Precise and reproducible automated cell seeding with VIAFLO 96 and VIAFLO 384 handheld electronic pipettes

  • Precise and reproducible automated cell seeding with VIAFLO 96 and VIAFLO 384 handheld electronic pipettes

    Mouse embryonic stem cell passaging protocol with VIAFLO 96 and VIAFLO 384 pipettes

    Cell culture is a technique in which cells are grown outside a living organism under well-defined, controlled conditions. It is important for basic biological research and a wide range of clinical studies. Mass culture of cells is indispensable for producing viral vaccines, enzymes, synthetic hormones, anticancer agents, recombinant proteins and antibodies, but also for studying the physiology and biochemistry of cells. Nowadays, cell culture is also used in agriculture for producing alternative foods. Preserving cell viability, avoidance of contamination, high experimental reproducibility, and accuracy are vital for manipulations carried out on cells during media changes, transfers, seeding and passaging. All these can be mastered with VIAFLO 96 and VIAFLO 384 handheld electronic pipettes.

  • Table of contents

    Cell culture is a technique in which cells are grown outside a living organism under well-defined, controlled conditions. It is important for basic biological research and a wide range of clinical studies. Mass culture of cells is indispensable for producing viral vaccines, enzymes, synthetic hormones, anticancer agents, recombinant proteins and antibodies, but also for studying the physiology and biochemistry of cells. Nowadays, cell culture is also used in agriculture for producing alternative foods. Preserving cell viability, avoidance of contamination, high experimental reproducibility, and accuracy are vital for manipulations carried out on cells during media changes, transfers, seeding and passaging. All these can be mastered with VIAFLO 96 and VIAFLO 384 handheld electronic pipettes.

Key benefits

  • This protocol is automated for maximum reproducibility. Reducing the hands-on time for laboratory personnel can help to diminish the risk of contamination.
  • The optimized z-height and mixing step set-up eliminates user variability. This is crucial to maximize viability during cell culture.
  • Pipetting is carried out at an accurate and consistent speed to minimize the shearing forces that cells are subjected to.
  • The VIAFLO 96 and VIAFLO 384 combine all the benefits of VIAFLO pipettes with the ability to pipette 24, 96 or 384 channels simultaneously, for unmatched throughput and precision.

Overview: mouse embryonic stem cell culturing

Experimental set-up 

In this protocol, ES-E14TG2 mouse embryonic stem cells (Sigma) are washed, harvested and transferred to a new plate with fresh media, using a VIAFLO 384 handheld electronic pipette with a three position stage (with positions A, A/B and B, Figure 1). For the aspiration steps, a VACUBOY vacuum hand operator is used in combination with a VACUSAFE aspiration system.
A 96 channel pipetting head (10-300 μl) is used together with 300 μl Sterile, Filter GripTips. Customized VIALINK programs are provided to perform cell washing, harvesting, seeding and splitting with a VIAFLO 384.

Note: The VIALINK programs provided can be easily adapted to culture other cell types by adding or removing steps from the protocols supplied.

Overview of the process

  • Step 1: Washing the cells
  • Step 2: Addition of detaching reagent
  • Step 3: Incubation and new media addition
  • Step 4: Splitting the cells
  • Step 5: Triplicating the cells
Figure 1: Three position stage of VIAFLO 96 and VIAFLO 384. a) position 1: position A and A/B can be reached, b) position 2: position A/B and B are reachable.
Figure 1: Three position stage of VIAFLO 96 and VIAFLO 384. a) position 1: position A and A/B can be reached, b) position 2: position A/B and B are reachable.

Downloads: App note and protocols for automated mouse embryonic stem cell seeding

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 customized with the VIALINK software.

Step-by-step procedure

1. Washing the cells

Washing the cells with 1x PBS.

Load a full box of 300 μl Sterile, Filter GripTips on the VIAFLO 384. Slide the three position stage to the left so it is in position 2 (A/B-B). Place a 300 ml automation friendly reagent reservoir with 1x PBS in position AB.
Aspirate the cell culture media (100 μl) from the 96 well culture plate using a VACUBOY in combination with a VACUSAFE (Figure 2).
After aspirating the cell culture media, place the plate on position B of the VIAFLO 384 and launch the custom VIALINK program 'PBS_WASH'. The pipette aspirates 80 μl 1x PBS from the automation friendly reservoir and dispenses it into the 96 well culture plate.

Figure 2: Aspirating the cell culture media using a VACUBOY vacuum hand operator.
Figure 2: Aspirating the cell culture media using a VACUBOY vacuum hand operator.

2. Addition of detaching reagent

Addition of accutase solution to detach the cells

Load a full box of 300 μl Sterile, Filter GripTips on the VIAFLO 384. Place the 300 ml automation friendly reagent reservoir with the accutase solution in position AB, with the stage in position 2.
Aspirate the 1x PBS from the culture plate using the VACUBOY with the VACUSAFE.
After aspirating the 1x PBS, place the 96 well culture plate on position B of the VIAFLO 384. Select and run the custom VIALINK program 'ACCUTASE_ADD'. The pipette aspirates 40 μl accutase solution from the automation friendly reagent reservoir and dispenses it into the 96 well cell culture plate.

3. Incubation and new media addition

Incubation of the cell culture plate and preparation of a new plate with fresh cell culture medium

Incubate the plate for 15 min at 37 °C with 8 % CO2 in the incubator to allow the accutase to detach the cells from the plate surface. During the incubation of the cell culture plate, a new 96 well culture plate with fresh cell culture medium can be prepared. Load a full box of Sterile, Filter 300 μl GripTips on the VIAFLO 384. Place the 300 ml automation friendly reagent reservoir with the fresh cell culture media on position AB, with the stage in position 2.
Take a gelatin-coated (either self-made or commercial) 96 well plate and remove the gelatin with the VACUBOY and the VACUSAFE. Place the new 96 well plate in position B of the VIAFLO 384. Launch the custom VIALINK program 'MEDIA_ADD'. The pipette aspirates 100 μl of fresh cell culture medium and dispenses it into the 96 well plate. After a 15 minute incubation, the 96 well culture plate should be examined under the microscope to check that cell detachment is complete. If the cell detachment is complete, there are two options of how to continue. In step four the user can passage the cells – splitting the cells in 1:3 ratio to a new plate. Or, in step five, the user can split the cells in 1:3 ratio, triplicating it into three new plates.

Tip:

  • The incubation parameters should be changed depending on the cell type and the detaching reagent.

4. Splitting the cells

Passaging the cells in 1:3 ratio

Load a full box of Sterile, Filter 300 μl GripTips on the VIAFLO 384. Place the 300 ml automation friendly reagent reservoir with the fresh cell culture media on position AB, with the stage in position 2.
Place the 96 well plate to be split on position B. Place a new 96 well plate on position A. Select and run the custom VIALINK program 'MIX_PLATE'. The VIAFLO 384 aspirates 80 μl fresh cell culture medium and dispenses it into the 96 well plate. It will be followed by a 30-cycle mixing step. When the mixing is finished, the VIAFLO 384 aspirates 40 μl of the cell suspension. The instrument will prompt the user to move the three position stage to position 1, so that position A can be reached. The pipette dispenses 40 μl of cell suspension to the new 96 well culture plate.

Tips:

  • In the mixing step, 150 μl of cell suspension is mixed, which is more than the volume in the wells. This creates bubbles, which helps to homogenize the cell suspension. This is a very efficient solution for ES-E14TG2 mouse embryonic stem cells but can be changed for some other cell lines.
  • The z-height limits are defined in the program to ensure optimal tip immersion depth, preventing the pipette tips from touching the bottom of the plate.

5. Triplicating the cells

Splitting the cells in 1:3 ratio onto three 96 well plates

Load a full box of Sterile, Filter 300 μl GripTips on the VIAFLO 384. Place the 300 ml automation friendly reagent reservoir with fresh cell culture medium on position AB, with the stage in position 2.
Place the plate to split in position B. Place the first new 96 well plate on position A. Keep the other two new 96 well plates close to the instrument, they will be placed in position A during the program. Launch the custom VIALINK program 'MIX_PLATE_TRI'. 95 μl of fresh cell culture medium will be aspirated and then dispensed into the 96 well plate. The cell culture medium and the cells will be mixed for 30 cycles. When this step is finished, the pipette aspirates 120 μl of cell suspension and alerts the user to change the three stage position, sliding it into position 1. The VIAFLO 384 dispenses 40 μl of cell suspension to a new 96 well plate, then prompts the user to change plate. This step will be repeated two times, resulting in the cells being split into three new plates.

Tip:

  • In this example, the ES-E14TG2 mouse embryonic stem cells were split in three. This can easily be set up differently for other cell lines, by changing the volumes of aspiration and dispensing.

Experimental results (Friedrich Miescher Institute for Biomedical Research) showed consistency between replicates of mouse embryonic cell samples with the same confluency. The mixing step was very efficient, allowing single cells to be seen under the microscope (Figure 3c).

Figure 3: Microscopy picture showing the different stages of the splitting protocol for four different wells.

Figure 3: Microscopy picture showing the different stages of the splitting protocol for four different wells. Pictures of mouse embryonic stem cells were taken a) after washing the cells with PBS, b) after the incubation with accutase, c) after splitting the cells, and d) 24 hours after splitting the cells. Wells 1 and 2 showed lower confluence, while wells 3 and 4 had higher confluence. An Axiovert 40 CFL (Zeiss) microscope was used with 2.5x magnification. (Photos courtesy of Julie Cramard, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.)

Remark

Partial plates:

Programs can be easily adapted to a different number of samples. The VIAFLO 384 is able to work with any number of tips loaded, giving you the benefit of simultaneous and accurate dispensing of a smaller number of samples.

Conclusion

  • The VIAFLO 384 offers a simple, accurate, reproducible, and fast pipetting solution for cell washing, transfer and seeding.
  • Prolonged manual pipetting tasks can lead to repetitive strain injury. This can be avoided by automating the cell culture steps with the VIAFLO 384, maximizing hands-free time.
  • With the optimized electronic mixing and perfect pipetting height control, homogenous cell suspensions are achieved, while minimizing the chance of cell shearing during passaging and splitting.
  • The VIAFLO 384 with a 96 channel pipetting head was used in this example, but the VIFALO 96 and VIAFLO 384 base units have easily interchangeable heads to allow 24, 96 and 384 channel pipetting, providing the right tool to match your throughput. The MINI 96 offers another option for this application, but in a much smaller and simpler package.

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  • Éva Mészáros, PhD

    Application Specialist

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Instruments and accessories

VIAFLO 96/ VIAFLO 384, Handheld Electronic Pipette Three position stage for 96 and 384 well plates 96 Channel Pipetting Head, 300 µl VACUSAFE VACUBOY Automation Friendly Reagent Reservoir, 300ml GripTip, 300 µl, Sterile, Filter (for automation systems) ECACC: Mouse Embriotic Stem Cells SIGMA/MERCK: Dulbecco's Phophate Buffered Saline SIGMA/MERCK: Accutase® solution SIGMA/MERCK: Glasgow Minimum Essential Medium SIGMA/MERCK: Gelatin from porcine skin SIGMA/MERCK: TPP® tissue culture plate

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

Three position stage for 96 and 384 well plates

This accessory expands available stage positions for microplates, reservoirs and tips on a VIAFLO 96 or VIAFLO 384 from 2 to 3 thereby enhancing workflow in a wide range of applications. Beneficially the Three Position Stage also features an indexing function to allow access to 384-well plates using a 96-channel pipetting head.

Part No. 6230

See all plate holder options
Three position stage for 96 and 384 channel pipetting

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

VACUSAFE

VACUSAFE combines vacuum pump, collection bottle, overflow protection and filter in an all-in-one liquid aspiration system. Such a closed aspiration system offers the most effective method for containment of biohazardous liquids by maximal personal protection.

Part No. 158310

More about VACUSAFE
VACUSAFE allows aspirating liquids form T-Flasks, well-plates and much more

VACUBOY

VACUBOY is a versatile aspiration tool that converts any vacuum source into a user-friendly aspiration system for aspirating liquids from virtually any laboratory container – at the touch of a button and in a controlled way. The hand piece, the core element of VACUBOY, is simply connected to any available vacuum source using silicone tubing and fitted with one of the different aspiration adapters. The perfect match is obtained when VACUBOY is connected to a VACUSAFE aspiration system.

Part No. 155500

More about VACUBOY
Scientist aspirating liquids from different labware types with VACUBOY hand operator

Automation Friendly Reagent Reservoir, 300ml

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 automation systems)

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
Integra-Relaunch_Website-Output-Produktebilder-Product_Overview-GripTips.png

ECACC: Mouse Embriotic Stem Cells

Pluripotent mouse embryonic stem cell line. ES-E14TG2a cells are deficient in in HPRT and are resistant to 0.06mM 6-thioguanine.

Part No. ES-E14TG2a

Source: Website supplier

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SIGMA/MERCK: Dulbecco's Phophate Buffered Saline

Dulbecco′s Phosphate Buffered Saline has been used for the dilution of fresh red blood cells to study the trapping of red blood cell aggregate. It has also been used to wash the wells of microtiter plate.

Part No. D8537

Source: Website supplier

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SIGMA/MERCK: Accutase® solution

Accutase® solution (in dulbecco′s phosphate-buffered saline (DPBS) without Ca++ or Mg++) contains proteolytic and collagenolytic enzymes for cell detachment. It is useful for the routine detachment of cells from standard tissue culture plasticware and adhesion coated plasticware.

Part No. A6964

Source: Website supplier

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SIGMA/MERCK: Glasgow Minimum Essential Medium

For use with adherent kidney cell lines such as baby hamster kidney cells (BHK).

Part No. G5154

Source: Website supplier

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SIGMA/MERCK: Gelatin from porcine skin

Gelatin is a hydrocolloid. Bovine or porcine bone and hide are its commercial sources. It is a collagen derivative and comprises protein as a major component with minerals and water.

Part No. G2500

Source: Website supplier

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SIGMA/MERCK: TPP® tissue culture plate

Gelatin is a hydrocolloid. Bovine or porcine bone and hide are its commercial sources. It is a collagen derivative and comprises protein as a major component with minerals and water.

Part No. G2500

Source: Website supplier

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Customer’s voice

With long term 96 well cultures, performing routine and frequent half media changes MANUALLY is laborious and fraught with scratch concerns. Using the VIAFLO 96 has made that culture easier and more robust. A must for anyone doing these kinds of cultures.

MARGOT BRICKELMAIER, Biogen