WO2023020599A1 - 一种类器官培养芯片和类器官培养方法 - Google Patents
一种类器官培养芯片和类器官培养方法 Download PDFInfo
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- WO2023020599A1 WO2023020599A1 PCT/CN2022/113449 CN2022113449W WO2023020599A1 WO 2023020599 A1 WO2023020599 A1 WO 2023020599A1 CN 2022113449 W CN2022113449 W CN 2022113449W WO 2023020599 A1 WO2023020599 A1 WO 2023020599A1
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Definitions
- the invention relates to the technical fields of tissue engineering and organ chips, in particular to an organoid culture chip and an organoid culture method.
- organoids are self-assembled by stem cells in vitro and grow into three-dimensional aggregates similar to human tissues or organs in structure and function, such as: brain organoids, blood vessel organoids, liver organs, kidney organoids and tumor organoids, etc.
- organoids On a global scale, organoids have shown their strong development potential, and a certain international competition pattern has been formed.
- the culture process of organoids mainly includes two steps: cell self-assembly into spheres and transfer or in situ differentiation culture.
- the culture process of cerebral cortical organoids mainly includes four stages: embryoid body formation, neuroectoderm induction, neuroepithelial differentiation, and cerebral organoid maturation.
- the culture of brain organoids it is necessary to coat Matrigel-coated embryoid bodies after neural induction, and then transfer them to low-adhesion culture plates or bioreactors for dynamic culture.
- the cells in the outer layer of organoids are vulnerable to mechanical damage and increase the chance of contamination.
- organoids tend to fuse with each other, and it is difficult to locate and observe.
- the above limitations lead to the limitations of the organoid culture process, such as complexity, great variability, low throughput, and difficulty in real-time monitoring.
- the purpose of the present invention is to provide an organoid culture chip and an organoid culture method, which can cultivate organoids in situ with high throughput, and the steps of the culture method are simple.
- the device has the advantages of low cost, easy operation, in situ imaging, and real-time monitoring, providing an innovative platform for simulating human organ development, mechanism research, toxicology testing, and drug screening.
- an organoid culture chip comprising:
- the organoid culture device is arranged in the cell culture plate; a culture medium reservoir is formed between the periphery of the organoid culture device and the cell culture plate; the organoid culture device includes: the organoid culture device body .
- the organoid culture chamber arranged in the body of the organoid culture device and the side holes arranged on the side walls on both sides of the body of the organoid culture device, the organoid culture chamber and the side walls on both sides
- the upper part is connected to form a perfusion channel, and the organoid culture chamber includes a sample well on the top and a microwell on the bottom, and both the sample well and the microwell are connected to the bottom of the cell culture plate Pass.
- multiple organoid culture chambers there are multiple organoid culture chambers, multiple pairs of side holes are provided on the side walls of the body of the organoid culture device, multiple organoid culture chambers and multiple pairs of side holes The holes are connected one by one to form a plurality of perfusion channels.
- the organoid culture chip includes a culture chip for constructing one of brain organoids, vascularized organoids, liver organoids, small intestine organoids, pancreatic organoids, kidney organoids and tumor organoids;
- the organoid culture chip is a vascularized organoid culture chip
- the organoid culture chip further includes: a film, arranged on the side hole.
- the sticking film includes a porous film or a filter screen, and the pore size of the porous film or filter screen is 20 ⁇ m to 200 ⁇ m. Its purpose is to enable the attachment of different types of cells to form a barrier structure.
- the side hole includes a first side hole and a second side hole
- the organoid culture device body is provided with a first side wall and a second side wall opposite to each other, a third side wall and a fourth side wall opposite to each other.
- Side wall; one or more of the first side holes are arranged on the first side wall of the organoid culture device body, and one or more of the second side holes are arranged on the organoid culture device body
- the first side hole and the second side hole are respectively arranged in one-to-one correspondence with the organoid culture chamber.
- first side wall of the organoid culture device body and the cell culture plate form a first medium reservoir, and the first medium reservoir is connected to the first side hole through the first side hole.
- the organoid culture chamber is connected;
- the second side wall of the organoid culture device body and the cell culture plate form a second medium reservoir, and the second medium reservoir passes through the second side hole and the organoid
- the culture chambers are connected.
- the first medium reservoir is communicated with the organoid culture chamber through the first side hole; the second medium reservoir is connected with the organoid culture chamber through the second side hole
- the chambers are connected, the purpose of which is to enable the liquid reservoirs on both sides to fully exchange nutrients and oxygen and remove necrotic cells during the perfusion culture process.
- third side wall and the fourth side wall are respectively adapted to the inner shape of the cell culture plate, and the third side wall and the fourth side wall are respectively compatible with the shape of the cell culture plate meet each other. Its purpose is to separate the cell culture plate into two separate reservoirs.
- the material of the organoid culture device is selected from quartz, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polycarbonate (PC), polyethylene terephthalate One or more of ester (PET) and resin;
- the bottom material of the organoid culture chamber is hydrophobic or treated with hydrophobicity. The purpose is that the cells in the organoid culture chamber can self-assemble into cell pellets on the surface of the hydrophobic material.
- the shape of the organoid culture chamber is one of rectangular, circular, trapezoidal, triangular and hemispherical; if the organoid culture chamber is rectangular, the length is 0.10 mm to 6.00 mm, and the width is 0.10mm-6.00mm; if the organoid culture chamber is circular, the diameter is 0.10mm-6.00mm;
- the shape of the side hole is one of rectangle, circle, trapezoid, triangle and hemispherical; if the side hole is a rectangle, its length is 0.10mm-6.00mm, and its width is 0.10mm-6.00mm; if the The side hole is circular with a diameter of 0.10mm-6.00mm.
- the height of the organoid culture device is 5.00mm-15.00mm;
- the interval length between the plurality of organoid culture chambers is 0.10 mm to 10.00 mm;
- the thickness of the side wall of the organoid culture device body is 0.10 mm to 6.00 mm;
- the area of the microwell at the bottom of the organoid culture chamber is 0.785 mm 2 to 100 mm 2 ;
- the area of the side hole is 1mm 2 -50mm 2 .
- the area of the side hole is 1 mm 2 -50 mm 2 .
- the area of the side hole is 1 mm 2 -12 mm 2 .
- a method for culturing organoids using the organoid culture chip comprising: adding cell suspension or matrigel-wrapped embryoid body EBs to the sterilized
- the organoid culture chamber in the organoid culture chip is cultured.
- the matrigel-wrapped embryoid body EBs are embryoid body EBs cultured in 1-10 mg/mL matrigel medium NIM for 11-15 days.
- the seed cells cultured by the above-mentioned organoid culture chip are not limited to human pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells), and are also applicable to other stem cells, including but not limited to human adult stem cells, tumor stem cells, and animal-derived stem cells.
- the cultured objects are not limited to organoids, but also applicable to three-dimensional spheres formed by other types of cells.
- the organoid culture chip provided by the present invention can cultivate organoids in situ with high throughput, and the steps of the culture method are simple; specifically:
- the materials used in the present invention are relatively common materials on the market, and the price is low.
- the production tools involved in the present invention are of low value, and have better advantages for common laboratories and mass production.
- the present invention can satisfy two-dimensional culture, three-dimensional culture and dynamic culture at the same time, simplifies the operation steps in the culture process of organoids; reduces the learning cost for beginners; Risk of contamination from transferring organoids.
- the present invention can prepare several to hundreds of organoids at the same time, and can realize high-throughput organoid preparation and culture requirements; during the culture process, the replacement of the medium does not require direct contact with the organoids. Organs reduce the risk of damage during the culture process.
- the chip is highly integrated with existing cell culture well plates and has good compatibility with existing bio-related optical instruments.
- the present invention has high consistency in the organoid culture process and can effectively reduce the differences between samples.
- the organoids cultured in the present invention are all Grow in separate chambers with low interference between samples.
- the device can be fully adapted to existing industrialized high-throughput drug screening systems, and has extremely high application potential in related fields such as research on tissue development, disease modeling, toxicology testing, and drug development.
- Fig. 1 is the structural representation of the organoid culture chip of embodiment 1;
- Example 2 is a three-dimensional structure diagram of the organoid culture chip in Example 1;
- FIG. 3 is a structural diagram of the organoid culture device in the organoid culture chip of Example 1;
- Fig. 4 is an in situ bright field picture of the brain organoids of Example 1 in the culture device, wherein the scale bars of D1, D7 and D14 are 200 ⁇ m, and the scale bars of D21, D28 and D35 are 500 ⁇ m;
- Example 5 is a top view, a section view and an assembly view of a brain organoid culture device based on a 12-well plate in Example 1;
- Figure 6 is an enlarged view of the culture device in Example 1, used to describe the specific dimensions of the culture chamber;
- Fig. 7 is the structural diagram of culture device in embodiment 1;
- Fig. 8 is an in situ bright field picture of the brain organoid of Example 8 in the culture device;
- Fig. 9 is a statistical diagram of QPCR identification of different cell markers in the brain organoid cultured in the brain organoid chip of Example 8.
- Figure 10 is the size and uniformity characteristics of the brain organoids cultured in the brain organoid chip of Example 8.
- Fig. 11 is a statistical chart of different cell markers of vascularized brain organoids identified by QPCR when the vascularized brain organoids were cultured in the brain organoid chip of Example 8;
- Figure 12 is the result of immunofluorescence identification of cell types of brain organoids in the chip; the reference numbers in the figure are:
- the body of the organoid culture device 211.
- the culture medium storage tank 31. The first culture medium storage tank; 32. The second culture medium storage tank;
- Figure 13 is a cell type diagram of brain organoids in the immunofluorescence identification chip of Example 8.
- Fig. 14 is a graph showing the cell types of brain organoids in the chip identified by immunofluorescence and QPCR in Example 8.
- an organoid culture chip is provided, as shown in Figures 1-3, including:
- An organoid culture device 2 is arranged in the cell culture plate 1; a culture medium reservoir 3 is formed between the periphery of the organoid culture device 2 and the cell culture plate 1; the organoid culture device 2 includes : an organoid culture device body 21, an organoid culture chamber 22 disposed in the organoid culture device body 21 and a side hole 23 disposed on the side wall of the organoid culture device body 21, the organoid culture The chamber 22 communicates with the side hole 23, and the organoid culture chamber 22 includes a sample addition hole 221 arranged at the top and a microwell 222 arranged at the bottom, and the sample addition hole 221 is connected to the microwell 222. All communicate with the bottom of the cell culture plate 1 .
- the cell suspension is added to the organoid culture chamber 22 through the sample injection hole 221 for cultivation, and the cultured organoids all grow and develop in independent chambers, and the interference between samples Sex is low.
- the culture medium needs only to be changed through the culture medium reservoir 3, and enters the organoid culture chamber 22 through the medium perfusion channel without directly contacting the organoid culture chamber 22. Organoids reduce the risk of damage during the culture process.
- the side holes 23 on both sides form a perfusion channel with the organoid culture chamber 22, and during the perfusion culture, factors such as fluid stimulation are used to simulate tissue growth in vivo
- the microenvironment can provide nutrients and oxygen exchange conditions in the process of stem cell culture, three-dimensional spheroid self-assembly, in situ differentiation and organoid maturation. It also realizes the one-step culture of stem cell differentiation into organoids, thus simplifying the culture process. Increase organoid throughput and reduce contamination risk.
- the device has the advantages of low cost, easy operation, in situ imaging, and real-time monitoring, providing an innovative platform for simulating human organ development, mechanism research, toxicology testing, and drug screening.
- multiple organoid culture chambers there are multiple organoid culture chambers, multiple pairs of side holes are provided on the side walls of the body of the organoid culture device, multiple organoid culture chambers and multiple pairs of side holes The holes are connected one by one to form a plurality of perfusion channels.
- one or more organoid culture chambers 22 can be provided; each organoid culture chamber 22 corresponds to two side holes 23; the medium perfusion channel is mainly used for medium exchange and organoid position fixation and the removal of dead cells in the culture chamber.
- the cell culture plate 1 can be a 96-well plate, a 48-well plate, a 24-well plate, a 12-well plate, a 6-well plate and various commonly used cell culture plates.
- a cell culture plate commonly used in the market at present can be used, and its length is 5 mm to 10 cm.
- the organoid culture chip of the present invention is modified based on the existing cell culture well plate, is highly combined with the existing cell culture well plate, and has good compatibility with the existing biological related optical instruments.
- the bottom material of the organoid culture chamber is hydrophobic or treated with hydrophobicity.
- the bottom material of the organoid culture chamber in the organoid culture device described in the embodiment of the present invention is a material with strong hydrophobicity, or is treated with hydrophobicity, in order to prevent the growth of cells attached to the wall.
- the first side wall of the organoid culture device body and the cell culture plate form a first medium reservoir, and the first medium reservoir passes through the first side
- the hole communicates with the organoid culture chamber;
- the second side wall of the organoid culture device body forms a second culture medium reservoir with the cell culture plate, and the second culture medium reservoir passes through the
- the second side hole communicates with the organoid culture chamber. Its purpose is to make the liquid reservoirs on both sides fully exchange nutrients and oxygen and remove necrotic cells during perfusion culture.
- the third side wall and the fourth side wall are respectively adapted to the shape of the inner side of the cell culture plate, and the third side wall and the fourth side wall are respectively compatible with
- the cell culture plates are abutted against each other. Its purpose is to separate the cell culture plate into two separate reservoirs.
- the material of the organoid culture device is an optically transparent material, including but not limited to quartz, glass, thermoplastic polymers, solidified polymers, and solvent-volatile polymers, such as: quartz, PDMS, One or more of PMMA, PC, PT, and resin agarose are used in combination.
- optically transparent materials are conducive to subsequent observation. For example: during the culture process, if you need to observe the cultured organoids, you can place the culture chip under a microscope for observation; For subsequent testing of organs, such as immunofluorescence identification, organoids can be processed in situ within the chip and observed microscopically.
- the height of the organoid culture device 2 is 5.00 mm to 15.00 mm; the reason for adopting this height range: it can make the medium in the reservoirs on both sides form a liquid level difference, which is a culture chamber.
- the organoids in the medium provide a dynamic culture environment. If the height is too small, the liquid level difference formed is small, which is not conducive to the exchange of nutrients and oxygen during the culture of organoids. If the height is too high, the medium will easily overflow during the dynamic culture process. ;
- the shape of the organoid culture chamber 22 in the organoid culture device 2 can be other shapes such as rectangle and circle, and its purpose is to provide an independent spatial culture environment for the cultured cell pellets or organoids. If the organoid culture chamber 22 is rectangular, its length is 2.00 mm to 6.00 mm, and its width is 2.00 mm to 6.00 mm; if the organoid culture chamber 21 is circular, its diameter is 2.00 mm to 6.00 mm ; The height of the organoid culture chamber is 5.00mm-15.00mm.
- the shape of the side hole 23 of the organoid culture chamber in the organoid culture device 2 can be other shapes such as rectangle and circle, and its purpose is to communicate with the reservoir (the first culture medium reservoir 31 and the second culture medium
- the liquid reservoir 32 provides a continuous perfusion culture environment for the cultured organoids.
- the side hole is provided with an adjustable baffle, and the side hole is blocked by adjusting the baffle.
- the size of the hole is used to adjust the pore diameter so as to realize the adjustment of perfusion speed and perfusion cycle.
- the area of the side holes ranges from 1 to 12 mm 2 ; if the side holes 23 of the organoid culture chamber are rectangular, the length is 0.10 mm to 6.00 mm, and the width is 0.10 mm to 6.00 mm; if the organoid culture chamber The side hole 23 of the chamber is circular, with a diameter of 0.10 mm to 6.00 mm;
- the organoid culture chamber interval 24 in the organoid culture device 2 has a length of 0.10 mm to 10.00 mm; the number of organoid culture chambers in the organoid culture device is 1 to 10 mm. 10.
- the thickness of the first side wall 211 and the second side wall 212 of the organoid culture chamber in the organoid culture device 2 is 0.1mm-6mm;
- the area of the side hole is 1 mm 2 -12 mm 2 .
- the range does not include the culture chip used to construct vascularized organoids; if the area of the side hole is less than 1mm 2 , it is difficult to fully exchange nutrients and oxygen during the culture process, and in the long-term culture process The generated dead cells are difficult to remove, affecting the growth and differentiation of organoids, making it difficult to grow normally; if the area of the side hole is greater than 12mm 2 , it cannot provide a limiting function for the organoids, allowing them to grow freely, and it is difficult to ensure uniformity. Area coefficient of variation >40%;
- the side view of the organoid culture chamber in the organoid culture device can be in other shapes such as rectangle, trapezoid, triangle and hemisphere.
- the sample hole 221 on the top of the organoid culture chamber 22 is rectangular, trapezoidal, triangular, hemispherical and other shapes; the area of the sample hole 221 ranges from 1 mm to 6 mm. operation and provide enough growth space for organoids, too small an area is not conducive to the actual operation of the operator, and too large an area makes it easy for organoids to grow arbitrarily in the culture chamber, which has no function of limiting the organoids, uniform decreased sex;
- the microwells 222 at the bottom of the organoid culture chamber 22 are other shapes such as rectangle, trapezoid, triangle and hemisphere; the bottom of the organoid culture chamber in the organoid culture device is a microwell array, and the openings of the microwells are selected from but Not limited to polygons such as circles, ovals, triangles, and rectangles; the area of the micropores 222 is 0.785mm 2 to 100mm 2 ; It is difficult to fully exchange substances and oxygen, and it is difficult to remove dead cells produced during the long-term culture process, which affects the growth and differentiation of organoids. If the area is too large, it will not be able to provide a limiting function for organoids and allow them to grow freely , it is difficult to ensure uniformity; more preferably, the micropore area is 4mm 2 -33mm 2 ;
- the bottom material of the organoid culture chamber 22 in the organoid culture device 2 is an optically transparent material, including but not limited to quartz, glass, thermoplastic polymer, curable polymer and solvent-volatile polymer, etc., for example: quartz , PDMS, PMMA, PC, PT, resin agarose in one or more combination.
- vascularized brain organoid culture chip is provided, as shown in Figures 5-7, including:
- An organoid culture device 2 is arranged in the cell culture plate 1; a culture medium reservoir 3 is formed between the periphery of the organoid culture device 2 and the cell culture plate 1;
- the organoid culture device 2 includes : organoid culture device body 21, a plurality of organoid culture chambers 22 arranged in the organoid culture device body 21 and a plurality of side holes 23 arranged on the side wall of the organoid culture device body 21, the The organoid culture chambers 22 communicate with the side holes 23 in one-to-one correspondence, and a plurality of the organoid culture chambers 22 include a sample-filling hole 221 at the top and a microwell 222 at the bottom. Both the sample well 221 and the microwell 222 are connected to the bottom of the cell culture plate 1;
- the sticking film is arranged on the side hole; the sticking film includes a porous film or a filter screen, and the pore diameter of the porous film or filter screen is 20 ⁇ m to 200 ⁇ m.
- the cell suspension is added to the organoid culture chamber 22 through the sample injection hole 221 for cultivation, and the cultured organoids all grow and develop in independent chambers, and the interference between samples Sex is low.
- the culture medium needs only to be changed through the culture medium reservoir 3, and enters the organoid culture chamber 22 through the medium perfusion channel without directly contacting the organoid culture chamber 22.
- Organoids reduce the risk of damage during the culture process. Since the organoid culture chamber 22 communicates with the side holes 23 one by one, the side holes 23 on both sides form a perfusion channel with the organoid culture chamber 22.
- the perfusion culture factors such as fluid stimulation are used to simulate The microenvironment of tissue growth in vivo can provide nutrients and oxygen exchange conditions in the process of stem cell culture, three-dimensional spheroid self-assembly, in situ differentiation and organoid maturation, and also realizes the one-step culture of stem cell differentiation into organoids, thus simplifying Culture workflows, increase organoid throughput, and reduce contamination risk.
- the device has the advantages of low cost, easy operation, in situ imaging, and real-time monitoring, providing an innovative platform for simulating human organ development, mechanism research, toxicology testing, and drug screening. Since matrigel is required for vascularized brain organoid culture, the medium perfusion well is encapsulated with a film (porous film or filter) for nutrient transport and hydrogel in the culture chamber to provide support .
- the medium perfusion channel is mainly used for medium exchange, position fixation of organoids, removal of dead cells in the culture chamber, and the like.
- the pore diameter of the porous film is 20 ⁇ m to 200 ⁇ m. This range of pore size is conducive to the attachment of cells and the sufficient exchange of nutrients and oxygen during organoid culture. If the pore size is too small, the liquid on both sides cannot be fully exchanged due to the tension of the liquid, which will cause hypoxia and other effects on the culture of organoids; If it is too large, it is not conducive to the attachment of related cells in the later stage of vascularization, and cannot form a stable vascular network structure;
- multiple organoid culture chambers there are multiple organoid culture chambers, multiple pairs of side holes are provided on the side walls of the body of the organoid culture device, multiple organoid culture chambers and multiple pairs of side holes The holes are connected one by one to form a plurality of perfusion channels.
- one or more organoid culture chambers 22 can be provided; each organoid culture chamber 22 corresponds to two side holes 23; the medium perfusion channel is mainly used for medium exchange and organoid position fixation and the removal of dead cells in the culture chamber.
- the cell culture plate 1 can be a 96-well plate, a 48-well plate, a 24-well plate, a 12-well plate, a 6-well plate and various commonly used cell culture plates.
- a cell culture plate commonly used in the market at present can be used, and its length is 5mm-10cm.
- the vascularized brain organoid culture chip of the present invention is modified based on the existing cell culture well plate, is highly combined with the existing cell culture well plate, and has good compatibility with the existing biological related optical instruments.
- the bottom material of the organoid culture chamber is hydrophobic or treated with hydrophobicity.
- the bottom material of the organoid culture chamber in the organoid culture device described in the embodiment of the present invention is a material with strong hydrophobicity, or is treated with hydrophobicity, in order to prevent the growth of cells attached to the wall.
- the first side wall of the organoid culture device body and the cell culture plate form a first medium reservoir, and the first medium reservoir passes through the first side
- the hole communicates with the organoid culture chamber;
- the second side wall of the organoid culture device body forms a second culture medium reservoir with the cell culture plate, and the second culture medium reservoir passes through the
- the second side hole communicates with the organoid culture chamber. Its purpose is to make the liquid reservoirs on both sides fully exchange nutrients and oxygen and remove necrotic cells during perfusion culture.
- the third side wall and the fourth side wall are respectively adapted to the shape of the inner side of the cell culture plate, and the third side wall and the fourth side wall are respectively compatible with
- the cell culture plates are abutted against each other. Its purpose is to separate the cell culture plate into two separate reservoirs.
- the material of the organoid culture device is an optically transparent material, including but not limited to quartz, glass, thermoplastic polymers, solidified polymers, and solvent-volatile polymers, such as: quartz, PDMS, One or more of PMMA, PC, PET, and resin agarose are used in combination.
- optically transparent materials are conducive to subsequent observation. For example: during the culture process, if you need to observe the cultured organoids, you can place the culture chip under a microscope for observation; For subsequent testing of organs, such as immunofluorescence identification, organoids can be processed in situ within the chip and observed microscopically.
- the height of the organoid culture device 2 is 5.00 mm to 15.00 mm; the reason for adopting this height range: in order to make the culture medium in the reservoirs on both sides form a liquid level difference, the culture chamber The organoids in the chamber provide a dynamic culture environment. If the height is too small, the liquid level difference formed is small, which is not conducive to the exchange of nutrients and oxygen during the culture of organoids. overflow;
- the shape of the organoid culture chamber 22 in the organoid culture device 2 can be other shapes such as rectangle and circle, and its purpose is to provide an independent spatial culture environment for the cultured cell pellets or organoids. If the organoid culture chamber 22 is rectangular, its length is 2.00 mm to 6.00 mm, and its width is 2.00 mm to 6.00 mm; if the organoid culture chamber (2-1) is circular, its diameter is 2.00 mm. mm ⁇ 6.00mm; the height of the organoid culture chamber is 5.00mm ⁇ 15.00mm.
- the shape of the side hole 23 of the organoid culture chamber in the organoid culture device 2 can be other shapes such as rectangle and circle, and its purpose is to communicate with the reservoir (the first culture medium reservoir 31 and the second culture medium
- the liquid reservoir 32 provides a continuous perfusion culture environment for the cultured organoids.
- the side hole is provided with an adjustable baffle, and the side hole is blocked by adjusting the baffle.
- the size of the hole is used to adjust the pore diameter so as to realize the adjustment of perfusion speed and perfusion cycle.
- the side hole 23 of the organoid culture chamber is rectangular, its length is 0.10 mm to 6.00 mm, and its width is 0.10 mm to 6.00 mm; if the side hole 23 of the organoid culture chamber is circular, its diameter 0.10mm ⁇ 6.00mm;
- the organoid culture chamber interval 24 in the organoid culture device 2 has a length of 0.10 mm to 10.00 mm; the number of organoid culture chambers in the organoid culture device is 1 to 10 mm. 10.
- the thickness of the first side wall 211 and the second side wall 212 of the organoid culture chamber in the organoid culture device 2 is 0.1mm-6mm;
- the area of the side hole used to construct the culture chip of vascularized organoids is 1 mm 2 to 50 mm 2 ; if the area of the side hole is less than 1 mm 2 , the nutrients and oxygen during the culture process It is difficult to fully exchange, and the dead cells produced during the long-term culture process are difficult to remove, affecting the growth and differentiation of organoids, making it difficult to grow normally; if the area of the side hole is greater than 50mm 2 , it cannot provide a limit for organoids function, let it grow freely, it is difficult to ensure uniformity, and the coefficient of variation of the area is >40%;
- the side view of the organoid culture chamber in the organoid culture device can be in other shapes such as rectangle, trapezoid, triangle and hemisphere.
- the sample hole 221 on the top of the organoid culture chamber 22 is rectangular, trapezoidal, triangular, hemispherical and other shapes; the area of the sample hole 221 ranges from 1 mm to 6 mm. operation and provide enough growth space for organoids, too small an area is not conducive to the actual operation of the operator, and too large an area makes it easy for organoids to grow arbitrarily in the culture chamber, which has no function of limiting the organoids, uniform decreased sex;
- the microholes 222 at the bottom of the organoid culture chamber 22 are rectangular, trapezoidal, triangular, hemispherical and other shapes; the bottom of the organoid culture chamber in the organoid culture device is a microwell array, and the openings of the microwells are selected from but not Limited to polygons such as circles, ellipses, triangles, and rectangles; preferably, the micropore area is 0.785 mm 2 to 100 mm 2 ; the reason for choosing this range of micropore area is: if the micropore size is too small, the nutrients It is difficult to fully exchange with oxygen, and the dead cells produced during the long-term culture process are difficult to remove, which affects the growth and differentiation of organoids. If the area is too large, it will not be able to provide a limiting function for organoids, allowing them to grow freely. It is difficult to guarantee uniformity;
- the bottom material of the organoid culture chamber 22 in the organoid culture device 2 is an optically transparent material, including but not limited to quartz, glass, thermoplastic polymer, curable polymer and solvent-volatile polymer, etc., for example: quartz , PDMS, PMMA, PC, PT, resin agarose in one or more combination.
- Embodiment 1 an organoid culture chip and its preparation method and culture method
- an organoid culture chip provided by an embodiment of the present invention includes:
- An organoid culture device 2 is arranged in the cell culture plate 1; a culture medium reservoir 3 is formed between the periphery of the organoid culture device 2 and the cell culture plate 1; the organoid culture device 2 includes : organoid culture device body 21, a plurality of organoid culture chambers 22 arranged in the organoid culture device body 21 and a plurality of side holes 23 arranged on the side wall of the organoid culture device body 21, the The organoid culture chambers 22 communicate with the side holes 23 in one-to-one correspondence, and a plurality of the organoid culture chambers 22 include a sample-filling hole 221 at the top and a microwell 222 at the bottom. Both the sample well 221 and the microwell 222 are in communication with the bottom of the cell culture plate 1 .
- the height of the organoid culture device is 10mm;
- the interval length between a plurality of said organoid culture chambers is 1 mm;
- the thickness of the side wall of the organoid culture device body is 1 mm;
- the microwells at the bottom of the organoid culture chamber have an area of 12.56 mm 2 .
- the prepared brain organoid culture chamber is pasted to the bottom of the six-well plate with PDMS, and the surrounding gap is filled with PDMS, so that the 12-well plate is separated into two independent reservoirs.
- the culture method of the organoid culture chip described in embodiment 1 comprises the following steps:
- the organoid culture chip is sterilized, and the sterilization methods include but are not limited to the following methods: radiation, ultraviolet light, and gas sterilization;
- the fluid perfusion methods include but not limited to pressure perfusion and gravity perfusion.
- the organoid culture chip can be placed on the Perfusion culture is carried out on a plate shaker, and the deflection angle can be 0-25 degrees; according to the culture conditions of different organoids, different deflection angles can be selected to provide suitable exchange of nutrients and oxygen during the culture process.
- the culture chip can be placed under a microscope for observation;
- the organoids can be processed in situ in the chip and observed under a microscope.
- organoid culture chip shown in Example 1 was used for culture, and the embryoid body formation and brain organoid colonization culture steps were as follows:
- Sterilization and cleaning of the culture device irradiate the above-mentioned brain organoid culture device with ultraviolet light for 30 minutes, and clean the culture chamber and liquid storage tank of the brain organoid 2-3 times with sterilized deionized water.
- Embryoid body formation On the first day, prepare EBs: digest stem cells into single cells, use EBs formation medium to adjust the cell density to a cell suspension of 6 ⁇ 10 4 cells/mL, and place in the organoid culture chamber Add 150 ⁇ L of cell suspension and culture in a 37°C incubator to form EBs.
- the EBs formation medium was replaced with neural induction medium NIM.
- the basic component of the NIM medium is DMEM/F12, and additionally add 1 ⁇ N2 (100 ⁇ ), 1 ⁇ GlutaMax (100 ⁇ ), 1 ⁇ NEAA (Non-Essential Amino Acid, 100 ⁇ ), 1 ⁇ g/mL heparin, 1 ⁇ penicillin-streptomycin (100 ⁇ ), 0.05mM ⁇ -Mercaptoethanol.
- the medium addition process 1 mL of NDM was added to reservoir 1, and 400 ⁇ L of NDM was added to reservoir 2.
- the deflection angle of the seesaw shaker is set at 25°.
- the deflection time of the rocker shaker is 5s.
- the basic components of the NDM medium are 50% DMEM/F12 and 50% Neurobasal Medium by volume, plus 1 ⁇ N2 (100 ⁇ ), 1 ⁇ B27-vitamin A (50 ⁇ ), 1 ⁇ GlutaMax (100 ⁇ ) , 1 ⁇ NEAA (Non Essential Amino Acid, 100 ⁇ ), 1 ⁇ g/mL heparin, 1 ⁇ penicillin-streptomycin (100 ⁇ ), 0.05mM ⁇ -Mercaptoethanol.
- This stage mainly differentiates to each cortex of the brain.
- Figure 4 shows brain organoids at different growth stages.
- the height of the organoid culture device is 5 mm; the interval length between a plurality of organoid culture chambers is 0.1 mm; the thickness of the side wall of the organoid culture device body is 0.1 mm; The area of the microhole at the bottom of the organ culture chamber is 0.785mm 2 ; the area of the side hole is 2.355mm 2 ; the others are the same as in Example 1.
- the height of the organoid culture device is 15 mm; the interval length between a plurality of organoid culture chambers is 0.1 mm; the thickness of the side wall of the organoid culture device body is 0.1 mm; The area of the microhole at the bottom of the organ culture chamber is 100 mm 2 ; the area of the side hole is 2.355 mm 2 ; the others are the same as in Example 1.
- the area of the micropores is changed to 4 mm 2 ; the others are the same as in Embodiment 1.
- the area of the micropores is changed to 33 mm 2 ; the others are the same as in Embodiment 1.
- the area of the side hole is changed to 1 mm 2 ; the others are the same as in Embodiment 1.
- the area of the side hole is changed to 12mm 2 ; the others are the same as in Embodiment 1.
- the organoid culture chip does not contain side holes, and the others are the same as in Example 1.
- Example 2 the micropore area is 0.2 mm 2 , and the others are the same as in Example 1.
- Example 3 the micropore area is 200 mm 2 , and the others are the same as in Example 1.
- the area of the side hole is 0.5 mm 2 , and the others are the same as in example 1.
- the area of the side hole is 50 mm 2 , and the others are the same as in example 1.
- Example 8 A vascularized brain organoid culture chip and its preparation method and culture method
- a vascularized brain organoid culture chip provided by an embodiment of the present invention includes:
- An organoid culture device 2 is arranged in the cell culture plate 1; a culture medium reservoir 3 is formed between the periphery of the organoid culture device 2 and the cell culture plate 1;
- the organoid culture device 2 includes : organoid culture device body 21, a plurality of organoid culture chambers 22 arranged in the organoid culture device body 21 and a plurality of side holes 23 arranged on the side wall of the organoid culture device body 21, the The organoid culture chambers 22 communicate with the side holes 23 in one-to-one correspondence, and a plurality of the organoid culture chambers 22 include a sample-filling hole 221 at the top and a microwell 222 at the bottom. Both the sample well 221 and the microwell 222 are connected to the bottom of the cell culture plate 1;
- the film is arranged on the side hole; the film comprises a porous film or a filter screen, and the porous film or filter screen has a pore size of 70 ⁇ m.
- the height of the organoid culture device is 10mm;
- the interval length between a plurality of said organoid culture chambers is 1 mm;
- the thickness of the side wall of the organoid culture device body is 1mm;
- the microwells at the bottom of the organoid culture chamber have an area of 12 mm 2 .
- the prepared brain organoid culture chamber is pasted to the bottom of the six-well plate with PDMS, and the surrounding gap is filled with PDMS, so that the 12-well plate is separated into two independent reservoirs.
- the culture method of the vascularized brain organoid culture chip described in embodiment 3 comprises the following steps:
- the basic component of the NIM medium is DMEM/F12, and 1 ⁇ N2 (100 ⁇ ), 1 ⁇ GlutaMax (100 ⁇ ), 1 ⁇ NEAA (Non Essential Amino Acid, 100 ⁇ ), 1 ⁇ g/mL heparin, 1 ⁇ penicillin-streptomycin (100 ⁇ ), 0.05mM ⁇ -Mercaptoethanol.
- vascularization-related cells were used to provide growth scaffolds and microenvironments, and neural differentiation medium NDM was added to the reservoir, and cultured on a rocker shaker.
- the medium addition process 1 mL of NDM was added to reservoir 1, and 400 ⁇ L of NDM was added to reservoir 2.
- the deflection angle of the seesaw shaker is set at 10-25°.
- the angle maintenance time of the seesaw shaker is 10h-24h.
- the basic components of the NDM medium are 50% DMEM/F12 and 50% Neurobasal Medium by volume, plus 1 ⁇ N2 (100 ⁇ ), 1 ⁇ B27-vitamin A (50 ⁇ ), 1 ⁇ GlutaMax (100 ⁇ ) , 1 ⁇ NEAA (Non Essential Amino Acid, 100 ⁇ ), 1 ⁇ g/mL heparin, 1 ⁇ penicillin-streptomycin (100 ⁇ ), 0.05mM ⁇ -Mercaptoethanol.
- the basic components of the NIM medium of the NMM are 50% DMEM/F12 and 50% Neurobasal Medium by volume, and 1 ⁇ N2 (100 ⁇ ), 1 ⁇ B27+vitamin A (50 ⁇ ), 1 ⁇ GlutaMax ( 100 ⁇ ), 1 ⁇ NEAA (Non Essential Amino Acid, 100 ⁇ ), 1 ⁇ g/mL heparin, 1 ⁇ penicillin-streptomycin (100 ⁇ ), 0.05mM ⁇ -Mercaptoethanol.
- Figure 8 shows the bright field image of the brain organoids formed, and the QPCR results shown in Figure 9 and Figure 11 identify the expression of markers in different cells of the brain organoids.
- Figure 12 is the immunofluorescence identification of the cell types of brain organoids in the chip.
- the brain organoids cultured in the chip can express neurons (Tuj1), astrocytes (GFAP) and microglial cells (Iba1 ) biomarker results;
- Figure 10 is the size and uniformity characteristics of the brain organoids cultured in the brain organoid chip of Example 8; it shows that the organoid chip and culture method of the present invention can obtain uniform brain organoids;
- Figure 13 is the identification of the cell types of brain organoids in the chip by immunofluorescence.
- the brain organoid chip cultured in the chip can express early neurons (Tuj1), neural stem cells (SOX2 and Nestin) and mature neurons (MAP2) The results of biomarkers;
- Figure 14 is immunofluorescence and QPCR identification of the cell type of the brain organoid chip, it can be observed that the brain organoid chip cultured in the chip can express the cortical structure represented by PAX6 and Nestin (VZ area and CP Region) biomarker results, as well as forebrain and hindbrain (ISL1) biomarkers.
- PAX6 and Nestin VZ area and CP Region
- the pore diameter of the porous film is 70 ⁇ m
- the height of the organoid culture device is 5 mm
- the interval length between a plurality of the organoid culture chambers is 0.1 mm
- the side wall of the organoid culture device body The thickness of the micropore is 0.1mm
- the area of the micropore at the bottom of the organoid culture chamber is 0.785mm 2
- the area of the side hole is 12mm 2 ; the others are the same as in Example 8.
- the pore diameter of the porous film is 70 ⁇ m
- the height of the organoid culture device is 15 mm
- the interval length between a plurality of the organoid culture chambers is 10 mm
- the side wall of the organoid culture device body The thickness is 6mm
- the area of the microhole at the bottom of the organoid culture chamber is 100mm 2
- the area of the side hole is 12mm 2 ; the others are the same as in Example 8.
- the area of the microhole is 4 mm 2 ; the area of the side hole is 2.355 mm 2 ; the others are the same as in Embodiment 8.
- the area of the microhole is 33 mm 2 ; the area of the side hole is 2.355 mm 2 ; the others are the same as in Embodiment 8.
- the area of the side hole is 1 mm 2 ; the others are the same as in Embodiment 8.
- the area of the side hole is 50 mm 2 ; the others are the same as in Embodiment 8.
- the pore diameter of the porous film is 20 ⁇ m, and the others are the same as in Embodiment 8.
- the pore diameter of the porous film is 200 ⁇ m, and the others are the same as in Embodiment 8.
- Example 7 the micropore area is 50 ⁇ m 2 , and the others are the same as in Example 8.
- Example 8 the micropore area is 50 mm 2 , and the others are the same as in Example 8.
- Comparative Example 10 the pore diameter of the porous film is 50 ⁇ m, and the others are the same as in Example 8.
- the micropore area is 0.2 mm 2 , which is smaller than the range of 0.785 mm 2 to 100 mm 2 in the embodiment of the present invention. It is difficult to fully exchange nutrients and oxygen during the culture process, and the The resulting dead cells are difficult to remove, affecting the growth and differentiation of organoids, making it difficult to grow normally;
- the micropore area is 200 mm 2 , which is larger than the range of 0.785 mm 2 to 100 mm 2 in the embodiment of the present invention, and it cannot provide the function of limiting the organoid, allowing it to grow freely, and it is difficult to ensure uniformity.
- the coefficient of variation of the area >40%;
- the area of the side hole is 0.5 mm 2 , which is smaller than the range of 1 mm 2 to 12 mm 2 in the embodiment of the present invention. It is difficult to fully exchange nutrients and oxygen during the cultivation process, and the long-term cultivation process produces It is difficult to remove the dead cells, affecting the growth and differentiation of organoids, making it difficult to grow normally;
- the area of the side hole is 50 mm 2 , which is larger than the range of 1 mm 2 to 12 mm 2 in the embodiment of the present invention, and it cannot provide the function of limiting the organoid, allowing it to grow freely, and it is difficult to ensure uniformity, and the coefficient of variation of the area is >40 %;
- Example 1-Example 7 sufficient exchange of nutrients and oxygen, removal of dead cells, high uniformity of brain organoids, simple operation, etc. during organoid culture;
- the micropore area is in the range of 0.785 mm 2 -100 mm 2
- the side hole area is in the range of 1 mm 2 -12 mm 2 to obtain organoids.
- the micropore area is 50 ⁇ m 2 , which is smaller than the range of 0.785 mm 2 to 100 mm 2 in the embodiment of the present invention, and it is difficult to fully exchange nutrients and oxygen during the culture process, and the long-term culture process produces It is difficult to remove the dead cells, affecting the growth and differentiation of organoids, making it difficult to grow normally;
- the micropore area is 50 mm 2 , which is larger than the range of 0.785 mm 2 to 100 mm 2 in the embodiment of the present invention, and it cannot provide the function of limiting the organoid, allowing it to grow freely, and it is difficult to ensure uniformity.
- the coefficient of variation of the area >40%;
- the pore diameter of the porous film is 5 ⁇ m, which is smaller than the range of 20 ⁇ m to 200 ⁇ m in the embodiment of the present invention, and there are disadvantages that the medium in the reservoirs on both sides cannot smoothly exchange substances, and the growth of organoids is limited;
- the pore diameter of the porous film is 50 ⁇ m, which is larger than the range of 20 ⁇ m to 200 ⁇ m in the embodiment of the present invention, and there is a disadvantage that endothelial cells cannot attach to the porous film to form a complete blood vessel;
- Example 8-Example 16 meet the medium in the liquid reservoir in the chip to complete the material exchange, and provide stable shear force for the organoids in the intermediate culture chamber, and can provide stable support for vascularization-related cells, And form the advantages of blood-brain barrier structure and function;
- the micropore area is in the range of 0.785 mm 2 to 100 mm 2
- the area of the side holes is in the range of 1 mm 2 to 50 mm 2
- the pore diameter of the porous film is 20 ⁇ m to 200 ⁇ m in order to obtain homogeneous blood vessels. organ.
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Abstract
一种类器官培养芯片和培养方法,所述类器官培养芯片包括:细胞培养板(1);类器官培养装置(2),设于所述细胞培养板(1)内;所述类器官培养装置(2)与所述细胞培养板(1)之间形成培养基储液池(3);所述类器官培养装置(2)包括:类器官培养装置本体(21)、多个设于所述类器官培养装置本体(21)内的类器官培养腔室(22)和多个设于所述类器官培养装置本体(21)两侧侧壁上的侧孔(23),所述类器官培养腔室(22)与所述侧孔(23)一一对应相连通形成灌流通道,每个所述类器官培养腔室(22)包括设于顶部的加样孔(221)和设于底部的微孔(222),所述加样孔(221)与微孔(222)均与所述细胞培养板(1)底部相连通。所述类器官培养芯片和培养方法可实现高通量、一步法动态灌流培养形态均一的类器官,并可原位动态观察类器官生长、发育全过程。
Description
本发明涉及组织工程和器官芯片技术领域,特别涉及一种类器官培养芯片和类器官培养方法。
近年来,随着细胞生物学和组织工程学的发展,三维细胞模型正逐渐取代传统二维细胞模型。类器官作为一种新型的三维体外研究模型,是由干细胞在体外自组装,并生长发育成为与人体组织或器官结构和功能相似的三维聚集体,例如:大脑类器官、血管类器官、肝脏类器官、肾脏类器官和肿瘤类器官等。在全球范围内,类器官已经显现出其强大的发展潜力,国际上已经形成一定的竞争格局。此外,中国药品审评中心颁发的《基因修饰细胞治疗产品非临床研究与评价技术指导原则》(试行)中提到:“当缺少相关动物模型时,可采用基于细胞和组织的模型(如2D和3D组织模型、类器官和微流体模型)”,这些模拟人体内环境的类器官可为有效性和安全性的评价提供有价值的补充信息。在器官水平上为疾病模型的建立、药物研发及精准医疗等方面具有重要的应用潜力。
目前,类器官的培养过程主要包括两个步骤:细胞自组装成球和转移或原位分化培养。例如:大脑皮层类器官的培养过程主要包括四个阶段:拟胚体形成、神经外胚层诱导、神经上皮分化和大脑类器官成熟。在大脑类器官培养过程中,需要将经过神经诱导后的拟胚体进行包被Matrigel,随后转入至低粘附的培养板或生物反应器中进行动态培养。然而,在类器官的转移过程中,类器官外层细胞容易受到机械性损伤,并且增加了污染的机率。此外,在悬浮培养的过程中,类器官之间易发生相互融合的现象,并且难以定位观察。上述局限性导致了类器官的培养过程复杂、差异性大、通量低且不易于实时监测等局限。
因此,为了克服上述传统类器官培养手段通量低且复杂等技术问题,有必要开发一种高通量原位培养类器官培养芯片,以用于类器官的应用研究中。
发明内容
本发明目的是提供一种类器官培养芯片和类器官培养方法,可以高通量原位培养类器官,且培养方法的步骤简单。在类器官培养芯片的培养腔室中接种多能干细胞,通过类器官培养和流体刺激等因素,模拟体内组织生长的微环境,可提供干细胞培养、三维球体自组装、原位分化和类器官成熟等过程中的营养物质和氧气交换条件,还实现了干细胞向类器官分化的一步法培养,从而简化培养流程、提高类器官通量和降低污染风险。此外,该装置具有低成本、易操作、可原位成像和实时监测等优势,为模拟人类器官发育、机制研究、毒理测试和药物筛选等方面提供了一个创新性平台。
为了实现上述目的,本发明采用如下技术方案:
在本发明的第一方面,提供了一种类器官培养芯片,包括:
细胞培养板;
类器官培养装置,设于所述细胞培养板内;所述类器官培养装置的外周与所述细胞培养板之间形成培养基储液池;所述类器官培养装置包括:类器官培养装置本体、设于所述类器官培养装置本体内的类器官培养腔室和设于所述类器官培养装置本体两侧侧壁上的侧孔,所述类器官培养腔室与所述两侧侧壁上的相连通形成灌流通道,所述类器官培养腔室包括设于顶部的加样孔和设于底部的微孔,所述加样孔与所述微孔均与所述细胞培养板底部相连通。
进一步地,所述类器官培养腔室为多个,所述类器官培养装置本体两侧侧壁上设有多对所述侧孔,多个所述类器官培养腔室与多对所述侧孔一一对应相连通形成多个所述灌流通道。
进一步地,所述类器官培养芯片包括用于构建大脑类器官、血管化的类器官、肝脏类器官、小肠类器官、胰腺类器官、肾脏类器官和肿瘤类器官中的一种的培养芯片;
所述类器官培养芯片为构建血管化的类器官培养芯片时,所述类器官培养芯片还包括: 贴膜,设于所述侧孔上。
进一步地,所述贴膜包括多孔薄膜或滤网,所述多孔薄膜或滤网的孔径为20μm~200μm。其目的在于能使得附着不同类型的细胞形成屏障结构。
进一步地,所述侧孔包括第一侧孔和第二侧孔,所述类器官培养装置本体设有相对设置的第一侧壁和第二侧壁、相对设置的第三侧壁和第四侧壁;1个或多个所述第一侧孔设于所述类器官培养装置本体的第一侧壁上,1个或多个所述第二侧孔设于所述类器官培养装置本体的第二侧壁上,所述第一侧孔和所述第二侧孔分别与所述类器官培养腔室一一对应设置。
进一步地,所述类器官培养装置本体的所述第一侧壁与所述细胞培养板形成第一培养基储液池,所述第一培养基储液池通过所述第一侧孔与所述类器官培养腔室相连通;
所述类器官培养装置本体的所述第二侧壁与所述细胞培养板形成第二培养基储液池,所述第二培养基储液池通过所述第二侧孔与所述类器官培养腔室相连通。
所述第一培养基储液池通过所述第一侧孔与所述类器官培养腔室相连通;所述第二培养基储液池通过所述第二侧孔与所述类器官培养腔室相连通,其目的在于使得两边的储液池可在灌流培养过程中营养物质和氧气的充分交换和坏死细胞的清除。
进一步地,所述第三侧壁和所述第四侧壁分别与所述细胞培养板的内侧形状相适配,所述第三侧壁和所述第四侧壁分别与所述细胞培养板相抵接。其目的在于将细胞培养板隔离成两个独立的储液池。
进一步地,所述类器官培养装置的材料选自石英、聚二甲基硅氧烷(PDMS)、聚甲基丙烯酸甲酯(PMMA)、聚碳酸酯(PC)、聚对苯二甲酸乙二酯(PET)和树脂中的一种或多种;所述类器官培养腔室的底部材料特性为疏水性或经疏水性处理。其目的在于类器官培养腔室中的细胞能在疏水材料表面自组装成细胞小球。
进一步地,所述类器官培养腔室的形状矩形、圆形、梯形、三角形和半球形中的一种;若所述类器官培养腔室为矩形,则长度为0.10mm~6.00mm,宽度为0.10mm~6.00mm;若所述类器官培养腔室为圆形,直径为0.10mm~6.00mm;
所述侧孔的形状矩形、圆形、梯形、三角形和半球形中的一种;若所述侧孔为矩形,其长度为0.10mm~6.00mm,宽度为0.10mm~6.00mm;若所述侧孔为圆形,其直径为0.10mm~6.00mm。
进一步地,所述类器官培养装置的高度为5.00mm~15.00mm;
多个所述类器官培养腔室之间的间隔长度为0.10mm~10.00mm;
所述类器官培养装置本体侧壁的厚度为0.10mm~6.00mm;
所述类器官培养腔室底部的所述微孔的面积为0.785mm
2~100mm
2;
所述侧孔面积为1mm
2~50mm
2。
对于血管化类器官培养芯片,所述侧孔面积为1mm
2~50mm
2。对于其他类器官培养芯片,所述侧孔面积为1mm
2~12mm
2。
在本发明的第二方面,提供了一种采用所述类器官培养芯片的类器官培养方法,所述方法包括:将细胞悬液或者基质胶包裹的拟胚体EBs加入灭菌后的所述类器官培养芯片中的所述类器官培养腔室中培养。
所述基质胶包裹的拟胚体EBs为于1~10mg/mL的基质胶培养基NIM中培养了11-15天的拟胚体EBs。
上述类器官培养芯片所培养的种子细胞不局限于人多能干细胞(胚胎干细胞和诱导多功能干细胞),同样适用于其他干细胞,包括但不限于人的成体干细胞、肿瘤干细胞以及动物来源的干细胞。
所培养的对象不局限于类器官,同样适用于其他类型细胞形成的三维小球。
本发明实施例中的一个或多个技术方案,至少具有如下技术效果或优点:
本发明提供的一种类器官培养芯片可以高通量原位培养类器官,且培养方法的步骤简 单;具体地:
(1)成本低且易于制造:本发明所采用的材料均为市面上较为常见的材料,且价格低廉。此外,本发明所涉及的制作工具价值低,对于普通实验室和批量生产均有较好的优势。
(2)操作简单且污染风险低:本发明可以同时满足二维培养、三维培养以及动态培养,简化了类器官在培养过程中的操作步骤;降低了初学者的学习成本;减少了在培养过程中因转移类器官产生的污染风险。
(3)通量高且兼容性好:本发明可以同时制备几个至几百个类器官,可以实现高通量的类器官制备和培养需求;在培养过程中,更换培养基无需直接接触类器官,降低了在培养过程中的损伤风险,此外,该芯片与现有的细胞培养孔板高度结合,对现有的生物相关光学仪器均有良好的兼容性。
(4)用户友好性高:传统的器官芯片主要基于PDMS制作的微流控芯片,不适于生物学家的操作习惯,具有很高的学习成本。而本发明有别与传统的器官芯片,充分的与细胞培养板结合,充分考虑生物学家的操作习惯,降低学习成本,具有很高的用户友好性。
(5)疾病造模和药物筛选具有极高的应用潜力:本发明在类器官培养过程中,一致性较高,能有效减少样本之间的差异,此外,本发明中所培养的类器官均在独立的腔室中生长发育,样本之间的干扰性低。此外,该装置能充分的与现有的工业化高通量的药物筛选系统适配,在研究组织发育、疾病造模、毒理测试和药物研发等相关领域具有极高的应用潜力。
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作一简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图。
图1为实施例1类器官培养芯片的结构示意图;
图2为实施例1类器官培养芯片的立体结构图;
图3为实施例1类器官培养芯片中的类器官培养装置结构图;
图4为实施例1的大脑类器官在培养装置中的原位明场图片,其中D1、D7和D14的标尺为200μm,D21、D28和D35的标尺为500μm;
图5为实施例1中基于12孔板的大脑类器官培养装置的俯视图、剖面图和组装图;
图6为实施例1中培养装置的放大图,用于描述培养腔室的具体尺寸;
图7为实施例1中培养装置的结构图;
图8为实施例8的大脑类器官在培养装置中的原位明场图片;
图9为实施例8的大脑类器官芯片中培养的大脑类器官时,QPCR鉴定类器官不同细胞标志物统计图;
图10为实施例8的大脑类器官芯片中培养的大脑类器官的大小和均一性特征;
图11为实施例8的大脑类器官芯片中培养的血管化大脑类器官时,QPCR鉴定血管化大脑类器官不同细胞标志物统计图;
图12为免疫荧光鉴定芯片中大脑类器官的细胞类型结果;图中附图标记为:
1、细胞培养板;
2、类器官培养装置;
21、类器官培养装置本体;211、第一侧壁;212、第二侧壁;213、第三侧壁;214、第四侧壁;
22、类器官培养腔室;221、加样孔;222、微孔;
23、侧孔;231、第一侧孔;232、第二侧孔;
24、间隔;
3、培养基储液池;31、第一培养基储液池;32、第二培养基储液池;
4、贴膜。
图13为实施例8的免疫荧光鉴定芯片中大脑类器官的细胞类型图;
图14为实施例8的免疫荧光和QPCR鉴定芯片中大脑类器官的细胞类型图。
下文将结合具体实施方式和实施例,具体阐述本发明,本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解,这些具体实施方式和实施例是用于说明本发明,而非限制本发明。
在整个说明书中,除非另有特别说明,本文使用的术语应理解为如本领域中通常所使用的含义。因此,除非另有定义,本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾,本说明书优先。
需要说明的是,当元件被称为“固定于”或“设于”另一个元件上,它可以直接在另一个元件上或者间接设在另一个元件上;当一个元件被称为是“连接于”另一个元件,它可以是直接连接到另一个元件或间接连接至另一个元件上。
需要理解的是,术语“长度”、“宽度”、“上”、下”、“前”、“后”、“第一”、“第二”、“竖直”、“水平”、“顶”、“底”、“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系,仅是为了便于描述本申请和简化描述,而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作,因此不能理解为对本申请的限制。
此外,在本申请的描述中,“多个”、“若干个”的含义是两个或两个以上,除非另有明确具体的限定。
本发明提供的技术方案总体思路如下:
根据本发明实施例一种典型的实施方式,提供一种类器官培养芯片,如图1-图3所示,包括:
细胞培养板1;
类器官培养装置2,设于所述细胞培养板1内;所述类器官培养装置2的外周与所述细胞培养板1之间形成培养基储液池3;所述类器官培养装置2包括:类器官培养装置本体21、设于所述类器官培养装置本体21内的类器官培养腔室22和设于所述类器官培养装置本体21侧壁上的侧孔23,所述类器官培养腔室22与所述侧孔23相连通,所述类器官培养腔室22包括设于顶部的加样孔221和设于底部的微孔222,所述加样孔221与所述微孔222均与所述细胞培养板1底部相连通。
本发明使用时,通过所述加样孔221将细胞悬液加入到所述类器官培养腔室22中进行培养,所培养的类器官均在独立的腔室中生长发育,样本之间的干扰性低。在培养过程中,更换培养基只需通过培养基储液池3换液,通过所述培养基灌流通道进入所述类器官培养腔室22内,无需直接接触所述类器官培养腔室22中的类器官,降低了在培养过程中的损伤风险。由于所述类器官培养腔室22与所述侧孔23相连通,因此两侧的侧孔23与类器官培养腔室22形成灌流通道,在灌流培养时通过流体刺激等因素,模拟体内组织生长的微环境,可提供干细胞培养、三维球体自组装、原位分化和类器官成熟等过程中的营养物质和氧气交换条件,还实现了干细胞向类器官分化的一步法培养,从而简化培养流程、提高类器官通量和降低污染风险。此外,该装置具有低成本、易操作、可原位成像和实时监测等优势,为模拟人类器官发育、机制研究、毒理测试和药物筛选等方面提供了一个创新性平台。
优选地,所述类器官培养腔室为多个,所述类器官培养装置本体两侧侧壁上设有多对所述侧孔,多个所述类器官培养腔室与多对所述侧孔一一对应相连通形成多个所述灌流通道。上述技术方案中,类器官培养腔室22可以设置一个多个;每个类器官培养腔室22对应两个侧孔23;所述培养基灌流通道主要用于培养基交换、类器官的位置固定和培养腔室中死细胞的清除等作用。
上述技术方案中,细胞培养板1可以为96孔板、48孔板、24孔板、12孔板、6孔板 以及各种常用型号的细胞培养板。可采用目前市面上常用的细胞培养板,其长度为5mm~10cm。本发明的类器官培养芯片基于现有的细胞培养孔板进行改造,与现有的细胞培养孔板高度结合,对现有的生物相关光学仪器均有良好的兼容性。
优选地,所述类器官培养腔室的底部材料特性为疏水性或经疏水性处理。本发明实施例中所述类器官培养装置中的类器官培养腔室底部材料为疏水性强的材料,或经疏水性处理,是为了防止细胞贴壁生长。
作为一种优选的实施方式,所述类器官培养装置本体的第一侧壁与所述细胞培养板形成第一培养基储液池,所述第一培养基储液池通过所述第一侧孔与所述类器官培养腔室相连通;所述类器官培养装置本体的第二侧壁与所述细胞培养板形成第二培养基储液池,所述第二培养基储液池通过所述第二侧孔与所述类器官培养腔室相连通。其目的在于使得两边的储液池可在灌流培养过程中营养物质和氧气的充分交换和坏死细胞的清除。
作为一种优选的实施方式,所述第三侧壁和所述第四侧壁分别与所述细胞培养板的内侧形状相适配,所述第三侧壁和所述第四侧壁分别与所述细胞培养板相抵接。其目的在于将细胞培养板隔离成两个独立的储液池。
本发明实施例中,所述类器官培养装置的材料为光学透性材料,包括但不限于石英、玻璃、热塑性聚合物、固化型聚合物和溶剂挥发型聚合物等,例如:石英、PDMS、PMMA、PC、PT、树脂琼脂糖中的一种或多种联合使用。采用光学透性材料有利于后续的观察,比如:在培养过程中,如需要对培养的类器官进行观察,可以将培养芯片放置在显微镜下观察;待培养结束后,如果需要对芯片内的类器官进行后续测试,如免疫荧光鉴定,可在芯片内对类器官进行原位处理,并进行显微观察。
作为一种优选的实施方式,所述类器官培养装置2的高度为5.00mm~15.00mm;采用该高度范围的原因:能够使得两边储液池中的培养基形成液位差,为培养腔室中的类器官提供动态培养环境,高度过小有所形成的液位差较小,不利于类器官的培养过程中的营养物质和氧气交换,高度过大会使得在动态培养过程中培养基容易溢出;
所述类器官培养装置2中的类器官培养腔室22的形状可为矩形和圆形等其他形状,其目的在于为所培养的细胞小球或类器官提供独立的空间培养环境。若所述类器官培养腔室22为矩形,其长度为2.00mm~6.00mm,宽度为2.00mm~6.00mm;若所述类器官培养腔室21为圆形,其直径为2.00mm~6.00mm;类器官培养腔室的高度为5.00mm~15.00mm。
所述类器官培养装置2中的类器官培养腔室侧孔23的形状可为矩形和圆形等其他形状,其目的在于连通储液池(第一培养基储液池31和第二培养基储液池32),为所培养的类器官提供持续的灌流培养环境,在本发明另一种实施方式中,所述侧孔上设有可调节的挡片,通过调节挡片挡住所述侧孔的大小来调节孔径从而实现灌流速度和灌流周期调节。侧孔面积的范围为1~12mm
2;若所述类器官培养腔室的侧孔23为矩形,其长度为0.10mm~6.00mm,宽度为0.10mm~6.00mm;若所述类器官培养腔室的侧孔23为圆形,其直径为0.10mm~6.00mm;
作为一种优选的实施方式,所述类器官培养装置2中的类器官培养腔室间隔24长度为0.10mm~10.00mm;所述类器官培养装置中的类器官培养腔室的数量为1~10个。
作为一种优选的实施方式,所述类器官培养装置2中的类器官培养腔室的第一侧壁211和第二侧壁212的厚度为0.1mm~6mm;
作为一种优选的实施方式,所述侧孔面积为1mm
2~12mm
2。(所述范围不包括用于构建血管化类器官的培养芯片);所述侧孔面积若小于1mm
2,在培养过程中的营养物质和氧气难以充分交换,并且在长时程的培养过程中所产生的死细胞难以清除,影响类器官的生长和分化,难以正常生长;所述侧孔面积若大于12mm
2,无法对类器官提供限位的功能,任其自由生长,难以保证均一性,面积变异系数>40%;
所述类器官培养装置中的类器官培养腔室侧视图可为矩形、梯形、三角形和半球形等 其他形状。
类器官培养腔室22顶部加样孔221为矩形、梯形、三角形和半球形等其他形状;加样孔221的面积范围为1mm~6mm,选择该面积范围的原因:便于在类器官培养过程中的操作和为类器官提供足够的生长空间,面积过小不利于操作者实际操作,面积过大则使得类器官容易在培养腔室中任意生长,对类器官没有起到限位的功能,均一性降低;
类器官培养腔室22底部的微孔222为矩形、梯形、三角形和半球形等其他形状;所述类器官培养装置中的类器官培养腔室底部为微孔阵列,微孔的开口选自但不限于圆形、椭圆形、三角形、矩形等多边形;所述微孔222面积为0.785mm
2~100mm
2;选用该微孔面积范围的原因为:微孔尺寸过小则在培养过程中的营养物质和氧气难以充分交换,并且在长时程的培养过程中所产生的死细胞难以清除,影响类器官的生长和分化,面积过大则无法对类器官提供限位的功能,任其自由生长,难以保证均一性;更为优选地,微孔面积为4mm
2~33mm
2;
所述类器官培养装置2中的类器官培养腔室22底部材料为光学透性材料,包括但不限于石英、玻璃、热塑性聚合物、固化型聚合物和溶剂挥发型聚合物等,例如:石英、PDMS、PMMA、PC、PT、树脂琼脂糖中的一种或多种联合使用。
根据本发明实施例另一种典型的实施方式,提供一种血管化的大脑类器官培养芯片,如图5-图7所示,包括:
细胞培养板1;
类器官培养装置2,设于所述细胞培养板1内;所述类器官培养装置2的外周与所述细胞培养板1之间形成培养基储液池3;所述类器官培养装置2包括:类器官培养装置本体21、多个设于所述类器官培养装置本体21内的类器官培养腔室22和多个设于所述类器官培养装置本体21侧壁上的侧孔23,所述类器官培养腔室22与所述侧孔23一一对应相连通,多个所述类器官培养腔室22包括设于顶部的加样孔221和设于底部的微孔222,所述加样孔221与所述微孔222均与所述细胞培养板1底部相连通;
贴膜,设于所述侧孔上;所述贴膜包括多空薄膜或滤网,所述多孔薄膜或滤网的孔径为20μm~200μm。
本发明使用时,通过所述加样孔221将细胞悬液加入到所述类器官培养腔室22中进行培养,所培养的类器官均在独立的腔室中生长发育,样本之间的干扰性低。在培养过程中,更换培养基只需通过培养基储液池3换液,通过所述培养基灌流通道进入所述类器官培养腔室22内,无需直接接触所述类器官培养腔室22中的类器官,降低了在培养过程中的损伤风险。由于所述类器官培养腔室22与所述侧孔23一一对应相连通,因此两侧的侧孔23与类器官培养腔室22形成灌流通道,在灌流培养时通过流体刺激等因素,模拟体内组织生长的微环境,可提供干细胞培养、三维球体自组装、原位分化和类器官成熟等过程中的营养物质和氧气交换条件,还实现了干细胞向类器官分化的一步法培养,从而简化培养流程、提高类器官通量和降低污染风险。此外,该装置具有低成本、易操作、可原位成像和实时监测等优势,为模拟人类器官发育、机制研究、毒理测试和药物筛选等方面提供了一个创新性平台。由于血管化的大脑类器官培养中需要用到基质胶,所述培养基灌流孔外部使用贴膜(多孔薄膜或滤网)进行封装,用于营养物质运输和培养腔室中的水凝胶提供支撑。
上述技术方案中,所述培养基灌流通道主要用于培养基交换、类器官的位置固定和培养腔室中死细胞的清除等作用。
所述多孔薄膜的孔径为20μm~200μm。该孔径范围有利于细胞的附着和类器官培养过程中营养物质和氧气的充分交换,孔径过小有由于液体张力,两边液体无法充分的交换,对类器官的培养造成缺氧等影响;孔径过大则不利于后期血管化的相关细胞的附着,无法形成稳定的血管网络结构;
优选地,所述类器官培养腔室为多个,所述类器官培养装置本体两侧侧壁上设有多对所述侧孔,多个所述类器官培养腔室与多对所述侧孔一一对应相连通形成多个所述灌流通 道。上述技术方案中,类器官培养腔室22可以设置一个多个;每个类器官培养腔室22对应两个侧孔23;所述培养基灌流通道主要用于培养基交换、类器官的位置固定和培养腔室中死细胞的清除等作用。
上述技术方案中,细胞培养板1可以为96孔板、48孔板、24孔板、12孔板、6孔板以及各种常用型号的细胞培养板。可采用目前市面上常用的细胞培养板,其长度为5mm-10cm。本发明的血管化的大脑类器官培养芯片基于现有的细胞培养孔板进行改造,与现有的细胞培养孔板高度结合,对现有的生物相关光学仪器均有良好的兼容性。
优选地,所述类器官培养腔室的底部材料特性为疏水性或经疏水性处理。本发明实施例中所述类器官培养装置中的类器官培养腔室底部材料为疏水性强的材料,或经疏水性处理,是为了防止细胞贴壁生长。
作为一种优选的实施方式,所述类器官培养装置本体的第一侧壁与所述细胞培养板形成第一培养基储液池,所述第一培养基储液池通过所述第一侧孔与所述类器官培养腔室相连通;所述类器官培养装置本体的第二侧壁与所述细胞培养板形成第二培养基储液池,所述第二培养基储液池通过所述第二侧孔与所述类器官培养腔室相连通。其目的在于使得两边的储液池可在灌流培养过程中营养物质和氧气的充分交换和坏死细胞的清除。
作为一种优选的实施方式,所述第三侧壁和所述第四侧壁分别与所述细胞培养板的内侧形状相适配,所述第三侧壁和所述第四侧壁分别与所述细胞培养板相抵接。其目的在于将细胞培养板隔离成两个独立的储液池。
本发明实施例中,所述类器官培养装置的材料为光学透性材料,包括但不限于石英、玻璃、热塑性聚合物、固化型聚合物和溶剂挥发型聚合物等,例如:石英、PDMS、PMMA、PC、PET、树脂琼脂糖中的一种或多种联合使用。采用光学透性材料有利于后续的观察,比如:在培养过程中,如需要对培养的类器官进行观察,可以将培养芯片放置在显微镜下观察;待培养结束后,如果需要对芯片内的类器官进行后续测试,如免疫荧光鉴定,可在芯片内对类器官进行原位处理,并进行显微观察。
作为一种优选的实施方式,所述类器官培养装置2的高度为5.00mm~15.00mm;采用该高度范围的原因:为能够使得两边储液池中的培养基形成液位差,为培养腔室中的类器官提供动态培养环境,高度过小有所形成的液位差较小,不利于类器官的培养过程中的营养物质和氧气交换,高度过大会使得在动态培养过程中培养基容易溢出;
所述类器官培养装置2中的类器官培养腔室22的形状可为矩形和圆形等其他形状,其目的在于为所培养的细胞小球或类器官提供独立的空间培养环境。若所述类器官培养腔室22为矩形,其长度为2.00mm~6.00mm,宽度为2.00mm~6.00mm;若所述类器官培养腔室(2-1)为圆形,其直径为2.00mm~6.00mm;类器官培养腔室的高度为5.00mm~15.00mm。
所述类器官培养装置2中的类器官培养腔室侧孔23的形状可为矩形和圆形等其他形状,其目的在于连通储液池(第一培养基储液池31和第二培养基储液池32),为所培养的类器官提供持续的灌流培养环境,在本发明另一种实施方式中,所述侧孔上设有可调节的挡片,通过调节挡片挡住所述侧孔的大小来调节孔径从而实现灌流速度和灌流周期调节。
若所述类器官培养腔室的侧孔23为矩形,其长度为0.10mm~6.00mm,宽度为0.10mm~6.00mm;若所述类器官培养腔室的侧孔23为圆形,其直径为0.10mm~6.00mm;
作为一种优选的实施方式,所述类器官培养装置2中的类器官培养腔室间隔24长度为0.10mm~10.00mm;所述类器官培养装置中的类器官培养腔室的数量为1~10个。
作为一种优选的实施方式,所述类器官培养装置2中的类器官培养腔室的第一侧壁211和第二侧壁212的厚度为0.1mm~6mm;
作为一种优选的实施方式,用于构建血管化类器官的培养芯片的所述侧孔面积为1mm
2~50mm
2;所述侧孔面积若小于1mm
2,在培养过程中的营养物质和氧气难以充分交换,并且在长时程的培养过程中所产生的死细胞难以清除,影响类器官的生长和分化,难以正 常生长;所述侧孔面积若大于50mm
2,无法对类器官提供限位的功能,任其自由生长,难以保证均一性,面积变异系数>40%;
所述类器官培养装置中的类器官培养腔室侧视图可为矩形、梯形、三角形和半球形等其他形状。
类器官培养腔室22顶部加样孔221为矩形、梯形、三角形和半球形等其他形状;加样孔221的面积范围为1mm~6mm,选择该面积范围的原因:便于在类器官培养过程中的操作和为类器官提供足够的生长空间,面积过小不利于操作者实际操作,面积过大则使得类器官容易在培养腔室中任意生长,对类器官没有起到限位的功能,均一性降低;
类器官培养腔室22底部微孔222为矩形、梯形、三角形和半球形等其他形状;所述类器官培养装置中的类器官培养腔室底部为微孔阵列,微孔的开口选自但不限于圆形、椭圆形、三角形、矩形等多边形;优选地,微孔面积为0.785mm
2~100mm
2;选用该微孔面积范围的原因为:微孔尺寸过小则在培养过程中的营养物质和氧气难以充分交换,并且在长时程的培养过程中所产生的死细胞难以清除,影响类器官的生长和分化,面积过大则无法对类器官提供限位的功能,任其自由生长,难以保证均一性;
所述类器官培养装置2中的类器官培养腔室22底部材料为光学透性材料,包括但不限于石英、玻璃、热塑性聚合物、固化型聚合物和溶剂挥发型聚合物等,例如:石英、PDMS、PMMA、PC、PT、树脂琼脂糖中的一种或多种联合使用。
下面将结合附图对本申请的一种类器官培养芯片和类器官培养方法进行详细说明。
实施例1、一种类器官培养芯片及其制备方法和培养方法
一、类器官培养芯片
如图1-图3所示,本发明实施例提供的一种类器官培养芯片,包括:
细胞培养板1;
类器官培养装置2,设于所述细胞培养板1内;所述类器官培养装置2的外周与所述细胞培养板1之间形成培养基储液池3;所述类器官培养装置2包括:类器官培养装置本体21、多个设于所述类器官培养装置本体21内的类器官培养腔室22和多个设于所述类器官培养装置本体21侧壁上的侧孔23,所述类器官培养腔室22与所述侧孔23一一对应相连通,多个所述类器官培养腔室22包括设于顶部的加样孔221和设于底部的微孔222,所述加样孔221与所述微孔222均与所述细胞培养板1底部相连通。
所述类器官培养装置的高度为10mm;
多个所述类器官培养腔室之间的间隔长度为1mm;
所述类器官培养装置本体侧壁的厚度为1mm;
所述类器官培养腔室底部的所述微孔的面积为12.56mm
2。
二、类器官培养芯片的制备方法
(a)制备大脑类器官培养腔室:按照CAD图纸,利用激光切割机将PMMA板切割成器件单元,形成类器官培养腔室;并在器件单元的两侧打出1个间隔为0.20-3.00mm、直径为1.00-3.00mm的圆形侧孔,具体的,可在器件单元的两侧打出1个间隔为3mm、直径为3mm的圆形侧孔;
(b)大脑类器官培养装置组装:将类器官培养装置和细胞培养板进行封接;所述封接方法包括但不限于黏合、一体注塑成型。
具体地,本发明实施例将制作好的大脑类器官培养腔室,用PDMS粘贴至六孔板的底部,并用PDMS填充周围的间隙,将12孔板隔离成两个独立的储液池。
二、大脑类器官的培养方法
实施例1所述类器官培养芯片的培养方法,包括以下步骤:
(1)类器官培养芯片进行灭菌处理,灭菌手段包括但不限于一下方法:辐射、紫外线、气体灭菌;
(2)将灭菌后的类器官培养芯片用灭菌水进行清洗,并充分晾干;
(3)将一定细胞浓度的细胞悬液加入至类器官培养芯片中的类器官培养腔室中,轻轻摇晃是细胞悬液均匀的分布在腔室内,最后盖上细胞培养板盖子;
(4)通过静置或者低速离心的方法是细胞充分的沉降到细胞培养孔的底部,在放置在二氧化碳培养险种静置培养12-48小时,使沉降后的细胞自组装成细胞小球;本过程为静态培养,如需换液或者更换其他培养基,可通过侧边小孔进行更换,避免损伤小球;
(5)待细胞小球成型后,若需要对类器官进行灌流培养,所述的流体灌注方式包括但不限于压力灌流、重力灌流,可根据培养过程中的需要将类器官培养芯片放置在翘板摇床上进行灌流培养,其偏转角度可为0-25度;根据不同类器官的培养条件,选择不同的偏转角度,以提供培养过程中的合适的营养物质和氧气的交换。
(6)在培养过程中,如需要对培养的类器官进行观察,可以将培养芯片放置在显微镜下观察;
(7)待培养结束后,如果需要对芯片内的类器官进行后续测试,如免疫荧光鉴定,可在芯片内对类器官进行原位处理,并进行显微观察。
具体地,采用实施例1所示的类器官培养芯片进行培养,拟胚体形成以及大脑类器官定植培养步骤如下:
(1)培养装置灭菌及清洗:将上述大脑类器官培养装置用紫外光照30min,用灭菌后的去离子水清洗大脑类器官的培养腔室和储液池2-3遍。
(2)拟胚体形成:第1天,制备EBs:将干细胞消化成单细胞,使用EBs形成培养基将细胞密度调整为6×10
4cells/mL的细胞悬液,类器官培养腔室中加入150μL的细胞悬液,置于37℃培养箱中培养,以形成EBs。
(3)诱导EBs想神经上皮方向分化:第6天,将EBs形成培养基更换为神经诱导培养基NIM。所述NIM培养基的基础成分为DMEM/F12,另添加1×N2(100×),1×GlutaMax(100×),1×NEAA(Non-Essential Amino Acid,100×),1μg/mL heparin,1×penicillin-streptomycin(100×),0.05mM β-Mercaptoethanol。
(4)诱导EBs神经分化:第12天,使用1-10mg/mL的Matrigel将大脑类器官培养包裹,整个过程避免产生气泡,并确保低温操作,维持Matrigel的液体状态。将转移后的大脑类器官培养装置置于37℃培养箱中,孵育1h使得Matrigel充分交联,并向储液槽中添加神经分化培养基NDM,置于翘板摇床中培养。
所述培养基添加过程:储液槽1中添加1mL的NDM,储液槽2中添加400μL的NDM。
所述翘板摇床的偏转角度设为25°。
所述翘板摇床的偏转时间为5s。
所述NDM培养基的基础成分为占体积50%DMEM/F12和50%Neurobasal Medium,另添加1×N2(100×),1×B27-vitamin A(50×),1×GlutaMax(100×),1×NEAA(Non Essential Amino Acid,100×),1μg/mL heparin,1×penicillin-streptomycin(100×),0.05mM β-Mercaptoethanol。
(5)诱导EBs分化成熟:第15天,将NDM基更换为神经成熟培养基NMM。
该阶段主要向大脑各个皮层分化。图4所示,不同生长阶段的大脑类器官。
实施例2
该实施例中,类器官培养装置的高度为5mm;多个所述类器官培养腔室之间的间隔长度为0.1mm;所述类器官培养装置本体侧壁的厚度为0.1mm;所述类器官培养腔室底部的所述微孔的面积为0.785mm
2;侧孔面积为2.355mm
2;其他均同实施例1。
实施例3
该实施例中,类器官培养装置的高度为15mm;多个所述类器官培养腔室之间的间隔长度为0.1mm;所述类器官培养装置本体侧壁的厚度为0.1mm;所述类器官培养腔室底部的所述微孔的面积为100mm
2;侧孔面积为2.355mm
2;其他均同实施例1。
实施例4
该实施例中,所述微孔的面积改为4mm
2;其他均同实施例1。
实施例5
该实施例中,所述微孔的面积改为33mm
2;其他均同实施例1。
实施例6
该实施例中,所述侧孔的面积改为1mm
2;其他均同实施例1。
实施例7
该实施例中,所述侧孔的面积改为12mm
2;其他均同实施例1。
对比例1
该对比例中类器官培养芯片不含有侧孔,其他均同实施例1。
对比例2
对比例2中,微孔面积为0.2mm
2,其他均同实施例1。
对比例3
对比例3中,微孔面积为200mm
2,其他均同实施例1。
对比例4
对比例4中,侧孔面积为0.5mm
2,其他均同实施例1。
对比例5
对比例5中,侧孔面积为50mm
2,其他均同实施例1。
实施例8、一种血管化的大脑类器官培养芯片及其制备方法和培养方法
一、血管化的大脑类器官培养芯片
如图5-图7所示,本发明实施例提供的一种血管化的大脑类器官培养芯片,包括:
细胞培养板1;
类器官培养装置2,设于所述细胞培养板1内;所述类器官培养装置2的外周与所述细胞培养板1之间形成培养基储液池3;所述类器官培养装置2包括:类器官培养装置本体21、多个设于所述类器官培养装置本体21内的类器官培养腔室22和多个设于所述类器官培养装置本体21侧壁上的侧孔23,所述类器官培养腔室22与所述侧孔23一一对应相连通,多个所述类器官培养腔室22包括设于顶部的加样孔221和设于底部的微孔222,所述加样孔221与所述微孔222均与所述细胞培养板1底部相连通;
贴膜,设于所述侧孔上;所述贴膜包括多空薄膜或滤网,所述多孔薄膜或滤网的孔径为70μm。
所述类器官培养装置的高度为10mm;
多个所述类器官培养腔室之间的间隔长度为1mm;
所述类器官培养装置本体侧壁的厚度为1mm;
所述类器官培养腔室底部的所述微孔的面积为12mm
2。
二、血管化的大脑类器官培养芯片的制备方法
(i)制备大脑类器官培养腔室:按照CAD图纸,利用激光切割机将PMMA板切割成器件单元形成类器官培养腔室,在器件单元的两侧打出3mm×4mm的侧孔,并在侧孔外贴上孔径为70μm的滤网;
(ii)大脑类器官培养装置组装:将类器官培养装置和细胞培养板进行封接;所述封接方法包括但不限于黏合、一体注塑成型。
具体地,本发明实施例将制作好的大脑类器官培养腔室,用PDMS粘贴至六孔板的底部,并用PDMS填充周围的间隙,将12孔板隔离成两个独立的储液池。
三、大脑类器官的培养方法
实施例3所述血管化的大脑类器官培养芯片的培养方法,包括以下步骤:
血管化大脑类器官在装置中的分化发育和成熟培养,具体为:
(1)第1天,使用低粘附的96孔板制备EBs:将干细胞消化成单细胞,使用EBs形成培养基将细胞密度调整为6×10
4cells/mL的细胞悬液,在96孔板中加入150μL的细胞悬 液,置于37℃培养箱中培养,以形成EBs。
(2)第6天,诱导EBs想神经上皮方向分化,将EBs形成培养基更换为神经诱导培养基NIM。
所述NIM培养基的基础成分为DMEM/F12,另添加1×N2(100×),1×GlutaMax(100×),1×NEAA(Non Essential Amino Acid,100×),1μg/mL heparin,1×penicillin-streptomycin(100×),0.05mM β-Mercaptoethanol。
(3)第12天,诱导EBs神经分化,使用1-10mg/mL的Matrigel将分化后的EBs重悬,并转移至大脑类器官培养装置的培养腔室中,整个过程避免产生气泡,并确保低温操作,维持Matrigel的液体状态。将转移后的大脑类器官培养装置置于37℃培养箱中,孵育10min使得Matrigel充分交联,用1-10mg/mL的Matrigel将培养腔室中的空隙进行填充,使得侧孔完全被Matrigel封接,以血管化相关细胞提供生长支架和微环境,并向储液槽中添加神经分化培养基NDM,置于翘板摇床中培养。
所述培养基添加过程:储液槽1中添加1mL的NDM,储液槽2中添加400μL的NDM。
所述翘板摇床的偏转角度设置为10~25°。
所述翘板摇床的角度维持时间为10h~24h。
所述NDM培养基的基础成分为占体积50%DMEM/F12和50%Neurobasal Medium,另添加1×N2(100×),1×B27-vitamin A(50×),1×GlutaMax(100×),1×NEAA(Non Essential Amino Acid,100×),1μg/mL heparin,1×penicillin-streptomycin(100×),0.05mM β-Mercaptoethanol。
(4)第15天,诱导EBs分化成熟,将NDM基更换为神经成熟培养基NMM。
所述NMM所述NIM培养基的基础成分为占体积50%DMEM/F12和50%Neurobasal Medium,另添加1×N2(100×),1×B27+vitamin A(50×),1×GlutaMax(100×),1×NEAA(Non Essential Amino Acid,100×),1μg/mL heparin,1×penicillin-streptomycin(100×),0.05mM β-Mercaptoethanol。
该阶段主要向大脑各个皮层诱导分化。图8所示形成大脑类器官的明场图,图9所示和图11的QPCR结果鉴定大脑类器官不同细胞的标志物的表达情况。结果显示大脑类器官中出现与人脑组织相似的主要细胞谱系:神经元、星形胶质细胞和小胶质细胞。图12为免疫荧光鉴定芯片中大脑类器官的细胞类型,能够观察到在芯片中培养的大脑类器官芯片能够表达神经元(Tuj1)、星形胶质细胞(GFAP)和小胶质细胞(Iba1)的生物标记物的结果;图10为实施例8的大脑类器官芯片中培养的大脑类器官的大小和均一性特征;表明本发明的类器官芯片和培养方法能够得到均一的大脑类器官;图13为免疫荧光鉴定芯片中大脑类器官的细胞类型,能够观察到在芯片中培养的大脑类器官芯片能够表达早期神经元(Tuj1)、神经干细胞(SOX2和Nestin)和成熟神经元(MAP2)的生物标记物的结果;图14为免疫荧光和QPCR鉴定芯片中大脑类器官的细胞类型,能够观察到在芯片中培养的大脑类器官芯片能够表达PAX6和Nestin代表的皮层结构(VZ区和CP区)生物标记物的结果,以及前脑和后脑(ISL1)的生物标志物。
实施例9
该实施例中,多孔薄膜的孔径为70μm,所述类器官培养装置的高度为5mm;多个所述类器官培养腔室之间的间隔长度为0.1mm;所述类器官培养装置本体侧壁的厚度为0.1mm;所述类器官培养腔室底部的所述微孔的面积为0.785mm
2;侧孔面积为12mm
2;其他均同实施例8。
实施例10
该实施例中,多孔薄膜的孔径为70μm,所述类器官培养装置的高度为15mm;多个所述类器官培养腔室之间的间隔长度为10mm;所述类器官培养装置本体侧壁的厚度为6mm;所述类器官培养腔室底部的所述微孔的面积为100mm
2;侧孔面积为12mm
2;其他均同实施例8。
实施例11
该实施例中,微孔的面积为4mm
2;侧孔面积为2.355mm
2;其他均同实施例8。
实施例12
该实施例中,微孔的面积为33mm
2;侧孔面积为2.355mm
2;其他均同实施例8。
实施例13
该实施例中,侧孔面积为1mm
2;其他均同实施例8。
实施例14
该实施例中,侧孔面积为50mm
2;其他均同实施例8。
实施例15
该实施例中,多孔薄膜的孔径为20μm,其他均同实施例8。
实施例16
该实施例中,多孔薄膜的孔径为200μm,其他均同实施例8。
对比例6
该对比例6中,类器官培养芯片不含有侧孔,其他均同实施例8。
对比例7
对比例,7中,微孔面积为50μm
2,其他均同实施例8。
对比例8
对比例8中,微孔面积为50mm
2,其他均同实施例8。
对比例9
对比例9中,多孔薄膜的孔径为5μm,其他均同实施例8。
对比例10
对比例10中,多孔薄膜的孔径为50μm,其他均同实施例8。
实验例1
1、对上述实施例1-7和对比例1-5的类器官培养芯片进行类器官培养效果进行统计,如表1所示,其中面积的标准差变异系数的计算方法为:变异系数C·V=(标准偏差SD/平均值Mean)×100%
表1
由表1的数据可知,
对于类器官芯片:
对比例1中,没有灌流通道,类器官营养物质和氧气交换不充分,难以正常生长;
对比例2中,微孔面积为0.2mm
2,小于本发明实施例0.785mm
2~100mm
2的范围,在培养过程中的营养物质和氧气难以充分交换,并且在长时程的培养过程中所产生的死细胞难以清除,影响类器官的生长和分化,难以正常生长;
对比例3中,微孔面积为200mm
2,大于本发明实施例0.785mm
2~100mm
2的范围,无法对类器官提供限位的功能,任其自由生长,难以保证均一性,面积变异系数>40%;
对比例4中,侧孔面积为0.5mm
2,小于本发明实施例1mm
2~12mm
2的范围,在培养过程中的营养物质和氧气难以充分交换,并且在长时程的培养过程中所产生的死细胞难以清除,影响类器官的生长和分化,难以正常生长;
对比例5中,侧孔面积为50mm
2,大于本发明实施例1mm
2~12mm
2的范围,无法对类器官提供限位的功能,任其自由生长,难以保证均一性,面积变异系数>40%;
实施例1-实施例7,类器官培养过程中营养物质和氧气交换充分、死细胞清除和大脑类器官均一性高、操作简单等;
综上可知,对于类器官芯片,微孔面积为0.785mm
2~100mm
2的范围,侧孔面积1mm
2-12mm
2的范围才能培养获得类器官。
2、对实施例8-实施例16和对比例6-对比例10的血管化大脑类器官培养芯片进行血管化类器官的培养,效果统计如下:
表2
由表2的数据可知:
对比例6中,没有灌流通道,类器官营养物质和氧气交换不充分,难以正常生长;
对比例7中,微孔面积为50μm
2,小于本发明实施例0.785mm
2~100mm
2的范围,在 培养过程中的营养物质和氧气难以充分交换,并且在长时程的培养过程中所产生的死细胞难以清除,影响类器官的生长和分化,难以正常生长;
对比例8中,微孔面积为50mm
2,大于本发明实施例0.785mm
2~100mm
2的范围,无法对类器官提供限位的功能,任其自由生长,难以保证均一性,面积变异系数>40%;
对比例9中,多孔薄膜的孔径为5μm,小于本发明实施例20μm~200μm的范围,存在两边储液池中的培养基无法顺利的进行物质交换,类器官的生长受限的缺点;
对比例10中,多孔薄膜的孔径为50μm,大于本发明实施例20μm~200μm的范围,存在内皮细胞无法附着在多孔薄膜上形成完整血管的缺点;
实施例8-实施例16,满足芯片中储液池中的培养基完成物质交换,并为中间培养腔室中的类器官提供稳定的剪切力,能够为血管化相关细胞提供稳定的支撑,并形成具有血脑屏障结构和功能优点;
综上可知,对于血管化类器官芯片,微孔面积为0.785mm
2~100mm
2的范围,侧孔面积1mm
2-50mm
2的范围,多孔薄膜的孔径为20μm~200μm才能培养获得均一化血管类器官。
最后,还需要说明的是,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (20)
- 一种类器官培养芯片,其特征在于,包括:细胞培养板;类器官培养装置,设于所述细胞培养板内;所述类器官培养装置的外周与所述细胞培养板之间形成培养基储液池;所述类器官培养装置包括:类器官培养装置本体、设于所述类器官培养装置本体内的类器官培养腔室和设于所述类器官培养装置本体两侧侧壁上的侧孔,所述类器官培养腔室与所述两侧侧壁上的侧孔相连通形成灌流通道,所述类器官培养腔室包括设于顶部的加样孔和设于底部的微孔,所述加样孔与所述微孔均与所述细胞培养板底部相连通。
- 根据权利要求1所述的一种类器官培养芯片,其特征在于,所述类器官培养腔室为多个,所述类器官培养装置本体两侧侧壁上设有多对所述侧孔,多个所述类器官培养腔室与多对所述侧孔一一对应相连通形成多个所述灌流通道。
- 根据权利要求1或2所述的一种类器官培养芯片,其特征在于,所述类器官培养芯片包括用于构建大脑类器官、血管化的类器官、肝脏类器官、小肠类器官、胰腺类器官、肾脏类器官和肿瘤类器官中的一种的培养芯片;所述类器官培养芯片为构建血管化的类器官培养芯片时,所述类器官培养芯片还包括:贴膜,设于所述侧孔上。
- 根据权利要求3所述的一种类器官培养芯片,其特征在于,所述贴膜包括多孔薄膜或滤网,所述多孔薄膜或滤网的孔径为20μm~200μm。
- 根据权利要求1所述的一种类器官培养芯片,其特征在于,所述侧孔包括第一侧孔和第二侧孔,所述类器官培养装置本体设有相对设置的第一侧壁和第二侧壁、相对设置的第三侧壁和第四侧壁;1个或多个所述第一侧孔设于所述类器官培养装置本体第一侧壁上,1个或多个所述第二侧孔设于所述类器官培养装置本体第二侧壁上,所述第一侧孔和所述第二侧孔分别与所述类器官培养腔室一一对应设置。
- 根据权利要求5所述的一种类器官培养芯片,其特征在于,所述类器官培养装置本体的所述第一侧壁与所述细胞培养板形成第一培养基储液池,所述第一培养基储液池通过所述第一侧孔与所述类器官培养腔室相连通;所述类器官培养装置本体的所述第二侧壁与所述细胞培养板形成第二培养基储液池,所述第二培养基储液池通过所述第二侧孔与所述类器官培养腔室相连通。
- 根据权利要求5所述的一种类器官培养芯片,其特征在于,所述第三侧壁和所述第四侧壁分别与所述细胞培养板的内侧形状相适配,所述第三侧壁和所述第四侧壁分别与所述细胞培养板相抵接。
- 根据权利要求1所述的一种类器官培养芯片,其特征在于,所述类器官培养装置的材料选自石英、聚二甲基硅氧烷(PDMS)、聚甲基丙烯酸甲酯(PMMA)、聚碳酸酯(PC)、聚对苯二甲酸乙二酯(PET)和树脂中的一种或多种;所述类器官培养腔室的底部材料特性为疏水性或经疏水性处理。
- 根据权利要求1所述的一种类器官培养芯片,其特征在于,所述类器官培养腔室的形状矩形、圆形、梯形、三角形和半球形中的一种;若所述类器官培养腔室为矩形,则长度为0.10mm~6.00mm,宽度为0.10mm~6.00mm;若所述类器官培养腔室为圆形,直径为0.10mm~6.00mm;所述侧孔的形状矩形、圆形、梯形、三角形和半球形中的一种;若所述侧孔为矩形,其长度为0.10mm~6.00mm,宽度为0.10mm~6.00mm;若所述侧孔为圆形,其直径为0.10mm~6.00mm。
- 根据权利要求1所述的一种类器官培养芯片,其特征在于,所述类器官培养装置的高度为5.00mm~15.00mm;多个所述类器官培养腔室之间的间隔长度为0.10mm~10.00mm;所述类器官培养装置本体侧壁的厚度为0.10mm~6.00mm;所述类器官培养腔室底部的所述微孔的面积为0.785mm 2~100mm 2;所述侧孔面积为1mm 2~20mm 2。
- 根据权利要求1所述的一种类器官培养芯片,其特征在于,所述类器官培养芯片包括由多能干细胞、成体干细胞和肿瘤干细胞衍生的类器官培养芯片。
- 一种采用权利要求1-11任一所述类器官培养芯片的类器官培养方法,其特征在于,所述方法包括:将细胞悬液或者将基质胶包裹的拟胚体(EBs)加入灭菌后的所述类器官培养芯片中的所述类器官培养腔室中培养。
- 根据权利要求12所述类器官培养芯片的类器官培养方法,其特征在于,包括以下步骤:(1)类器官培养芯片进行灭菌处理,灭菌手段包括但不限于一下方法:辐射、紫外线、气体灭菌;(2)将灭菌后的类器官培养芯片用灭菌水进行清洗,并充分晾干;(3)将一定细胞浓度的细胞悬液加入至类器官培养芯片中的类器官培养腔室中,轻轻摇晃是细胞悬液均匀的分布在腔室内,最后盖上细胞培养板盖子;(4)通过静置或者低速离心的方法是细胞充分的沉降到细胞培养孔的底部,在放置在二氧化碳培养险种静置培养12-48小时,使沉降后的细胞自组装成细胞小球;本过程为静态培养,如需换液或者更换其他培养基,可通过侧边小孔进行更换,避免损伤小球;(5)待细胞小球成型后,若需要对类器官进行灌流培养,所述的流体灌注方式包括但不限于压力灌流、重力灌流,可根据培养过程中的需要将类器官培养芯片放置在翘板摇床上进行灌流培养,其偏转角度可为0-25度;根据不同类器官的培养条件,选择不同的偏转角度,以提供培养过程中的合适的营养物质和氧气的交换。(6)在培养过程中,如需要对培养的类器官进行观察,可以将培养芯片放置在显微镜下观察;(7)待培养结束后,如果需要对芯片内的类器官进行后续测试,如免疫荧光鉴定,可在芯片内对类器官进行原位处理,并进行显微观察。
- 一种根据权利要求1-11任一项所述的类器官培养芯片的制备方法,其特征在于,包括以下步骤:(a)制备大脑类器官培养腔室:按照CAD图纸,利用激光切割机将PMMA板切割成器件单元形成类器官培养腔室,并在器件单元的两侧打出1个间隔为0.20-3.00mm、直径为1.00-3.00mm的圆形侧孔;(b)大脑类器官培养装置组装:将类器官培养装置和细胞培养板进行封接;所述封接方法包括但不限于黏合、一体注塑成型。
- 根据权利要求14所述的类器官培养芯片的制备方法,其特征在于,类器官培养腔室用PDMS粘贴至六孔板的底部,并用PDMS填充周围的间隙,将12孔板隔离成两个独立的储液池。
- 一种大脑类器官培养芯片的制备方法,其特征在于,所述类器官培养芯片,包括:细胞培养板;类器官培养装置,设于所述细胞培养板内;所述类器官培养装置的外周与所述细胞培养板之间形成培养基储液池;所述类器官培养装置包括:类器官培养装置本体、设于所述类器官培养装置本体内的类器官培养腔室和设于所述类器官培养装置本体两侧侧壁上的侧孔,所述类器官培养腔室与所述两侧侧壁上的侧孔相连通形成灌流通道,所述类器官培养腔室包括设于顶部的加样孔和设于底部的微孔,所述加样孔与所述微孔均与所述细胞培养板底部相连通;所述类器官培养装置的高度为10mm;多个所述类器官培养腔室之间的间隔长度为1mm;所述类器官培养装置本体侧壁的厚度为1mm;所述类器官培养腔室底部的所述微孔的面积为12.56mm 2;其制备方法包括以下步骤:(a)制备大脑类器官培养腔室:按照CAD图纸,利用激光切割机将PMMA板切割成器件单元,形成类器官培养腔室,并在器件单元的两侧打出1个间隔为0.20-3.00mm、直径为1.00-3.00mm的圆形侧孔;(b)大脑类器官培养装置组装:将类器官培养装置和细胞培养板进行封接;所述封接方法包括但不限于黏合、一体注塑成型。
- 一种大脑类器官培养芯片的制备方法,其特征在于,所述类器官培养芯片,包括:细胞培养板;类器官培养装置,设于所述细胞培养板内;所述类器官培养装置的外周与所述细胞培养板之间形成培养基储液池;所述类器官培养装置包括:类器官培养装置本体、设于所述类器官培养装置本体内的类器官培养腔室和设于所述类器官培养装置本体两侧侧壁上的侧孔,所述类器官培养腔室与所述两侧侧壁上的侧孔相连通形成灌流通道,所述类器官培养腔室包括设于顶部的加样孔和设于底部的微孔,所述加样孔与所述微孔均与所述细胞培养板底部相连通;所述类器官培养装置的高度为10mm;多个所述类器官培养腔室之间的间隔长度为1mm;所述类器官培养装置本体侧壁的厚度为1mm;所述类器官培养腔室底部的所述微孔的面积为12.56mm 2;其制备方法包括以下步骤:(a)制备大脑类器官培养腔室:按照CAD图纸,利用激光切割机将PMMA板切割成器件单元,形成类器官培养腔室,并在器件单元的两侧打出1个间隔为3mm、直径为3mm的圆形侧孔;(b)大脑类器官培养装置组装:将类器官培养装置和细胞培养板进行封接;所述封接方法包括但不限于黏合、一体注塑成型。
- 根据权利要求16或17所述的大脑类器官培养芯片的制备方法,其特征在于,大脑类器官培养腔室用PDMS粘贴至六孔板的底部,并用PDMS填充周围的间隙,将12孔板隔离成两个独立的储液池。
- 一种血管化的大脑类器官培养芯片的制备方法,其特征在于,所述血管化的大脑类器官培养芯片,包括:细胞培养板;类器官培养装置,设于所述细胞培养板内;所述类器官培养装置的外周与所述细胞培养板之间形成培养基储液池;所述类器官培养装置包括:类器官培养装置本体、多个设于所述类器官培养装置本体内的类器官培养腔室和多个设于所述类器官培养装置本体侧壁上的侧孔,所述类器官培养腔室与所述侧孔一一对应相连通,多个所述类器官培养腔室包括设于顶部的加样孔和设于底部的微孔,所述加样孔与所述微孔均与所述细胞培养板底部相连通;贴膜,设于所述侧孔上;所述贴膜包括多空薄膜或滤网,所述多孔薄膜或滤网的孔径为70μm;所述类器官培养装置的高度为10mm;多个所述类器官培养腔室之间的间隔长度为1mm;所述类器官培养装置本体侧壁的厚度为1mm;所述类器官培养腔室底部的所述微孔的面积为12mm 2;其制备方法包括以下步骤:(i)制备大脑类器官培养腔室:按照CAD图纸,利用激光切割机将PMMA板切割成器件单元形成类器官培养腔室,在器件单元的两侧打出3mm×4mm的侧孔,并在侧孔外贴上孔径为70μm的滤网;(ii)大脑类器官培养装置组装:将类器官培养装置和细胞培养板进行封接;所述封接方法包括但不限于黏合、一体注塑成型。
- 根据权利要求19所述的血管化的大脑类器官培养芯片的制备方法,其特征在于,大脑类器官培养腔室,用PDMS粘贴至六孔板的底部,并用PDMS填充周围的间隙,将12孔板隔离成两个独立的储液池。
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CN117402818A (zh) * | 2023-12-15 | 2024-01-16 | 成都艾名迈德医学检验实验室有限公司 | 一种拟胚体封装材料、封装装置及封装装置的制备方法 |
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CN114657127A (zh) * | 2022-02-28 | 2022-06-24 | 武汉大学 | 一种大脑类器官模型及其制备方法与应用 |
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