CN114989977A - Tumor organoid chip for multi-cell interaction and drug screening and preparation method thereof - Google Patents
Tumor organoid chip for multi-cell interaction and drug screening and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a tumor organoid chip for multi-cell interaction and drug screening and a preparation method thereof, wherein the tumor organoid chip comprises a hard substrate layer and a PDMS layer arranged above the hard substrate layer; the PDMS layer comprises a fluid inlet, a fluid outlet, a fluid channel connected with the fluid inlet and the fluid outlet, and three culture chambers surrounded by the fluid channel, wherein the peripheries of the three culture chambers consist of a micro-column array; the chip has the advantages of ingenious structural design and convenient and quick sample addition, relatively independent perfusion and culture spaces are formed by utilizing the micro-column array, the same dynamic fluid culture condition can be endowed to cells in the cavity, the interaction of different cells can be researched, the chip can also be used for culturing the same cell, and the occurrence, growth and transfer of tumors can be observed in real time by adopting the chip, so that the actual effect of the medicine at different stages can be accurately evaluated, and the chip has a higher application prospect for the future individual accurate tumor treatment.
Description
Technical Field
The invention relates to the field of microfluidic chips and preparation methods thereof, in particular to a tumor organoid chip for multi-cell interaction and drug screening and a preparation method thereof.
Background
The evaluation of the anticancer drugs before clinical and the clinical drug test have important meanings, the traditional drug evaluation has high evaluation cost and general effectiveness, and because the traditional 2D cell plane culture and small animal model have great difference with the human body, the traditional 2D cell plane culture and small animal model are not enough for accurately researching the curative effect of the drugs and solving the problem of the drug resistance of clinical tumors; in addition, the tumors of different patients have great difference, and it is often difficult to carry out customized high-flux drug screening according to the patients clinically. The in vitro tumor model is a novel evaluation platform with potential, the tumor organoid is taken as a novel in vitro tumor model from a patient, the microenvironment characteristics and the gene mutation spectrum of the parental immune tumor are retained to the maximum extent, and the model is an in vitro model which can be efficiently applied to the research of tumor diseases and the drug screening. However, since the heterogeneous microenvironment in human tumor tissues is very complex, besides a large number of tumor cells, there are also cell types such as immune cells, fibroblasts, vascular cells, etc., and there are complex interactions between cells and cells, and between cells and extracellular matrix, the above complex tumor microenvironment has important significance for the disease development and drug sensitivity of tumors. However, the existing organoid culture method cannot truly simulate a real heterogeneous tumor microenvironment, and a tumor heterogeneous microenvironment model capable of simulating various cells in vivo is urgently needed, so that the organoid culture method is used for researching various cell interactions and tumorigenesis, metastasis and progression mechanisms. In addition, the in vivo tumor microenvironment is a dynamic process, including physicochemical factors such as fluid shear force, pH and gradient, and the like, and the traditional cell culture method is difficult to reconstruct a real tumor heterogeneous microenvironment.
Accordingly, there is a need for improvements and developments in the art.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a tumor organoid chip for multi-cell interaction and drug screening and a preparation method thereof, and aims to solve the problem that the existing organoid culture method cannot truly simulate a real heterogeneous tumor microenvironment.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a tumor organoid chip for multi-cell interaction and drug screening comprises a hard substrate layer and a PDMS layer arranged above the hard substrate layer;
the PDMS layer comprises a fluid inlet, a fluid outlet, a fluid channel connected with the fluid inlet and the fluid outlet, and three culture chambers surrounded by the fluid channel, wherein the peripheries of the three culture chambers consist of a micro-column array.
The tumor organoid chip for multi-cell interaction and drug screening is characterized in that the hard substrate in the hard substrate layer is one of PDMS, glass, PMMA, polypropylene and polyethylene.
The tumor organoid chip for multi-cell interaction and drug screening is characterized in that the fluid channel is connected with the fluid inlet and the fluid outlet through a serpentine-structure fluid channel, and the serpentine-structure fluid channel is 0.6-1.5mm wide and 0.1-0.5mm deep.
The tumor organoid chip for multi-cell interaction and drug screening is characterized in that the three culture chambers are distributed in an equidistant triangular space, the three culture chambers are connected with each other through rectangular channels, the three culture chambers are all circular cell culture chambers, and the diameter of each circular cell culture chamber is 0.5-1.5 mm.
The tumor organoid chip for multi-cell interaction and drug screening is characterized in that sample adding ports are respectively arranged above the three culture chambers.
The tumor organoid chip for multi-cell interaction and drug screening is characterized in that one of the three culture chambers is inoculated with a biological ink solution loaded with tumor cells, and the other two culture chambers are inoculated with a biological ink solution loaded with stromal cells.
The tumor organoid chip for multi-cell interaction and drug screening is characterized in that the solubility of the bio-ink solution is 1% -10%, and the bio-ink solution is a temperature-sensitive bio-ink solution or a photosensitive bio-ink solution.
The tumor organoid chip for multi-cell interaction and drug screening is characterized in that the temperature-sensitive bio-ink is one or more of type I collagen, matrigel, hyaluronic acid and fibrinogen; the photosensitive bio-ink is one or more of gelatin acrylate, hyaluronic acid acrylate, gellan gum acrylate and alginic acid acrylate.
The tumor organoid chip for multi-cell interaction and drug screening is characterized in that the stromal cells are one or more of fibroblasts, vascular endothelial cells, macrophages and lymphocytes.
The tumor organoid chip for multi-cell interaction and drug screening is characterized in that the microcolumns in the microcolumn array are circular, the diameter of each microcolumn is 0.05-0.1mm, and the array interval is 0.02-0.1 mm;
or the microcolumns in the microcolumn array are square, the side length of each microcolumn is 0.05-0.1mm, and the array interval is 0.02-0.1 mm.
Has the advantages that: the invention discloses a tumor organoid chip for multi-cell interaction and drug screening, which comprises a hard substrate layer and a PDMS layer arranged above the hard substrate layer; the PDMS layer comprises a fluid inlet, a fluid outlet, a fluid channel connected with the fluid inlet and the fluid outlet, and three culture chambers surrounded by the fluid channel, wherein the peripheries of the three culture chambers consist of a micro-column array. The microfluidic organ chip has the advantages of ingenious structural design, convenient and quick sample addition, relatively independent perfusion and culture spaces formed by the microcolumn array, no leakage and nutrient exchange meeting of the microcolumn, and the same dynamic fluid culture condition can be endowed to the cells in the chamber, the influence of different variable control on experimental results is overcome, the interference of unknown factors is reduced, the function difference among cells is reduced, not only can the interaction of different cells be researched, the complex heterogeneous tumor multicellular microenvironment is reconstructed, but also the heterogeneous tumor multicellular microenvironment can be used for culturing the same cells, the cell culture efficiency and the accuracy of drug screening results are improved, the occurrence, growth and transfer of tumors can be observed in real time by adopting the chip of the invention, therefore, the actual effect of the medicine at different stages can be accurately evaluated, and the method has a high application prospect for personalized and accurate tumor treatment in the future.
Drawings
FIG. 1 is a schematic top view of an exemplary tumor organoid chip for multi-cell interaction and drug screening in accordance with the present invention;
FIG. 2 is a schematic diagram of the overall structure of a tumor organoid chip for multi-cell interaction and drug screening according to an embodiment of the present invention;
FIG. 3 is a diagram of the whole tumor organoid chip encapsulated for multi-cell interaction and drug screening according to the embodiment of the present invention;
FIG. 4 is an enlarged schematic view of a culture chamber of a tumor organoid chip for multi-cell interaction and drug screening in accordance with an embodiment of the present invention;
FIG. 5 is a schematic diagram of a tumor organoid sphere of breast cancer formed inside a culture chamber of a tumor organoid chip for multi-cell interaction and drug screening according to an embodiment of the present invention.
Reference numerals are as follows: the device comprises a hard substrate layer 10, a PDMS layer 20, a fluid inlet 21, a serpentine fluid channel 22, a fluid channel 23, a culture chamber 24, a micro-column array 25, a fluid outlet 26, a rectangular connecting channel 27 and a sample adding port 28.
Detailed Description
The invention provides a tumor organoid chip for multi-cell interaction and drug screening and a preparation method thereof, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
The microfluid chip technology is a new technology which is rapidly developed in recent years, and can be used for simulating physiological conditions and functions of tissues and organs in vivo by virtue of the characteristics of accurate scale, high integration, various material manufacturing processes, flexible and changeable design, high controllability and the like.
Therefore, based on clinical patient tumor specimens, an engineered heterogeneous tumor organoid chip is constructed in vitro quickly, and has important significance for preclinical and clinical high-throughput drug screening. How to construct the tumor organoid 3D heterogeneous microenvironment participated by various cells on the microfluidic chip simultaneously, simulate the interaction among tumor cells in vivo, and be used for screening antitumor drugs, so that the realization of the personalized and accurate tumor treatment of patients still has the challenge.
Based on this, the invention provides a tumor organoid chip for multi-cell interaction and drug screening, which is shown in fig. 1-3 and comprises a hard substrate layer 10 and a PDMS layer 20 disposed above the hard substrate layer 10; wherein, the PDMS layer 20 includes a fluid inlet 21 and a fluid outlet 26 inside, a fluid channel 23 connected to the fluid inlet 21 and the fluid outlet 26, and three culture chambers 24 surrounded by the fluid channel 23, and the periphery of the three culture chambers 24 is composed of a micro-column array 25.
In particular, the microfluidic organ chip of the invention has smart structural design and convenient and rapid sample addition, forms relatively independent perfusion and culture spaces by utilizing the micro-column array, can meet the requirement of nutrient exchange while ensuring no leakage, and the same dynamic fluid culture condition can be endowed to the cells in the chamber, the influence of different variable control on experimental results is overcome, the interference of unknown factors is reduced, the function difference among cells is reduced, not only can the interaction of different cells be researched, the complex heterogeneous tumor multicellular microenvironment is reconstructed, but also the heterogeneous tumor multicellular microenvironment can be used for culturing the same cells, the cell culture efficiency and the accuracy of drug screening results are improved, the occurrence, growth and transfer of tumors can be observed in real time by adopting the chip of the invention, therefore, the actual effect of the medicine at different stages can be accurately evaluated, and the method has a high application prospect for personalized and accurate tumor treatment in the future.
In some embodiments, the hard substrate in the hard substrate layer is one of PDMS, glass, PMMA, polypropylene, polyethylene.
In some embodiments, the fluid channel is connected to both the fluid inlet and the fluid outlet by a serpentine fluid channel 22, the serpentine fluid channel 22 has a width of 0.6-1.5mm and a depth of 0.1-0.5mm, and the serpentine fluid channel is sized to meet the perfusion rate of the culture medium, thereby being closer to the real heterogeneous tumor microenvironment.
In some embodiments, the fluid channel 23 has a width of 1-2mm and a depth of 0.1-0.5 mm.
In some embodiments, the three culture chambers are distributed in an equidistant triangular space, two of the three culture chambers are connected by a rectangular channel 27, and the three culture chambers are all circular cell culture chambers with a diameter of 0.5-1.5 mm.
In some embodiments, a sample addition port 28 is disposed above each of the three culture chambers for adding tumor cells and stromal cells.
In some embodiments, one of the three culture chambers is inoculated with the tumor cell-loaded bio-ink solution, and the other two of the three culture chambers are inoculated with the stromal cell-loaded bio-ink solution.
In some embodiments, the bio-ink solution has a solubility of 1% to 10%, and the bio-ink solution is a temperature-sensitive bio-ink solution or a photosensitive bio-ink solution.
In some embodiments, the temperature-sensitive bio-ink is one or more of type I collagen, matrigel, hyaluronic acid, fibrinogen; the photosensitive bio-ink is one or more of gelatin acrylate, hyaluronic acid acrylate, gellan gum acrylate and alginic acid acrylate.
In some embodiments, the stromal cells are one or more of fibroblasts, vascular endothelial cells, macrophages, lymphocytes.
In some embodiments, the microcolumns in the microcolumn array are circular, the diameter of the microcolumns is 0.05 to 0.1mm, and the array pitch is 0.02 to 0.1 mm; or the microcolumns in the microcolumn array are square, the side length of each microcolumn is 0.05-0.1mm, and the array interval is 0.02-0.1 mm.
Specifically, a relatively independent perfusion and culture space is formed by utilizing a micro-column array, and the size of the micro-column can meet the requirement of nutrient exchange while ensuring no leakage of cell bio-ink.
In some embodiments, the method for preparing a tumor organoid chip for multi-cell interaction and drug screening mainly comprises the following steps:
(A) designing a micro-fluid chip structure: the microfluidic chip comprises two layers, wherein the upper layer is a PDMS layer, the lower layer is a hard substrate layer, two independent fluid channels and three circular culture chambers are separated from the interior of the PDMS layer on the upper layer by a circular micro-column array, and the chip is provided with a fluid channel and a fluid inlet and a fluid outlet;
(B) designing a flow channel of the microfluid chip: a fluid channel with a rectangular cross section is arranged in the chip, and the inlet and the outlet of the fluid channel are respectively connected with a fluid inlet and a fluid outlet through the fluid channel with the snake-shaped structure;
(C) design of cell culture chamber of microfluidic chip: three circular cell culture chambers are uniformly distributed on one side of a central fluid channel of the chip in an equidistant triangular structure, and the chambers are connected by a rectangular channel;
(D) microfluidic chip cell culture: and (3) slowly inoculating 10% matrigel bio-ink solutions loaded with tumor cells, fibroblasts and vascular endothelial cells into the three culture chambers in the step (C) through a sample port, incubating for 10 min at the temperature of 37 ℃, dynamically perfusing a culture medium in a fluid channel, and culturing for a specific time to obtain the tumor organoid chip for multi-cell interaction and drug screening.
The invention also provides a method for using the tumor organoid chip for multi-cell interaction and drug screening according to the above scheme of the invention, which comprises the following steps:
and (2) sterilizing the tumor organoid chip for multi-cell interaction and drug screening at high temperature, and adding the prepared three different cell-carrying biological ink suspensions into three different circular culture chambers respectively to ensure that the biological inks in the adjacent chambers are contacted with each other at the joint. Culturing with peristaltic pump dynamic perfusion culture medium under 37 deg.C and 5% CO in a ten-thousand-stage superclean laboratory and a ten-thousand-stage superclean bench 2 In the incubator, cell culture is carried out at a specific time, cell growth, proliferation, migration and gene and protein expression change conditions are observed, heterogeneous tumor organoids are finally formed, then candidate antitumor drugs are dynamically perfused into the organoid chips at the blood flowing speed, and the curative effect of the drugs is evaluated by means of live/dead fluorescent staining, histological sections, flow cytometry and immunohistochemistry, so that high-throughput screening of the drugs is carried out, clinical medication is guided, and the tumor development mechanism is revealed.
In some embodiments, the culturing is at a condition time of 10-30min and the perfusion rate is 0.05-1 mL/h.
In some specific embodiments, referring to fig. 4, an enlarged schematic diagram of a culture chamber of a tumor organoid chip for multi-cell interaction and drug screening according to an embodiment of the present invention is shown in fig. 5, in which a is a bio-ink matrix and b is a tumor organoid sphere, and a physical map of a breast cancer tumor organoid sphere is formed, as can be seen, a tumor organoid sphere cultured by using the chip of the present invention is closer to a real condition.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is clear that the described embodiments are only a part of the embodiments of the invention, not all embodiments, merely intended to illustrate the invention and in no way limit it. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
Example 1
A preparation method of a tumor organoid chip for multi-cell interaction and drug screening comprises the following steps:
(A) designing a micro-fluid chip structure: the microfluidic chip comprises two layers, wherein the upper layer is PDMS (polydimethylsiloxane), the lower layer is PDMS, two independent fluid channels and three circular culture chambers are separated from the interior of the upper layer PDMS by a circular micro-column array (the diameter is 0.1mm, and the distance between the two micro-column arrays is 0.1 mm), one fluid channel is designed on the chip, and a fluid inlet and a fluid outlet are configured on the chip;
(B) designing a flow channel of the microfluid chip: the chip is provided with a fluid channel with a rectangular cross section, the width is 2mm, the depth is 0.5mm, the inlet and the outlet of the flow channel are serpentine fluid channels, the width is 1.5mm, and the depth is 0.5 mm;
(C) design of cell culture chamber of microfluidic chip: three circular cell culture chambers are uniformly distributed on one side of a central fluid channel of the chip in an equidistant triangular structure, the diameter of each circular cell culture chamber is 1.5mm, and the chambers are connected by rectangular channels;
(D) micro-fluid chip cell culture: and (C) slowly inoculating 10% matrigel bio-ink solutions loaded with tumor cells, fibroblasts and vascular endothelial cells into the three culture chambers in the step (C) through a sample adding port, incubating for 10 min at 37 ℃, then dynamically perfusing a culture medium in a fluid channel at a perfusion speed of 0.1 mL/h, culturing for a specific time to study the change processes of cell transfer, cell activity, proliferation, morphology and tumor marker gene expression, and screening the anti-tumor drugs to obtain the tumor organ chip for multi-cell interaction and drug screening.
Example 2
A preparation method of a tumor organoid chip for multi-cell interaction and drug screening comprises the following steps:
(A) designing a micro-fluid chip structure: the microfluidic chip comprises two layers, wherein the upper layer is PDMS (polydimethylsiloxane), the lower layer is PMMA, two independent fluid channels and three circular culture chambers are separated from the interior of the upper layer PDMS by a circular micro-column array (the diameter is 0.05 mm, and the distance between the two circular culture chambers is 0.02 mm), and the chip is provided with one fluid channel and is provided with a fluid inlet and a fluid outlet;
(B) designing a flow channel of the microfluid chip: the chip is provided with a fluid channel with a rectangular cross section, the width of the fluid channel is 1mm, the depth of the fluid channel is 0.1mm, an inlet and an outlet of the flow channel are serpentine-structure fluid channels, the width of the fluid channel is 0.6 mm, and the depth of the fluid channel is 0.1 mm;
(C) design of a microfluid chip cell culture chamber: three circular cell culture chambers are uniformly distributed on one side of a central fluid channel of the chip in an equidistant triangular structure, the diameter of each circular cell culture chamber is 0.5mm, and the chambers are connected by rectangular channels;
(D) microfluidic chip cell culture: and (3) slowly inoculating 1% matrigel biological ink solution loaded with tumor cells, macrophages and vascular endothelial cells into the three culture chambers in the step (C) through a sample adding port, incubating for 30min at 37 ℃, then dynamically perfusing the culture medium in a fluid channel at a perfusion speed of 1 mL/h, culturing for a specific time to study the change processes of cell transfer, cell activity, proliferation, morphology and tumor marker gene expression, and screening the anti-tumor drugs to obtain the tumor organ chip for multi-cell interaction and drug screening.
Example 3
A preparation method of a tumor organoid chip for multi-cell interaction and drug screening comprises the following steps:
(A) designing a micro-fluid chip structure: the microfluidic chip comprises two layers, wherein the upper layer is PDMS (polydimethylsiloxane), the lower layer is polypropylene, two independent fluid channels and three circular culture chambers are separated from the interior of the upper layer PDMS by a circular micro-column array (the diameter is 0.07 mm, and the distance between the two circular culture chambers is 0.05 mm), and one fluid channel is designed on the chip and provided with a fluid inlet and a fluid outlet;
(B) designing a flow channel of the microfluid chip: the chip is provided with a fluid channel with a rectangular cross section, the width is 1.5mm, the depth is 0.3 mm, the inlet and the outlet of the flow channel are serpentine fluid channels, the width is 1mm, and the depth is 0.3 mm;
(C) design of cell culture chamber of microfluidic chip: three circular cell culture chambers are uniformly distributed on one side of a central fluid channel of the chip in an equidistant triangular structure, the diameter of each circular cell culture chamber is 1mm, and the chambers are connected by rectangular channels;
(D) micro-fluid chip cell culture: and (3) slowly inoculating 5% type I collagen biological ink solution loaded with tumor cells, macrophages and lymphocytes into the three culture chambers in the step (C) through a sample adding port respectively, incubating for 15 min at the temperature of 37 ℃, then dynamically perfusing a culture medium in a fluid channel at the perfusion speed of 0.5 mL/h, culturing for a specific time to study the change processes of cell transfer, cell activity, proliferation, morphology and tumor marker gene expression, and screening the anti-tumor drugs to obtain the tumor organ chip for multi-cell interaction and drug screening.
Example 4
A preparation method of a tumor organoid chip for multi-cell interaction and drug screening comprises the following steps:
(A) designing a micro-fluid chip structure: the microfluidic chip comprises two layers, wherein the upper layer is PDMS (polydimethylsiloxane), the lower layer is polyethylene, two independent fluid channels and three circular culture chambers are separated from the interior of the upper layer of PDMS by a square micro-column array (the side length is 0.1mm, and the distance between the two micro-column arrays is 0.06 mm), and one fluid channel is designed on the chip and provided with a fluid inlet and a fluid outlet;
(B) designing a flow channel of the microfluid chip: the chip is provided with a fluid channel with a rectangular cross section, the width is 1.6 mm, the depth is 0.4 mm, the inlet and the outlet of the flow channel are serpentine fluid channels, the width is 0.8 mm, and the depth is 0.4 mm;
(C) design of cell culture chamber of microfluidic chip: three circular cell culture chambers are uniformly distributed on one side of a central fluid channel of the chip in an equidistant triangular structure, the diameter of each circular cell culture chamber is 1.2 mm, and the chambers are connected by rectangular channels;
(D) micro-fluid chip cell culture: and (C) slowly inoculating 3% hyaluronic acid biological ink solutions loaded with tumor cells, fibroblasts and lymphocytes into the three culture chambers in the step (C) through a sample adding port, incubating for 12 min at 37 ℃, then dynamically perfusing a culture medium in a fluid channel at a perfusion speed of 0.3 mL/h, culturing for a specific time to study the change processes of cell transfer, cell activity, proliferation, morphology and tumor marker gene expression, and screening the anti-tumor drugs to obtain the tumor organ chip for multi-cell interaction and drug screening.
Example 5
A preparation method of a tumor organoid chip for multi-cell interaction and drug screening comprises the following steps:
(A) designing a micro-fluid chip structure: the microfluidic chip comprises two layers, wherein the upper layer is PDMS (polydimethylsiloxane), the lower layer is glass, two independent fluid channels and three circular culture chambers are separated from the interior of the upper layer PDMS by a square micro-column array (the side length is 0.05 mm, and the distance between the two micro-column arrays is 0.04 mm), and the chip is provided with one fluid channel and is provided with a fluid inlet and a fluid outlet;
(B) designing a flow channel of the microfluid chip: the chip is provided with a fluid channel with a rectangular cross section, the width is 1.3 mm, the depth is 0.2 mm, the inlet and the outlet of the flow channel are serpentine fluid channels, the width is 0.7 mm, and the depth is 0.2 mm;
(C) design of cell culture chamber of microfluidic chip: three circular cell culture chambers are uniformly distributed on one side of a central fluid channel of the chip in an equidistant triangular structure, the diameter of each circular cell culture chamber is 0.8 mm, and the chambers are connected by rectangular channels;
(D) micro-fluid chip cell culture: and (C) respectively and slowly inoculating 5% of fibrinogen biological ink solution loaded with tumor cells, fibroblasts and lymphocytes into the three culture chambers in the step (C) through a sample adding port, incubating for 20 min at 37 ℃, then dynamically perfusing a culture medium in a fluid channel at a perfusion speed of 0.4 mL/h, culturing for a specific time to study the change process of cell transfer, cell activity, proliferation, morphology and tumor marker gene expression, and screening the anti-tumor drugs to obtain the tumor organ chip for multi-cell interaction and drug screening.
Example 6
A preparation method of a tumor organoid chip for multi-cell interaction and drug screening comprises the following steps:
(A) designing a micro-fluid chip structure: the microfluidic chip comprises two layers, wherein the upper layer is PDMS (polydimethylsiloxane), the lower layer is glass, two independent fluid channels and three circular culture chambers are separated from the interior of the upper layer PDMS by a square micro-column array (the side length is 0.07 mm, and the distance between the two micro-column arrays is 0.09 mm), and one fluid channel is designed on the chip and provided with a fluid inlet and a fluid outlet;
(B) designing a flow channel of the microfluid chip: the chip is provided with a fluid channel with a rectangular cross section, the width is 1.9 mm, the depth is 0.4 mm, the inlet and the outlet of the flow channel are serpentine fluid channels, the width is 1.1 mm, and the depth is 0.2 mm;
(C) design of cell culture chamber of microfluidic chip: three circular cell culture chambers are uniformly distributed on one side of a central fluid channel of the chip in an equidistant triangular structure, the diameter of each circular cell culture chamber is 1.3 mm, and the chambers are connected by rectangular channels;
(D) micro-fluid chip cell culture: and (C) slowly inoculating 8% gelatin acrylate biological ink solutions loaded with tumor cells, fibroblasts and lymphocytes into the three culture chambers in the step (C) through a sample adding port, incubating for 10 min at 37 ℃, then dynamically perfusing a culture medium in a fluid channel at a perfusion speed of 0.4 mL/h, culturing for a specific time to study the change processes of cell transfer, cell activity, proliferation, morphology and tumor marker gene expression, and screening the anti-tumor drugs to obtain the tumor organ chip for multi-cell interaction and drug screening.
In summary, the invention discloses a tumor organoid chip for multi-cell interaction and drug screening and a preparation method thereof, and the tumor organoid chip comprises a hard substrate layer and a PDMS layer arranged above the hard substrate layer; the PDMS layer comprises a fluid inlet, a fluid outlet, a fluid channel connected with the fluid inlet and the fluid outlet, and three culture chambers surrounded by the fluid channel, wherein the peripheries of the three culture chambers consist of a micro-column array. The microfluid organ chip of the invention has smart structure design and convenient and rapid sample adding, forms relatively independent perfusion and culture spaces by utilizing the micro-column array, ensures that the micro-column has no leakage, can meet the requirement of nutrient exchange, and the same dynamic fluid culture condition can be endowed to the cells in the chamber, the influence of different variable control on experimental results is overcome, the interference of unknown factors is reduced, the function difference among cells is reduced, not only can the interaction of different cells be researched, the complex heterogeneous tumor multicellular microenvironment is reconstructed, but also the heterogeneous tumor multicellular microenvironment can be used for culturing the same cells, the cell culture efficiency and the accuracy of drug screening results are improved, the occurrence, growth and transfer of tumors can be observed in real time by adopting the chip of the invention, therefore, the actual effect of the medicine at different stages can be accurately evaluated, and the method has a high application prospect for personalized and accurate tumor treatment in the future.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A tumor organoid chip for multi-cell interaction and drug screening is characterized by comprising a hard substrate layer and a PDMS layer arranged above the hard substrate layer;
the PDMS layer comprises a fluid inlet, a fluid outlet, a fluid channel connected with the fluid inlet and the fluid outlet, and three culture chambers surrounded by the fluid channel, wherein the peripheries of the three culture chambers consist of a micro-column array.
2. The tumor organoid chip for multi-cell interaction and drug screening according to claim 1, wherein the hard substrate of the hard substrate layer is one of PDMS, glass, PMMA, polypropylene and polyethylene.
3. The tumor organoid chip for multicellular interaction and drug screening of claim 1 wherein the fluid channel is connected to both the fluid inlet and the fluid outlet by a serpentine-structured fluid channel having a width of 0.6-1.5mm and a depth of 0.1-0.5 mm.
4. The tumor organoid chip for multi-cell interaction and drug screening according to claim 1, wherein the three culture chambers are distributed in an equidistant triangular space, every two of the three culture chambers are connected by a rectangular channel, the three culture chambers are all circular cell culture chambers, and the diameter of each circular cell culture chamber is 0.5-1.5 mm.
5. The tumor organoid chip for multi-cell interaction and drug screening according to claim 4, wherein sample injection ports are respectively disposed above the three culture chambers.
6. The tumor organoid chip for multi-cell interaction and drug screening of claim 1, wherein one of the three culture chambers is inoculated with a bio-ink solution loaded with tumor cells, and the other two of the three culture chambers are inoculated with a bio-ink solution loaded with stromal cells.
7. The tumor organoid chip for multicellular interaction and drug screening according to claim 6, wherein the solubility of the bio-ink solution is 1% to 10%, and the bio-ink solution is a temperature-sensitive bio-ink solution or a photosensitive bio-ink solution.
8. The tumor organoid chip for multicellular interaction and drug screening according to claim 7, wherein the temperature-sensitive bio-ink is one or more of type I collagen, matrigel, hyaluronic acid, fibrinogen; the photosensitive bio-ink is one or more of gelatin acrylate, hyaluronic acid acrylate, gellan gum acrylate and alginic acid acrylate.
9. The tumor organoid chip for multi-cell interaction and drug screening according to claim 6, wherein the stromal cells are one or more of fibroblasts, vascular endothelial cells, macrophages, lymphocytes.
10. The tumor organoid chip for multi-cell interaction and drug screening according to claim 1, wherein the microcolumns in the microcolumn array are circular, the diameter of the microcolumns is 0.05-0.1mm, and the array pitch is 0.02-0.1 mm;
or the microcolumns in the microcolumn array are square, the side length of each microcolumn is 0.05-0.1mm, and the array interval is 0.02-0.1 mm.
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