Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis
Summary
We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.
Abstract
The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.
Introduction
The epididymis in the male reproductive tract is an organ lined with a layer of mosaic epithelial cells. As in other epithelial tissues, the various cell types of the epididymal epithelium, including principal cells, clear cells, basal cells and cells from the immunological and lymphatic systems, work in a concerted manner to function as the barrier at the tubule frontline and as the supporting cells for sperm maturation and physiology1,2,3. Thus, these epithelial cells play an essential role in reproductive health.
Epithelial cells are generally regarded as non-excitable cells that are unable to generate all-or-none action potentials in response to depolarizing stimuli, due to a lack of voltage-gated Na+ or Ca2+ channels4,5. However, epithelial cells express unique sets of ion channels and transporters that regulate their specialized physiological roles, such as secretion and nutrient transportation6. Different epithelial cells therefore possess characteristic electrical properties. For example, the principal cells express the CFTR for fluid and chloride transportation and express the TRPV6 for calcium reabsorption, whereas the clear cells express the proton pump V-ATPase for luminal acidification1,7,8,9. Some transporters and ion channels that regulate the physiological features of the epididymal epithelial cells have been reported, but the functional properties of epididymal epithelial cells are largely not yet understood10,11,12,13.
Whole-cell patch-clamp recording is a well-established technique for examining the intrinsic properties of both excitable and non-excitable cells, and is particularly helpful for studying the functions of primarily dissociated cells in heterogeneous cell samples; the voltage-clamp is used for measuring the passive membrane properties and the ionic currents of single cells14,15. The passive membrane properties include input resistance and capacitance. The former parameter indicates the intrinsic membrane conductance, while the latter implies the surface area of the cell membrane (a phospholipid bilayer, where ion channels and transporters are located, that serves as a thin insulator separating extracellular and intracellular media). The membrane capacitance is directly proportional to the cell membrane's surface area. Together with the membrane resistance that is reflected by the input resistance, the membrane time constant, which indicates how fast the cell membrane potential responds to the flow of ion channel currents, can be determined. In this regard, by combining the current response characteristics from a series of voltage steps applied to the cells, the biophysical kinetics and properties of the cells are determined15,16,17,18.
In the present paper, we describe the procedures for isolating epithelial cells from the rat cauda epididymis and the steps for measuring the membrane properties of different cell types in the dissociated cell mixture using the whole-cell patch-clamp. We show that the epididymal principal cells exhibit distinct membrane electrophysiological properties and that the conductances can be readily identified from other cell types.
Protocol
Representative Results
Discussion
In this protocol, the enzymatic dispersion of the rat cauda epididymides consistently yielded healthy epithelial cells. The quality of the epididymal epithelial cells for the patch-clamp experiments is dependent on a few critical steps in the protocol. For instance, the centrifugation of the cell mixture at a low centrifugal force (30 x g) is important for removing the spermatozoa and the epididymal luminal content; the epididymal epithelial cells become unhealthy in the presence of the spermatozoa in the cell culture. I…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Christopher Antos for helpful comments on the text. This work was supported by start-up funding from ShanghaiTech University awarded to Winnie Shum and by the funding from the National Natural Science Foundation of China (NNSFC no. 31471370).
Materials
| Instrument of AXON system | |||
| Computer controlled amplifier | Molecular Devices – Axon | Multiclamp 700B patch-clamp amplifier | |
| Digital Acquisition system | Molecular Devices – Axon | Digidata 1550 converter | |
| Microscope | Olympus | BX-61WI | |
| Micromanipulator | Sutter Instruments | MPC-325 | |
| Recording chamber and in-line Heater | Warner Instruments | TC-324C | |
| Instrument of HEKA system | |||
| Patch Clamp amplifier | Harvard Bioscience – HEKA | EPC-10 USB double | |
| Microscope | Olympus | IX73 | |
| Micromanipulator | Sutter Instruments | MPC-325 | |
| Recording chamber and in-line Heater | Warner Instruments | TC-324C | |
| Other Instrument | |||
| Micropipette Puller | Sutter Instrument | P-1000 | |
| Recording Chamber | Warner Instruments | RC-26G or homemade chamber | |
| Borosilicate capillary glass with filament | Sutter Instrument / Harvard Apparatus | BF150-86-10 | |
| Vibration isolation table | TMC | 63544 | |
| Digital Camare | HAMAMASTU | ORCA-Flash4.0 V2 C11440-22CU | |
| Reagents for isolation | |||
| RPMI 1640 medium | Gibco | 22400089 | |
| Penicillin/Streptomycin | Gibca | 15140112 | |
| IMDM | ATCC | 30-2005 | |
| IMDM | Gibco | C12440500BT | |
| Collagenase I | Sigma | C0130 | |
| Collagenase II | Sigma | C6885 | |
| 5-α-dihydrotestosterone | Medchemexpress | HY-A0120 | |
| Fetal bovine serum | capricorn | FBS-12A | |
| Micropipette internal solutions (K+-based solution) (pH 7.2, 280-295 mOsm) | |||
| KCl, 35mM | Sigma/various | V900068 | |
| MgCl2 · 6H2O, 2mM | Sigma/various | M2393 | |
| EGTA, 0.1mM | Sigma/various | E4378 | |
| HEPES, 10mM | Sigma/various | V900477 | |
| K-gluconate, 100mM | Sigma/various | P-1847 | |
| Mg-ATP, 3mM | Sigma/Various | A9187 | |
| The standard external recording physiological salt solution (PSS) (pH 7.4, 300-310 mOsm) | |||
| NaCl, 140mM | Sigma/various | V900058 | |
| KCl, 4.7mM | Sigma/various | V900068 | |
| CaCl2, 2.5mM | Sigma/various | V900266 | |
| MgCl2 · 6H2O, 1.2mM | Sigma/various | M2393 | |
| NaH2PO4, 1.2mM | Sigma/various | V900060 | |
| HEPES, 10mM | Sigma/various | V900477 | |
| Glucose, 10mM | Sigma/various | V900392 | |
| For pH adjustment | |||
| NaOH | Sigma/various | V900797 | Purity >=97% |
| KOH | Sigma/various | 60371 | Purity >=99.99% |
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