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Three dimensional cell culture construct and apparatus for its making

A three-dimensional cell and cell culture technology, applied in the field of forming and manufacturing the insert, porous three-dimensional cell culture insert, and culturing mammalian cells, can solve the problem of difficult control of fiber size, difficult determination of matrix pore size and shape, and difficult operation And other issues

Inactive Publication Date: 2008-08-20
刘青
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantages of these fibrous polystyrene matrices are: the size of the fibers is difficult to control; the pore size and shape of the matrix are not easily determined; soft in the state, making further manipulation of the matrix difficult without deformation

Method used

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  • Three dimensional cell culture construct and apparatus for its making
  • Three dimensional cell culture construct and apparatus for its making
  • Three dimensional cell culture construct and apparatus for its making

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0149] Example 1: Method of Manufacturing Cell Culture Inserts

[0150] Fabricate cell culture inserts using polystyrene material. The components of the cell culture insert shown in Figure 5 were used for assembly in the cell culture insert. These parts are injection molded according to design. After the parts are made, the first layer is first placed into the assembly guide, and then the second, third and fourth layer of parts are sequentially placed in the guide. Thus, the total number of parts is four. These 4 parts are then joined together using a polystyrene fiber connector as shown in Figure 7. The ends of the connector are further secured by knotting or deforming the ends so that they cannot escape from the holes of the insert. After assembly, the cell culture inserts were plasma treated in an argon atmosphere using a Polaron PT7300 RF plasma barrel etcher (Quorum Technology, East Sussex, UK). Its RF power, pressure and processing time are fixed at 296 watts, 1×1...

Embodiment 2

[0152] Example 2: Using Cell Culture Inserts to Cultivate Cells

[0153] The present invention also provides a method for culturing living cells using the cell culture insert in a tissue culture polystyrene plate. The cell culture inserts used for this study had dimensions of 10 mm wide by 10 mm long by 0.3 mm thick, with square pores of 200 μm and fiber diameters of 400 μm. Inoculate smooth muscle cells using the static inoculation method: pipette 500 μl of smooth muscle cell suspension (1×10 5 cells / ml) from the upper surface of the insert, allowing the cells to attach for 2 hours at 37°C before perfusing more cell culture medium. After seeding the cells, the cell culture inserts were placed in multi-well plates containing cell culture medium and incubated at 37°C in a 90% humidity chamber containing 5-10% CO. 2 cultured in an incubator in an air atmosphere. The growth medium consisted of Dulbecco's Modified Eagle's Medium (DMEM) containing 5% (v / v) fetal bovine serum. ...

Embodiment 3

[0156] Example 3: Cultivating cells in bioreactors using cell culture inserts

[0157] The present invention also provides a method for culturing living cells in a bioreactor using the cell culture insert. The cell culture inserts used here are disc-shaped (10 mm diameter disc, 0.8 mm thick, 80% porosity, and 200 μm fiber diameter) and fit the bioreactor as described.

[0158] Rat bone marrow stromal cells (MSCs) were first statically seeded on the cell culture insert. 500 μL of MSC suspension containing 250,000 rat MSCs was pipetted from the upper surface of the cell culture insert, cells were allowed to attach at 37° C. for 2 hours, and then flushed with cell culture medium. After seeding the cells, the seeded cell culture inserts were maintained in flow perfusion cell culture vessels. The cell culture inserts seeded with cells were submerged in a complete osteodifferentiation medium and incubated at 37[deg.]C, 90% humidity in an air atmosphere containing 5-10% carbon di...

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Abstract

The present invention relates to a three dimensional construct formed from non-biodegradable and non-cytotoxic polymers that provide an internal and external space for living cells to attach, proliferate and differentiate. The construct is composed of polymer struts and / or fibers which are joined together in a designed 3 dimensional pattern. The 3 dimensional cell culture construct (cell culture insert) is intended to be used together with cell / tissue culture plate, tissue culture flask, bioreactor and the like under normal cell culture conditions. The invention further provides methods of making the 3 dimensional cell culture construct. Finally, the invention provides kits comprising one or more 3 dimensional porous cell culture construct in a package together with other cell culture supplies, such as tissue culture plate and flasks.

Description

technical field [0001] The invention relates to a porous three-dimensional cell culture insert for living cell attachment, proliferation and differentiation. The invention also relates to methods of forming and manufacturing the inserts, and methods of using the inserts to culture mammalian cells in cell culture plates (e.g., plastic culture dishes and multiwell tissue / cell culture plates), cell culture flasks, and bioreactors method. The invention belongs to the field of cell and tissue culture. Background technique [0002] In the prior art, the main cell culture methods routinely used include the following two types: [0003] 1) Culture cells in a monolayer two-dimensional manner [0004] Cell culture is a very useful and widely used technique in the fields of drug discovery, cell biology, toxicology, bioengineering, and tissue engineering. Routine cell culture is carried out in cell culture plates such as 2, 4, 6, 24, 96 well cell culture plates made of non-degradabl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M3/00C12M1/26B29C69/00B29C71/04
CPCC12M25/14
Inventor 刘青
Owner 刘青
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