Density Gradient-Based Separation of Testicular Cell Types Using a STA-PUT Apparatus

Abstract

Mammalian spermatogenesis is a complex differentiation process, and testis tissue comprises numerous somatic cell types as well as germ cells at different developmental steps along the spermatogenic process. Isolation and purification/enrichment of somatic cells (Leydig, Sertoli, myoid, or epithelial) and spermatogenic cells (spermatogonia, spermatocytes, round spermatids, elongating and condensing spermatids, testicular spermatozoa) is therefore a prerequisite for many of the biochemical, molecular, and cellular assays used to study spermatogenesis. A classical and cost-effective method to achieve this is the STA-PUT apparatus method that employs standard gravity-based separation of a testicular cell suspension through a linear BSA gradient to collect fractions enriched for individual cell types according to cell size. Those fractions can either immediately be used for analysis, or they can be followed up with differential plating to further purify individual cell types of interest. While some of the current alternative methods like fluorescence-activated cell sorting (FACS), elutriation, or single-cell analysis can produce a higher degree of cell-type purity, spermatogenic tissue analysis-purification technique (STA-PUT) is a reliable and cost-effective method that is available also to laboratories with limited access to high-cost specialized equipment and that yields very high cell number numbers. In addition, the STA-PUT apparatus can also be used to separate different cell types from other tissues based on their size or density.

Keywords: BSA gradient; Cell fractionation; Cell isolation; Cell separation; Density gradient; STA-PUT; Sertoli cell; Spermatid; Spermatocyte; Spermatogenesis; Testis.