Name: isolation of sertoli cells and peritubular cells from rat testes
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Description: Watch this Scientific Journal Video about Isolation of Sertoli Cells and Peritubular Cells from Rat Testes at JoVE.com

The authors would like to thank Guido Verhoeven and Ludo Deboel, Leuven, who were extremely helpful in establishing the isolation of primary Sertoli cells in the Meinhardt lab in Giessen. Monika Fijak, Gie&#223;en, is acknowledged for her help with figure 1A and advice. Studies were supported by the Deutsche Forschungsgemeinschaft through grant BH 93/1-1 (S.B.) and funding of the International Research Graduate College JLU Gie&#223;en (Germany)/Monash University (Melbourne, Australia) GRK 1871 (S.B.). Support was also obtained from a grant of the State of Hessen within the program &#34;Landes-Offensive zur Entwicklung Wissenschaftlich-&#246;konomischer Exzellenz&#34; (LOEWE) called &#34;M&#228;nnliche Infertilit&#228;t bei Infektion &#38; Entz&#252;ndung&#34; (MIBIE).

1. Animal Ethics Statement
Experiments described here were performed according to the guidelines of the Regierungspr&#228;sidium Giessen, Germany, as the local authority and confirm to the German Code of Practice for the Care and Use of Animals for Experimental Purposes (Permission no: GI 20/23 Nr. A 31/2012).
2. Preparation of Media, Enzyme Solutions and Animals
<ol>
	<li>Prepare 1% iodine alcohol by dissolving 1 g of iodine in 100 ml ethanol.</li>
	<li>Prepare 2x 500 ml Dulbecco&#180;...

<ol>
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W., Dym, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+normal+development+of+the+blood-testis+barrier+and+the+effects+of+clomiphene+and+estrogen+treatment.">The normal development of the blood-testis barrier and the effects of clomiphene and estrogen treatment.</a> Anat. Rec. 176, 331-344 (1973).</li><li>Dorrington, J. H., Roller, N. F., Fritz, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+follicle-stimulating+hormone+on+cultures+of+Sertoli+cell+preparations.">Effects of follicle-stimulating hormone on cultures of Sertoli cell preparations.</a> Mol. Cell. Endocrinol. 3, 57-70 (1975).</li><li>Tung, P. S., Skinner, M. K., Fritz, I. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+induced+by+follicle-stimulating+hormone+or+dibutyryl+cyclic+AMP+on+presumptive+Sertoli+cells+in+culture.">Structural changes induced by follicle-stimulating hormone or dibutyryl cyclic AMP on presumptive Sertoli cells in culture.</a> Proc. Natl. Acad. Sci. U. S. A. 72, 1838-1842 (1975).</li><li>Teng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+Sertoli+cells+and+their+effects+on+the+function+of+co-cultured+allogeneic+islets+in+vitro.">Isolation and culture of adult Sertoli cells and their effects on the function of co-cultured allogeneic islets in vitro.</a> Chin. Med. J. (Engl.). 118, 1857-1862 (2005).</li><li>Kohno, S., Ziparo, E., Marek, L. F., Tung, K. 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D., Bartke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+Sertoli+cells+from+the+testes+of+adult+Siberian+hamsters:+analysis+of+proteins+synthesized+and+secreted+by+Sertoli+cells+cultured+from+hamsters+raised+in+a+long+or+a+short+photoperiod.">Isolation and culture of Sertoli cells from the testes of adult Siberian hamsters: analysis of proteins synthesized and secreted by Sertoli cells cultured from hamsters raised in a long or a short photoperiod.</a> Biol. Reprod. 52, 658-666 (1995).</li><li>Zhang, H., Liu, B., Qiu, Y., Fan, J., Yu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pure+cultures+and+characterization+of+yak+Sertoli+cells.">Pure cultures and characterization of yak Sertoli cells.</a> Tissue Cell. 45, 414-420 (2013).</li><li>Ziparo, E., Geremia, R., Russo, M. A., Stefanini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+interaction+in+vitro+between+Sertoli+cells+and+germ+cells+at+different+stages+of+spermatogenesis.">Surface interaction in vitro between Sertoli cells and germ cells at different stages of spermatogenesis.</a> Am. J. Anat. 159, 385-388 (1980).</li><li>Anway, M. D., Folmer, J., Wright, W. W., Zirkin, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Sertoli+cells+from+adult+rat+testes:+an+approach+to+ex+vivo+studies+of+Sertoli+cell+function.">Isolation of Sertoli cells from adult rat testes: an approach to ex vivo studies of Sertoli cell function.</a> Biol. Reprod. 68, 996-1002 (2003).</li><li>Scarpino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+Sertoli+cell+isolation+by+DSA+lectin,+allowing+mitotic+analyses.">A rapid method of Sertoli cell isolation by DSA lectin, allowing mitotic analyses.</a> Mol. Cell. Endocrinol. 146, 121-127 (1998).</li><li>Mather, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+two+distinct+mouse+testicular+epithelial+cell+lines.">Establishment and characterization of two distinct mouse testicular epithelial cell lines.</a> Biol. Reprod. 23, 243-252 (1980).</li><li>Korbutt, G. S., Elliott, J. F., Rajotte, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotransplantation+of+allogeneic+islets+with+allogeneic+testicular+cell+aggregates+allows+long-term+graft+survival+without+systemic+immunosuppression.">Cotransplantation of allogeneic islets with allogeneic testicular cell aggregates allows long-term graft survival without systemic immunosuppression.</a> Diabetes. 46, 317-322 (1997).</li><li>Fritz, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+cell-germ+cell+relationships+in+mammalian+testes+during+development+and+spermatogenesis.">Somatic cell-germ cell relationships in mammalian testes during development and spermatogenesis.</a> Ciba Found. Symp. 182, 271-281 (1994).</li><li>Whaley, P. D., Chaudhary, J., Cupp, A., Skinner, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+specific+response+elements+of+the+c-fos+promoter+and+involvement+of+intermediate+transcription+factor(s)+in+the+induction+of+Sertoli+cell+differentiation+(transferrin+promoter+activation)+by+the+testicular+paracrine+factor+PModS.">Role of specific response elements of the c-fos promoter and involvement of intermediate transcription factor(s) in the induction of Sertoli cell differentiation (transferrin promoter activation) by the testicular paracrine factor PModS.</a> Endocrinology. 136, 3046-3053 (1995).</li><li>Bhushan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uropathogenic+Escherichia+coli+block+MyD88-dependent+and+activate+MyD88-independent+signaling+pathways+in+rat+testicular+cells.">Uropathogenic Escherichia coli block MyD88-dependent and activate MyD88-independent signaling pathways in rat testicular cells.</a> J. Immunol. 180, 5537-5547 (2008).</li><li>Meinhardt, A., Hedger, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological,+paracrine+and+endocrine+aspects+of+testicular+immune+privilege.">Immunological, paracrine and endocrine aspects of testicular immune privilege.</a> Mol. Cell. Endocrinol. 335, 60-68 (2011).</li><li>Fijak, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Testosterone+on+Inflammatory+Response+in+Testicular+Cells+and+Expression+of+Transcription+Factor+Foxp3+in+T+Cells.">Influence of Testosterone on Inflammatory Response in Testicular Cells and Expression of Transcription Factor Foxp3 in T Cells.</a> Am. J. Reprod. Immunol. , 12-25 (2015).</li><li>Mamidi, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+passing+mesenchymal+stem+cells+through+smaller+bore+size+needles+for+subsequent+use+in+patients+for+clinical+or+cosmetic+indications.">Impact of passing mesenchymal stem cells through smaller bore size needles for subsequent use in patients for clinical or cosmetic indications.</a> J. Transl. Med. 10, 229 (2012).</li><li>Ito, S., Karnovsky, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde-glutaraldehyde+fixatives+containing+trinitro+compounds.">Formaldehyde-glutaraldehyde fixatives containing trinitro compounds.</a> J. Cell. Biol. 39, 168-169 (1968).</li><li>Galdieri, M., Ziparo, E., Palombi, F., Russo, M. A., Stefanini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pure+Sertoli+cell+cultures:+a+new+model+for+the+study+of+somatic-germ+cell+interactions.">Pure Sertoli cell cultures: a new model for the study of somatic-germ cell interactions.</a> J. Androl. 5, 249-254 (1981).</li><li>Fijak, M., Meinhardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+testis+in+immune+privilege.">The testis in immune privilege.</a> Immunol. Rev. 213, 66-81 (2006).</li><li>Jegou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sertoli+cell+in+vivo+and+in+vitro.">The Sertoli cell in vivo and in vitro.</a> Cell Biol. Toxicol. 8, 49-54 (1992).</li><li>Pineau, C., Le Magueresse, B., Courtens, J. L., Jegou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+in+vitro+the+phagocytic+function+of+Sertoli+cells+in+the+rat.">Study in vitro the phagocytic function of Sertoli cells in the rat.</a> Cell Tissue Res. 264, 589-598 (1991).</li><li>Ren, Y., Savill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis:+the+importance+of+being+eaten.">Apoptosis: the importance of being eaten.</a> Cell Death Differ. 5, 563-568 (1998).</li><li>Ducray, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+mouse+Sertoli+cell+line+producing+rat+androgen-binding+protein+(ABP).">Establishment of a mouse Sertoli cell line producing rat androgen-binding protein (ABP).</a> Steroids. 63, 285-287 (1998).</li><li>Konrad, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Sertoli+cells+express+epithelial+but+also+mesenchymal+genes+after+immortalization+with+SV40.">Rat Sertoli cells express epithelial but also mesenchymal genes after immortalization with SV40.</a> Biochim. Biophys. Acta. 1722, 6-14 (2005).</li></ol>

The seminiferous tubules are bounded by a circular layer of peritubular cells and a basal lamina on the luminal side. Sertoli cells are resting on the basal lamina, establish the blood testis barrier through formation of occluding junctions between adjacent cells and provide the structural framework for the organisation of the seminiferous epithelium. Sertoli cells and adjacent spermatogenic cells maintain intimate contacts throughout germ cell development providing physical contact and communication alike. Therefore, wh...

Testes produce male gametes and sexual hormones, i.e., androgens. The organ is composed of two compartments. In the interstitial compartment, that represents about 10-12% of total testicular volume1, steroidogenesis takes place within the Leydig cells. The tubular compartment represents about 60-80% of the testicular volume1 and contains germ cells and two types of somatic cells - peritubular cells and Sertoli cells. The testis is divided by connective tissue septa into 250-300 lobules, each containing 1-3 highly convoluted seminiferous tubules. These tubules are enclosed by a basal membrane, a sheet of collagen, and a circumferential layer of peritubular cells (Figure 1A).
The germinal epithelium is located on the luminal side of the basal membrane. Sertoli cells are large elongated cells that span the whole germinal epithelium from the basal membrane to the lumen. They are strongly attached to the basal membrane and form a continuous cellular sheet through basolateral organized tight junctions that occludes the germinal epithelium from the interstitium and represents the blood-testis barrier. Sertoli cells have prominent cytoplasmic projections and ramifications that enable them to get into tight morphological and functional contact with a species-specific but constant number of germ cells. Diploid germinal stem cells proliferate and differentiate into spermatogonia.
During meiosis I short-lived tetraploid spermatocytes are generated that develop further into four haploid spermatids during meiosis II. All germ cells are interconnected by cytoplasmic bridges so that they form a cellular net. The principal event during maturation of spermatids is the extrusion of large parts of the cytoplasm, forming residual bodies, in a process called spermiogenesis. Residual bodies are phagocytosed by Sertoli cells. Late spermatids are then released into the tubular lumen and transported into the epididymis for further maturation. Sertoli cells and germ cells appear to mutually coordinate spermatogenesis topographically and functionally.
Preparation of individual testicular cell types started almost a century ago when small testicular pieces were cultivated and cell types identified by microscopy2. By careful dissection of the tubules after opening the tunica albuginea using fine-tipped forceps it was later possible to separate tubular and interstitial compartments3. In 1975 Welsh and Wiebe introduced a collagenase treatment in order to free the tubules from adhering interstitial tissue and a pancreatin treatment for removal of the outer peritubular cell layer4. From early on young immature rats (around 20 days old) had been used in which the Sertoli cells comprise a large fraction of the tubular cell population because spermatogenesis has not begun yet. At this age rat Sertoli cells cease to divide, and tight junctions between neighboring cells form so that the blood-testis barrier is established5.
Independent of Welsh and Wiebe Dorrington et al. introduced a combination of trypsin and deoxyribonuclease followed by soybeen trypsin inhibitor and collagenase treatment that was published in the same year6. Both groups also used mechanical force (drawing the digested tubular fragments repeatedly through a syringe needle or a Pasteur pipette respectively) in order to produce a homogeneous cell suspension for plating that contains approximately 70% Sertoli cells. After 3 days in culture, using a medium that is serum-free, the percentage of Sertoli cells increases to approximately 90%. This could be largely attributed to the death of contaminating germ cells. Residual peritubular cells (PTCs), however, stay firmly attached via their extracellular matrix. PTCs, but not Sertoli cells, are known to produce fibronectin that can serve as a marker protein for estimating contamination with PTCs. Therefore, Tung et al. reasoned that an additional hyaluronidase treatment might improve the purity of the Sertoli cell fraction7. Indeed they could show that an additional treatment was able to reduce contamination by PTCs approximately 20-fold, which becomes particularly evident when in comparison a serum-containing medium is used for cultivating purified Sertoli cells. From then on the improved procedure of Tung et al. became the prevailing protocol and was extensively used by other major groups in the field8 (Figure 1B).
During collagenase treatment the majority of PTCs are released and can be isolated in parallel to Sertoli cells. Whereas the PTCs vigorously proliferate and do not respond to follicle-stimulating hormone (FSH), Sertoli cells do not undergo mitosis anymore and respond to FSH by characteristic morphological changes and an increase in cyclic adenosine monophosphate (cAMP) concentration9. Very similar enzymatic digestion protocols can be used for the isolation of primary Sertoli cells from other animals like man10, mouse11,12, Siberian hamster13 or yak14. For the removal of large amounts of contaminating germ cells a hypotonic shock can be employed at the end of the isolation procedure15. This allows also the efficient isolation of Sertoli cells from adult rat testes16. An enriched Sertoli cell suspension can also be separated from germ cells by plating the suspension on lectin dishes coated with Datura Stramonium agglutinin17. Only Sertoli cells and a few residual PTCs adhere to the lectin plates.
Primary Sertoli cell cultures, mainly from the rat, have been used initially for investigating responsiveness to hormones or establishing cell lines like the mouse Sertoli cell line TM418. This cell line has been investigated in more than a hundred studies until today. In a translational approach Sertoli cells have been utilized for immuno-protection of co-cultured cells and tissues like in the co-transplantation of xeno- or allogeneic pancreatic islets for long-term graft survival without systemic immunosuppression19. Isolated Sertoli cells have been also used in co-culture experiments for studying epithelial-mesenchymal (Sertoli cells - PTCc) and somatic-germ cell (Sertoli cells - germ cells) interactions20,21. Recently primary Sertoli cells have been employed for investigating the expression of Toll-like receptors and the secretion of proinflammatory cytokines as well as the signal transduction cascades leading to cytokine expression following infection with nonpathogenic and uropathogenic E. coli22. Other recent investigations were using Sertoli cells for studying testicular immune privilege23 and demonstrated that testosterone pre-treatment suppresses the LPS-induced inflammatory response24.

<table border="0" cellpadding="0" cellspacing="0" width="813">
	<tbody>
		<tr height="80">
			<td height="80" style="height:80px;width:289px;">Actin (smooth muscle) antibody clone 1A4</td>
			<td style="width:111px;">Dako</td>
			<td style="width:124px;">M0851</td>
			<td style="width:289px;">Monoclonal mouse anti human antibody. 
			Use 1:100 dilution for immunofluorescence.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Albumin bovine fraction V 
			Standard grade, lyophilized</td>
			<td style="width:111px;">Serva</td>
			<td style="width:124px;">11930.04</td>
			<td style="width:289px;">Filter (0.45 &#181;m) BSA solutions used for immunofluorescence.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Corning&#8482; Cell Strainers 70&#160;&#181;m</td>
			<td style="width:111px;">Corning</td>
			<td style="width:124px;">431751</td>
			<td>White color</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Collagenase A from Clostridium histolyticum</td>
			<td style="width:111px;">Roche</td>
			<td style="width:124px;">10103586001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">DAPI mountant ProLong&#174; Gold Antifade</td>
			<td style="width:111px;">Life technologies</td>
			<td style="width:124px;">P-36931</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">D-glucose</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">G8644</td>
			<td>100 g/l</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Dulbecco&#180;s PBS without Ca2+/Mg2+</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">DNase I</td>
			<td style="width:111px;">Roche</td>
			<td style="width:124px;">10104159001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Electron microscope</td>
			<td style="width:111px;">Zeiss</td>
			<td>EM 109S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Fluorescence microscope</td>
			<td style="width:111px;">Zeiss</td>
			<td style="width:124px;">Axioplan 2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Hyaluronidase from bovine testis</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">H3506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Inverted light microscope</td>
			<td style="width:111px;">Olympus</td>
			<td style="width:124px;">CKX41</td>
			<td>Routine cell culture microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Mouse IgG / IgM (H+L) polyclonal 
			secondary Antibody&#160;</td>
			<td style="width:111px;">Life technologies</td>
			<td style="width:124px;">A-10684</td>
			<td>Alexa Fluor&#174; 488 conjugate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Oil Red O</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">O0625</td>
			<td style="width:289px;">Has replaced Sudan III and Sudan IV 
			because of brighter color.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Paraformaldehyde</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">P6148</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Penicillin (5,000&#160;U/ml)/ 
			Streptomycin (5,000 &#181;g/ml)</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">15070-063</td>
			<td>100x solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">RPMI-1640</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">21875-034</td>
			<td>Contains 300 mg/L L-glutamine</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Trypsin from porcine pancreas</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">T5266</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Trypan Blue Stain (0.4%)</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:289px;">Trypsin inhibitor from soybean</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">T6522</td>
			<td style="width:289px;">The Sigma product is considerably cheaper than the previously used 
			BPTI (Aprotinin) from Roche.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:289px;">Vimentin antibody (V9)</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">V6630</td>
			<td style="width:289px;">Monoclonal mouse anti pig vimentin antibody.&#160; Use 1:100 dilution for immunofluorescence.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Wistar WU rats</td>
			<td style="width:111px;">Charles River</td>
			<td style="width:124px;">N/A</td>
			<td>Should be 19 days old on day of experiment.</td>
		</tr>
	</tbody>
</table>

isolation of sertoli cells and peritubular cells from rat testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

The described procedure allows isolation of approximately 12 x 107 Sertoli cells from 10 rat testes. 3 x 106 cells are plated per well on a 6-well plate so that six to seven 6-well plates are available for experiments on day 7. The Trypsin-DNase I digestion is the most critical step during Sertoli cell isolation. If digestion at this point is advancing too far, the ratio of germ cells/Sertoli cells will disproportionally increase up to the end. During day 3-6 of cult...

Watch this Scientific Journal Video about Isolation of Sertoli Cells and Peritubular Cells from Rat Testes at JoVE.com

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

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