Hielscher – Ultrasound Technology

Ultrasonic Lysis: Cell Disruption & Extraction

Cell disintegration or lysis is a common part of daily sample preparation in biotech laboratories. The goal of lysis is to disrupt parts of the cell wall or the complete cell to release biological molecules. The so-called lysate can consist in e.g. plasmid, receptor assays, proteins, DNA, RNA etc. Following steps of the lysis are fractionation, organelle isolation or/and protein extraction and purification. The extracted material (= lysate) has to be separated and is subject to further investigations or applications, e.g. for proteomic research. Ultrasonic homogenizers are a common tool for successful cell lysis. As the ultrasonic intensity can be leveled by adjusting the process parameters, the optimal sonication intensity from very soft to very hard can be set individually for each substance and medium to meet the specific application requirement.

Cell Structure

Cells are protected by a semi-permeable plasma membrane which consists in a phospho-lipid bilayer (also protein-lipid bilayer; formed by hydrophobic lipids and hydrophilic phosphorus molecules with embedded protein molecules) and creates a barrier between the cell interiors (cytoplasm) and the extracellular environment. Plant cells and prokaryotic cells are surrounded by a cell wall. Due to multiple layers thick cell wall of cellulose, plant cells are harder to lyse than animal cells. The cell interior, such as organelles, nucleus, mitochondrion, is stabilized by the cytoskeleton.
By lysing the cells, it is aimed at extracting and separating the organelles, proteins, DNA, mRNA or other biomolecules.

Ultrasonicators are a commonly used tool for the sample preparation of cell matter.

Fig. 1: Ultrasonic extraction from cells: the microscopic transverse section (TS) of apical stem of mint (Mentha piperita) shows the mechanism of actions during ultrasonic extraction from cells (magnification 2000x) [resource: Vilkhu et al. 2011]

Methods

There are several methods to lysate cells, which can be divided into mechanical and chemical methods, which include the use of detergents or solvents, the application of high pressure, or the use of a bead mill or of a french press. The most problematic disadvantage of these methods is the difficult control and adjustment of process parameters and thereby impact.
The main disadvantages of common lysis methods:

Different lysis techniques

Table: Conventional methods of cell lysis have major disadvantages

On the contrary, sonication is a very efficient and reliable tool for cell disintegration that allows for a complete control over the sonication parameters. This ensures a high selectivity on materials release and product purity. [Balasundaram et al. 2009] It is suitable to all cell types and easily applicable in small and large scale. Ultrasonicators are easy to clean. An ultrasonic homogenizer always features clean-in-place (CIP) and sterilize-in-place (SIP) function. The sonotrode consists in a massive titanium horn which can be wiped or flushed in water or solvent (depending on the working medium). The maintenance of ultrasonicators is due to their robustness almost neglectable.

Lysis

Lysis is a sensitive process. During the lysis the protection of cell membrane is destroyed, however the inactivation, denaturation and degradation of the extracted proteins by an unphysiological environment (deviation from pH-value) must be prevented. Therefore, in general lysis is carried out in a buffer solution. Most difficulties arise from uncontrolled cell disruption resulting in an untargeted release of all intracellular material or/ and the denaturation of the target product.

Ultrasonic cell lysis can be used for sample preparation in the lab and for production of large volumes. Click to enlarge!

Fig. 1: 200 watts powerful ultrasonic homogenizer UP200Ht with digital control and automatic data recording for reliable and reproducible cell lysis.

Ultrasonic Lysis

Generally, the lysis of samples in the lab will take between 15 seconds and 2 minutes. As the intensity of sonication is very easy to adjust by amplitude setting an sonication time as well as by choosing the right equipment, it is possible to disrupt cell membranes very gently or very abruptly, depending on the cell structure and on the purpose of lysis (e.g. DNA extraction requires softer sonication, complete protein extraction of bacteria requires a more intense ultrasound treatment). The temperature during the process can be monitored by an integrated temperature sensor and can be easily controlled by cooling (ice bath or flow cells with cooling jackets) or by sonication in pulsed mode. During pulse-mode sonication, short sonication burst cycles of 1-15 seconds duration allow for heat dissipation and cooling during the longer intermittent periods.
All ultrasound-driven processes are completely reproducible and linearly scalable.

Ultrasonic homogenizer VialTweeter for the simultaneous sample preparation of up to 10 test tubes. (Click to enlarge!)

The ultrasonic device VialTweeter allows for simultaneous sample preparation of up to 10 vials under same process conditions.

Ultrasonic Homogenizers

Various types of ultrasonic devices allow for matching the sample preparation goal and to assure user-friendliness and operation comfort. Probe-type ultrasonicators are the most common devices in the lab. They are most suitable for the preparation of small and mid-size samples with volumes of 0.1mL up to 1000mL. Different power sizes and sonotrodes allow to adapt the ultrasonicator to the sample volume and the vessel for most effective and efficient sonicating results. The ultrasonic probe device is the best choice when single samples have to be prepared.

If more samples have to be prepared, e.g. 8 vials of cell solution, devices such as the VialTweeter or an ultrasonic cuphorn are the most suitable homogenizer for lysis. Several vials are sonicated at the same time, at the same intensity. This saves not only time, but ensures also the same treatment of all samples, which makes the results among the samples reliable and comparable. Furthermore, cross-contamination by immersing the ultrasonic sonotrode (also known as ultrasonic probe, horn, tip, or finger) is avoided. Since vials are used, time-consuming clean-up and sample loss due to decanting of vessels are omitted.
For higher volumes, e.g. for the commercial production of cell extracts, continuous ultrasonic systems with a flow cell reactor are most suitable. The continuous and even flow of the processed material assures an even sonication. All parameters of the ultrasonic disintegration process can be optimized and adjusted to the requirements of the application and the specific cell material.

Exemplary procedure for ultrasonic lysing of bacterial cells:

  1. Preparation of the cell suspension: Cell pellets must be completely suspended in a buffer solution by homogenizing (choose your buffer solution compatible with following analysis, e.g. specific chromatography method). Add lysozymes and/ or other additives, if needed (they must be also compatible with separation/ purification means). Mix/ homogenize the solution gently under mild sonication until complete suspension is achieved.
  2. Ultrasonic lysis: Place the sample in an ice bath. For cell disruption, sonicate the suspension at 60-90 second bursts (using your ultrasonicator’s pulse mode).
  3. Separation: Centrifuge the lysate (e.g. 10 min. at 10,000 x g; at 4degC). Separate the supernatant from the cell pellet carefully. The supernatant is the total cell lysate. After filtration of the supernatant, you obtain a clarified fluid of the soluble cell protein.



The most common applications for ultrasonicators in biology and biotechnology are:

  • Cell extract preparation
  • Disruption of yeast, bacteria, plant cells, soft & hard cell tissue, nucleic material
  • Protein extraction
  • Preparation and isolation of enzymes
  • Production of antigens
  • DNA extraction and/ or targeted fragmentation
  • Liposome preparation
High power ultrasound homogenizers like the UIP1000hd are used for cell disruption and extraction in batch or continuous flow-though mode. (Click to enlarge!)

Ultrasonic benchtop systems such as the UIP1000hd (1kW) can process larger volumes of biological material.

The manifold applications of ultrasound branches out in the sectors of biotechnology, bioengineering, microbiology, molecular biology, biochemistry, immunology, bacteriology, virology, proteomics, genetics, physiology, cellular biology, hematology, and botany.

For evaluation and optimization of customers’ applications, Hielscher Ultrasonics offers a fully equipped ultrasonic process laboratory. Please contact us for further information!

Literature/References

  • Balasundaram, B.; Harrison, S.; Bracewell, D. G. (2009): Advances in product release strategies and impact on bioprocess design. Trends in Biotechnology 27/8, 2009. pp. 477-485.
  • Vilkhu, K.; Manasseh, R.; Mawson, R.; Ashokkumar, M. (2011): Ultrasonic Recovery and Modification of Food ingredients. In: Feng/ Barbosa-Cánovas/ Weiss (2011): Ultrasound Technologies for Food and Bioprocessing. New York: Springer, 2011. pp. 345-368.

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