Multiorgan microdevices

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Guided By:-

Prof. P. V. Devrajan By:-

Ashish Singh Rajput 14PHP2003

M.Pharm(Pharmaceutics) Institute of Chemical Technology, Mumbai.

Multiorgan microdevices for ADME evaluation and Drug design

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Contents•Introduction

•Design and Fabrication

•Microfabricated Organ Models

Lungs on Chip

Liver on chip

Gut Epithelium on chip

Cardiac System on chip

•Contibution in Drug development process.

•Challenges

•Bibliography.


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Introduction:- Multiorgan micro-devices are in-vitro set up of animal cells to simulate the same

physiological environment and study the effect of drug on different cells and organs.

These systems are capable of simulating human metabolism.



The devices have the potential to predict potential toxic side effects with higher accuracy before a drug enters the expensive and time consuming phase of clinical trials.

Since single organ devices are testing platforms for tissues that can later be combined with other tissues within multi-organ devices

Multi-organ micro-devices can be seen as physical representations of Physiologically based pharmacokinetic models in which the organs are represented by an actual compartment.

Devices could be a way for the development of individualized medicine.

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Human PBPK model showing different Organs.

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Why use microfluidics?The science of manupulating small amounts of

fluids in Microfabricated hollow channels.

Sample savings – nL of enzyme, not mL

Faster analyses – can heat, cool small volumes quickly

Integration – combine lots of steps onto a single device

Novel physics – diffusion, surface tension, and surface effects dominate

This can actually lead to faster reactions!


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Device Development and Fabrication:- Photolithography is a core microfabrication technique used to transfer

microscale patterns to photosensitive materials by selective exposure to optical radiation.

A silicon wafer is spin coated with a thin uniform film of a photosensitive material

( Photoresist ) Photomask with a pattern defined covers the photosensitive material.


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Exposure of the photoresist to high-intensity UV light through the photomask which protects some regions and exposes others based on the design of the pattern.

Soft lithography involves fabrication of elastomeric stamps using liquid prepolymer of PDMS is cast against the pattern of photoresist.

the PDMS stamp is inked with protein solution, dried and brought in conformal contact with a surface for a period ranging from 30 s to several minutes

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Upon removal of the stamp, a pattern is generated on the surface that is defined by the raised structure of the stamp.

In 3d Cell culture different ECM gel , Hydrogels and Agarose are used as base mould which enable them to grow equivallently in all direction.

PDMS:Poly dimethylsiloxaneECM : Fibronectin, collagen

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Microfabricated Organ Models:-

Lungs on Chip Liver on chip Gut Epithelium on chip Cardiac System on chip


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Lungs on chip:-

The device is made using human lung and blood vessel cells and it can predict absorption of airborne nanoparticles and mimic the inflammatory response triggered by microbial pathogens.

Human lung alveolar pithelial cells are cultured at an air–liquid interface on one side while human lung capillary endothelial cells are grown on the opposite side .

Human lung-on-a-chip device consists of two PDMS microfluidic channel layers separated by a thin (10 mm), flexible, ECM-coated PDMS membrane with micro engineered pores (10 mm in diameter) that mimics the alveolar–capillary interface of the living lung

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• Three PDMS layers are aligned and irreversibly bonded to form two sets of three parallel microchannels separated by a 10-mm-thick PDMS membrane containing an array of through-holes with an effective diameter of 10 mm.

• After bonding, PDMS etchant is flowed through the side channels. Etching of the membrane layers produces two large side chambers to which vacuum is applied to cause mechanical stretching.

• Actual lung- on-a-chip microfluidic device can be seen on Fig. E

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Casting PDMS Membrane

Pre polymered the layers

Photolithography of microchannels

Coat with the binding layer and

incubate at 65 “c overnight

Bound irreversibly with

the two layers

Etching the membrane with TBAF & NMP

Apply Hydrostatic Pressure

&Vaccume

Run the etchant solution

Workflow

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A Microfluidic device was used to model the airway architecture to simulate abnormal obstruction of airways and to study the effect of liquid propagation and rupture on the alveolar epithelial cells lining the alveoli.

The two layer device was designed to allow controlled mechanical stretching of the endothelial–epithelial bilayer, mimicking the mechanical cues present in the lung during breathing.

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Liver on Chip Liver and kidney are the major organ responsible for detoxification of toxins

and metabolism of drug.

Organ to organ interaction often seen with liver which change the metabolite of drug.

The Co-culture pattern of Rat primary hepatocytes and stromal cells improved various liver-specific functions which were very close to actual liver physiology.

The functional unit of the liver, the acinus, produces different set of proteins depending on the locations within the unit as per the O2 gradient in tissue.


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Liver and Kidney Interaction (Liver cell

models HepaRG was used)

Ca+2 release in Kidney

Active metabolite shows Anticancer

activity

Liver and Kidney Interaction

(Liver cell line HepG2/C3a was

used)

Less Bioactivation of Drug and

Perturbation in cell differentiation

Less Bioactivation of Drug and

Perturbation in cell differentiation

(Ifosfamide activated by CYP450)

Ifosfamide activated by CYP450)

Less Ca+2

release.

Some special types of liver cells are responsible for

bioactivation of drug

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GIT on ChipThe in vivo environment of the GI tract is extremely complex consisting of

circular tissues and metered length.

The lumen is separated by several layers of tissues containing mucosa, muscle, and blood vessels.

The inside lining of the epithelial layer is covered with villi, which increase the absorptive surface area.

The two major in vitro methods for predicting drug absorption are the Caco-2 model and the parallel artificial membrane permeability assay (PAMPA).They mainly test the permeability of drugs based on passive diffusion.


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The Caco-2 cell monolayer model was able to predict the absorption coefficient of rapidly and completely absorbed drugs, while the prediction for slowly and incompletely absorbed drugs were inaccurate.

Sung et al. developed a novel hydrogel microfabrication method to create collagen scaffold mimicking the shape of intestinal villi, and cultured Caco-2 cells into a 3-dimensional villi shape.

Using this 3D villi scaffold, permeability coefficients were measured and were shown to be closer to in vivo values than the conventional 2D model.

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Heart On chipThe Muscular Thin Film (MTF) assay measures contractile stresses generated by

anisotropic muscular tissue engineered on top of a deformable elastic thin film.

The contractility of the engineered tissue is derived from the observation of the three-dimensional (3D) deformations of MTFs

The assay has been employed for evaluating contractility and tissue structure from multiple tissues .

The microdevice has a heated metallic base for maintaining physiological temperatures, transparent top for optically recording MTF deformation and embedded electrodes for electrical field stimulation of the tissue


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An MTF chip was brought from the incubator and placed in the aluminum chamber.

The polycarbonate top was tightly fastened creating a fluidic seal between the top and PDMS coated MTF chip.

The Infusions of 10 ml at a flow rate of 1 ml /min were employed for complete flush out of the system.

Microtissues were electrically stimulated at 2 Hz with 10– 15 V of a bipolar square pulse of 10 ms duration to stimulate membrane depolarization.

After completion of contractility experiments, MTF chip is immunostained for nuclei, actin, and sarcomeric Actin to directly compare the stress generated by each tissue on the chip relative to its sarcomere organization.

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Contibution in Drug development process

Cost effective drug discovery

Predicting Drug efficacy and Toxic side Effects.

Testing drug Interaction & combinations.

Predicting the bioavailability of Drug.

Drug specific treatment and Individualized medicine.


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Challenges1) Development of suitable Culture Media.Typical cell culture media contain a mixture of defined nutrients dissolved in

a buffered physiological saline solution.

Cell-cultures are designed to mimic the relevant in vivo environment. A temperature of 37 °C relevant to body temperature, and a controlled humidified gas mixture of 5% CO2 and 95% O2 are the standard physical conditions.

Media depends upon the organ and cells because each organ is specific in terms of nutrients intake .

E.g.- The Promo Cell Skeletal Muscle Cell Growth Medium is a low-serum (5% V/V) medium optimized for the expansion of human skeletal muscle cells.


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One media could not be suitable for two different type of cells so it is very hard to select the commom media in multiorgan devices.

E.g.- The tissues were stimulated with TGF-ß1, . TGF-ß1( transforming growth factor beta ) supported the growth of A549 lung cells, but inhibited the growth of HepG2/C3A liver cells.

This response highlights the difficulty of finding a common medium with growth factors that support the viability of all cell types.

Some common culture medias are Low serum (5% v/v )or Serum free medium Fetal bovine Serum ES cult Basal medium (Marketed Product)


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Cell Sources:- Primary human cell , such as skin ,

skeletal muscle , and blood ,are relatively easy to obtain. Acquisition of others, such as neurons, is more problematic .

In such cases investigators are often limited to cadaver tissue as a cell source.

Alternative methods like iPPC culture, stem cells propagation and 3D cell culture best suited for culture of cell and highlights the importance of novel in vitro platforms for developing new therapies


Methods to cell culture

InducedPluirepotent cell culture technique

Genetic Engineerin

g

3D cell culture

Techniques

Stem cell propagatio

n

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Thank You

Health & Medicine

Multiorgan microdevices