Cell seeding on BIOMIMESYS®Hepatocyte hydrogels with the VIAFLO 96/384 channel handheld electronic pipette

  • Cell seeding on BIOMIMESYS®Hepatocyte hydrogels with the VIAFLO 96/384 channel handheld electronic pipette

    Creating 3D cell spheroids suitable for high content screening (HCS)

    Creating three-dimensional (3D) spheroids suitable for high content screening (HCS) may be difficult and is often an expensive process. Here, Celenys – now part of HCS Pharma – and INTEGRA describe how a 96 channel pipette can help seeding and maintenance of a 3D cell culture with BIOMIMESYS® Hepatocyte hydrogels in microplates. The method achieves physiologically relevant cellular models in a high throughput screening (HTS) format.

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      Creating three-dimensional (3D) spheroids suitable for high content screening (HCS) may be difficult and is often an expensive process. Here, Celenys – now part of HCS Pharma – and INTEGRA describe how a 96 channel pipette can help seeding and maintenance of a 3D cell culture with BIOMIMESYS® Hepatocyte hydrogels in microplates. The method achieves physiologically relevant cellular models in a high throughput screening (HTS) format.

    Key benefits

    • The accurate z-height and pipetting speed set-up of the VIAFLO 96/384 handheld electronic pipette enables worry-free cell seeding, removal of culture media and drug compound testing.
    • The 96 channel head processes all the microplate wells at once, saving a lot of time. Fewer steps are required compared to a multichannel pipette, which minimizes the risk of errors and improves the reproducibility of cell cultures.
    • The small footprint VIAFLO 96/384 fits easily into a laminar flow cabinet, making the device ideal for all kinds of cell culture applications.

    Overview: BIOMIMESYS hydrogels for 3D culture 

    BIOMIMESYS is a range of new patented hyaluronic acid scaffolds for 3D cell culture. Celenys scaffold is made of RGDS and galactosamine-grafted hyaluronic acid, adipic acid dihydrazide crosslinker, and extracellular matrix (ECM) proteins (collagen type I and IV). BIOMIMESYS is suitable for robotic tests due to the thickness of the scaffold – around 600 μm – with an average porosity ranging from 100 to 200 μm (see Figure 1).

    Figure 1: BIOMIMESYS synthesis and SEM observation of a section of the final hydrogel product
    Figure 1: BIOMIMESYS synthesis and SEM observation of a section of the final hydrogel product

    Experimental set-up

    The VIAFLO 96/384 is used for seeding and renewing cell medium. The device is equipped with a 96 channel 300 μl pipetting head and 300 μl Sterile, Filter GripTips are used. The entire application is performed in a laminar flow cabinet.

    Figure 2: HepG2 seeding on a 96 well plate containing BIOMIMESYS Hepatocyte hydrogels using a 96 channel head on the VIAFLO 96/384
    Figure 2: HepG2 seeding on a 96 well plate containing BIOMIMESYS Hepatocyte hydrogels using a 96 channel head on the VIAFLO 96/384

    Step-by-step procedure

    1. Cell seeding on BIOMIMESYS hydrogel with the VIAFLO 96/384

    Homogenize HepG2 cells and seed on the ready-to-use BIOMIMESYS Hepatocyte hydrogel. This step takes less than two minutes.

    HepG2 cells are homogenized in an automation friendly reagent reservoir (150 ml or 300 ml) by aspiration and dispensing. This is achieved using the Pipet/mix mode of the VIAFLO 96/384. Then, cells are transferred into a 96 well plate containing the BIOMIMESYS Hepatocyte hydrogel. Selecting the right height (z-height limit: 2.9 mm) and pipetting speed (set on 2) settings on the VIAFLO 96/384 is essential to guarantee homogenous seeding of the cells on the hydrogel (see Figure 2). Cell medium (150 μl) is added by setting the z-height limit to 3.2 mm and the pipetting speed to 2.

    2. Chlorpromazine treatment of HepG2 cell line

    Remove culture medium and add the hepatotoxicity-inducing drug chlorpromazine.

    Carefully remove 100 μl of medium and add 100 μl of the drug, using a pipetting speed of 1 to prevent harming the cells. A z-height limit of 3 mm is set to ensure spheroids are not accidently aspirated or destroyed by the pipette tip.

    3. HepG2 viability assay

    Determine the metabolic activity of HepG2 cells using a WST-1 assay.

    Remove 100 μl of culture medium using the normal Pipet mode at speed 2 and a z-height limit of 3 mm. Add 10 μl of the WST-1 reagent using the same pipette settings described in step 2.

    4. Biliary canaliculi activity of HepG2

    Biliary canaliculi activity of HepG2 cells treated with chlorpromazine was examined using 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDFDA) (21884, Sigma).

    Remove 100 μl of culture medium and add 200 μl of PBS to the HepG2 cells, using the normal Pipet mode with the same z-height and speed described in step 2. Wash the HepG2 cells by adding and removing 200 μl of PBS (repeat this step five times). The convenient three position stage of the VIAFLO 96/384 accommodates the waste and reagent reservoirs (positions A and AB, respectively) needed for the washing step, and a target sample plate (position B).

    Conclusion

    • Easy liquid handling, time savings and high accuracy in generating identical data sets.
    • Very low variability in cellular metabolic activity between wells.
    • Treatment, seeding and viability assay are all performed with the VIAFLO 96/384.
    • Easy observation using a microscope.

    April 2020, by

    • Lydiane Saucède, PhD

      Application Scientist

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    • Jan-Peter Baldin, PhD

      Product Manager

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    Any questions? I'm happy to help!

    • Tom Bentivegna

      Product Manager

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    Used instruments and accessories

    VIAFLO 96/384, Handheld Electronic Pipette 96 Channel Pipetting Head, 300 µl Plate holder, spring loaded, with slide function Automation Friendly Reagent Reservoirs, 150 ml GripTip, 300 µl, Sterile, Filter Hepatocyte plate

    VIAFLO 96/384, Handheld Electronic Pipette

    With this 24, 96 and 384 channel pipette, INTEGRA meets the need for an affordable, compact and easy to use pipette that provides increased productivity. The unique operating concept makes VIAFLO 96/384 as easy to use as any traditional handheld pipette.

    Part No. 6001 / 6031

    See how it work
    The VIAFLO 96/384 is handheld, electronic pipette

    96 Channel Pipetting Head, 300 µl

    Users can simply change the pipetting head of their VIAFLO 96 and VIAFLO 384 to guarantee optimal matching of the available volume range to the application performed. Changing a pipetting head of VIAFLO 96 and VIAFLO 384 is easy and takes less than one minute.

    Part No. 6103

    Find out more about VIAFLO 96/384 Pipetting Heads Specifications
    VIAFLO 96/384 96 channel pipetting head

    Plate holder, spring loaded, with slide function

    For 24, 96 and 384 channel pipetting.Available for deck position A (6215) and B (6220), these plate holders feature a spring load mechanism to secure microplates in place. Both holders also feature the slide function to access 384 well plates with a 96 channel head.

    Part No. 6215 / 6220

    Overview all about plate holder options

    Automation Friendly Reagent Reservoirs, 150 ml

    150 ml Automation Friendly Clear Advantage™ Reservoirs (Polystyrene) which are made of crystal clear 100 % virgin polystyrene and fit into a reusable automation friendly base with bold, crisp, clearly visible graduation markings.

    The SureFlo™ Array located on the flat bottom reservoir prevents pipette tip seal off. This allows users to rest the pipette tips directly on the bottom of the reservoir offering an incredibly low dead volume of less than 3 ml!

    Find out more about Automation Friendly Reagent Reservoirs
    Reservoir for automation with SureFlo™ anti-sealing array

    GripTip, 300 µl, Sterile, Filter

    GripTip pipette tips perfectly match the multi-lobe tip fitting, snapping firmly on during loading. This guarantees a perfect seal on every tip, preventing them from loosening, leaking or completely falling off. All tips are precisely aligned horizontally, enabling accurate touch-offs, even when pipetting with 384 tips.

    Part No. 6435

    Learn more about GripTips pipette tips for Benchtop Pipetting Systems
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    Hepatocyte plate

    BIOMIMESYS® Liver hydroscaffold is made of RGDS and galactosamine-grafted Hyaluronic Acid, Adipic Acid dihydrazide crosslinker and extracellular matrix (ECM) proteins (collagen type I and collagen type IV) to mimic liver-ECM composition. 

    Source: Website HCS Pharma

    Visit supplier website

    Customer’s voice

    The best pipette we ever bought! The VIAFLO 96 has transformed the speed and accuracy of all of our plate based assays. Not sure what we did before we had it. The instrument is robust and reliable so that all members of the team can use it. It is portable and can be moved in and out of hoods as needed. Importantly, it has saved us a huge amount of time.

    Farhat Khanim, University of Birmingham