From smaller-scale assays to industrial-scale screening operations, cell-based assays are critical for applications that include elucidating basic mechanisms of cell function, target validation and compound screening. Cell-based assays provide valuable information about the effect of a cell perturbation more quickly than most other means of evaluating response to a compound or other alteration. Conventional techniques for transferring cells into these assays are less than ideal—manual protocols are too tedious and error-prone for large-scale assay pipelines, while automated instruments can introduce cell stress or damage, which alters experimental results and makes data less reflective of biology. The Echo liquid handler (Figure 1) from Labcyte (Sunnyvale, Calif.) is a reliable, automated platform that treats cells gently and reproducibly to ensure consistent assay conditions and accurate downstream data.
Applications of cell-based assays
Figure 1 – Echo liquid handler.The explosion of interest in stem cells after researchers figured out how to induce pluripotency in adult cells has made it an especially active area for cell-based assay use, helping elucidate how these cells behave both in their pluripotent state and as they find their niche and differentiate into mature cells of specific lineage. Cell-based assays are used across drug discovery and development for target identification and validation, compound screening, antibody or peptide engineering and monitoring cellular events for efficacy and biosafety. They serve as a proving ground between initial target development and animal testing, allowing scientists to observe the effects of investigational compounds in human cells and remove likely failures from the pipeline before moving ahead to more expensive testing.
With the wealth of applications for cell-based assays, many of them related to human health, it is imperative that results are as accurate and biologically relevant as possible. Ultimately, this means keeping cells happy and healthy from the time they are cultured until they are queried or measured to generate assay data.
Cell transfer challenges
The value of information from a cell-based assay depends almost entirely on the fitness of the cell being studied. When that cell is performing as it would in its native environment, the results it produces when introduced to a compound, for example, are highly useful for assessing how that compound would affect similar cells in a person. If that cell has been altered in some way, that is, stressed or damaged, possibly without the researcher’s knowledge, then its interaction with the compound may not reflect native biology at all.
One of the pivotal points in building cell-based assays is the transfer process used to move cells out of their growth medium, whether via a culture plate or suspension tube or flask, and into the microtiter plate where the experiment will be performed. This single step can carefully preserve or damage cells; unfortunately, it is nearly impossible to determine which has occurred before the assay is conducted, or sometimes even later in data analysis.
For cells, the journey from growth medium to assay plate is much more perilous than it looks. Typical options for transferring cells include automated liquid handlers that use peristaltic or solenoid pumps, pipette tips or valves. The pumps tend to apply shear stress on the cells, and even minimal levels of this stress can quickly alter cell gene expression or metabolism. Pipette tips add more stress, and can create friction as the cell bumps against plasticware.1 Instruments using valves may be the most damaging; cells often bang into these devices during transfer. Each application of stress or friction risks a cascade of reactions inside the cells, which dramatically changes their internal function and viability.
Dispensing a consistent number of cells into each well for every assay is another major challenge in cell transfer. When cell numbers are not normalized, reactions become imbalanced, with some wells requiring more target or more reagents. Because these assays tend to be performed on a large scale—at least 96 wells per plate, but more often 384 and in some cases even 1536—they function best when all wells are treated the same, getting the same amount of target and reagents. If the cell numbers vary significantly, then well-to-well results are not comparable, resulting in misleading data about compound efficacy or potency.
Most problems caused by cell transfer cannot be detected at the time of assay or corrected during data analysis with bioinformatics tools. The only viable solution is to implement a cell transfer technology that treats cells gently and consistently.
An acoustic alternative
Labcyte liquid handlers feature acoustic droplet ejection, that is, they deploy low-energy sound waves to propel minute quantities of fluid from a source to the assay microtiter plate. Because there is no contact with the sample (Echo automated liquid handlers do not use tips, nozzles or valves), the cells are maintained at optimal viability and fitness, with minimal effect from the transfer process and significantly reduced chances of cross-contamination. The technology is precise, accurate and rapid, allowing users to move even the smallest volumes of sample efficiently and consistently.
The Echo liquid handler was tested on several cell types and densities to evaluate its ability to transfer cells without causing damage. Cell lines included Hep-G2, an adherent liver cancer cell line with epithelial morphology from frozen stock; PC-3, an adherent human prostate cancer cell line with epithelial morphology; and THP-1, an acute monocytic leukemia cell line derived from peripheral blood. In all cases, acoustic transfer did not induce cell stress, and thus had no impact on cell viability at a point where conventional methods can do harm, and transfer occurred with consistent cell counts.2,3 This work demonstrated that the Echo liquid handler can transfer cells safely and gently, regardless of whether they were fresh or frozen, adherent or suspension.
Conclusion
By harnessing the power of sound waves, it is possible to implement cell-based assay procedures that keep cells safe during transfer. This approach ensures consistent volume and cell density during transfer to maximize assay reliability and reproducibility of results.
References
- Xie, Y.; Wang, F. et al. Pipetting causes shear stress and elevation of phosphorylated stress-activated protein kinase/jun kinase in pre-implantation embryos. Mol. Reprod. Dev. 2007, 74, 1287–94; doi: 10.1002/mrd.20563.
- Lesnick, J. et al. Technical note. High-precision cell dispensing with the Labcyte Echo® liquid handler. Labcyte Inc., Sunnyvale, Calif., Sept 2016.
- Kulesskiy, E.; Saarela, J. et al. Precision cancer medicine in the acoustic dispensing era: ex vivo primary cell drug sensitivity testing. JALA 2016, 21(1), 27–36; doi:10.1177/2211068215618869.
John Lesnick is senior application scientist, Labcyte Inc., 1190 Borregas Ave., Sunnyvale, Calif. 94089, U.S.A.; tel.: 408-747-2000; e-mail: [email protected]; www.labcyte.com