Cell culture container with protein immobilized modifying function

A cell culture and protein technology, which is applied in the field of protein immobilized and modified cell culture containers, can solve the problems of cumbersome experimental operations, high economic costs, and affecting the effect of cell culture differentiation, so as to simplify experimental operations and avoid instability , the effect of reducing uncontrollable factors

Pending Publication Date: 2018-07-24
上海晶诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] When a certain protein needs to be added during the cell culture process, researchers often need to express and purify the protein, and the purified protein needs to be added every time the medium is changed, which is relatively expensive economically and experimentally. more cumbersome
In addition, due to the stability of the product after protein expression and purification, protein packaging and storage time, etc., it will affect the effect of cell culture and differentiation.

Method used

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  • Cell culture container with protein immobilized modifying function
  • Cell culture container with protein immobilized modifying function
  • Cell culture container with protein immobilized modifying function

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of pET28a-hGMCSF-IL4 and pET28a-hIL4-GMCSF plasmids

[0040] The pET28a-hGMCSF-IL4 plasmid and the pET28a-hIL4-GMCSF plasmid were constructed by the seamless cloning method, and relevant primers were designed according to the requirements of the seamless cloning experiment method to contain the plasmids pET50b-hGMCSF and pET50b-hIL4 containing the GMCSF and IL4 genes respectively (Constructed by Shanghai Jingnuo Biotechnology Co., Ltd.) as a DNA template for seamless cloning after PCR and gel recovery. The nucleotide sequence of GMCSF is shown in SEQ ID NO: 7, and its amino acid sequence is shown in SEQ ID NO: 1; The nucleotide sequence of IL4 is shown in SEQ ID NO: 8, and its amino acid sequence is shown in SEQ ID NO: 2.

[0041] 1. Amplify hGMCSF-linker, linker-IL4, hIL4-linker and linker-GMCSF fragments respectively.

[0042] (1) PCR amplification of hGMCSF-lingker and linker-GMCSF fragments Use pET50b-hGMCSF as a DNA template, and the primer...

Embodiment 2

[0079] Example 2 The cell culture container whose inner wall is glass

[0080] 1. Expression and purification of hGMCSF-IL4 and hIL4-GMCSF fusion proteins

[0081] The positive clones with correct sequencing in Example 1 were selected and cultured overnight at 37° C. and 200 rpm with 5 ml of LB containing Kana antibiotics. On the next day, transfer the overnight culture solution to 50 mL of liquid LB containing Cannabidiol at a ratio of 1:100, culture at 37°C, 200 rpm, for 3 hours (cultivate in large quantities until OD600=0.6 to 0.8 or so). Then transfer to 1L LB medium containing kana antibiotics, culture at 37°C, 200rpm for about 5 to 6 hours, then cool down to 16°C for culture, add 0.5mmol / L final concentration of IPTG to induce expression, and culture overnight. Centrifuge at 4000rpm for 10min to collect overnight expressed bacteria, resuspend the bacteria in 40ml of equilibrium solution (20mM pH8.0Tris-Cl, 0.5MNaCl, 40mM imidazole), sonicate on ice at 40% power, sonicat...

Embodiment 3

[0087] Example 3 Culture of differentiation of monocytes into dendritic cells

[0088] After the fusion protein is immobilized on the glass cell culture dish, it can be directly used for cell culture.

[0089] (1) collecting human peripheral blood mononuclear cells with a blood cell separator;

[0090] (2) further purify mononuclear cells (PBMC) by density gradient centrifugation of lymphocyte separation medium;

[0091] (3) Wash twice with serum-free culture medium to obtain PBMCs with a purity above 90%.

[0092] The prepared peripheral blood mononuclear cells were cultured with the glass cell culture dish prepared in Example 2 and immobilized with the fusion protein, the cell culture medium was 10% FCS+serum-free medium, and the culture conditions of the cell culture box were 37°C, The concentration of carbon dioxide was 5%, the cell culture medium was changed every other day, and cultured for 6-7 days, the formation of pseudopods could be observed in the cells under the ...

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Abstract

The invention discloses a cell culture container with a protein directional immobilized modifying function. The cell culture container of which the inner wall is enveloped with protein is built for fusion protein of GMCSF (granulocyte-macrophage colony-stimulating factor) and IL4 (interleukin 4). A preparation method of the cell culture container comprises the following steps of adding a His tag for the protein; performing silanization treatment on the inner wall of the cell culture container; performing AB-NTA (agrose bead-nitrilotriacetic acid) hatching modifying on the inner wall of the cell culture container; adding an NiCl2 (nickel chloride) solution to couple AB-NTA and Ni (nickel); adding the protein containing the His tag, performing affinity treatment together with nickel ions inthe AB-NTA, and immobilizing to the inner wall of the cell culture container. The cell culture container has the advantages that in the cell culture process, the enveloped protein is not lost when a culture solution is replaced, so that the re-adding of the protein for multiple times is not needed, the consistency of the protein quality is ensured, the number of uncontrollable factors is reduced in the whole culture process, and the cell culture and differentiation effects are ensured; the reversible effect is realized, and the cell culture container can be utilized for multiple times.

Description

technical field [0001] The invention relates to the technical field of cell culture, and more specifically, to a protein-immobilized modified cell culture vessel. Background technique [0002] Cell culture, also known as cell cloning technology, is an essential process in cell biology research and the production of related biological products. Through different treatments such as gene editing, drug treatment, and induced differentiation of cells, cell culture can achieve the purposes of cell life process research, disease and drug mechanism, and directed differentiation into other cells. Large-scale cell culture can also be used to prepare cells. Factors, antibodies and other biological protein drugs. Cell culture is an essential technology in the biological field. [0003] When a certain protein needs to be added during the cell culture process, researchers often need to express and purify the protein, and the purified protein needs to be added every time the medium is ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12M3/00C12M1/22
CPCC12M23/10C12N5/0639C07K14/535C07K14/5406C07K2319/21C12N2506/115C12N2501/22C12N2501/2304
Inventor 周亚凤王绪德申兆兴刘雪宾
Owner 上海晶诺生物科技有限公司
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