WO2013075392A1 - Procédé et dispositif de construction d'un microenvironnement cellulaire en trois dimensions sur la base d'un échafaudage spongieux transparent - Google Patents

Procédé et dispositif de construction d'un microenvironnement cellulaire en trois dimensions sur la base d'un échafaudage spongieux transparent Download PDF

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WO2013075392A1
WO2013075392A1 PCT/CN2012/001013 CN2012001013W WO2013075392A1 WO 2013075392 A1 WO2013075392 A1 WO 2013075392A1 CN 2012001013 W CN2012001013 W CN 2012001013W WO 2013075392 A1 WO2013075392 A1 WO 2013075392A1
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sponge
scaffold
prepolymer solution
polyethylene glycol
dimensional
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PCT/CN2012/001013
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English (en)
Chinese (zh)
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杜亚楠
赵姗
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清华大学
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Priority to CN201280042758.0A priority Critical patent/CN104053459B/zh
Publication of WO2013075392A1 publication Critical patent/WO2013075392A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture

Definitions

  • the invention relates to a method and a device for constructing a three-dimensional microenvironment, in particular to a method as simple and convenient to operate as the traditional two-dimensional cell culture technology (easy as 2D), realizing a three-dimensional cell microenvironment including soluble factors, biological materials and cells.
  • the method and device for construction belong to the field of biomedical engineering.
  • Two-dimensional cell culture (based on commercial petri dishes or multi-well plates) has been developed for more than 100 years and is widely used in life science basic research, pharmaceutical industry, medical research and other fields.
  • the two-dimensional cell culture environment is far removed from the real environment in which cells grow in the body. Therefore, this simplified two-dimensional microenvironment does not simulate and reproduce the three-dimensional microenvironment in the body.
  • the research process that relies on in vivo experiments in animals is complex and different from human response. Therefore, three-dimensional cell culture technology has been greatly developed in recent years.
  • the three-dimensional cell culture technique refers to planting different kinds of cells into a material carrier having a three-dimensional structure, so that the cells can migrate, grow, and function in the three-dimensional spatial structure of the carrier.
  • the goal of this technique is to mimic the cell growth environment in vivo.
  • the core factor is the interaction between cells and the three-dimensional microenvironment, namely the interaction between cells and molecules, cells and extracellular matrix (ECM), and cells and cells.
  • ECM extracellular matrix
  • the three-dimensional cell microenvironment simulates and reproduces the cell growth environment and state (growth, differentiation, polarization, cell-cell interaction, etc.) in vitro, and the cells are in two dimensions in terms of gene expression, matrix secretion, and cell function. There are significant differences in culture.
  • hydrogel culture method is to cross-link a suspension of cells and materials into a hydrogel under certain conditions, and the cells are three-dimensionally cultured in a hydrogel crosslinked network system.
  • Commonly used gelling methods are: temperature transitions (such as collage, matrigel), H transitions (such as chitosan), addition of ions (such as alginate), light exposure (such as hyaluronic acid or dextran-containing vinyl groups), and the like.
  • the stent culture method refers to planting a cell or a suspension of cells-material into a three-dimensional scaffold material that has been formed to realize three-dimensional culture.
  • Static planting generally drops cell or cell-material suspension directly onto the scaffold; dynamic planting uses external power (such as rotary planting, surface ultrasonic implant, centrifugal planting, magnetic field planting, etc.) to allow cells to penetrate the stent more efficiently and evenly.
  • external power such as rotary planting, surface ultrasonic implant, centrifugal planting, magnetic field planting, etc.
  • the cells need to undergo a crosslinking process together with the prepolymer during the gelation process, which is inevitably damaged; and about 90% of the components in the hydrogel system are Water, poor mechanical properties, not suitable for long-term fluid exchange culture and quantitative detection.
  • hydrogels have good optical properties and can be used for real-time unlabeled observation of cell growth under ordinary white light microscopy in the same way as two-dimensional culture. It has wide applications in angiogenesis and oncogenesis.
  • the static implantation method is simple and widely used, but it is also the least effective.
  • the dynamic implantation method has the potential danger of damage to cells by external mechanical force, and the optical performance of the stent culture system is poor, the distribution and growth of cells in the stent system, Changes such as migration cannot be observed in real time, and it is necessary to use fluorescence labeling, fixed staining, etc., which undoubtedly increases the complexity and technical difficulty of the experiment.
  • the stent has good mechanical properties and can be used for long-term fluid-changing and proliferating culture of cells as in the two-dimensional culture method. Nutrient, widely used in tissue engineering, clinical testing and other fields.
  • three-dimensional cell culture technology has developed rapidly. More than ten kinds of three-dimensional cell culture products (such as Alvetex, AlgiMatrix GEM, Microtissues, RAFT, n3D, etc.) have been introduced in the market, all from the United States, Britain, Switzerland and other places. There are two main types of products: hydrogel type and stent type. E.g.
  • qgelbio company launched QGel TM PEG is a powder, it is mixed with the cell suspension and QGel TM buffer, QGel TM Disc Caster in a mold, three-dimensional hydrogel preparation containing a sheet of cells; the company launched 3Dbioteck 3D InsertTM-PS and 3D InsertTM-PCL are in-line porous scaffolds based on multi-well plates.
  • the cell suspension is directly added to the interconnected porous scaffold for three-dimensional culture.
  • the pre-preparation process of the hydrogel-type three-dimensional cell culture method is complicated, and requires multiple steps.
  • the cells and the prepolymer are crosslinked into a gel, which has the risk of damage, and requires a matching mold, and the price is high, but Since the hydrogel has good optical properties, the microscope can be used to observe the cell state under white light in the subsequent experiments; while the porous scaffold type three-dimensional cell culture mode has a simple operation process and low technical difficulty, but the optical performance of the porous scaffold is poor, and it cannot be used in ordinary white light microscopy.
  • the cell state is directly observed, and the cells are required to be fluorescently labeled and fluorescently imaged.
  • DDW Drug Discovery World
  • the three-dimensional cell research method should be consistent with the traditional two-dimensional cell research method in the whole experimental system: no special tools and techniques are needed for the culture method, and the detection method can rely on the off-the-shelf instruments and methods for real-time detection. Detection.
  • a three-dimensional cell culture system requires consideration of multiple factors in actual research. Such as the source of the material matrix (natural or synthetic materials), the physical properties of the material matrix (chemical compatibility, mechanical properties, degradability, structural properties, etc.), the biological activity of the material matrix (adhesion sites, induction signals, etc.), The equipment, conditions of use, scope of application, cell packaging method, culture method, detection method, etc. required for three-dimensional culture technology.
  • the ideal three-dimensional cell culture system enables imaging and characterization during cell planting, culture, proliferation, and passage, as well as traditional two-dimensional cell culture (easy as 2D).
  • the ideal three-dimensional cell culture system can also meet microscale and high-throughput. Research requirements.
  • the ideal three-dimensional cell culture system should also have patterning, microstructure controllability, easy to monitor (monitoring) And so on, the establishment of a bionic in vitro model with more complex and fine structure.
  • the apparatus for constructing a three-dimensional microenvironment provided by the present invention is a device based on a three-dimensional transparent sponge stent.
  • the sponge scaffold is a transparent sponge scaffold
  • the transparent sponge scaffold is the sponge scaffold having a transparency of 50% or more.
  • the sponge stent is made of a biological material and has a plurality of small holes; the pores have a pore diameter of 1 nm - 999 ⁇ , a pore spacing of 1 ⁇ - 999 ⁇ , and a porosity formed by the pores on the sponge stent (porous The ratio of the pore volume of the object to the total volume of the object is 70% - 99.9%; the volume of the sponge scaffold is 0. ⁇ m 3 - 1000 cm 3 .
  • the pore size of the pores may specifically be 1 ⁇ -150 ⁇ , 1 ⁇ -85 ⁇ 10 ⁇ -150 ⁇ or ⁇ -125 ⁇ ; the porosity may specifically be 82.4%-94.2%, 82.4%-93.3% or 93.3% -94.2% (such as 82.4% 93.3% or 94.2%); the volume of the sponge holder may be 0.2355mm 3 -37.56mm 3 , 0.2355mm 3 -4.82 mm 3 , 6.26mm 3 -37.56mm 3 , 0.603 mm 3 -4.82 mm 3 or 0.2355 mm 3 -0.603 mm 3 (specifically eg 0.2355mm 3 , 0.603 mm 3 , 1.204 mm 3 , 1.809 mm 3 , 2.415 mm 3 , 3.026 mm 3 , 3.620 mm 3 , 4.22 mm 3 , 4.82 mm 3 , 6.26mm 3 12.52 mm 3 , 25
  • the biomaterial is a crosslinkable synthetic biomaterial and/or a crosslinkable natural biomaterial;
  • the synthetic biomaterial is at least one of the following: polyethylene glycol, polyethylene glycol derivative , polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd copolymer, polydimethylsiloxane, polyanhydride, polyester, polyamide, polyamino acid, polyacetal , polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, and polyethylene oxide;
  • the natural biomaterial is at least one of the following: gelatin, Gelatin derivatives, alginate, alginate derivatives, agar, matrigel, collagen, proteoglycans, glycoproteins, hyaluronic acid, laminin and fibronectin.
  • the apparatus for constructing a three-dimensional microenvironment comprises two devices of the following A1) and A2): A1) the transparent sponge scaffold or an array of sponge scaffolds composed of two or more transparent sponge scaffolds; A2) a substrate A for loading a sample liquid;
  • the substrate A carrying the sample liquid is a hydrophilic substrate or a hydrophobic substrate; the hydrophilic substrate or the hydrophobic substrate is prepared by various modification methods, including chemical modification, physical modification, and combination thereof. .
  • the hydrophilicity or hydrophobicity of the substrate causes the sample liquid to form a thin layer of liquid on the surface of the substrate; the thickness of the liquid layer is usually 1 ⁇ - 200 ⁇ , which can form a thin layer of cells without gravity Aggregation occurs underneath, resulting in uneven distribution.
  • the micromachining technique of the hydrophilic or hydrophobic substrate is at least one of the following: photolithography, microcontact printing, microfluidic patterning layer Laminar flow patterning stencil patterning Imprint lithography, flow lithography, etc.
  • the thickness of the liquid thin layer may specifically be ⁇ - ⁇ or 10 ⁇ -50 ⁇ , such as ⁇ or 50 ⁇ .
  • the crosslinking method of the biomaterial is at least one of the following: a photocrosslinking method, a chemical cross-linking method, a physical cross-linking method, a radiation cross-linking method, an enzyme-catalyzed cross-linking method, an activated microbead cross-linking method, and the like.
  • the pore-forming technique of the pores is at least one of the following: porogen filtration, phase separation, emulsion freeze-drying, solvent evaporation, gas foaming, fiber bonding, and the like.
  • the patterning of the sponge scaffold can be achieved by using micromachining techniques, and/or high throughput of the sponge scaffold array can be achieved.
  • the micromachining technology of the three-dimensional microenvironment is at least one of the following: photolithography, microcontact printing, microfluidic patterning, laminar flow patterning. ), stencil patterning, imprint lithography, fluid lithography, etc.
  • a patterned three-dimensional sponge holder and a high-throughput three-dimensional sponge stent array are obtained by photocrosslinking.
  • the sponge scaffold array is composed of more than three of the sponge scaffolds to form the high throughput three dimensional sponge scaffold array.
  • the sponge scaffolds constituting the sponge scaffold array may specifically be 16-192, such as 16, 24, 64, 192.
  • the transparent sponge scaffold is prepared by a photocrosslinking method and a chemical cross-linking method.
  • the transparent sponge scaffold is prepared by photocrosslinking, comprising the steps of: bl) polymer monomer polyethylene glycol diacrylate and photoinitiator 2-hydroxy-4-( Dissolving 2-hydroxyethoxy)-2-methylpropiophenone in a mixed solution of the clearing agent and water to obtain a photocrosslinkable prepolymer solution A;
  • step B2 irradiating the photocrosslinkable prepolymer solution A obtained in step bl) with an ultraviolet light source to cause a cross-linking reaction of the photocrosslinkable prepolymer solution A to obtain a hydrogel;
  • the hydrogel obtained in the step b2) is immersed in ultrapure water to remove the uncrosslinked polymer monomer polyethylene glycol diacrylate, the clearing agent and impurities.
  • the content of the polymer monomer polyethylene glycol diacrylate in the photocrosslinkable prepolymer solution A is per 100 ml of the photocrosslinkable prepolymer solution A. Containing 1-50 g of the polymer monomer polyethylene glycol diacrylate; the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in the photocrossing
  • the content of the prepolymer solution A is 0.1-10 g of the 2-hydroxy-4-(2-hydroxyethoxy)-2-methyl group per 100 ml of the photocrosslinkable prepolymer solution A.
  • the phenylacetone; the volume fraction of the transparent agent in the mixed solution of the transparent agent and water needs to be 0.01% or more and less than 100%.
  • the transparent agent may be 1,2,4-butanetriol, ethylene glycol, 1,3-butanediol, glycerol, 1,2-propanediol, 1,3-propanediol, pentaerythritol, cis-1, At least one of a polyhydric alcohol such as 2-cyclopentanediol, tetramethylene alcohol or pentaerythritol.
  • a polyhydric alcohol such as 2-cyclopentanediol, tetramethylene alcohol or pentaerythritol.
  • the polymer monomer polyethylene glycol diacrylate is contained in the photocrosslinkable prepolymer solution A in an amount of the photocrosslinkable prepolymerized per 100 ml.
  • Solution A contains 10 g of the polymer monomer polyethylene glycol diacrylate; the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone is The content of the photocrosslinked prepolymer solution A is 0.5 g per 100 ml of the photocrosslinkable prepolymer solution A.
  • the 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone; the transparent agent is specifically 1,2,4-butanetriol, the 1,2,4-butane
  • the volume percentage of the alcohol in the mixed solution of the clearing agent and water is specifically 60% (i.e., the volume ratio of the 1,2,4-butanetriol to the water is 3:2).
  • the transparent sponge scaffold is prepared by chemical cross-linking, comprising the steps of:
  • step C2 after obtaining the prepolymer solution B in step cl), adding it to a mold for preparing a sponge scaffold, and subjecting the prepolymer solution B to a chemical cross-linking reaction (at room temperature) to obtain a hydrogel;
  • the hydrogel obtained in the step c2) is immersed in ultrapure water to remove the uncrosslinked polymer monomer polyethylene glycol diacrylate, the clearing agent and impurities.
  • the polymer monomer polyethylene glycol diacrylate may be contained in the prepolymer solution B in an amount of 1 to 50 g of the polymer monomer per 100 ml of the prepolymer solution B.
  • a polyethylene glycol diacrylate; the ammonium persulfate content in the prepolymer solution B may be 0.01-lg of the ammonium persulfate per 100 ml of the prepolymer solution B;
  • ⁇ , ⁇ ', ⁇ '-tetramethyldiethylamine may be contained in the prepolymer solution B in an amount of 0.01-lg per 100 ml of the prepolymer solution B.
  • ⁇ '-tetramethyldiethylamine the volume percentage of the clearing agent in the mixed solution of the transparent agent and water needs to be 0.01% or more and less than 100%.
  • the transparent agent may be 1,2,4-butanetriol, ethylene glycol, 1,3-butanediol, glycerol, 1,2-propanediol, 1,3-propanediol, pentaerythritol, cis-1, At least one of a polyhydric alcohol such as 2-cyclopentanediol, tetramethylene alcohol or pentaerythritol.
  • a polyhydric alcohol such as 2-cyclopentanediol, tetramethylene alcohol or pentaerythritol.
  • the polymer monomer polyethylene glycol diacrylate is in the prepolymer solution
  • the content of the cerium may be 10 g of the polymer monomer polyethylene glycol diacrylate per 100 ml of the prepolymer solution; the content of the ammonium persulfate in the prepolymer solution B may be Each 100 ml of the prepolymer solution B contains 0.05 g of the ammonium persulfate; the content of the ruthenium, osmium, iridium, ⁇ '-tetramethyldiethylamine in the ruthenium of the prepolymer solution may be Each 100 ml of the prepolymer solution B contained 0.5 g of the hydrazine, hydrazine, hydrazine, ⁇ '-tetramethyldiethylamine.
  • the transparent agent is specifically 1,2,4-butanetriol, and the volume percentage of the 1,2,4-butanetriol in the mixed solution of the transparent agent and water is 60% (ie, the The volume ratio of 1,2,4-butanetriol to the water is 3: 2).
  • the mold for preparing the sponge stent may be specifically made of a biological material, which is a synthetic biomaterial and/or a natural biomaterial;
  • the artificial biomaterial is at least One: polymethacrylate, polyethylene glycol, polyethylene glycol derivative, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid copolymer, polydimethyl Silicone, polyanhydride, polyacrylate, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polyethylene, polycarbonate, and polyethylene oxide;
  • the natural biomaterial is at least one of the following: gelatin, gelatin derivatives, alginate, alginate derivatives, agar, matrigel, collagen, proteoglycan, glycoprotein, hyaluronic acid, laminin and fiber Connexin.
  • the uncrosslinked polymer monomer polyethylene glycol diacrylate is removed as described in steps b3) and c3)
  • the hydrogel is further frozen at -200 ° C ⁇ 0 ° C for 1-72 h, and then dried for l-72 h to obtain the sponge scaffold or the sponge scaffold array.
  • the hydrogel is frozen at -20 ° C. 4-5h, and then dried at -50 ° C, 20 Pa, for 12 hours, to obtain the sponge stent or the sponge stent array.
  • the apparatus for constructing a three-dimensional microenvironment provided by the present invention further includes a bezel mounted on the substrate A.
  • the frame is made of a biological material, which is a synthetic biomaterial and/or a natural biomaterial;
  • the synthetic biomaterial is at least one of the following: polyethylene glycol, polyethylene glycol derived , polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd copolymer, polydimethylsiloxane, polyanhydride, polyester, polyamide, polyamino acid, polycondensation An aldehyde, a polycyanoacrylate, a polyurethane, a polypyrrole, a polyester, a polymethacrylate, a polyethylene, a polycarbonate, and a polyethylene oxide;
  • the natural biomaterial is at least one of the following: gelatin , gelatin derivatives, alginate, alginate derivatives, agar, matrigel, collagen, proteoglycans, glycoproteins, hyaluronic acid, laminin and fibronectin.
  • the substrate A is specifically a hydrophilic substrate; the frame on the substrate A is specifically made of polymethyl methacrylate.
  • the device further includes a substrate B supporting the sponge holder or the sponge holder array as needed.
  • the substrate B is a slide that holds the sponge holder or the sponge holder array.
  • the use of the sponge scaffold or the device in constructing a three-dimensional microenvironment is also within the scope of the present invention.
  • the method for constructing a three-dimensional microenvironment provided by the present invention is based on the sponge scaffold (three-dimensional transparent sponge scaffold), and is capable of realizing a method comprising a soluble factor, a biological material and a cell, as well as a conventional two-dimensional cell culture method.
  • the construction of three-dimensional cell microenvironment, and at the same time meet the research purposes of patterning, high-throughput, label-free real-time monitoring. (figure 1 )
  • the planting method of the present invention which is as simple as the conventional two-dimensional cell culture method is based on the conventional dropping method and the liquid thin layer method.
  • the conventional dropping method means that the sample liquid is directly dropped on the surface or the side of the stent, and the sample liquid is automatically sucked into the inside of the stent.
  • the liquid thin layer method refers to contacting a three-dimensional sponge scaffold on a thin layer of liquid covering molecules, materials and cells, and using the automatic adsorption of the sponge scaffold, the molecules, materials and cells are automatically dispersed into the sponge scaffold to realize High-throughput simultaneous loading completes the construction of molecules, materials and cellular three-dimensional microenvironments.
  • the liquid thin layer method for constructing a three-dimensional microenvironment is specifically to construct a three-dimensional microenvironment by using the device, which may include the following steps:
  • step a2) covering the substrate A forming the thin layer of the liquid in step a1 on the sponge or sponge scaffold array of the device, or covering the sponge scaffold or the sponge scaffold array in step a) Forming the thin layer of the liquid on the substrate A, after the sample liquid is dispersed into the sponge scaffold or the sponge scaffold array, achieving three-dimensional loading of the sample liquid to complete the three-dimensional microenvironment Construct;
  • the thin layer of liquid has a thickness of ⁇ - 200 ⁇ , which can form a thin layer of cells without forming agglomeration under the action of gravity, resulting in uneven distribution.
  • the thickness of the liquid thin layer may specifically be ⁇ - ⁇ or 10 ⁇ - 50 ⁇ , such as ⁇ or 50 ⁇ .
  • the sample liquid may specifically include any one of the following a) - d): a) various points Any one or a mixture of any of the sub-substances (eg, small molecule compounds, drugs, nucleic acids, proteins, etc.); b) various natural and synthetic materials (eg, extracellular matrices, polymeric materials, microbeads, etc.) Any one or a mixture of any of the following; c) any one or a mixture of any of a variety of cells and microorganisms (eg, eukaryotic/prokaryotic cells, viruses, microorganisms, etc.); d) a) -c) a mixture of several of them.
  • the sub-substances eg, small molecule compounds, drugs, nucleic acids, proteins, etc.
  • various natural and synthetic materials eg, extracellular matrices, polymeric materials, microbeads, etc.
  • Any one or a mixture of any of the following c) any one or a mixture of
  • all of the above three-dimensional microenvironments include the three-dimensional microenvironment described in any one of the following a) - d): a) various molecular substances (such as small molecule compounds, drugs, nucleic acids, proteins, etc.) Any one or a mixture of any of the following; b) any one or a mixture of any of a variety of natural and synthetic materials (eg, extracellular matrices, polymeric materials, microbeads, etc.); c) various cells And a mixture of any one or any of microorganisms (such as eukaryotic/prokaryotic cells, viruses, microorganisms, etc.); d) a mixture of any of a)-c).
  • various molecular substances such as small molecule compounds, drugs, nucleic acids, proteins, etc.
  • Any one or a mixture of any of the following b) any one or a mixture of any of a variety of natural and synthetic materials (eg, extracellular matrices, polymeric materials, microbe
  • the above three-dimensional cellular microenvironment research applications are extensive, including but not limited to: molecular/material/cell microarrays for studying molecules/cells, materials/cells, cell/cell interactions; drug screening; in vitro model building; tissue engineering; regeneration Medicine; pathology research and so on.
  • Figure 1 is a general view of the method design.
  • A-1 to A-3 are two-dimensional cell culture techniques. Specifically, A-1 is a cell dropping and tiling in a culture dish, A-2 is a microscopic observation of the cell state on the culture dish, and A-3 is subculture. After A-3, various cell studies can be performed as needed.
  • B-1 to B-5 are three-dimensional cell culture techniques.
  • B-1 is the cell that is automatically adsorbed into the transparent porous sponge scaffold
  • B-2 is the microscopic observation of the internal cell state of the transparent sponge scaffold
  • B-3 is subcultured
  • B -4 is a high-throughput automatic loading of liquid thin layer method (1-liquid thin layer; 2-sponge holder array; 3- hydrophobic frame)
  • B-5 is micro-machining technology for micro-scale, patterned design (1-TMP Modified slides; 2-cover slides; 3-OTS modified slides; 4-UV light; 5-patterned photomask; 6-prepolymer), after B-5, various cells can be used as needed the study.
  • Figure 2 shows the preparation and characterization of a transparent sponge scaffold.
  • A is a sponge stent mold prepared by chemical crosslinking method.
  • B is a physical diagram of the sponge scaffold prepared by the chemical cross-linking method (front view and side view) and its electron micrograph.
  • C and D are photo-crosslinking methods for the preparation of sponge scaffolds (cylindrical and toroidal) and their electron micrographs (C large aperture, D small aperture).
  • E and F are the pore size distribution maps of the sponge scaffold prepared by photocrosslinking (E large pore size, F small pore diameter).
  • Figure 3 shows a transparent sponge scaffold that automatically loads molecules, materials, and cells.
  • a and B are cylindrical sponge scaffold arrays that automatically load molecules (phenol red solution), A for loading and B for loading.
  • C and D are regular hexagonal sponge bracket arrays for automatic loading of materials (gelatin), C for loading and D for loading.
  • E is a regular hexagonal and rectangular sponge scaffold array of autoloaded cells (HeLa).
  • F-1 to F-9 are dynamic maps of the fluorescent microspheres (fluorescent microsphere diameter ⁇ ) automatically loaded on the sponge scaffold.
  • Figure 4 shows the optical performance test of the transparent sponge holder. ⁇ Observe the text for the transparent sponge scaffold (0.5mm) prepared by chemical cross-linking method (the two samples on the left are 0% 1,2,4-butanetriol, and the two samples on the right are 60% 1,2, 4-butanetriol).
  • B is a transparent sponge scaffold of different heights prepared by chemical cross-linking method.
  • C is a sponge scaffold prepared by photocrosslinking. The text (0% 1,2,4-butanetriol) is observed.
  • D is a transparent sponge scaffold prepared by photocrosslinking to observe the text (60% 1,2,4-butanetriol).
  • E is a quantitative diagram of the transparency of the transparent sponge scaffold with different volume ratios of 1,2,4-butanetriol.
  • F is a quantitative plot of the transparency of a transparent sponge scaffold (60% clear 1,2,4-butanetriol) at different heights.
  • Figure 5 shows the growth of HeLa cells in a transparent sponge scaffold to form tumor microspheres.
  • A-1 to A-5 Microscopic observation of HeLa cells grows in a transparent sponge scaffold over time to form tumor microspheres.
  • B is a microscope to observe HeLa tumor microspheres Fluorescent picture.
  • C is an electron micrograph of HeLa tumor microspheres.
  • D is a three-dimensional fluorescence picture of HeLa tumor microspheres observed by laser confocal microscopy.
  • Figure 6 shows the high survival rate of HeLa tumor microspheres subculture.
  • A is a microscopic picture of HeLa tumor microspheres in a transparent scaffold.
  • B is a HeLa tumor microsphere that is digested and washed out of the scaffold.
  • C and D are tumor microspheres dead/live staining fluorescence pictures, C is low magnification 2x, and D is high magnification 10x.
  • Figure 7 shows a liquid thin layer method for automatically loading molecules and materials into a high-throughput, patterned sponge scaffold.
  • A-1 and A-2 form a hydrophilic substrate for plasma cleaning
  • A-1 is the contact angle of water before washing
  • A-2 is the contact angle of water after washing.
  • B is a micromachining technology (photolithography) to prepare a high-throughput sponge stent array.
  • C is a thin layer of different dye liquids for plasma cleaning and microcontact printing.
  • D is a liquid thin layer method for high throughput automatic loading of different dye liquids into the sponge scaffold array.
  • E is a liquid thin layer method for automatically loading Doxorubicin drugs and fluorescent microspheres into micromachining technology (photolithography) to prepare a patterned sponge scaffold (Tsinghua University Centennial Celebration icon).
  • Figure 8 is a liquid thin layer method for automatically loading HeLa cells into a sponge scaffold array.
  • A is a liquid thin layer method to automatically load HeLa cells into an array of circular sponge stents.
  • B-1 to B-3 are automatic loading effects of liquid layers at different cell concentrations.
  • C is a three-dimensional scan of a laser confocal microscope that automatically loads a thin layer of liquid at different cell concentrations into the interior of the sponge scaffold.
  • D is the cell Titer-Blue test liquid thin layer method to automatically load different concentrations of HeLa cells into the sponge scaffold and the residual amount of liquid in the thin layer of liquid.
  • E is the Cell Titer-Blue quantitative fluorescence intensity-live cell number standard curve.
  • F is the ratio of the concentration of HeLa cells entering the sponge scaffold by the quantitative liquid thin layer method according to the standard curve E map.
  • the porosity referred to in the following examples refers to the ratio of the pore volume of the porous body to the total volume of the object.
  • Example 1 Preparation of a patterned transparent three-dimensional sponge scaffold by chemical cross-linking method and photocrosslinking method respectively
  • the biomaterial for preparing a transparent sponge scaffold provided in this embodiment is polyethylene glycol diacrylate (PEGDA4000).
  • Polyethylene glycol diacrylate has the advantages of good biocompatibility, mechanical properties, non-degradability and various crosslinking methods, and is suitable as a biological material for preparing a transparent sponge stent in this embodiment.
  • Polyethylene glycol diacrylate synthesis method Under nitrogen, 5g of polyethylene glycol (PEG4000) powder is stirred and dissolved in 50ml of dichloromethane solution, slowly adding 0.76ml of triethylamine solution and 0.47ml The acryloyl chloride solution was stirred at room temperature for 24 hours. Thereafter, it was washed by adding 100 ml of a 2 M potassium carbonate solution, and allowed to stand for separation, and the lower dichloromethane mixture was collected. The methylene chloride mixed droplets were precipitated by adding to 500 ml of anhydrous ether solvent, and the white powder was collected by filtration and dried at room temperature to give a white PEGDA solid powder.
  • PEG4000 polyethylene glycol
  • a polymethyl methacrylate (PMMA) plate having a thickness of 0.5 mm, 1 mm, 2 mm, and 3 mm was formed by a Rayjet laser engraving machine to form a mold.
  • the template design is done by the software AutoCAD: The template is 75mm long and 25mm wide. It is evenly distributed with 3 X 14 micropores with a diameter of 4mm as the support model. The center distance of each micro hole is 1000 m.
  • the main processing parameters of the laser engraving machine are: cutting energy 100%, cutting times 2, cutting line speed 10%. See Figure 2- ⁇ . 2. Preparation of hydrogel by chemical crosslinking
  • the pre-polymer solution B prepared by preparing the hydrogel by the chemical cross-linking method of the invention is characterized by the addition of a transparent agent, thereby satisfying the research of label-free imaging for the three-dimensional microenvironment in the fields of biology, pharmacy and medicine.
  • Useful clearing agents are mainly polyols such as ethylene glycol, 1,3-butanediol, 1,2,4-butanetriol, glycerol, 1,2-propanediol, 1,3-propanediol, pentaerythritol. , cis-1,2-cyclopentanediol, tetrabutyl alcohol, pentaerythritol, and the like.
  • the volume percentage of the clearing agent polyol in the final prepolymer solution B is from 0.01% to 100%.
  • the preparation of the prepolymer solution B and the preparation method of the hydrogel will be described below using 1,2,4-butanetriol as a transparent agent: 10% (w/v) polyethylene glycol diacrylate It was dissolved in a mixed solution of 1,2,4-butanetriol and water (60/40 by volume) at 60 °C.
  • the above hydrogel was prepared into a porous scaffold by freeze drying.
  • the specific process is as follows: The above hydrogel-loaded mold is immersed in ultrapure water to remove impurities such as uncrosslinked monomers and 1,2,4-butanetriol, and replaced with water for 4-5 times. Thereafter, it was frozen at -20 ° C for 4-5 h, and then transferred to a freeze dryer (-50 ° C, 20 Pa) for 12 hours to obtain a white porous sponge scaffold.
  • a sponge scaffold is prepared, and the physical map and the electron microscope image are shown in Fig. 2-B.
  • the photomask was designed using AutoCAD drawing software.
  • Design size The photomask size is 76.2mmx25.4mm, and the pattern can be freely designed according to requirements.
  • Printed a film photomask at a printing house (Tsinghua University Printing Factory).
  • a major feature of the photocrosslinkable prepolymer solution A of the present invention is the addition of a clearing agent, thereby meeting the requirements for the implementation of label-free imaging in a three-dimensional microenvironment in the fields of biology, pharmacy, and medicine.
  • Useful clearing agents are mainly polyols such as ethylene glycol, 1,3-butanediol, 1,2,4-butanetriol, glycerol, 1,2-propanediol, 1,3-propanediol, pentaerythritol. , cis-1,2-cyclopentanediol, tetrabutyl alcohol, pentaerythritol, and the like.
  • the volume percentage of the clearing agent polyol in the final photocrosslinkable prepolymer solution A is from 0.01% to 100%.
  • the preparation method of the photocrosslinkable prepolymer solution A will be described below using 1,2,4-butanetriol as a transparent agent: 10% (w/v) polyethylene glycol diacrylate and 0.5% (w/v) 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone dissolved in 1,2,4-butanetriol and water at 60 ° C (volume ratio In the mixed solution of 60/40), the photocrosslinkable prepolymer solution A was obtained after cooling (the concentration of each of the above substances was the final concentration in the photocrosslinkable prepolymer solution A). Mix and store in 4 ° C environment for use. In the experiment, a control in which the clearing agent 1,2,4-butanetriol was not added was simultaneously set.
  • the specific coating method is as follows: 1) Place the slide on the staining rack, soak in the aqueous detergent solution, and sonicate for 20 minutes; 2) immerse the slide staining rack in 10% sodium hydroxide solution for 60 minutes; 3) Re-immersion in ultrapure water for 30 minutes, drying; 4) Immerse the slide staining rack in a solution of 5% (V/V) OTS in n-hexane for 30 minutes at room temperature, or contain 1% ( V/V) TMP in ethanol (containing 3% acetic acid) for 3 minutes; 5) Soak the OTS-coated slide in toluene for 10 minutes, soak the TMP-coated slide in absolute ethanol 10 minutes; 6) Finally, the slides are dried and stored in a refrigerator at 4 °C for use.
  • the ultraviolet light source is the Canadian Omnicure S2000 UV cross-linking instrument; the specific UV cross-linking parameters are: ultraviolet light intensity 20mW/cm 2 , illumination time 8.5s.
  • the hydrogel was prepared into a porous scaffold by freeze drying.
  • the specific process is as follows: The combination of the cross-linking of the above step 4 is gently disassembled, and the hydrogel-fixed glass slide (ie, the substrate B supporting the sponge scaffold or the sponge scaffold array) is immersed in ultrapure water. The uncrosslinked monomer, 1,2,4-butanetriol and impurities were removed and replaced with 4-5 times of water. Thereafter, it was frozen at -20 ° C for 4-5 h, and then transferred to a freeze dryer (-50 ° C, 20 Pa) for 12 hours to obtain a white three-dimensional patterned sponge scaffold. According to the above method, different sizes of sponge scaffolds were prepared. The physical map and the electron micrograph are shown in C and D in Fig. 2.
  • the size of the sponge scaffold is 0. 2355 ⁇ 1.
  • the volume of the sponge scaffold of D in Fig. 2 is 0.603 ⁇ 1, 1.204 ⁇ 1, 1.809 ⁇ 1, 2.415 ⁇ 1, 3.026 ⁇ 1, 3.620 ⁇ 1, 4.22 ⁇ 1, 4.82 ⁇ 1, respectively.
  • the pore size of the sponge scaffold shown in Figure 2C is 10 ⁇ -125 ⁇ ; the pore spacing is ⁇ -999 ⁇ , and the percentage distribution of pore size distribution is (see Fig.
  • the pore size of the sponge scaffold shown in D in Fig. 2 is 1 ⁇ -85 ⁇ ; the pore spacing is ⁇ -999 ⁇ , and the percentage distribution of pore size distribution is (see F in Fig.
  • the automatic loading method for molecules, materials and cells is the traditional dropping method (B-1 in Fig. 1).
  • the conventional dropping method means that the sample liquid is directly dropped on the surface or the side of the stent, and the sample liquid is automatically sucked into the inside of the stent.
  • the sample solution may be any one or a mixture of a small molecule compound, a drug, a nucleic acid, a protein, an extracellular matrix component, a polymer material, a microbead, a eukaryotic cell, a prokaryotic cell, a virus, a microorganism, or any one of several.
  • the ⁇ indicator phenol red is used as an example of a molecular sample in the sample liquid ( ⁇ and B in Fig. 3), and lmg/ml gelatin is used as an example of a material sample in the sample liquid (C and D in Fig. 3), 5 X 10 6 / ml of HeLa cells (medium The Basic Medical Cell Center of the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences, 3111C0001CCC000011) was automatically loaded into the three-dimensional patterned "sponge holder" prepared by the photocrosslinking of Example 1 as a cell sample (E in Fig. 3).
  • the sponge stent array is composed of 64 sponge stents.
  • the regular hexagonal and rectangular sponge holders in Figures 3-C and 3- ⁇ were prepared by the photocrosslinking method described in Example 1.
  • the sides of the regular hexagon were 500 ⁇ m, and the length of the rectangle was ⁇ , and the width was 500 ⁇ ; sponge holder height is 300 ⁇ ; sponge holder pore size 10 ⁇ -125 ⁇ ; pore spacing 1 ⁇ -999 ⁇ ; porosity 93.3%, good connectivity; water absorption is 3.5-5 times the theoretical volume; They are all composed of 16 sponge scaffolds.
  • the dynamic process of automatically loading 5 ⁇ 10 6 /ml of fluorescent microspheres (diameter ⁇ ) with a fluorescent microscope was recorded by fluorescence microscopy (F in Fig. 3).
  • F-1 is 1.5 seconds
  • F-2 is 2.5 seconds
  • F-3 is 3.5 seconds
  • F-4 is 4.5 seconds
  • F-5 is 5.5 seconds
  • F-6 is 7 seconds
  • F-7 is 8.5 seconds
  • F-8 is 11 seconds
  • F-9 is 13 seconds.
  • the sponge holder with a water absorption of 5 ⁇ 1 completed the automatic loading process and took 13 seconds.
  • text observation and transparency measurement are used to detect the transparent optical properties of the sponge holder.
  • the sponge scaffold (Fig. 2, 3, Fig. 3, C) prepared according to the chemical cross-linking method and the photocrosslinking method of Example 1 was immersed in water and placed in a 37 ° C incubator for 2-3 days to remove air bubbles inside the scaffold. Place them on a glass slide and observe the text through the holder (A, C and D in Figure 4).
  • the sponge scaffold prepared by the method of the present invention has good optical properties after repeated water absorption, and the text under the slide glass can be clearly seen. Samples without a clearing agent had poor optical properties and could not clearly see the text under the slide.
  • HeLa cells were selected as a case observation to record their growth in a transparent sponge scaffold.
  • 5 X 10 6 /ml HeLa cell suspension was added to the sponge scaffold and cultured in a 37 ° C, 5% CO 2 , saturated humidity incubator.
  • the culture medium was added with 10% (v/v) FBS.
  • Ordinary white light microscopy observation showed that HeLa cells gradually grew to form tumor microspheres in a transparent sponge scaffold (the sponge scaffold prepared by chemical cross-linking in Example 1) (A-1 to A-5 in Fig. 5).
  • A-1 to A-5 are recorded pictures after cell planting for 0h, 4h, 26h, 50h, lOOh.
  • D is a laser confocal microscope to observe the tumor microsphere picture (nucleus: blue-DAPI, cytoskeleton: Red-rhodamine ), further demonstrating that HeLa cells are
  • the growth state in the three-dimensional environment is three-dimensional growth, which is different from the single-cell layer growth in the traditional two-dimensional environment.
  • Example 5 Subculture of cells in a sponge scaffold
  • HeLa cells were used as a case for subculture.
  • the HeLa tumor microspheres (Fig. 6 A) in the transparent stent of Example 4 were subjected to 0.25% trypsin digestion for 2 min, and the culture solution was washed 3-4 times to obtain a HeLa cell suspension (B in Fig. 6).
  • C-1 and C-2 in Figure 6 the cells were observed to maintain near 100% survival by fluorescence microscopy.
  • C-1 is the low magnification lens 2X observation picture
  • C-2 is the high magnification lens 10 X observation picture.
  • Example 6 Liquid thin layer method automatically loading drug molecules and materials into a high-flux, patterned sponge scaffold array.
  • a high-throughput, patterned auto-loading method for molecules and materials is a liquid thin layer method (in FIG. 1).
  • the liquid thin layer method refers to covering a patterned sponge scaffold array on a thin liquid layer or covering a thin layer of liquid on the patterned sponge scaffold array, and utilizing the adsorption of the sponge and the easy flow separation of the liquid thin layer, automatically introducing the liquid It is sucked into the sponge holder to achieve three-dimensional micro-scale loading of the sample solution.
  • the following relates to the fabrication of the substrate A for the device for constructing the three-dimensional microenvironment, the fabrication of the hydrophobic frame on the substrate A, and the high-throughput, patterned automatic loading of the molecules and materials using the liquid thin layer method. . details as follows:
  • the slides were modified to a hydrophilic substrate using an HPDC basic plasma cleaning system, i.e., substrate A for making a device for constructing a three-dimensional microenvironment.
  • substrate A for making a device for constructing a three-dimensional microenvironment.
  • the specific process is as follows: 1) Place the slide into the cabin of the washing machine, open the vacuum oil pump in the plasma cleaning system, and vacuum for 5 minutes; 2) When the purple glow is generated in the cabin, the time is cleaned for 1 minute; The effect is shown in Figure 7 for A-1 and A-2 (A-1 contact angle of water before washing, A-2 contact angle of water after washing).
  • the liquid can form a thin layer on the substrate.
  • the polygonal sponge scaffold array of B in Fig. 7 is prepared according to the photocrosslinking method described in Example 1, the outer diameter of the polygon is ⁇ ; the height of the sponge scaffold It is 300 ⁇ ; its sponge scaffold has a pore size of 1 ⁇ -85 ⁇ ; pore spacing is 1 ⁇ -999 ⁇ ; porosity is 82.4%, connectivity is good; water absorption is 1-2 times theoretical volume; the sponge scaffold array is composed of 192 sponge scaffolds Composition.
  • the laser engraving machine (Rajet) cuts the PMMA plate with a thickness of 0.5mm to obtain the area division with the sponge stent array.
  • Liquid thin layer method automatically loads molecules and materials into high-throughput, patterned sponge scaffolds
  • the Doxorubicin drug (red fluorescence) and the fluorescent microsphere (green, diameter ⁇ ) were simultaneously loaded into the patterned sponge scaffold using the liquid thin layer method in the above three steps (Fig. 7 ⁇ ). Achieve simple simultaneous loading of multiple liquids.
  • the Tsinghua Centennial Icon-like Sponge Scaffold in Figure 7 was prepared according to the photocrosslinking method described in Example 1.
  • the height of the sponge scaffold was 300 ⁇ ; the pore size of the sponge scaffold was 10 ⁇ -125 ⁇ ; the pore spacing was 1 ⁇ -999 ⁇
  • the porosity is 93.3%, and the connectivity is good; the water absorption is 3.5-5 times the theoretical volume.
  • Example 7 Automatic loading of liquid thin layer method HeLa cells were planted into a high-throughput sponge scaffold array
  • Calcein-AM reagent (sigma, C1359) was used to detect whether the high-throughput sponge scaffold array can automatically load HeLa cells. Then, Cell Titer-Blue reagent (Promega, G8080) was used to study the liquid thin layer method Qualcomm. The efficiency of loading HeLa cells automatically.
  • the HeLa cell suspension was gently pipetted evenly, and the cells were treated with Calcein-AM (the method of use is referred to the reagent instructions), and 200 ⁇ l suspensions of different concentrations were uniformly spread on different hydrophilic slide substrates (base).
  • the high-throughput sponge scaffold On the ⁇ ), gently place the high-throughput sponge scaffold on the slide glass (the substrate ⁇ and the sponge holder array fixed on it, and the circular sponge support of ⁇ in Figure 8 is prepared according to the photocrosslinking method described in Example 1
  • the inner diameter of the ring is 2400 ⁇ , the outer diameter is 3400 ⁇ ; the height of the sponge stent is 300 ⁇ ; the pore size of the sponge stent is 10 ⁇ -125 ⁇ ; the pore spacing is 1 ⁇ -999 ⁇ ; the porosity is 93.3%, the connectivity is good; It is 3.5-5 times of the theoretical volume; the sponge scaffold array consists of 24 sponge scaffolds.)
  • Covered in a thin layer of cell liquid (thick layer thickness of 50 ⁇ m), after the cell solution is dispersed into the inside of the sponge scaffold, light Lightly remove the scaffold array slides to achieve high-throughput three-dimensional loading of HeLa cells ( Figure 8, A, HeLa cells: green-Calcein-AM).
  • Cell Titer-Blue reagent was used to quantify the efficiency of the liquid thin layer method for autoloading cells.
  • a series of concentrations of HeLa cells were implanted in a 96-well plate at 4 ⁇ 10 4 /well, 8 ⁇ 10 3 /well, 1.6 ⁇ 10 3 /well, 320/well, and 3 samples were repeated for each concentration.
  • ⁇ culture solution and 20 ⁇ l of Cell Titer-Blue reagent were added to each well, and incubated at 37 ° C in a 5 C0 2 saturated humidity incubator for 1 h. The fluorescence intensity was measured with a microplate reader to establish a standard curve (E in Figure 8).
  • Suspensions of different cell concentrations were automatically loaded into the high-throughput sponge scaffold array (substrate B and the sponge scaffold array immobilized thereon, in the circular sponge scaffold shown in A of Fig. 8) according to the above liquid thin layer method.
  • a mixture of ⁇ culture solution and 200 ⁇ l of Cell Titer-Blue reagent was separately added, at 37 Incubate for 1 h in °C, 5 C0 2 saturated humidity incubator (Fig. 8, Dl: 5 X 10 6 /ml, D-2: 1 X 10 6 /ml, D-3: 2X 10 5 /ml) .
  • the invention proposes a method for constructing a three-dimensional cell microenvironment based on the transparent sponge scaffold material, which is as simple and convenient as the two-dimensional cell culture method, and satisfies the patterning, high-throughput, real-time without Marking monitoring and other research purposes. 1)
  • the automatic and simple loading of cells or cells-materials is achieved by the porosity of the three-dimensional sponge scaffold material.
  • the cell or cell-material suspension can be automatically adsorbed into the porous sponge scaffold to form a three-dimensional microenvironment system; 2) the planting cells can realize free three-dimensional growth, proliferation, and easy passage in the three-dimensional microenvironment; 3) Using the excellent optical properties of the transparent sponge scaffold material, the conventional equipment (such as ordinary optical microscope) can be used to perform unmarked observation on the cells; 4)
  • the transparent sponge scaffold material can effectively combine the three-dimensional micro-machining technology to realize the micro-environment of the three-dimensional micro-environment , patterned to form a high-throughput three-dimensional microenvironment array; 5)
  • We have also invented a simple, widely applicable liquid thin layer method that enables rapid, non-destructive realization of the three-dimensional microenvironment of molecules, materials and cells, and mixtures thereof. High-throughput synchronous loading.
  • the method of the invention combines engineering, chemistry, physics, material science and other subject knowledge to prepare a three-dimensional transparent sponge porous stent, utilizing the porosity, optical transparency, mechanical of the stent Elasticity, cross-linking controllability, etc., provide a simple and widely applicable platform for the study of precise and controllable and high-throughput three-dimensional cellular microenvironment in the fields of biology, pharmacy, medicine, etc. 2.
  • the present invention proposes The three-dimensional microenvironment construction method is as simple as the traditional two-dimensional cell culture method (easy as 2D), using the off-the-shelf instruments to satisfy the rapid planting, free growth, long-term proliferation, and easy of cells in a three-dimensional system.
  • Basic culture requirements such as passage and real-time monitoring; 3.
  • the transparent sponge stent concept proposed by the present invention fills a gap in the field of material properties.
  • the method for preparing a transparent sponge stent provided by the invention effectively improves the problem of poor optical performance of the traditional three-dimensional stent, and can meet the requirements of real-time unmarked imaging research on the three-dimensional micro-environment in the fields of biology, pharmacy, medicine, etc., and greatly reduces the requirement.
  • the liquid thin layer loading method proposed by the present invention can realize high-throughput synchronous construction of a three-dimensional cellular micro-environment without special expertise and means and expensive equipment, It reduces the use requirements of personnel skills, environmental space and other aspects during operation, has broad application prospects, and simple operation steps reduce damage to living cells; 5.
  • the method of the present invention can be combined with various existing research techniques (such as three-dimensional micro-machining technology, cell dynamic planting technology, automated operating system, etc.) combined to achieve patterning, microstructure controllability, real-time monitoring (monitoring) and other purposes to meet different research areas Multiple needs for different research purposes 6.
  • the present invention can be finally applied to the development of various three-dimensional cell application products, providing an off-the-shelf platform for three-dimensional cellular microenvironment research to realize the three-dimensional cellular products in the fields of traditional biology, pharmacy and medicine. Barriers are widely used.
  • the method of the invention is simple to operate for those familiar with conventional two-dimensional cell culture and research, without the need for other specialized techniques and means (such as micromachining techniques and special synthetic materials) and expensive equipment (such as automation, micromachining equipment). Ultimately, it is widely used in the field of traditional biology, pharmacy and medicine.

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Abstract

La présente invention concerne un procédé et un dispositif de construction d'un microenvironnement cellulaire en trois dimensions. Le procédé comprend les étapes suivantes, consistant à : 1) établir un échafaudage spongieux et transparent en trois dimensions ou un réseau d'échafaudages spongieux et transparents en trois dimensions ; 2) établir une fine couche liquide d'échantillon contenant des molécules, une substance, des cellules, et un mélange de celles-ci ; 3) combiner l'échafaudage spongieux et transparent ou le réseau d'échafaudages spongieux et transparents du point 1) avec la fine couche liquide du point 2), et charger un échantillon de liquide, construisant ainsi un microenvironnement en trois dimensions. Le procédé et le dispositif fournissent une plateforme simple et pratique pour la recherche sur la croissance, la prolifération, l'observation sans marqueur, et l'analyse fonctionnelle de cellules dans un microenvironnement en trois dimensions dans des domaines tels que la recherche biomédicale et la recherche et le développement de médicaments, et permettent de réaliser rapidement et sans perte la reproduction du microenvironnement tridimensionnel des molécules, d'une substance, des cellules, et d'un mélange de celles-ci, le chargement synchrone à flux élevé, et l'observation en temps réel sans marqueur.
PCT/CN2012/001013 2011-11-23 2012-07-27 Procédé et dispositif de construction d'un microenvironnement cellulaire en trois dimensions sur la base d'un échafaudage spongieux transparent WO2013075392A1 (fr)

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Publication number Priority date Publication date Assignee Title
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CN110628757B (zh) * 2019-09-29 2022-03-04 北京科技大学 一种基于超声体波在毛细管中三维细胞培养的方法
CA3163247A1 (fr) * 2019-12-03 2021-09-30 Massachusetts Institute Of Technology Adhesifs tissulaires resistant aux fluides corporels
CN113476664A (zh) * 2021-07-07 2021-10-08 深圳大学 兼具开放大孔和全连通微通道的生物支架及其制备方法
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CN115850729B (zh) * 2021-09-23 2023-11-14 四川大学 光固化多孔水凝胶材料及其制备方法
CN116077741A (zh) * 2022-08-24 2023-05-09 深圳先进技术研究院 一种可固定细胞并在体内实现细胞增殖和释放的微米级3d细胞生物支架及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101862475A (zh) * 2009-10-10 2010-10-20 广州市创伤外科研究所 一种ⅱ型胶原透明质酸复合海绵支架及其用途

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2076105U (zh) * 1990-10-11 1991-05-01 中山医科大学孙逸仙纪念医院 一次性用细菌、真菌培养盒
JPH1052268A (ja) * 1996-05-01 1998-02-24 Kanebo Ltd 微生物担持体及びその製造方法
KR100520691B1 (ko) * 2003-03-14 2005-10-12 한상배 비표면적 및 생체친화성이 개선된 고정상 생물막 담체
CN1868553A (zh) * 2006-04-04 2006-11-29 杨玉民 宫颈皮肤组织工程支架及制作方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101862475A (zh) * 2009-10-10 2010-10-20 广州市创伤外科研究所 一种ⅱ型胶原透明质酸复合海绵支架及其用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TAKAHIRO, OHNO ET AL.: "Effect of type I and type II collagen sponges as 3D scaffolds for hyaline cartilage-like tissue regeneration on phenotypic control of seeded chondrocytes in vitro", MATERIALS SCIENCE AND ENGINEERING C, vol. 24, 2004, pages 407 - 411, XP055069818 *

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