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. Ultrasonic homogenizers are widely used for successful cell lysis. The major advantage of sophisticated ultrasonicators is the precise control over process parameters such as intensity and temperature, which allows for a gentle, yet highly efficient cell disruption and extraction.

Cell Lysis using Ultrasound

Ultrasonic cell lysis and extraction is a method used to break open cells and extract the contents using high-frequency sound waves, i.e. ultrasound. Sonication is a lysis technique, which is widely used as an established and reliable methods of cell disruption and extraction of intracellular material. Ultrasonic lysis is a reliable technique to prepare lysate containing e.g. plasmid, receptor assays, proteins, DNA, RNA etc. As the ultrasonic intensity can be levelled by adjusting the process parameters, the optimal sonication intensity from very soft to intense can be set individually for each substance and medium to meet specific application requirements. 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.

Compared to other cell lysis and extraction methods, ultrasonic cell lysis has several advantages:

1. Speed: Ultrasonic cell lysis and extraction is a fast method that can break open cells in a matter of seconds. This is much faster than other methods such as homogenization, freeze-thawing or bead-milling.
2. Efficiency: Ultrasonic cell lysis and extraction can be used to treat small, large or multiple samples at once, making it more efficient than other methods that require individual processing of small samples.
3. Chemical-free: Ultrasonic cell lysis and extraction is a non-invasive method that does not require the use of harsh chemicals or enzymes. This makes it ideal for applications where the integrity of the cell contents needs to be maintained. Unwanted contamination of samples can be avoided.
4. High yield: Ultrasonic cell lysis and extraction can extract a high yield of cellular contents, including DNA, RNA, and proteins. This is because the high-frequency sound waves break open the cell walls and release the contents into the surrounding solution.
5. Temperature Control: Sophisticated ultrasonciators allow for precise temperature control of the sample. Hielscher digital sonicators are equipped with a pluggable temperature sensor and temperature monitoring software.
6. Reproducible: Protocols for ultrasonic cell lysis can be easily reproduced and even matched to different larger or smaller sample volumes by a simple linear scale-up.
7. Versatile: Ultrasonic cell lysis and extraction can be used to extract a wide range of cell types, including bacteria, yeast, fungi, plant and mammalian cells. It can also be used to extract different types of molecules, including proteins, DNA, RNA, and lipids.
8. Simultaneous preparation of numerous samples: Hielscher Ultrasonics offers several solutions to comfortably process numerous samples under the exactly same process conditions. This makes the sample preparation step of lysis and extraction highly efficient and time-saving.
9. Easy to use: Ultrasonic cell lysis and extraction equipment is easy to use and requires minimal training. The equipment is also economical as it is a single investment with no requirement for re-purchasing of disposals. This makes it attractive for a wide range of researchers and laboratories.

Overall, ultrasonic cell lysis and extraction is a fast, efficient, precisely controllable, and versatile method for extracting cellular contents. Its advantages over alternative methods make it an attractive choice for a wide range of research and industrial applications.

Working Principle of Ultrasonic Cell Lysis

Ultrasonic cell lysis and extraction uses high-frequency sound waves to disrupt cells and extract their contents. The sound waves create pressure changes in the surrounding liquid, causing small bubbles to form and collapse in a process known as cavitation. These bubbles generate localized highly intense mechanical forces that can break open cells and release their contents into the surrounding solution.

Cell lysis using an ultrasonicator commonly involves the following steps:

• The sample is placed in a tube or container with a liquid buffer.
• An ultrasonic probe is inserted into the sample, and high-frequency sound waves with approx. 20-30 kHz are applied.
• The ultrasound waves cause oscillation and cavitation in the surrounding liquid, generating localized forces that break open cells and release their contents.
• The sample is centrifuged or filtered to remove any cellular debris, and the extracted contents are collected for downstream analysis.

Powerful ultrasound waves disrupt the cell matrix of biological structures and release the bioactive compounds. Mass transfer between the plant material and the solvent (e.g., buffer) is intensified. (graphic: ©Vilkhu et al., 2006)

Disadvantages of Common Lysis Methods

During your work in laboratories, you might have already experienced the hassle of cell lysis using traditional mechanical or chemical lysis protocols.

• Mechanical lysis: Mechanical lysis methods, such as grinding with mortar and pestle or homogenization using a french press, bead mill, or rotor-stator system often lack precice control and adjustment options. This means the use of milling and grinding can quickly generate heat and shear forces that can damage the sample and denature proteins. They can also be time-consuming and require large amounts of starting material.
• Chemical lysis: Chemical lysis methods, such as detergent-based lysis, can damage the sample by disrupting the lipid bilayer and denaturing proteins. They can also require multiple steps and may leave residual contaminants that interfere with downstream applications. Finding the optimum dosage of detergent is an additional challenge.
• Freeze-thaw cycles: Freeze-thaw cycles can cause cell membranes to rupture, but repeated cycles can also cause protein denaturation and degradation. This method can also require multiple cycles, which can be time-consuming and often results in lower yields.
• Enzymatic lysis: Enzymatic lysis methods can be specific to certain cell types and require multiple steps, making them time-consuming. They also generate waste and require careful optimization to avoid degradation of the sample. Enzymatic lysis kits are often expensive. If your current enzymatic lysis procedure gives insufficient results, sonication as synergistic method can be applied to intensify cell disruption.

In contrast to conventional mechanical and chemicals cell lysis methods, 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. [cf. Balasundaram et al., 2009]
It is suitable to all cell types and easily applicable in small and large scale – always under controlled conditions. 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.

Ultrasonic Lysis and Cell Disruption

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 Homogenizers for Cell Lysis and Extraction

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. 8-10 vials of cell solution, an intense indirect sonication with ultrasonic systems such as the VialTweeter or an ultrasonic cuphorn are the most suitable homogenization method for an efficient 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, during indirect sonication cross-contamination by immersing the ultrasonic sonotrode (also known as ultrasonic probe, horn, tip, or finger) is avoided. Since individually to sample size matched vials are used, time-consuming clean-up and sample loss due to decanting of vessels are omitted. For the uniform sonication of multiwell or microtiter plates, Hielscher offers the UIP400MTP.
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:

• 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.
• 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).
• 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 or hard cell tissue, nucleic material
• Protein extraction
• Preparation and isolation of enzymes
• Production of antigens
• DNA extraction and/ or targeted fragmentation
• Liposome preparation