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Cell disruption methodologies

Written by Maja Hunter, Ph.D.

Humans have tailored their surroundings to suit their needs for centuries. We have domesticated animals and cultivated plants through breeding programs that employ artificial selection and hybridization. Nowadays, the time-consuming but rewarding processes can be done quicker and more precisely using a genetic engineering approach. Modifications of living organisms are vital for natural sciences research and enable gathering knowledge of biological processes. Biotechnological industries use such findings to create products that simplify and improve our everyday life. A lot of important work is done in microorganisms, for example in Escherichia coli and yeast, however, often other microorganisms are used for industrial production. Is the desired product exported outside of the producer microorganism or cell, or should the cells be disrupted in order to access it?

Cell disruption is a frequently used method for releasing biological molecules from inside a cell. Different types of cells, e.g., mammalian, bacterial, yeast, plant cells and algal cells have unique characteristics and therefore different cell disruption techniques are applied. Chemical and enzymatic methods require chemicals and reagents, whereas mechanical methods require a mechanical device to disrupt cells.

Bead-beating

One of the mechanical methods is bead-beating. Glass, ceramic or steel beads, 0.1-2 mm in diameter, are used to disrupt very small sample sizes of all types of cellular material – from spores to animal and plant tissues, and in a short amount of time. Beads are added to a cell suspension in a vial, which is placed on a shaker that oscillates at about 2000 oscillations per minute. The energy produced from bead-beating warms up the sample, which can cause damage to heat-sensitive proteins, if the sample is not cooled during the process [1].

Freeze-thaw method

The freeze-thaw method breaks the bacterial and mammalian cells, due to swelling of the cells and formation of ice crystals during the freezing cycle. This approach is effective for the release of recombinant proteins from the cytoplasm of the cell but multiple cycles are necessary for efficient lysis, so the process is time consuming [2].

Ultrasonification

For extraction of compounds from plants, microalgae, seaweeds [3] and Gram-negative bacterial cells, e.g., E. coli, ultrasonification is usually used. The applied sound energy agitates particles in a sample and causes the cell disruption. However, yeast cells and the cell walls of Gram-positive bacteria are much harder to break and high pressure needs to be applied in order to disrupt them [1].

French Pressure Cell Press

French Pressure Cell Press, invented in the 1940s, utilizes high pressure to force cells through a narrow orifice, which causes cells to lyse due to the shear forces experienced across the pressure differential [4, 5]. Before processing, the sample needs to undergo a pre-processing step to remove cell clumps from the suspension that may clog the valve. This technology is not suitable for large sample volumes [1].

High-Shear Processing

High-pressure homogenization underwent a revolution with introduction of fixed geometry microchannels. The multiplier pump generates high pressures that forces the preprocessed sample through microchannels, generating high shear rates that rupture cells. Compared to the narrow orifice, the sample has a longer residence time inside the microchannels, therefore, every mL of the sample experiences the same pressure. Consequently, the results are consistent and reproducible. The temperature of the sample raises during processing, 1.7 °C per 1,000 psi or 69 bar, due to the supplied energy through shear and impact. The processed sample that exits the microchannels needs to be quickly and efficiently cooled down. Dyhydromatics ShearJet® processors have a heat-exchanger installed directly downstream the Reaction Chamber® module, which ensures high protein yields, and temperature-sensitive proteins remain active. After the first pass through the Reaction Chamber® module, the processed cell lysate contains large cell wall fragments (450 nm), therefore, the viscosity stays rather low [6]. The second pass further reduces the length of cell wall fragments.

High-shear processing has been proven throughout the years to fit into research laboratories just as well as into industrial production departments. Such technology is used for various applications in addition to cell disruption, such as nanoemulsions, nanosuspensions, nanodispersions, drug delivery vehicles and polysaccharide size reduction, just to name a few. The process that best suits your application in the research lab is linearly scalable to suit the production process, due to the Reaction Chamber® technology.

At Dyhydromatics, we have years of experience in the field of high-shear processors. Our hydraulic lab as well pilot scale high-pressure processors, (the Dyhydromatics ShearJet® HL60 and HP1200 processors), operate in the pressure range of 5-30 Kpsi (345-2068 bar) and were designed to simplify sample processing, to preserve sample volume and save valuable time.

  • Small volume. A 5-mL sample can be easily processed on the ShearJet® HL60 processor using syringes, as well as higher volumes using a glass or stainless-steel reservoir.

  • Practical. There is a sample recirculation option, if more passes are needed; and programable process duration – the pulse mode, where after the selected number of pulses the processing automatically stops.

  • Safe. The installed emergency stop (E-stop) is a safety feature that activates if the pressure reaches 40,000 psi. This happens if the Reaction Chamber® module is clogged, hydraulic oil is low, the temperature too high, or if the filter is clogged.

  • GMP-environment friendly. Dyhydromatics ShearJet® processors are able to fulfill strict requirements of the pharmaceutical industry as the procedures are readily recorded and the equipment can be cleaned or steamed in place (CIP/SIP). The product contact parts can also be autoclaved.

  • Easy to maintain. The ShearJet® HL60 processor contains no O-rings that need to be exchanged every year and contains only 5 high pressure connections, which reduces the possibility of leaking.

  • Efficient cooling. The jacketed helical coil is used for cooling the sample in combination with a chiller, which gives you total control of the sample temperature and therefore more reproducible results.

  • Quiet operation. Quieter than the competitive equipment - 68 db.

  • Linear scalability. Due to Reaction Chamber® technology, the processing conditions optimized in lab research can be used on a pilot or production scale. If you want to fine-tune the existing production process on the lab scale, the linear scalability rule applies as well.

 

With Dyhydromatics processors, 95 % lysis of E. coli cells is achieved in 1 pass through the 87 µm microchannels of the Reaction Chamber® module at 18,000 psi. Successful disruption of yeast cells, up to 95 % lysis, requires 4-5 passes (depending on the strain) and higher pressures, up to 30,000 psi through 87-100 µm microchannels. We offer proof-of-concept testing and look forward to finding the optimal processing conditions for your application!

If you are interested in our equipment or you want to learn more about our technology, don’t hesitate to reach out to our friendly customer service. 

Figure 1. Yeast cell disruption. (A) Before the processing and (B) after 10 passes at 30,000 psi 95 % lysis was achieved. The number of passes required depends on the microchannel diameter of the Reaction Chamber® module, temperature, yeast strain and applied pressure.

Literature

[1] Wikipedia. Cell disruption. https://en.wikipedia.org/wiki/Cell_disruption# [Last edited 9 November 2022], [Retrieved 17 February 2023].

[2] Thermo Fisher Scientific. Traditional Methods of Cell Lysis for Protein Extraction. https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/traditional-methods-cell-lysis.html [Last edited in 2022], [Retrieved 17 February 2023].

[3] Garcia-Vaquero, M.; Rajauria, G.; O'Doherty, J.V.; Sweeney, T. (2017-09-01). "Polysaccharides from macroalgae: Recent advances, innovative technologies and challenges in extraction and purification". Food Research International. 2017; 99(3): 1011–1020. doi:10.1016/j.foodres.2016.11.016. hdl:10197/8191.

[4] French CS, Milner HW, Koenig ML, and Macdowall FDH: The photochemical activity of isolated chloroplasts. Carn. Inst. Wash. Yearb. 1948; 47:91-93

[5] French CS, Milner HW: V. Carbon Dioxide Fixation and Photosynthesis. 232-250. Cambridge University Press, New York

[6] Agerkvist, Iréne and S O Enfors. “Characterization of E. coli cell disintegrates from a bead mill and high pressure homogenizers.” Biotechnology and Bioengineering 36 (1990): n. pag.

Posted December 14, 2023

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