Method for efficiently and sequentially inducing macrophages to be polarized in vitro

A macrophage and sequential technology, applied in the field of efficient sequential induction of macrophage polarization in vitro, can solve the problems of insignificant differences in protein expression, uncertain macrophage status, and inability to accurately differentiate THP-1 cells. Achieve accurate research and eliminate the effect of macrophage polarization

Inactive Publication Date: 2021-01-12
AFFILIATED HOSPITAL OF ZUNYI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows scientists to quickly stimulate large numbers of tiny red blood cells called Theonella monocytes through an enzyme process without affecting their ability to divide them correctly. By doing this they could help researchers better identify specific subpopulations of white blood cells like neutrocyte intermediating zone plasma cells. These isolated populations were then used to investigate how these special properties might change during inflammations caused by various diseases including autoimmune disorders.

Problems solved by technology

This patented technical problem addressed in this patents relates to improving the efficiency at producing large amounts of certain macromolecular substances called extracelluarbodies (Ebs). These EBs play key roles in both innate and adaptive immunocompetentiating processes involving inflammations, cancer development, autoimmune response, and regulation of various biological reactions like wound healing and tissue injury. Current techniques involve modifying these materials with chemical agents or enhancer compounds, but their effects on production remain uncertain due to lack of knowledge regarding how much each material contributes towards its own activity.

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  • Method for efficiently and sequentially inducing macrophages to be polarized in vitro
  • Method for efficiently and sequentially inducing macrophages to be polarized in vitro
  • Method for efficiently and sequentially inducing macrophages to be polarized in vitro

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Experimental program
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Embodiment 1

[0030] Embodiment 1: the sequential induction method of the present invention

[0031] see figure 1 , the embodiment of the present invention is a method for efficiently and sequentially inducing the polarization of macrophages in vitro, comprising the following steps:

[0032] S1 expansion and proliferation culture: THP-1 cells are expanded and proliferated. Specifically, THP-1 cells were routinely recovered and subcultured using RPMI1640 medium containing 10% FBS and 0.05 nM β-mercaptoethanol to ensure the activity and high proliferation of THP-1 cells in an undifferentiated state. In the expansion and proliferation culture stage of THP-1 cells, avoid using human HPL for cell culture, if human HPL is used, it will cause abnormal adhesion of THP-1 cells.

[0033] In this example, THP-1 cells were purchased from the American Type Culture Collection (ATCC) Corporation, and cryopreserved in liquid nitrogen until use. RPMI 1640 medium containing 10% heat-inactivated FBS, 1% pe...

Embodiment 2

[0043] Embodiment 2: analysis verification

[0044] 1. Materials and methods

[0045] 1. THP-1 cell culture

[0046] THP-1 cells were purchased from the American Type Culture Collection (ATCC) and kept cryopreserved in liquid nitrogen until use. RPMI 1640 medium containing 10% heat-inactivated FBS, 1% penicillin / streptomycin, 1% amphotericin B and 0.05 nM 2-mercaptoethanol was used to expand THP-1 cells. Place cells at 75cm 2 flasks and cultured in 5% CO 2 , 37 ℃ humid incubator. THP-1 cells were centrifuged at 1400 rpm for 5 min at 8°C for cell passage. THP-1 cells at passage 16 were used for macrophage induction experiments.

[0047] 2. Comparison of PMA concentration and subsequent cytokine induction time during THP-1 macrophage differentiation

[0048] THP-1 cells were induced to differentiate into M0 macrophages using PMA. will be 6×10 5 THP-1 cells were resuspended in 10% FBS RPMI1640 medium containing different concentrations of PMA (5nM, 25nM, 150nM), and then...

Embodiment 3

[0068] Embodiment 3: application experiment

[0069] Experiment of co-cultivating periosteal mesenchymal stem cells and M1 macrophages: Mesenchymal stem cells have a very powerful immunoregulatory effect and can strongly inhibit the inflammatory response. The applicant found that when the jaw bone periosteum stem cells were co-cultured with the above-mentioned THP-1 M1 macrophages, the jaw bone periosteum mesenchymal stem cells showed reduced expression of CD80 and CD86 (inflammation-related proteins) in M1 type macrophages ( Image 6 ), the expression changes of related proteins indicate that periosteal mesenchymal stem cells can reduce the polarization of M0 to M1 macrophages, and have a tendency to polarize to M2 macrophages. This shows that the macrophages obtained by the above scheme can be effectively used in the study of the secretion regulation of mesenchymal stem cells in the co-culture system; similarly, they can also be used for M0 / M1 / M2 macrophages to regulate the ...

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Abstract

The invention discloses a method for efficiently and sequentially inducing macrophages to be polarized in vitro. The method comprises the following steps of: S1, carrying out multiplication culture onTHP-1 cells by using an RPMI1640 culture medium containing 10% of FBS and beta-mercaptoethanol; S2, carrying out pretreatment before induction on the THP-1 cells by using PBS containing HPL and a culture medium; S3, in an RPMI1640 culture medium which contains 5% of HPL and does not contain the beta-mercaptoethanol, slowly inducing the THP-1 cells by adopting low-concentration PMA so as to enablethe THP1 cells to be differentiated into M0 macrophages in an adherence mode, and then removing the PMA; and S4, in an RPMI1640 culture medium which contains 5% of HPL and does not contain the beta-mercaptoethanol, inducing M0 macrophages for more than 72 hours by using LPS/IFN-gamma, so that the M0 macrophages are polarized into M1; or inducing the M0 macrophages for more than 72 hours by usingIL-4/IL-13, so that the M0 macrophages are polarized into M2 macrophages. Compared with the prior art, the method disclosed by the invention has the advantages that firstly, the THP-1 cells are slowlyinduced by adopting the low-concentration PMA, so that the immatureness state of the M0 type macrophages differentiated from the THP1 cells can be guaranteed; and then the optimal induced polarization time of the subsequent M1/M2 type macrophages is over 72 hours, so that the polarization state of the M1/M2 type macrophages is guaranteed. Meanwhile, the differentiation process is carried out in an environment without animal-derived components, so that the polarization efficiency of the macrophages can be effectively ensured.

Description

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Claims

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Application Information

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Owner AFFILIATED HOSPITAL OF ZUNYI UNIV
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