582 BIOLOGY OF REPRODUCTION 61, 582–589 (1999) Magnetic Cell Sorting Is a Fast and Effective Method of Enriching Viable Spermatogonia from Djungarian Hamster, Mouse, and Marmoset Monkey Testes 1 Viktoria von Scho ¨nfeldt, Hanumanthappa Krishnamurthy, Luca Foppiani, and Stefan Schlatt 2 Institute of Reproductive Medicine of the University Mu ¨nster, 48149 Mu ¨nster, Germany ABSTRACT Germ cell transplantation, which offers promising new ap- proaches for research and clinical applications, has focused in- terest on spermatogonia. This paper describes a procedure that permits the isolation of large quantities of viable spermatogonia. The immunomagnetic isolation procedure was applied to testic- ular cell suspensions from photoinhibited and photostimulated Djungarian hamsters, mice, and marmoset monkeys. The cells were incubated with a polyclonal rabbit anti-c-kit IgG, binding of which was characterized by immunohistochemical staining. For magnetic labeling, a secondary anti-rabbit IgG conjugated to ferromagnetic microbeads was used. Separation columns al- lowed the retention of magnetically labeled cells within the ma- trix. The magnetic fractions were eluted after removal of the column from the magnetic field. All fractions were analyzed for cellular morphology and by flow cytometry. The final enrich- ment of c-kit-positive cells in the magnetic fraction using fully active testes was in the range of 25–55% with a viability rate of 80–90%. The magnetic fractions of all three species were characterized by high numbers of diploid cells. Cytological anal- ysis revealed a strong enrichment of spermatogonia. No haploid cells were retained in the magnetic fraction. In comparison to conventional procedures, magnetic cell separation is an efficient and fast approach for isolation of spermatogonia. INTRODUCTION The basic aspects of spermatogonial morphology and physiology were described in the 1960s and 1970s (for re- view see [1, 2]). In recent years spermatogonia have re- gained the interest of reproductive biologists as new tech- niques have led to novel applications for using these cells. For example, homologous and xenologous germ cell trans- plantation has been described in mice [3–5], based on the regrowth of germ cells from stem spermatogonia after transfer into a recipient testis. Recently it has been shown that cryopreserved or cultured spermatogonia allow the re- population of the recipient testis [6, 7]. Germ cell trans- plantation offers new routes for the production of male gametes, for the introduction of transgenes into the male germ line, and for the gonadal protection of oncological patients [8, 9]. Another advance is the development of tech- niques for the culture of spermatogonia, which helped to elucidate the interaction of spermatogonia with Sertoli cells in coculture [10]. The revitalized interest in spermatogonial stem cells prompted us to develop a new technique for their isolation. Magnetic cell separation has become a widely used method for separating many different types of cells. Meth- Accepted March 31, 1999. Received December 29, 1998. 1 This work was supported by a grant from the Deutsche Forschungs- gemeinschaft (Ni130/17–1). This work was part of a Diploma thesis (V.v.S.) at the faculty of biology of the University of Mu ¨nster. 2 Correspondence: Stefan Schlatt, Institute of Reproductive Medicine of the University, Domagkstr. 11, D-48149 Mu ¨nster, Germany. FAX: 49 251 835 6093; e-mail: schlats@uni-muenster.de ods have been described for the isolation of human lym- phocytes [11], dendritic cells [12], fetal cells from maternal blood [13], granulocytes [14], and megakaryotic cells [15], as well as natural killer cells, T cells, tumor cells, and ep- ithelial cells. Blood stem cell technology has profited sig- nificantly from the introduction of magnetic cell sorting [16]. Negative and positive selection strategies using de- pletion columns or positive selection columns have been implemented; and for many applications, multi-sort options are available from the manufacturer of the magnetic beads and separation columns (Miltenyi Biotech, Auburn, CA). In this study we applied commercially available IgGs directed against the c-kit receptor protein to the immuno- magnetic isolation of spermatogonia. Binding of the anti- body to c-kit was characterized by immunohistochemical procedures on testicular tissue sections. The percentage of c-kit/fluorescein isothiocyanate (FITC)-positive cells and the DNA content of the sorted cell fractions were analyzed by flow cytometry. MATERIALS AND METHODS Animals Fully regressed testes from photoinibited hamsters (n = 27) that had been kept in short photoperiods (16L:8D) for 8–14 wk were used for initial experiments to develop the basic principles of the digestion protocol and isolation pro- cedure, since these testes present a physiologically enriched source of spermatogonia [17, 18]. In subsequent experi- ments, normal testes showing the full germ cell comple- ment from photostimulated adult hamsters (n = 10), adult mice (n = 9), and adult marmoset monkeys (n = 8) were used. All animals were derived from colonies at our insti- tute and were treated in accordance with the Federal Ger- man Law on the handling of experimental animals. Cell Isolation from the Seminiferous Epithelium The testes were excised and decapsulated. Testicular tis- sue was minced using fine scissors and transferred into cul- ture medium (Gibco, Gaithersburg, MD; Dulbecco’s mod- ified Eagle’s medium [DMEM]/F12) containing collagenase type I (Sigma Chemical Co., St. Louis, MO; 1 mg/ml) and DNase (Sigma; 0.5 mg/ml). Digestion was performed at 37°C for 10 min in a shaking water bath operated at 110 cycles/min. Interstitial cells were separated by sedimenta- tion at unit gravity for 10 min and washed in DMEM/F12. A final digestion of the basal lamina components was car- ried out in a mixture of collagenase type I (Sigma; 1 mg/ ml), DNase (Sigma; 0.5 mg/ml), and hyaluronidase (Sigma; 0.5 mg/ml) under the same conditions as for the first di- gestion step. The single-cell suspension obtained was washed successively with medium and PBS containing 2 mM EDTA (Sigma) and 0.5% fetal calf serum (FCS; Gib- co). Undigested remains of the tunica albuginea were elim- inated by filtering the cell suspension through a 50-m nylon Downloaded from https://academic.oup.com/biolreprod/article/61/3/582/2734490 by guest on 24 June 2022