Name: isolation of sertoli cells and peritubular cells from rat testes
Uploaded: 2016-02-08T13:00:00.000+00:00
Duration: 11 min 11 s
Description: Watch this Scientific Journal Video about Isolation of Sertoli Cells and Peritubular Cells from Rat Testes at JoVE.com

笔者想感谢圭多范霍文和鲁Deboel，鲁汶，谁是在吉森的迈进实验室建立初级睾丸支持细胞的分离极有帮助。莫妮卡Fijak，吉森，承认她与图1A和咨询帮助。研究由德意志研究联合会授予通过BH 93 / 1-1（SB）和资助国际研究研究生院吉林大学吉森（德国）/莫纳什大学（澳大利亚墨尔本）1871年GRK（SB）的支持。从黑森州州的赠款被称为"MännlicheInfertilität贝Infektion＆Entzündung"（MIBIE）节目"兰德斯进攻性楚发展协会Wissenschaftlich-ökonomischerExzellenz"（LOEWE）中也获得支持。

1.动物伦理声明这里描述的实验按照Regierungspräsidium德国吉森准则执行，因为地方当局并确认实践照顾和动物用于实验目的德语代码（许可号:GI 20/23上午十时正A 31 / 2012）。 2.媒体，酶溶液和动物的制备<ol><li>由1克碘溶在100ml乙醇中制备1％碘醇。 </li><li>制备2×500毫升Dulbecco&#39;s磷酸盐缓冲盐水（PBS）不含Ca 2+和Mg 2+ 7.5毫升D-葡萄糖（100克/升）和5毫升100×青霉素/链霉素原液（15毫克/毫升ð补充各葡萄糖，50 U / ml的青霉素和50μg/ ml的链霉素最终）。 </li><li>补充1×500毫升罗斯韦尔园区纪念研究所（RPMI）用5ml 100×青霉素/链霉素原液（50单位/毫升青霉素和50微克/毫升链霉素鳍1640培养基人）。 </li><li>制备胰蛋白酶DNA酶I溶液（2.5毫克/毫升胰蛋白酶和10微克/毫升DNA酶I）加入25毫克胰蛋白酶和0.1毫升DNA酶I（1毫克/毫升DNA酶I）至9.9毫升PBS中。 </li><li>溶解在5ml PBS中的胰蛋白酶50mg的胰蛋白酶抑制剂的抑制剂溶液（10毫克/毫升）。溶解25mg的胰蛋白酶抑制剂在10毫升PBS中的胰蛋白酶抑制剂B液（2.5毫克/毫升）。 </li><li>对于胶原酶透明质酸酶 - 脱氧核糖​​核酸酶I（DNA酶I）溶液溶解10毫克胶原酶A（1毫克/毫升终浓度），10毫克透明质酸酶（1mg / ml的终浓度）和0.1毫升DNA酶I（1毫克/毫升DNA酶I）（ 10微克，10毫升的PBS / ml的终浓度）。 </li><li>通过加入10毫克透明质酸酶（1mg / ml的终浓度）和0.1毫升DNA酶I溶液（1毫克/毫升）（10微克/毫升终浓度）至9.9毫升的PBS制备透明质酸酶DNA酶I溶液。 </li><li>准时交货Wistar大鼠，使得它们在塞尔托利细胞分离的日龄19天。 注意:所述的协议被设计为10只，但是可以修改为至少5和最多20只大鼠。所有溶液的体积必须作相应的调整。 </li><li>通过在100毫升异丙醇溶解0.35克油红O制备油红O原液。搅拌O / N，通过0.2微米和储存在室温为长达一年的筛选。 </li><li>通过混合6毫升油红O贮存液用4ml水制备油红O染色溶液（3:2）。通过后0.2微米的10-20分钟的过滤器。接下来的2小时内使用。 </li><li>制备4％（重量/体积）通过加入4g多聚甲醛粉末（PFA）向80ml 1×PBS中的搅拌的同时轻轻地在通风橱甲醛溶液。热到〜60℃，加入1毫升的1摩尔的NaOH，并继续搅拌，直到多聚甲醛溶解。让溶液冷却至室温，调整用1M HCl将pH至7.4并定容至100毫升，1×PBS中。过滤溶液（0.45微米），并使用在等分新鲜或储存在-20℃下几个月。 注:聚合甲醛（PFA）解聚至单体甲醛水。这个过程是通过适度加热和弱碱性pH催化。刚准备所有的酶溶液和过滤灭菌（0.2μM）他们在使用当天。如果一个不同的制造商的酶（ 参见材料/设备 表 ）时，小心地调整其浓度/活性。 在下面的协议的"吸管"代表血清吸管。 </li></ol> 3.曲细精管的制备<ol><li>麻醉10只雄性Wistar大鼠逐一在使用CO 2的干燥器，并且取代每分钟腔室体积的至少20％的流速。通过挤压一脚垫等到呼吸停止，测试麻醉，并且如果不存在反应通过颈脱位安乐死每只大鼠。流水下切片颈静脉和颈动脉（阴道carotica）沥血。通过用70％乙醇擦拭消毒腹部。 </li><li>使用镊子解除从腹部亩皮肤克莱斯和切出一个从耻骨联合到胸骨（图2A）延伸的椭圆形皮肤瓣，向上拍打它放到胸前。接下来，抢腹肌，并从耻骨联合到胸骨使内侧切口。 <ol><li>挤压与骨盆拇指腹部，以推动睾丸从下骨盆向上。挑附睾脂肪垫（图2B）用钳子用于进一步拉起睾丸。切断精索（图2B），留下白膜完整，并收集所有的睾丸在20毫升PBS在50毫升的锥形管（图2C）。 </li></ol></li><li>收集所有睾丸后，通过添加1％的等体积（20毫升）（重量/体积）的乙醇碘消毒他们。倒置该管的两倍，并立即倒出上清液。迅速用25ml的PBS每个（图2D）洗睾丸两次。 </li><li>转移睾丸至培养皿含有ING15毫升PBS（图2E）。一端牢牢把握每一个睾丸被镊子，做一个小切口进入在另一端的白膜，并挤掉使用封闭的剪刀片作为一种工具刮肾小管。 注意:肾小管仍应形成紧凑睾丸形束，只有少数小管延伸到所述溶液中，以使均匀的酶促消化（图2F）。 </li><li>传输解封装睾丸含有10ml胰蛋白酶DNA酶I溶液的100毫升螺旋盖瓶中。在32℃（120振荡/分钟）执行在振荡水浴中消化。 4分钟后，评价与小管的分散肉眼肾小管。一旦管开始分散到溶液中，立即停止消化。如果需要的话，继续孵育达2分钟。 </li><li>通过加入5ml胰蛋白酶抑制剂溶液A停止胰酶消化吹打起来，充分搅拌溶解上下3-4时间S使用10毫升吸管，并将其转移到50毫升锥形管中。 </li><li>孵育5分钟后观察小管通过单位重力沉淀，仔细用10毫升吸管从步骤3.6去除上清，加入10毫升胰蛋白酶抑制剂B液，以全面停止胰酶消化。通过上下吹打2-3次，重悬管。 </li><li>为了除去污染质细胞洗管9次用25毫升的PBS每个。用25毫升吸管重悬在每洗管，让他们通过单位重力10分钟解决。始终使用相同的25毫升吸管移液PBS，再悬浮和除去上清液。 </li></ol>管周细胞4.移动/隔离（PTC）的<ol><li>传输所有小管到100毫升螺旋盖瓶中，并添加胶原酶透明质酸酶DNA酶I溶液（图3A）。在32℃在振荡水浴中10分钟进行消化（120振荡/分钟）。 </li><li>吸管悬浮液在载玻片上的下降，快速检查的倒置光学显微镜的常规细胞培养下的小管在放大100倍。如果消化不够先进持续增加2分钟孵化（不计所需显微镜检查的时间）。 注意:在此步骤期间所有管获得缩短的长度和细管边缘应该变得粗糙（图3B 和 C）。粗糙的边缘是指示为管周细胞的释放。 </li><li>用25毫升吸管从步骤3.8与消化小管转移悬浮液到50毫升锥形管中。用10ml PBS洗100毫升瓶装和它在锥形管添加到管。允许消化小管到由单位重力10分钟沉降，小心地吸出上清液，将含有的PTC，再次使用25毫升吸管，并将其转移到一个新的锥形管中。在过去的几毫升的愿望使用奥德5毫升吸管R以防止管的任何结转。 </li><li>添加20毫升RPMI补充有10％热灭活的胎牛血清（FBS）的1640培养基中收集到的小管的上清液，在300×g离心在RT 10分钟不使用断混合和离心。 </li><li>小心倒出上清液并在20ml重悬丸状的PTC的RPMI在含有每行16毫升培养基5 T75烧瓶补充有10％热灭活FBS 1640培养基和种子各4mL。在5％CO 2气氛中 （图4A）在37℃。 </li><li>在培养的第 3天trypsinize PTC和分裂他们1:在含有补充有10％热灭活FBS16毫升RPMI1640培养基的T75烧瓶2各自（产率共10瓶）（图4B）。在5％CO 2气氛中于37℃继续温育。 </li><li>在培养的第 5天再次trypsinize PTC和板他们上取决于实验6孔或24孔板（每板1 T75瓶）。实验可以在第6天进行。 </li></ol> 5.隔离支持细胞（SCS） <ol><li>用25毫升吸管悬浮步骤4.3彻底在25毫升PBS的解决管，让他们通过单位重力12分钟解决。使用相同的25毫升吸管移液PBS，小管和去除上清液再悬浮。重复洗3次（4次洗涤总共）。 </li><li>最后一次洗涤消化小管转移至另外100之后ml旋盖含透明质酸酶的DNase I溶液瓶。在32℃（120振荡/分钟）执行在振荡水浴中消化。 </li><li> 5分钟后再次检查管通过放大100倍的光学显微镜检查，如步骤4.2所述。 注:短管，管聚集和释放的细胞应该是可见的（图3D）。 <ol><li>允许管状聚集体沉降10分钟，吸出上清液我们ING 25毫升吸管。洗涤聚合用25毫升的PBS和10分钟的每个洗涤步骤（图3E）沉降时间4倍。 </li></ol></li><li>添加20毫升的RPMI 1640培养基中，并通过装在有20毫升注射器18G的针通过该悬浮液的10倍。避免气泡产生。 注意:拉动悬浮液中并通过针头被认为是一个道次。细胞通过流体动力剪切破坏和均化是在倾向于聚集细胞的制备的标准技术。细胞的穿过小内径的皮下注射针是不太可能对他们的功能特性产生影响25。 图5C 和 D示出了纯化的支持细胞在培养第4天的正常超微结构。 <ol><li>吸管悬浮液在载玻片上并快速地检查一个倒置光学显微镜的常规细胞培养下的小管在10倍的放大倍率，如果AGG一滴regates都分散（图3F）。过滤通过70微米的细胞过滤细胞悬液，以获得在滤液纯单细胞悬液。 </li></ol></li><li>离心操作5.4在200 XG在室温下10分钟的滤液不使用休息。用25毫升吸管小心吸出上清液，并悬浮支持细胞在40ml的RPMI 1640培养基（无血清）。 </li><li>混合100μl的细胞悬浮液用100μl台盼蓝染色，并用一个纽鲍尔改进腔室计数细胞。根据该计数结果调整细胞浓度为3×10 6个细胞/ ml。种子1毫升，每孔在6孔板（= 1天）。如果油红O染色（第6步）计划把盖玻片到一个或播种支持细胞前的几个井。 注意:在悬浮Sertoli细胞迅速沉降使得管应的小区等分试样采取用吸管之前进行简单振摇每一次。直到4他们紧紧ATT天ACH到底层。 </li><li>为了除去在第4天的浮动或粘附生殖细胞（图4C）洗涤用于电镀孤立塞尔托利细胞（步骤5.6），用PBS（图4D）的6孔板的每个孔中。吸出介质，加入1-2毫升的PBS至各孔中，水平摇上剧烈的表，但没有溢出PBS中的6孔板。重复洗涤3次，并在第5天及6（图4E）执行相同的洗涤程序。 注意:在第4天塞尔托利细胞已牢固地连接并显示像倒置光学显微镜（图4C）根据图案的鹅卵石。实验可以从起7天来执行。 <ol><li> （替代对步骤5.7）为免除的浮动和粘附生殖细胞接种于6孔板之后执行低渗休克3天。取下中，加入1ml 20毫米的Tris pH值7.5，直接取出冰箱里的。吸出Tris缓冲液后90〜120秒，但不后来。洗涤细胞两次，用PBS，并加2ml的RPMI 1640培养基（无血清）中。任选地，进行实验的第二天。 注意:低渗休克的较长的持续时间越生殖细胞可被删除，但更Sertoli细胞迷路以及。所述第一PBS洗涤应该为了不取代任何支持细胞很快但小心进行。后直接不同尺寸的低渗处理大量液泡可以在相差显微镜细胞质中观察到。液泡消失低渗处理后的几个小时内。 </li></ol></li></ol> 6，油红O染色<ol><li>在执行RT的所有步骤。第7天洗支持细胞生长在盖玻片（步骤5.6），用PBS两次，用PBS 10％福尔马林固定。 10分钟后代替甲醛与新鲜溶液，并继续固定另一个60分钟。 注意:所有解决方案都加入到孔中（多个）用盖玻片下一步骤之前吸出。 </l我&gt; <li>吸甲醛，用清水冲洗细胞两次，并添加60％的异丙醇。 5分钟后，使细胞在空气中干燥几分钟。 </li><li>加入每孔1毫升油红O染色解决方案，并孵育10分钟。吸出溶液，并用水洗涤细胞立即4次和挂接盖玻片在甘油的PBS（9:1）。采取与常规光学显微镜（图4F）亮场图像。 </li></ol> 7.免疫荧光染色<ol><li> （来自步骤4.5）或生长在用PBS盖玻片两次塞尔托利细胞（来自步骤5.5）洗涤PTC和用4％PFA固定它们在室温10分钟。 </li><li>用在PBS中5％BSA在RT 1小时孵育。丢弃BSA-PBS溶液，并添加含有肌动蛋白（平滑肌）新鲜的BSA-PBS溶液（对PTC）的或波形蛋白（用于支持细胞）初级抗体（稀释1:100为两种抗体）并在4℃CO / N孵育。 注意:孵化用BSA块非特异性的第一抗体的结合。 </li><li>洗涤盖玻片5分钟3次在PBS-0.1％吐温20和绿色荧光染料缀合的山羊抗小鼠二抗（稀释1:1000）孵育在室温下在黑暗中1小时。 </li><li>洗涤盖玻片5分钟3次在PBS-0.1％吐温20和DAPI安装介质装入它们。 </li></ol> 8.超微结构分析<ol><li>生长在用PBS将6cm细胞培养板洗涤塞尔托利细胞（来自步骤5.6）和在2.5％戊二醛，2.5％多聚甲醛和0.05％苦味酸在67毫二甲砷酸缓冲液中的混合物修复它们为2小时，在4℃（根据伊藤和Karnovsky 26 pH为7.4）。 </li><li>在1％的四氧化锇随后的O / N培养在0.3％乙酸双氧铀溶于50mM马来酸盐缓冲液（pH 5）执行后固定。嵌入样品中根据标准程序嵌入媒体。切薄片，用柠檬酸铅对比它们并用电子显微镜检查。 </li></ol>

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作者什么都没有透露。

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, when Sertoli cells are isolated using the procedure described here they get deprived of their intimate contacts to neighboring PTCs and spermatogenic cells. Nevertheless they appear to retain many properties that are characteristic for them in vivo like hormone responsiveness and expression of marker genes although in a time-dependent manner and on a lower level. 
Primary Sertoli cells develop large neutral lipid droplets after six days of culture. One of the vital functions of Sertoli cells is phagocytosis of degenerating and apoptotic germ cells and of germ cell material like the so called residual bodies29. It is known that residual bodies and cytoplasts from elongated spermatids adhere to isolated Sertoli cells and get phagocytosed30. As it is impossible to remove all germ cells during the isolation procedure we therefore hypothesize that contaminating germ cells can also adhere to Sertoli cells after plating and get phagocytosed. Since residual bodies contain small lipid droplets it is easily conceivable that Sertoli cells acquire large lipid droplets after extensive phagocytosis of adhering and damaged germ cells. There is also evidence that phagocytotic clearance of apoptic cells actively downregulates inflammatory and immune responses31 which could be important for establishing and maintaining the testis as an immune priviliged organ.
When isolated Sertoli cells are plated on top of a layer of primary peritubular cells both cell types survive longer and do form a basal lamina between them. Morphologically they are able to form structures reminiscent of seminiferous tubules indicating that both cell types have retained their capacity to properly interact7. Like freshly isolated primary Sertoli cells the frequently used mouse Sertoli cell line TM4 does respond to FSH with increased production of cAMP18, but contrary to primary cells it is not able to produce androgen binding protein32. Moreover it has been shown that the mouse Sertoli cell line 1 lacks the immunoprotective properties associated with primary Sertoli cells12. 
Similarly the rat Sertoli cell line SCIT-C8 expresses the epithelial markers transferrin, clusterin and stem cell factor but has lost androgen receptor expression and androgen responsiveness whereas it has aquired expression of some mesenchymal markers like fibronectin and entactin-133. Therefore, primary Sertoli cells, although expensive and tedious to isolate, appear to retain significantly more qualitative in vivo properties during cell culture than cell lines do. 
Future applications of primary Sertoli cells will focus on immuno-protection of co-cultured or co-transplanted cells, on experiments studying cell-cell interactions, and on investigating the immune privilege of testis.

睾丸产生雄配子和性激素， 即雄激素。器官是由两个车厢。在间质车厢，代表总睾丸容积1的约10-12％，类固醇取睾丸间质细胞内进行。管状隔室表示睾丸体积1的约60-80％，并含有生殖细胞和两种类型的体细胞-管周细胞和支持细胞。睾丸是由结缔组织隔膜分成小叶250-300，每个都包含1-3高度错综复杂的曲细精管。这些细管是由基底膜，胶原片，和管周细胞的周层（图1A）包围。 生殖上皮位于基底膜的腔侧。支持细胞是跨越从基底膜管腔整个生殖上皮大细长细胞。他们强烈ATT痛的基底膜，形成通过基底组织紧密连接，从间质闭塞生发上皮和表示血睾屏障的连续的蜂窝片。支持细胞有显着的细胞质预测和后果，使他们能够进入与种属特异性，但固定数量的生殖细胞紧密形态和功能联系。二倍体胚干细胞增殖和分化成精原细胞。 在减数分裂我短暂的四倍体精母细胞产生减数分裂II期间进一步分为四个单倍体精子细胞发展。所有生殖细胞通过细胞质桥，使得它们形成一个蜂窝网互连。精子细胞的成熟过程中的主要事件是细胞质的大部分挤出，形成残体，在一个叫做精子的过程。残体被支持细胞吞噬。然后晚期精子细胞被释放到管状内腔和输送到附睾进一步成熟。支持细胞和生殖细胞似乎相互配合地形精子发生和功能。 个体睾丸细胞类型的启动准备近一个世纪前，当小睾丸片进行培养和细胞类型鉴定显微镜2。通过打开用细尖镊子白膜后肾小管小心剥离它以后可能分离肾小管及间质车厢3。在1975年威尔士和Wiebe的，以便从附着间质组织和胰酶处理以除去外管周细胞层 4的释放管引入一个胶原酶处理。从早期年轻幼鼠（约20天）就在其中所使用的支持细胞构成，因为精子还没有开始的肾小管上皮细胞人口的很大一部分。在这个年龄段大鼠塞尔特OLI细胞停止分裂，和相邻小区之间的紧密连接形成，使血睾屏障建立5。 独立的威尔士和韦博Dorrington 等人介绍的胰蛋白酶结合，并脱氧核糖核酸其次soybeen胰蛋白酶抑制剂和胶原酶的治疗，发表于同年6。两组以便产生用于电镀的均匀的细胞悬浮液，它包含大约70％的支持细胞也使用机械力（通过注射器针头或分别巴斯德吸管吸取消化管状片段反复）。后在培养3天后，使用介质是无血清的，支持细胞的百分比增加至大约90％。这可以在很大程度上归因于污染的生殖细胞的死亡。残余肾小管周围细胞（PTC）的，但是，坚定不移地通过自己的细胞外基质连接。的PTC，但不支持细胞，已知会产生纤连蛋白可以作为标志物蛋白用于与PTC的推定污染。因此，董建华等。理由是，一个附加的透明质酸酶治疗可以改善塞尔托利细胞级分7的纯度。事实上，他们可以表明，附加的处理是能够由PTC的，以减少污染约20倍，当在比较的含有血清的培养基中用于培养纯化塞尔托利细胞，其变得特别明显。从那时起Tung等的改进的过程。成为普遍的协议和外地8（图1B）被广泛地使用由其它主要基团。 在胶原酶处理多数的PTC的被释放并且可以并行地隔离，以支持细胞。而PTC的剧烈增殖，并且不向卵泡刺激素（FSH）反应，塞尔托利细胞不再进行有丝分裂和通过特性的形态变化到FSH响应和增加环磷酸腺苷（cAMP）的浓度9。非常相似的酶消化的协议可用于初级塞尔托利细胞从其他动物，如人10，鼠标11,12，西伯利亚仓鼠13或牦牛14的隔离。用于除去大量污染生殖细胞低渗休克可以在分离步骤15的端部可以采用。这也允许支持细胞的高效分离从成年大鼠睾丸16。一个富集塞尔托利细胞悬浮液，也可以从生殖细胞通过电镀涂有曼陀罗凝集素17凝集素菜悬浮液中分离。仅支持细胞和一些残留的PTC粘附到凝集素板。 原发性睾丸支持细胞培养，主要是从大鼠，已初步用于研究响应激素或建立细胞系，如鼠标支持细胞里NE TM4 18。该细胞系已在一百多个研究被调查直至今日。在平移的方法支持细胞已被用于共培养细胞和类似的在长期移植物存活异种或同种异体胰岛的共移植组织的免疫保护无全身免疫抑制19。分离的塞尔托利细胞已在共培养实验中被还用于研究上皮-间质（Sertoli细胞- PTCC）和体生殖细胞（支持细胞-生殖细胞）的相互作用20,21。最近主要支持细胞已被用于研究Toll样受体的表达和炎性细胞因子的分泌，以及信号转导级联，导致细胞因子感染后非致病性和肾盂肾炎大肠杆菌表达大肠杆菌 22。最近其他调查采用支持细胞为研究睾丸的免疫privilege 23和表明睾酮预处理抑制LPS诱导的炎症反应24。

<table><tbody><tr><td>Actin (smooth muscle) antibody clone 1A4</td><td>Dako</td><td>M0851</td><td>Monoclonal mouse anti human antibody.&lt;br/&gt; Use 1:100 dilution for immunofluorescence.</td></tr><tr><td>Albumin bovine fraction V&lt;br/&gt; Standard grade, lyophilized</td><td>Serva</td><td>11930.04</td><td>Filter (0.45 &amp;micro;m) BSA solutions used for immunofluorescence.</td></tr><tr><td>Corning Cell Strainers 70&amp;nbsp;&amp;micro;m</td><td>Corning</td><td>431751</td><td>White color</td></tr><tr><td>Collagenase A from&lt;em&gt; Clostridium histolyticum&lt;/em&gt;</td><td>Roche</td><td>10103586001</td><td></td></tr><tr><td>DAPI mountant ProLong Gold Antifade</td><td>Life technologies</td><td>P-36931</td><td></td></tr><tr><td>D-glucose</td><td>Sigma</td><td>G8644</td><td>100 g/L</td></tr><tr><td>Dulbecco&amp;acute;s PBS without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>Electron microscope</td><td>Zeiss</td><td>EM 109S</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Zeiss</td><td>Axioplan 2</td><td></td></tr><tr><td>Hyaluronidase from bovine testis</td><td>Sigma</td><td>H3506</td><td></td></tr><tr><td>Inverted light microscope</td><td>Olympus</td><td>CKX41</td><td>Routine cell culture microscope</td></tr><tr><td>Mouse IgG / IgM (H+L) polyclonal&lt;br/&gt; secondary Antibody&amp;nbsp;</td><td>Life technologies</td><td>A-10684</td><td>Alexa Fluor 488 conjugate</td></tr><tr><td>Oil Red O</td><td>Sigma</td><td>O0625</td><td>Has replaced Sudan III and Sudan IV because of brighter color.</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Penicillin (5,000&amp;nbsp;U/ml)/&lt;br/&gt; Streptomycin (5,000 &amp;micro;g/ml)</td><td>Gibco</td><td>15070-063</td><td>100x solution</td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>21875-034</td><td>Contains 300 mg/L L-glutamine</td></tr><tr><td>Trypsin from porcine pancreas</td><td>Sigma</td><td>T5266</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Trypsin inhibitor from soybean</td><td>Sigma</td><td>T6522</td><td>The Sigma product is considerably cheaper than the previously used&lt;br/&gt; BPTI (Aprotinin) from Roche.</td></tr><tr><td>Vimentin antibody (V9)</td><td>Sigma</td><td>V6630</td><td>Monoclonal mouse anti pig vimentin antibody.&amp;nbsp; Use 1:100 dilution for immunofluorescence.</td></tr><tr><td>Wistar WU rats</td><td>Charles River</td><td></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

睾丸，特别是雄配子，挑战以独特的方式免疫系统，因为精子的分化先出现在青春期的时候 - 建立全身免疫耐受十余年后。精细胞表达了许多可能被视为由免疫系统非自身蛋白质。然后在睾丸必须能够建立耐受这些新抗原一方面，但仍然能够保护自身免受感染和肿瘤的发展，另一方面。因此睾丸是体内该容忍外来抗原没有唤起一个有害的炎性免疫应答的少数免疫特权部位之一。支持细胞中发挥关键作用的维持睾丸的这种免疫特权环境，并且还延长cotransplanted细胞存活在外国的环境。因此主要支持细胞是研究睾丸无法通过电子邮件的免疫特权的重要工具通过建立的细胞系或其它细胞模型asily取代。在这里，我们提出了一个详细而全面的协议，用于支持细胞的分离 - 如果需要的管周细胞 - 从大鼠睾丸一天之内。

所描述的过程允许从10大鼠睾丸约12×10 7个塞尔托利细胞的分离。 3×10 6个细胞每孔接种于6孔板，使六至七个6孔板可用于在第7天实验胰DNA酶I消化是在塞尔托利细胞分离中最关键的步骤。如果在这点上消化正在推进太远，生殖细胞的比率/ Sertoli细胞会不成比例增加至结束。在培养3-6天它小心洗掉尽可能多的非粘性或松散连接的生殖细胞尽可能是很重要的。作为4天以上的替代广泛洗涤生殖细胞可以通过在第3天27低渗处理被除去。这种方法的一个优点是，塞尔托利细胞培养物的时间被保持在最小以便塞尔托利细胞特异性功能是潜在的更好的保留比文化的一个长时间之后。 （图5B）来证明。因为管周细胞有很强的增殖潜力，塞尔托利细胞培养在无血清的执行。在10％的存在血清的PTC将后培养6天后构成所有细胞的大约20％，而它的分数可以预计低于1％，在不存在血清7。几乎所有的分离Sertoli细胞是存活通过台盼蓝染色来判断（未示出）。围绕培养的第7天的分离的塞尔托利细胞至少污染生殖细胞和小管周围的细胞（ 图4），从而使实验应围绕这天可能的话来执行。对于重复性好它重复实验是非常重要的总是对文化的同一天。如果转染计划他们应该在第4天或5来执行，并且细胞提取物应在第5天或6制成。 过在无血清条件支持细胞至少两周是响应于FSH和生产更多的雄激素结合蛋白，纤维蛋白溶酶原活化剂和转时的FSH治疗。对于较长的培养是必需的管周细胞或血清的饲养层。四周后文化恶化，细胞从基质7分离。 文化最支持细胞的第6天已获得脂滴。在大多数细胞的单个大液泡已经形成（图5C和5D）。中性脂质含量可以通过油红O染色（图4F）容易地可视化。 除了塞尔托利CEL<html> LS移除的PTC可以培养以及（图4A 和 4B）。的PTC从10睾丸收率可期望形成后培养2天后汇合层5 T75瓶。像分离的支持细胞新鲜分离的PTC是由可通过传代在很大程度上除去生殖细胞污染。通过标准胰蛋白酶2，使得10瓶将在第5天分离之后汇合:烧瓶拆分1。孤立的PTC的纯度为&gt; 95％，并且可以使用α平滑肌肌动蛋白免疫标记（图5A）来确认。从这一天起的PTC可以用于实验。一个瓶就足够了大约6孔板，和水井应在第二天汇合。细胞可传代多次，但是实验应该总是在相同的通道以确保细胞在可重复的方式表现进行。 上传/ 53389 / 53389fig1.jpg"/&gt; 图1. 形态程序的睾丸和工作流程中。（A）与相邻的间质组织的一个曲细精管的部门示意性地表示。曲细精管是由基底膜（BM）和肌样细胞管周（PTC）的圆周层封闭。生殖上皮位于基底膜的腔侧。支持细胞（SC），跨越整个生发上皮强烈连接到BM，并通过代表血睾屏障（BTB）基底定位遮挡路口互连。其不规则核（N）示出了一个或多个裂缝状的凹槽，并包含一个核仁​​。生殖细胞（GC）是与在其发展的各个阶段旺亲密接触。肾小管间质隔室包含睾丸间质细胞（LC）和免疫细胞如巨噬细胞（MΦ）以及血管（BV）。数字改编自Fijak和迈进28。的程序（B）的工作流程;颜色编码就像是（A）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53389/53389fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53389/53389fig2.jpg" /> 图2. 从Wistar大鼠睾丸切下并解封装。如文中所述（A）中的腹膜腔是通过一个纵向切口打开。星号（*）表示epidydimal脂肪垫。 （B）的睾丸通过切割在含有20毫升的PBS 50ml锥形管中收集精索和 （C）中除去。当所有睾丸已收集它们消毒在20毫升1％（重量/体积）的乙醇碘。以除去碘它们很快用25ml洗涤两次PBS每个。第一PBS洗涤后（D）睾丸。如文中描述的（E）的睾丸被转移至培养皿（右），以及细精管被封闭的剪刀刀片的装置挤出打开白膜（在左侧）。 （F）的解封装睾丸仍形成单管突出的紧凑睾丸形肿块。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53389/53389fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53389/53389fig3.jpg" /> 图3. 睾丸支持细胞的分离。（A）去除间质细胞经胰蛋白酶DNA酶I消化和洗涤用9倍PBS管后成为动员和看起来很干净。 （B）在胶原酶透明质酸酶的DNase I处理肾小管周围细胞从外层和生殖细胞s的释放。肾小管得到缩短并获得外观粗糙。 （C）用PBS洗涤4倍后管状片段。 （D）的透明质酸酶DNA酶I处理释放残余管周细胞以及生殖细胞。管得到进一步的缩短，管状聚集形成。用PBS洗涤4次后，（E）管聚合。 （F）的单塞尔托利细胞通过通过18G针传递集合体10倍制备。在（AE）比例尺= 200微米，在（F）= 50微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53389/53389fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53389/53389fig4.jpg" /> 管周细胞的图4.培养（AB）和支持细胞（CF）和除去铅污染的<html> ING生殖细胞。（A）从步骤4.5将重悬的PTC立即接种第二天拍照。 （B）与生殖细胞每天3污染分裂后降低。 （C）在第4天支持细胞表现出典型的鹅卵石状图案。浮动并秉承生殖细胞是用PBS洗涤前可见。 （D）在用PBS洗涤3次后，同样良好。污染的生殖细胞的数目减少。洗涤三次，用PBS重复在第5天及6（E）的在培养的几乎所有的生殖细胞已被删除6天。支持细胞已形成自己独有寻找细胞层，大部分细胞已获得一个大液泡。 （F）油红O染色显示，这些空泡是包含基本上中性脂脂滴。在（AD）= 200微米，以（E）= 50微米，在（F）的比例尺= 25微米。.JPG"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/53389/53389fig5.jpg" /> 图5. 免疫荧光染色。纯化的原管周细胞染色与肌动蛋白（平滑肌）抗体（A）和睾丸支持细胞与波形蛋白抗体（B）。没有污染的细胞是在视这两个领域可见。在（A）和（B）比例尺= 10微米。 （C）和在培养4天Sertoli细胞（D）的电子显微镜照片。 （℃）注如图 4C看出导致鹅卵石状图案相邻小区（箭头）的密切接触。单脂滴（L）中的每个细胞的细胞质中观察到。 （D）的最丰富的细胞器线粒体（M）和高尔基体（G）。细胞核（N）包含一个核仁​​（NC）和示出了典型的裂缝状压痕。在（C）= 1微米比例尺和（D）= 0.25微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53389/53389fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

需要进行其免疫特性的炎症或感染，和利用在学习睾丸免疫特权，信号转导原塞尔托利细胞。在这里，我们描述了高度纯化的主支持细胞和从大鼠睾丸管周细胞的分离的基于酶的协议。

从大鼠睾丸支持细胞的分离和管周细胞

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 ...

isolation-of-sertoli-cells-and-peritubular-cells-from-rat-testes

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

22.4K 次观看.  Justus-Liebig-Universität Gießen. 需要进行其免疫特性的炎症或感染，和利用在学习睾丸免疫特权，信号转导原塞尔托利细胞。在这里，我们描述了高度纯化的主支持细胞和从大鼠睾丸管周细胞的分离的基于酶的协议。 

视频: Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

分离的原发性上皮细胞从附睾的全细胞贴片钳记录

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

科研

发育生物学

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

用小鼠模型对精子发生细胞学分析的精管壁球技术

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

小鼠血睾丸屏障完整性的一种活体研究方法

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

使用标准实验室设备从小鼠测试中浓缩帕西滕苯细胞和精子

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

基于细胞基和细胞外矩阵的Murine睾丸器官生成

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

使用紫罗兰兴奋细胞渗透DNA结合染料分离穆林精子生成细胞

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

生殖细胞的移植和睾丸组织在小鼠异种移植

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

生物学

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

在文化序列富集的精原干细胞和祖细胞（SSCS）推导长期成年小鼠SSC线

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

简单和高效的睾丸细胞悬液的制备技术

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

生精细胞类型使用STA把速度沉降分离

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...