Micro-fluidic lung chip for simulating physiological characteristics of lung of child

Abstract

The invention belongs to the technical field of micro-fluidic chips, and provides a micro-fluidic lung chip for simulating child lung physiological characteristics, and particularly aims at simulating specific respiratory frequencies of children at different growth stages, simulating a tissue barrier interface of pulmonary epithelium and endothelium, and simulating a fluid microenvironment. The micro-fluidic chip is mainly composed of two layers of micro-channels, the channels are separated by a polydimethylsiloxane porous membrane, two sides of each channel are respectively provided with a vacuum cavity, each layer of channel is provided with an inlet and an outlet, and a dynamic environment is formed through fluid. Lung epithelial cells and endothelial cells are respectively inoculated in the upper channel and the lower channel on the chip to form an alveolar-capillary interface on the chip. By applying negative pressure to the vacuum cavity, the respiratory frequencies of children in different growth stages are simulated. The invention provides a multi-factor participated tissue level research system for children lung diseases and children medicine research.

Description

Technical Field

[0001] This invention belongs to the technical field of microfluidic chips, specifically relating to a microfluidic lung chip that simulates the physiological characteristics of children's lungs, its preparation method, and its application. Background Technology

[0002] Disease models play a crucial role in studying the processes and mechanisms of disease development, drug screening and development, and drug efficacy and mechanisms of action. The lack of disease models is a significant reason for the shortage of pediatric drugs. Currently, pediatric drug research and development typically uses young animal models. However, model animals differ considerably from humans at the genetic level and in terms of biological barriers, leading to situations where animal models cannot filter out harmful or ineffective drugs, and potentially effective drug candidates fail to enter clinical trials. To bridge the species gap, human cells are a valuable tool for studying disease phenotypes and pathological mechanisms. However, 2D cell culture differs completely from the in vivo cell growth environment, lacking high-level tissue structure and failing to reflect cell-to-cell interactions and material exchange responses to drugs, resulting in low accuracy in predicting human responses from drug trial results.

[0003] Pneumonia is a common respiratory disease in pediatric clinics and a frequent cause of hospitalization in children in my country. Existing pneumonia disease models significantly limit research on pediatric diseases and drugs. To overcome the limitations of simple traditional models, using microfluidic organ-on-a-chip technology to simulate models with the physiological characteristics of children's lungs is a key technology for accelerating the development of drugs to treat pediatric lung diseases. Organ-on-a-chip technology uses microfluidic chips as carriers and biomaterials such as hydrogels to simulate the three-dimensional microenvironment of cell survival in vitro. It accurately simulates the human body's response to drugs or various external stimuli, making it a good alternative for predicting the human body's response to drugs and various external stimuli.

[0004] However, most existing organ-on-a-chip systems are still designed based on adult physiological parameters, and their key parameters such as mechanical stimulation (e.g., respiratory rate) do not reflect the specific characteristics of the pediatric population. Children's physiological functions differ systematically from adults. Taking the respiratory system as an example, children (especially infants) have a significantly higher respiratory rate than adults. The resting respiratory rate of newborns can reach 30-60 breaths / minute (0.5–1.0 Hz), while that of toddlers is approximately 24-40 breaths / minute, far exceeding the 12-20 breaths / minute (0.2–0.33 Hz) of adults. This difference not only affects gas exchange efficiency but is also closely related to processes such as airway dynamics, particulate matter deposition, drug delivery, and inflammatory responses. Directly using chip models based on adult physiological conditions to study pediatric diseases and drug responses makes it difficult to accurately simulate the microenvironment and pathological state of children's lungs, thus affecting the predictive value of drug screening and toxicological evaluation.

[0005] Therefore, the present invention aims to provide a microfluidic lung chip that simulates the physiological characteristics of children's lungs, simulating the high respiratory rate unique to children, constructing the tissue barrier interface between the lung epithelium and endothelium, simulating the fluid microenvironment, and studying the effects of drugs on lung cell function, tissue barrier, and release of inflammatory factors in a pneumonia model. Summary of the Invention

[0006] The purpose of this invention is to provide a microfluidic lung chip that simulates the physiological characteristics of children's lungs, simulates the respiratory rate of children at different growth stages, constructs the tissue barrier interface between the lung epithelium and endothelium, simulates the fluid microenvironment, and studies the effects of drugs on lung cell function, tissue barrier, and release of inflammatory factors in a pneumonia model.

[0007] The microfluidic lung chip comprises an upper chip and a lower chip, which are separated by a porous polydimethylsiloxane (PDMS) membrane and bonded together by oxygen plasma treatment, forming a rectangular chip shape.

[0008] The upper chip consists of a lung channel and a lung epithelial cell inlet pool 2 and a lung epithelial cell outlet pool 5, while the lower chip consists of a vascular channel and a vascular endothelial cell inlet pool 1 and a vascular endothelial cell outlet pool 6.

[0009] Vacuum chambers are provided on both sides of the channel. Applying negative pressure in the vacuum chambers can achieve mechanical stretching of the intermediate PDMS porous membrane.

[0010] Both the upper and lower substrates are PDMS. The lung channel has a cross-sectional height of 0.25 mm and a width of 1 mm, and the vascular channel has a cross-sectional height of 0.25 mm and a width of 1 mm. The vacuum cavities on both sides of the channels have a height of 0.25 mm and a width of 1.18 mm. The PDMS porous membrane is 25 μm thick and has pores with a diameter of 8 μm and a pore spacing of 40 μm. The PDMS porous membrane is coated with a mixture of type I collagen and matrix gel to provide a near-physiological growth microenvironment for cells.

[0011] Furthermore, the fabrication method of the microfluidic lung chip includes the following steps: (1) Chip mold design and fabrication: A microfluidic lung chip mold was designed using SoldWorks and fabricated using a 3D printer; the mold was then placed in a UV curing chamber and cured for 30 min. (2) Chip fabrication: The base material and curing agent were mixed at a weight ratio of 10:1 and degassed in a vacuum for 30 min. The degassed base material and curing agent mixture was then poured into molds with multiple channel patterns. The mixture was then heat-cured in an oven at 60 ℃ for 1.5 h. The upper and lower chip structures with channels were obtained by demolding. Holes were drilled in the upper chip structure, and the upper chip and PDMS porous membrane were cleaned with oxygen plasma for 90 seconds. The cleaned upper chip and PDMS porous membrane were then bonded together and heat-cured in a 60 ℃ oven for 8-12 hours. The upper chip and PDMS porous membrane composition were further subjected to oxygen plasma cleaning for 90 s with the lower chip. The cleaned composition and the lower chip were then bonded together and thermocured in an oven at 60 ℃ for 8 h-12 h. (3) Chip preprocessing: After sterilization by UV irradiation for 30 min, the chip channels were modified with 50 μg / mL type I collagen and 300 μg / mL matrix gel for 8 h-12 h, and then washed 2-3 times with PBS. (4) Cell inoculation and culture: Adjust the number of lung epithelial cells to 2.5 × 10⁻⁶. 5 A cell suspension of 100 cells / mL was seeded into the upper channel of the chip and incubated at 37°C for 8-10 h; the endothelial cell count was adjusted to 2.5 × 10⁶ cells / mL. 5 Cell suspensions of 100 cells / mL were seeded into the lower channel of the chip. The chip was placed upside down in an incubator for 8-10 hours and then placed upright. Continuous perfusion was performed using a microfluidic driving method. A syringe pump was connected to a vacuum chamber, and the pressure change in the vacuum chamber was controlled to stretch the porous membrane. In addition, the pumping and perfusion frequency of the syringe pump was changed to achieve mechanical stretching of the porous membrane at different frequencies, simulating the respiratory frequency of children at different growth stages.

[0012] The microfluidic lung chip simulating the physiological characteristics of children's lungs as described in this invention, or the microfluidic lung chip simulating the physiological characteristics of children's lungs prepared by the preparation method described in this invention, is used in the preparation of in vitro models for studying the effects of drugs for treating children's lung diseases on lung cell function, tissue barrier, and release of inflammatory factors.

[0013] Preferably, the in vitro model is a pediatric pneumonia in vitro model.

[0014] The present invention has the following beneficial effects: This invention relates to a microfluidic lung chip that can be used for research on pediatric lung diseases and drugs. First, a lung organ-on-a-chip is constructed, incorporating the tissue barrier functions of the lung epithelium and endothelium, a simulated fluid microenvironment, and a lung organ-on-a-chip mimicking the high respiratory rate characteristic of children. This platform is then used to study the effects of drugs on lung cell function, tissue barrier function, and the release of inflammatory factors in a pediatric lung disease model. Attached Figure Description

[0015] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. Hereinafter, embodiments of the invention will be described in detail with reference to the accompanying drawings, wherein: Figure 1 This is a schematic diagram of the overall structure of the microfluidic lung chip of the present invention; Among them, 1 is the vascular endothelial cell inlet pool, 2 is the lung epithelial cell inlet pool, 5 is the lung epithelial cell outlet pool, 6 is the vascular endothelial cell outlet pool, and the two-layer chip channel is separated by a PDMS porous membrane; 3 and 4 are the vacuum chamber and the injection pump connection hole.

[0016] Figure 2 This is a cross-section of a lung chip model; Among them, 7 is the lung channel, 8 is the lung epithelial cells, 9 is the PDMS porous membrane, 10 is the endothelial cells, 11 is the vascular channel, and 12 is the vacuum chamber.

[0017] Figure 3 This is a simulation diagram of the flow velocity and shear stress of fluids with different flow rates in the channel according to Embodiment 1 of the present invention.

[0018] Figure 4 This is a schematic diagram of the simulation results of Embodiment 1 of the present invention. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. 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. Unless otherwise specified, the materials and reagents used are commercially available.

[0020] Example 1: A microfluidic lung chip simulating the physiological characteristics of children's lungs The microfluidic chip designed and fabricated using SoldWorks and a 3D printer is shown in the figure. Figure 1The microfluidic chip is composed of two layers of chips bonded together. The upper chip includes a lung epithelial cell inlet pool 2, a lung epithelial cell outlet pool 5, a vascular endothelial cell inlet pool 1, a vascular endothelial cell outlet pool 6, and vacuum chamber and injection pump connection holes 3 and 4, all of which are externally connected to perfusion tubing. The lung channel has a cross-sectional height of 0.25 mm and a width of 1 mm, and the vascular channel has a cross-sectional height of 0.25 mm and a width of 1 mm. The vacuum cavities on both sides of the channels have a height of 0.25 mm and a width of 1.18 mm. The PDMS porous membrane has a thickness of 25 μm and has pores with a diameter of 8 μm and a pore spacing of 40 μm.

[0021] The specific preparation method is as follows: The base material and curing agent were mixed at a weight ratio of 10:1 and degassed in a vacuum for 30 min. The degassed base material and curing agent mixture was then poured into molds with multiple channel patterns. The mixture was then heat-cured in an oven at 60 ℃ for 1.5 h. The upper and lower chip structures with channels were obtained by demolding. Holes are drilled in the upper chip structure, and the upper chip and PDMS porous membrane are cleaned with oxygen plasma for 90 seconds. The cleaned upper chip and porous membrane are then bonded and heat-cured in a 60 ℃ oven for 8-12 hours. The upper chip and PDMS porous membrane composition were further subjected to oxygen plasma cleaning for 90 s with the lower chip. The cleaned composition and the lower chip were then bonded together and thermocured in an oven at 60 ℃ for 8 h-12 h. Sterilize by UV irradiation, modify chip channels with 50 μg / mL type I collagen and 300 μg / mL matrix gel for 8 h-12 h, and wash with PBS 2-3 times; Adjust the number of lung epithelial cells to 2.5 × 10⁻⁶. 5 Cell suspensions of 100 cells / mL were seeded into the upper channels of the chip and incubated at 37°C for 8-10 h; the endothelial cell count was adjusted to 2.5 × 10⁶ cells / mL. 5 Cell suspensions of 100 cells / mL were seeded into the lower channel of the chip. The chip was placed upside down in an incubator for 8-10 hours and then placed upright. Continuous perfusion was performed using a microfluidic actuation method. A syringe pump was connected to a vacuum chamber to control the pressure changes in the vacuum chamber and mechanically stretch the porous membrane at 0.5 Hz to simulate the respiratory rate of children aged 1-3 years.

[0022] Example 2: Simulation of internal parameters of lung organ-on-a-chip perfusion flow rate and shear stress in lung organ-on-a-chip, such as Figure 3 and Figure 4As shown, the biomimetic conditions of the internal physical parameters of the lung organ-on-a-chip were simulated using finite element analysis (COMSOL MultiPhysical 5.6), and a suitable perfusion flow rate was determined. The perfusion flow rate of the lung epithelial channel can be set to 27 μL / h, producing 0.05 dynes / cm². 2 To match the shear stress experienced by human lung epithelial cells, the vascular perfusion flow rate can be set to 270 μL / h, producing 0.5 dynes / cm². 2 It conforms to the shear stress experienced by vascular endothelial cells.

[0023] Example 3: To demonstrate that the microfluidic lung chip described in this invention has physiological differences from that of adults, a comparative study was conducted on the chip's inflammatory stress response. (1) Experimental groups: ① Simulated child respiratory rate group: 0.5 Hz periodic mechanical stretching was applied to the PDMS porous membrane to simulate the respiratory rate of children aged 1-3 years.

[0024] ② Simulated adult respiratory rate group: Periodic mechanical stretching of 0.2 Hz was applied to the intermediate PDMS porous membrane to simulate the respiratory rate of an adult.

[0025] (2) Content: To simulate lung infection, 1 μg / mL of bacterial lipopolysaccharide (LPS) was added to the perfusion fluid of the chip vascular channel in all groups for 24 hours to stimulate them, while maintaining their respective mechanical stretching frequencies.

[0026] After the stimulation ended, the effluent from the vascular channels of each chip was collected.

[0027] The concentration of interleukin-6 (IL-6), a key pro-inflammatory factor, in the effluent was detected using an enzyme-linked immunosorbent assay (ELISA) kit.

[0028] (3) Results: After LPS stimulation, the IL-6 content released by the chip in the children's frequency group (0.5 Hz) (646.48 pg / mL) was significantly higher than that released by the chip in the adult frequency group (0.2 Hz) (389.106 pg / mL). This result indicates that under the same pathogen stimulation, the chip simulating the respiratory rate of children produced a more severe inflammatory response, which is highly consistent with the clinical observation that children with lung infections often have a more rapid and intense inflammatory response.

[0029] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several modifications or equivalent substitutions can be made to the technical solution without departing from the principle of the present invention, and these modifications or equivalent substitutions should also be considered within the scope of protection of the present invention.

Claims

1. A microfluidic lung-on-a-chip that mimics the physiological characteristics of a child's lung, characterized in that, The microfluidic chip simulates the respiratory frequency of children in different growth stages by constructing a tissue barrier interface of lung epithelium and endothelium and simulating a fluid microenvironment.

2. The microfluidic lung-on-a-chip simulating the physiological features of children's lung according to claim 1, characterized in that, The microfluidic lung chip simulates the respiratory frequency of children in different growth stages by applying negative pressure to the two vacuum cavities to realize mechanical stretching of the middle porous membrane at a frequency of 0.5-0.75 Hz.

3. The microfluidic lung-on-a-chip simulating the physiological features of children's lung according to claim 2, characterized in that, The porous membrane is mechanically stretched at a frequency of 0.5 Hz to simulate the respiratory frequency of children aged 1-3 years.

4. The microfluidic lung-on-a-chip simulating the physiological features of children's lung according to claim 2, characterized in that, The microfluidic chip is bonded by an upper chip and a lower chip, and the upper chip comprises a lung epithelial cell inlet pool (2), a lung epithelial cell outlet pool (5), a vascular endothelial cell inlet pool (1), a vascular endothelial cell outlet pool (6), and vacuum cavity and injection pump connection holes (3) and (4), which are all externally connected to perfusion tubes.

5. The microfluidic lung-on-a-chip simulating the physiological features of children's lung according to claim 4, characterized in that, The microfluidic chip comprises a lung channel and a blood vessel channel, and the two channels are separated by a polydimethylsiloxane (PDMS) porous membrane, and the cells comprise lung epithelial cells and vascular endothelial cells.

6. The microfluidic lung-on-a-chip simulating the physiological features of children's lung according to claim 5, characterized in that, The cross-sectional height of the lung channel is 0.25 mm, and the width is 1 mm; the cross-sectional height of the blood vessel channel is 0.25 mm, and the width is 1 mm; the vacuum cavities on both sides of the channels have a height of 0.25 mm and a width of 1.18 mm; the PDMS porous membrane has a thickness of 25 μm, and has pores with a diameter of 8 μm and a pore spacing of 40 μm; and the PDMS porous membrane is coated with a mixture of type I collagen and Matrigel to provide a growth microenvironment close to the physiological state for the cells.

7. The method of claim 1 to 6 for the fabrication of a microfluidic lung-on-a-chip mimicking the physiological features of the child's lung, characterized in that, The preparation method comprises the following steps: (1) Chip mold design and preparation: Use SoldWorks to design a microfluidic lung chip mold, and use a 3D printer to make the mold; place the mold in a UV curing box and cure for 30 min; (2) Chip preparation: Mix the base material and the curing agent in a weight ratio of 10:1, and degas in a vacuum for 30 min; pour the degassed base material and the curing agent mixture onto the mold with a plurality of channel patterns pre-set thereon; heat and solidify in a 60 ℃ oven for 1.5 h; and demold to obtain an upper chip structure and a lower chip structure with channels; Punch holes in the upper chip structure, and perform oxygen plasma cleaning on the upper chip and the PDMS porous membrane for 90 s; bond the cleaned upper chip and the PDMS porous membrane, and heat and solidify in a 60 ℃ oven for 8-12 h; Further oxygen plasma clean the upper chip and the PDMS porous membrane composition and the lower chip for 90 s, and bond the cleaned composition and the lower chip, and heat and solidify in a 60 ℃ oven for 8-12 h; (3) Chip pretreatment: Ultraviolet irradiation sterilization for 30 min, modify the chip channel with 50 μg / mL type I collagen and 300 μg / mL Matrigel for 8-12 h, and wash with PBS for 2-3 times; (4) Cell inoculation and culture: Pulmonary epithelial cells were adjusted to 2.5 x 10 5 cells / mL cell suspension and seeded on the upper channel of the chip and incubated at 37 °C for 8-10 h; endothelial cells were adjusted to 2.5 x 10 5 cells / mL cell suspension and seeded on the lower channel of the chip, the chip was placed upside down in the incubator for 8-10 h and then placed right side up; microfluidic driving method was used for continuous perfusion; a syringe pump was connected to the vacuum chamber to control the change of air pressure in the vacuum chamber, thereby realizing the stretching of the porous membrane; in addition, changing the pumping frequency of the syringe pump realized the mechanical stretching of the porous membrane at different frequencies, simulating the breathing frequency of children at different growth stages.

8. The method of claim 7, wherein: The microfluidic lung chip realizes mechanical stretching of the middle porous membrane at a frequency of 0.5-0.75 Hz by applying negative pressure to the two side vacuum cavities, thereby simulating the breathing frequency of children at different growth stages, the lung epithelial channel perfusion flow is set to 27 μL / h, and 0.05 dyne / cm 2 The shear stress conforms to that borne by human lung epithelial cells, and the vascular channel perfusion flow is set to 270 μL / h, and 0.5 dyne / cm 2 The shear stress conforms to that borne by vascular endothelial cells.

9. Use of the microfluidic lung-on-a-chip simulating the physiological features of a child's lung according to any one of claims 1 to 6 or of the microfluidic lung-on-a-chip simulating the physiological features of a child's lung prepared by the method according to claim 7 or claim 8 for the preparation of an in vitro model for the study of the effects of drugs for the treatment of lung diseases in children on lung cell function, tissue barrier, inflammatory factor release.

10. Use according to claim 9, characterized in that: The in vitro model is an in vitro model of child pneumonia.

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