Sample transfer from one format of microplate to another has been necessary from the time that microplates came into vogue as the method of choice for storage and assay of compounds.
While miniaturized assay formats have been developed to enable the use of reagents and compounds in a more cost-effective way, they are not always compatible with all microplate formats. Some ADME tests can be run only in a 96-well plate, for instance.
Therefore, it becomes essential to ensure rapid but efficient sample transfer from one format to another to streamline compound management and the work of assays in laboratories.
The Fluent system from Tecan is a pioneer in this area, being built to deliver speed and high throughput, with increased capacity and more free time away from the workstation. The end result is higher productivity and reliable work standards.
Figure 1: The Fluent compound management solution. A Fluent 1080 equipped with an eight-channel Flexible Channel Arm, a Multiple Channel Arm and a Robotic Gripper Arm is shown. A Carousel is integrated onto the right-hand side of the instrument.
Materials and methods
Fluent Laboratory Automated Workstation
Tecan has brought out several high-efficiency liquid management automated solutions of which the Fluent platform is the most recent offering. As shown in Figure 1, Fluent is capable of standardized and rapid pipetting using both the Flexible Channel Arm (FCA) with its eight channels, and the Multiple Channel Arm (MCA).
The Dynamic Deck™ technology patented by Tecan enables the user to exploit the workstation optimally by integrating many Tecan modules simultaneously. Some useful tools utilized here include the Carousel, the Stacker, carriers for nested or isolated microplates, troughs and single-use tips, which can be stacked up to six layers deep, as seen in Figure 2.
Figure 2: Typical deck layout for the Fluent compound management solution. Microplates can be stored up to six deep on one deck segment.
The MCA also offers the facility of switching adapters automatically during a run, so that it can lift and use either 96 or 384 tips in the same experiment. The ability to carry out independent and task-appropriate procedures with the arms means that processes can be performed in parallel and arranged to fit into the schedule with greater speed and efficiency.
Each Fluent platform for compound management has the following tools:
- An FCA with eight channels, having either single-use or reusable fixed steel tips to gain access to a single selected tube or well, so as to easily perform explicit distribution of samples and controls and to achieve serial dilutions
- An MCA which allows the user to switch between 96- and 384-channel pipetting instantaneously during a single run, and thus allows easy distribution of reagent and replication of the plate
- Five adapter plates to increase the ease of adaptation of the process for handling different compounds
- The COMBO adapter plate which can pipette with 384 single-use tips in parallel, in several columns of 16 tips each or rows of 24 tips each, from the MCA 384 tip box.
- The non-COMBO adapter which can pipette with 384 single-use tips in parallel.
- The Extended Volume Adapter (EVA) capable of mapping four channels into a single one and which can pipette target volumes of up to 500 μl using MCA 96 single-use tips, from several columns of eight tips each or rows of 12 tips each, using an MCA 96 disposable tip box or other boxes with tips arranged in 4 columns by 6 rows or even 12 columns by 8 tips.
- The non-EVA adapter when four-to-one mapping is unnecessary, can aspirate volumes up to 125 μl using the MCA single-use tips, from several columns of eight or rows of 12 tips each, from an MCA 96-tip box or other box shapes, as above.
- Lastly, the 96 out of 384 adapter, which can pipette a maximum volume of 125 μl, by utilizing 96 disposable tips in a quadrant from an MCA 384 disposable tip box. Multiple rows of 16, or columns of 8 can be easily and simultaneously picked up from a 384 disposable tip box.
- Plates and consumables are rapidly and smoothly transferred by a Robotic Gripper Arm (RGA), between integrated devices, storage modules and the worktable with no interruption to pipetting.
It is possible to load different plate formats on the system, ranging from 96 to 1,536-well plates. The FCA can transfer up to 8 samples simultaneously while the MCA can transfer whole plates at a time.
Three methods have been described to reformat a plate with 96 wells. This holds whether the 96-well plate is to be copied to another similar plate or whether four such plates are to be copied into a single plate with 384 wells. The method is chosen with regards to its benefits. Another possibility is reformatting four 384-well plates to one 1,536-well plate or preparing three plates with 384 wells each.
In this method the MCA is used with an EVA/nonEVA adapter to reformat four 96-well plates into one single 384-well plate. Four 96-disposable tip boxes are used and may be placed on the Carousel or the worktable itself.
The four source plates containing the samples, and the target 384-well plate may, again, be either on the worktable or on the Carousel. The advantage of using the Carousel is that the Robotic Gripper Arm can then pick up each box or plate and take it to its transfer station on the worktable, with the plate barcode being read on the way.
Plate or box lids are also removed during the transfer to be kept for the time being in an empty carrier or a hotel on the worktable.
The MCA collects whichever adapter plate is selected and picks up 96 tips from the box, as shown in Figure 3. It now sucks up the target volume from the first 96-well source plate and puts it into quadrant 1 of the target plate.
The tips are then released into the empty tip box, or if the worktable is so set, thrown into the waste. This cycle is repeated until all source plates have been dispensed into successive quadrants of the target plate, and the adapter is then placed back in the holder.
If required, the microplate as well as box lids are now put back on, and the plates may be placed on the Carousel to clear the worktable, if needed, or just left on the worktable. Figure 4 outlines this process.
The primary benefit of this technique is its ease of use – the user needs only to press the button to start the run once the labware is placed on the worktable.
Figure 3: Top: The Multiple Channel Arm equipped with an EVA adapter plate picks up 96 disposable tips from a 96 disposable tip box. Bottom: The Robotic Gripper Arm moves a Multiple Channel Arm 96 disposable tip box from the Carousel transfer station to the worktable.
Figure 4: Plate layouts and pipetting schemes for reformatting four 96-well plates into one 384-well plate using an EVA or Non EVA adapter plate on the Multiple Channel Arm.
Here the single-use tips are nested in eight layers of MCA 96 tips per nest, making 768 tips altogether. This saves space on the worktable, since only an entry stack with nested tips and an empty stack for discharge of used tips is placed on it.
The MCA with either an EVA or nonEVA adapter is used to pick up 96 tips at a time, suck up fluid from the first 96-well source plate and place it in quadrant 1 of the empty target plate. The top layer of the stack being now empty, the Robotic Gripper Arm picks it up and puts it on to the exit stack.
The used tips are now placed on to this empty layer before a fresh set is picked up from the second layer of the entry stack. This goes on until all source plate contents have been transferred, while the entry stack now contains four layers of nested tips for the next run. This process is represented in Figure 5.
This method is recommended when space is at a premium as the entry stack stores tips for two pipetting sequences and two positions are saved on the worktable. This can be cut down further if the tips are not put into an exit stack but are directly discharged to waste via a point at the front or through the worktable.
Figure 5: The Multiple Channel Arm equipped with an EVA adapter plate picks up 96 disposable tips from a nested stack. After use, the tips can be discharged into the empty disposable tip box in the front.
The only difference in this method is that the MCA now picks up the 96 of 384 adapter plate. This means that one quadrant of single-use tips, 96 in number, is now removed by the MCA from the 384-tip box. The box is now put on the MCA active carrier by hand or picked up the Robotic Gripper Arm and placed there, if it was previously in the Carousel.
The rest of the process runs as before, with sucking up of source fluid from the first source plate of 96 wells, to be placed in quadrant 1 of the 384-well plate. The used tips are discharged to the first quadrant of the tip box.
Repetition of these steps leads to complete transfer of all the four source plates into the target plate. In the case of the 384-tip box, it has individual tip compartments so that the used tips do not cross-contaminate the unused ones. Figure 7 represents the process.
This method has the benefit of requiring only one SBS position on the worktable for tip storage during this experiment. The use of only one type of tip throughout an experiment, for all transfers, is essential in some tests to ensure comparability, and in general, this makes testing easier for logistical reasons. However, filtered single-use tips may be used, which is not possible in the second method. Also, 96-well plates may be changed for 384-well plates if required.
Figure 6: The Multiple Channel Arm equipped with the 96 out of 384 adapter plate and 96 disposable tips transfers liquid from a 96- to a 384-well plate.
Four plates with 384 wells may also be placed into one 1,536-well plate. For this process, all the labware needed is put on the worktable from the hotels or the Carousel. As needed, the Robotic Gripper Arm can lift off plate and box lids and store them in a hotel or an empty worktable carrier.
The MCA picks up the COMBO adapter plate and a set of 15 μl MCA 384 single-use tips, and sucks up the target fluid from the first source plate to place it into quadrant 1 of the bigger target plate. As Figure 8 shows, the used tips are put into the empty tip box which the Robotic Gripper Arm then takes away from the carrier, putting in its place a fresh MCA 384-tip box.
Repetition of this process fills up all quadrants of the target plate. The final step is to put back the MCA COMBO plate in the holder, and to replace plate lids if any. The entire method is shown in Figure 9.
This method can be used to fill three 384-well plates with samples of a buffer, DMSO or other solution used to dissolve a compound for assay, from a trough, commonly before a serial dilution is performed.
The lab equipment is put on the worktable while the solution is used to fill the trough. The MCA picks up the COMBO plate and a complete 384-tip set of 125 μl capacity each. These suck up the target compound of desired volume from the source plate and discharge it into the first 384-well plate as shown in Figure 10.
This is repeated using the same set of 384 tips until all three plates are filled. The adapter and tips are put back on the holder, ready to be used for the next cycle.
Figure 7: Plate layouts and pipetting schemes for the reformatting four 96-well plates into one 384-well plate using the 96 out of 384 adapter plate on the Multiple Channel Arm.
Figure 8: The Multiple Channel Arm equipped with the COMBO adapter plate and 384 disposable tips transfers liquid from a 384- to a 1,536-well plate.
Figure 9: Plate layouts and pipetting schemes for reformatting four 384-well plates into one 1,536-well plate using the COMBO adapter plate on the Multiple Channel Arm
Figure 10: Pipetting scheme for filling three 384-well plates with buffer solution or DMSO.
In addition, the time taken for each process method was recorded, as shown in Tables 1 to 4.
Table 1: Timelines for transfer of labware between the Carousel and the worktable.
Table 2: Timelines for reformatting four 96-well plates into one 384-well plate.
Table 3: Timeline for reformatting four 384-well plates into one 1,536-well plate.
Table 4: Timeline for filling empty plates with buffer solution or DMSO.
This experiment shows the Fluent system can easily and rapidly deal with plate reformatting with the ability to be modified for individual applications. The use of the MCA and the capacity to change adapter plates during the process run simplifies and speeds up the procedure of reformatting from plate to plate or copying into plates with 96, 384 or 1,536 wells, to maximize the throughput.
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