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Chapter 1: Cell on a Chip I (cell behavior control)

Basic cell structure

1. Cell membrane
2. Cycloskelton
3. DNA and RNA
4. Organelles
Cells in the In Vivo environments
Ways to control microenvironment
1. Cell culture technologies
- Microfluidic (scale down)
- Static (almost impossible to control microenvironment)
- Bioreactor (scale up)
2. Microenvironmental Stimuli
- Single stimulations
- Biochemical, mechanical, surface, electrical, cellular etc
- Multiple stimulations
- Chemotaxis (the movement of an organism in response to
chemical stimulus)
Diffusion in tissue
- Gradient generation by Transwell
Transwell - used to study chemotaxis in vitro. Transwell
assays - to show cancer cell migration towards chemokines.
A Transwell consists of a multi-well plate and small inserts having
thin porous membranes at the base. To conduct a Transwell assay, a
cell suspension is added into the inserts, and media containing
chemoattractants is added to the (bottom) wells in the plate. The
inserts are placed in the plate wells, and the two are incubated
together for a defined amount of time, during which some cells
migrate from the top-side of the membrane to its underside through
pores in the membrane. After incubation, the inserts are removed
from the multi-well plate, washed, fixed and stained. Before imaging
the fixed inserts, Q-tips or cotton swabs are used to wipe the topside of the membrane gently to remove cells that have not
migrated, leaving migrated cells untouched. Multiple regions of each
insert are imaged, and the numbers of cells in each image are
averaged to estimate cell migration for that insert.
- Gradient generation by microplatform
A gradient was formed across the channel, perpendicular to
the direction of flow.

Gradient generators
1. Cross channel gradient generator
2. Microfluidic Butterfly
3. AIM Biotech

New gradient generation chip diffusion

Solution A and B containing different conc of different chemicals
were introduced at very slow speed into the main channel -> when
they meet, 2 phased flow was created -> if flow speed is sufficiently
slow, wide gradient band can be created.
Challenge of gradient chip: control, long term, little volume, and no
peripheral device
How to generate ultra slow flow?
Due to osmotic pressure between PEG and water, osmosis driven
flow was generated.
Microfluidic system operatable inside cell incubator
Seeding of neural progenitors Human ES
Neural progenitors: Neural progenitors are cells that are capable of
dividing a limited number of times and have the capacity to
differentiate into a restricted repertoire of neuronal and glial cell
Durotaxis: Durotaxis is a form of cell migration in which cells are
guided by rigidity gradients, which arise from differential structural
properties of the extracellular matrix (ECM). Most normal cells
migrate up rigidity gradients (in the direction of greater stiffness).[1]
1. Extension
2. Adhesion
3. Translocation
4. De-adhesion
Electrotaxis: movement of an organism or system of cells in
an electric field
Asymmetric excitation caused by receptor gradient
Difference between eletrotaxis and chemotaxis
A: in chemotaxis a gradient of ligand stimulates activation of more
receptors on one side of the cell than the other. The result is
increased cytoplasmic signaling on one side of the cell relative to
the other, even if the receptor density is the same on each side. The
side of the cell with higher signaling becomes the leading edge, and
the cell migrates up the ligand gradient. B: in electrotaxis,
asymmetric signaling results from an increase in density of
membrane receptors (or membrane) on the cathode-facing side of
the cell. The intracellular signal is higher on the cathode-facing side

of the cell, which becomes the leading edge, and the cell migrates
toward the cathode.
2D and 3D cell culture
2D cell culture in microchip

Cell culture under the gradient

Cell culture under the flow (is it impt or sth?)
Braille display: pin patterns can be changed to protrude up or down
-> control cellular directions
Patterned cell culture and cell-cell interaction
1. Microcontact printing: Microcontact printing (or CP) is a form
of soft lithography that uses the relief patterns on a
master polydimethylsiloxane (PDMS) stamp to form patterns of selfassembled monolayers (SAMs) of ink on the surface of
a substrate through conformal contact as in the case ofnanotransfer
printing (nTP).
2. Electrical micropatterning: the cytophobic nature of albumin
coated substrates rapidly switched to a cell-adhesive one when
exposed to an oxidizing agent such as hypobromous acid which can
be produced by the electrochemical oxidation of bromide ion in aq
3. Micropatterning on a mechanical structure:
(i) micromechanical substrates enable micrometer-resolution cell
positioning. E.g. Microfabricated silicon parts can be fully separated
locked tgt with comb fingers in contact or slightly separated
(ii) Reconfigurable cell culture. Cultures can be reversibly switched
to iniate or to eliminate contact between 2 cell populations;
individual populations can also be removed and replaced
4. Patterning by dielectrophoresis (DEP): the translational motion of
polarizable matter (neutral or charged) within a nonuniform electric
field. The DEP force moves particles toward regions of high field
intensity (positive DEP or +DEP) or low field intensity (negative DEP

or DEP), depending on electrical properties of the particle and

suspending medium
5. Patterning by microfluidic channel: using medium flow to pattern
6. Patterning by standing surface acoustic waves (SSAW)
we use pairs of slanted-finger interdigital transducers (SFITs)
acoustic tweezers" to generate a tunable standing surface acoustic
wave field, which in turn patterns microparticles or cells in one- or
two-dimensional arrays inside the microfluidic channels--all without
the assistance of fluidic flow.
A surface acoustic wave (SAW) is an acoustic wave traveling
along the surface of a material exhibiting elasticity, with
an amplitude that typically decays exponentially with depth into the
Cell sorting
Active systems generally use external fields (e.g., acoustic, electric,
magnetic, and optical (light), pressure difference) to impose forces
to displace cells for sorting.
Passive systems use inertial forces, filters, and adhesion
mechanisms to purify cell populations.
E.g. Cell docking within engraved microwells by capillarity-drive
flow, by receding meniscus
Chapter 2: Chip for cell assay
Assay: assessment/evaluation
In vitro roughly means in test tube, while in vivo means roughly
in life. In vivo assays involve the use of animals. In vitro assays
like cell based assays and receptor binding assays involve tissue
derived from animals.
Culture -> treatment -> selection -> lysis -> separation -> analysis
Cell sorting
1. Removal of undesired contaminants
e.g. removal of tumor cells in autologous bone marrow transplant
removal of contaminating fibroblasts in a cartilage biopsy
2. Purification of cells
3. Depletion: removal of unwanted cell types

4. Enrichment: positive selection of a desired cell type

1. Fluorescence-activated cell sorter (FACS)
- to count, examine, sort microscopic particles suspended in a
stream of fluid
It provides a method for sorting a heterogeneous mixture of
biological cells into two or more containers, one cell at a time, based
upon the specific light scattering and fluorescent characteristics of
each cell.
2. Cell sorting by structure
-only if there is a diff between sizes of cell
(i) Weir type filters size exclude cellular components while allowing
flow of smaller cells and molecules to pass through a planar slit
(ii) Pillar type filters are arrays of pillars which exclude cells larger
than the spacing of the pillars
(iii) cross flow filters are arranged perpendicular to pri channel flow
to avoid probs associated with obstructed flow
3. Hydrodynamic separation methods: pinched flow fractionation,
hydrodynamic filtration, and deterministic lateral displacement
4. Blood cell sorting using microfiltration membrane
crossflow filtration: the device consisted of top and cottom
microfluidic channels and PMM sandwiched in between. Cells smaller
than membrane pore size would pass through the PMM and exit
through the bottom while cells larger than the membrane pore
would stay in the top channel.
5. DEP (dielectrophoretic separation method)
- can be positive or negative which affects where cells are
positioned within a field. DEP has been utilized in microfluidic
systems in a variet of arrangement.
Dielectrophoresis is a phenomenon in which a force is exerted on a
dielectric particle when it is subjected to a non-uniform electric field.
This force does not require the particle to be charged.

Four electrodes create an electric field that induces a dipole in

a biological cell. The orientation of the induced dipole causes
the cell to experience a negative DEP force towards the
center, trapping the cell.

6. Optical cell sorting

- sample input passed through the center, cells are analyzed and
then switched based on their detected fluorescence. Target cells are
directed by the laser to the collection output while all other cells
flow to the waste output

7. Cell sorting by ultrasonic standing waves

The principle of USW manipulation is based on steady-state acoustic
radiation forces, which typically drive particles to the pressure nodes
of the standing wave.
8. Cell sorting by focusing ridge
To induce cell rolling, a surface needs to be coated with a ligand
specific to the target cell type. The rolling target cells need to be
focused, so slanted ridges are added to the bottom of the channel.
When the target cell comes into contact with the surface of the
coated ridges, it will begin to roll along it and eventually turn the
corner into the space between ridges. By following the path of the
direction of the ridges, the targeted cells will be focused on one side
of the channel known as the gutter. The non-target cells should not
adhere and roll along the ridges, allowing them to be spatially
differentiated from the target cells.
Cell lysis
Lysis: the breaking down of a cell often by viral, enzymic or osmotic
mechanisms that compromise its integrity. A fluid containing the
contents of lysed cells is called lysate.
Mechanical methods
1. Sonication uses sonochemistry: the effect of sonic waves on
chemical systems. In the case of sonication for cell lysis, ultrasound
(high-frequency) energy is applied to samples to agitate and disrupt
the cell membranes.
2. Electrical lysis device with integrated microelectrodes
3. Mechanical lysis device with sharp knife life protrusion
Non-mechanical methods
1. chemical: using organic solvents to disrupt the cell wall, and
permeate through the cell membrane
Separation (DNA Purification)
- After PCR (polymerase chain reaction) produces many copies
of DNA molecules, there is a need to separate the DNA
molecules from similar sized molecules
- Multiplex PCR may produce (i) more than 20 diff products (ii)
some only 1 or 2 base pairs apart
1. Electric charge
- pull on the DNA molecules
2. Matrix with pores
- Separate the molecules based on the size of the DNA and the size
of the pores

A solution of DNA molecules is placed in a gel. Because each DNA molecule is negatively charged, it
can be pulled through the gel by an electric field. Small DNA molecules move more quickly through
the gel than larger DNA molecules.
The result is a series of bands, with each band containing DNA molecules of a particular size. The
bands furthest from the start of the gel contain the smallest fragments of DNA. The bands closest to
the start of the gel contain the largest DNA fragments.

Batch separation vs continuous separation

(i) batch: injection a small amount of sample into sep column and
detection after sep has been completed
(ii) continuous: sample is injected continuously together and
detection takes place simultaneously
DNA sep through electrophoresis using nanoposts
- sep in a DNA prism, consisting a regular array of posts. A large
electric field is applied diagonally, a small electric field is
applied horizontally. By switching between the 2 fields, DNA
molecules of different length are separated.
Immunoassay: a procedure for detecting or measuring specific proteins or
other substances through their properties as antigens or antibodies.
Concept of his microchip-based immunosorbent assay

Huma carcinoembryonic antigen (CEA) which is one of the most

widely used tumor markers for serodiagnosis of colon cancer was
assayed with this system.
How genetic sequencing works
1. det chemical structure of fragment
2. separate strands
3. introduce sample
4. identify result
Further application
1. Cells on a post

micromechanical regulation of
cell function
2. Caenorhabditis elegans The worm is conceived as a single cell
which undergoes a complex process of development,
starting with embryonic cleavage, proceeding through
morphogenesis and growth to the adult.
It has a nervous system with a brain (the circumpharyngeal nerve
It exhibits behavior and is even capable of rudimentary learning.
C. elegans is an excellent model organism for studying behavior at
the neuronal level. However, its very small so it is challenging to
deliver stimuli to C. elegans and monitor neuronal activity in a
controlled env. => developed 2 microfluidic chips, behavior chip and
olfactory chip for imaging of neuronal and behaviorual responses in
C. elegans.

Chapter 3: Cell on a Chip III
3D Cell culture
Fabricate 3D tissues
Spheroid formation
1. Single cell/multilayers -> spontaneously cluster
2. Rotary culture
3. Rocked culture
4. Methylcellulose
5. Matrices
1. Hanging drop

2. Microwell
- a flat plate with multiple "wells" used as small test tubes.
- cells not uniform
- a challenge of scale up
3D Hepatocyte co-culture for artificial liver (Rat)
- A hepatocyte is a cell of the main parenchymal tissue of the
liver. Hepatocytes make up 70-85% of the liver's mass.

Hepatic stellate cell -> needed for injury healing and secreting
collagen scar tissue
3D cells in microfluidic devices
- Using microfluidic devices, cell spheroids can be cultured in a normal
incubator and a particular spheroid can be monitored for certain periods of
- A multilayer microfluidic device with a porous membrane was employed to achieve
both the spheroid formation and in-situ culture.
- A microfluidic array platform containing concave microwells and flat cell culture
chambers for EB formation and its culture was also developed
- Formation of cell spheroids using gravity oriented microfluidic device was also

New method using PDMS

we develop a bi-layer microfluidic device with polydimethylsiloxane (PDMS) for cell
spheroid formation and culture. The bottom layer of this device is equipped with
several rows of cubical cavities as cell culture chambers, and the top layer has a
channel covering all the cell culture chambers.
Scaffold based 3D tissue engineering
- Cells are often 'seeded' into these structures
- Scaffolds mimic the native extracellular matrix of the native tissue, recapitulating the in
vivomilieu and allowing cells to influence their own microenvironments
Methods to make scaffold
1. Hydrogels
2. Electrospun nanofibers
3. Gas foamed
4. Self leached
5. Freeform fabrication
6. Topography library


Cell sheet

cell sheets to avoid the limitations of tissue reconstruction using

biodegradable scaffolds or single cell suspension injection.
Cell sheets are prepared using temperature-responsive culture dishes.
Temperature-responsive polymers are covalently grafted onto the
dishes, allowing various types of cells to adhere and proliferate at
The cells spontaneously detach when the temperature is reduced
below 32C without the need for proteolytic enzymes.
Penetration from perphery: slow detachment
Penetration from perphery and bottom: rapid
With hydrophillic PEG chains: rapid detachment

Application of cell sheet

- able to fabricate thick tissues and organs
- e.g. Lung: seal pulmonary air leak using dermal fibroblast cell
- Ear: inner ear regeneration therapy using nose mucosal cell
- Liver: liver regenerative medicine using highly functional gene
transferred sheets
Engineering of Organs using 3D tissues

3D brain tissue on a chip

3D nervous system

3D Pancreas Tissue
- encapsulated islets
- to camouflage islets by encasing them in protective barriers.
- for example, encapsulating cells in hydrogels
- immune protection by encapsulation
Para-thyroid function recovery using human T-MSC
TMSC: human tonsil-derived mesenchymal stem cells
- differentiated TMSC resemble parathyroid cells
extranotes: Compared with undifferentiated control TMSC, TMSC differentiated with activin A
and soluble sonic hedgehog induced a significant release of PTH as early as day 7, with
increased PTH release occurring in response to lower calcium levels and vice versa.

Chapter 4

model is requird?
screening of drug, toxicity, food etc
mechanism of disease
physiophathological study

animal models: systemic but not human

static 2D and 3D human cell culture: human but not systemic
therefore both are poorly predictive
core technology for organ on chip
- well defined cells: harvesting of primary cell, stem cell
- control of microenvironments: physical cues, chemical

3D organ (tissue): 3D spheroid, 3D printing

Detection: molecular imaging, nano tech, biosensors

2D organ on a chip
Liver on a chip
- the alternate radial patterned HUVECs mimic the shape and the
function of sinusoid-like vascular endothelial lining cells that are
shown in the classic real hepatic lobule.
2D brain on a chip

a microfluidic chip based on three-dimensional (3D) neurospheroids that more closely

mimics the in vivo brain microenvironment by providing a constant flow of fluid that is
readily observed in the interstitial space of the brain.
- Uniform neurospheroids, with cell-cell interactions and contacts in all directions, were
formed in concave microwell arrays, and a slow interstitial level of flow was
maintained using an osmotic micropump system.

Heart on a chip
Human iPSC-based heart on a chip
Organ on 3D microplatform
Gut on a chip
Lung on a chip
Kidney on a chip
Blood brain barrier on a chip
Cancer on a chip
General platform for organ on a chip
Built in vasculature for organ on a chip
3D gastrointestinal (GI) tract model
Bone marrow on a chip
3D printing of tissue
Chapter 5: electronics for man machine
1. BCI brain computer interface
2. BMI brain machine interface
- bypasses the brains normal pathways of peripheral nerves and
Classification of neural electrode
Non-Invasive electrode
1. Hard materials: silicone electrode
2. Soft materials
Invasive (implantable)
1. Cuff electrode

2. Sieve electrode
3. Depth electrode
4. Planar electrode
electrocorticography: multichannel brain-surface recording and stimulation
with probe electrode arrays (Monkey)
Epicranial Auditory cortex recording (rat)
Skin electrodes
1. Long term wearable (skin and tissue compatible)
2. Flexible and stretchable
3. Robust to motion and sweat
4. Easy interface to conventional device
epidermal electronice
applications: health monitoring, energy harvesting
capacity coupling