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Method for obtaining extracellular vesicles through mouse lung microvascular endothelial cell immortalization

A technology of endothelial cells and microvessels, applied in the field of cell culture, can solve problems such as research bias and large differences in vesicle characteristics, and achieve the effects of reducing efficiency and cost, improving loss, and improving cell aging problems

Pending Publication Date: 2022-03-08
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, vesicles are highly heterogeneous, with widely varying vesicle properties from different sources
Therefore, using vesicles of other endothelial cell origin to explore the role of PMECs-EVs in lung disease may lead to research bias

Method used

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  • Method for obtaining extracellular vesicles through mouse lung microvascular endothelial cell immortalization
  • Method for obtaining extracellular vesicles through mouse lung microvascular endothelial cell immortalization
  • Method for obtaining extracellular vesicles through mouse lung microvascular endothelial cell immortalization

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Embodiment Construction

[0057] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

[0058] S1. Isolation of primary mouse pulmonary microvascular endothelial cells

[0059] Preparation of S1.1 Mouse Lung Marginal Single Cell Suspension

[0060] After sacrificing the mice, take out both lungs from a sterile petri dish, peel off the visceral pleura, cut off the margins of the lungs on both sides, place them in DMEM-F12 basal medium, rinse 3 times, and transfer them into a 5ml centrifuge tube (Eppendorf tube ), add an appropriate amount of type I co...

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Abstract

The invention discloses a method for obtaining extracellular vesicles through mouse lung microvascular endothelial cell immortalization, which comprises the following steps: S1, mouse lung microvascular endothelial primary cell separation: separating newborn mouse marginal lung tissues, cutting the tissues into small blocks by ophthalmic scissors, preparing a single-cell suspension, removing CD45 +, CD90.2 + and CD326 + cells through magnetic bead negative selection, and preparing the single-cell suspension; screening endothelial cell colonies for in-vitro proliferation; s2, endothelial cell immortalization: transfecting six lentiviruses, screening, and selecting an immortalized cell strain according to the growth rate and form of the cell; s3, extracting vesicles: extracting the vesicles by adopting an ultrasonic centrifugation method. According to the invention, through magnetic bead negative selection and circling hand sections, the damage of traditional magnetic bead positive selection to extract cells is avoided, and the loss of endothelial cells is greatly improved; according to the method, the lentivirus is transfected to the primary cell to construct the immortalized cell, the immortalized cell secretes the vesicles, the characteristics of the vesicles secreted by the primary cell are reserved, and the extraction cost of the vesicles is greatly reduced.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for obtaining extracellular vesicles by immortalizing mouse lung microvascular endothelial cells. Background technique [0002] Pulmonary microvascular endothelial cells (PMECs) are an important component of the alveolar-capillary barrier and participate in gas exchange. They play important roles in lung homeostasis and inflammatory responses. Given the important role of PMECs, culturing PMECs in vitro is crucial for elucidating the molecular and cellular mechanisms of lung diseases. The study of mouse pulmonary microvascular endothelial cells (pMPMECs) is limited by cell culture. The most common methods for isolating pMPMECs that have been published so far include enzymatic hydrolysis of lung tissue and immunobinding of pMPMECs to CD31 or CD102 magnetic beads. However, positive magnetic bead selection of pMPMECs leads to early senescence of cells with low prolif...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/071C12N15/867
CPCC12N5/069C12N15/86C12N2510/04C12N2509/00C12N2740/15043
Inventor 邱海波刘旭夏飞萍郭凤梅刘玲
Owner SOUTHEAST UNIV