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Harrick Plasma is thrilled to announce a new collaboration effort with Alvéole, makers of PRIMO: Bioengineering Technology. PRIMO is a maskless photopatterning platform that offers precise control over the cellular microenvironment. Applications of Primo include Cryo-ET, cell culture, microfluidics, and spheroid formation.
Harrick Plasma’s benchtop plasma cleaners work well with PRIMO. Plasma treatment removes organic contamination and increases substrate hydrophilicity, enabling smooth, consistent deposition of polar coatings, such as Poly-L-lysine (PLL) or Alveole’s patented PLPP gel. Substrates cleaned with plasma prior to coating include Cryo-EM grids, glass slides or dishes, 96-well plates, PDMS, and much more.
Below you will find brief summaries and process conditions for PRIMO & Harrick Plasma applications. For more information on PRIMO, visit Alveole’s website or contact an Alveole representative.
Cryo-Electron Tomography (Cryo-ET)
Cryo-ET is a tool with growing importance in the study of cell biology as it enables researchers to study vitrified cells in near living condition. Using Alveole’s PRIMO platform, researchers can increase the number of cells available for analysis by controlling cell distribution. With random cell seeding, cells may adhere to metal grid bars or edges, where analysis is difficult if not impossible. By micropatterning the EM grids with ECM proteins, the cells can be centered in the grids to improve throughput and image quality. For more information, visit Alveole’s page on Cell Positioning for Cryo-ET.
Plasma cleaning is often reported to improve vitrification quality, enhancing Cryo-EM results. Please visit Harrick Plasma’s Cryo-EM page for more details.
Grid Preparation Steps:
- Plasma cleaning
- Power: High
- Time: 15-30 seconds
- Pressure: 200-1,000mTorr
- Gas: air or oxygen
- Place grids on coverslip with PDMS stencil for stability
- Coat slides with Poly-L-lysine (PLL) for 30 minutes
- Wash with HEPES (0.1 M, pH 8.5).
- Dissolve mPEG-SVA (5 kDa) at 100 mg/ml in HEPES (0.1 M, pH 8.5) and incubate for one hour
- Wash with mQ water
- Mix 1 μl of Alveole PLPP Gel with 2 μl of mQ water, apply to the grids and wait until dry
Through this process, m-PEG is grafted to PLL, making the grids cell and protein repellant. The resulting grids are prepared for micropatterning using PRIMO. For further process details, consult Alveole’s application note “ Increasing cryogenic electron microscopy experiment output with PRIMO” or contact Alveole customer support.
The Extracellular Matrix (ECM)
Cells depend on their surroundings for vital signals, relying heavily on chemical, topographical, and mechanical cues. By recapitulating the extracellular matrix (ECM) using precise and reproducible micropatterns, researchers can improve cell culture quality and enhance cell studies. The Alveole Primo platform enables researchers to finely control the deposition of biomolecules, and thus tailor substrates with specific cues. To learn more, visit Alveole’s page on Controlling the Cell Microenvironment.
Harrick Plasma cleaners can be used to clean glass slides and standard 96-well glass bottom plates prior to the deposition of poly-lysine (PLL) and ECM proteins. Plasma benefits cell studies by improving cell adhesion and biocompatibility.
Process Conditions
- Substrate: 96 Well Plates, Glass Bottom Dish, PDMS Gel
- Power: High, Med
- Time: 15-60s
- Pressure: 200-1,000mTorr
- Gas: Ambient Air, oxygen
Alveole & Harrick Plasma ECM Articles
Donker L, Houtekamer R, Vliem M, Sipieter F, Canever H, Gomez-Gonzalez M, Bosch-Padros M, Pannekoek W, Trepat X, Borghi N, and Gloerich M. “A mechanical G2 checkpoint controls epithelial cell division through E-cadherin-mediated regulation of Wee1-Cdk1”. Cell Reports 2022 41(2), 111475. 10.1016/j.celrep.2022.111475
Garbett D, Bisaria A, Yang C, et al. “T-Plastin reinforces membrane protrusions to bridge matrix gaps during cell migration”. Nature Communications 2020 11, 4818. 10.1038/s41467-020-18586-3
Guillamat P, Blanch-Mercader C, Pernollet G, et al. “Integer topological defects organize stresses driving tissue morphogenesis”. Nature Materials 2022 21, 588–597. 10.1038/s41563-022-01194-5
Melero C, Kolmogorova A, Atherton P, Derby B, Reid A, Jansen K, and Ballestrem C. “Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins”. JOVE 2019 doi:10.3791/60092
Versaevel M, Alaimo L, Seveau V. et al. “Collective migration during a gap closure in a two-dimensional haptotactic model”. Scientific Reports 2021 11, 5811. 10.1038/s41598-021-84998-w
Zhou W, Niu J, Xiao W, and Ou L. “Adsorption of bulk nanobubbles on the chemically surface-modified muscovite minerals”. Ultrason. Sonochem. 2019 51: 31-39 10.1016/j.ultsonch.2018.10.021
Microfluidics
The PRIMO platform and Harrick Plasma cleaners can be used to rapidly prototype microfluidic devices, eliminating long and complicated microfabrication steps. Poly(dimethylsiloxane) (PDMS) is an inexpensive, inert, and transparent material used widely to create microfluidic devices or chips. PDMS can easily be patterned with microchannels using soft lithography. However, one bottleneck in microfluidic device fabrication is development of a master mold, often a patterned silicon wafer or glass slide.
PRIMO photopatterning offers an opportunity to generate new master molds for microfluidics easily and reproducibly in lab on a daily bases. As a result, researchers can easily tune their designs and make changes as needed to improve their design. The devices have a wide range of applications including cell culture, organ-on-a-chip, and single cell sequencing. For more information, visit Alveole’s page on Microfabrication.
Plasma cleaning is an essential tool for microfluidic device fabrication as it is required for PDMS bonding. Air or oxygen plasma oxidize the surfaces of materials to be bonded, leaving reactive silanol (SiOH) groups. When two plasma treated surfaces are placed in contact, the silanol groups react to form bridging Si-O-Si bond at the interface, creating an irreversible seal. Visit Harrick Plasma’s PDMS Bonding page for more.
Process Conditions
Substrate: PDMS, Glass Slides
Harrick Plasma Equipment was featured in a Journal of Visual Experiments (JOVE) video showing surface preperation for Alveole PRIMO