Closed-loop electrical stimulation system for cell cultures

• US 20050282149A1
• Filed: 05/24/2005
• Published: 12/22/2005
• Est. Priority Date: 05/28/2004
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Kovacs, Gregory T. A., Whittington, R. Hollis
• Granted Patent: US 7,993,906 B2
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: G01N 33/4836 using multielectrode arrays

Abstract

The present invention provides a sensitive system for measuring the physiological response of an in-vitro cell culture to an environmental parameter. An electrical property of the cell culture is measured as a control signal, and a parameter of a stimulus is adjusted in real time to maintain the control signal at a specified value as the environment of the cell culture is altered, for example, pharmacologically. Artifact reduction and real-time control methods are two key aspects of preferred embodiments of the invention, and enable highly accurate determination of pulse parameters which elicit a desired response. Both aspects must be highly robust to the natural variations inherent in a biological system. This system is beneficial for studying the effects of environmental alterations because extremely small changes in the physiological response can be measured over time, revealing the magnitude and time-dependence of the impact of these alterations on the cell culture.

Claims

1. A method for measuring a response of a cell culture to changes in an environmental parameter of the cell culture, the method comprising:

a) providing an in vitro electrically active cell culture having an electrical cellular response to an electrical stimulus;

b) providing the electrical stimulus to the cell culture;

c) measuring the electrical cellular response of the cell culture to the electrical stimulus;

d) controlling the electrical stimulus to set the measured electrical cellular response to a selected value;

e) recording a time dependence of a culture parameter of the cell culture; and

f) altering the environmental parameter of the cell culture.

2. The method of claim 1, wherein said cell culture comprises a tissue selected from the group consisting of:

cardiac tissue, neural tissue, smooth muscle tissue, skeletal muscle tissue, pancreatic islet tissue and combinations thereof.

3. The method of claim 1, wherein said electrical cellular response is a response of substantially all of said cell culture.

4. The method of claim 1, wherein said electrical cellular response is a response of a fraction of said cell culture.

5. The method of claim 1, wherein said electrical cellular response is selected from the group consisting of:

voltage waveform;

current waveform;

impedance;

conduction velocity;

conduction direction;

conduction speed; and

time delay between said stimulus and said response.

6. The method of claim 1, wherein said electrical cellular response comprises efficacy of said stimulus for producing an electrical response.

7. The method of claim 1, wherein said providing the electrical stimulus comprises providing charge-balanced electrical pulses.

8. The method of claim 1, wherein said measuring the electrical cellular response comprises:

a) detecting events which are beyond a selected threshold;

b) classifying the detected events as stimulation artifact events or non-artifact events based on temporal coincidence;

c) selecting the threshold;

d) nulling and padding the stimulation artifact events to provide a modified data stream; and

e) filtering the modified data stream.

9. The method of claim 8, wherein said threshold is selected to maximize a number of non-artifact events.

10. The method of claim 8, wherein said selecting the threshold comprises selecting the threshold from a predetermined set of threshold values.

11. The method of claim 1, wherein said controlling comprises altering a stimulus parameter selected from the group consisting of:

amplitude;

duration;

waveform;

polarity;

pulse rate;

pulse train duration;

multi-electrode timing; and

multi-electrode spatial pattern.

12. The method of claim 1, wherein said controlling comprises:

a) calculating a new step increment proportional to a product of a current step increment and an error signal;

b) incrementing the electrical stimulus by the new step increment.

13. The method of claim 1, wherein said controlling comprises:

a) incrementing the electrical stimulus by a step increment having a sign dependent on a difference between said measured electrical cellular response and said selected value;

b) increasing a magnitude of the step increment if the difference has the same sign for a number of consecutive iterations greater than a predetermined search threshold;

c) decreasing the magnitude of the step increment if the difference has an alternating sign for a number of consecutive iterations greater than a predetermined oscillation threshold.

14. The method of claim 1, wherein said culture parameter is said electrical stimulus.

15. The method of claim 1, wherein said culture parameter is selected from the group consisting of:

action potential amplitude;

action potential duration;

action potential waveform;

conduction velocity;

conduction direction;

conduction speed;

time delay between said stimulus and said response;

impedance;

capture fraction of stimulation;

production of a protein;

expression of a gene;

concentration of a chemical element or compound; and

pH.

16. The method of claim 1, wherein said culture parameter is an optical response selected from the group consisting of:

radiation from a voltage-sensitive dye;

radiation from an ion-sensitive dye;

radiation from a fluorescent probe; and

radiation from the cell culture.

17. The method of claim 1, wherein said environmental parameter is selected from the group consisting of:

concentration of a chemical element or compound;

pH;

mechanical stress;

mechanical strain;

acoustic radiation;

temperature;

electromagnetic radiation;

particulate radiation;

electric field; and

magnetic field.

18. A system for measuring the response of an in vitro electrically active cell culture to changes in an environmental parameter of the cell culture, the system comprising:

a) an electrical source providing an electrical stimulus to the cell culture;

b) an electrical sensor measuring an electrical cellular response of the cell culture to the electrical stimulus; and

c) a controller adjusting the electrical stimulus to set the measured electrical cellular response to a selected value,

wherein the environmental parameter of the cell culture is varied, and

wherein a time dependence of a culture parameter of the cell culture is recorded.

19. The system of claim 18, wherein said electrical source comprises a planar microelectrode or a planar microelectrode array.

20. The system of claim 19, further comprising a ground electrode in proximity to said electrical source.

21. The system of claim 18, wherein said electrical sensor comprises a planar microelectrode or a planar microelectrode array.

22. The system of claim 21, further comprising a ground electrode in proximity to said electrical sensor.