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Vector for cell biomass production and cell culture device comprising same

A cell culture and carrier technology, applied in biochemical cleaning devices, enzymology/microbiology devices, methods of supporting/immobilizing microorganisms, etc., can solve the needs of recovering cells that cannot meet the requirements of high cell density in 3D culture, difficult cells, fibroblast contamination

Pending Publication Date: 2022-07-22
CESCO BIOENGINEERING CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, collecting cells with the BioNOC II carrier has the following disadvantages: 1. The holes randomly stacked in the fibers will form a multi-layered structure of more than twenty layers, and the holes therein have a pore size ranging from a few microns to hundreds of microns, based on the above With a physical obstacle of more than 20 layers of fibers, the release of cells cannot be as direct as on a flat surface; 2. During the process of collecting cells, fibers are also easily released simultaneously and cause cell contamination; 3. The above The released fibers are of similar size to the cells and are therefore difficult to separate from the collected cells
[0019] Although a variety of carriers for cell culture are disclosed in the aforementioned patents, none of these carriers can meet the requirements of high cell density in 3D culture, easy cell recovery, and no release of additional particles when collecting cells

Method used

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  • Vector for cell biomass production and cell culture device comprising same
  • Vector for cell biomass production and cell culture device comprising same
  • Vector for cell biomass production and cell culture device comprising same

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0085] Example 1: Material Preparation

[0086] This example is the growth of cells on the support structures provided by the present invention.

[0087] Materials and methods: Prepare a polypropylene (PP) mesh layer with a mesh size of 3 mm × 3 mm, and a polyethylene terephthalate (PET) mesh layer with a mesh size of 200 μm × 200 μm. The size of an entire mesh layer is 43 cm (length) x 3 cm (width). Two layers of PET mesh layer are arranged on one layer of PP mesh layer, then the periphery is heat-sealed by hot pressing, and then this structure is subjected to surface hydrophilic treatment, and then it is curled into a column with a diameter of about 4cm and a height of 3cm Body type carrier 200. The column carrier 200 was placed in a BelloCell culture flask (culture chamber 400) (CESCO Bioengineering Co., Ltd.), and sterilized using gamma rays in a dose range of 25 kGy to 35 kGy, such as Figure 11 shown. Curl the carrier structure into a columnar microstructure such as ...

example 2

[0088] Example 2: Cell Culture

[0089] Use about 3 x 10 mesenchymal stem cells (MSCs) isolated from human Wharton's Jelly 7 Quantity is in the substratum of 100ml, and it is seeded (seed) in the above-mentioned BelloCell bottle with carrier structure of the present invention 3 hours; After confirming that planting rate (seeding efficiency) exceeds 90%, add the fresh substratum of 400ml The total medium was made to be 500 ml; the above BelloCell bottle was then mounted to a BelloStage-3000 machine (CESCO Bioengineering Co., Ltd.), and the following parameters were used: Ascent speed: 1 mm / s; T_H: 10 seconds; descent speed: 1 mm / s ;B_H: 30 seconds at 37°C and 5% CO 2 Cell growth was checked on the 4th and 8th day of culture, and after confirming that the cell growth had reached confluency, the cell harvest (harvest) was performed on the 8th day.

[0090] The type of the carrier protected by the present invention can also be a stack body formed by random stacking of a pluralit...

example 3

[0093] Example 3: Cell / tissue release

[0094]After cells grown to confluence, the growth surface was rinsed 3 times with D-PBS, and then trypsin / EDTA was submerged for 5 minutes; the trypsin / EDTA solution was removed before cells were detached from the growth surface; tap and Shake the BelloCell bottle and hit the growth surface against the walls of the BelloCell bottle to force the cells to detach, then rinse with D-PBS, repeat the tap / shake and rinse 3 times; then collect all product, centrifuge, resuspend (re-suspend), and check the cell number and viability. Results are shown in Figure 17 . According to the data, the density of cells grown in each BelloCell flask using the carrier structure of the present invention can reach 9×10 7 . Here, a carrier with two PET mesh layers and one PP mesh layer (group 1) and a carrier with three PET mesh layers and one PP mesh layer (group 2) are used, both of which are in diameter 4cm and height 3cm. Cell recovery was calculated ...

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Abstract

The present invention provides a 3D porous growth surface comprising a fabric having a plurality of reticular layers that can form a cylindrical fixed bed by crimping each reticular layer, or a fixed bed of other shape by stacking or filling the multilayer reticular structure. The invention also provides a method for enhancing cell growth by maintaining the aperture and structure of the reticular layer. The invention also provides a method for enhancing the cell recovery rate, which uses the reticular layer, has fewer obstacles compared with a carrier made of non-woven fabrics or other porous materials, and can enhance the cell recovery rate. The invention further provides a method for reducing particle generation in the cell collection process, the periphery of the multi-layer net-shaped structure is subjected to edge sealing so as to prevent fibers or particles from being released, and the diameter of the yarn on the net-shaped layer is larger than the wall size of the cells so as to facilitate filtration and separation. The growth surface provided by the invention can be easily made into a fixed bed by simply curling a plurality of reticular layers, so that not only can the manufacturing cost be reduced, but also the large-scale manufacturing of a carrier of a fixed bed bioreactor is facilitated.

Description

[0001] This application claims priority to US Provisional Patent Application No. 62 / 953,575, filed 2019.12.25, entitled "A Cell Culture Substrate for Mass Production of Biomass," the disclosure of which is incorporated herein by reference. technical field [0002] The invention relates to the field of cell culture substrates, in particular to a cell culture substrate for eukaryotic cell biomass production. Background technique [0003] With the rapid development of biotechnology, cell culture techniques for prokaryotic or eukaryotic cells have become increasingly important. In general, eukaryotic cells grow slowly and are susceptible to damage from shear stress and contamination, whereas most eukaryotic cells are anchorage-dependent, requiring attachment to a growth surface for growth. In order to cultivate these adherent eukaryotic cells, a variety of culture carriers with growth surfaces have been developed. Culture carriers, porous substrates made of polyurethane or poly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/00C12N5/00
CPCC12N5/0062C12N5/0068C12M25/14C12M25/02
Inventor 张景明
Owner CESCO BIOENGINEERING CO LTD