Methods and apparatus for detecting the effect of cell affecting agents on living cells

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BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for detecting the effects of cell affecting agents on living cells and to apparatus adapted to the practice of such methods. Solutions or suspensions of cell affecting agents are flowed over cells and the effects of these agents are measured.

2. Description of the Background of the Invention

Studies of the effect of various cell affecting agents on living cells have been reported in the literature. See, e.g., Meisner, H. and Tenny, K. (1977) "pH as an indicator of free fatty acid release from adipocytes," J. Lipid Research, 18:774-776; Nilsson, N. and Belfrage, P. (1979) "Continuous monitoring of free fatty acid release from adipocytes by pH-stat titration," J. Lipid Research 20:557-560; Reuss, L., Weinman, S. and Constantin, J. (1984) "H.sup.+ and HCO.sup.-.sub.3 transport at the apical membrane of the gallbladder epithelium," pp. 85-96 of Forte, J., Warnock, D. and Rector, F. Jr. (eds.) Hydrogen Ion Transport in Epithelia, Wiley-Interscience; Zeuthen, T. and Machen, T. (1984) "HCO.sup.-.sub.3 /CO.sub.2 stimulates NA.sup.+ /H.sup.+ and Cl.sup.- /HCO.sup.-.sub.3 exchange in Necturus gallbladder," pp. 97, 108 (ibid.); Handler, J. S., Preston, A. S. and Steele, R. E. (1984) "Factors affecting the differentiation of epithelial transport and responsiveness to hormones," Federation Proceeding 43:2221-2224; and Simmons, N. L., Brown, C. D. A. and Rugg, E. L. (1984) "The action of epinephrine on Madin-Darby canine kidney cells," Federation Proceedings, 43:2225-2229. These references disclose the detection of changes in pH and other electrical potentials by the addition of cell affecting agents to cells disposed in a relatively large amount of medium, i.e., a bulk medium. A disadvantage of these techniques is that the pH and other electrical potential measurements are taken from the bulk medium and do not necessarily reflect the actual values immediately adjacent to the cellular membranes of the living cells. Also, the high ratio of bulk volume to cell volume inevitably dilutes the effects of the cells on the properties of the extracellular medium. Accordingly, sensitivity is lost or greatly reduced.

Photoresponsive sensors for measuring biochemical systems are disclosed in various patent documents owned by the assignee of the present invention. See. e.g., U.S. Pat. Nos. 4,591,550 (Hafeman et al.) and 4,704,353 (Humphries et al.); and European Patent Application No. 213,825 (Hafeman et al.). U.S. Pat. No. 4,519,890 discloses a flow pH chamber. These patent publications disclose the use of microorganisms to measure changes in the environment of the solution to be measured. There is no disclosure in these publications of cells in micro flow chambers used to measure of the effects of cell affecting agents. See, also, U.S. Pat. Nos. 4,737,464 (McConnell et al.) and 4,741,619 (Humphries et al.), which are likewise owned by the assignee of the present invention.

Various ways of using fluorescence to measure extracellular effects of living cells and analytes are disclosed in the literature. See. e.g., Briggs et al. (1985) "Fiber Optic Probe Cytometer" J. Immunological Methods, 81:73-81; Hafeman et al. (1984) "Superoxide Enhances Photo Bleaching During Cellular Immune Attack Against Fluorescent Lipid Monolayer Membranes" Biochemica Biophysica Acta 772:20-28; and U.S. Pat. Nos. 4,560,881 and 4,564,598.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide methods for detecting the effects of cell affecting agents on living cells with greater accuracy and precision.

It is an additional object of the present invention to provide methods for detecting the effects of cell affecting agents on living cells that avoid the use of large volume, bulk media.

It is a specific object of the present invention to provide methods for detecting an effect of a cell affecting agent on living cells by: (a) providing living cells that are retained in a micro flow chamber adapted for continuous or intermittent flow of a solution or suspension containing the cell affecting agent in intimate contact with the cells so that the amount of cell affecting agent in contact with the cells may be controlled; (b) flowing a solution or suspension containing the cell affecting agent in intimate contact with the cells, thereby producing a cell mediated extracellular effect or change in pH, redox potential, cell surface potential or trans-cellular potential; and (c) measuring the effect of the cell affecting agent by a means for detecting pH, redox potential, cell surface potential or trans-cellular potential that is operably associated with the micro flow chamber.

It is a further object of the present invention to provide apparatus particularly adapted to practice the inventive methods.

In a preferred embodiment of the invention, the cells may be retained in the micro flow chamber by spontaneous or natural adhesion of the cells to the internal surface of the flow chamber or on a porous membrane or microcarrier contained in the flow chamber. Alternatively, the cells may be retained in the micro flow chamber by means of a binding agent that is biologically compatible with the cells. A preferred example of such a binding agent is agarose.

In another preferred embodiment of the present invention, the living cells may be retained in the micro flow chamber by providing the micro flow chamber with a surface having a plurality of wells or depressions that act to physically trap the cells on the surface of the micro chamber, preferably by gravitational sedimentation. These wells should be of a sufficient width and depth such that the cells remain in the micro flow chamber during ordinary flow rates. The cells may then be removed from the trapping wells by any appropriate means, including the use of high flow rates through the micro flow chamber that wash the cells out of the wells or by inversion of the chamber to dislodge the cells from the wells into the flow stream. In an alternative embodiment, the cells may be retained in the flow chamber by trapping them within a compartment of the flow chamber separated by a porous membrane.

Some features of the preferred geometry of the micro flow chamber used in the present invention are common to most applications of the device. Chief among these is the volume-to-surface ratio in the chamber, which controls the maximum concentration of adherent cells. The pH change per proton excreted by a cell is inversely proportional to the cell concentration and, therefore, to the volume-to-surface ratio. For the planar slab geometry of the chamber as described in FIGS. 1-4, this ratio is simply the chamber height, typically 100 .mu.m.

The design tradeoff involving volume-to-surface ratio is principally one of sensitivity versus ease of fluid handling. With our present preferred apparatus, sensitivity becomes seriously degraded for ratios above approximately 200 .mu.m. For thicknesses below approximately 50 .mu.m we anticipate significant fluid-handling problems that might offset sensitivity gains. However, instrumental improvements, such as chambers micro-machined into the sensor surface, might allow an integration of fluidics and chamber on a much smaller scale, permitting the analysis of one or a few living cells at ratios of 10-50 .mu.m.

The preferred volume of the micro flow chamber used in the present invention will depend to some extent on the intended application of the device. For example, if the number of cells available is a limitation, then subomicroliter chamber volumes are preferred (e.g., 1 mm.sup.2 surface area and 100 .mu.m height, or 100 nL, containing about 10.sup.3 cells). For other applications involving a larger number of cells, chamber volumes on the microliter or even milliliter scale can be envisioned (surface areas on the order of square centimeters, with about 10.sup.5 cells/cm.sup.2, but still maintaining approximately 100 .mu.m chamber height). Thus, the preferred micro flow chamber has both a small total volume achievable in the chamber and/or a small height or volume-to-surface ratio.

The type of micro flow chamber that is preferably used in the inventive method is of the type disclosed in U.S. Pat. No. 4,591,550, the disclosure of which is incorporated herein by reference. An argon and/or helium/neon laser may be used as a source of energy in place of the light emitting diodes disclosed in U.S. Pat. No. 4,591,550. Alternatively, the micro flow chamber useful in the present invention may be of the type disclosed in pending U.S. patent application Ser. No. 876,925, filed Jun. 20, 1986, which is owned by the assignee of the present invention, the disclosure of which is incorporated herein by reference application Ser. No. 876,925 is now U.S. Pat. No. 4,915,812 granted Apr. 10, 1990, Most preferably the micro flow chamber to be used in the present invention includes a silicon semiconductor electrode on or near which the living cells are retained. By means of this electrode, the various electrical effects caused by the cell affecting agent may be detected or measured. The micro flow chamber to be used in the present invention should preferably provide for both intermittent and continuous flow of solutions or suspensions, since either intermittent or continuous flow of solutions or suspensions may be used in practicing the present invention.

The present invention may be used in conjunction with either eukaryotic or prokaryotic cells, so long as the particular cells are capable of being retained in the micro flow chamber. Genetically transfected cells may also be used. In addition, a wide variety of cell affecting agents may be used, including irritants, drugs, toxins, other cells, receptor ligands, receptor agonists, immunological agents, viruses, pathogens, pyrogens, and hormones.

A wide variety of effects caused by the cell affecting agents may be detected or measured according to the present invention. Preferred effects include the pH, redox potential, and other electrical properties of the solution or suspension that flows in intimate contact with the living cells in the micro flow chamber, such as cell surface potential and transcellular potential. These effects may be measured or detected by a variety of conventional means. For example, pH can be detected by measuring the fluorescence or absorbance of a pH sensitive dye such as fluorescein or phenol red in the extracellular medium or fixed to a part of the chamber. In a similar manner other dyes can be used to detect redox potential.

The present invention also includes methods of identifying microbes, methods of screening for the activity of drugs, methods for detecting toxic substances and methods for detecting intercellular reactions. In these various methods, solutions or suspensions containing the desired cell affecting agent are flowed in intimate contact with the living cells retained in the micro flow chamber. The effect(s) of the cell affecting agent on the cells are then measured and provide the means by which bacteria may be identified, drugs screened, and toxins and intercellular reactions detected.

The present invention has several advantages over other methods of measuring metabolic activity, such as oximetry and microcalorimetry. Specifically, the present invention possesses rapid measurement time, enhanced sensitivity, noninvasiveness, generality and the potential for further miniaturization and integration.

Certain preferred embodiments of the present invention are discussed below in more detail in connection with the drawings and the detailed description of the preferred embodiments. These preferred embodiments do not limit the scope or nature of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 represent schematic cross-sectional views of micro flow chambers useful in practicing the preferred inventive methods.

FIG. 4 represents a schematic top perspective view of a micro flow chamber useful in practicing the preferred inventive methods.

FIG. 5 represents a schematic diagram of apparatus useful in practicing the preferred inventive methods.

FIG. 6 represents a schematic diagram of a degasser useful in practicing the preferred inventive methods.

FIG. 7 represents a schematic side view of adherent cells located in a micro flow chamber.

FIG. 8 represents a schematic side view of cells retained in wells in a micro flow chamber.

FIG. 9 represents a diagram of a circuit useful in the present invention.

FIG. 10 represents a typical determination of cellular metabolic rates that may be made by use of the present invention.

FIGS. 11-23 represent the experimental results obtained in connection with Examples 1, 2, 4, 6, 9, 11 and 17.

FIG. 24 represents results of exposing gram negative bacteria to an antibiotic which has an effect and one that has no effect on the gram negative bacteria.

FIG. 25 represents a schematic plan view of wells for retaining cells useful in practicing the preferred inventive methods.

FIGS. 26-46 represent the experimental results obtained in connection with Examples 19-23.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to FIG. 5, the instrumental setup of the preferred embodiment preferably consists of one or more syringe drives or pumps, a degassing chamber, a selection valve or injection loop valve, a flow chamber, a reference electrode reservoir, and the associated electronics. required for the silicon semiconducting electrode sensor and data processing.

The syringe drives provide the solutions or suspensions (i.e., the medium) to the flow chambers at controlled flow rates. Both the flow rate and the on/off cycle of the syringe drives can be controlled by means of a computer during the data acquisition phase. Alternatively, other types of pumps, such as peristaltic pumps, can be used in place of the syringe drives.

Referring to FIG. 6, the degassing chamber consists of the length of silicone rubber tubing through which the solutions or suspensions pass. The outside of the tubing is maintained in a reduced pressure air atmosphere (approximately 2/3 atmospheric pressure). A degassing chamber is preferred because the solutions or suspensions in the syringes and tubing are at room temperature and the flow chamber is commonly at 37.degree. C. As the room temperature medium reaches the flow chamber it warms to 37.degree. C. resulting in the formation of bubbles in the chamber. Bubbles in the flow chamber interfere with measurements in several ways. They can cause a high resistance path to the reference electrode resulting in an increase in noise in the photocurrent signal. They can also alter the aqueous volume in the chamber thus changing the buffer capacity of the chamber. For a given cellular metabolic rate, the rate of change of pH in the chamber will vary with the number and size of the bubbles. Bubbles form in the chamber and grow with time. When they reach a certain size they dislodge and are carried away in the flowing stream. This can create low frequency noise in the buffer capacity of the flow chamber.

The selection valve allows the user to direct the flow from one of two syringes into the flow chamber. An alternative is an injection loop valve. This allows the user to inject a bolus of solution or suspension into the flow stream without interrupting the flow. Typically when the injection loop valve is used, one syringe is loaded with medium and used to perfuse the stream by means of the injection loop. The volume of the agent solution or suspension and the time of exposure of the cells to the agent can be varied by varying the injection loop size and the flow rate of the perfusion stream.

FIGS. 1-4 illustrate a preferred micro flow chamber 1 having a silicon sensor 2 with an inlet port 3 and an outlet port 4. In FIG. 1, cells 5 are adhered in a monolayer to the upper surface of silicon sensor and the response on the silicon sensor is modulated by laser light 6. Alternatively, the cell can be retained adjacent the silicon sensor by a permeable membrane. In operation, a solution containing a cell affecting agent enters through inlet port 3 and flows over cells 5 in the micro flow chamber 1. The solution exits outlet port 4 where the exiting solution is in electrical contact with a reference electrode. The local response on the upper surface of the silicon sensor 2 is modulated by laser light 6 and measured. FIG. 2 illustrates wells 7 that trap or retain therein cells during slow flow rates and from which the cells may be flushed out of the flow chamber during fast flow rates.

The flow chamber is preferably a thin channel bounded on the bottom by the silicon sensor 2, and on the top by an indium-tin-oxide (ITO) coated glass cover slip 8. Adherent cells may also be grown on the surface of the cover slip instead of the silicon semiconductor electrode. The spacing between the silicon sensor and the cover slip is approximately 100 .mu.m. The spacing between the sensor 2 and the cover slip 8 may be achieved by means of a Teflon spacer 14 that has an appropriate channel cutout that forms the flow chamber. A controlling electrode 13, preferably a platinum wire, penetrates the plastic housing of the flow chamber to make electrical contact with the ITO controlling electrode. Electrical contact with the silicon sensor is made via the metal baseplate 9 of the flow chamber. Teflon inlet and outlet tubes, 3 and 4, penetrate the plastic housing and allow medium to flow through the flow chamber. The outlet tube 4 terminates in a reservoir that contains a Ag/AgCl reference electrode. Thus the outlet tube 4 acts as a salt bridge to allow measurement of the potential of solutions or suspensions in the flow chamber. The entire flow chamber is mounted on a hollow metal support that is maintained at constant temperature, typically 37.degree. C., by a temperature controlled circulating water bath 10. The cover slip 8 is removable and may be maintained in operative position by means of removable silicon rubber gaskets 11 and retaining members 12.

Two different instruments may be preferably used for the purpose of illuminating the silicon sensor to generate a suitable photoresponse: a laser instrument and a light emitting diode (LED) instrument.

In the laser instrument, the flow chamber is mounted on a light microscope stage. The beam from a 150mW argon ion laser is directed through a 10 kHz mechanical chopper and into a beam expander that generates a beam approximately 2.5 cm in diameter. The beam then passes through a polarizing filter (variable attenuator) and is directed by mirrors through a quadruple knife edge adjustable aperture into the barrel of the microscope. Alternatively, in one version of the apparatus, the chamber is mounted on a microscope stage and the beam from a 10 mW HeNe laser is projected through an epi-illumination system onto the sensor. In these configurations, one can see the cells in the flow chamber through the microscope and can direct a square or rectangular probe beam of any desired size to any region of the silicon surface in the flow chamber. This is particularly useful when nonconfluent cell cultures are used. It allows one to direct the probe beam to regions of greatest cell density. In addition one can control for instrumental drift by directing the beam to regions where no cells are visible.

In the LED instrument, two flow chambers are mounted side by side on a temperature controlled plate. For illumination, four fiber optic light guides for each chamber penetrate the temperature controlled plate to illuminate the silicon sensor from underneath (the side opposite the surface in contact with the medium). The four fibers are aligned so as to have one near the inlet tube, one near the outlet tube, and two equally spaced between the inlet and the outlet. This instrument is preferably used with cells that grow to a reasonably consistent and uniform density. However, it still gives some latitude in picking regions of greatest cell density. One end of the optical fibers butts up against the silicon sensor and the other end is coupled to an infrared LED. Each fiber is 1.5 mm in diameter. Data can be collected from eight sites (four for each of the two chambers) simultaneously every four seconds.

There may be three primary electrical connections to the chamber: (1) a controlling electrode connected to the indium-tin oxide coating on the cover slip via a thin platinum ribbon or wire; (2) a Ag/AgCl reference electrode in an external well that measures the potential of solution in the flow chamber via a salt bridge comprising the chamber's outlet tube; and (3) an ohmic connection to the base of the silicon chip. A personal computer with a custom circuit board may handle the acquisition, analysis, and display of data in ways that are well known in that art.

A preferred circuit for use in the present invention is disclosed in FIG. 9. The sensor electrode system is shown comprising a silicon sensor, a reference electrode (R. E.) and a controlling electrode (C. E.). The controlling electrode maintains the solution at an arbitrary potential with respect to ground via a low impedance contact to ground. The arbitrary potential is defined by the surface potential at the electrode/solution interface and the various contact potentials between the electrode material and ground. The reference electrode is of the salt bridge type, and thus measures the potential of solution independent of solution composition. The purpose of the electronic circuit is twofold. First, it allows for varying the potential from the solution to the electrical contact to the silicon. Second, it converts a photogenerated alternating current to an alternating voltage (the output signal). U2A is configured as a conventional voltage follower whereby the reference electrode potential is buffered. U1 is a digitally controlled bipolar current source. A connection between pins 18 and 19 forms a current loop. The sign and magnitude of the current in this loop is controlled by the digital word applied to pins 1 through 13. The potential on pins 18 and 19 floats with respect to ground. As configured in the circuit, the combination of U1 and U2A allows the voltage at pin 5 of U2B to be offset from the voltage at pin 3 of U2A. The sign and magnitude of this voltage offset are determined by the digital word applied to U1, and the values of R1 and R2. U2B is used both as a voltage follower to apply a known offset voltage through R3 and R4 to the silicon, and as a current to voltage converter to convert the alternating photocurrent to an alternating voltage at pin 7. The signal gain of U2B is determined by R3. R4 is used to match the impedance of the circuit to that of the sensor. The voltage from silicon to solution is swept by applying a series of incrementally increasing words to U1. The signal amplitude (alternating photocurrent) is read out at U2B pin 7 as a function of potential applied from solution to silicon.

Living cells may be adhered to the chamber in a variety of ways. As shown in FIG. 7, naturally adherent cells may be grown either on the surface of the silicon sensor or the cover slip in an incubator or both. The flow chamber is assembled while keeping the cells moist. Flow is established quickly to keep the cells from over acidifying the medium or consuming all of the oxygen in the chamber. For non-adherent cells, the cells may be mixed with an agarose solution at 37.degree. C. and plated onto the surface of the silicon sensor or cover slip in an approximately 50 .mu.m thick layer in a humid atmosphere. The silicon sensor is then refrigerated for about 15 min. to solidify the agarose. The flow chamber is then assembled and used.

Collagen and gelatin may also be used in conjunction with or in place of agarose. Fibronectin, chondronectin, laminin or other similar substances may optionally be used in adhering the living cells to the surface of the micro flow chamber. Alternatively, the living cells may be dispersed on or in biologically compatible microcarrier beads.

In another embodiment, cells may be grown on or in a porous membrane. This membrane may be inserted into the flow chamber between the controlling electrode and the sensor surface so that culture medium flows along at least one side of the membrane. Some cells, such as epithelial cells, form a monolayer with differential apical and basolateral surfaces. In such cases the properties of both sides of the cell sheet can be studied separately by controlling which side of the membrane is closer to the sensor surface.

The generally preferred inventive method may be practiced as follows. The chamber is assembled with living cells retained therein and attached to the fluidics in the instrument. Flow (typically 100 .mu.L/min) of a low buffer capacity solution or suspension is established and the signal from the sensor is allowed to stabilize. Normal cell-culture medium may be used, including serum if desired, except that bicarbonate and buffers such as HEPES should be excluded. Some drift is seen initially due to warming of the chamber and equilibration of the chamber with the new medium. Once stable, flow in the chamber is halted and the silicon sensor signal is monitored. A decrease in potential reflects the decrease of pH in the chamber due to cellular metabolism. The rate or slope of pH decrease is a measure of the metabolic rate of the cells. The pH is allowed to drop far enough to obtain enough data points to accurately determine the slope of the line (within a few percent error). Typically this reflects a pH drop of approximately 0.1 to 0.5 pH units and requires approximately 1 to 4 minutes. Flow is then restarted and the pH returns to the initial value. This sequence is repeated until reproducible slopes are obtained. A typical determination of metabolic rate is shown in FIG. 10.

In another preferred embodiment, medium is flowed continuously over the cells and the potential is measured at a plurality of sites. If one site is located upstream of the direction of flow from another and there are intervening cells, there will be a pH difference between the two sites with the upstream site being less acidic. This pH difference is due to the metabolic action of the cells on the medium as it traverses the space between the sites. The magnitude of this pH difference, as detected by the silicon semiconductor electrode, is a measure of the metabolic rate of the cells located between the two sites.

For testing cell affecting agents, two strategies may be advantageously employed. In one approach, two syringe drives are used. First metabolic rates are determined as described above with a control medium. Then the syringe drive with the control medium is turned off and a syringe drive with the same medium plus the cell affecting agent is turned on. An appropriate selection valve directs the flow from either syringe drive to the flow chamber. Metabolic rate measurements are then taken in the pres