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

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.

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.

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.

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).

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.