Magnetic cell sorting is a fast and effective method of enriching viable spermatogonia from Djungarian hamster, mouse, and marmoset monkey testes.
Germ cell transplantation, which offers promising new approaches for research and clinical applications, has focused interest on spermatogonia. This paper describes a procedure that permits the isolation of large quantities of viable spermatogonia. The immunomagnetic isolation procedure was applied to testicular 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 allowed the retention of magnetically labeled cells within the matrix. 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 enrichment 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 analysis 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.
von Schönfeldt
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Institute of Reproductive Medicine of the University Münster, 48149 Münster, Germany.
Krishnamurthy
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H
Foppiani
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Schlatt
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S
D000818:Animals; D002144:Callithrix; D006224:Cricetinae; D005434:Flow Cytometry; D007074:Immunoglobulin G; D007150:Immunohistochemistry; D018189:Immunomagnetic Separation / Q000379:methods*; D008027:Light; D008297:Male; D051379:Mice; D008863:Microspheres; D017111:Phodopus; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis; D011817:Rabbits; D013093:Spermatogonia / Q000166:cytology*; D013737:Testis / Q000166:cytology*
D007074:Immunoglobulin G; D019009:Proto-Oncogene Proteins c-kit