CN112852706A - 3D (three-dimensional) organ engineering method based on aqueous two-phase droplet microfluidics - Google Patents
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Abstract
The invention provides a 3D organ engineering method based on aqueous two-phase droplet microfluidics. The method comprises cell loading, islet organoid engineering, liver organoid engineering and the like of the aqueous two-phase hydrogel microcapsule. The invention mainly combines the emerging 3D organoid and droplet microfluidic technology of human pluripotent stem cell sources, and is used for constructing the in vitro model of the 3D organoid of the stem cell sources by controllably synthesizing the aqueous two-phase hydrogel microcapsule with good biocompatibility, determined components and uniform size. The invention has the advantages of simple operation, good controllability, reduced organoid variability, high flux generation and the like, and provides a powerful new platform for the organoids in the aspects of biomedical application such as disease simulation, drug screening, in-vivo transplantation and the like.
Description
Technical Field
The invention belongs to the fields of regenerative medicine and the like, and particularly relates to a 3D organ engineering method based on aqueous two-phase droplet microfluidics.
Background
The in vitro construction of 3D tissue or organ models can simulate human physiology and is of great significance in biomedical applications. Organoids from various stem cell sources (including adult stem cells, embryonic stem cells ESC and induced pluripotent stem cells hiPSC) have been a major technological breakthrough in recent years, representing a novel in vitro organ model. Organoids are 3D multicellular complex structures formed by stem cells undergoing specific differentiation and self-organization in 3D culture, and can summarize key structures and functions of corresponding organs in vivo. Many organoids, such as intestinal, brain, liver, kidney, retinal organoids, have been successfully developed and have shown great potential in research on tissue and organ development, disease simulation, drug screening, and transformation medicine applications. Despite the potential advantages and applications of such techniques, a number of limitations and deficiencies are still encountered. In general, organoid production relies primarily on the self-organization of stem cells embedded in a matrix of animal origin (e.g., Matrigel). However, due to matrix composition uncertainty and lot-to-lot variation, these traditional methods often produce organoids with significant variability, poor controllability, lower yields, and the like. By combining the existing engineering means such as biological material engineering and microfluidic technology, the organoid technology is hopeful to be optimized.
The hydrogel material is used as a cell 3D culture scaffold and has been widely applied to the aspects of stem cell induced differentiation, cell co-culture system, organoid model establishment and the like. However, the hydrogel block obtained by 3D cell culture by using the traditional technology has the defects of small specific surface area, unsmooth cell nutrient exchange, poor controllability of material size and morphology and the like, so that the phenotype and function of cells or tissues are influenced. There is therefore a need for a method for preparing a hydrogel scaffold material with a controlled microstructure, which can meet these requirements and is beginning to be applied in the synthesis of micro-hydrogel (microgel) materials. Hydrogel microcapsules have been considered as suitable 3D cell culture scaffolds and transplantation carriers in tissue engineering due to their homogeneous morphology, good permeability and the ability to be produced in large quantities. At present, the synthesized hydrogel microcapsules mainly depend on a microfluidic droplet technology, and the related hydrogel types comprise artificially synthesized polyethylene glycol (PEG) materials, natural alginate, gelatin, agar and the like. Different from the traditional oil-water system, the micro-fluidic droplet system based on the double-water-phase system is rapidly developed in recent years. The aqueous two-phase system utilizes the incompatible nature of the polymers, so that solutions of two different polymers may self-stratify after mixing to form a two-phase system. The two-phase solution of the system is aqueous solution, has better biocompatibility, and avoids the possible cytotoxicity risk brought by the traditional water-in-oil emulsion. The microcapsules prepared by the technology and the materials have great application value in the aspects of cell 3D culture, stem cell induced differentiation and in-vitro tissue or organ construction. The hydrogel material and the microfluidic droplet technology are combined, so that the tissue shape and size can be better controlled to be uniform, and the 3D micro-tissue can be generated at high flux. However, the existing double-aqueous-phase microfluidic droplet technology is used for organoid engineering, and the optimization of the formation and operation of in-vitro organoids is still blank.
Disclosure of Invention
The invention aims to develop a novel aqueous two-phase droplet microfluidic system for preparing hydrogel microcapsules in one step, and aims to provide a 3D organoid engineering method. The established droplet microfluidic system comprises different functional units: a multiphase fluid inlet, a droplet generation and microcapsule generation unit. The system can be used for cell encapsulation, 3D culture, differentiation and organoid formation by forming hydrogel microcapsules with uniform size in a controllable manner, is favorable for reducing the variability of organoids, and has the characteristics of easiness in operation, good biocompatibility, high flux and high controllability. Provides a new strategy and technical platform for multi-organoid engineering and organoid models in disease research, drug screening, transplantation treatment and other aspects.
The invention provides a 3D organoid engineering method based on aqueous two-phase droplet microfluidics, which combines the 3D organoid from human pluripotent stem cells and the droplet microfluidics technology, and is used for constructing an in vitro model of the 3D organoid from the stem cells by controllably synthesizing aqueous two-phase hydrogel microcapsules with good biocompatibility, determined components and uniform size; comprises the following steps:
step one, adopting a double-aqueous-phase droplet microfluidic chip to generate a double-aqueous-phase hydrogel microcapsule and complete cell loading;
step two, 3D organoid engineering: when human pluripotent stem cells are gathered to a certain growth density, a commercialized mTESR1 culture medium for culturing the stem cells is replaced by an endoderm differentiation culture medium, and the tissue precursor cells are subjected to static culture and then induced to differentiate to finally produce the 3D organoid.
The growth density of the human pluripotent stem cells is 50% -70%.
The endoderm differentiation medium is one of the following two:
endoderm differentiation medium 1: the basic components are commercial DMEM/F12 culture medium, B27supplement (50 x) accounting for 1% of the total volume, KnockOut Replacement (KSR) accounting for 1% of the total volume, GlutaMax (100 x) accounting for 1% of the total volume, penicillin-streptomycin (100 x) accounting for 1% of the total volume, and a factor of Human Activin-A accounting for 80-100 ng/ml;
endoderm differentiation medium 2: the base component was commercial RPMI-1640 medium, added with B27supplement (50X) at 1% of the total volume, KnockOut Replacement (KSR) at 1% of the total volume, GlutaMax (100X) at 1% of the total volume, penicilin-streptomycin (100X) at 1% of the total volume, and factor of Human Activin-A at 80-100 ng/ml.
The generation and cell loading of the aqueous two-phase hydrogel microcapsule are specifically as follows: the hydrogel material selects a double water phase system consisting of polyethylene glycol (PEG) -glucan with good biocompatibility and stability, and the molecular weight range of the PEG is as follows: 8000-: 10-50%, dextran molecular weight range: 70k-500kDa, concentration range: 10 to 30 percent; CaCl with the concentration of 0.5-4% is introduced into the chip outlet2The solution is quickly crosslinked with sodium alginate in situ to form hydrogel microcapsules;
the viscosity range of sodium alginate used is: 55-1000cps, concentration range: 0.1-2%; the flow rate of the dispersed phase is 0.01-1 mul/min, the flow rate of the continuous phase is 0.5-5 mul/min, and the switching period of a pump valve is 0.1-1 s;
the human pluripotent stem cells are directionally differentiated into precursor cells of liver or pancreatic islets, and 1 × 107～5×107cells/mL precursor cells are digested into single cells by 0.25% trypsin-EDTA, a cell suspension and a disperse phase solution containing sodium alginate are fully and uniformly mixed, the mixture is introduced into a disperse phase channel (5) through a disperse phase inlet (3) as a whole, a droplet containing the cells is stably formed in a main channel (8) through the separation of a pump valve and the maintenance of a continuous phase, and the sodium alginate is further rapidly crosslinked in situ to form the aqueous two-phase microcapsule hydrogel loading the cells.
The 3D organoid is a pancreatic islet organoid or a liver organoid.
The method for engineering the 3D organoid into the islet organoid comprises the following specific steps:
(1) endoderm induced differentiation: replacing mTESR1 culture medium of human pluripotent stem cells with 50% -70% of the density in the culture plate with endoderm differentiation culture medium 1, and standing and culturing for 5 days;
(2) induction of pancreatic endoderm differentiation: replacing DMEM/F12 culture medium with high-sugar DMEM culture medium, adding B27supplement (50 x) accounting for 0.5% of the total volume, and standing and culturing for 6 days, wherein the final concentration is 2 mu M dorsomorphin, 2 mu M retinoic acid, 10 mu M SB431542,5ng/mL basic fiber growth factor (bFGF) and 250nM SANT-1;
(3) induction of pancreatic endocrine precursor cell differentiation: the DMEM medium needs to be added with B27supplement (50 x) accounting for 0.5 percent of the total volume, and the final concentration is 2 mu M dorsomorphin small molecular compound, 10 mu M SB431542 small molecular compound, 50 mu g/mL ascorbic acid (ascorbyl acid) and 10 mu M DAPT (gamma-secretase inhibitor) for standing culture for 5 days;
(4) islet organoid production: to promote further differentiation into islet cells, the DMEM medium was replaced with commercial CMRL 1066 medium, and additionally 0.5% by volume of B27supplement (50X) was added to the total volume to a final concentration of 25mM glucose (glucose), 10mM nicotinamide (nicotinamide), 10. mu.M SB431542 small molecule compound, 50. mu.g/mL ascorbic acid (ascorbic acid) and 2. mu.M dorsomorphin small molecule compound; digesting and centrifuging islet cells cultured for 15-23 days, fully and uniformly mixing a cell suspension and a dispersed phase solution containing sodium alginate, introducing the mixture into a chip to form a cell-loaded hydrogel microcapsule, and transferring the microcapsule into a pore plate for continuous culture; the cells are aggregated into spheres in the microcapsules on the next day, and are further differentiated and developed into islet organoids in an induction culture medium; then long-term culture can be carried out in the culture medium, liquid is changed every 1-3 days during the culture, and the cell viability and the insulin secretion function of the islet organoid are identified.
The method for engineering the 3D organoid into the liver organoid comprises the following specific steps:
(1) endoderm induced differentiation: replacing mTESR1 culture medium of human pluripotent stem cells with 50% -70% of the density in the culture plate with endoderm differentiation culture medium 2, and standing and culturing for 5 days;
(2) induction of hepatic precursor cell differentiation and proliferation: replacing Activin-A in the endoderm differentiation medium 2 in the step (1) with HGF and bFGF factors, and standing and culturing for 5 days; the final concentration of HGF is 20-30ng/ml, and the final concentration of bFGF is 10-20 ng/ml;
(3) liver organoid production: in order to promote the further differentiation of the hepatic precursor cells, the culture Medium in the step (2) is replaced by a commercial Hepatocyte Culture Medium (HCM), and OSM factors and dexamethasone (Dex) are added for standing culture; the final concentration of OSM is 10-20ng/ml, and the final concentration of Dex is 10-7-10-6M; digesting and centrifuging the liver precursor cells cultured for 10-15 days, fully and uniformly mixing the cell suspension and the dispersed phase solution containing sodium alginate, introducing the mixture into a chip to form a cell-loaded hydrogel microcapsule, and transferring the hydrogel microcapsule into a pore plate for continuous culture; the cells are aggregated into balls in the microcapsules on the next day, and are further differentiated and developed into liver organoids in an induction culture medium; after 15 days, removing OSM factors from the culture medium, replacing the culture medium with HCM culture medium containing Dex only, and then carrying out long-term culture, wherein the culture medium is replaced every 1-3 days, and cell viability and function identification of liver organoid are carried out.
The hydrogel material used is any substance that can be rapidly cross-linked; in particular to one of sodium alginate, chitosan, photopolymerisable gelatin and PEGDA.
The human pluripotent stem cell is a human induced pluripotent stem cell (hiPSC) or an embryonic stem cell (hESC), and the cell seeding density ranges from 2 x 103～6×106cells/ml。
The invention is realized by the following technical scheme: the production of aqueous two-phase hydrogel microcapsules and the cell loading; islet organoid engineering; liver organoid engineering.
The production and cell loading of the aqueous two-phase hydrogel microcapsule specifically comprise the following steps: (1) the prepared micro-fluidic droplet chip of the integrated pump valve mainly comprises a continuous phase, a dispersed phase inlet, a pneumatic pump valve, a droplet generation unit and a microcapsule generation unit. Selecting a double water phase system consisting of polyethylene glycol (PEG) -dextran, wherein the molecular weight range of PEG is as follows: 8000-: 10-50%, glucanSugar molecular weight range: 70k-500kDa, concentration range: 10 to 30 percent; CaCl with the concentration of 0.5-4% is introduced into the chip outlet2The solution is quickly crosslinked with sodium alginate in situ to form a hydrogel microcapsule, and the viscosity range of the sodium alginate is as follows: 55-1000cps, concentration range: 0.1-2%; the flow rate of the dispersed phase is 0.01-1 mul/min, the flow rate of the continuous phase is 0.5-5 mul/min, and the on-off period of the pump valve is 0.1-1s, and the size, the distance and other parameters of the liquid drops can be controlled by adjusting the flow rate of the dispersed phase, the flow rate of the continuous phase and the on-off period of the pump valve.
(2) The human pluripotent stem cells are directionally differentiated into precursor cells of liver or pancreatic islets, and 1 × 107～5×107Digesting the cells/mL precursor cells into single cells by using 0.25% trypsin-EDTA, fully and uniformly mixing a cell suspension and a disperse phase solution containing sodium alginate, introducing the mixture into a disperse phase channel, separating the mixture through a pump valve and maintaining a continuous phase to stably form liquid drops containing the cells in a main channel, and further quickly crosslinking the sodium alginate in situ to form the cell-loaded aqueous two-phase hydrogel microcapsule.
The islet organoid engineering comprises the following specific steps:
(1) endoderm induced differentiation: replacing mTESR1 culture medium of human pluripotent stem cells with DMEM/F12 culture medium, wherein the mTESR1 culture medium of human pluripotent stem cells with 50% -70% of the density in the culture plate is collected, B27supplement (50 x) accounting for 1% of the total volume, KSR accounting for 1% of the total volume, GlutaMax accounting for 1% of the total volume and factors of Activin-A with the concentration of 80-100ng/ml are added, and the culture plate is subjected to standing culture for 5 days.
(2) Induction of pancreatic endoderm differentiation: DMEM/F12 medium was replaced with high-sugar DMEM medium, and B27supplement (50X) was added at a concentration of 0.5% by volume, to give a final concentration of 2. mu.M dorsomorphin, 2. mu.M retinic acid, 10. mu.M SB431542,5ng/mL basic fibrous growth factor (bFGF) and 250nM SANT-1, and allowed to stand for 6 days.
(3) Induction of pancreatic endocrine precursor cell differentiation: DMEM medium was cultured in a static culture for 5 days with the addition of 0.5% by volume of B27supplement (50X) to a final concentration of 2. mu.M dorsomorphin, 10. mu.M SB431542, 50. mu.g/mL ascorbic acid and 10. mu.M DAPT.
(4) Islet organoid production: to promote further differentiation into islet cells, DMEM medium was replaced with commercial CMRL 1066 medium, and additionally 0.5% by volume of B27supplement (50X) was added to the total volume to a final concentration of 25mM glucose, 10mM nicotinamide, 10. mu.M SB431542, 50. mu.g/mL ascorbic acid and 2. mu.M dorsomorphin; digesting and centrifuging islet cells cultured for 15-23 days, fully and uniformly mixing a cell suspension and a dispersed phase solution containing sodium alginate, introducing the mixture into a chip to form a cell-loaded hydrogel microcapsule, and transferring the microcapsule into a pore plate for continuous culture; the cells are aggregated into spheres in the microcapsules on the next day, and are further differentiated and developed into islet organoids in an induction culture medium; then long-term culture can be carried out in the culture medium, liquid is changed every 1-3 days during the culture, and the cell viability and the insulin secretion function of the islet organoid are identified.
Compared with the prior art, the invention has the following beneficial effects:
the invention combines stem cells, biological materials and engineering technology, is better used for organoid generation, improves the physiological relevance of organoids, and provides a powerful technical support for in vitro multi-organoid engineering and biomedical research thereof.
Drawings
Fig. 1 is a schematic structural diagram of a two-aqueous-phase droplet microfluidic chip, wherein: a general diagram; b partial diagram of pump valve structure; c a schematic diagram of hydrogel-loaded cells,
wherein: 1 continuous phase inlet; 2 a gas inlet; 3 inlet of disperse phase; 4 an outlet for droplets; 5 channels of the dispersed phase; 6 a gas channel; 7 continuous phase channel; 8, a main channel; 9 pneumatic pump valve.
Fig. 2 is a schematic diagram of organoid generation in a droplet microfluidic system. Wherein: a, generating a schematic diagram by using hydrogel microcapsules; b schematic diagram of hydrogel microcapsule for cell-loading and organoid engineering,
fig. 3 is a representation of islet organoid engineering in hydrogel microcapsules, wherein: a bright field representation (scale: 50 μm) and 7 day islet organoid size distribution plot; b, a dead-live staining map and a cell viability fluorescence quantitative characterization map within 7 days; and c, immunofluorescence staining patterns of islet organoids in the microcapsules.
Fig. 4 is a representation of liver organoid engineering in hydrogel microcapsules, wherein: a bright field characterization and dead staining (scale: 50 μm); b immunofluorescence staining pattern of liver organoids in microcapsules.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1
The aqueous two-phase droplet microfluidic system is used for islet organoid engineering.
The islet organoid engineering comprises the following specific steps:
(1) endoderm induced differentiation: replacing mTESR1 culture medium of 50% density human pluripotent stem cells in the culture plate with DMEM/F12 culture medium, adding B27supplement accounting for 1% of the total volume, KSR accounting for 1% of the total volume, GlutaMax accounting for 1% of the total volume and factor of Activin-A with concentration of 80ng/ml, and standing and culturing for 5 days.
(2) Induction of pancreatic endoderm differentiation: DMEM/F12 medium was replaced with high-sugar DMEM medium, and B27supplement was added at a concentration of 0.5% of the total volume, to give a final concentration of 2. mu.M dorsomorphin, 2. mu.M retinoic acid, 10. mu.M SB431542,5ng/mL basic fiber growth factor (bFGF) and 250nM SANT-1, and allowed to stand for 6 days.
(3) Induction of pancreatic endocrine precursor cell differentiation: DMEM medium was added with 0.5% of B27supplement, 2. mu.M dorsomorphin, 10. mu.M SB431542, 50. mu.g/mL ascorbic acid and 10. mu.M DAPT, and allowed to stand for 5 days.
(4) Islet organoid production: to promote further differentiation into islet cells, DMEM medium was replaced with commercial CMRL 1066 medium, and B27supplement was added in an amount of 0.5% of the total volume to a final concentration of 25mM glucose, 10mM nicotinamide, 10. mu.M SB431542, 50. mu.g/mL ascorbic acid and 2. mu.M dorsomorphin;
digesting the islet cells cultured for 15 days into single cells by using 0.25% trypsin-EDTA, and obtaining the islet cells with the density of 1 × 107cell suspension and disperse phase solution containing sodium alginate are fully and uniformly mixed, introduced into a disperse phase channel, separated and connected through a pump valveAnd (3) under the maintenance action of continuous phase, stably forming liquid drops containing cells in the main channel, and further quickly crosslinking sodium alginate in situ to form the aqueous two-phase hydrogel microcapsule loaded with the cells. Transferring into a pore plate for continuous culture, aggregating cells in the microcapsule into spheres in the next day, and further differentiating and developing into islet organoids in an induction culture medium; then long-term culture can be carried out in the culture medium, the culture medium is changed every 1 day, and the cell viability and the insulin secretion function of the islet organoid are identified.
The schematic diagram and the preliminary characterization diagram of the islet organoid engineered by using the two-aqueous-phase droplet microfluidic system according to the above steps are respectively shown in fig. 2 and fig. 3, which shows that islet cells can be successfully encapsulated and differentiated in hydrogel microcapsules to form the islet organoid.
Example 2
The aqueous two-phase droplet microfluidic system is used for islet organoid engineering.
The islet organoid engineering comprises the following specific steps:
(1) endoderm induced differentiation: replacing mTESR1 culture medium of human pluripotent stem cells with DMEM/F12 culture medium, wherein the mTESR1 culture medium is collected in the culture plate at a density of 70%, B27supplement accounts for 1% of the total volume, KSR accounts for 1% of the total volume, GlutaMax accounts for 1% of the total volume, and Activin-A factors with a concentration of 100ng/ml are added, and the mixture is subjected to standing culture for 5 days.
(2) Induction of pancreatic endoderm differentiation: DMEM/F12 medium was replaced with high-sugar DMEM medium, and B27supplement was added at a concentration of 0.5% of the total volume, to give a final concentration of 2. mu.M dorsomorphin, 2. mu.M retinoic acid, 10. mu.M SB431542,5ng/mL basic fiber growth factor (bFGF) and 250nM SANT-1, and allowed to stand for 6 days.
(3) Induction of pancreatic endocrine precursor cell differentiation: DMEM medium was added with 0.5% of B27supplement, 2. mu.M dorsomorphin, 10. mu.M SB431542, 50. mu.g/mL ascorbic acid and 10. mu.M DAPT, and allowed to stand for 5 days.
(4) Islet organoid production: to promote further differentiation into islet cells, DMEM medium was replaced with commercial CMRL 1066 medium, and B27supplement was added in an amount of 0.5% of the total volume to a final concentration of 25mM glucose, 10mM nicotinamide, 10. mu.M SB431542, 50. mu.g/mL ascorbic acid and 2. mu.M dorsomorphin;
digesting islet cells cultured for 23 days into single cells by using 0.25% trypsin-EDTA, and obtaining the islet cells with the density of 5 × 107The cell suspension and the dispersed phase solution containing sodium alginate are fully and uniformly mixed, the mixture is introduced into a dispersed phase channel, liquid drops containing cells are stably formed in a main channel through the separation of a pump valve and the maintenance of a continuous phase, and the sodium alginate is further rapidly crosslinked in situ to form the cell-loaded aqueous two-phase hydrogel microcapsule. Transferring into a pore plate for continuous culture, aggregating cells in the microcapsule into spheres in the next day, and further differentiating and developing into islet organoids in an induction culture medium; then long-term culture can be carried out in the culture medium, the culture medium is changed every 1 day, and the cell viability and the insulin secretion function of the islet organoid are identified.
The schematic diagram and the preliminary characterization diagram of the islet organoid engineered by using the two-aqueous-phase droplet microfluidic system according to the above steps are respectively shown in fig. 2 and fig. 3, which shows that islet cells can be successfully encapsulated and differentiated in hydrogel microcapsules to form the islet organoid.
Example 3
The aqueous two-phase droplet microfluidic system is used for liver organoid engineering.
The liver organoid engineering comprises the following specific steps:
(1) endoderm induced differentiation: mTESR1 medium in which 50% density of human pluripotent stem cells were pooled in the culture plate was replaced with 1640+ B27 medium, and Activin-A at a concentration of 80ng/ml was added thereto, followed by static culture for 5 days.
The base component of the 1640+ B27 culture medium is commercial RPMI-1640 culture medium, and B27 accounting for 1% of the total volume needs to be added.
(2) Induction of hepatic precursor cell differentiation and proliferation: adding HGF and bFGF factors into a 1640+ B27 culture medium, and standing and culturing for 5 days;
the final concentration of HGF is 20ng/ml, and the final concentration of bFGF is 10 ng/ml.
(3) Liver organoid production: to promote further differentiation of hepatic precursor cells, 1640+ B27 cultureReplacing the medium with a commercial hepatocyte culture medium HCM, adding OSM factor and dexamethasone Dex, and standing for culture; the final concentration of the OSM is 10ng/ml, and the final concentration of the Dex is 10-7M。
Digesting hepatic precursor cells cultured for 10-15 days into single cells with 0.25% trypsin-EDTA, and concentrating to 1 × 107The cell suspension and the dispersed phase solution containing sodium alginate are fully and uniformly mixed, the mixture is introduced into a dispersed phase channel, liquid drops containing cells are stably formed in a main channel through the separation of a pump valve and the maintenance of a continuous phase, and the sodium alginate is further rapidly crosslinked in situ to form the cell-loaded aqueous two-phase hydrogel microcapsule. Transferring into a pore plate for continuous culture; the cells are aggregated into balls in the microcapsules on the next day, and are further differentiated and developed into liver organoids in an induction culture medium; after 15 days, the culture medium is removed of OSM factors, and is replaced by HCM culture medium containing Dex only, long-term culture can be carried out subsequently, liquid is replaced once every 1 day during the culture period, and cell viability and function identification of liver organoid are carried out.
The schematic diagram and the preliminary characterization diagram of liver organoid engineering by using the aqueous two-phase droplet microfluidic system according to the above steps are respectively shown in fig. 2 and fig. 4, which shows that liver precursor cells can be successfully encapsulated and differentiated in hydrogel microcapsules to form liver organoids.
Example 4
The aqueous two-phase droplet microfluidic system is used for liver organoid engineering.
The liver organoid engineering comprises the following specific steps:
(1) endoderm induced differentiation: mTESR1 medium in which 70% density of human pluripotent stem cells were pooled in the culture plate was replaced with 1640+ B27 medium, and Activin-A at a concentration of 100ng/ml was added thereto, followed by static culture for 5 days.
The base component of the 1640+ B27 culture medium is commercial RPMI-1640 culture medium, and B27 accounting for 1% of the total volume needs to be added.
(2) Induction of hepatic precursor cell differentiation and proliferation: adding HGF and bFGF factors into a 1640+ B27 culture medium, and standing and culturing for 5 days;
the final concentration of HGF is 30ng/ml, and the final concentration of bFGF is 20 ng/ml.
(3) Liver organoid production: in order to promote the further differentiation of the hepatic precursor cells, the 1640+ B27 culture medium is replaced by a commercial hepatic cell culture medium HCM, and an OSM factor and dexamethasone Dex are added for standing culture; the final concentration of the OSM is 20ng/ml, and the final concentration of the Dex is 10-6M。
Digesting hepatic precursor cells cultured for 10-15 days into single cells with 0.25% trypsin-EDTA, and adjusting the density to 5 × 107The cell suspension and the dispersed phase solution containing sodium alginate are fully and uniformly mixed, the mixture is introduced into a dispersed phase channel, liquid drops containing cells are stably formed in a main channel through the separation of a pump valve and the maintenance of a continuous phase, and the sodium alginate is further rapidly crosslinked in situ to form the cell-loaded aqueous two-phase hydrogel microcapsule. Transferring into a pore plate for continuous culture; the cells are aggregated into balls in the microcapsules on the next day, and are further differentiated and developed into liver organoids in an induction culture medium; after 15 days, the culture medium is removed of OSM factors, and is replaced by HCM culture medium containing Dex only, long-term culture can be carried out subsequently, liquid is replaced once every 1 day during the culture period, and cell viability and function identification of liver organoid are carried out.
The schematic diagram and the preliminary characterization diagram of liver organoid engineering by using the aqueous two-phase droplet microfluidic system according to the above steps are respectively shown in fig. 2 and fig. 4, which shows that liver precursor cells can be successfully encapsulated and differentiated in hydrogel microcapsules to form liver organoids.
Claims (9)
1. A3D organoid engineering method based on aqueous two-phase droplet microfluidics is characterized in that: the method combines the 3D organoid and droplet microfluidic technology of human pluripotent stem cell source, and is used for constructing the in vitro model of the 3D organoid of the stem cell source by controllably synthesizing the aqueous two-phase hydrogel microcapsule with good biocompatibility, determined components and uniform size; comprises the following steps:
step one, adopting a double-aqueous-phase droplet microfluidic chip to generate a double-aqueous-phase hydrogel microcapsule and complete cell loading;
step two, 3D organoid engineering: when human pluripotent stem cells are gathered to a certain growth density, a commercialized mTESR1 culture medium for culturing the stem cells is replaced by an endoderm differentiation culture medium, and the tissue precursor cells are subjected to static culture and then induced to differentiate to finally produce the 3D organoid.
2. The bi-aqueous droplet microfluidics-based 3D organoid engineering method of claim 1, wherein: the growth density of the human pluripotent stem cells is 50% -70%.
3. The bi-aqueous droplet microfluidics-based 3D organoid engineering method of claim 1, wherein: the endoderm differentiation medium is one of the following two:
endoderm differentiation medium 1: the basic components are commercial DMEM/F12 culture medium, B27supplement (50 x) accounting for 1% of the total volume, KnockOut Replacement (KSR) accounting for 1% of the total volume, GlutaMax (100 x) accounting for 1% of the total volume, penicillin-streptomycin (100 x) accounting for 1% of the total volume, and a factor of Human Activin-A accounting for 80-100 ng/ml;
endoderm differentiation medium 2: the base component was commercial RPMI-1640 medium, added with B27supplement (50X) at 1% of the total volume, KnockOut Replacement (KSR) at 1% of the total volume, GlutaMax (100X) at 1% of the total volume, penicilin-streptomycin (100X) at 1% of the total volume, and factor of Human Activin-A at 80-100 ng/ml.
4. The bi-aqueous droplet microfluidics-based 3D organoid engineering method of claim 1, wherein:
the generation and cell loading of the aqueous two-phase hydrogel microcapsule are specifically as follows: the hydrogel material selects a double water phase system consisting of polyethylene glycol (PEG) -glucan with good biocompatibility and stability, and the molecular weight range of the PEG is as follows: 8000-: 10-50%, dextran molecular weight range: 70k-500kDa, concentration range: 10 to 30 percent; CaCl with the concentration of 0.5-4% is introduced into the chip outlet2The solution is quickly crosslinked with sodium alginate in situ to form hydrogel microcapsules;
the viscosity range of sodium alginate used is: 55-1000cps, concentration range: 0.1-2%; the flow rate of the dispersed phase is 0.01-1 mul/min, the flow rate of the continuous phase is 0.5-5 mul/min, and the switching period of a pump valve is 0.1-1 s;
the human pluripotent stem cells are directionally differentiated into precursor cells of liver or pancreatic islets, and 1 × 107～5×107cells/mL precursor cells are digested into single cells by 0.25% trypsin-EDTA, a cell suspension and a disperse phase solution containing sodium alginate are fully and uniformly mixed, the mixture is introduced into a disperse phase channel (5) through a disperse phase inlet (3) as a whole, a droplet containing the cells is stably formed in a main channel (8) through the separation of a pump valve and the maintenance of a continuous phase, and the sodium alginate is further rapidly crosslinked in situ to form the aqueous two-phase microcapsule hydrogel loading the cells.
5. The bi-aqueous droplet microfluidics-based 3D organoid engineering method of claim 1, wherein: the 3D organoid is a pancreatic islet organoid or a liver organoid.
6. The bi-aqueous droplet microfluidics-based 3D organoid engineering method of claim 5, wherein: the method for engineering the 3D organoid into the islet organoid comprises the following specific steps:
(1) endoderm induced differentiation: replacing mTESR1 culture medium of human pluripotent stem cells with 50% -70% of the density in the culture plate with endoderm differentiation culture medium 1, and standing and culturing for 5 days;
(2) induction of pancreatic endoderm differentiation: replacing DMEM/F12 culture medium with high-sugar DMEM culture medium, adding B27supplement (50 x) accounting for 0.5% of the total volume, and standing and culturing for 6 days, wherein the final concentration is 2 mu M dorsomorphin, 2 mu M retinoic acid, 10 mu M SB431542,5ng/mL basic fiber growth factor (bFGF) and 250nM SANT-1;
(3) induction of pancreatic endocrine precursor cell differentiation: the DMEM medium needs to be added with B27supplement (50 x) accounting for 0.5 percent of the total volume, and the final concentration is 2 mu M dorsomorphin small molecular compound, 10 mu M SB431542 small molecular compound, 50 mu g/mL ascorbic acid (ascorbyl acid) and 10 mu M DAPT (gamma-secretase inhibitor) for standing culture for 5 days;
(4) islet organoid production: to promote further differentiation into islet cells, the DMEM medium was replaced with commercial CMRL 1066 medium, and additionally 0.5% by volume of B27supplement (50X) was added to the total volume to a final concentration of 25mM glucose (glucose), 10mM nicotinamide (nicotinamide), 10. mu.M SB431542 small molecule compound, 50. mu.g/mL ascorbic acid (ascorbic acid) and 2. mu.M dorsomorphin small molecule compound; digesting and centrifuging islet cells cultured for 15-23 days, fully and uniformly mixing a cell suspension and a dispersed phase solution containing sodium alginate, introducing the mixture into a chip to form a cell-loaded hydrogel microcapsule, and transferring the microcapsule into a pore plate for continuous culture; the cells are aggregated into spheres in the microcapsules on the next day, and are further differentiated and developed into islet organoids in an induction culture medium; then long-term culture can be carried out in the culture medium, liquid is changed every 1-3 days during the culture, and the cell viability and the insulin secretion function of the islet organoid are identified.
7. The bi-aqueous droplet microfluidics-based 3D organoid engineering method of claim 5, wherein: the method for engineering the 3D organoid into the liver organoid comprises the following specific steps:
(1) endoderm induced differentiation: replacing mTESR1 culture medium of human pluripotent stem cells with 50% -70% of the density in the culture plate with endoderm differentiation culture medium 2, and standing and culturing for 5 days;
(2) induction of hepatic precursor cell differentiation and proliferation: replacing Activin-A in the endoderm differentiation medium 2 in the step (1) with HGF and bFGF factors, and standing and culturing for 5 days; the final concentration of HGF is 20-30ng/ml, and the final concentration of bFGF is 10-20 ng/ml;
(3) liver organoid production: in order to promote the further differentiation of the hepatic precursor cells, the culture Medium in the step (2) is replaced by a commercial Hepatocyte Culture Medium (HCM), and OSM factors and dexamethasone (Dex) are added for standing culture; the final concentration of OSM is 10-20ng/ml, and the final concentration of Dex is 10-7-10-6M; digesting and centrifuging liver precursor cells cultured for 10-15 days, fully and uniformly mixing a cell suspension and a dispersed phase solution containing sodium alginate, introducing into a chip to form a cell-loaded hydrogel microcapsule,transferring into a pore plate for continuous culture; the cells are aggregated into balls in the microcapsules on the next day, and are further differentiated and developed into liver organoids in an induction culture medium; after 15 days, removing OSM factors from the culture medium, replacing the culture medium with HCM culture medium containing Dex only, and then carrying out long-term culture, wherein the culture medium is replaced every 1-3 days, and cell viability and function identification of liver organoid are carried out.
8. The bi-aqueous droplet microfluidics-based 3D organoid engineering method of claim 1, wherein: the hydrogel material used is any substance that can be rapidly cross-linked; in particular to one of sodium alginate, chitosan, photopolymerisable gelatin and PEGDA.
9. The bi-aqueous droplet microfluidics-based 3D organoid engineering method of claim 4, wherein: the human pluripotent stem cell is a human induced pluripotent stem cell (hiPSC) or an embryonic stem cell (hESC), and the cell seeding density ranges from 2 x 103～6×106cells/ml。
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| CN114214267A (en) * | 2021-12-06 | 2022-03-22 | 大连大学 | Organoid matrigel microspheres and preparation method and application thereof |
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