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The advantages of combined microplate washing and dispensing

The success of liquid handling for tissue culture experiments, especially when using microplates, depends on a variety of factors including a sterile working environment, correct fluid dynamics, and flexible solution dispensing. Often several different instruments are required to accomplish the necessary tasks. In this article, the benefits of using an integrated solution for washing cells and dispensing liquids are discussed.
by Paul Held


FLUID PATH
The fluid path must be sterile in order to prevent bacterial contamination. While sterile single-use devices have been employed, a reusable autoclavable fluid path is more desirable as the high temperature and pressure of an autoclave will effectively kill organisms, and the process can be verified through quality control procedures. Unfortunately, some liquid handling processes include materials that cannot withstand the high temperatures, pressure and water vapour associated with steam. Other sterilisation methods including gas or chemical sterilisation are less aggressive than autoclaving, however, they are generally not as reliable, and verification of sterility is often not possible. Autoclaving is the preferred method for readily available, reliable, fast and verifiable sterility of a fluid path.

FLUID DYNAMICS

Fluid dynamics must be appropriate to the task. When washing cells, liquid dispense rate and pressure must be appropriate for the cell type yet still provide accurate dispensing and complete washing. Specifically, when washing cells grown in a monolayer, the fluid dispensing and aspiration rates should be variable enough to provide adequate washing, and also gentle enough such that cells are not dislodged from the microwell surface. Vigorous dispense rates may denude the bottom of a microplate well, particularly if the cells are loosely adherent. Fluid dynamics are also critically important to maintain the viability of cells in suspension during the seeding process. Additionally, a variety of cell types and experimental conditions often are performed on a single microplate, so dispensing a number of solutions to different strips simultaneously may be required. The ability to dispense different cell types, or to skip microplate rows or columns when dispensing, is a benefit in that it allows experimental flexibility. Rapid, accurate and precise addition of multiple reagents allows biochemical assays to be performed on plated tissue culture cells. For example, cells can be lysed and assayed directly in a microplate well using the BCA method of protein determination. The only requirement is that specific volumes of lysis reagent followed by working BCA reagent be added to the microplates.

COMBINATION WASHING AND DISPENSING
With these criteria in mind, the EL406 Microplate Washer Dispenser from BioTek Instruments combines several existing fluid handling technologies into one instrument. A patented washing manifold provides full microplate washer functionality in both 96- and 384-well microplates, and an ultrasonic bath provides simple and complete cleaning of the manifolds aspiration and dispense tubes. Washing speed is variable and optimised even for loosely adherent cell layers. Immediately adjacent to the microplate washer manifold is an eight-channel peristaltic pump dispenser manifold. This 96- and 384-well microplate-capable dispenser uses autoclavable cassettes in different sizes to optimise fluid dispense accuracy and precision in the range from 1 L to 3mL. In addition to a low dead volume, reagents can be recovered by reversing the direction of the peristaltic pump. These cassettes and the appropriate sized tubing may be autoclaved to ensure complete sterility of the fluid path. Two optional syringe pump dispensers can also be utilised to dispense additional reagents; a total of three reagents can be dispensed without operator intervention.


Figure 1. Bifurcated washer fluid path of the EL406.
Table 1. Dispense accuracy into 96-well plates. Individual 8-well microplate strips were weighed before and after a syringe pump dispenser had dispensed fluid into each well of 12 strips (96 wells). Data represent the mean and average of twelve determinations. The percent deviation is the ratio of the difference between the calculated and expected values to the expected values.

PATENTED WASHING MANIFOLD
Dispense and aspiration manifolds are physically separated and are arranged on top of each other. The lower dispense manifold allows the tube from the upper aspiration manifold to pass through and enter the microplate well. In order to dispense fluid into a small well while aspirating from the same well, as seen in overflow and bottom washing, the dispense tube is tilted from vertical and offset from the centre of the well. This canted design also has the added benefit of a swirled fluid path for a more vigorous wash. In addition, the EL406 incorporates a dual fluid path and software control designed specifically to lower the dispense rate to the lowest possible flow, without affecting dispense accuracy and precision [Figure 1]. When low flow rates are selected, the flow control valve directs all fluid movement through the low flow line. When standard rates are selected, the flow control valve opens and allows full fluid movement through the system.

PERISTALTIC PUMP DISPENSER

The integrated peristaltic pump dispenser is compatible with 96- and 384-well microplates. Rollers are fixed to a rotating rotor, which is encircled by stretched flexible tubing that is compressed at the points where the tubing meets the rollers. As the rotor revolves, the fluid in the tubing is forced through the tubing. The volume pumped is dependent on the distance between the rollers, rotor position, tubing tension, inner diameter of the tubing and the geometry of the dispenser tip. By controlling these variables, accurate and precise liquid dispensing can be performed. The EL406 uses a customised cassette design, optimised for volume and performance, in three sizes (1 L, 5 L, and 10 L) to dispense in full increments. The cassettes are autoclavable, and because of the materials used, do not require regular recalibration after the sterilisation process. The dispense head automatically adjusts for microplate height.

SYRINGE PUMP DISPENSER
The EL406s microprocessor controlled syringe pump dispenser is also compatible with 96- and 384-well microplates with a high degree of accuracy and precision, yet never requires recalibration. As seen in Table 1, when the minimum setting for 96-well microplates (10 L) was selected, the dispense-volume, determined gravimetrically, was found to deviate from the expected rate by 11.7%. The deviation rapidly diminished with larger volumes to less than 1%. In all cases, the dispense volume was quite precise. Additionally, the coefficient of variance (%CV)  was found to be less than 7% at 10 L per well. The %CV also decreased rapidly with larger volumes to less than 1% at volumes above 50 L per well.

SUMMARY
The integrated combination of washing, peristaltic pump dispensing, and syringe pump dispensing provides a complete solution for a wide range of liquid handling applications from gentle to vigorous. Multifunctional liquid handling in microplate format is now available when using the flexible and compact EL406 Microplate Washer Dispenser.

THE AUTHOR
Paul Held,
BioTek Instruments,
Winooski, Vermont, USA


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