High-Precision Dispensing of Live Cells in a 1536-Well Format
by Mary Cornett and Anca Rothe, Innovadyne Technologies, Inc.
Overview
Objective: Improving the dispensing of live cells in a high-throughput environment
Why: The increasing demand for live cell assays due to their greater biological relevance.
How: Using a fast, reliable automated liquid handler with simple flow paths and low maintenance.
Some of the variables that can be optimized:
Pressure of dispense (backing pressure)
Speed of dispense (dispense mode)
Speed of aspiration
Size of nozzle orifice
Dispensing materials:
Assay: CellTiter-Glo Luminescent Cell Viability
Greiner 1536-well plates (Lumitrac 200)
CHO-K1 cells
Liquid handler: Nanodrop II (Innovadyne Technologies, Inc.)
Results: Excellent precision and viability in a high density 1536-well plate format
Introduction/Background
Cell-based assays have become indispensable in the drug screening industry for testing interesting pharmaceutical compounds for their effects on live cells. The recent rapid increase in popularity and use of cell-based assays in high-throughput screening laboratories is based on the fact that cell-based assays offer a more accurate and complete representation of the real-life model. Since live cells are used, more biologically relevant data are acquired, revealing the complex cell biology information that surround the target compound in the cells.
Although hand-pipetting cells had been useful in the low-throughput past, the need for fast, automated, reliable, dispensing of live cells is clearly apparent if the throughput of these assays is to match that of traditional assays. Challenges affected the quality and speed of assays performed with live cells include:
Maintaining cell viability in the often harsh environment of liquid handling robots
Settling of cells in suspensions over time
Maintaining acceptable precision of these assays
Here we present appropriate conditions for dispensing live CHO-K1 cells in order to achieve the same quality in these assays as in traditional assays.
Materials and Methods
CHO-K1 cells were used for all the assays and were dispensed at a density of 1000 cells/well into white 1536 Greiner plates. The CellTiter-Glo Luminescent Cell Viability Assay from Promega was used as a homogenous assay and the signal was detected on the LEADseeker Multimodality Imaging System (Amersham Biosciences).
For each plate, 2 µL cells and 2 µL CellTiter-Glo reagent were added to each well. In the first row of each plate, cells were replaced with PBS buffer in order to get signal to noise ratios. Tryptan Blue exclusion was used in our viability studies.
An 8-tip Nanodrop CAS from Innovadyne Technologies, Inc. (see Fig. 1a) was used as the automated liquid handler for both the cell and reagent dispensing.
| Nanodrop CAS Liquid Handler |
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Fig. 1a: Nanodrop 2-plate stage and fluidics module
| Paddle Wheel Stirrer |
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Fig. 1b: Paddlewheel stirrer and wider bore nozzles
Innovadyne’s “non-contact” pipetting systems isolate the aspirating syringes and solenoid dispense valves from the sample flow (see Figs. 2a and 2b). This ensures that cells are not in contact with the actuator and allows dispensing without stressing of the cells that occurs with flow-through solenoid or peristaltic technologies.| Flow Path of a Tip |
Fig. 2: Sample flow path of a single tip -- prime, aspirate, dispense and wash
Cells were maintained in suspension using a ‘paddlewheel’ type stirrer customized by E&K Scientific to fit on the Nanodrop stage (Fig. 1b). From a variety of available nozzle sizes compatible with the Nanodrop, those with the largest diameter orifice (200 µm) were employed in order to minimize the shearing forces on the cells during the dispensing process so the health and viability of the cells would be preserved.
In order to reduce reader and plate artifacts, a background correction was performed on the raw data from the luminescence reader by using an averaged background plate of all plates read in each set (5 plates for all data presented here). The validity of the normalization procedure was confirmed by reading some plates backwards and noting that the precision did not change dramatically by changing the reading direction.
Results
Cell Viability
Studies by Tryptan Blue exclusion found that cell viability was best using the wider-bore 200 µm nozzles available specifically for cell dispensing and other custom applications. In this study, cells that were hand pipetted directly from the cell stock solution were compared with those that had been dispensed with the Nanodrop CAS at 12 psi and then hand-pipetted from a microtiter plate. The cell viability between these two sets of cells was comparable to within about 5%.
Dispensing Pressure
In order to determine the best pressure for cell based dispensing, an evaluation was done to compare plates dispensed with a backing pressure of 12 psi and those dispensed with a pressure of 20 psi. While higher pressure dispensing would increase the shear stress on the cells, the additional ejection velocity also has the ability to more cleanly cleave drops, particularly those of cell and bead suspensions, as well as more viscous or sticky solutions. As can be seen in the following representative graphs (Figs. 3 & 4), the higher pressure did result in slightly better precision while maintaining almost identical signal to noise ratios (51-53 and 50 respectively), suggesting the density of viable cells was similar for both sets of plates.
| Dispensing Pattern of Live Cells into a 1536-Well Plate - 12 psi |
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Fig. 3. Relative luminescence for each well of a 1536-well plate using CellTiter-Glo Luminescent Cell Viability Assay reagents and CHO-K1 cells (1000 cells/well). Other conditions: 12 psi backing pressure, 200 µm nozzles, 14 µL/s aspirate rate.
| Dispensing Pattern of Live Cells into a 1536-Well Plate - 20 psi |
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Fig. 4. Relative luminescence for each well of a 1536-well plate using CellTiter-Glo Luminescent Cell Viability Assay reagents and CHO-K1 cells (1000 cells/well). Here the dispense is done at 20 psi.
Other Dispensing Conditions
A chart of other plates dispensed under various conditions, including those of the previous graphs, appears below. Other observations are also listed below.
Table 1 - Whole-Plate Cv’s Under Various Dispensing Conditions | ||
Condition |
12 psi |
20 psi |
PBS suspension |
4.5%, 4.1% |
3.6%, 3.8% |
F12K media |
4.1%, 4.3% |
- |
Fast aspiration*/PBS suspension |
4.2%, 4.3% |
- |
|
*The standard aspiration speed is 14 µL/s. A faster aspiration speed of 20 µL/s is used for these plates. | ||
No decrease in performance with increasing aspiration rate is seen, however, it might be expected that less hardy cell lines would benefit from a slower aspirate rate.
Similar Cvs were found with cells suspended in both PBS buffer or F12K media.
Conclusions
We have demonstrated the following:
We are able to dispense live cells in a 1536-well plate environment with excellent precision and very little loss of viability.
The option to choose from the variables listed above allows the user to optimize cell dispensing for his or her individual needs such as the use of different cell types and different cell assays.
This liquid handler and methodology easily lend themselves to working in an automated high-throughput environment, in part because operating in an environment in which reagents do not flow through moving parts keeps the assay results consistent over time and minimizes the need for maintenance and downtime.
Slightly higher than standard dispensing pressures improved precision with no apparent loss of cell viability.
Acknowledgements
We would like to thank DiscoveRx Corp. for their time and the use of their cell culture facilities.
Applications