CN112143642B

CN112143642B - Vascularized tumor micro-fluidic organ chip for in vitro culture and preparation method thereof

Info

Publication number: CN112143642B
Application number: CN202010884867.3A
Authority: CN
Inventors: 王晓林; 李钦宇
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2020-08-28
Filing date: 2020-08-28
Publication date: 2022-06-14
Anticipated expiration: 2040-08-28
Also published as: CN112143642A

Abstract

The invention provides a vascularized tumor micro-fluidic organ chip for in vitro culture and a preparation method thereof, wherein the chip comprises: the PMMA module is provided with three through holes penetrating through the PMMA module in the thickness direction; the upper surface of the glass sheet and the periphery of the lower surface of the PMMA module are bonded into a whole through a bonding layer, so that a first chamber for containing the tumor pellets, a second chamber for storing a culture solution and a third chamber are formed by the three through holes respectively; the middle area of the lower surface of the PMMA module and the glass sheet form a hollow tissue chamber; the static pressure difference generated by the culture solution with the liquid level height difference can promote the generation of a three-dimensional capillary network, and the recruitment of capillaries can be realized through the co-culture of the three-dimensional capillary network and the tumor pellets, so that the three-dimensional vascularized tumor micro-tissue is constructed. The invention overcomes the defects of time consumption and high cost of the prior microfluidic organ chip manufacturing, has simple process, and especially provides application value for the research of in vitro vascularization tumor diseases.

Description

Vascularized tumor micro-fluidic organ chip for in vitro culture and preparation method thereof

Technical Field

The invention relates to the technical field of biomedical engineering, in particular to a vascularized tumor microfluidic organ chip for in vitro culture and a preparation method thereof.

Background

The vascular system is important in the transport of nutrients, oxygen and waste products within the human body as a facilitator of the exchange of substances between local tissues and the systemic circulation. Due to its own functionality and complexity, with the development of tissue engineering, the study of the vascular system has profound applications in the therapeutic engineering of various vascular-related diseases, and simultaneously provides a basis for potential drug screening methods.

At present, cancer is one of the biggest challenges facing the health field all over the world, and the drug efficacy evaluated by the traditional animal model cannot predict the influence of the drug efficacy on the human body, so that the development of a new platform for treating tumor diseases in vitro is urgent. The nutrients required by tumors are usually transported by the blood stream in their peripheral blood vessels, and the mechanism of constructing a fused tumor vascular network has become one of the leading directions in the field of cancer treatment. In constructing a vascularized tumor network, endothelial cells are a common cell type that transport nutrients and oxygen to the tumor cells via the blood stream. Therefore, there is a need for a way to build up Tumor Microenvironment (TME) in three-dimensional culture of tumor cells in vitro. In a specific in-vitro three-dimensional environment, cancer cells can be enriched into spheroids, which have high similarity with tumors in a human body in aspects of morphology, cell density and physical environment, are favorable for shortening the research and development period of new anti-cancer drugs, and the appearance of vascularized tumor chips provides a platform for reconstructing in-vitro tumor microenvironment models, and has profound application in the treatment of vascular related diseases and the screening of anti-cancer drugs.

The traditional biochip manufacturing technology is widely applied to constructing in-vitro three-dimensional blood vessel networks based on hydrogel, and PDMS has become one of the most applied materials in microfluidic chips due to the advantages of good light transmittance, easy processing and forming, high biocompatibility, excellent air permeability and the like, and angiogenesis is cooperatively induced by micro-patterning, growth factor gradient and cell co-culture, so that the physiological function in vivo can be better simulated. By co-culturing human lung fibroblast (NHLF) and Human Umbilical Vein Endothelial Cell (HUVEC), the vascular network can be formed by the proliferation, differentiation and connection of HUVEC under the induction action of Vascular Endothelial Growth Factor (VEGF), and the generation of vascular lumen can be realized after in vitro culture for several days.

The proliferation and differentiation of cells need to be cooperatively controlled by various external complex factors, and Organ-on-Chips (OoC) combine the advantages of the microfluidic technology and the cell biology field, thereby having outstanding advantages in the aspect of controlling the external factors and parameters. Because of following the most basic principle of constructing organ chips, the functional OoC can greatly shorten the time of drug development, design specific vascularization chip structures aiming at different organoids, and reflect and analyze the physiological functions of the organoids.

Through the search of the existing microfluidic chip process, chips for simulating lung respiration, kidney, liver tissue, heart, eyeball, brain tissue and the like are developed and used for realizing the accurate control of biochemical signals and electrophysiological signals in a human body by combining a microelectronic technology and a micro-processing technology in recent years, but how to balance time and cost in a manufacturing method is still a big problem. The common traditional PDMS chip process relates to an MEMS photoetching process and a subsequent molding method, and has long time consumption and high cost; the 3D printing method based on the biocompatible material greatly shortens the problem of time consumption in manufacturing, overcomes the defect that PDMS absorbs small molecules, and is still expensive in cost, especially in the process of pursuing high-precision chips.

In the previous reports, the vascularized chip platform for drug screening has received much attention, but improvements to the chip preparation method are still quite limited. Duc t.t.phan, Brianna m.craver et al in Lab Chip,2017, 17: 511-520 written "avasculated and saturated organ-on-a-chip platform for large-scale drug screening", and proposed a high-throughput in vitro 3D organ chip platform capable of combining with a 96-well plate, wherein the surface of a PDMS chip and the bottom of the well plate are aligned and bonded by chemical surface treatment, angiogenesis mode under static hydraulic pressure difference stimulation is realized by using different heights of culture solution in the well, and FDA-approved anti-tumor drugs are delivered by a vascular network, so that large-scale drug screening can be performed on a plurality of cancer cells.

The construction of personalized tumor organ chips has been the focus of attention of researchers in this field, and y.nashimoto, t.hayashi et al, integrated Biology,2017,9(6):506-518, written above, "integrated durable vascular networks with a high-molecular-dimensional tissue a microfluidic device", describes a microfluidic platform perfusable by vascular networks, which proves that the vascular networks transport small molecular substances into the interior of the spherical tissues, but it still follows the PDMS chip process, and the content of the study is limited to angiogenesis (angiogenesis) in which endothelial cells grow from both side channels, and lacks related studies on angiogenesis (vasculogenesis) around the tumor tissues.

Because the process of the traditional PDMS Chip is time-consuming, and the PDMS material is easy to absorb small molecules to influence drug screening, J.Ko, J.Ahn et al write a word "Tumor-on-a-Chip: a-stabilized microfluidic culture platform for inducing Tumor angiogenesis" on Lab Chip,2019,19:2822-2833 by an injection molding method, so as to construct an open vascularized Tumor platform capable of being combined with a 96-well plate, and solve the problem of small molecule absorption of PDMS, each structural unit of the platform is provided with two culture solution reservoirs and a circular through hole for placing a 3D Tumor pellet, so that the accurate positioning of a Tumor and the angiogenesis induced by the Tumor can be realized, but the whole platform is limited by higher printing and manufacturing cost.

In summary, ideal compromise between time and cost is not realized in the manufacturing of the in vitro vascularized tumor microfluidic platform reported at present, the traditional process and the emerging 3D printing technology have own defects, and meanwhile, a personalized in vitro vascularized chip structure is urgently needed to be realized for different tumor spheres cultured in 3D.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a vascularized tumor microfluidic organ chip for in vitro culture and a preparation method thereof.

The invention provides a vascularized tumor microfluidic organ chip for in vitro culture, which comprises:

the device comprises a Polymethyl Methacrylate Module (PMMA), wherein the PMMA module is provided with a first through hole, a second through hole and a third through hole which penetrate through the PMMA module in the thickness direction, and the second through hole and the third through hole are respectively arranged at two sides of the first through hole;

the glass sheet is arranged below the polymethyl methacrylate module, the upper surface of the glass sheet and the periphery of the lower surface of the polymethyl methacrylate module are bonded into a whole through a bonding layer, and a first cavity for containing the tumor pellets, a second cavity for storing a culture solution and a third cavity are formed by the first through hole, the second through hole and the third through hole respectively; the middle area of the lower surface of the polymethyl methacrylate module and the glass sheet form a hollow tissue cavity, and fibrin gel or collagen gel is filled in the tissue cavity; the tissue chamber communicates the first chamber with the second chamber and the bottom of the third chamber; by injecting culture solution with different heights into the second chamber and the third chamber, the static pressure difference generated by the culture solution with liquid level height difference can promote the generation of a three-dimensional capillary network in the tissue chamber, and the recruitment of capillaries can be realized through the co-culture of the three-dimensional capillary network and the tumor pellets, so that the three-dimensional vascularized tumor micro-tissue is constructed.

Preferably, the periphery of the bottom of the polymethyl methacrylate module is encapsulated by PDMS to prevent liquid leakage.

Preferably, the polymethylmethacrylate module has a rectangular structure.

Preferably, a culture cover is disposed on the top of the pmma module and covers the first through hole, the second through hole and the third through hole to prevent the tumor pellet or the culture solution from directly contacting with the external environment to cause contamination.

Preferably, the number of the first through holes is multiple, and the first through holes are used for accommodating different types of tumor pellets.

Preferably, the cross section of the first through hole is circular or elliptical.

Preferably, the tissue chamber is circular or rectangular.

Preferably, the thickness of the glass sheet is 0.5mm to 1.5 mm.

Preferably, the thickness of the adhesive layer is 100 μm to 500 μm.

Preferably, the thickness of the polymethyl methacrylate module is 6mm to 15 mm.

The second aspect of the present invention provides a method for preparing the vascularized tumor microfluidic organ chip for in vitro culture, which comprises:

the manufacturing method comprises the following steps of (1) manufacturing a polymethyl methacrylate module by utilizing laser cutting of a polymethyl methacrylate plate, namely forming a first through hole in the middle, and a second through hole and a third through hole which are respectively positioned at two sides of the first through hole on the polymethyl methacrylate module;

soaking the cut polymethyl methacrylate module in deionized water, and carrying out ultrasonic cleaning;

drying the cleaned polymethyl methacrylate module, and then wiping the module with alcohol for disinfection;

cleaning and drying the glass sheet by adopting ultrasonic waves;

tightly attaching a bonding layer to the upper surface of the cleaned glass sheet, imaging the bonding layer, and cutting off the bonding layer in the middle area through the imaged bonding layer to form a hollow quadrangle;

aligning a polymethyl methacrylate module to the patterned bonding layer, tightly bonding the periphery of the lower surface of the polymethyl methacrylate module to the upper surface of the glass sheet, bonding the polymethyl methacrylate module and the glass sheet into a whole, and reserving a set height between the middle area of the lower surface of the polymethyl methacrylate module and the glass sheet as a tissue chamber to obtain the tumor vascularization microfluidic chip for in vitro culture.

Preferably, after obtaining the vascularized tumor microfluidic organ chip for in vitro culture, the method further comprises:

uniformly mixing Polydimethylsiloxane (PDMS) prepolymer with a curing agent according to a set proportion, and removing bubbles in the mixture in a vacuum pump to obtain a polydimethylsiloxane packaging material;

and coating a polydimethylsiloxane packaging material on the periphery of the bottom of the polymethyl methacrylate module for sealing, and then putting the module into an oven to cure the polydimethylsiloxane packaging material.

Preferably, after the polymethyl methacrylate plate is cut and shaped by using laser, the method further comprises the following steps:

and manufacturing a culture cover, namely cutting a polymethyl methacrylate plate by adopting laser to form the culture cover, and covering the culture cover above the polymethyl methacrylate module to prevent the tumor pellets or culture solution from directly contacting with the external environment to cause pollution.

Compared with the prior art, the invention has at least one of the following beneficial effects:

the micro-fluidic chip is composed of a glass sheet and a polymethyl methacrylate module, wherein the polymethyl methacrylate module is provided with a first through hole for placing a tumor pellet, a second through hole and a third through hole for storing a culture solution, a static pressure difference generated by the culture solution with a certain liquid level height difference can promote the generation of a three-dimensional capillary network, and the recruitment of capillary vessels can be realized through the co-culture of the three-dimensional capillary network and the tumor pellet, so that the three-dimensional vascularized tumor micro-tissue is finally constructed; the microfluidic chip overcomes the defects of time consumption and high cost in the conventional microfluidic organ chip manufacturing process, and has the advantages of simple processing technology, high operation flexibility and the like. Meanwhile, PMMA does not absorb small drug molecules, so that the PMMA is more favorable for application in drug screening. In addition, based on the organ chip design of the open microfluidic technology, the flexible taking and placing of the tumor pellets and high-flux automatic operation can be realized. In the biomedical engineering field, especially provides innovative application value for the in vitro vascularized tumor disease model construction and the related research of novel anti-tumor drug screening.

According to the micro-fluidic chip, the double-sided adhesive tape (the bonding layer) is arranged on the surface of the glass sheet and cut into a specific shape, and the double-sided adhesive tape is bonded with the polymethyl methacrylate module, so that the height of the tissue cavity between the middle area of the polymethyl methacrylate module and the glass sheet is ensured; and further packaging the periphery of the chip by PDMS packaging material to prevent liquid leakage.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a three-dimensional process flow diagram of a vascularized tumor microfluidic organ chip for in vitro culture according to a preferred embodiment of the present invention;

FIG. 2 is a schematic diagram of the two-dimensional cross-sectional structure of the vascularized tumor microfluidic organ chip for in vitro culture according to a preferred embodiment of the present invention;

FIG. 3 is a schematic structural diagram of a four vascularized tumor PMMA module after laser cutting according to a preferred embodiment of the present invention;

FIGS. 4 a-4 d are 4-fold enlarged photographs of the structure of the blood vessel cultured by using four structural units of the vascularized tumor microfluidic organ chip according to a preferred embodiment of the invention;

FIG. 5a is a photograph of a vascular structure at 40 times magnification in accordance with a preferred embodiment of the present invention;

FIG. 5b is a photograph of the vascular structure at 20 times magnification in accordance with a preferred embodiment of the present invention;

FIG. 6a is a diagram showing the connection of tumor globules and the peripheral vascular network after 4 times of amplification of the vascularized tumor microfluidic organ chip according to a preferred embodiment of the present invention;

FIG. 6b is a drawing of the tumor globules connected to the peripheral vascular network at 10 times magnification corresponding to day 9 of FIG. 6 a;

the scores in FIG. 2 are respectively expressed as: 1 is PMMA module, 2 is glass piece, 3 is first through-hole, 4 is the second through-hole, 5 is the third through-hole, 6 is the tie coat, 7 is the tissue cavity, 8 is the tumour pellet, 9 is the culture solution.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will aid those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any manner. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

Example 1

Referring to fig. 2, a schematic diagram of a two-dimensional cross-sectional structure of a vascularized tumor microfluidic organ chip for in vitro culture according to the present invention is shown, wherein the schematic diagram comprises: PMMA (polymethyl methacrylate) module 1 and glass piece 2, the periphery of the lower surface of PMMA module 1 and the upper surface of glass piece 2 bond as an organic whole.

Referring to fig. 2, the PMMA module 1 is provided with a first through hole 3, a second through hole 4 and a third through hole 5 penetrating through the thickness direction thereof, and the second through hole 4 and the third through hole 5 are respectively disposed at two sides of the first through hole 3.

Referring to fig. 2, the glass sheet 2 is arranged below the PMMA module 1, and the upper surface of the glass sheet 2 and the periphery of the lower surface of the PMMA module 1 are bonded into a whole through the bonding layer 6, so that the lower ends of the first through hole 3, the second through hole 4 and the third through hole 5 are closed, and a first chamber for accommodating the tumor pellet 8, a second chamber for storing the culture solution 9 and a third chamber are respectively formed; the middle area of the lower surface of the PMMA module 1 and the glass sheet 2 form a hollow tissue cavity 7, fibrin gel or collagen gel is filled in the tissue cavity 7, and the tissue cavity 7 provides a space for the generation of a three-dimensional capillary network; the height of the tissue cavities 7 may be 100 μm-500 μm, i.e. the thickness of the adhesive layer 6. The tissue cavity 7 enables the first cavity to be communicated with the bottoms of the second cavity and the third cavity; by injecting the culture solution 9 with different heights into the second chamber and the third chamber, the static pressure difference generated by the culture solution 9 with the liquid level height difference can promote the generation of a three-dimensional capillary network in the tissue chamber 7, and the recruitment of capillaries can be realized through the co-culture of the three-dimensional capillary network and the tumor beads 8, so that the three-dimensional vascularized tumor micro-tissue is constructed. In specific implementation, fibrin gel or collagen gel is filled in the tissue cavity 7 along the bottom of the PMMA module 1 by the action of capillary force, so that non-ideal leakage cannot be generated; after the fibrin gel or the collagen gel is solidified, the tumor globules 8 in the first through holes 3 can slowly settle under the action of gravity; referring to fig. 2, the hydraulic height in the second and third chambers for storing the culture solution 9 is 7mm, the pressure difference generated by the culture medium with the difference in liquid level can stimulate the growth of blood vessels of co-cultured cells in fibrin gel, and the blood capillaries surround and are connected around the tumor globules 8, so as to realize the screening of the antitumor drugs.

The micro-fluidic chip is based on a polymethyl methacrylate (PMMA) material with high biocompatibility, a second chamber and a third chamber for storing the culture solution 9 are formed by laser cutting, and the micro-fluidic chip and the first chamber for placing the tumor pellet 8 can adopt a mode of bonding a 3M double-sided adhesive tape and the glass sheet 2, so that the gel can be ensured to be perfused to the bottom tissue culture chamber by utilizing capillary force.

Referring to fig. 1, the vascularized tumor microfluidic organ chip for in vitro culture can be prepared by the following method, including the following steps:

s1, as shown in fig. 1 (a), washing the glass sheet: and sequentially ultrasonically cleaning the glass sheet for 8min according to the sequence of acetone, ethanol and water, taking out after each cleaning, and drying at 70 ℃.

S2, as shown in fig. 1 (b), a double-sided adhesive tape (adhesive layer) with a thickness of 200 μm is cut according to the designed PMMA module structure size, and the double-sided adhesive tape is closely attached to the upper surface of the cleaned glass sheet.

S3, as shown in fig. 1 (c) and (d), patterning the double-sided adhesive tape according to the designed chip structure unit, specifically, cutting off the middle area of the double-sided adhesive tape by using a scalpel, that is, forming an area with a certain height of middle space by the upper surface of the glass sheet and the double-sided adhesive tape as a tissue cavity, wherein the height of the tissue cavity matches with the thickness of the double-sided adhesive tape; in the implementation, the shape of the patterned double-sided adhesive tape is determined by the shape of the laser-cut PMMA module.

S4, as shown in fig. 1 (e), removing the protective film on the upper surface of the double-sided adhesive, aligning the PMMA module with the patterned double-sided adhesive, and tightly pressing the PMMA module on the glass surface until no bubble or trace bubbles exist in the double-sided adhesive area; when the step is implemented specifically, the PMMA module and the glass sheet are tightly pressed to the greatest extent, so that air bubbles are removed to prevent the culture solution from leaking. The shape of the tissue cavity formed by the PMMA module and the glass sheet can be designed according to requirements in a personalized mode, such as a circular ring shape, a rectangular shape and the like. The PMMA module is made by cutting a polymethyl methacrylate plate by laser, namely a first through hole in the middle, a second through hole and a third through hole which are respectively positioned at two sides of the first through hole are formed in the PMMA module.

S5, as shown in (f) of figure 1, uniformly mixing the PDMS prepolymer and the curing agent according to a ratio of 10:1, removing bubbles in the mixture in a vacuum pump to obtain a PDMS packaging material, coating the PDMS packaging material around the bottom of the PMMA module for sealing to ensure that the culture solution has no leakage phenomenon, and then placing the PMMA packaging material into a 60 ℃ oven for 4h to cure the PDMS packaging material. In particular embodiments, the PDMS prepolymer and the curing agent may be mixed in other ratios.

The preparation method adopted by the embodiment is a vascularized tumor micro-fluidic organ chip for in vitro culture prepared based on a self-assembly method, overcomes the defects of time consumption and high cost of the existing micro-fluidic organ chip preparation, is simple in process, the prepared environment belongs to a fully open environment, the operation flexibility is high, PMMA does not absorb small molecules, drug screening is facilitated, PMMA modules with different shapes and sizes can be freely and flexibly integrated according to specific application, and personalized design and high-flux automatic operation are realized. The preparation method is completely different from the traditional common manufacturing process, the PMMA module is manufactured firstly, namely, the PMMA module is subjected to laser cutting to form a structure with a specific size, then the PMMA module is bonded with the glass sheet through the patterned double-sided adhesive tape, so that the strong bonding force is ensured, the tissue cavity with a certain height is formed spontaneously, and finally the periphery of the chip is packaged by the PDMS, so that the sealing performance of the whole structure is ensured, and the liquid leakage phenomenon is prevented.

Example 2

Referring to fig. 3, four PMMA module structures after laser cutting can be prepared by the following method, so as to realize the diversity of the PMMA module after laser cutting. The PMMA block can be prepared by the following steps:

and S10, designing and drawing a two-dimensional cutting layout by using AutoCAD drawing software, wherein the shape and the size of the structure can be customized according to needs.

S20, cutting the PMMA board by laser according to the designed structure of S10 to obtain a first round through hole for placing a tumor pellet, two second through holes for storing culture solution and a third through hole, and obtaining a PMM A module; the three through holes obtained in the step are respectively penetrated in the thickness direction of the PMMA plate; in one embodiment, the thickness of the polymethylmethacrylate plate is 8mm, the PMMA module after laser cutting has a size parameter of 20mm × 20mm × 8mm (length × width × height), the diameter of the first circular chamber for placing the tumor pellet is 1mm, and the size parameters of the second and third chambers are 6mm × 6mm × 8mm (length × width × height).

And S30, soaking the obtained PMMA module in deionized water, carrying out ultrasonic cleaning for 30min, then taking out the PMMA module, drying, and dipping 75% alcohol with a cotton ball for wiping and disinfection.

Referring to fig. 3, four PMMA modular structures shown in (a) - (d) can be used for vascularized tumor culture, wherein: in FIG. 3 (a), a circular first through hole with a diameter of 1mm is included, the tissue cavity has a rectangular structure, and the width of the tissue cavity is 3mm, so that the influence of a single tumor globule on the three-dimensional capillary network can be studied. The principle of the figure 3 (b) is similar to that of the figure 3 (a), the diameter of the first through hole is 1mm or 4mm, the tissue cavity is in a ring shape with the width of 1mm or 1.5mm, the ring-shaped three-dimensional capillary network culture can be realized, the angiogenesis mechanism of the iris of the eyeball can be simulated, and a new blood vessel culture platform is provided for exploring eye diseases. Referring to fig. 3 (c), an athletic field-shaped first through hole with a width of 3mm (the cross section of the first through hole is an ellipse) is included in the figure, a platform is provided for placing tumor tissues or organoid bodies with larger size and volume, and the width of the tissue cavity is 1mm or 1.5 mm. In fig. 3, (d) is a double-through-hole structural unit, which includes two circular first through holes with a diameter of 1mm, and the PMMA module of the structure can study the effect of different kinds of tumor pellets on blood vessels under the same microenvironment and the mutual influence between two kinds of tumor pellets through a three-dimensional capillary network.

Based on the four PMMA modules with different structures (as shown in fig. 3), four microfluidic chips with different structures can be prepared, and the specific preparation method can be performed by the preparation method of example 1. The practical culture effect of the microfluidic chips with four different structures is explained below to prove the feasibility of the vascularized tumor microfluidic organ chip for in vitro culture and the preparation process thereof.

Preferably, a cover having air holes is disposed at each of the four PMMA blocks to prevent contamination of the culture solution and cells in each PMMA block.

Referring to fig. 4 a-4 d, there are shown the blood vessel growth pictures of the microfluidic chip with different structures under the microscope, which correspond to the PMMA modules with four structures in fig. 3 (a) - (d), respectively. Respectively filling fibrin gel into the tissue cavities of the microfluidic chips with four different structures, wherein the cells contained in the fibrin gel are Human Umbilical Vein Endothelial Cells (HUVEC) and human lung fibroblasts (NHLF), and the initial inoculation concentration of the cells is 7 multiplied by 10⁶/mL。

Referring to fig. 4a, a blood vessel growth diagram from day 0 to day 4 corresponding to a rectangular microfluidic chip is shown, the size of a tissue cavity is 3mm × 3mm, the diameter of a middle circular first through hole is 1mm, culture solution in a second through hole and a third through hole is sucked out every two days, fresh culture solution is added, and the direction of the height difference of the culture solution is changed to uniformly promote the growth of capillary vessels in the tissue cavity; as can be seen, HUVECs on day 2 had begun to exhibit fragmented growth and form junctions upon induction of NHLF; lumen formation began and thickened gradually on day 4, creating a three-dimensional capillary network. Referring to fig. 5a, by the growth of 5 days, the diameter of the lumen of the generated three-dimensional capillary network of the local area can reach 50 μm after the three-dimensional capillary network is amplified by 40 times, and a channel is provided for the transfer of small drug molecules.

Referring to fig. 4b, which is a blood vessel growth diagram of the annular microfluidic chip on day 3, the diameter of the middle circular first through hole is 1.5mm, the width of the tissue cavity is 1mm, and the culture solution and the flow direction are changed every two days; as can be seen from the figure, the vessels are tightly connected at day 3, the growth direction of the vessels is gradually concentrated towards the first through hole in the middle, and the effect of angiogenesis (angiogenesis) generated by the inner ring simulates the mechanism of angiogenesis. Referring to fig. 5b, a dense three-dimensional capillary network is shown from a 20-fold magnification of the localized area visible in the figure.

Referring to fig. 4c, a blood vessel growth diagram of the field-shaped micro-fluidic chip on day 6 is shown, a three-dimensional capillary network is formed in the diagram, the vertical distance between the first through holes is 5mm, the left-right distance is 3mm, the widths of the left tissue chamber and the right tissue chamber in the diagram are respectively 1mm and 1.5mm, culture solution flows to the second through holes and the third through holes on the two sides from the middle first through hole or reversely flows, the solution is changed every two days, the flowing direction is changed, the operability is improved due to the large size of the first through hole, and various tumor tissues or organ-like bodies can be placed in the first through hole in the center to simulate the real physiological environment in a human body. The first through hole of the track-and-field type micro-fluidic chip structure is large in size, and in order to effectively promote angiogenesis in the chamber, culture solution can be placed in the first through hole, so that the direction of pressure difference is changed from the first through hole to the second through hole and the third through hole on two sides or vice versa.

Referring to fig. 4d, which is a blood vessel growth diagram of the two-through-hole micro-fluidic chip on day 2, the up-down distance of the tissue cavity is 3mm, the left-right distance is 5.5mm, the diameter of the first through hole in the middle is 1mm, and the liquid and the flow direction are changed every two days; as can be seen from the figure, a tightly connected three-dimensional capillary network is formed between the blood vessels on day 2, and different types of tumor pellets can be placed in the two first through holes for the subsequent study of the inter-tumor effect and the drug screening.

As can be seen from FIGS. 4a to 4d, the different structures of the vascularized tumor microfluidic organ chip for in vitro culture demonstrate the angiogenesis effect, and to further verify the interaction between the three-dimensional capillary network and the tumor pellet in the designed structure, on day 0, after the tissue chamber is filled with the gel with HUVEC and NHLF, the mixed gel of the tumor pellet co-cultured with lung cancer cells (A549), HUVEC and NHLF is implanted into the bottom of the first through hole by using a dispensing needle, and the initial inoculation amount ratios of the three are respectively 5 × 10³:10⁴:10⁴. Referring to fig. 6a, the process of connecting and forming the three-dimensional capillary network and the tumor globule from day 0 to day 9 is recorded, the diameter of the tumor globule is about 500 μm, the co-cultured tumor globule and the surrounding three-dimensional capillary network gradually form connection, the three-dimensional capillary network on the side close to the tumor globule is denser, and the three-dimensional capillary network slowly migrates into the tumor globule. Reference toFig. 6b shows a field image of the tumor pellet at day 9 after 10 times magnification, which shows that the effect of blood vessel anastomosis in the PMMA annular chip is successfully achieved, and provides a basis for the next blood vessel perfusability operation.

In specific implementation, a plurality of chips can be integrated on the same glass sheet, mass production is realized, time is saved, production is efficient, and a new platform is provided for constructing high-flux in-vitro vascularization culture and tumor drug screening.

The microfluidic organ chip disclosed by the embodiment provides innovative application values in the field of biomedical engineering, particularly for the relevant researches of in-vitro vascularized tumor disease model construction and novel anti-tumor drug screening.

The foregoing description has described specific embodiments of the present invention. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims

1. A vascularized tumor microfluidic organ chip for in vitro culture, comprising:

the device comprises a polymethyl methacrylate module, a first connecting piece and a second connecting piece, wherein the polymethyl methacrylate module is provided with a first through hole, a second through hole and a third through hole which penetrate through the polymethyl methacrylate module in the thickness direction, and the second through hole and the third through hole are respectively arranged at two sides of the first through hole; the polymethyl methacrylate module is made of a polymethyl methacrylate plate by laser cutting;

the glass sheet is arranged below the polymethyl methacrylate module, the upper surface of the glass sheet and the periphery of the lower surface of the polymethyl methacrylate module are bonded into a whole through a bonding layer, and a first cavity for containing the tumor pellets, a second cavity for storing a culture solution and a third cavity are formed by the first through hole, the second through hole and the third through hole respectively; the middle area of the lower surface of the polymethyl methacrylate module and the glass sheet form a hollow tissue cavity, and fibrin gel or collagen gel is filled in the tissue cavity; the tissue chamber communicates the first chamber with the second chamber and the bottom of the third chamber; by injecting culture solution with different heights into the second chamber and the third chamber, the static pressure difference generated by the culture solution with liquid level height difference can promote the generation of a three-dimensional capillary network in the tissue chamber, and the recruitment of capillaries can be realized through the co-culture of the three-dimensional capillary network and the tumor pellets to construct a three-dimensional vascularized tumor micro-tissue;

the upper surface of the glass sheet is provided with a bonding layer, the bonding layer is cut into a specific shape and is bonded with the polymethyl methacrylate module, so that the height of a tissue cavity between the middle area of the polymethyl methacrylate module and the glass sheet is ensured; and the periphery of the bottom of the polymethyl methacrylate module is encapsulated by PDMS (polydimethylsiloxane) so as to prevent liquid leakage.

2. The vascularized tumor microfluidic organ chip for in vitro culture according to claim 1, wherein the polymethylmethacrylate module has a rectangular structure.

3. The microfluidic tumor organ chip for in vitro culture according to claim 1, wherein the top of the polymethylmethacrylate module is provided with a culture cover covering the first through hole, the second through hole and the third through hole to prevent the tumor pellet or the culture solution from directly contacting with the external environment to cause contamination.

4. The vascularized tumor microfluidic organ chip for in vitro culture according to claim 1, wherein the number of the first through holes is multiple for accommodating different types of tumor pellets.

5. The vascularized tumor microfluidic organ chip for in vitro culture according to claim 1, wherein has one or more of the following characteristics:

-the first through hole is circular or elliptical in cross section;

-the tissue chamber is circular or rectangular.

6. The vascularized tumor microfluidic organ chip for in vitro culture of claim 1, wherein having one or more of the following features:

-the thickness of the glass sheet is between 0.5mm and 1.5 mm;

-the thickness of the bonding layer is from 100 μm to 500 μm;

-the thickness of the polymethylmethacrylate module is 6mm-15 mm.

7. The method for preparing the vascularized tumor microfluidic organ chip for in vitro culture according to claim 1, which comprises:

cleaning and drying the glass sheet by adopting ultrasonic waves;

8. The method for preparing the vascularized tumor microfluidic organ chip for in vitro culture according to claim 7, wherein after obtaining the vascularized tumor microfluidic organ chip for in vitro culture, the method further comprises:

uniformly mixing the polydimethylsiloxane prepolymer and a curing agent according to a set proportion, and removing bubbles in the mixture in a vacuum pump to obtain a polydimethylsiloxane packaging material;

9. The method for preparing the microfluidic organ chip for vascularized tumors cultured in vitro according to claim 7, wherein the step of forming the polymethyl methacrylate plate by laser cutting further comprises: