3D In Vitro Cell Cultures

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Из курса от партнера Johns Hopkins University

Toxicology 21: Scientific Applications

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Johns Hopkins University

Toxicology 21: Scientific Applications

14 оценки

This course familiarizes students with the novel concepts being used to revamp regulatory toxicology in response to a breakthrough National Research Council Report “Toxicity Testing in the 21st Century: A Vision and a Strategy.” We present the latest developments in the field of toxicology—the shift from animal testing toward human relevant, high content, high-throughput integrative testing strategies. Active programs from EPA, NIH, and the global scientific community illustrate the dynamics of safety sciences.

Из урока

Module 4

The first lesson in this module will explain the concept of read-across, why it is important for regulatory risk assessment, and how it can be implemented by industry in response to REACH legislation. The organotypic cultures lesson will teach you about existing in vitro cell-based models starting from simple monolayer cultures of cell lines and ending with complex combined organ-on-a-chip and human-on-a-chip approaches.

Paradigm Shift in Toxicology7:50

2D In Vitro Cell Culture8:53

3D In Vitro Cell Cultures8:27

Organ-on-a-Chip11:40

Познакомьтесь с преподавателями

Lena Smirnova
Research Associate
Center for Alternatives to Animal Testing
Thomas Hartung
Professor
Environmental Health and Engineering

This section is talking about three-dimensional in vitro cell cultures.

There are several different techniques used to generate three-dimensional in

vitro cell cultures.

Tissue slices, what you can see here is a hippocampus tissue slide.

You take the organ and cut it in thin slices.

They can be maintained in cultures for at least 24 hours.

They are spontaneous aggregating cultures, for example,

the picture here showing embryonic bodies.

The cells are spontaneously aggregating together and

form this three-dimensional structure.

We have the hanging drop techniques.

You put cells in a suspension that forms a hanging drop.

Due to gravity, the cells are clustering together in the bottom and

form a spheroid or an aggregate.

Scaffolds, this is an engineering part where

the cells are seeded in an extracellular matrix compartment.

We also have layered cultures and reaggregating cultures.

And I'm going to go through these two techniques a little bit more details.

For layered cultures, I have the example of skin, the organ structure of skin.

It consists of many different cell types such as keratinocytes,

melanocytes, Langerhans, and dendritic cells.

This little figure shows the complexity of the skin.

There are many more cell types present, but

these are the main that people are trying to mimic in the in vitro system.

The function of skin.

Skin is the larger organ of the human body.

It's a protective barrier.

It should protect us from chemicals as well as protecting us from not get

dehydrated.

Some of the functions you want to be able to measure in

an in vitro system is absorption, biotransformation,

as well you want to be able to measure skin sensitization irritation response.

Skin model is the in vitro model that has been most developed and

are actually used in a regulatory basis.

Challenges, to incorporate the dendritic cells, which are very important for

the skin sensitization and irritation response.

You also need to include the hair follicles.

They're large inter-individual differences, and

there's a lot of diseases that are affecting the skin.

You need an air and a media compartment to be able to mimic the physiological

situation in vivo, and the skin morphology varies during the lifetime.

This figure are illustrating reconstruction of human skin.

In step one, normal fibroblasts and keratinocytes are isolated from,

for example, human foreskin, and they're expanded in a monolayer culture.

Fibroblasts are embedded in to a collagen matrix, and

the collagen is allowed to control growth.

Keratinocytes are then sitting on top at the medium is changed in

differentiation media.

This happens in step 2 in the figure.

Finally, the culture is lifted to the air medium interface.

Step 3, so

part of the cells will be in contact with the media while part of the cells

will be in contact with air, exactly the way we have it in the in vivo situation.

The total culture duration is about 14 days.

In the figure to the right, you can see an histology of the epidermis.

First, you can see an in vivo, and secondly, an in vitro.

And what you can see is that they are very, very similar

showing that the in vitro system is very good in mimicking the in vivo situation.

I now want to talk about reaggregating cultures.

My example here is the brain, organ structure of the brain.

The brain consists of numerous different cell types.

It has many subtypes of neurons that consist of astrocytes,

oligdendrocytes, as well as microglia, the immune cells.

Brain is the most sensitive organ to xenobiotic perturbations.

The function that you want to mimic in an in vitro system is the electrical firing,

which is the communication between neurons.

You want to be able to measure calcium waves in neurons and astrocytes.

Myelination is an important function.

And especially in the brain, cell-cell interactions are of major importance.

There are many challenges with modelling the brain,

it is the most complex organ in the human body.

It also can have a blood brain barrier that is protecting the brain from

xenobiotic perturbation.

This is very difficult to mimic in the same system.

Cerebrospinal fluid in the brain is very difficult to mimic.

Our medium is normally very complex, however,

it does not mimic the physiological situation in vivo.

The brain has different extracellular matrix than most other organs.

And this makes a challenge if you want to do the engineering part and

uses scaffolds for your brain system.

And now I want to show you the technique of the reaggregrating cultures.

In this example, I'm showing you human induced pluripotent stem cells.

The induced pluripotent stem cells are differentiated

into neural precursor cells.

This is done two-dimensional.

The cells are then scraped off into a single cell suspension.

And they are cultured during gyratory shaking,

constant gyratory shaking illustrated on this picture.

Due to the movement, the cells are clustering together and

form a three-dimensional brain culture, we call this, Mini Brains.

Due to the shaking, you can control the size of the aggregates.

This is very important.

If the aggregate grows too big, you have be difficult to have nutritions and

oxygen penetrating the core.

These cells can later be differentiated into neurons,

astrocytes, oligodendrocytes.

This aggregating brain culture have shown nice organ function,

measured by microelectrode arrays,

the spontaneous electrical activities to the communication between neurons.

This model has also shown response to neurotransmitters with an increase in

intracellular calcium when treated with neurotransmitter glutamate.

Finally, this model has also shown function of the myelination.

Myelination of axons are occurring by oligodendrocytes.

In J and K, you can see a three-dimensional reconstruction.

In I and M, you can see an electron microscopy.

If you're looking in the M picture, you can see how the myelin sheath is wrapping

around the axon, exactly the way it looks in vivo.

Advantages is that they reproduce much better the complexity of the organ

architecture.

It gives an improved cell-cell and cell-matrix interaction.

This leads to an increased cell survival, as far as an increased differentiation,

and finally leads to an increased function.

However, there are limitations and challenges.

Endpoint is generally needed to be optimized from 2D.

Most of our endpoints in toxicology have been developed for

two-dimensional cultures, and they cannot just be applied to the 3D culture.

Of course if a system is more complex, it often has lower reproducibility.

It's a very big challenge to study on a single cell level

as this more mimics a tissue.

You can have limit perfusion of essential nutrients and

oxygen if the organ is too big.

And it also lacks continuous exchange for supply and removal of molecules.

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