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 physiology^1,2,3. Thus, these epithelial cells play an essential role in reproductive health.

Epithelial cells are generally r....

Protocol

All animal experiments are carried out in accordance with the guidelines of the ShanghaiTech University Institutional Animal Care and Use Committee, which fulfill the local and international requirements.

1. Experimental Animals

1. Use adult male Sprague-Dawley rats (~300-450 g) between 8-12 weeks old. At this age in the rats, the sperm have arrived in the cauda epididymides.

2. Isolation of Epithelial Cells from Rat Cauda Epididymides

NOTE: The following steps are performed under non-aseptic conditions unless otherwise stated.

1. Preparation of dissect....

Results

The described enzymatic digestion procedure for the isolation of epithelial cells from the rat cauda epididymides is a modified protocol from our previous studies^9,12. This method produces a mixture of single cells with over 90% viability and without surface blisters or swollen cell volume. The heterogeneous cell mixture consists mainly of principal cells, clear cells and basal cells, as we have described previously

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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%