Appendix E.  Cell Fractionation & Centrifugation

Introduction

 

Image Source: Garland Publishing

Cell fractionation is the process of producing relatively pure fractions of cellular components. The process involves two basic steps: disruption of the tissue and lysis of the cells, followed by centrifugation.

Tissue Disaggregation and Cell Lysis

The first step in cell fractionation is tissue disruption and cell lysis. The objective is to disaggregate the cells and gently break them open with minimum damage to the cellular components. This can be accomplished in several ways: 1) homogenization, 2) sonication, and 3) chemical / osmotic lysis. The particular method one chooses depends on the tissue and cell type and the particular cell fraction of interest.

Homogenization is probably the most widely used cell disruption technique. Homogenization is a process by which fluid shearing is used to break open plasma membranes. It involves the use of a mechanical homogenizer, like a blender or a motorized mortar and pestle. The process is a very sensitive one, and care must be taken to prevent mechanically damaging the very components one is trying to isolate. Therefore, to get desirable and reproducible results, care must be taken to control the homogenization conditions. Three different types of homogenizers are in common use. A Dounce Homogenizer consists of a glass pestle and a glass mortar. A Potter-Elvehjem Homogenizer consists of a teflon pestle and a glass pestle.

Sonication involves the use of high frequency sound waves to lyse cells. Sonication is often used when prokarytic cells are to be lysed. The sound waves are delivered using an apparatus with a vibrating probe that is immersed in the liquid cell suspension. Mechanical energy from the probe initiates the formation of microscopic vapor bubbles that form momentarily and implode, causing shock waves to radiate through a sample.

Osmotic lysis is a method that can be used to disrupt some cells, such as mammalian red blood cells. In this method, a buffered hypotonic solution followed by simple mechanical agitation can effectively lyse the cells by osmotically swelling the cells to the point of lysis. Mechanical agitation is employed to break open swollen cells that have not lysed.


Centrifugation

Most of the cellular components in a cell lysate will eventually, given time, settle to the bottom of a tube. To accelerate this process, the lysate can be subjected to centrifugation. In centrifugation, the lysate produced from tissue aggregation is rotated at a high speed, imposing a force on the particles perpendicular to the axis of rotation. The force is called a relative centrifugal force (RCF), expressed as a multiple of the force of Earth's gravitational force (x g). For example, an RCF of 1000 x g is a force 1000 times greater than Earth's gravitational force. When a particle is subjected to centrifugal force, it will migrate away from the axis of rotation at a rate dependent on the particle's size and density.

Differential Centrifugation

Several centrifugation methods are used by biologists. Differential centrifugation is a centrifugation method whereby the sequential centrifugation of a cell lysate at progressively increasing centrifugation force is used to isolating cellular components of decreasing size and density. The separation of the cellular components is based solely on their sedimentation rate through the centrifugation medium, which, in turn, is dependent on the size and shape of the cellular components. Each centrifugation step results in the production of a pellet, usually containing a mixture of cellular components of the same size and/or density. The fluid resting above the pellet, the supernatant, can be removed and subjected to additional centrifugations to generate pellets containing other cellular components. The primary advantages of the technique are that it is relatively rapid and simple and it usually requires a high-speed centrifuge, which is commonly found in laboratories. The primary disadvantage of the process is that it only separates cellular components that differ signifigantly in size. Therefore the fractions are crude.

 

Image Source: Garland Publishing

Density Gradient Centrifugation

The nucleus is the largest and the most dense of all the subcellular structures of the hepatocyte. Therefore, it is relatively easy to isolate the nuclear fraction using density gradient centrifugation. Density gradient centrifugation is generally regarded as the preferred method of purifying nuclei and organelles. In density-gradient centrifugation, the homogenate is centrifuged through a column of centrifugation medium of increasing density, separating the nuclei and organelles based on size, mass and density.

There are two basic types of density gradient centrifugation: Rate-zonal centrifugation and Isopycnic centyrifugation. Rate-zonal centrifugation separates structures based primarily on particle size and mass. In isopycnic centrifugation, particles are separated based primarily in bouyant density.

The standard method of nuclear fractionation developed by Blobel and Potter, 1966, uses isopycnic purification through a hyperdense, hyperosmotic 2.25 M sucrose solution. In this technique, a crude pellet of nuclei is first produced through differential centrifugation. The pellet is then added to the 2.25 M buffered sucrose solution and centrifuges at 100,000 x g for 1 hour to purify the nuclear fraction. The major disadvantage of this method is that the 2.25 M sucrose solution is very hyperosmotic. The nuclei tend to lose water through osmosis and shrink as they are centrifuged through the hyperosmotic solution. Furthermore, high viscosity sucrose solutions can be difficult to prepare and measure out accurately.

Image Source: Axis-Shield

An alternate density gradient centrifugation method that overcomes these problems is a rate-zonal density gradient centrifugation method using a discontinuous density gradient made of iodixanol. Iodixanol (C35H44I6N6O15) is a nonionic hydrophilic compound, first developed as a contrast medium for coronary angiography. Its major advantage to density gradient centrifugation is that relatively high density iodixanol soutions can be made that are isoosmotic and low viscosity. For nuclear fractionation, the iodixanol gradient is prepared by carefully layering a 25% iodixanol solution containing the homogenate on top of a 30% iodoxanol solution, which rests on a 35% iodixanol solution. When centrifuged at 10,000 g for 20 minutes, the nuclei fall through the 30% solution to rest on top of the 35% solution.

To obtain satisfactory fractionation using any centrifugation scheme, four centrifugation conditions must be controlled:

  • a) fluid density
  • b) type of centrifugation rotor
  • c) relative centrifugation force (RCF) and duration of its application
  • d) centrifugation temperature.

The unit of RCF is the gravitational unit (g). The RCF produced by a centrifuge depends directly on the rotor size and the speed of rotation. Knowing the RCF that needs to be produced and the rotor size, one can calculate the rotation speed (RPM) using the following formula:

RCF = 0.00001118 x r x RPM2

where r is the radius of the rotor in cm.


 

 

Image Source: Youngstown State University

Image Source: LabX

Centrifuges and Centrifuge Rotors

There are three basic types of centrifuges used routinely by biologists. They differ in, among other things, the rotational speed and relative centrifugal force that can be generated. Microcentrifuges are table-top centrifuges used to process small volumes. They can attain speeds up to approximately 12,00013,000 rpm. The are typically used in cell culture, microbiology and molecular biology. High-speed centrifuges handle larger volumes and can attain higher speeds, up to approximately 30000 rpm. They come in both table-top and floow models. Ultracentrifuges are designed to process moderate volumes of sample at speeds in excess of 70,000 rpm. Ultracentrifuges are generally employed to isolate small particles, such as ribosomes and viruses and macromolecules, such as proteins. They are also used in cell fractionation techniques that require centrifugation of cellular components through relative high-density centrifugation media.

Centrifuge rotors are the highly-engineered devices that hold the centrifugation tubes as they are spun. There are two basic types of rotors routinely used by biologists: fixed angle rotors and swinging bucket rotors.

Fixed angle rotors hold the centrifugation tubes at a fixed angle (generally 20 - 40 degrees) as they are spun. These are the most commonly used rotors in the cell biology laboratory. In a fixed angle rotor, the materials are forced against the side of the centrifuge tube, and then slide down the wall of the tube, resulting in a faster separation of particles. They generally have no moving parts.

Swinging bucket rotors have buckets that are free to swing out on a pivot perpendicular to the axis of rotation. They are they rotor of choice when using a density gradient centrifugation medium. Moreover, if there is a danger or scraping off an outer shell of a particle (such as the outer membrane of a chloroplast), then the swinging bucket is the rotor of choice. Swinging bucket rotors have hinges that hold separate buckets, making this type of rotor more prone to mechanical failure.


CellBiologyOLM is authored by Stephen Gallik, Ph. D.| Copyright © 2011 by Stephen Gallik, Ph. D. | Licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License