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 ...

The overall goal of this procedure is to isolate highly purified primary Sertoli cells and peritubular cells, or PTCs, from rat testes. This method can help answer key questions in the male reproductive immunology field such as how do Sertoli cells influence testicular macrophages, or how is autophagy regulated in Sertoli cells? Harvesting Sertoli cells and peritubular cells is tedious and requires expensive reagents.The isolated primary cells retain significantly more qualitative in vivo properties during cell culture, as compared to cell lines. Implications of this technique extend to our diagnosis of male infertility, as Sertoli cells play crucial role in the development of the spermatogenic cells. Generally, individuals new to this method will struggle, because it can be difficult to assess whether the seminiferous tubules have been properly digested.Visual demonstration of this method is critical, as the entire procedure involves many steps that require proper execution. Small mistakes can add up to failure. Begin by disinfecting the abdomens of ten exsanguinated male Wistar rats with 70%ethanol.Then, for each animal in turn, use forceps to lift the skin from the abdominal muscles, and cut out an oval-shaped skin lobe extending from the pubic symphysis to the sternum. Lift the skin upwards onto the chest. Then grasp the abdominal muscles, and make a medial incision from the pubic symphysis to the sternum.Now squeeze the abdomen with the thumbs from the pelvis upwards to push the testes out of the lower pelvis. Using forceps, pull on the epididymal fat pad to extend the testes further. Then cut the spermatic cord, leaving the tunica albuginea intact.And collect the testes in 20 milliliters of PBS in a 50 milliliter conical tube. After collecting all of the testes, disinfect the organs with an equal volume of 1%iodine in ethanol. And decant the supernatant immediately.Quickly wash the testes twice with 25 milliliters of PBS each, then transfer the organs to a Petri dish containing 15 milliliters of fresh PBS. Next, holding each testis firmly with forceps at one end, make a small incision into the tunica albuginea at the opposite end. Squeeze out the tubules using closed scissor blades as a scraping tool.The tubules should still exhibit a compact testicle-shaped bundle, with only a few tubules extending into the saline solution. When all of the testes have been decapsulated, transfer the denuded organs into a 100 milliliter screw cap bottle containing 10 milliliters of trypsin-DNase 1 solution, and digest the decapsulated testes in a shaking water bath at 32 degrees Celsius. After four minutes, evaluate the tubules with the naked eye for the dispersion of the tubules.As soon as the tubules begin to deseminate into the solution, immediately stop the digestion with five milliliters of Trypsin Inhibitor Solution A, pipetted up and down three to four times. Transfer the tissue slurry to a 50 milliliter conical tube. And let the tubules settle by unit gravity.After five minutes, use the same 10 milliliter pipette to carefully remove the supernatant, then add 10 milliliters of Trypsin Inhibitor B solution, pipetting the tubules up and down two to three times to completely stop the digestion. To remove the contaminating interstitial cells, use a 25 milliliter pipette to resuspend the tubules in 25 milliliters of PBS. Then let the tubules settle by gravity for 12 minutes.And repeat the PBS wash nine more times as just demonstrated. To isolate the peritubular cells, transfer the PBS washed tubules into a 100 milliliter screw cap bottle containing a collagenase hyaluronidase DNAse 1 solution, and digest them for another ten minutes in a shaken water bath at 32 degrees Celsius. At the end of the incubation, transfer a drop of the suspension onto a glass slide, and quickly check the tubules under an inverted light microscope at a 100x magnification.The tubules will appear shortened in length with rough edges, indicative of the release of the peritubular cells. Using the same 25 milliliter pipette, transfer the digested tubules into a 50 milliliter conical tube, and rinse the 100 milliliter bottle with 10 milliliters of PBS. Add the PBS wash to the suspension.After allowing the isolated tubule fragments to settle for 10 minutes, use the 25 milliliter pipette to carefully transfer the supernatant, containing the PTCs, into a new conical tube. To prevent the carry over of any tubules, use a five milliliter pipette to aspirate the last few milliliters of solution. Now mix 20 milliliters of RPMI 1640 medium supplemented with 10%heat inactivated FPS, with the supernatant.And spin down the cells. Carefully decant the supernatant, and resuspend the peritubular cell pellet in 20 milliliters of fresh medium. Then seed four milliliters of PTCs in each of five T75 flasks containing 16 milliliters of medium at 37 degrees Celsius and 5%carbon dioxide.To isolate the Sertoli cells, use a new 25 milliliter pipette to resuspend the settled tubules thoroughly in 25 milliliters of PBS. After allowing the tubules to settle for another 12 minutes, use the same pipette to wash the tubules three more times in the same way. Then transfer the tubules into a new 100 milliliter screw cap bottle.And add the hyaluronidase DNase 1 solution. Further digest the tubules in a shaking water bath at 32 degrees Celsius for five minutes. Take an aliquot of the suspension by light microscope inspection at a 100x magnification.Short tubules, tubular aggregates, and released cells should be visible. Allow the tubular aggregates to settle for 10 minutes, then use a new 25 milliliter pipette to remove the supernatant and wash the aggregates four times in 25 milliliters of PBS, with ten minutes of sedimentation between each wash. The contaminating PTC population will have been reduced.After the last wash, add 20 milliliters of RPMI 1640 medium to the cells, and pass the suspension through an 18G needle mounted on a 20 milliliter syringe 10 times. Next, quickly confirm that all of the aggregates have been dissociated, and filter the cell suspension through a 70 micron cell strainer to obtain a pure single-cell suspension. Spin down the filtrate.Using the 25 milliliter pipette to carefully aspirate the supernatant. Then resuspend the isolated Sertoli cells in 40 milliliters of serum-free RPMI 1640 medium. Then count the number of viable cells by Trypan blue exclusion, and adjust the cell concentration to three times ten to the six cells per milliliter.Finally, feed one milliliter of cells per well in a six-well plate. The purity of the isolated Sertoli cells is greater than 95%as demonstrated by Vimentin immunolabeling. On day six of culture, most of the Sertoli cells have acquired lipid droplets, with a single large vacuole observed in the majority of cells.The neutrolipic content of the droplets can be easily visualized by Oil Red O staining. The isolated Sertoli cell culture is the least contaminated with germ and peritubular cells around day seven, so experiments should be performed as close to this day as possible. The PTCs can be cultured as well.Reaching a purity of 95%by day five after isolation, as confirmed by smooth muscle actin immunolabeling. The procedure can be completed in one day. Using ten rats provides enough Sertoli cells for six or seven six-well plates, and enough peritubular cells for ten conifer T75 flasks.When attempting this step for the first time, remember it is important to monitor carefully the three enzymatic digestion, with either naked eye or an inverted light microscope. Initially, it is also important to assess the purity of the isolated Sertoli cells and PTCs using immunolabeling with Vimentin and smooth muscle actin antibodies, respectively. In presence of serum, the peritubular cells proliferate relatively fastly, whereas Sertoli cells stop dividing.Keep in mind that Sertoli cell and peritubular cells culture are contaminated with germ cell that have to be removed by washing or passaging, respectively. This technique has paved the way for endocrinologists and immunologists to explore the interactions between testicular cells and hormones, to establish testicular cell lines, and to investigate the testicular immune privilege. Well, after watching this video, you should have a good understanding of how to isolate rat Sertoli cells and peritubular cells, and how to avoid technique pitfalls like over-digestion of tubules by Trypsin DNase 1.

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