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来自 University of California, Irvine 的课程

Basics of Extracellular Vesicles

104 个评分

University of California, Irvine

Basics of Extracellular Vesicles

104 个评分

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).

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

与讲师见面

Cecilia Lasser
Researcher
Krefting Research Centre, University of Gothenburg

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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