A device and method for achieving homogeneous growth of cell colonies

A cell and pluripotent stem cell technology, applied in tissue cell/virus culture devices, biochemical equipment and methods, methods of supporting/immobilizing microorganisms, etc., can solve the problem of unable to adjust the size of colonies, uncontrollable spontaneous differentiation, and affecting embryonic stem cells Proliferation and self-renewal issues, to achieve a good sterile environment, reduce handling and damage, and the effect of simple preparation process

Active Publication Date: 2016-01-06
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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  • A device and method for achieving homogeneous growth of cell colonies
  • A device and method for achieving homogeneous growth of cell colonies

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 fluid: every 250ml MEF culture fluid contains DMEM (11965092, Invitrogen) 225ml, fetal calf serum (10099141, Invitrogen) 25ml, glutalanine dipeptide (GlutamaxI) (35050061, Invitrogen) 2.5ml, MEM non-essential amino acid (MEMNEAA ) (11140050, Invitrogen) 2.5ml, penicillin / streptomycin (Pen / strep) (100X (15140122, Invitrogen) 2.5ml.

[0071] Human embryonic stem cell culture medium: every 250ml culture medium contains knockoutDMEM (10829018, Invitrogen) 200ml, Knockoutserumreplacer (10828028, Invitrogen) 50ml, glutalanine dipeptide (GlutamaxI) (35050061, Invitrogen) 2.5ml, MEM non-essential amino acid (MEMNEAA ) (11140050, Invitrogen) 2.5ml, penicillin / streptomycin (Pen / strep) (15140122, Invitrogen) 2.5ml, human basic fibroblast growth factor (hbFGF) (25μg / m1) (233-FB-025, R...

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Abstract

The invention discloses a cell colony growth device. The device provided by the invention includes a micro-patterned template with several micro-patterned cavities and a viscous hydrogel located under the micro-patterned template. The two are cross-linked and adhered as a whole; each of the micro-patterned cavities has Shape and size determine the physical space in which cell colonies grow. The material used as the viscous hydrogel consists of natural biological materials and / or synthetic biological materials that can be cross-linked to form a hydrogel, and a cross-linking agent. The platform provided by the invention has a simple preparation process and is easy to use with a culture dish; it uses micropatterning technology to construct a variety of cell co-culture environments, providing a convenient and practical means for high-throughput model construction and screening; at the same time, it can also be implemented Independent regulation of cell colony shape and size, mechanical strength and chemical composition of adhesion substrates, growth factors and chemical signals, co-cultured cells and other factors; as well as in-situ cell proliferation culture and induced differentiation culture.

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 (Inducedpluripotent stem cells, iPS). [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 differentiated to form multipo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12M3/00
CPCC12M23/10C12M25/14C12N2535/00C12M23/34C12N2502/13C12N2533/30C12N2533/54C12N2535/10C12N5/0606
Inventor 杜亚楠姚睿王婧宇
Owner TSINGHUA UNIV
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