Apparatus and method for realizing homogeneous growth of cell colony

A cell and totipotent stem cell technology, applied in tissue cell/virus culture devices, biochemical equipment and methods, methods of supporting/immobilizing microorganisms, etc., can solve uncontrollable spontaneous differentiation, affect embryonic stem cell proliferation and self-renewal, cannot Adjust the colony size and other issues to achieve a good sterile environment, reduce handling and damage, and facilitate the preparation process

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

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Problems solved by technology

The spontaneous differentiation of embryonic stem cells can be rescued by individually regulating the activation of Smad1, suggesting that the size of hESC colonies can independently regulate the differentiation and self-renewal of stem cells, but the colony size cannot be adjusted under classical culture conditions. Under the joint action of the microenvironment, there must be uncontrollable spontaneous differentiation, which affects the proliferation and self-renewal of embryonic stem cells

Method used

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  • Apparatus and method for realizing homogeneous growth of cell colony
  • Apparatus and method for realizing homogeneous growth of cell colony

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, the manufacture of cell colony in vitro culture device

[0058] 1. Preparation of Micropatterned Templates

[0059] The upper micropatterned template was prepared by laser cutting method. The specific operation is as follows: use a Rayjet laser engraving machine to cut a polymethyl methacrylate plate (Zunbao Technology) with a thickness of 0.5 cm to form an upper micro-patterned template. according to figure 1 As shown, the micro-pattern template is designed by the software AutoCAD: the template is circular, with a diameter of 22.1mm, and the center is a square with 8×8 cavities with a diameter of 300 μm, 500 μm, 1000 μm or 1500 μm as the micro-pattern. In the cell (such as totipotent stem cell) culture space, the distance between the centers of two adjacent cavities is 1500 μm. The main processing parameters of the laser engraving machine are: cutting energy 100%, cutting times 2, cutting line speed 10%.

[0060] The processed polymethyl methacrylate ...

Embodiment 2

[0068] Example 2, using the cell colony in vitro culture device of Example 1 to cultivate human embryonic stem cells

[0069] 1. Proliferation and culture of human embryonic stem cells

[0070] MEF culture medium: every 250ml MEF culture medium contains DMEM (11965092, Invitrogen) 225ml, fetal bovine serum (10099141, Invitrogen) 25ml, glutamate dipeptide (Glutamax I) (35050061, Invitrogen) 2.5ml, MEM non-essential amino acid (MEM NEAA) (11140050, Invitrogen) 2.5ml, penicillin / streptomycin (Pen / strep) (100X (15140122, Invitrogen) 2.5ml.

[0071] Human embryonic stem cell culture medium: Each 250ml culture medium contains 200ml of knock out DMEM (10829018, Invitrogen), 50ml of knockout serum replacer (10828028, Invitrogen), 2.5ml of glutamate dipeptide (Glutamax I) (35050061, Invitrogen), MEM Non-essential amino acid (MEM NEAA) (11140050, Invitrogen) 2.5ml, penicillin / streptomycin (Pen / strep) (15140122, Invitrogen) 2.5ml, human basic fibroblast growth factor (hbFGF) (25μg / m1) (...

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Abstract

The invention discloses an apparatus for growth of a cell colony. The apparatus provided by the invention comprises a micro graphical pattern template with a plurality of micro graphical cavities and a viscous hydrogel disposed under the micro graphical pattern template; the micro graphical pattern template and the viscous hydrogel are cross-linked and adhered into a whole; the shape and size of each micro graphical cavity determines a physical space of the growth of the cell colony. Materials used for the viscous hydrogel can be composed of a natural biomaterial and/or an artificially synthesized biomaterial capable of cross-linkedly forming the hydrogel and a cross-linking agent. A platform provided by the invention is simple in preparation process, is simple and practical for use matching with a culture dish. A plurality of co-culture environments are constructed by using the micro graphical technology, so that convenient and practical means are provided for construction and screening of high-throughput model; at the same time, independent control a plurality of factors such as the shape and size of the cell colony, mechanical strength and chemical components of an adhesive substrate, growth factors and chemical signals, co-culture cells and the like; and cell proliferation culture and induced differentiation culture can be carried out in-situ.

Description

technical field [0001] The invention relates to a cell colony growth device, in particular to a device for realizing the homogeneous growth of totipotent stem cell colonies, and also to a method for using the device to regulate the self-renewal and directional differentiation of totipotent stem cells. Background technique [0002] Totipotent stem cells (TSC) refer to stem cells that have unlimited differentiation potential and can differentiate into all tissues and organs, mainly including embryonic stem cells (embryonic stem cells, ESC) and induced pluripotent stem cells (Induced pluripotent stem cells, iPS) Two categories. [0003] Embryonic stem cells are isolated and cultured from early mammalian embryos, which are characterized by developmental totipotency, participate in the development of the entire organism, and constitute various tissues and organs of the human body. Embryonic stem cells are obtained from blastocysts at the gastrula stage, and further differentiate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M3/00
CPCC12N5/0606C12M23/10C12M23/34C12M25/14C12N2502/13C12N2533/30C12N2533/54C12N2535/00C12N2535/10
Inventor 杜亚楠姚睿王婧宇
Owner TSINGHUA UNIV
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