Method for constructing three-dimensional neural stem cell model in two steps by adopting micro-fluidic technology

A microfluidic technology and neural stem cell technology, applied in the field of microfabrication technology and tissue engineering, can solve problems such as no standardized experimental methods, and achieve the effects of saving labor, good repeatability, and reducing cell culture costs

Active Publication Date: 2013-06-12
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the research on the construction of three-dimensional neural tissue derived from NSCs in vitro is

Method used

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  • Method for constructing three-dimensional neural stem cell model in two steps by adopting micro-fluidic technology
  • Method for constructing three-dimensional neural stem cell model in two steps by adopting micro-fluidic technology
  • Method for constructing three-dimensional neural stem cell model in two steps by adopting micro-fluidic technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Design and fabrication of a microfluidic chip suitable for three-dimensional culture of neural stem cells

[0037] 1. Design of microfluidic chip

[0038] In order to provide a good microenvironment for the growth of NSCs-collagen complexes, this study designed a microfluidic chip suitable for the growth of NSCs-collagen complexes in vitro. The overall design of the chip is as follows: figure 1Shown: in the figure 1 is the left cell injection hole with a diameter of 2mm; 2 is the right cell injection hole with a diameter of 3mm; 3 is the waste liquid outlet with a diameter of 3mm; 4 is the cell culture chamber. The length of the cell culture chamber is 200 mm, the width is 1 mm, and the depth is 150 μm. Its internal structure is as figure 2 Shown: the side wall of the culture chamber is a palisade-like structure composed of several micropillars with a length of 100 μm and a width of 50 μm, and each microcolumn is spaced at a distance of 20 μm. There is a 4...

Embodiment 2

[0058] The screening of embodiment 2 NSCs in vitro serum-free expansion medium

[0059] This example aims to realize the massive expansion of NSCs in vitro under serum-free culture conditions, thereby providing sufficient cell numbers for the construction of a three-dimensional model of NSCs. The mixed culture solution of DMEM / F12 / RPMI1640 (1:1:1, V:V:V) was selected as the basic medium in the experiment, and four key components were selected from the commonly used additives: glucose (Glucose), glutamine ( Glutamine), lipids (Lipids), bovine serum albumin (BSA), 4 factors and three levels of orthogonal experiments were established to determine the optimal dosage of these 4 components in the expansion medium and their expansion of NSCs increase the effect.

[0060] Orthogonal experiments were divided into 9 groups, which were carried out on 24-well culture plates, and 3 wells were selected for parallel experiments in each group. The experimental cells were derived from the th...

Embodiment 3

[0068] Example 3 Construction of NSCs-collagen three-dimensional complex on microfluidic chip

[0069] In this example, the NSCs-collagen hydrogel mixture was first inoculated into the cell culture chamber of the microfluidic chip, and then the three-dimensional growth of NSCs in the collagen scaffold was realized by replacing the medium in a two-step method. The specific method is as follows:

[0070] 1. Preparation of single cell suspension

[0071] Transfer the third-generation NSCs "neurospheres" from the culture bottle to a 15ml centrifuge tube, centrifuge at 1000rpm for 5min, discard the supernatant, and collect the suspended neurospheres; add an equal volume of Accutase to the collection TM For enzymes, after shaking in a water bath at 37°C for 15 minutes, add 5ml of fresh medium, centrifuge at 1000rpm for 5min; discard the supernatant, resuspend the cells with 1ml of fresh medium, and place a polished Pasteur pipette against the end of the centrifuge tube At the bott...

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Abstract

The invention relates to a method for constructing a three-dimensional neural stem cell model in two steps by adopting the micro-fluidic technology. The method is characterized in that a rat tail collagen I is used as a three-dimensional support, a micro-pillar array type micro-fluidic chip is used as a culture platform, and a neural stem cell is cultured in two steps, wherein in the early culture stage, a culture medium for promoting the amplification of the neural stem cell is injected into a cell culture chamber, and in the later culture stage, a conditioned medium suitable for the growth of the neural stem cell and the daughter cells thereof is used, and a three-dimensional composite structure which is similar to a nerve tissue is formed by simulating the microenvironment of different neurogenesis stages in the body. The method provided by the invention is good in repeatability and can be used for construction a plurality of groups of samples. The adopted microfluidic culture system is in a microliter volume and can be regulated accurately, thus the amount of various high-cost cell growth factors, immunologic fluorescent antibodies and cell hormones used in the process of culturing the cell can be reduced greatly, and the cell culture cost can be lowered. The three-dimensional neural stem cell model is expected to be a nerve tissue substitute for screening a novel medicament or monitoring an environmental toxin.

Description

technical field [0001] The invention belongs to the field of microprocessing technology and tissue engineering, and relates to a method for constructing a three-dimensional cell model of neural stem cells in vitro using a microfluidic cell culture system. Background technique [0002] The in vitro culture methods of neural stem cells (NSCs) are mainly divided into suspension culture method and monolayer culture method. Suspension-cultured NSCs grow in the form of "neurospheres" in vitro, and a large number of NSCs and their differentiated daughter cells co-exist inside the neurospheres. As the neurospheres continue to grow, it will be difficult to transfer nutrients to the interior of the neurospheres, resulting in a large number of cells in the inner core withering, necrosis, and even a hollow phenomenon. The monolayer culture method has played an important role in the research on the morphological characteristics, growth characteristics of NSCs, and the characteristics of...

Claims

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Application Information

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IPC IPC(8): C12N5/0797C12M3/00
Inventor 刘军山葛丹刘天庆马学虎刘冲
Owner DALIAN UNIV OF TECH
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