated onto the wafer through a multi-step spin coating process. After preheating at 65°C for 30 seconds, followed by pre-baking at 95°C for 3 minutes and 30 seconds, the pre-treated silicon wafer is exposed to ultraviolet light for 12 seconds. After exposure, the channels designed in 1.1 will solidify on the silicon wafer. Subsequently, the silicon wafer is placed on a 95°C heating plate for 4 minutes and 30 seconds for post-baking. Finally, the silicon wafer is developed to remove any uncured photoresist around the edges. The prepared silicon wafer template must undergo hydrophobic treatment for subsequent demolding processes: The silicon wafer is placed in a sealed container, and 10 μl of fluorosilane solution is added to the container. The container is then sealed and placed in a 120°C oven for evaporation deposition for 2 hours. Chip fabrication and bonding: PDMS prepolymer and its crosslinking catalyst were thoroughly mixed (10:1, w / w). Air bubbles in the mixture were removed using a vacuum drying oven. The de-bubbled mixture was then slowly poured onto a silicon wafer template. Once poured to a suitable height, the template was placed in an oven and cured for 2 hours at 80°C to obtain the PDMS layer. The cured chip was cut out using a No. 3 scalpel, and after drilling holes, the PDMS layer was bonded to the glass substrate using an oxygen plasma system. The substrate was then baked in an 80°C oven for at least 4 hours. The microfluidic chip was sterilized by exposing it to high-intensity ultraviolet light.

[0043] Example 2: Preparation of neutrophil-like cell drug delivery carriers using the microfluidic chip prepared in Example 1:

[0044] 2.1 HL-60 cell differentiation was induced with 1.25% DMSO. DMSO was added directly to the HL-60 cell suspension cultured in RPMI-1640 complete medium and the cells were differentiated for 5 days in a constant temperature incubator at 37°C containing 5.0% CO2. During this period, the medium did not need to be changed.

[0045] 2.2. The mixture of neutrophils prepared in 2.1 and the drug to be loaded is injected into the microfluidic chip prepared in Example 1. When the cells pass through narrow channels smaller than the cell diameter within the chip, the cells are compressed and undergo rapid mechanical deformation, thereby creating transient pores on the cell membrane surface, which promotes the passive diffusion of the drug to be loaded into the cells, thus obtaining a drug delivery carrier based on neutrophils.

[0046] The microfluidic chip has two cylindrical channels, inlet 1 and outlet 5, both with a diameter of 1 mm. Both inlets and outlets are connected using PTFE capillaries with an inner diameter of 0.8 mm and an outer diameter of 1.2 mm. At the inlet, the PTFE capillary is connected to a flat-head needle with an outer diameter of 0.9 mm. The cell volume is approximately 5 × 10⁻⁶ cells / day. 6 A suspension of neutrophils was mixed with the drug to be loaded and loaded into a syringe. The syringe was then connected to a flat-nosed needle to form a pathway. A syringe pump was used to push the liquid into the inlet 1 of the microfluidic chip at a flow rate of 150 μL / min. After passing through the first transition zone 2, the liquid entered the microfluidic channel array 3. After undergoing a cyclic mechanical stimulation of "squeeze-elastic recovery-re-squeeze-re-elastic recovery" by the microfluidic channel array 3, the liquid collected from the second transition zone 4 to the outlet 5. Cells were collected at the outlet 5 using centrifuge tubes. All instruments and materials used in the experiment needed to be sterilized by exposing them to high-intensity ultraviolet light before the experiment. The cell loading process took 8 minutes.

[0047] 2.3 Comparative experiment on the size of the compression slit and the flow rate of the medicine:

[0048] For cell delivery technologies based on mechanical deformation to achieve drug delivery, the key is the minimum compression size (slit size) of the cell compression zone. According to literature reports, when cells pass through a contraction zone 30%–80% smaller than their diameter, mechanical deformation occurs, inducing transient pores on the cell membrane under compressive and shear forces, thus effectively facilitating the entry of exogenous substances into the cell. To ensure optimal delivery efficiency with acceptable cell recovery and viability, we used HL-60 cells as a model and optimized the conditions by varying the slit size and flow rate. In Example 1, four microfluidic chips with slit sizes of 4µm, 6µm, 8µm, and 10µm were prepared. In preliminary experiments, it was found that when using a chip with a slit size of 4µm for drug delivery, less than 3% of the recovered cells were intact under microscopic examination, and there were a large number of cell debris. For HL-60 cells with a cell diameter in the range of 13.58±1.64µm, using a chip with a slit size of 4µm would cause excessive compression, leading to cell death and the generation of a large number of cell debris, which could easily cause microchannel blockage, thus affecting the stability and reproducibility of the experiment. Therefore, the 4µm slit chip was not further optimized.

[0049] In this embodiment, FITC-Dextran with a molecular weight of 4 kDa and a final concentration of 300 μg / mL was used for drug delivery. Comparative experiments were conducted using three microfluidic chips with slit sizes of 6 µm, 8 µm, and 10 µm, and four drug flow rates of 100 µL / min, 125 µL / min, 150 µL / min, and 175 µL / min. Flow cytometry results are as follows: Figure 4 As shown:

[0050] Figure 4 The experiment illustrates the delivery of FITC-Dextran (4 kDa molecular weight) to HL-60 cells at a final concentration of 300 µg / mL using three independent microfluidic chips, each with slit sizes of 6 µm, 8 µm, and 10 µm, at four different flow rates: 100 µL / min, 125 µL / min, 150 µL / min, and 175 µL / min. Figure 4 The control group consisted of HL-60 cell suspensions containing FITC-Dextran with a final concentration of 300 µg / mL and a molecular weight of 4 kDa, but without microfluidic chip processing.

[0051] from Figure 4 As can be seen, when using a chip with a slit width of 6 μm, at a flow rate of 175 µL / min, the delivery efficiency can reach 95.8%, and the survival rate of intact cells can reach 99.8%.

[0052] When using a chip with a slit width of 8 μm, at a flow rate of 175 µL / min, the delivery efficiency can reach 84.1%, and the survival rate of intact cells can reach 99.5%.

[0053] When using a chip with a slit width of 10 μm, at a flow rate of 175 µL / min, the delivery efficiency can reach 60.2%, and the survival rate of intact cells can reach 99.3%.

[0054] The statistical results of delivery efficiency for each comparison group in this embodiment are as follows: Figure 5 As shown:

[0055] When the slit size is 10 μm, the cell deformation is small and the change in membrane permeability is limited, resulting in low delivery efficiency of target molecules. Except for the flow rate of 175 µL / min, the average values ​​are less than 35%, namely 16.27±6.48%, 23.7±15.02%, and 32.33±3.93%, respectively.

[0056] When the slit size is 8 μm, the cells are subjected to moderate mechanical compression, which significantly enhances membrane permeability and increases delivery efficiency to over 45%. At flow rates of 125 µL / min, 150 µL / min, and 175 µL / min, the efficiency can reach 64.43 ± 22.44%, 74.3 ± 9.02%, and 69.8 ± 18.11%, respectively.

[0057] When the slit size was 6 μm, cell deformation reached its maximum, and the delivery efficiency increased to 84.4±7.54%, 87.87±4.54%, and 91.33±3.89% at flow rates of 125 µL / min, 150 µL / min, and 175 µL / min, respectively. This indicates that within a certain flow rate range, a smaller microchannel width can enhance cell deformation, increase membrane permeability, and thus improve molecular delivery efficiency.

[0058] In this embodiment, we consider the FITC positivity rate as delivery efficiency (P delivery) and take into account the effect of cell recovery rate (P recovery) in subsequent statistics, thus defining loading efficiency (P loading = P delivery × Precovery). Further comprehensive consideration of cell loading efficiency is then given. The statistical results of cell loading efficiency for each comparative group in this embodiment are as follows: Figure 6 As shown, the cell loading efficiency is relatively high (0.38%) when the slit size is 8 μm and the flow rate is between 125 µL / min and 175 µL / min. ~ 0.44).

[0059] Example 3: Preparation of neutrophil drug delivery carriers using FITC-BSA as the drug to be loaded:

[0060] 3.1. HL-60 cell differentiation was induced using 1.25% DMSO. DMSO was directly added to the HL-60 cell suspension cultured in RPMI-1640 complete medium. Differentiation was carried out for 5 days in a 37°C incubator containing 5.0% CO2, without changing the medium during this period. Figure 7 As shown, HL-60 cells transformed into neutrophil-like cells (dHL-60) after 5 days of DMSO chemical induction. Neutrophil-like cells (dHL-60) serve as a neutrophil replacement model. The expression level of CD11b in HL-60 cells after DMSO induction was detected by flow cytometry, and the results are shown below. Figure 8As shown in the figure, CD11b is one of the characteristic markers of neutrophils, mediating their chemotaxis and phagocytosis. The CD11b-positive cell rate of the treated neutrophil-like cells (98.1%) was much higher than that of the unstained control group and the HL-60 isotype control group, indicating that HL-60 cells were induced to transform into neutrophils. This transformation method is a classic method for obtaining mature neutrophils using HL-60 cells, and has been reported in previous literature.

[0061] 3.2 A microfluidic chip with an 8 μm slit size, prepared in Example 1, was used to prepare a drug delivery carrier for neutrophils at a flow rate of 150 µL / min. 300 µg / mL FITC-BSA was selected as the drug to be loaded, and the drug delivery to neutrophils was detected. The cell flow cytometry results are as follows: Figure 9 As shown in the figure. The results showed that FITC-positive neutrophils accounted for 53.1% of the results in a single experiment, and the average delivery efficiency across multiple experiments was 55.93 ± 19.7%.

[0062] Example 4: Preparation of neutrophil drug delivery carriers using PTX-ALB as the drug to be loaded:

[0063] 4.1 Preparation of neutrophil-like cells is the same as in 3.1.

[0064] 4.2 A neutrophil-based drug delivery carrier was prepared using a microfluidic chip with an 8 μm slit size, fabricated in Example 1, at a flow rate of 150 µL / min. 300 µg / mL PTX-ALB was selected as the drug to be loaded. The drug loading capacity of neutrophils was estimated by detecting the ALB content in the cell lysate using ELISA. The PTX-ALB loading capacity of neutrophils was 784.20 ± 74.6 ng / 10⁻¹². 5 In cells, the mass ratio of ALB to PTX in the drug PTX-ALB to be loaded is 10:1, and the content of the antitumor active ingredient PTX is approximately 0.09 nmol / 10 cells. 5 Individual cells. For example... Figure 10 As shown.

[0065] PTX-ALB is a special formulation of paclitaxel, primarily achieved by binding paclitaxel to albumin nanoparticles. Paclitaxel possesses broad-spectrum antitumor activity and can be used to treat various malignant tumors, including pancreatic cancer and glioma. PTX-ALB can improve the solubility and bioavailability of paclitaxel, thereby further enhancing its antitumor effects.

[0066] Example 5: Treatment of glioma cells at the cellular level using a neutrophil-based drug delivery vehicle:

[0067] The operation flow diagram of this embodiment is as follows: Figure 11 As shown in the figure. This embodiment evaluated the antitumor activity of PTX-ALB-loaded neutrophils and U87-eGFP glioma cells under co-culture conditions. Since neutrophils are suspension cells, they can be removed by washing after co-incubation, and adherent U87-eGFP cells can be viable using the standard MTT assay. In this embodiment, we define one unit (1U) of neutrophils as approximately 4 × 10⁻⁶ cells. 5 100 µL of cells per well. After calculation, 1 U of PTX-ALB-loaded neutrophilic cells is equivalent to a PTX concentration of approximately 3.6 µM (2.5 U of cells is equivalent to a PTX concentration of approximately 9 µM).

[0068] We measured the relative cell viability of U87-eGFP after treatment with 1 U cells for 48 hours, 2.5 U cells for 48 hours, and 1 U cells for 72 hours, respectively. Figure 12 As shown in the results, when unloaded neutrophils were added, the relative activity of U87-eGFP cells was not significantly different from that of the control group, indicating that unloaded neutrophils had little effect on tumor cells. Figure 12 The vertical axis represents the percentage of relative cell activity, from... Figure 12 It is evident that extending the treatment time of PTX-ALB-loaded neutrophils to 72 hours or increasing the number of drug-loaded cells to 2.5 U significantly reduced the relative activity of U87-eGFP cells, by 62±3% and 80±7%, respectively. This indicates that PTX-ALB-loaded neutrophils have a therapeutic effect on U87-eGFP glioma cells.

[0069] Simultaneously, cell morphology and green fluorescent protein expression were observed using fluorescence microscopy, and the results are as follows: Figure 13 As shown, it can also be seen that U87-eGFP cells in the group with added unloaded neutrophils (as shown) Figure 13 The middle image (of which) still shows clear morphology and bright green fluorescent protein expression. However, when 1U of PTX-ALB-loaded neutrophils (…) were added… Figure 13 (See right figure) After 48 hours of treatment, the relative viability of U87-eGFP cells decreased, but not significantly (e.g., ...). Figure 12 As shown), and the expression of green fluorescent protein in U87-eGFP cells is weakened ( Figure 13 (The right image is shown), but it is not significant, and cell morphology can still be observed.

[0070] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0071] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A microfluidic channel for squeezing cells, characterized in that, The microfluidic channel is composed of several cell extrusion units arranged continuously. Each cell extrusion unit consists of a main channel and an extrusion zone (8). The main channel consists of a connecting section (6) and a curved section (7). The connecting section (6) and the curved section (7) have the same width. One end of the curved section (7) is connected to the connecting section (6), and the other end is connected to the extrusion zone (8). The extrusion zone includes an extrusion section one (9), an extrusion slit (11), and an extrusion section two (10). The curved section (7) is connected to the extrusion section one (9). One end of the extrusion slit (11) is connected to extrusion section one (9), and the other end is connected to extrusion section two (10); the width of extrusion section one (9) gradually decreases from the main channel to the minimum at extrusion slit (11), and then gradually increases at extrusion section two (10) until it is consistent with the main channel; one end of extrusion section two (10) is connected to extrusion slit (11), and the other end is connected to the main channel connecting section of the next extrusion unit; the extrusion slit (11) is rectangular, with a width of 4-10μm and a length of 20-60μm.

2. The microfluidic channel for squeezing cells according to claim 1, characterized in that, The curved section (7) is a serpentine channel formed by several semi-circular channels arranged alternately and connected vertically.

3. The microfluidic channel for squeezing cells according to claim 1, characterized in that, The width of the extrusion section 1 (9) and extrusion section 2 (10) gradually decreases in a step-like manner, divided into five levels. The first level is connected to the main channel and has the same width as the main channel. The fifth level (15) has an opening at one end connected to the extrusion slit (11). From the first level to the fifth level, the width gradually decreases.

4. The microfluidic channel for squeezing cells according to claim 1, characterized in that, The microfluidic channel is composed of 10 cell compression units arranged in a continuous sequence.

5. The microfluidic channel for squeezing cells according to claim 1, characterized in that, The width of the main channel is 100μm.

6. The microfluidic channel for squeezing cells according to claim 2, characterized in that, The semi-circular channel has a radius of 200 μm and a width of 100 μm.

7. The microfluidic channel for squeezing cells according to claim 3, characterized in that, The first stage of the extrusion section 1 (9) and the second stage of the extrusion section 2 (10) has a width of 100 μm; the second stage (12) has a width of 80 μm and a length of 50 μm; the third stage (13) has a width of 60 μm and a length of 50 μm; the fourth stage (14) has a width of 40 μm and a length of 50 μm; and the fifth stage (15) has a width of 20 μm and a length of 50 μm.

8. A microfluidic chip for squeezing cells, characterized in that, The microfluidic chip consists of an inlet (1), a first transition region (2), a microfluidic channel array (3), a second transition region (4), and an outlet (5); the microfluidic channel array (3) is formed by several microfluidic channels as described in claim 1 arranged in parallel.

9. The use of the microfluidic channel for squeezing cells as described in any one of claims 1 to 7 in the preparation of a cell drug delivery carrier.

10. The application of the microfluidic chip for squeezing cells as described in claim 8 in the preparation of cell drug delivery carriers.

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