The pharmaceutical industry has widely recognized patch clamping as the "gold standard" to provide the highest information content in the drug discovery screening process by directly measuring the current passing through ion channels. This technology, developed 30 years ago by the German researchers Neher and Sakmann, allows direct voltage control of a cell and thus the ability to study ion channel pharmacology in detail.
However, manual patch clamping is time consuming, needs an experienced operator, and yields low throughput. Glass has proven to be the best substrate for patch clamping and does not suffer from capacitance problems like silicon chips. Using standard borosilicate glass electrodes resulted in high quality patch clamp data.
The same patch electrodes as in basic research were used to invent the principle of the Flip-the-Tip technology, i.e., flushing cells into the pipette from the back and forming a GigaSeal directly at the tip inside a patch pipette.1
Flyion's (www.flyion.com) approach was to utilize standard components for the Flyscreen 8500 robot to guarantee high quality recordings and to make the system affordable for smaller pharmaceutical companies and CRO firms alike.
Depending on throughput, the user can choose a version with either 3 or 6 measurement channels. The 3-channel version of the Flyscreen robot can be upgraded to the 6-channel version at any time. The robot runs completely unattended for several hours because only intra- and extracellular solution, 2 mL of cell suspension to be placed in the cell hotel, a rack of FlipTips, and a standard 96-well compound plate have to be provided.
When so-called sticky compounds are to be tested a glass vial plate holder can be mounted instead of a polystyrene microtiter plate. The FlipTip consumables are packed in 48 units per rack. Operation is asynchronous and parallel. Thus, every recording tip socket is treated as its own entity and its performance is independent of other tip sockets.
The FlipTip consists of a standard borosilicate patch pipette glued into a plastic jacket. The inner chamber of a recording socket is prefilled with intracellular solution before the FlipTip is inserted tightly into the socket and backfilled with extracellular solution. A cell suspension of only a few hundred to a thousand cells is automatically taken up from a cell hotel and dispensed into the back of the FlipTip.
Simply by gravity, the cells travel toward the tip of the pipette. Subsequent gentle suction draws a single cell into the end of the tip, forming a classical GigaSeal of 15 GOhm, equal to the manual patch clamp method. Further suction pulses are then applied to establish the open whole-cell configuration and, under voltage-clamp conditions, the response of an ion channel can be measured either by voltage or ligand activation (Figure 1).
Alternatively the cell membrane can be perforated by using pore-forming agents, allowing perforated patch clamp recordings. FlipTip and liquid handling, seal formation, the establishment of whole-cell configuration, and drug application are all performed automatically by the Flyscreen robot.
Data Flow Handling
Data is stored in a MySQL database on an FTP-server which can be installed either locally on the PC performing the operation or on a network server. Further, several Flyscreen machines can be connected to the same database. This allows the user to analyze the results basically from any PC where the Flyscreen software is installed.
The Flyscreen Suite software communicates with the PatchMaster software (Heka Instruments) and uses its functionality to stimulate, analyze, and display raw data. In general, the user modifies a predefined sequence.
The Sequence File holds the entire description of a patch clamp experiment and information about the ion channel to be screened by calling downstream files such as Segment Templates, Pulse Files, and Online Analysis Files. Predefined Segment Templates control the robot's functions, such as pump movements, suction, washing cycles, volume of compound application, and can be used to compile a customized Sequence.
The Pulse File defines the actual voltage pulse pattern to evoke the ionic current. The Online Analysis File extracts the defined parameter(s) of interest. This could be the mean, Min/Max, peak amplitude, or slope value of the current. The results are displayed in online analysis graph windows and stored in the database (Figure 2). The user can modify the content and hence, can customize the predefined library of sequences, specific for every ion channel to be studied.
Data can be further analyzed with respect to seal and whole-cell success rates, experiment duration, drug action, and biophysical parameters such as RSeries, Cslow, CFast, using Flyion's FlyStat software. The MicrosoftAccess-based FlyStat software comes with the system.
The Flyscreen Suite software allows the user to completely design an automated patch clamp experiment. Upon starting the software a dialog window appears allowing entry of cell information, intra- and extracellular solution in use, the FlipTip package number, compound information, type of experiment (whole-cell or perforated mode), number of experiments and available FlipTips, number of compound applications, and the Sequence to be used. Running a batch simply means loading these parameters and pressing the start button (Figure 3).
The user defines the number of experiments which is, in essence, independent of the number of FlipTips. Assuming a single compound application per cell, and a triplicate replication of 6 concentrations to achieve a valid dose-response curve, a minimum number of 18 FlipTips are required.
Usually, success rates range between 5060%, so 36 FlipTips have to be provided to successfully finish the described batch. However, the Flyscreen Suite keeps track of available FlipTips and will ask the user to either refill the FlipTip rack or to abort the batch, in case the experiment runs short on FlipTips.
The same functionality concerns liquid handling. The user specifies the volume of all liquids in use (including compounds), and the software will notify the user to either refill solutions or abort an ongoing batch in case a liquid compartment runs empty.
The main goal was to automate manual patch clamping while retaining high quality data. A definition of a successful experiment encompasses the following parameters: a true GigaSeal of more than 1 GOhm, a series resistance of less than 5 MOhm in whole-cell mode, a continued total resistance between 0.5 and 1 GOhm, and a duration of these parameters of at least 15 min.
Given a 3 or 6 channel robot, a data volume of around 140280 data points per day can be collected, given a twofold compound application per FlipTip and a recording time of 20 min.
Further, the FlipTips have a much lower stray capacitance (<10 pF) compared to chip-based systems, making them useful for studying fast voltage-activated ion channels.
The Flyscreen software uses an internal scheduler for intelligent robot control. This scheduler optimizes the throughput by maximizing the parallel execution of experimental steps. In essence, electrophysiological operations are performed in multiple recording channels while another experiment is using the robot arm on a different recording tip socket channel.
Similar to manual patch clamping, a task is finished or cancelled once a user-defined flag condition has been met or not. As an example, a seal segment is set to last 5 minutes to reach a user-defined seal resistance. If the value has been reached before the specified time period, the scheduler shifts all following segments forward in time. This is performed for every recording tip socket independently.
In panel A of Figure 4, a completed batch of 15 FlipTips is shown, and individual segments are depicted in different colors. Most notably are the red segments for RPip validation and control measurements (channel expression test), yellow seal segments, and green segments indicating compound application. Among the three tip sockets, it is evident that the segments do not all occur at the same time, reflecting the action of the scheduler. This batch took 1 h 40 min to be completed, and showed a success rate of 80% (seal and whole-cell formation).
Panel B of Figure 4 shows the successive blockade of the Kv1.5 potassium channel expressed in an LTK cell line by 5 mM 4-Aminopyridine, and panel C depicts the time course of a blockade shown in an online analysis window. The user can export all the data points in an online analysis window easily as ASCII values to construct dose/ response and I/V relationships. Several on-line analysis windows can be opened at the same time to analyze different aspects of the data trace. Thus a multi-parameter analysis can be performed with a single experimental run.
Panel D shows a FlyStat analysis over a time period of 18 days, revealing the success rate for valid FlipTips (RPip), seal and whole-cell state, and the recording time.
In conclusion, automation of manual patch clamping using classical borosilicate glass pipettes was achieved. The use of standard components makes the Flyscreen8500 affordable for big and small pharma and will boost ongoing secondary screening and lead optimization studies in preclinical drug candidate evaluation.
In particular, the focus of Flyion was to not only improve data throughput compared to manual operation but clearly to retain the high data quality a user can achieve by using standard patch glass pipettes. The concept of the FlipTip has been proven to show equal data compared to classical patch clamp and to yield higher data throughput at an exceptional quality level.