Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Summary

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Abstract

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.

Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 μm diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.

Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Protocol

1. Pulling Glass Micropipettes

Equipment: Micropipette Puller, Microforge.

Glass: Boroscillicate glass capillaries (~1.5 mm external diameter, ~1.4 mm internal diameter).

1. Micropipettes are pulled using the same basic approach that is used to prepare glass microelectrodes for electrophysiology recordings. Briefly, a glass capillary is heated in the middle and when the glass starts to melt the two halves of the capillary are pulled apart generating two micropipettes. Multiple commercial pullers are available to perform this process ranging from rela....

Discussion

Microaspiration provides a simple and highly reproducible method to estimate cell stiffness/deformability by applying negative pressure to a cell membrane and measuring membrane deformability in response to well-defined pressure. It was first developed by Mitchison and Swann (1954) to characterize the elastic properties of sea-urchin eggs to provide insights into the mechanisms of cell division ²¹ and then to look at the mechanical properties in red blood cells ¹. This method has been used in multip.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sutter pipette puller	Sutter Instruments	P-97
Microforge	Narishige	MF-830
Inverted Fluorescent Microscope	Zeiss	Axiovert 200M	The microscope should be preferably equipped with 3D/deconvolution capabilities.
Videocamera	Zeiss	AxioCam MRm
Image Acquisition sotware	Zeiss	AxioVision
Pneumatic Pressure Transducer	BioTek	DPM-1B	DPM1B Pneumatic Transducer Tester can now be found by FLUKE.
Pipette glass	Richland	Customized glass	Pipettes were customized with a 1.2 inner diameter and 1.6 outer diameter.
DiI Dye	Invitrogen	D282	Dissolves well in DMSO