Pancreatic-like structural body and construction method and application thereof

A construction method and structure technology, applied in the field of pancreas-like structure and its construction, can solve the problems of poor controllability, inability to achieve high-throughput stable preparation and high-throughput screening, etc.

Active Publication Date: 2020-05-26
REGENOVO BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Current organoid construction methods all use hydrogel material encapsulation technology, which is easy to operate, but the controllability is very poor, and high-throughput stable preparation and high-throughput screening cannot be achieved.

Method used

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  • Pancreatic-like structural body and construction method and application thereof
  • Pancreatic-like structural body and construction method and application thereof
  • Pancreatic-like structural body and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1 Biological material stock solution preparation

[0083] Gelatin solution: mix gelatin powder (Sigma, G1890) with 0.9% sodium chloride solution at a mass ratio of 15:100, vortex the solution for 1 minute, heat at 60°C for 2 hours, and repeat the shaking The operation of vortex and heating was performed twice to finally make it dissolve evenly, then aliquoted and stored at 4°C. Incubate in the cell culture incubator for 10 minutes before each use to make it melt into a homogeneous solution.

[0084] Sodium alginate solution: mix sodium alginate powder (Sigma, A2033) with 0.9% sodium chloride solution at a mass ratio of 4:100, vortex the solution for 1 minute, and heat at 60°C for 2 hours. Repeat the operation of shaking, vortexing and heating twice again to finally dissolve it evenly, and store it at 4°C.

[0085] Matrigel: Matrigel (Corning, 356234) was thawed at 4°C, aliquoted on ice, and stored at -20°C. Thaw at 4°C before use and use within 24 hours. ...

Embodiment 2

[0086] Example 2 Stem cells and vascularized cell culture

[0087] Human adipose-derived stem cell (Adipose-derived stem cell, ADSC) (Sciencell, 7510) was cultured in the ADSC cell expansion medium, and the ADSC cell expansion medium was carried out according to the MSCM medium kit (Sciencell, 7501). Preparation; the culture bottle was pre-primed with PLL solution (Sigma, P4832) diluted 8 times with sterile water, and the bottom was primed for 1 hour; when the cells were 90% confluent, they were passaged at a ratio of 1:3, and the culture medium was replaced every 2-3 days.

[0088] Human umbilical vein endothelial cells (human umbilical vein endothelial cell, HUVEC) (ATCC, PCS-100-010) were cultured in HUVEC cell expansion medium. HUVEC cell expansion culture medium was prepared according to the EBM-2 medium kit (LONZA, CC-3162). When the cells were 90% confluent, they were subcultured at a ratio of 1:3, and the culture medium was changed every 2-3 days.

Embodiment 3

[0089] Example 3 Stem cells are pre-differentiated in a multi-well plate

[0090] ADSC cells of passage 4 were used for differentiation. Use the method above to prime common 24-well plates before differentiation. After ADSC cells were digested with 0.25% trypsin / EDTA (Gibco, 25200) digestion solution, the cell pellet was collected by centrifugation at 1200rpm for 5min, and the cell pellet was collected at 2×10 5Cells / ml were suspended in ADSC cell differentiation medium to obtain ADSC cell solution, and 1ml of ADSC cell solution was added to each well of a 24-well plate. The composition of the differentiation medium is in the mixed DMEM medium (Gibco, 11965) and DMEM / F-12 medium (Gibco, 11320) of 1:1 volume ratio, add 10mM Nicotinamide (Sigma, 72340), 2nM Activin A (R&D, 294-HG), 10nM Exendin-4 (Sigma, E7144), 10nM Pentagastrin (Sigma, B1636), 100pM hepatocyte growth factor (Sigma, SRP6014), 2% B-27 supplement (Gibco, 17504), 1% N-2 supplement (Gibco, A13707), 1% penicillin...

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Abstract

The invention provides a pancreatic-like structure and a construction method thereof. The construction method comprises the following steps: A, inducing stem cells and / or progenitor cells in vitro todifferentiate into pancreatic-like cell clusters; B, mixing the vascularized cells, the pancreatic-like cell clusters and a hydrogel material, and performing biological printing to obtain a pre-gel three-dimensional structure body; and C, culturing the pre-gel three-dimensional structure body by adopting a multi-cell culture solution and / or a bioreactor to obtain the pancreas-like structure body.The pancreatic-like structure provided by the invention is composed of vascularized cells and pancreatic-like tissue cells, and has the form, phenotypic characteristics and physiological functions ofnatural tissues. The method can be applied to the aspects of organoid construction, tissue / organ / human body chips, tissue engineering, regenerative medicine, in-vitro physiological model / pathologicalmodel / pharmacological model construction, cytobiology or drug research and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a pancreas-like structure and its construction method and application. Background technique [0002] Organoids are a class of three-dimensional cell cultures that contain some of the key properties of their representative organs. Such artificial structures cultured in vitro include stem cell populations and / or progenitor cell populations with self-renewal ability, can differentiate into corresponding organ-specific cell types, have a similar spatial structure to corresponding organs, and can reproduce part of the functions of corresponding organs , thus providing a highly physiologically relevant in vitro system. At present, there have been reports on the use of organoid technology to construct intestines, liver, pancreas, kidneys, prostate, lungs, optic cups, and brains, and have demonstrated excellent structures and functions similar to natural tissues beyond traditional technique...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/0775A61L27/38A61L27/22A61L27/20A61L27/24A61L27/16A61L27/18A61L27/52
CPCC12N5/0697C12N5/0676A61L27/3804A61L27/222A61L27/20A61L27/24A61L27/227A61L27/16A61L27/18A61L27/52C12N2502/22C12N2502/28C12N2533/74C12N2533/54C12N2506/1384
Inventor 姚睿徐铭恩
Owner REGENOVO BIOTECH
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