Mieke Geens1, Ellen Goossens, Herman Tournaye. 1. Biology of the Testis Research Group, Department of Embryology and Genetics, Medical School and Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium. mieke.geens@uzbrussel.be
Abstract
OBJECTIVE: To investigate whether a selective matrix adhesion-based protocol can enrich germ cells and deplete cancer cells from contaminated human testicular cell suspensions. DESIGN: Artificially contaminated and control testicular cell suspensions were selectively cultured. After each experimental step, the presence of B cells was evaluated by fluorescence-activated cell sorting (FACS) and/or polymerase chain reaction (PCR). SETTING: Academic medical center. PATIENT(S): Five vasectomy reversal patients willing to donate testicular tissue for research after informed consent. INTERVENTION(S): In vitro culture, magnetic-activated cell sorting (MACS), matrix adhesion, FACS, and PCR. MAIN OUTCOME MEASURE(S): FACS analysis for CD49f and human leukocyte antigen (HLA) class I expression; PCR analysis for a 120-bp fragment of the B-cell receptor. RESULT(S): With the use of a matrix adhesion-based protocol, CD49f(+) HLA class I(-) cells could be highly enriched, but HLA class I(+) CD49f(-) cells could still be detected. PCR proved that the cell suspensions contained malignant cells originating from the cell line used to contaminate them. CONCLUSION(S): Even though an efficient enrichment of germ cells was achieved, the resulting population was not pure. Malignant cells were detected in selected cell suspensions of all five patients, demonstrating that the efficacy of this protocol is insufficient for clinical application.
OBJECTIVE: To investigate whether a selective matrix adhesion-based protocol can enrich germ cells and deplete cancer cells from contaminated human testicular cell suspensions. DESIGN: Artificially contaminated and control testicular cell suspensions were selectively cultured. After each experimental step, the presence of B cells was evaluated by fluorescence-activated cell sorting (FACS) and/or polymerase chain reaction (PCR). SETTING: Academic medical center. PATIENT(S): Five vasectomy reversal patients willing to donate testicular tissue for research after informed consent. INTERVENTION(S): In vitro culture, magnetic-activated cell sorting (MACS), matrix adhesion, FACS, and PCR. MAIN OUTCOME MEASURE(S): FACS analysis for CD49f and human leukocyte antigen (HLA) class I expression; PCR analysis for a 120-bp fragment of the B-cell receptor. RESULT(S): With the use of a matrix adhesion-based protocol, CD49f(+) HLA class I(-) cells could be highly enriched, but HLA class I(+) CD49f(-) cells could still be detected. PCR proved that the cell suspensions contained malignant cells originating from the cell line used to contaminate them. CONCLUSION(S): Even though an efficient enrichment of germ cells was achieved, the resulting population was not pure. Malignant cells were detected in selected cell suspensions of all five patients, demonstrating that the efficacy of this protocol is insufficient for clinical application.