MT-0.6081 Microfluidics and BioMEMS

Organs-on-a-chip:Microfludic organ models

3.4.2014Ville Jokinen

Organs-on-a-chip- Miniaturized, microchip based models of organs- Consist of biological (cells/tissue) and non-biological parts- Does not mean fully functional mini organs.- Related fields: implantable chips, regenerative tissue engineering- Current status: proof-of-concept studies, basic biomedical research- In future, high hopes for use in pharmaceutical development

Why organs-on-a-chip?• Basic research: Possibility for experimentation at a level intermediate to cell culture

models and animal models.• Pharmaceutical industry: Need for more efficient screening prior to clinical trials.

• Animal models: + Direct experimentation on in vivo conditions - Ethical issues, time and cost. Biological complexity can be overwhelming.

• Cell culture models: simplicity, lack of architecture+ Simplicity- Far removed from in vivo conditions

• Organ-on-a-chip models:+ A middle ground between cell culture and animal models?- Largely undemonstrated for actual biological or biomedical research.

Basic features• One or more different types of cells cultured on a chip in a specific architecture.

Often the cell culture is directional/polarized.• Each cell population (or tissue side) has a controlled environment, e.g. O2

concentration, media, temperature.• Possibility for individually addressing different areas of the cell cultures• Controlled mechanical properties (rigid, soft) and movement (static, “breathing”,

flow)• Controlled interaction between cells: physical contact, soluble factor

communication• Integrated sensors, actuators, stimulating components

What kind of cells?• Pieces or slices of actual tissue (brain slice, blood vessel)• Primary cells (taken from a subject)+ Closest to in vivo conditions- Require test animal sacrifices

• Immortal cell lines. For example, HeLa cells (cervical cancer cells taken from Henrietta Lachs in 1951)

• Stem cell lines.+ Standardized across laboratories+ Do not require test animals- Cell line deviations and contaminations- Probably less accurate models for in vivo processes

• Patient derived stem cell lines, induced pluripotent cells+ Patient specificity- Difficult biology- Still very much in early development stages

Lung model- A coculture of human alveolar epithelial cells and human pulmonary microvascular endothelial cells-PDMS membrane that is periodically stretched to model breathing.-Breathing motion makes a critical difference for e.g. particle uptake.

Gut model- PDMS chip, polyester semipermeable membrane, Caco-2 cells (model cells forthe small intestine)- Integrated magnetic stirrers for continuous media flow and optical fiber insertsfor - Cells seeded from one side only- Polarized transportation achieved, modeling apical (AP) and basolateral (BL) sides.

Directional transport of rhodamine from the basolateral to the apical side.

Kidney model-Primary rat inner medullary collecting duct cells seeded on porous polyester membrane- The cell membrane is polarized, and both sides can be fluidically addressed.- Fluidic shear has many effects on the function of the kidneys, but it is not wellunderstood.

Cells on the kidney chip have a totally different form and arrangement as comparedto traditional cell culture on a glass dish.

Drug studies: a) effect of vasopressin on osmolarity, b) effect of aldosterone on Na transport

Heart model- Cardiomyocytes from neonatal rats-2-dimensional model based on an elastomeric PDMS film and a patterned layeror cardiomyocytes.-Optical detection of synchronized contraction and “heartbeat”

Contractile stresses with 2 Hz pacingStress range similar as previously measured fromisolated muscles.

Spontaneous activity and effect of epinephrine (adrenaline)Dose response observed, results in line with those from isolated muscles

Cancer models- Many aspects of cancer modeled on chip, e.g. tissue heterogeneity (this example)and entry of tumor cells into blood (student case).-The chip is made out of PDMS and glass and has channels and a filter for cellretention.

Chemotherapy drug doxorubicin diffusion

In future, maybe cancer model chips can be like this. For now, it is just a drawing.

Case study: Brain-on-a-chip- Historical perspective: Squid giant axons (up to 1 mm in diameters) were used in experiments that lead to the discovery of the mechanism of action potentials. -Macroscopic axons could be interfaced with macroscopic tools. -Human axons are ≈ 1 µm in diameter, suggesting micro/nano sized tools.

- Most important cell types for central nervous system (CNS): neurons and glial cells(non neuron support cells of CNS).- In vitro studies: brain slices or primary neurons and glial cells are commonly used. - Immortal cell lines with neuron like properties also exist, but are less common- In future, patient derived induced pluripotent cells differentiated into neurons?

Axon isolation

Somas

Axons in 3 µm high microchannels

Soma≈ 10 µm Axon

≈ 1 µm

Jokinen et al. J. Neurosci. Methods, 2013

- Most common component for neuron chips: isolation of axons from somas.

Axonal isolation by surface patterning- Chemical cues can also be used for axonal isolation.- Neurons typically do not grow on many things. Special neuroadhesive coatings need to be used, most commonly poly-L-lysine PLL- PLL can be patterned by e.g. stenciling or microcontact printing

Directional network - In vivo, central nervous system is directional exhibiting clearly differing pre- andpostsynaptic neural populations-Axon diode based on the axonal tendency to grow mostly straight.- Valving is also a possibility for achieving directionality.

Neuron-glia coculture- Glial cells are important supporting cells that act in tight collaboration with neuronsin vivo. Cocultures can be created.

-Somal and axonal sections as previously. In addition, glial cell patterning througha stencile mask on the axonal side.

Fluidic isolation- Important for compartmentalization. Different cell populations can undergodifferent biochemical treatments.-Based on hydrostatic pressure difference to drive fast enough slow to counteractdiffusion.

Electrophysiology- Neurons are electrically active cells. Measurement by either microelectrodes orpatch clamping.

Perfusion- Localized chemical stimulation for e.g. phamaceutical application or causing trauma.- High temporal and spatial resolutions challenging.

Axotomy- Trauma is one of the most studied pathologies on neuro chips. Based on physical damaging of the axons. CNS does not regenerate well from trauma. Why?- Trauma by mechanical damage, chemical treatments or heat.

Brain slices- One step higher in the complexity hierarchy compared to neural populations: brain slices on chip

Individual neurons on chip- One step lower in the complexity hierarchy compared to neural populations:individual neurons on chip

-Individual neurons seeded on wells on top of a multielectrode array. -Channels for axons are then drawn in situ with a laser on agarose matrix. -The resulting network is directional.