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3D organoid engineering method based on two-aqueous-phase droplet microfluidics

An organoid, aqueous two-phase technology, applied in the field of regenerative medicine, to achieve the effect of improving physiological relevance

Pending Publication Date: 2021-05-28
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of aqueous two-phase microfluidic droplet technology for organoid engineering and optimization of the formation and operation of in vitro organoids is still blank.

Method used

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  • 3D organoid engineering method based on two-aqueous-phase droplet microfluidics
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  • 3D organoid engineering method based on two-aqueous-phase droplet microfluidics

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Effect test

Embodiment 1

[0044] Aqueous two-phase droplet microfluidic system for islet organoid engineering.

[0045] The specific steps for engineering the islet organoids are:

[0046] (1) Induction of endoderm differentiation: Replace the mTESR1 medium containing human pluripotent stem cells at a density of 50% in the culture plate with DMEM / F12 medium, add 1% of the total volume of B27 supplement, and 1% of the total volume of KSR, GlutaMax accounting for 1% of the total volume and Activin-A factors at a concentration of 80ng / ml were added, and cultured statically for 5 days.

[0047] (2) Induce pancreatic endoderm differentiation: replace DMEM / F12 medium with high-glucose DMEM medium, add 0.5% of the total volume of B27 supplement, the final concentration is 2μM dorsomorphin, 2μM retinoic acid, 10μM SB431542, 5ng / mL basic Fibroblast growth factor (bFGF) and 250nM SANT-1 were cultured statically for 6 days.

[0048] (3) Induction of differentiation of pancreatic endocrine precursor cells: DMEM ...

Embodiment 2

[0053] Aqueous two-phase droplet microfluidic system for islet organoid engineering.

[0054] The specific steps for engineering the islet organoids are:

[0055] (1) Induction of endoderm differentiation: replace the mTESR1 medium containing human pluripotent stem cells at a density of 70% in the culture plate with DMEM / F12 medium, add 1% of the total volume of B27 supplement, and 1% of the total volume of KSR, GlutaMax accounting for 1% of the total volume and Activin-A factors at a concentration of 100 ng / ml were added and cultured statically for 5 days.

[0056] (2) Induce pancreatic endoderm differentiation: replace DMEM / F12 medium with high-glucose DMEM medium, add 0.5% of the total volume of B27 supplement, the final concentration is 2μM dorsomorphin, 2μM retinoic acid, 10μM SB431542, 5ng / mL basic Fibroblast growth factor (bFGF) and 250nM SANT-1 were cultured statically for 6 days.

[0057] (3) Induction of differentiation of pancreatic endocrine precursor cells: DMEM...

Embodiment 3

[0062] Aqueous two-phase droplet microfluidic system for liver organoid engineering.

[0063] The specific steps for engineering the liver organoids are:

[0064] (1) Endoderm induced differentiation: replace the mTESR1 culture medium of human pluripotent stem cells at a density of 50% in the culture plate with 1640+B27 medium, add 80ng / ml of Activin-A factor, and culture for 5 sky.

[0065] The basic component of the 1640+B27 medium is commercialized RPMI-1640 medium, and 1% of the total volume of B27 needs to be added.

[0066] (2) Inducing the differentiation and proliferation of hepatic precursor cells: add HGF and bFGF factors to the 1640+B27 medium and culture for 5 days;

[0067] The final concentration of HGF is 20 ng / ml, and the final concentration of bFGF is 10 ng / ml.

[0068] (3) Production of liver organoids: In order to promote the further differentiation of liver precursor cells, the 1640+B27 medium was replaced with commercial hepatocyte medium HCM, and addit...

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Abstract

The invention provides a 3D organoid engineering method based on two-aqueous-phase droplet microfluidics. The method comprises the steps of cell loading of the aqueous two-phase hydrogel microcapsules, pancreas islet organ engineering, liver organ engineering and the like. According to the invention, an emerging human pluripotent stem cell-derived 3D organ and a droplet microfluidic technology are mainly combined, and the two-aqueous-phase hydrogel micro-capsule with good biocompatibility, definite components and uniform size is controllably synthesized and is used for constructing a stem cell-derived 3D organ in-vitro model. The method has the advantages of simplicity in operation, good controllability, reduction of the variability of the organoid, high throughput generation and the like, and provides a powerful new platform for the organoid in biomedical application aspects of disease simulation, drug screening, in vivo transplantation and the like.

Description

technical field [0001] The invention belongs to the fields of regenerative medicine and the like, and in particular relates to a 3D organoid engineering method based on two-phase liquid droplet microfluidics. Background technique [0002] The construction of 3D tissue or organ models in vitro can simulate human physiology, which is of great significance in biomedical applications. Organoids from multiple stem cell sources (including adult stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (hiPSCs)) have been a major technological breakthrough in recent years and represent a new type of in vitro organ model. Organoids are 3D multicellular complex structures that are specifically differentiated and self-organized by stem cells in 3D culture, which can summarize the key structures and functions of corresponding organs in vivo. At present, a variety of organoids have been successfully developed, such as intestinal, brain, liver, kidney, and retinal orga...

Claims

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Application Information

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IPC IPC(8): C12N5/00C12N5/071
CPCC12N5/0062C12N5/0671C12N2533/30C12N2533/54C12N2533/74C12N2533/76C12N2506/45C12N2506/02
Inventor 秦建华王亚清刘海涛
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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