Single Cell Manipulator

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Single Cell Manipulator
Mentor Dr. Erik Herzog Dept. of
Biology Washington University in St. Louis
Presented by Amy Y. Chen February 4,
2002 Teammates Eileen Chou Julie Kim
Photo from http//biomed.wustl.edu/faculty/sakiyam
a/Sakiyama_Lab-Home.htm
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Background

• Mammals have a biological clock (i.e. circadian
  rhythm) that is usually regulated by light/dark
  cycles
• Without environmental cues, this clock can still
  keep 24 hour time
• Hardin, P.E. From biological clock to
  biological rhythms. Genome Biology, 2000 1(4)
  reviews 1023.1-1023.5
• Need to understand the cellular and molecular
  basis for this behavior

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Motivation for Research

• Age-related deterioration of clock function
• Aujard, F., Herzog, E.D., Block, G.D. Circadian
  rhythms in firing rate of individual
  suprachiasmatic nucleus neurons from adult and
  middle-aged mice. Neuroscience, 2001 106(2)
  255-261

• Dosage prescriptions of (cancer treatment) drugs
• Jet lag
• Transportation/shift work
• Doctors hours
• http//www.cbt.virginia.edu/tutorial/tutorialmain.
  html

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Region Under Study

• Suprachiasmatic nucleus (SCN) of the anterior
  hypothalamus
• Klein, D.C., Moore, R.Y., Reppert, S.M. (1991)
  In Suprachiasmatic Nucleus The Minds Clock.
  New York, Oxford UP

• Questions to explore
• Which cells are responsible for pacemaking?
• Can one cell keep time, or is a group of cells
  needed?
• How does a group of cells cooperate to drive
  this clockwork behavior?

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Focused Approach

• Study the circadian properties of isolated cells
  (the pacemaking ability of a single neuron)

• Need a device to precisely move and place one
  cell onto a multielectrode array for recording
  over several weeks time

Firing rate of one SCN neuron from a young adult
mouse. From Aujard, F., et. al. Full reference on
Slide 3.
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Procedure
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Specifications

• Move one neuron
• Minimal damage to the cell (pN range of force)
• x,y,z coordinate movement
• coarse dxdy3.3 cm dz2 cm
• fine 1 micron
• No drift when releasing the cell (temperature,
  humidity dependent)

• Easily sterilized probe
• Easily detachable probe
• Size/bulk of device compatible with microscope
• Right- or left-handed use
• Obliterate other cells in the vicinity

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Existing Solutions

• Laser tweezers use of IR to trap cells by
  optical forces
• Least invasive, wont compromise sterility
• Heat from IR damages conduction in neurons
• Can only use thin substrates for good penetration

• Capillary suction pick up a cell and eject it
  using hydrostatic forces
• Forces may damage the cell
• Cell may stick to the glass / sides of capillary
  tube

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Existing Solutions

• Putter bend a capillary tube and push the cell
  along (used like a hockey stick)
• Would not be picking up the cell
• Not very precise

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Design Alternatives
Probe Isolate and pick up / target one neuron
Movement control Ensure micron precision
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Probe Design

• Should be able to move a sphere 10-20 ? in
  diameter
• Mechanical
• Ring that pivots
• Scoop that parts, allows cell to drop

• Hydrostatic
• Fluid flow pressure system

• www.stanford.edu/xjzhang/papers/2000/DARPA_1102.
  pdf

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Probe Design

• Electrical
• Sequentially switch on a field at the bottom of
  the dish
• Use of dielectrophoretic forces to trap cells
• Rutten, W. et al. Neuroelectronic interfacing
  with culture multielectrode arrays toward a
  cultured probe. Proc. IEEE (2001) 89(7),
  1013-1029)
• Generate a small field at the desired location

• Bioengineering
• Adhere neuron to polystyrene bead coated with
  laminin
• Magnetic bead, magnetic probe
• Silicon chips coated with laminin

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Movement Design

• Should have 1 ? resolution in 3 dimensions
• Micromanipulator
• Manual, joystick control
• Automated
• (World Precision Instruments, Leica, Narishige,
  Geneq, ASI Imaging)
• Stage stepper motor (www.cartesiantech.com/sq_tech
  .html)
• DNA microarray technology (www.Gene-Chips.com)

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Preliminary Analysis
Bioengineering concept Sources Dr. Sakiyama,
Protocols for Neural Cell Culture 3rd ed., Humana
Press, 2001
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Preliminary Analysis

• Obstacles
• Availability of desired bead size (Spherotech,
  Polysciences)
• Long preparation time polylysine, laminin
  coating takes several hours
• Complete adhesion of neuron to bead 1 day
• Detach cell using trypsin and/or EDTA wash (5-30
  minutes)
• Neuron floats away after washing, not in precise
  location

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Design Schedule
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Team Responsibilities
Julie vendor search/contact for suitable
micromanipulators, beads, other parts Eileen
research electric fields concept, effects on
neural conduction Amy refine bioengineering
concept, develop a protocol