Field of the Invention

This invention relates generally to membrane transport studies, and more specifically to improvements in apparatus for carrying out such studies.

Background of the Invention In vitro evaluation of ion or solute transport and regulation of these transport processes across a membrane are important in determining the permeability of drug molecules, including peptides and proteins, in the evaluation of delivery systems and formulation excipients, in the evaluation of specific transport mechanisms, and in the study of the pathologic effects of drugs or drug delivery systems on the mucosa. Transport studies have been carried out in the past using a device known as an Ussing Chamber. The Ussing Chamber comprises two separable parts, which come together to form a closed chamber. At the interface between the two separable parts, a layer of tissue, e.g., excised intestinal tissue from a rat, is mounted on pins which extend across the interface between the two parts of the chamber. Thus, the tissue separates the chamber into two halves, each of which contains a quantity of buffer solution in contact with the tissue layer. The buffer solution is circulated through each chamber half by an air lift pump, and temperature is maintained at a constant level by a temperature-controlled water jacket surrounding a part of the buffer recalculation path. In me Ussing chamber, transport across the membrane is typically determined by using radioactive material as a tracer. Other analytical techniques (e.g., HPLC, RIA and ELISA) can be used to measure transport. In addition other tracers, such as fluorescent materials, can be used. While various techniques are available to determine transport across a tissue membrane, it is also necessary to determine the

viability of the membrane. An electrical technique is often used. A first pair of electrodes is arranged with the tips of the electrodes located in the chamber, and on opposite sides of, and in close proximity to, the membrane. These electrodes are connected to a millivolt meter for determining the spontaneous potential difference. A second pair of electrodes is arranged with the tips of the electrodes in the respective portions of the chamber. The electrodes of the second pair are connected to an adjustable current source in series with a microammeter. The current source is controllable, through a feedback circuit, in response to the voltage measured by the millivolt meter, so that the measured voltage can be held at any value including zero. The electrical apparatus which includes the feedback circuit is known generally as a "voltage clamp."

In carrying out electrical measurement using the voltage clamp, the potential difference across the membrane is first measured by measuring the voltage between the first pair of electrodes while the current between the second pair of electrodes is zero. Then, current is applied to the path between the electrodes of the second pair in a direction such as to oppose the potential difference across me membrane. The current required to bring the measured potential difference to zero is recorded as a "short circuit" current. Trans-membrane electrical resistance can be determined by Ohms Law.

The Ussing chamber has been used in measurement of transport across excised animal tissue, but is not suitable for use with cultured cell monolayers.

One approach to membrane transport studies using cultured cell monolayers has been to use a so-called "diffusion chamber" of the kind available from Precision Instrument Design, 205 Alpine Meadows Rpad #4, P. O. Box 1242, Tahoe City, California 96145. A typical diffusion chamber comprises two vertically elongated acrylic "half-cells" which can be clamped together on either side of a membrane to provide a solution-containing cavity on opposite sides of the membrane. A modified version of the diffusion chamber is available in which a polycarbonate filter is

insertable between the two acrylic half -cells. The polycarbonate filter serves as a substrate for a cultured cell monolayer. In both versions of die diffusion chamber, electrodes are suspended from caps fitted to the top of each half-cell. The electrodes are used to determine the potential difference across the cell layer, or alternatively to supply current for the measurement of trans-membrane electrical resistance. The solution in each cavity is circulated and oxygenated through a reservoir and passages integrally formed in the half-cell. Thus, these and similar configurations, referred to in me art as "single-unit" diffusion chambers, cannot be adapted to commercially available and inexpensive glass recirculation devices for both tissue and cultured cell monolayers. In addition, access to the passages for cleaning a single unit configuration is difficult, isotopes in d e solution circulated through the unit tend to adhere to plastic, and temperature control is difficult and inaccurate due to thermal lag in the plastic block and the volume limited of circulating solution and proximity to the chamber. Consequendy, large fluctuations in solution concentration and fluctuation in measurements from experiment to experiment can occur. The principal object of this invention is to provide a low-cost diffusion chamber system for determining d e transport phenomena of permeable membranes utilizing a Ussing-type diffusion chamber in combination with a conventional solution circulating device which is easy to maintain and clean. Another object of d e invention is to provide a diffusion chamber system which will maintain the required temperature and concentration of die solution and which will improve reproducibility of results in like experiments. Still another object of die invention is to provide for more effective, more accurate, and more readily reproducible measurement of me electrical parameters of membrane barriers in transport studies. Ano er important object of the invention is to provide a more practical apparatus for testing transport phenomena of cultured cell monolayers and animal tissues.

Brief Summary of the Invention

The apparatus in accordance with the invention comprises, as one of its elements, an insert having a tubular wall with opposite ends, an opening in each end, an internal passage extending from the opening at one end to the opening at the other end, and means, at a location within the passage, for supporting a membrane barrier such as animal tissue or a monolayer of epithelial cells grown on a porous substrate. The membrane has two opposite faces and extends transversely over die cross- section of the passage, so that any molecule moving through die passage passes through die membrane.

Another element of the apparatus is a chamber for containing buffer solution. The chamber comprises two separable bodies, each having a face, a hollow interior space, and an opening in its face communicating with its hollow interior space. The two bodies are positioned with their faces in mutually abutting relationship. At least one of d e two bodies receives d e cell culture insert in its opening.

Sealing means are provided for cooperation with die insert and with the chamber, to prevent flow of buffer solution around die exterior of d e insert from die hollow interior space of one of die two bodies to die hollow interior space of die other.

Means are provided for circulating buffer solution through the hollow interior spaces of each of die two bodies. A first pair of electrodes is provided. One electrode of die first pair is located within the hollow interior space of one of the two bodies and the odier electrode of die first pair is located within die hollow interior space of die other of die two bodies.

A second pair of electrodes is also provided. One electrode of the second pair extends through d e hollow interior space of one of the two bodies and has a tip located in close proximity to die porous substrate, within the internal passage of die cell culture insert, on one side thereof. The other electrode of die second pair extends through die hollow interior space of the other of die two bodies and has a tip

located in close proximity to the porous substrate on the other side thereof. At least one of die electrodes of the second pair extends d rough the opening in one of die ends of d e tubular wall of the cell culture insert

The body receiving the cell culture insert has a cylindrical recess in its opening for receiving die cell culture insert. In a preferred version of d e apparatus, both bodies have recesses for receiving die cell culture insert, a part of die insert being received in die recess of one body, and another part of die insert being received in die recess of the other body.

The porous substrate is preferably spaced from both ends of the internal passage of d e insert, and die electrodes of the second pair preferably extend through die respective openings in the opposite ends of die tubular wall of the insert into close proximity to the porous substrate.

Each of die separable bodies comprises a side wall at least in part defining its hollow interior space, and at least one of die electrodes of said second pair preferably extends obliquely dirough die side wall of one of die two separable bodies and dirough die hollow interior space thereof, into die interior of d e insert.

In a preferred embodiment of the invention, in which a porous substrate is spaced from both ends of said internal passage, each of die electrodes of the second pair extends obliquely dirough die side wall of one of d e two separable bodies and dirough a hollow interior space into the interior of die insert. The apparatus preferably includes a voltage clamp connected to die first and second pairs of electrodes, for applying an electrical current to the electrodes of die first pair in response to the potential difference between die electrodes of the second pair.

Where he electrode of die second pair extends obliquely through the side wall of one of d e two separable bodies, a seal is provided between the electrode and die side wall dirough which it extends. The electrode extends through an oblique passage which has an internal shoulder and is internally threaded on the portion of d e

passage on die side of the shoulder toward the exterior of the side wall. The seal comprises a resilient O-ring seated against die shoulder of die passage and surrounding die electrode. An externally threaded hollow fitting, having an internal passage for receiving the electrode, is engaged widi the threads of the oblique passage and presses axially on the O-ring, so that d e O-ring is compressed axially against the shoulder and bears radially inwardly on the electrode.

Further objects, details and advantages of die invention will be apparent from d e following detailed description, when read in conjunction with the drawings.

Brief Description of the Drawings

FIG. 1 is a cross-sectional view of an apparatus in accordance with die invention, showing the modified Ussing chamber and the liquid circulation system;

FIG. 2 is a schematic view showing the modified Ussing chamber with a voltage clamp; and FIG. 3 is an exploded cross-sectional view of an insert for use in the modified

Ussing chamber of FIG. 1.

Detailed Description

The apparatus, as shown in FIG. 1, comprises a modified Ussing chamber 10 supported by clamping screws 12 and 14 on a yoke 16. The modified Ussing chamber 10 is provided with a buffer solution circulating and temperature regulating device 18, for circulating buffer solution within die Ussing chamber.

The modified Ussing chamber 10 comprises two halves 20 and 22, which, in die embodiment described, are essentially identical to each other. These chamber halves are in face-to-face relationship with each other, meeting at a planar interface 24. Each chamber half is formed from a syndietic resin such as polymethylmethacrylate, known commercially as Plexiglas or Lucite. Chamber half 20 has a hollow interior space 26, a portion of which has a frusto-conical wall 28, d e diameter of which increases in die direction toward die opposite chamber half. A cylindrical space 30 is provided at the small-diameter end of die conical wall, and a cylindrical recess 32 is provided at die large diameter end of die conical wall. This recess is slighdy larger in diameter than die large-diameter end of the conical wall, and is open to the face of die chamber half at interface 24. Since chamber half 22 is similar to chamber half 20, and has a recess 34 similar to recess 32, when die two halves come together, recesses 32 and 34 together form a continuous cylindrical space for receiving a cell culture insert 36, which, in die assembled device, is situated partly in recess 32 and partly in recess 34. Insert 36 may be of die kind commercially

marketed under the name Snapwell™ by Costar Corporation, Cambridge,

Massachusetts. Pins 38 are provided to align the two chamber halves so that the cylindrical walls of recesses 32 and 34 are aligned with each other, and so diat the two chamber halves can be separated and reassembled readily.

A passage 40, formed in chamber half 20, provides a connection between die interior space 26, and a tube 42. Tube 42 is connected dirough a lengdi 44 of flexible tubing, to a vertically extending tube 46 inside a jacketed glass vessel 48, which contains buffer solution 50, preferably a mixture of potassium, sodium, calcium and magnesium chlorides and bicarbonate. The surface 52 of die buffer is at a level above die opening 54 at d e upper end of tube 46. A gas, for example, a mixture of oxygen and carbon dioxide, from a supply tube 56 is introduced into tube 46 dirough a small diameter tube 58, which extends dirough die side wall of tube 46 at a level below opening 54. Gas bubbles 60 emitted from tube 58 decrease d e effective density of d e buffer solution in the upper portion of tube 46, and induce an upward flow in tube 46, so that liquid is drawn out of space 26 through passage 40. A liquid return path is provided by tube 62, flexible tube 64, tube 66 and passage 68. The action of bubbles 54 causes buffer solution to be recirculated between space 26 and vessel 48. A similar arrangement is provided to recirculate buffer solution between die interior space of chamber half 22 and a vessel 70. A flow of water at a controlled temperature, e.g. 37°C, is maintained dirough a jacket 72 surrounding vessels 48 and 70, to maintain a constant temperature in die recirculating buffer solution. The temperature-controlled water is introduced into d e jacket through passage 74, and flows outwardly from the jacket through passage 76. Air vents, extending dirough the walls of vessels 48 and 70 and dirough the wall of jacket 72, are provided at 78 and 80. Referring now to FIG. 3, die cell culture insert 36 comprises two principal parts: a sleeve 82, having a dirough passage with a shoulder 84, and a cell growth member 86. Member 86 has a porous surface 88, of polycarbonate, which serves as a

substrate for supporting a cell monolayer. Member 86 fits snugly into cylindrical space 90 in sleeve 82, and comes into engagement with shoulder 84. Flange 92 of member 86 comes into close proximity to face 94 of the sleeve, and allows member 86 to be removed from d e sleeve. When parts 82 and 86 are assembled, porous surface 86 is situated approximately midway between the end faces 94 and 96 of sleeve 82.

Preferably, porous surface 88 is circular in shape, and its diameter is d e same as that of the openings of die chamber halves of a conventional Ussing chamber used for animal tissue measurements. Typically, die area of die conventional opening is 1.13 cm^., and the area of porous surface 88 is similarly 1.13 cm^. The recesses 32 and 34 in die chamber halves make it possible for the diameter of die porous surface 88 to be approximately equal to the diameters of die large ends of die frusto-conical internal walls of die chamber halves, so mat die electrical performance of die modified chamber in accordance with tiris invention is similar to mat of d e conventional Ussing chamber. O-rings 98 and 100 are provided on reduced end portions of d e sleeve to establish a seal between d e insert 36 and die walls of d e recesses in the chamber halves, to prevent buffer solution from flowing from one interior space to die other around die outside of the cell culture insert.

The schematic view of die apparatus, in FIG. 2, shows die electrical connections to d e modified Ussing Cell. A voltage clamp 102 applies a controlled D.C. voltage to electrodes 104 and 106, which extend into die interior spaces of die respective chamber halves at die narrow ends of the frusto-conical walls. The electrodes are typically in the form of "bridges" consisting of hollow polyethylene tubes containing a conductive gel, e.g. a mixture of Agar and KC1 solution. Electrical connections are made from metal electrical leads to the bridges through cells 107 and 108, each of which contains a conductive solution, e.g. KC1 solution. A

microammeter 110 is connected in line 112 between the voltage clamp and cell 107 to register the current in the current path between electrodes 104 and 106.

In order to measure the electrical potential across the cell monolayer in die cell culture insert, potential electrodes 114 and 116 are provided. These electrodes are also preferably "bridge" electrodes, and are connected to the voltage clamp 102 dirough calomel cells 118 and 120 and lines 124 and 126. A millivolt meter 122 is connected across lines 124 and 126 to register the potential difference across the cell monolayer.

The voltage clamp 102 is a well-known device used for transport studies on in vitro epidielial tissues. The voltage clamp is fundamentally a device which applies an electrical current to the path between electrodes 104 and 106, and which utilizes feedback to control die magnitude of the current in response to die potential difference measured by electrodes 114 and 116. The feedback loop in the voltage clamp makes it possible to apply a current at a level such as to hold d e potential difference across die tissue layer at any desired level, including zero. One important parameter to be measured is PD, die potential difference across die cell layer. PD is registered direcdy by the millivolt meter 122, when die current in die patii between electrodes 104 and 106 is zero. Another important parameter to be measured is die "short-circuit" current Igc, the current required to nullify die potential difference PD. I _sc is measured by setting the voltage clamp so that die voltage registered by millivolt meter 122 is held at zero. The current registered by microammeter 110 is die short circuit current

A preferred voltage clamp is the Model VCC600 Voltage-Current Clamp, available from Precision Instrument Design, 205 Alpine Meadows Road #4, P. O. Box 1242, Tahoe City, CA 96145. In order to achieve accurate and reproducible measurements, the tips of die potential measuring electrodes 114 and 116 should be as close as possible to the cell monolayer, and are preferably centered on the cell monolayer. Therefore, electrodes

114 and 116 are arranged as shown in FIG. 1, with the electrodes extending obliquely through die walls of the chamber halves 20 and 22, and through the openings at the ends of die cell culture insert so that the electrode tips 128 and 130 are in close proximity to, and centered on, die cell layer on porous substrate 88 (FIG. 3). Electrodes 114 and 116 are removably secured in place by externally threaded, hollow screws 132 and 134, which have central passages dirough which die electrodes extend. The screws are threaded into internally threaded recesses in d e walls of die chamber halves, and clamp resilient O-rings 136 and 138 axially against shoulders within d e recesses so d at die O-rings, as tiiey are axially compressed, press radially inwardly against the electrodes to provide a liquid-tight seal. Screws 132 and 134 allow d e electrodes to be removed for replacement and also allow the positions of d e electrodes to be adjusted. The angles of die axes of die passages dirough which die electrodes extend are such that die electrodes clear the edges of die openings of the cell culture insert 36 and so that die tips 128 and 130 are centered on the cell layer when in very close proximity, i.e. less man 1.0 mm, to the cell layer and its supporting substrate 88.

The apparatus described herein makes it possible to monitor cell culture integrity and viability using a voltage clamp, and to achieve effective, accurate, and reproducible measurement of the electrical parameters of a cultured cell monolayer in transport studies. By making it possible to achieve effective measurement of die electrical parameters of cell monolayers, die invention also reduces the need for animal tissue in transport studies. Otiier advantages of die invention include die fact that the modified Ussing chamber utilizes a cell culture insert which has a cross- section the same as d at for a standard Ussing chamber, so diat comparisons can be made between animal tissue and cell cultures. The modified Ussing diffusion cell in accordance with the invention is applicable both to academic and industrial studies, and can be used in studies of a wide variety of tissues used in drug delivery, including nasal, pulmonary, buccal, oral,

stomach and intestinal routes of delivery. By utilizing a Ussing-type diffusion cell in combination widi a conventional solution circulating device, the assembly is more accessible for cleaning and maintenance. With the circulating system separate from the diffusion cell, a greater volume of the solution provides better temperature and concentration control for better reproducibility of results in repeated experiments and tests.

Various modifications can be made to the apparatus described herein. For example, aldiough the cell culture insert 36 is preferably as shown in FIG. 3, with the porous substrate midway between the ends of sleeve 82, the insert can be designed so diat the porous substrate is located at one end of the sleeve, and the potential electrodes can be arranged so at one extends into die sleeve with its tip in close proximity to one face of die substrate, while die tip of the other electrode is in close proximity to die opposite face of die substrate, but entirely outside the sleeve of die insert. Various other modifications can be made to die apparatus as described herein without departing from the scope of the invention as defined in die following claims.