A kind of preparation method and application of immobilized cell

A technology of cells and cell suspensions, applied in biochemical equipment and methods, methods based on microorganisms, immobilized on/in organic carriers, etc., can solve problems such as solid phase of living cells that have not been reported, and avoid long-term Injection, good biocompatibility, good stability

Active Publication Date: 2019-02-19
长春力太生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Since pectin can be degraded by the pectinase secreted by the bacteria in the human colon, pectin was used as the anion component, chitosan and calcium ions were used as the cation component, and the skeleton micropellets were prepared by the solid-phase method in liquid to Preparation of drug colon-localized release formulations has been extensively reported, but not for immobilization of living cells

Method used

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  • A kind of preparation method and application of immobilized cell
  • A kind of preparation method and application of immobilized cell
  • A kind of preparation method and application of immobilized cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Preparation of solid-phased cell pellets

[0031] Cell culture: CHO cells that can express the humanized monoclonal antibody Herceptin against human Her-2 antigen (see Chinese patent application CN201510368641.7 for the construction method) were inoculated in CD-Forti-CHO medium (Invitrogen, product number: A11483- 01), 37°C, 5% CO 2 , shake culture at 150rpm until the cell density reaches 5×106 / ml, centrifuge at room temperature at 1000rpm for 5 minutes, discard the supernatant, hang the cells with fresh medium, and adjust the cell density to 10 7 / ml, spare.

[0032] Preparation of calcium chloride cross-linking solution: calcium chloride 3% (g / v, concentration range: 0.5-10%), 0.5% (0.1-5%) chitosan, dissolved in 80ml CD-Forti-CHO medium , use 1% phosphoric acid to adjust the pH value to 5.5 (2.5-7.0), until the solution becomes completely clear, supplement the volume to 100ml, and filter to sterilize with a 0.22um filter.

[0033] The preparation steps...

Embodiment 2

[0039] Example 2. Fed-batch culture and engineering application of solid-phased cells

[0040] Suspend the prepared calcium pectin CHO cell microspheres in CD-Forti-CHO serum-free medium with a volume ratio of 1:2 (microspheres:medium), 37°C, 5% CO 2 Culture, shaking culture at 150rpm, on the 3rd, 5th, 7th, 9th, 11th and 13th day, respectively detect the concentration of glucose, various amino acids and lactic acid in the medium, add glucose and Feed C-ATG (Invitrogen A14420- 01) The concentration of feed is 4.5 g / L and 5-10%, and at the same time, adjust the pH value to 7.2 with 0.1 mol / L NaOH, continue to cultivate for 14 days, filter and recover the medium, and resuspend with fresh medium For cell microspheres, continue to culture the cell microspheres for 3-5 days. The concentrations of glucose, various amino acids and lactic acid in the medium were detected every day, and the medium was recovered by filtration on the fifth day, the microspheres were resuspended with fres...

Embodiment 3

[0042] Example 3. Detection of antibody concentration in culture medium:

[0043] 1. Coating: Dilute the Her-2 antigen to 10ug / ml with PBS, 100ul per well, and place at 4°C overnight; the next day, discard the solution in the well, and wash 3 times with washing buffer PBST, 3 minutes each time ;

[0044] 2. Sealing: add 200ul of freshly prepared 1% BSA to each well, place at 37°C for 1 hour; then, discard the solution in the well and wash with PBST 3 times, 3 minutes each time;

[0045] 3. Adding samples: Add 0.1ml of a certain dilution of the sample to be tested to the above-mentioned coated reaction well, incubate at 37°C for 1 hour, and then wash 3 times; the concentration (ng / ml) of the standard Herceptin is as follows:

[0046] 0, 0.488, 0.9765, 1.9531, 3.906, 7.8125, 15.625, 31.5, 62.5, 125;

[0047] 4. Add enzyme-labeled antibody: Add 0.1ml of freshly diluted HRP-labeled rabbit anti-human IgG to each reaction well; incubate at 37°C for 1 hour, then wash with PBST 3 ti...

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Abstract

The invention discloses a preparation method of immobilized cells. The method comprises the following steps: a pectin solution is added to a suspension containing to-be-immobilized cells, a calcium chloride and chitosan solution is added, and a pectin-containing cell microspherical suspension is obtained. According to the method, the cell activity can be maintained, nutrients of the cells are allowed to enter pellets, regular growth and division of the cells are kept, the pellets can be secreted by metabolites of the cells, and accordingly, related recombinant proteins are separated from a culture medium and purified. The invention further discloses applications of the preparation method to the immobilized cell engineering and drug preparation.

Description

technical field [0001] The invention relates to a solid phase cell and its application in the field of bioengineering. Background technique [0002] Immobilized Cells technology (Immobilized Cells) is a new technology developed in the 1960s. It has been widely used in the fields of chemical industry, energy, environmental protection and medicine. It refers to the use of various physical and chemical methods to locate free cells in In a specific space area, increase the concentration of cells or enzymes, and maintain biological activity and repeated use of technology. Compared with the traditional suspension biological treatment method, solid-phase cells have the advantages of increased bacterial density per unit volume, easy recovery of bacteria, and enhanced environmental tolerance, and solid-phase bacteria can increase the substrate or product to the cell membrane. Permeability and thermostability of enzymes. Solid-phase cells maintain the original state and natural envi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/10C12P21/08A61K39/395A61K9/00A61P35/00C12R1/91
Inventor 宋海鹏李敏王庆东
Owner 长春力太生物技术有限公司
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