It has long been known that cells can be induced to migrate by the application of small direct current electric fields, a phenomenon referred to as galvanotaxis. We recently reported some significant effects of electric signals of physiological strength in guiding cell migration and wound healing. We present here protocols to make “electrotactic chambers”, and to apply an electric field to cells or tissues cultured in the chamber. The chamber can be built to allow controlled medium flow to prevent the potential development of chemical gradients generated by the electric fields. It can accommodate cells on planar culture or in 3-dimensional cultures in gels, with modification to maintain small pieces of tissue and organ in vitro, as well as small embryos. Mounted on an inverted microscope, this setup allows close and well controlled observation of cellular responses to electric signals. As similar electric fields are widely present during development and wound healing, this experimental system can be used to simulate and study cellular and molecular responses to electric signals. In order to test the effect of electric signals in vivo, we have also developed a pharmacological approach to modulate endogenous wound electric fields in a cornea wound-healing model in vivo.
Endogenous direct current (DC) electric fields (EFs) occur naturally in vivo. This was first demonstrated at wounds by Emil Du-Bois Reymond(reference 1). More than 150 years ago, he measured electric currents of ~1uA flowing out of a cut he made in his own finger. Using various modern techniques, including micro-glass electrodes and vibrating probes, others and we have detected a similar electric current flow at wounds of skin and cornea of every species we have studied, including human skin. In cornea and skin, a laterally-oriented, wound-induced EF is generated instantaneously when the epithelium is damaged and it persists until re-epithelialisation restores the electrical resistance barrier function of the epithelium. These EFs are estimated to be at least 40-50 mV/mm at cornea wounds and 100-150 mV/mm at skin wounds(Reference 2-6). Cumulating experimental evidence suggests an important role for such electric signals in directing cell migration in wound healing(Reference 6-14).
Endogenous DC EFs have been measured during development, regeneration and following damage to non-epithelial tissues(reference 10,11,14-20). These EFs arise because of spatial and temporal variations in epithelial transport of charged ions such as Na+, K+ and Cl-, and spatial variations in the electrical resistance of epithelial sheets. Disruption of the endogenous electrical gradients during development induces skeletal and neural abnormalities(reference 16,19,21). It has been shown that the spinal cord responds to damage by generating large and persistent electrical signals, and in turn applied electric stimulation can promote spinal cord repair in human and other mammals(reference 22-25).
Based on the facts that there are endogenous EFs and disruption of these EFs disrupts wound healing and development, there is a lasting but contained research on cellular response to EFs for several decades. Amongst various signals suggested to guide cell migration and division in development and wound healing, electric signal is much less well studied. The biological and medical research community at large are not familiar with possible roles of electric fields as a directional signal in guiding cell migration to heal a wound. The experimental techniques to study electric signals controlled cellular behaviours are known very limited to a small research community.
Many in vitro experiments show that EFs of strength equivalent to those measured in vivo control important cell behaviours such as directional cell migration (galvanotaxis or electrotaxis) and cell division orientation(reference 14,26-34). Our recent letter to Nature provides further experimental evidence that the electric signal as a directional cue probably plays a far more important role in directing cell migration in wound healing of epithelium than previously believed. We also reported in the letter two genes important for EF-induced cellular response(reference 35). Following the publication, there is a strong demand that we make our “technological expertise widely available so that others may embark on these investigations”(reference 36).
The protocols we used are based on those pioneered and used by a handful laboratories to apply EFs to cells in vitro (reference 27-29,31,32,37-44). We have modified and used electrotactic chambers to accommodate cells growing in planar culture or in three-dimensional (3D) gels, en bloc tissue cultures in 3D and possible small embryos, such as that from frog and zebra fish. Therefore it is also possible to apply electric fields to in vivo systems. The EF is applied to the cells or tissues cultured in a customer designed electrotactic chamber via agar salt bridges, Steinberg’s solution and Ag/AgCl electrodes. The depth of the electrotactic chamber is adjustable to accommodate different thickness of the samples, while maintaining a reasonably stable voltage and current flow, temperature and pH in the chamber. It is possible to apply electric fields to cells and tissues for extended period of time up to several days while cell behaviours can be monitored continuously. Modification to the system will allow high-resolution imaging using cover glass-based dishes as well as lower magnification imaging for big cell sheet movement in tissue or organ culture with 3D tracking of cell migration and cell division in vitro and ex vivo.
Migration rates of different types of cells vary significantly. Some fast-moving cells, for instance neutrophils or Dictyostelium Discoideum, migrate at migration rates of 20 – 30 um/min(reference 35, 45). Electrotactic experiment with those types of cells is relatively easy, since the temperature and pH are normally very stable within 30 – 60 minutes duration of experiments. Some other types of cells migrate very slowly, which requires longer time to record a detectable distance change and to quantify the rate and direction of cell migration. It is therefore crucial to be able to maintain stability of the temperature and pH in the chamber. In our hands, embryonic stem cells, progenitor cells and neurones migrate slowly, hence the required time-lapse recording is normally taken for approximately 5 – 8 hours. A temperature control unit was designed on the imaging system to provide a stable temperature required (see PROCEDURE). The depth of the electrotactic chamber should be kept minimal in order to reduce the Joule heating effect (see PROCEDURE). When exposed in the air (low atmospheric CO2, 0.04%), pH changes in the chamber can be observed when high voltage is applied and electric current is strong. Routine cell culture media have relied on sodium bicarbonate as the primary buffering system to stabilize pH(reference 46). As most experiments with EF application were done outside CO2 incubators, we have used the following methods to control pH in the electrotactic chamber: 1). An incubator around the microscope with additional 95% O2 / 5% CO2 supply; 2). Using commercially available CO2-independent medium if possible; and 3). Addition of HEPES buffer into the medium (see PROCEDURE below). Optimal experimental conditions can be achieved by a combination of temperature control unit with CO2 supply, and CO2 independent medium / HEPES buffered medium.
Application of EFs to 3D cultures, tissue blocks or embryos is technically demanding, especially when there is a need to experiment for a long period of time. A deeper electrotactic chamber is required to accommodate thicker tissues to be studied. This inevitably increases heat generation in the chamber due to the Joule effect. We have used several configurations for 3D tissue culture to minimize the temperature rise due to the increase in the depth of the chamber. Depending on the thickness of the tissue to be studied, the depth of the side panels of the electrotactic chamber can be adjusted (details see PROCEDURE below). Another common problem of 3D culture is en bloc movement of the tissue / organ in the chamber. The tissue / organ tends to move or slide away from the original position if not immobilized, which makes the tracking of cell behaviour in the block difficult and unreliable. We have developed techniques to stabilize tissue blocks with either Matrigel or fibrinogen/aprotinin/thrombin gel (see PROCEDURE).
Application of EFs can generate chemical gradients in the culture chamber. Although the interaction of chemical cues and electrical cues are highly likely in vivo, it is critical to exclude or minimize this interaction in electrotactic experiment. This can be achieved using a “cross-flow” electrotactic chamber, so that a continuous medium flow can be maintained to disrupt possible chemical gradient build up(reference 28) (see PROCEDURE).
To test the effect of electric signals in vivo, we developed an alternative approach in addition to that achieved by using electrodes. Wounding the tissue instantly generates an endogenous electric current which could be measured directly(reference 4,6,7,22,47). Pharmacological or chemical agents which modulate ion transportation are applied to a wound to either enhance or decrease ion transportation, thus to enhance and decrease endogenous wound electric fields accordingly (reference 6,28,33,35). The effects of such modification on wound healing can be studied in vivo. Our protocols for applying drugs to modulate EFs are also provided. Cornea wound is used as a model system in this protocol. This protocol can be completed in 2 days.