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Breast Milk

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Basics of Extracellular Vesicles

加州大学尔湾分校

4.8（166 个评分） | 9.4K 名学生已注册

This course aims to provide the basic knowledge about extracellular vesicles (EV) a generic term including exosomes, microvesicles, microparticles, ectosomes, oncosomes, prostasomes, and many others. It covers areas such as EV history, nomenclature, biogenesis, EV cargo as well as the release and uptake mechanisms, collection and processing prior to isolation, different isolation methods, characterization and quantification techniques. This course is divided into five modules. Module 1 is an introduction to the field and will cover the nomenclature and the history of EVs. Module 2 will focus on the biogenesis, release and uptake mechanisms of EVs as well as the different EV cargos (RNA, protein, lipids). In Module 3, we will focus on the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Module 4 and 5 will present different isolation methods and characterization/quantification techniques, respectively. Here differential ultracentrifugation, size exclusion chromatography, density gradient, kit based precipitation, electron microscopy (EM), cryo-TEM, flow cytometry, atomic-force microscopy and nanoparticle tracking analysis will be presented. The recommended prerequisites are college-freshman-level biology and biochemistry. After a completed course you should be able to: + Discuss the nomenclature and subgroups of extracellular vesicles. + Describe the RNA, protein and lipid content of extracellular vesicles. + Describe the basic concepts about the most common isolation and characterization techniques and how these techniques are used in the EV field. + State the benefits and limitations of the most common isolation and characterization techniques for extracellular vesicles. + Explain the considerations that are important during the collection and isolation of EVs from different body fluid. + Describe the release and uptake mechanisms of extracellular vesicles All lectures are given in English. Each of the five modules will be followed by an exam. All exams will be in the format of multiple choice questions. The course is organized in collaboration between the International Society for Extracellular Vesicles (ISEV), University of California Irvine (USA), University of Gothenburg (Sweden) and Pohang University of Science and Technology (South Korea).

查看授课大纲

您将学习的技能

Biology, Sample Preparation, Sample Collection, Cell Biology

审阅

4.8（166 个评分）

5 stars
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FI

Jun 30, 2019

I loved this course! Very complete, well balanced and dynamic lectures. I learned a lot about EVs and can't wait to start working with them!

JK

Apr 24, 2017

I enjoyed how the course touched on all important aspects of EVs in depth providing relevant and supportive literature

从本节课中

Collection and Processing of Cell Culture Media and Body Fluids Prior to Isolation of Extracellular Vesicles

In this module, we will focus on the collection and processing of cell culture media and body fluids prior to isolation of extracellular vesicles. We will discuss some of the considerations and guidelines that are important during the collection of staring material and isolation of extracellular vesicles. This module will help you with the different choices, such as anticoagulants, collection time points and protein inhibitors, which are important for the outcome when working with a particular body fluid compared to conditional media or other body fluids. This module will also illustrate some example of studies on extracellular vesicles from body fluid such as blood, urine, breast milk and cerebrospinal fluid and why it is of interest to analyse extracellular vesicles from these body fluids.

Cell Culture Media13:18

Blood Plasma and Serum15:54

Breast Milk8:55

Cerebrospinal Fluid15:49

教学方

Cecilia Lasser
Researcher

脚本

Hello, my name is Esther Nolte-'t Hoen. I am Principal Investigator at Utrecht University, the Netherlands, and this course is about how to collect and process human breast milk in order to isolate extracellular vesicles from these body fluids. I will start with the short introduction on the importance of breast milk for the health of newborns and the potential role of breast milk extracellular vesicles. The next part of the course will be on effects of breast milk storage on extracellular vesicle isolation, the vesicle isolation itself and characterization of breast milk EV. Breast milk is highly important for the growth and development of neonates. Besides the fact that breast milk has a high nutritional value, breast milk also provides the neonate with factors that support, function and the development of the babies immune system. It is known that passive immunity is transferred via breast milk from mother to child. This means that breast milk contains ready to use immunological components like immunoglobulins, antimicrobials, and cytokines, and chemokines that are immediately active after ingestion of the milk. However, breast milk is also thought to be involved in instruction of the adaptive immune response of the neonate, to help it discriminate between material to which it should respond might pathogens and material to which tolerance should be induced, like food antigens and cell antigens. It is still largely unclear which breast milk components can instruct the infant's adaptive immune system, but it may depend on transfer of antigen across the memory epithelium and the presence of immune modulatory factors. A few years ago, extracellular vesicles or EV were found to be present in breast milk. This little bilayer and closed nanosites vesicles can contain multiple targeting and sickling components and can therefore carry both antigens and immune modulatory factors. In addition, there is evidence that milk EV has immune modulatory capacity. Milk EV may therefore play a role in instructing the infants immune system via the gastrointestinal tract. So how can breast milk be isolated and stored for isolation effects of extracellular vesicles. Breast milk can be expressed with a manual or electric breast pump. The use of breast milk for scientific purposes needs approval from the institute's ethics committee. Our laboratory has investigated if and how milk samples can be stored prior to extracellular vesicle isolation. To understand what happens when breast milk is stored at low temperature, we need to have a closer look at the composition of milk. Milk contains a large variety of different lipid-enclosed structures. It contains different cell types like macrophages, neutrophils, T cells, dendritic cells, and epithelial cells. But besides cells milk contains a large number of milk fat globules. These are the fat storage units of milk containing large amounts of triglycerides. They are formed by the breast epithelial cells as plasma membrane enclosed lipid droplets and are therefore lipid trilayer enclosed. They very largely incise. The extracellular vesicles are a third lipid enclosed entity present in breast milk. They may be of various cellular origins, are lipid bilayer enclosed, and the majority of them is 50 to 200 nanometers in size. If you store breast milk at low temperatures as is for example done with milk in milk banks, the problem arises that cells present in breast milk die as you can see here in this graph. We found that this does not only happen at below 0 temperatures, but also at 4 degrees Celsius. The cell death leads to cellular fragmentation, and cell death induced vesicle formation which contaminates the naturally formed extracellular vesicle population present in milk. The next part of the talk describes how extracellular vesicles can be isolated from fresh breast milk. The first steps in such an extracellular vesicle isolation protocol should be aimed at removing unwanted components, such as cells and milk fat globules. In our published protocol, fresh breast milk is processed within 20 minutes after expression by centrifugation steps at increasing g-force cells are removed as well as most of the milk fat globules which form a cream layer. The extracellular vesicles are left in the so called milk supernatants. After two centrifugation steps to remove cells and pellet, the cell and cream-free supernatants can either be used for immediate vesicle isolation. Or stored at minus 80 degrees Celsius for extracellular vesicle isolation at a later time point. When performing high-speed ultracentrifugation of the milk supernatant in order to directly pellet extracellular vesicles, you run into the risk that a non-resuspendable pellet is formed. This is most likely due to the fact that also large globular proteins, like casein, spin down at high g-forces. It is very hard to efficiently recover extracellular vesicles from such a solid pellet. As an alternative extracellular vesicles can be spun by ultracentrifugation from the cell and cream-free supernatant into a sucrose density gradient. In this gradient extracellular vesicles are separated from other components based on buoyant density. Fractions of the spun gradient can be analyzed for proteins, lipids, or RNA content. When using western blotting to compare the protein content of the extracellular vesicles containing fractions with higher and lower density fractions, you can find enrichment for proteins characteristic to extracellular vesicle populations. So just the tetra-spanning CD9 and CD63. Moreover, new relevant proteins such as HLA class II are present in these fractions. When using cryo Electromicroscopy to study the breast milk extracellular vesicle population in the near native state. We observed that extracellular vesicles in milk were diverse in size. Although, the majority of vesicles were smaller than 200 nanometers. And we found at the subset of vesicles exposed most of it revealed searing on the outside, which could be visualized using gold labels annexin V. To summarize this course, when you start doing research on breast milk extracellular vesicles you should consider the following points. There is a large variation in breast milk composition of different mothers. Dark adverse first effects of cold storage of complete breast milk like milk cell death and cell fragments contaminate extracellular vesicle populations. Furthermore, centrifugation of large, globular milk proteins, like casein can entrap extracellular vesicles. Recommendations for the research on breast milk extracellular vesicles are therefore to take into account that parity, time after delivery, and time of day may affect the number and type of milk extracellular vesicles. Other recommendations are to store breast milk only after removable of cells and cream and to further purify milk extracellular vesicles by top-down centrifugation of milk supernatant into a density gradient. Thank you very much for listening.

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