Establishing Protocols for Automated Harvesting

Introduction

Culturing of a variety of stable cell lines – including:

  • Hybridoma
  • Insect
  • Bacterial
  • Yeast
  • Mammalian lines (including stem cells)

Is a key job in the pharmaceutical industry. The cells must be maintained and grown on for utilization in cell-based assays, such as clone selection, siRNA and high content screening and ADME studies. Usually, cells are cultured in flasks, but in an automated environment, cells can be grown in multiwell plates with six, 12 or 24 wells for example.

This article details an automated procedure (which relieves laboratory workers from monotonous manual processing work and gives more time for other important jobs) for harvesting frequently used animal and human cell lines cultivated in multi-well plates.

Methods and Materials

Observations were undertaken in sterile conditions; the workstation was housed in a biological safety cabinet and the liquid system thoroughly sterilized. Automated harvesting was carried out by employing a Freedom EVO® workstation (Figure 1) equipped with a Liquid Handling Arm™ (LiHa) with fixed tips, a Robotic Manipulator Arm™ (RoMa) and a bespoke tilting rack with a Variomag® Teleshake (Thermo Scientific).

The tilting rack permits complete liquid uptake from large diameter wells, e.g. from a six-well plate, and also shaking for mixing and cell detachment throughout cell dissociation (Figure 2).

System overview. 1. Biosafety cabinet; 2. LiHa; 3. Te-Shake™ with chamber slides; 4. Reagent troughs and LiHa wash station; 5. Sterile system liquid; 6. RoMa; 7. Flask flipper; 8. Te-Shake with tubes; 9. Tilting rack and Variomag Teleshake; 10. Centrifuge; 11. Liquid waste. Not all components are required for this application.

Figure 1: System overview. 1. Biosafety cabinet; 2. LiHa; 3. Te-Shake™ with chamber slides; 4. Reagent troughs and LiHa wash station; 5. Sterile system liquid; 6. RoMa; 7. Flask flipper; 8. Te-Shake with tubes; 9. Tilting rack and Variomag Teleshake; 10. Centrifuge; 11. Liquid waste. Not all components are required for this application.

Tilting rack and Variomag Teleshake with six and 24-well plates.

Figure 2: Tilting rack and Variomag Teleshake with six and 24-well plates.

Cell Lines

Four cell lines with differing adherence properties were selected (Table 1 and Figure 3) – HeLa, SaOs, CHO and HEK.

Table 1: Cell lines used for the experiments.

Cell lines used for the experiments.

Microscopy images of the cell lines used, x40.

Figure 3: Microscopy images of the cell lines used, x40.

Table 2: Parameters for harvesting of cell lines in multi-well plates.

Parameters for harvesting of cell lines in multi-well plates.

Experimental conditionsStandard six- and/or 24-well plates (growth area 57 and 45.6 cm2 respectively) were used to grow cells in until around 70% confluency was reached. Cells are in the exponential growth phase and can be split or utilized for assays at this confluency. Optimal harvesting parameters, such as incubation time, reagent volume and mixing, were first calculated manually for each of the cell types. Afterward, the protocol was given to the Freedom EVO liquid handling platform and the automated procedure was optimized (Table 2).

Experiments were executed side by side using both the manual and automated methods, utilizing three to six microplates. Cells from every well on a plate were merged and the total viability, cell number, and aggregation rate established with a Cedex® cell counter (Roche CustomBiotech). Plates were scrutinized under a microscope to check that all cells had detached and been gathered post harvesting.

Discussion and Results

Automated cell harvesting obtained cell counts between 92 and 105%, with viability ranging between 80 and 100%. The cell count for manual harvesting was standardized at 100%, and results from the automated procedure were measured in relation to this value. Yet, HeLa cells harvested from 24-well plates with a confluency of 90-100% showed a viability of only 60%.

The low viability could be the result of cells reaching the stationary phase because of nutrient depletion. For HEK cells the aggregation rate was highest. Though this was not because of inadequate mixing; this cell type is recognized to build aggregates. More detailed findings are illustrated in Figure 4.

Automated harvesting was as good as manual harvesting in the areas of aggregation rate, cell count and viability (Figure 4).

Cell count, viability and aggregation rate of the manual and automated protocols using multi-well plates.

Cell count, viability and aggregation rate of the manual and automated protocols using multi-well plates.

Figure 4: Cell count, viability and aggregation rate of the manual and automated protocols using multi-well plates.

Crucial parameters for an effective cell harvest with sufficient viability and density were

  • Pipetting speed
  • Confluency
  • Dissociation procedure

Protocols were optimized for a confluency of approximately 70%, unless stated otherwise. An automated confluency check can help calculate the optimal harvesting time, and for non-invasive brightfield or fluorescence analysis of cell culture microplates, Tecan has integrated cell imaging systems such as Spark® (Tecan) the CELLAVISTA® (SYNENTEC) onto the Freedom EVO platform.

Releasing cells that are dissociated by tapping the microplate is another crucial step when moving a manual procedure to an automated system. The Teleshake was able to suspend cells, but parameters like time and the shaking frequency had to be optimized for each cell line.

The speed was altered so that the whole well surface was covered with reagent and agitated throughout incubation. The dissociation reaction was halted by the inclusion of medium containing 10% serum, and the following shaking step significantly increased the harvesting rate (data not shown). To give harvesting results that were comparable, the cell suspension pipetting speed and uptake of partition volume were crucial.

Extensive mixing cycles and high pipetting speeds could result in a lower number of cells being harvested, so the pipetting speed has to be moderate, even if this lengthens the procedure for large volumes.

It is necessary to keep up good maintenance procedures, with extensive, regular cleaning of the liquid system – even if it is unused, to obtain reproducible results and maintain sterility.

Conclusions

Automated and manual procedures conducted in multi-well plates showed comparable cell harvesting results for aggregation rate, cell count, and viability. Separate protocols have been designed and the automation parameters have been optimized for each of the four cell lines selected.

This process must be replicated and adjusted for other cell lines. The four cell lines studied were chosen for their variety of properties, such as aggregation rate and strength of adherence, so the protocol development for other cell lines with characteristics that are similar will be easier.

Acknowledgements

We would like to thank Prof Dr Ursula Graf-Hausner* and Dr Nicola Franscini**, Institute of Chemistry and Biological Chemistry, Zurich University of Applied Sciences, Wädenswil, Switzerland, for the collaboration and for performing the experiments and establishing the protocols.*

Now owner of Graf 3dcellculture, **now at Swissmedic.

About TecanTecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world


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Last updated: Sep 12, 2018 at 6:21 AM

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