A method for cell co-culture inducing directed differentiation of stem cells in vitro

A culture method and stem cell technology, applied in the field of cell co-culture induced stem cell directional differentiation in vitro, can solve the problems of induced cell and stem cell pollution, high price, increased experimental cost, etc., to avoid expensive materials and processes, improve differentiation efficiency, and differentiate Effect of effect improvement

Active Publication Date: 2016-09-14
源创吉因(重庆)细胞应用技术研究院有限公司
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Induced cells and stem cells are mixed and cultured in a direct contact co-culture system. Although this system is closer to the microenvironment of in vivo cells, the induced cells and stem cells are contaminated with each other and cannot be used in clinical practice.
Although the Transwell co-culture system uses a porous flat membrane to separate the two types of cells, which avoids the problem of cell separation and reflects the interaction between cells and cells and between cells and factors, it lacks the influence of extracellular matrix components and stiffness. Moreover, the concentration distribution of soluble factors between the two types of cells cannot be adjusted, thus limiting the differentiation efficiency of stem cells and the improvement of the functional level of differentiated cells, and this system is expensive, which greatly increases the cost of experiments
[0005] Due to the above-mentioned main defects in the existing cell co-culture system, which severely limits the further development and application of stem cell directed differentiation technology in vitro, it is urgent to develop a convenient and practical multi-angle simulation of cells between cells, cells and extracellular matrix in the microenvironment of cells in vivo A new method of in vitro cell co-cultivation based on the interaction between cells and soluble factors, in order to realize the regulation of stem cell directional pre-differentiation in vitro, and further promote the application of stem cells in the field of regenerative medicine

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A method for cell co-culture inducing directed differentiation of stem cells in vitro
  • A method for cell co-culture inducing directed differentiation of stem cells in vitro
  • A method for cell co-culture inducing directed differentiation of stem cells in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Cell co-culture promotes directed differentiation of human mesenchymal stem cells into neural precursor cells

[0041] The 4th generation human umbilical cord mesenchymal stem cells were divided into 2 × 10 4 cells / cm 2 The density is inoculated in the inner area of ​​the ring gasket with an inner diameter of 35 mm, and the thickness of the gasket is 300 μm, and DMEM culture solution containing 10% FBS is added to culture overnight.

[0042] Add agarose powder into water and pressurize at 121°C for 20 minutes to obtain a 3% (W / V, g / ml) agarose storage solution. Sodium alginate (molecular weight 430kDa, ratio of guluronic acid to mannuronic acid 1.5) powder was dissolved in physiological saline to obtain a 1.5% (W / V, g / ml) solution. Dilute the preheated 3% (W / V, g / ml) agarose storage solution and 1.5% (W / V, g / ml) sodium alginate solution with water to prepare the final agarose concentrations of 0.35, 0.7 and 1.4% respectively (W / V, g / ml) mixture, the final ...

Embodiment 2

[0044] Example 2: Cell co-culture promotes directed differentiation of human embryonic stem cells into cardiac progenitor cells

[0045] The 32nd generation human embryonic stem cell line H9 cells were inoculated in the inner area of ​​the ring gasket with an inner diameter of 35mm, and the thickness of the gasket was 100, 500 and 1000 μm, respectively, and DMEM culture medium containing 10% FBS was added to culture overnight.

[0046] Add the agarose powder into the water, pressurize at 121°C for 20 minutes, and obtain a 3% (W / V, g / ml) agarose storage solution. Sodium alginate (molecular weight 430kDa, ratio of guluronic acid to mannuronic acid 1.5) powder was dissolved in physiological saline to obtain a 1.5% (W / V, g / ml) solution. Dilute the preheated 3% (W / V, g / ml) agarose storage solution and 1.5% (W / V, g / ml) sodium alginate solution with water to prepare agarose with a final concentration of 0.7 (W / V, g / ml), the final concentration of sodium alginate is 0.4% (W / V, g / ml) ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
thicknessaaaaaaaaaa
thicknessaaaaaaaaaa
molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention relates to a method for cell co-cultivation to induce stem cell directional differentiation in vitro. An annular spacer is installed at the bottom of the culture dish, stem cells are inoculated in the annular area, cultivated in an incubator, and then the agarose / Sodium alginate mixed solution is added to the annular area, after the solution is solidified, add CaCl 2 The solution is calcified, and chitosan solution is added to react to form a layer of agarose / sodium alginate / chitosan slab composite gel on the stem cells. Induced cells were seeded on the surface of the composite gel. The co-cultivation of induced cells and stem cells in the present invention can simulate the interaction between cells in vivo and between cells and soluble factors, and the polysaccharide-based agarose / sodium alginate / chitosan slab composite gel can simulate the extracellular matrix in vivo, Its stiffness and thickness can regulate the directional differentiation of stem cells, and at the same time realize the isolation and co-culture of stem cells and induced cells, making the differentiated cells easy to harvest, and will play an important role in the application of regenerative medicine.

Description

technical field [0001] The invention relates to the field of regenerative medicine, and relates to a method for co-cultivating cells to induce directional differentiation of stem cells in vitro. Background technique [0002] Stem cells are ideal seed cells for regenerative medicine due to their ability to self-renew and differentiate into various types of cells such as nerve, cardiac muscle, liver, islet, osteoblast, cartilage or fat. However, due to the uncontrollable spontaneous differentiation of stem cells, when stem cells are implanted into damaged tissues in the body, stem cells not only differentiate into the required cell types and participate in tissue repair, but also differentiate into other types of cells, and even have high tumorigenicity. sex. Pre-differentiation of stem cells in vitro, so that they can be pre-differentiated into the desired type of cells or their precursor cells before transplantation, can improve the efficiency of directed differentiation, r...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0735C12N5/0775C12N5/0797C12N5/0789C12N5/071C12N5/079C12N5/077
Inventor 马小军刘洋连建春孙广炜贺欣
Owner 源创吉因(重庆)细胞应用技术研究院有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap