DAD-12 Superfusion: Powerful Delivery of Drugs to Cells

By Alan Kriegstein, ALA Instruments, Inc.

One of the simplest ways to determine the effect of a substance, be it a drug or a chemical, on a living organism is to apply that substance to the organism and see what happens. In the case of an individual cell, or small group of cells, this is complicated by the inherent small geometry. Yet the need to see the effect of drug application on the cellular level creates a demand for equipment to do this kind of work, and indeed has established a market for it.

In modern neuroscience research, cells are typically impaled with a sharp electrode (<1µm tip) or tapped with a patch electrode (a smooth tipped glass capillary with a 1-5µm tip) which in turn is attached to an amplifier specifically designed for the task of amplifying the picoamp signals of the cell. In either case, the electrode, which is made from a glass capillary tube, is filled with a saline solution capable of making an electrical connection with the cell. Over the years, many specific ion pathways and membrane channels have been discovered in neurons and other cell types. Each ionic current has a specific signature that can be described as the normal ionic current. This current is characterized by a waveform seen on an oscilloscope which represents the flow of current into, or out of, the cell depending on the phase of the current. Thus, one of the simplest ways to evaluate the effect of a drug on a neuron is to see how it alters the specific waveform of a known ionic current because this directly effects the cell's ability to transmit signals.

There are several methods now available to apply drugs to cells under study. The cells can be shot with beads that contain the drug in micro pores, the cells can be micro injected, the cell can be perfused externally, and internally, the drug can be added to the cell bath and absorbed, or it can be puffed directly onto the cell.

The idea of puffing the drug onto the cell goes back to the beginning of cell physiology studies. Researchers took advantage of the same micro capillaries that they made the electrodes out of to make puffing tubes to apply the drugs to the cells they were studying. One of the problems that arose with the typical "puffer pipette," as they are often referred to, was that the small hole in the tip frequently caused the applied substance to spread out quickly and become diluted. This is not a problem when one is using strong agonists or toxins where tiny amounts have great effects; but what about dose response curves where the data are dependent on precise application concentrations?

The simplest way to insure the concentration of the drug being applied to the cell is to present the cell with a large stream of solution that completely engulfs the cell in the applied solution. This is commonly known as the "sewer pipe effect" wherein the swath of the applied solution completely engulfs the cell forming what can also be referred to as a "concentration clamp" around the cell. That is to say, that for the time that the cell is in the flow, it sees nothing but the substance being applied and is in essence isolated in the flow stream.

The ability to change the applied substance rapidly seems a natural extension. Sending a 100µm stream of solution at a cell does not accomplish more than if one takes the same substance and adds it to the cell bath, i.e. no special equipment would be required to accomplish the same experiment. We can open up whole new areas of research when we are able to switch, in rapid time course, the stream of solution flowing over the cell. Since signals on the cellular level operate in the msec time range, we need to approach this speed range to catch certain events, and simulate, or stimulate others.

The DAD-12 Superfusion System is the basic workhorse for these types of experiments particularly on neurons. (DAD stands for Drug Application Device, and 12 for twelve possible solutions.) The word superfusion is used because the system applies the substance around the cell, and perfusion really refers to passing the substance through the cell. It should be noted that these types of systems can also be referred to as focal drug application systems.

The DAD incorporates our proprietary Micromanifold which forms the front end of the system. The Micromanifold is made of fused silica capillaries that form a conduit in which 13 applied substances (one channel is reserved for a wash solution) flow together and exit a single output tube of 100 or 200µm. The Micromanifold is small enough to be mounted on a micromanipulator and the tip can be brought near, and pointed at, the cell under study.

The DAD-12 is designed to deliver one drug at a time. While mixing might be helpful, we cannot insure that a homogenous mixture will come out. Therefore only one drug at a time can be delivered. If the user wants to test a mixture it should be prepared beforehand and added to a reservoir. The Micromanifold combined with other features of the system minimize the amount of substance mixing in the system. Control of the output is set via computer which, through an interface, controls the timing of valves and the pressurization of substance reservoirs that determines the flow rate. The DAD is not calibrated to put out specific amounts, but it can be calibrated by the end user in a simple procedure. Figure 1 shows a typical calibration curve of output of plain water.

A typical experiment is carried out under the microscope where the investigator finds the cell to be studied. The cell is then patched or otherwise impaled by an electrode that is connected to an amplifier. The Micromanifold of the DAD is pointed at the cell. The investigator puffs different drugs or drug concentrations that were loaded into the DAD reservoirs onto the cell. The entire sequence is recorded using the amplifier and a histogram of the response is produced after several applications.

A focal drug application system offers additional advantages over other application methods. Among these added benefits are that cellular exposure to the applied substances can be limited. The cell bath can be set up with a cross current perfusion so that any drugs applied to one cell, are washed downstream of the cell under study, and removed from the bath. Any other cells up stream of the application point are not exposed and thus left fresh for a new experiment. Also the Micromanifold is built to include an additional 13th tube that carries a wash solution down into the inner part of the Micromanifold where all the solutions come together.

This way a continuous wash can be applied to a cell under study. The continuous wash dilutes any of the other substances that might leach out from their respective tubes. Its presence also puts static pressure on all the other tubes to reduce mixing or leaching in the first place. The wash can also be used to detect if the flow has a negative effect on a cell. Some cells may be affected by the tiny force of the stream which can alter their response causing a "flow artifact." (A valve in the DAD system stops the wash flow whenever a test substance valve opens, and returns the wash flow when the test substance is stopped.)

Computer control of the DAD is simple. The program sets up a spread sheet style list of steps. Each step calls a different valve and the user can set the on-time of the valve and set any delays that may be necessary. Other menus allow the user to select the pressure applied to each substance as it is emitted (this sets the flow rate), the voltage sent to a particular valve including an 'on' and 'holding' voltage and other parameters. A separate 'on' and 'holding' voltage allows a valve to be spiked on at higher voltage to open faster, then stepped to a lower voltage to prevent over heating. System configuration and sequence files can all be saved for later recall.

Overall, the DAD system provides a turnkey approach to focal drug application. It combines many important features in one package to simplify the user's set-up. One can take advantage of the DAD-12 system's flexibility to set up other types of perfusion styles, such as the "j" tube or "U" tube. These methods take advantage of specific flow characteristics of small tubing to create high-speed (µsec-msec) exchange systems. In addition, the DAD-12 can be used as a valve control system to regulate flow to a whole chamber where cells or tissue are kept viable for a period of time while under study. While it is possible to build a home-made focal applicator system, the DAD-12 System incorporates so many useful features in one package, it would be difficult even for the technically gifted to duplicate in his or her spare time.

About the Author

Alan Kriegstein has been president of ALA Scientific Instruments Inc. for the last 10 years. His primary function as president is targeting and managing new product development. Kriegstein received a B.S. in biology from Union College in Schenectady, NY, and did graduate work in Neuroscience at the School of Public Health Science in Albany, NY. He is also president of Lptek Corp. a start-up company which manufactures computer parallel port interfaces for test, measurement, and control applications.

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For more information: Alan Kriegstein, President, ALA Scientific Instruments, Inc., 1100 Shames Dr., Westbury, NY 11590. Tel: 516-997-5780. Fax: 516-997-0528. Email: sales@alascience.com.