Semi-automated medium exchange in Akura™ 96 and 384 Spheroid Microplates with the VIAFLO 96 and VIAFLO 384

  • Semi-automated medium exchange in Akura™ 96 and 384 Spheroid Microplates with the VIAFLO 96 and VIAFLO 384

    Efficient and precise liquid handling protocol with the VIAFLO 96 and VIAFLO 384

    3D human in vitro cell models are gaining increased importance in the drug discovery and development cycle of the pharmaceutical industry as they mimic the human in vivo responses of newly developed substances more precisely than 2D models. They can help to bridge the gap between the traditional 2D in vitro models and whole-animal systems as the traditional models do not effectively recapitulate the more complex signalling and cellular interactions found in vivo. Spheroids are scaffold-free 3D cell culture models that are aggregated from one or more organ-specific cell types.

    InSphero has developed 3D cell-based assay solutions in the fields of liver toxicology, metabolic diseases, and oncology. Industrial use of these complex in vitro systems requires a perfect match of the tissue model, cell culture plates and liquid handling instrumentation. InSphero’s Akura™ 96 and 384 Spheroid Microplates have been engineered for the generation, long-term cultivation, observation and testing of spheroids in 96 and 384 well formats in a simple, flexible, and automation-compatible platform. Its scalable format requires liquid handling systems for convenient, yet quick, reliable, and precise medium exchange, sampling, dosing, and spheroid transfer from multiple plates. The semi-automated VIAFLO 96 and VIAFLO 384 electronic pipettes are ideal for these routine lab tasks, offering 96 and 384 channel pipetting in a compact and easy-to-use platform, for increased productivity.

  • Table of contents

    3D human in vitro cell models are gaining increased importance in the drug discovery and development cycle of the pharmaceutical industry as they mimic the human in vivo responses of newly developed substances more precisely than 2D models. They can help to bridge the gap between the traditional 2D in vitro models and whole-animal systems as the traditional models do not effectively recapitulate the more complex signalling and cellular interactions found in vivo. Spheroids are scaffold-free 3D cell culture models that are aggregated from one or more organ-specific cell types.

    InSphero has developed 3D cell-based assay solutions in the fields of liver toxicology, metabolic diseases, and oncology. Industrial use of these complex in vitro systems requires a perfect match of the tissue model, cell culture plates and liquid handling instrumentation. InSphero’s Akura™ 96 and 384 Spheroid Microplates have been engineered for the generation, long-term cultivation, observation and testing of spheroids in 96 and 384 well formats in a simple, flexible, and automation-compatible platform. Its scalable format requires liquid handling systems for convenient, yet quick, reliable, and precise medium exchange, sampling, dosing, and spheroid transfer from multiple plates. The semi-automated VIAFLO 96 and VIAFLO 384 electronic pipettes are ideal for these routine lab tasks, offering 96 and 384 channel pipetting in a compact and easy-to-use platform, for increased productivity.

Key benefits

  • The ultra-low attachment Akura 96 and 384 Spheroid Microplates allow convenient scaffold-free formation of spheroids – one per well – below 750 μm via cellular self-assembly.
  • The special well design featuring the SureXchange™ tapered ledge enables an easy medium exchange and near-complete removal in a single pipetting step, preventing spheroid loss and increasing yield during long-term spheroid growth and analysis.
  • The 1 mm diameter flat-bottom observation window enables simple spheroid observation, and a greater distance between the observation windows of adjacent wells reduces well-to-well imaging crosstalk compared to standard 96 and 384 well plates.
  • An accurate pipetting system, such as the VIAFLO 96 and VIAFLO 384, is crucial to ensure seeding of the same number of cells into all wells throughout the plate and produce highly uniform spheroid models. It also enables the addition of the correct concentrations of test compounds and guarantees an equal medium exchange between different wells to ensure responses with a high reproducibility.
  • Several user-defined position settings are available for delicate pipetting operations such as medium exchange. The Z height can be set to ensure that all subsequent operations are performed at the same height and the same amount of medium is removed without inadvertently aspirating the spheroid. The pipette tip can be precisely positioned above the spheroid for a reliable transfer without accidental damage.
  • A very helpful feature of INTEGRA’s VIAFLO 96 and VIAFLO 384 is the wide range of adjustable pipetting speeds. This ensures a safe medium exchange without the risk of strong turbulences that could cause spheroids to be damaged by shearing forces or to float.

Overview: How to perform medium exchange in Akura 96 and 384 Spheroid Microplates with VIAFLO 96 and VIAFLO 384

Experimental set-up

The VIAFLO 96 and VIAFLO 384 are used for medium exchange in Akura 96 and 384 plates. The liquid handling steps are optimized to ensure there is no spheroid loss. The VIAFLO 96 is equipped with a 96 channel pipetting head (10-300 µl) and both spring loaded and standard plate holders. 300 µl Sterile, Filter GripTips are used. The spheroids are cultured in Akura 96 plates with 70 µl of medium. The VIAFLO 384 is set up with a 384 channel pipetting head (5-125 µl) and both spring loaded and standard plate holders. 125 µl Sterile, Filter GripTips are used. Spheroids are cultured in Akura 384 plates with 50 µl of medium. Customized VIALINK programs are provided for every step of the protocol.

 

Downloads: App note and protocols for the Akura™ 96 and 384 Spheroid Microplates with the VIAFLO 96 and VIAFLO 384

Here you can download the complete protocols used in this application and use them on your VIAFLO 96 and VIAFLO 384. The protocol can be modified and transferred directly to the pipette with the VIALINK software.

Step-by-step procedure

Overview of the process:

  • Program 1: Medium aspiration – Akura 96
  • Program 2: Medium dispensing – Akura 96
  • Program 3: Medium aspiration – Akura 384
  • Program 4: Medium dispensing – Akura 384

1. Medium aspiration – Akura 96

Medium is aspirated from four Akura 96 plates and discarded or sampled

Load 300 µl Sterile, Filter GripTips onto the VIAFLO 96. Place a 300 ml automation friendly reagent reservoir on position B. Place an Akura 96 lid with the opening facing down on position A. When fixing the lid in position, make sure that its orientation notch does not face the spring of the plate holder (see video 1). Place the Akura 96 plate containing the spheroids onto the lid (see video 1). This allows the plate position to be manually adjusted to create a small horizontal offset (1-2 mm) with respect to the tips (Figure 1a and Figure 2). This ensures that the pipette tips are positioned on the SureXchange ledge of the well, preventing unintended aspiration of spheroids.

Video 1: How to place an InSphero Akura 96 Spheroid Microplate on the VIAFLO 96.
Graphic showing the correct pipette tip positioning on the SureXchange ledge of an Akura 96 plate during medium aspiration and dispense.
Figure 1: a) Correct pipette tip positioning on the SureXchange ledge of an Akura 96 plate during medium removal and b) medium addition.

Select the custom program 'M_ASP_96_MAN'. The pipette aspirates 75 µl of medium at speed 1. This low speed minimizes the shearing force that the spheroid could be subjected to. The aspiration volume is set higher than the volume of the well (70 µl) to ensure complete removal of the medium. The special design of the well protects the spheroids and leaves a defined residual volume of 5-7 µl. This step is repeated 3 times, changing the plate containing the spheroids in between each step. When the last aspiration step is finished, move the pipette head to position B. A prompt on the pipette screen informs the user that the medium needs to be purged to the waste reservoir or into a collection plate. The purge step is performed with speed 8. Eject and discard the tips afterwards. The last step in the program is a loop, which means that the 4 aspiration steps and the final purging step are repeated up to 100 times, allowing processing of up to 400 plates. Abort the program once all the plates have been processed.

If the medium is intended to be sampled for downstream analysis, it must be dispensed after each aspiration step into a respective sampling plate.

The Akura 96 plate is adjusted to have a small horizontal offset with respect to the tips.
Figure 2: Horizontal offset of the Akura 96 plate on the VIAFLO 96 with respect to the tips.

Tips:

  • Use Sterile, Filter GripTips for contamination-free processing.
  • The Run button is pressed twice to purge the liquid when waste disposal is required. This warning step supports the user and makes sure that the waste is not dispensed back into the wells and is an important feature when exchanging the medium on several plates at once.

2. Medium dispensing – Akura 96

Fresh medium is dispensed into the wells of four Akura 96 plates from the reservoir

Discard the used tips and load new 300 µl Sterile, Filter GripTips onto the VIAFLO 96. Place an automation friendly reagent reservoir with fresh medium on position B. Allow up to 10 ml of extra medium to prevent aspiration of air.

Place an Akura 96 plate onto an Akura 96 lid in position A. Adjust the plate manually to create a small horizontal offset (1-2 mm). Select and run the custom program 'M_DISP_96_MAN'. Move the pipette head to the reservoir on position B. The pipette aspirates 290 µl of fresh medium, which is sufficient to fill four plates with 70 µl medium plus 10 µl of excess volume. Move the pipette head to position A and gently immerse the pipette tips into the wells of the Akura 96 plate until the minimum height is reached (Figure 1b). The pipette dispenses 70 µl medium into each well at a slow speed. The dispensing step is repeated 3 times, changing the plate after each step. The pipette will prompt the user to purge the medium that remains in the tip. The aspiration and dispensing process of four plates is repeated up to 100 times due to a loop at the end of the program.

Tip:

  • Low pipetting speed can be set up in the VIALINK program to avoid damaging the delicate spheroids.

3. Medium aspiration – Akura 384

Medium is aspirated from one or more Akura 384 plates and discarded or sampled

Load 125 µl Sterile, Filter GripTips on the VIAFLO 384. Place an empty 300 ml automation friendly reagent reservoir on position B.

Place an Akura 384 lid with the opening facing down on position A. To fixing the lid in position, make sure that the orientation notch of the lid does not face the spring of the plate holder (see video 2). Place the Akura 384 plate containing the spheroids onto the lid (see video 2). This allows the plate to be manually adjusted towards the user (‘Bottom of plate’, Figure 3), creating a small horizontal offset (1-2 mm) with respect to the tips. This ensures that the pipette tips are positioned on the SureXchange ledge of the well, preventing unintended aspiration of spheroids (Figure 4a).

Video 2: How to place an InSphero Akura 384 Spheroid Microplate on the VIAFLO 384.

Select the custom program 'M_ASP_384_MAN'. The pipette aspirates 70 µl at speed 1. This low speed minimizes the shearing force that the spheroid could be subjected to. The aspiration volume is set higher than the volume of the well (50 µl) to ensure complete removal of the medium. The special design of the well protects the spheroids and leaves a defined residual volume of 2-3 µl. A prompt on the pipette screen informs the user that the medium needs to be purged to the waste reservoir or into a collection plate. The last step in the program is a loop, which means that the required pipetting steps can be easily repeated for multiple plates without the need to restart the program. Abort the program, eject, and discard the tips once all the plates have been processed.

Graphic showing the specific well design of the Akura 384 plate with the SureXchange ledge.
Figure 3: Well design of the Akura 384 plate showing the SureXchange ledge.
The correct pipette tip positioning on the SureXchange ledge of an Akura 96 plate during medium aspiration and dispensing shown graphically.
Figure 4: Correct pipette tip positioning on the SureXchange ledge of an Akura 384 plate during a) medium removal and b) medium addition.

4. Medium dispensing – Akura 384

Fresh medium is dispensed into two Akura 384 plates from the reservoir

Discard the used tips and load new 125 µl Sterile, Filter GripTips onto the VIAFLO 384. Place an automation friendly reagent reservoir with fresh medium on position B. Allow up to 10 ml of extra medium to prevent aspiration of air.

Place an Akura 384 plate onto an Akura 384 lid in position A. Adjust the plate manually towards the user (‘Bottom of plate’) to create a small horizontal offset (1-2 mm). Select and run the custom program 'M_DISP_384_MAN'. Move the pipette head to the reservoir on position B. The pipette aspirates 110 µl fresh medium, which is sufficient to fill two plates with 50 µl medium plus 10 µl of excess volume. Move the pipette head to position A and gently immerse the pipette tips into the wells of the Akura 384 plate until the minimum height is reached (Figure 4b). The pipette dispenses 50 µl medium into each well at slow speed. The dispensing step is repeated once more for a second well plate. The pipette will prompt the user to purge the medium that is remaining in the tip. The dispensing process for two plates is repeated up to 100 times due to a loop at the end of the program.

Tip:

  • Z heights may vary from system to system, depending on the calibration. Check the correct height using a dummy plate. Pipette tips need to be positioned 0.2-0.3 mm above the spheroid. Lower pipette positioning could damage the spheroids.

Three independent experiments showed <1 % spheroid loss from Akura 384 plates during nine consecutive medium exchanges (Figure 5).

>1 % spheroid loss after 9 medium exchanges in an Akura 384 plate using the VIAFLO 384
Figure 5: Nine consecutive medium exchanges were performed with InSphero’s Human Islet Spheroids in Akura 384 plates using INTEGRA’s VIAFLO 384. Three independent experiments aspirating >90 % of the medium showed <1 % spheroid loss.

Remarks

  • Partial plates: Programs can be adapted to a different number of samples at any time, giving laboratories total flexibility to meet current and future demands.
  • Automatic mode: The VIAFLO 96 and VIAFLO 384 handheld electronic pipettes can operate in an automatic mode, ensuring that all the liquid handling steps are performed in the same way. The customized automatic programs can be found in the download section. IMPORTANT: When using the automatic programs there is no need to place the lid below the Akura 96 and 384 plates.
  • Spheroid collection and transfer: The special coating of the Akura plates prevents adherence of the spheroids to the bottom of the well. This aids the collection of microtissues for transfer into another plate format, or for further processing. The customized program for spheroid collection and transfer (TRANS_TO_384) is available in the download section.

Conclusion

  • The VIAFLO 96 and VIAFLO 384 handheld electronic pipettes ensure precise and accurate medium exchange in Akura 96 and 384 plates with minimal spheroid loss.
  • Setting the Z height and the Tip Align on the VIAFLO 96 and VIAFLO 384 operator-induced pipetting minimizes variability.
  • The motor-assisted movements of the VIAFLO 96 and VIAFLO 384 reduce strain on the arms and wrists during pipetting of multiple plates.
  • The compact design of the VIAFLO 96 and VIAFLO 384 fits into most laminar flow cabinets when sterile conditions are required.
  • GripTips deliver a precise and consistent tip seal. This ensures that all tips on a multichannel pipette are at exactly the same height – increasing the accuracy and precision of each multichannel dispense – and never accidentally fall off.
  • The time to result can be drastically reduced when using the 96 or 384 channel pipettes.

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  • Éva Mészáros, PhD

    Application Specialist

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Instruments and accessories

VIAFLO 96/ VIAFLO 384, Handheld Electronic Pipette Plate holder, spring loaded, with slide function Standard plate holder 96 Channel Pipetting Head, 300 µl 384 Channel Pipetting Head 125 µl Automation Friendly Reagent Reservoir, 300 ml GripTip, 300 µl, Sterile, Filter for 96 Channel Pipetting Heads GripTip, 125 µl, Sterile, Filter for 384 Channel Pipetting Heads InSphero: Akura 96 InSphero: Akura 384

VIAFLO 96/ VIAFLO 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 works
The VIAFLO 96/384 is handheld, electronic pipette

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 plate holder options
Spring loaded plate holder A for 96 and 384 well plates, with slide function

Standard plate holder

For 24 and 96 channel pipetting. The standard plate holder (6205) is used for simple 24 and 96 well transfers. The plate holder with slide function (6210) enables a grid offset to access 384 well plates with a 96 channel head.

Part No. 6205

Find out more about the standard plate holder
Standard plate holder

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

384 Channel Pipetting Head 125 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. 6132

Find out more about 384 Channel Pipetting Heads
384 Channel Pipetting Heads

Automation Friendly Reagent Reservoir, 300 ml

INTEGRA offers two types of automation friendly reagent reservoirs:

  • 150 ml and 300 ml Clear Advantage™ reagent reservoirs which are made of both crystal clear virgin polystyrene and polypropylene. Both options fit into a reusable automation friendly base with bold, crisp, clearly visible graduation markings.
     
  • The 300 ml open well, 12 column and 8 row polypropylene reservoir blocks offer improved chemical compatibility.
Find out more about Automation Friendly Reagent Reservoirs
300 ml Reservoir filled

GripTip, 300 µl, Sterile, Filter for 96 Channel Pipetting Heads

Precise and consistent tip seal ensures that all tips on a VIAFLO 96 are  at an identical height for reliable touch offs and consistent pipetting results. An additional tip straightness inspection process ensures all pipette tips access wells even when dispensing to a 384 well plate.

Part No. 6435

Find out more about GripTips for 96 Channel Pipetting Heads
Sterile, Filter GripTips for 384 Channel Pipetting Heads

GripTip, 125 µl, Sterile, Filter for 384 Channel Pipetting Heads

Precise and consistent tip seal ensures that all tips on VIAFLO 96 and VIAFLO 384 are  at an identical height for reliable touch offs and consistent pipetting results. An additional tip straightness inspection process ensures all pipette tips access wells even when dispensing to a 384 well plate.

Part No. 6465

Find out more about GripTips for 384 Channel Pipetting Heads
Sterile, Filter GripTips for 384 Channel Pipetting Heads

InSphero: Akura 96

Previously GravityTRAP™ ULA Plate.

The one-and-only 96-well spheroid plate with InSphero’s unique features specially designed for lossless spheroid/organoid formation and handling as well as reliable culturing and analysis.

Part No. CS-09-004-03

Source: Website supplier

More about InSphero's Akura 96
Akura™ 96 Spheroid Microplate

InSphero: Akura 384

High throughput – Secure handling – Superior imaging

The ultimate spheroid screening plate unifying robust liquid and spheroid handling with automated high-resolution imaging. The flat bottom and black walls of the InSphero 384 well plate facilitates high-content imaging while the lower cavity of the wells allows medium exchange, dosing and endpoint determination in the same plate saving time and resources.

Part No. CS-09-003-02

Source: Website supplier

More about InSphero's Akura 384
The-ultimate-spheroid-screening-plate-unifying-robust-liquid-and-spheroid-handling-with-automated-high-resolution-imaging

Customer’s voice

The Viaflo 96/384 let us for faster plate operation in medium-throughput applications. Especially important is the programming of the positions in space for precise pipetting in desired position of the well.

MICHAL RUDNIK, InSphero AG