PT1: 28/09 Flashcards
What is Micropipette aspiration
a simple technique used to study cell mechanics by applying a suction pressure on the cell surface using a capillary tube
What is micropipette aspiration used for
- measuring the cell’s Young’s modulus and membrane elasticity, apparent viscosity, cortical tension
- studying nuclear deformation in stem cell differentiation
- Characterize the time-dependent bulk and shear moduli of structures
What is a typical MPA setup
3 main components:
1. micromanipulator responsible for controlling the position of the micropipette tip in 3D on cell’s surface
2. pressure generator such as a microfluidic pump, relies on pneumatic pressure system
3. microscope with a camera to image and record deformation and perform the analysis
What is the overall pressure at the micropipette tip defined by?
deltaP = Pinitial - (rho)gh, where h is the distance between the cell culture dish and the reservoir (see image)
Why must the micropipette be free of air bubbles
air bubbles can affect the accuracy of the pressure
What are the main steps in the MPA procedure?
(Set-up & Calibrate first)
1. Cell aggregate culturing and preparation
2. micropipette fabrication
3. aspiration measurement
4. analysis of mechanical parameters
Briefly describe the cell aggregate culturing and preparation
- transfect cell line with cDNA
- Culture cells in medium
- Incubate for 24h in 96 well plate
- cell aggregates with a diameter between 250um and 400um are extracted
- Cells treated to prevent cell adhesion and placed in gyratory shaker for 24h
- aggregates are suspended in culture medium
Briefly describe the fabrication and preparation of micropipettes
- Fabricated suing borosilicate capillaries (sorry?)
a) laser based puller which uses CO2
b) quartz tiles used to size micropipettes
c) incubate in solution to prevent cell adhesion on pipette walls.
A unique micropipette is made for each experiment because the radius must be specific
What must the radius of a manufactured micropipettes be, in relation to cell size?
The radius of the pipette must be on average three times larger than the cells, but around 1/3 of the cell aggregate size
What determines the magnitude of the applied pressure steps (deltaP)
The pressure applied must be high enough for the tissue to flow inside the pipette, but small enough to not damage the tissue
Describe the process of getting micropipette aspiration measurements
- introduce pipette into an imaging chamber oriented towards cell aggregates.
- Critical pressure, Pc, must be determined:
- low pressure applied to lift tissue from bottom of culture dish
- increase of pressure stepwise until the aspirated section of the cell forms a hemisphere (R = Rp). This is the Pc. - Apply steps of deltaP (delta P > Pc) for 3h, and take images every few seconds to inverted microscope.
- Set deltaP to 0 and take images every few seconds
- displacement of front of cell aggregate tongue is measured with respect to the pipette tip opening
How is the surface tension calculated
Laplace law, using critical pressure Pc, radius of pipette Rp, radius of aspirated cells, and surface tension (gamma).
Which mechanical parameters can be extracted
Surface tension
Viscosity
Elasticity
How is viscosity extracted?
It is determined as a function of aspiration and retraction rates (Lasp and Lret), applied pressure, and pipette radius. the rates are determined from aspiration and retraction curves
when does elastic deformation of the material end
when the material enters a viscoelastic creeping flow regime with constant velocity. this displacement can be modeled via a modified maxwell model.
