b.tech. Biotechnology Notes (2)

8/6/2019 b.tech. Biotechnology Notes (2)

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ASSIGNMENTON

CELLDISTRUPTIONTECHNIQUES

SUBMITTED BY:-MUDIT MISRAB.TECH(B.T.)


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SECTION-AROLL No.-33

Cell disruption

Cell disruption is a method or process for releasingbiological molecules from inside a cell.

Choice of disruption methodThe production of biologically-interesting moleculesusing cloning and culturing methods allows the studyand manufacture of relevant molecules.Except forexcreted molecules, cells producing molecules ofinterest must be disrupted. This page discusses

various methods.

Major factorsSeveral factors must be considered.

Volume or sample size of cells to be disruptedIf only a few microliters of sample are available, caremust be taken to minimize loss and to avoid cross-contamination.

Disruption of cells, when hundreds or even thousandsof liters of material are being processed in aproduction environment, presents a differentchallenge. Throughput, efficiency, and reproducibilityare key factors.

How many different samples need to be disrupted atone time?Frequently when sample sizes are small, there aremany samples. As sample sizes increase, fewer

samples are usually processed. Issues are samplecross contamination, speed of processing, andequipment cleaning .

How easily are the cells disrupted?As the difficulty of disruption increases (e.g. E. coli),more force is required to efficiently disrupt the cells.

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http://en.wikipedia.org/wiki/E._colihttp://en.wikipedia.org/wiki/E._coli


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For even more difficult samples (e.g. yeast), there is aparallel increase in the processor power and cost. Themost difficult samples (e.g. spores) requiremechanical forces combined with chemical or

enzymatic efforts, often with limited disruptionefficiency.

What efficiency of disruption is required?Over-disruption may impact the desired product. Forexample, if subcellular fractionation studies areundertaken, it is often more important to have intactsubcellular components, while sacrificing disruption

efficiency.For production scale processes, the time to disruptthe cells and the reproducibility of the methodbecome more important factors.

How stable is the molecule(s) or component thatneeds to be isolated?In general, the cell disruption method is closelymatched with the material that is desired from thecell studies. It is usually necessary to establish the

minimum force of the disruption method that willyield the best product. Additionally, once the cells aredisrupted, it is often essential to protect the desiredproduct from normal biological processes (e.g.proteases) and from oxidation or other chemicalevents.

What purification methods will be used following celldisruption?

It is rare that a cell disruption process produces adirectly usable material; in almost all cases,subsequent purification events are necessary. Thus,when the cells are disrupted, it is important toconsider what components are present in thedisruption media so that efficient purification is notimpeded.

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LysisFor easily disrupted cells such as insect andmammalian cells grown in culture media, a mildosmosis-based method for cell disruption (lysis) iscommonly used. Quite frequently, simply lowering theionic strength of the media will cause the cells toswell and burst. In some cases it is also desirable toadd a mild surfactant and some mild mechanicalagitation or sonication to completely disassociate thecellular components. Due to the cost and relativeeffort to grow these cells, there is often only a smallquantity of cells to be processed, and preferredmethods for cell disruption tend to be a manual

mechanical homogenizer, nitrogen burst methods, orultrasound with a small probe. Because thesemethods are performed under very mild conditions,they are often used for subcellular fractionationstudies.For cells that are more difficult to disrupt, such asbacteria, yeast, and algae, hypotonic shock alonegenerally is insufficient to open the cell and strongermethods must be used, due to the presence of cell

walls that must be broken to allow access tointracellular components. These stronger methods arediscussed below.

Laboratory-scale methods

Enzymatic methodThe use of enzymatic methods to remove cell walls iswell-established for preparing cells for disruption, orfor preparation of protoplasts (cells without cell

walls) for other uses such as introducing cloned DNAor subcellular organelle isolation. The enzymes aregenerally commercially available and, in most cases,were originally isolated from biological sources (e.g.snail gut for yeast or lysozyme from hen egg white).The enzymes commonly used include lysozyme,

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lysostaphin, zymolase, cellulase, mutanolysin,glycanases, proteases, mannase etc.Disadvantages include:

Not always reproducible.

In addition to potential problems with the enzymestability, the susceptibility of the cells to the enzymecan be dependent on the state of the cells. Forexample, yeast cells grown to maximum density(stationary phase) possess cell walls that arenotoriously difficult to remove whereas midloggrowth phase cells are much more susceptible toenzymatic removal of the cell wall.

Not usually applicable to large scale.Large scale applications of enzymatic methods tend

to be costly and irreproducible.The enzyme must be removed (or inactivated) toallow cell growth or permit isolation of the desiredmaterial.

Bead methodA second common laboratory-scale mechanicalmethod for cell disruption uses glass or ceramicbeads and a high level of agitation. At the lowest

levels of the technology, the beads are added to thecell suspension in a tube and the sample is mixed ona common laboratory vortex mixer. This processworks for easily disrupted cells, is inexpensive, andmultiple samples can be conveniently processed. Themore sophisticated level, bead-based methods use aclosed container holding the sample and the beadswith an electric motor to provide vigorous agitation.When larger samples are processed, some form ofcooling is provided (typically liquid CO2) as thesample heats significantly due to the extremeagitation. Another configuration uses an inert, rapidlyrotating rotor in a small container containing the cellsand beads.Disadvantages include:

Limited ability to scale to larger samples.

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Variability in product yield and purity Occasional problems with foaming and sample

heating, especially for larger samples

SonicationA third common laboratory-scale method for celldisruption applies ultrasound (typically 20-50 kHz) tothe sample (sonication). In principle, the high-frequency is generated electronically and themechanical energy is transmitted to the sample via ametal probe that oscillates with high frequency. Theprobe is placed into the cell-containing sample andthe high-frequency oscillation causes a localized highpressure region resulting in cavitation and impaction,

ultimately breaking open the cells. Although the basictechnology was developed over 50 years ago, newersystems permit cell disruption in smaller samples(including multiple samples under 200 L inmicroplate wells) and with an increased ability tocontrol ultrasonication parameters.Disadvantages include:

Heat generated by the ultrasound process mustbe dissipated.

High noise levels (most systems require hearingprotection and sonic enclosures)

Yield variability Free radicals are generated that can react with

other molecules.

Detergent methodsDetergent-based cell lysis is an alternative to physicaldisruption of cell membranes, although it issometimes used in conjunction with homogenization

and mechanical grinding. Detergents disrupt the lipidbarrier surrounding cells by disrupting lipid:lipid,lipid:protein and protein:protein interactions. Theideal detergent for cell lysis depends on cell type andsource and on the downstream applications followingcell lysis. Animal cells, bacteria and yeast all have

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differing requirements for optimal lysis due to thepresence or absence of a cell wall. Because of thedense and complex nature of animal tissues, theyrequire both detergent and mechanical lysis to

effectively lyse cells.In general, nonionic and zwitterionic detergents aremilder, resulting in less protein denaturation uponcell lysis, than ionic detergents and are used todisrupt cells when it is critical to maintain proteinfunction or interactions. CHAPS, a zwitterionicdetergent, and the Triton X series of nonionicdetergents are commonly used for these purposes. Incontrast, ionic detergents are strong solubilizingagents and tend to denature proteins, thereby

destroying protein activity and function. SDS, andionic detergent that binds to and denatures proteins,is used extensively for studies assessing proteinlevels by gel electrophoresis and western blotting.In addition to the choice of detergent, otherimportant considerations for optimal cell lysis includethe buffer, pH, ionic strength and temperature.

The 'cell bomb'

A fifth laboratory-scale system for cell disruption israpid decompression or the "cell bomb" method. Inthis process, cells in question are placed under highpressure (usually nitrogen or other inert gas up toabout 25,000 psi) and the pressure is rapidlyreleased. The rapid pressure drop causes thedissolved gas to be released as bubbles thatultimately lyse the cell.Disadvantages include:

Only easily disrupted cells can be effectively

disrupted (stationary phase E. coli, yeast, fungi,and spores do not disrupt well by this method).

Large scale processing is not practical. High gas pressures have a small risk of personal

hazard if not handled carefully.

High-shear mechanical methods.

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High-shear mechanical methods for cell disruption fallinto three major classes: rotor-stator disruptors,valve-type processors, and fixed-geometryprocessors. (These fluid processing systems also are

used extensively for homogenization anddeaggregation of a wide range of materials usesthat will not be discussed here.) These processors allwork by placing the bulk aqueous media under shearforces that literally pull the cells apart. Thesesystems are especially useful for larger scalelaboratory experiments (over 20 mL) and offer theoption for large-scale production.

Rotor-stator Processors

Most commonly used as tissue disruptors.Disadvantages include:

Do not work well with difficult-to-lyse cells likeyeast and fungi

Often variable in product yield. Poorly suited for culture use.

Valve-type processorsValve-type processors disrupt cells by forcing themedia with the cells through a narrow valve underhigh pressure (20,00030,000 psi or 140210 MPa). Asthe fluid flows past the valve, high shear forces in thefluid pull the cells apart. By controlling the pressureand valve tension, the shear force can be regulated tooptimize cell disruption. Due to the high energiesinvolved, sample cooling is generally required,especially for samples requiring multiple passesthrough the system. Two major implementations ofthe technology exist: the French pressure cell press

and pumped-fluid processors.French press technologyuses an external hydraulicpump to drive a piston within a larger cylinder thatcontains the sample. The pressurized solution is thensqueezed past a needle valve. Once past the valve,the pressure drops to atmospheric pressure and

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generates shear forces that disrupt the cells.Disadvantages include:

Not well suited to larger volume processing. Awkward to manipulate and clean due to the

weight of the assembly (about 30 lb or 14 kg).Mechanically pumped-fluid processors function byforcing the sample at a constant volume flow past aspring-loaded valve.Disadvantages include:

Requires 10 mL or more of media. Prone to valve-clogging events. Due to variations in the valve setting and

seating, less reproducible than fixed-geometryfluid processors.

Fixed-geometry fluid processorsFixed-geometry fluid processors are marketed underthe name of Microfluidizer processors. Theprocessors disrupt cells by forcing the media with thecells at high pressure (typically 20,00030,000 psi or140210 MPa) through an interaction chambercontaining a narrow channel. The ultra-high shearrates allow for:

Processing of more difficult samples Fewer repeat passes to ensure optimum sample

processingThe systems permit controlled cell breakage withoutthe need to add detergent or to alter the ionicstrength of the media. The fixed geometry of theinteraction chamber ensures reproducibility.Especially when samples are processed multipletimes, the processors require sample cooling.Cell disruption by rapid decompression is one ofseveral methods of cell disruption and is also calledexplosive decompression or cell bomb.

Cell disruption by nitrogen decompressionLarge quantities of nitrogen are first dissolved in thecell under high pressure within a suitable pressure

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vessel. Then, when the gas pressure is suddenlyreleased, the nitrogen comes out of the solution asexpanding bubbles that stretch the membranes ofeach cell until they rupture and release the contents

of the cell.Nitrogen decompression is claimed to be moreprotective of enzymes and organelles than ultrasonicand mechanical homogenizing methods and tocompare favorably to the controlled disruptive actionobtained in a PTFE and glass mortar and pestlehomogenizer. While other disruptive methods dependupon friction or a mechanical shearing action thatgenerate heat, the nitrogen decompression procedureis accompanied by an adiabatic expansion that cools

the sample instead of heating it.The blanket of inert nitrogen gas that saturates thecell suspension and the homogenate offers protectionagainst oxidation of cell components. Although othergases: carbon dioxide, nitrous oxide, carbonmonoxide and compressed air have been used in thistechnique, nitrogen is preferred because of its non-reactive nature and because it does not alter the pHof the suspending medium. In addition, nitrogen is

preferred because it is generally available at low costand at pressures suitable for this procedure. Oncereleased, subcellular substances are not exposed tocontinued attrition that might denature the sample orproduce unwanted damage. There is no need to watchfor a peak between enzyme activity and percentdisruption. Since nitrogen bubbles are generatedwithin each cell, the same disruptive force is applieduniformly throughout the sample, thus ensuringunusual uniformity in the product. Cell-freehomogenates can be produced.

ApplicationsThese techniques are used to:

Homogenize cells and tissues Release intact organelles

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Prepare cell membranes Release labile biochemicals Produce uniform and repeatable homogenates

without subjecting the sample to extreme

chemical or physical stress.According to manufacturers of nitrogendecompression devices, the method is particularlywell suited for treating mammalian and othermembrane bound cells. It has also been usedsuccessfully for treating plant cells, for releasingvirus from fertilized eggs and for treating fragilebacteria. It is not recommended for untreatedbacterial cells. Yeast, fungus, spores and othermaterials with tough cell walls do not respond well to

this method.

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b.tech. Biotechnology Notes (2)