CN112961822B - 一种睾丸类器官及其构建方法与应用 - Google Patents

一种睾丸类器官及其构建方法与应用 Download PDF

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CN112961822B
CN112961822B CN202110210528.1A CN202110210528A CN112961822B CN 112961822 B CN112961822 B CN 112961822B CN 202110210528 A CN202110210528 A CN 202110210528A CN 112961822 B CN112961822 B CN 112961822B
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赵小阳
万聪
姚昭锴
罗芳
李朝晖
常港
任少芳
饶琪
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Southern Medical University
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Abstract

本发明公开了一种睾丸类器官及其构建方法与应用。该睾丸类器官的构建方法,包括如下步骤:(1)用聚合球培养液重悬睾丸细胞,得到的睾丸细胞重悬液置于34~37℃下培养40~72h,得到聚合睾丸细胞的聚合球;(2)将步骤(1)得到的聚合球置于琼脂块上,加入精原干细胞培养液,34~37℃下培养4~6天;(3)除去精原干细胞培养液,加入睾丸分化液,34~37℃下培养18~25天。该构建方法所得到的睾丸类器官与体内睾丸相似,该睾丸类器官中存在各类生殖细胞(精原干细胞、精母细胞)、体细胞;采用本发明提供的构建方法,可以在体外得到具有功能的单倍体细胞。

Description

一种睾丸类器官及其构建方法与应用
技术领域
本发明属于生物医学领域,具体涉及一种睾丸类器官及其构建方法与应用。
背景技术
建立体外精子发生的研究平台,是研究不育症的疾病发生机制的首要阶段。目前,小鼠精子发生的体外分化平台已经建立,但该体系还存在获得的精子样细胞未能形变成熟、获得子代的效率低下、发生机制尚未完全阐明等缺陷。鉴于目前体外二维的培养条件还未能模拟体内三维的培养环境,而体内细胞分化伴随复杂的信号通路参与和多种表观修饰变化,因此,模拟体内生殖细胞发育的微环境,对研究临床不育尤为重要。
研究表明,支持细胞、曲精小管和基底膜等在内的三维空间睾丸微环境对体内精子发生过程非常重要。它们不仅提供结构性支持,还有形成血睾屏障、为生殖细胞提供微环境、运送精子发生的重要激素等作用。一直以来,科学家尝试在体外模拟睾丸微环境,其中一种方法是将生殖细胞培养在二维培养板上或共培养在一种单层饲养层细胞上。虽然生殖细胞与饲养层细胞(例如支持细胞)之间紧密接触。但该方法并未形成曲细精管结构及睾丸微环境。为了在体外模拟接近于体内的睾丸微环境,研究者们构建了多种三维培养方法,例如将原代青春期大鼠和猪的睾丸细胞培养在支持细胞基质中和/或移植到免疫缺陷小鼠皮下,可以发现新生曲精小管状结构的形成。其他研究人员将青春期前的小鼠睾丸组织培养在琼脂糖基质中,也发现类似结构的形成。然而,《In Vitro Reconstruction of MouseSeminiferous Tubules Supporting Germ Cell Differentiation》提及:体外三维琼脂培养只能分化到精母细胞阶段,并未得到具有功能的单倍体细胞及健康的后代。
发明内容
为了克服现有技术的不足,本发明的第一方面的目的,在于提供一种睾丸类器官的构建方法。
本发明的第二方面的目的,在于提供第一方面的构建方法得到的睾丸类器官。
本发明的第三方面的目的,在于提供第一方面的构建方法和/或第二方面的睾丸类器官的应用。
为了实现上述目的,本发明所采取的技术方案是:
本发明的第一个方面,提供一种睾丸类器官的构建方法,包括如下步骤:
(1)用聚合球培养液重悬睾丸细胞,得到的睾丸细胞重悬液置于34~37℃下培养40~72h,得到聚合睾丸细胞的聚合球;
(2)将步骤(1)得到的聚合球置于琼脂块上,加入精原干细胞培养液,34~37℃下培养4~6天;
(3)除去精原干细胞培养液,加入睾丸分化液,34~37℃下培养18~25天。
优选的,步骤(1)中所述聚合球培养液包含以下成分:MEMα、KOSR(knockout serumreplacement,血清替代物)、L-谷氨酰胺、非必需氨基酸、丙酮酸钠、β-巯基乙醇和GDNF(glial cell-derived neurotrophic factor,胶质细胞系衍生的神经营养因子);进一步的,所述聚合球培养液以MEMα与KOSR的混合液为基础培养液,其中,MEMα与KOSR的体积比为(88~92):(8~12)、L-谷氨酰胺的终浓度为0.1~2.5mM、非必需氨基酸的终浓度为0.5~2mM、丙酮酸钠的终浓度为0.5~2mM、β-巯基乙醇的终浓度为50~100mM、GDNF的终浓度为10~30ng/mL。
优选的,步骤(1)中所述睾丸细胞采用两步酶消化法得到,具体如下:取动物睾丸,去除睾丸包膜后,与胶原酶IV混合,消化,离心去上清;然后与胰蛋白酶混合,消化,加入DMEM+10%FBS终止消化,离心去上清,得到睾丸细胞。
优选的,所述动物为小鼠。
优选的,步骤(1)中所述睾丸细胞重悬液中睾丸细胞的浓度为600~900个细胞/uL;进一步为700~800个细胞/uL;更进一步为700个细胞/uL。
优选的,步骤(1)中所述睾丸细胞重悬液置于容器中培养。
优选的,步骤(1)中所述睾丸细胞重悬液的培养过程为:睾丸细胞重悬液置于34~37℃下培养6~8h后,拍打装有睾丸细胞重悬液的容器的四周,使容器中的睾丸细胞聚合更紧致,然后继续培养32~66h。
优选的,步骤(2)中所述琼脂块为用MEMα浸泡12~36h后的琼脂块。
优选的,步骤(2)中所述聚合球置于琼脂块上的密度为2~5个/cm2;进一步为3~4个/cm2;更进一步为4个/cm2
优选的,步骤(2)中所述精原干细胞培养液的加入量为到达琼脂块高度的70~98%;进一步为80~95%;更进一步为90%。
优选的,步骤(2)中所述精原干细胞培养液包含以下组分:StemPro34基础培养液、非必需氨基酸、丙酮酸钠、L-谷氨酰胺、β-巯基乙醇、stemPro34 supplement、bFGF(碱性成纤维细胞生长因子)、LIF(白血病抑制因子)、EGF(表皮细胞生长因子)、GDNF(胶质细胞系衍生的神经营养因子);进一步的,所述精原干细胞培养液还包含以下组分:、葡萄糖、BSA、生物素、β-雌二醇、孕酮、抗坏血酸、FBS(胎牛血清);更进一步的,所述精原干细胞培养液以StemPro34基础培养液与stemPro34 supplement的混合液为基础培养液,其中,StemPro34基础培养液与stemPro34 supplement的体积比为(95~98):(2~5),非必需氨基酸的终浓度为0.5~2mM、丙酮酸钠的终浓度为0.5~2mM、L-谷氨酰胺的终浓度为0.1~2.5mM、β-巯基乙醇的终浓度为50~100mM、bFGF的终浓度为5~15ng/mL、LIF的终浓度为100~1500U/mL、EGF的终浓度为5~15ng/mL、GDNF的终浓度为10~30ng/mL、葡萄糖的终浓度为0~8mg/mL、BSA的终浓度为0~10mg/mL、生物素的终浓度为0~12ng/mL、β-雌二醇的终浓度为0~40ng/mL、孕酮的终浓度为0~70ng/mL、抗坏血酸的终浓度为0~150mM、FBS的终浓度(体积浓度)为0~1.5%。
优选的,步骤(3)中所述睾丸分化液的加入量为到达琼脂块高度的70~98%;进一步为80~95%;更进一步为90%。
优选的,步骤(3)中所述睾丸分化液包含以下组分:MEMα、KOSR(knockout serumreplacement,血清替代物)、L-谷氨酰胺、非必需氨基酸、丙酮酸钠、β-巯基乙醇;进一步的,所述睾丸分化液以MEMα与KOSR的混合液为基础培养液,其中,MEMα与KOSR的体积比为(88~92):(8~12)、L-谷氨酰胺的终浓度为0.1~2.5mM、非必需氨基酸的终浓度为0.5~2mM、丙酮酸钠的终浓度为0.5~2mM、β-巯基乙醇的终浓度为50~100mM。
本发明的第二个方面,提供一种睾丸类器官,通过第一方面的构建方法得到。
本发明的第三方面,提供第一方面的构建方法和/或第二方面的睾丸类器官在疾病模型构建及药物筛选中的应用。
本发明的有益效果是:
本发明提供的构建方法所得到的睾丸类器官与体内睾丸相似,该睾丸类器官中存在各类生殖细胞(精原干细胞、精母细胞)、体细胞;采用本发明提供的构建方法,可以在体外得到具有功能的单倍体细胞;同时,本发明提供的构建方法操作简便、安全可靠、成本低廉。
本发明首次体外建立睾丸类器官,为将来研究临床男性不育及药物筛选提供平台。
附图说明
图1是实施例1中小鼠睾丸类器官的构建方法流程图。
图2是实施例2中不同类型精原细胞的免疫荧光染色图:其中,A为精原干细胞的免疫荧光染色图;B为分化中的精原细胞的免疫荧光染色图;C为已分化的精原细胞的免疫荧光染色图。
图3是实施例2中不同时期的精母细胞的染色体铺片图:A为细线期的精母细胞的染色体铺片图;B为偶线期的精母细胞的染色体铺片图;C为粗线期的精母细胞的染色体铺片图;D为双线期的精母细胞的染色体铺片图。
图4是实施例2中体细胞的免疫荧光染色图:其中,A是肌样细胞(ACTA2阳性)与支持细胞(SOX9阳性)共染图;B是Leydig细胞(3βHSD阳性)与支持细胞(SOX9阳性)共染图。
图5是实施例2中精子细胞的免疫荧光染色图。
图6是实施例3中单倍体分选图:其中,A为对照组的单倍体流式分选图;B为实验组的单倍体流式分选图;C为实验组得到的单倍体白光图。
图7是实施例3中实验组分选得到的单倍体圆形精子胞浆注射(ROSI)后的囊胚发育图:
其中,A为2细胞期图;B为4细胞期图;C为桑葚期图;D为囊胚期图。
图8是实施例3中体外精子来源(实验组)的子代图。
图9是实施例3中实验组的子代交配后生产的小鼠图。
具体实施方式
以下通过具体的实施例对本发明的内容作进一步详细的说明。
本实施例中所使用的材料、试剂等,如无特别说明,为从商业途径得到的试剂和材料。
实施例1小鼠睾丸类器官的构建
一种小鼠睾丸类器官的构建方法,其流程图如图1所示,具体如下:
(1)将新生小鼠(出生3天,ICR小鼠,购于广东省医学实验动物中心)处死,用75%酒精棉球擦洗小鼠身体;剖腹取双侧睾丸,将睾丸置于含含MEMα基础液的直径为3.5cm的平皿中,去除睾丸组织的包膜;用镊子将睾丸撕成小块,PBS清洗2遍后,加入2mL胶原酶IV(2mg/mL),37℃消化3分钟,然后1000rpm离心3min,弃上清,PBS洗涤1次;加入2mL0.05%胰蛋白酶,37℃消化5分钟,加入2mL DMEM(12800017,Life Technologies)+10%FBS(SE200-ES,VISTECH)终止消化,1000rpm离心3min,弃上清,得到睾丸细胞。
(2)将步骤(1)得到的睾丸细胞用聚合球培养液(聚合球培养液的成分及含量如表1所示)重悬,得到浓度为700个细胞/uL的睾丸细胞重悬液,按每孔70uL的量用排枪吸取睾丸细胞重悬液至U96孔板中,将U96孔板放入37℃培养箱中培养培养6小时后取出,用手轻轻敲打U96孔板四侧3次,使U96孔板中的细胞更好的聚合成聚合球,然后放入培养箱中继续培养42h。
表1聚合球培养液的成分及含量
体积 终浓度 货号 品牌
MEMα 45mL 12571063 Thermo Fisher
KOSR 5mL A3181502 Thermo Fisher
非必需氨基酸 500μL 1mM 11140050 Gibco
丙酮酸钠 500μL 1mM 11360-070 Gibco
L-谷氨酰胺 500μL 2mM 25030081 Gibco
β-巯基乙醇 50μL 5×10<sup>-5</sup>M 21985023 Thermo Fisher
GDNF 5μL 20ng/mL 512-GF-050 R&D
(3)制备琼脂块(在100mL水中加入1g琼脂粉(39346-81-1,Biosharp)后,摇匀,加热至100℃,冷至50~60℃后,用移液管吸取35mL至国产10cm皿中,4℃下凝固30min即可使用,用小刀将10cm皿中的琼脂切成小块(大小为1cm x 1cm),用镊子将琼脂块转移到24孔板中,每孔加入500uL MEMα浸泡6小时,去除MEMα,每个琼脂块放4个聚合球(聚合球在转移前,用200uL移液枪(移液枪的枪头尖部被剪)先吸取少许培养液,然后再吸取聚合球,吸取前可轻轻吹打聚合球,去除少许死细胞),每孔加入500uL精原干细胞培养液(精原干细胞培养液的成分及含量如表2所示)使精原干细胞培养液到达琼脂块高度的90%,并在24孔板各孔间隙之间入少许蒸馏水。放入37℃培养箱培养4天,每隔4天置换等体积的新鲜精原干细胞培养液。
表2精原干细胞培养液的成分及含量
Figure BDA0002952105900000051
Figure BDA0002952105900000061
(4)除去精原干细胞培养液,每孔加入500uL睾丸分化液(睾丸分化液的成分及含量如表3所示)使精原干细胞培养液到达琼脂块高度的90%,并将24孔板置于34℃培养箱培养18天,每隔4天置换等体积的新鲜睾丸分化液,得到睾丸类器官。
表3睾丸分化液的成分及含量
体积 终浓度 货号 品牌
MEMα 45mL 12571063 ThermoFisher
KOSR 5mL A3181502 ThermoFisher
非必需氨基酸 500μL 1mM 11140050 Gibco
丙酮酸钠 500μL 1mM 11360-070 Gibco
L-谷氨酰胺 500μL 2mM 25030081 Gibco
β-巯基乙醇 50μL 5×10<sup>-5</sup>M 21985023 Thermo Fisher
实施例2小鼠睾丸类器官的鉴定
1.鉴定睾丸类器官精原细胞类型
将实施例1得到的睾丸类器官(聚合球)用4%多聚甲醛固定,然后进行石蜡包埋切片,对组织切片进行免疫荧光染色,具体为:分别添加一抗(anti-PLZF(SC-28319,Santacruz)与anti-DDX4(ab13840,abcam)、anti-KIT(3074T,Cell SignalingTechnology)与anti-DDX4(a b13840,abcam)、anti-STRA8(ABN1656,Millipore)与PCNA(SC-56,Santacruz),抗体稀释比均为1:400,放置于4℃冰箱过夜;添加二抗:Alexa Fluor488(115-545-003,Jackson),Alexa Fluor 594(111-585-003,Jackson),抗体稀释比1:400,室温静置1h;Hoechst33342(H3570,Thermo Fisher)染核,稀释比l:1000,室温染10min,观察,结果如图2所示:D DX4与PLZF共染,表明类器官中存在精原干细胞;DDX4与KIT共染,表明类器官中存在正在分化的精原干细胞;STRA8与PCNA共染,表明类器官中存在已分化的精原干细胞。
2.鉴定睾丸类器官精母细胞类型
用1mL枪头吸取实施例1得到的睾丸类器官(聚合球)至国产3.5cm皿中,PBS洗涤一次,用1mL一次性注射器将睾丸类器官切成小块,加入2mL胶原酶IV(4mg/mL),消化10min,中途用移液枪吹打,然后加入4mL DMEM+10%FBS终止消化,将细胞消化得到的细胞悬液转移至15mL离心管中,1300rpm离心3min,弃上清,用低渗液HEB(HEB的成分及含量如表4所示)处理半个小时后,再放置100mM的蔗糖中终止;用1%多聚甲醛固定2小时后再进行免疫荧光染色,具体如下:分别添加一抗(anti-SYCP3(ab15093,abcam与anti-γH2AX(ab26350,abcam)、anti-RAD51(ab133534,abcam)与anti-SYCP3(ab15093,abcam)、a nti-SYCP1(ab15090,abcam)与anti-SYCP3(ab15093,abcam),抗体稀释比均为1:400,放置于4℃冰箱过夜;添加二抗:Alexa Fluor 488(115-545-003,Jackson),Alexa Fluor 594(111-585-003,Jackson),抗体稀释比1:400,室温静置1h;Hoechst33342(H3570,ThermoFisher)染核,稀释比l:1000,室温染10min;观察,结果如图3所示:通过染色体铺片,根据染色体形态(细偶期染色体成细长的线状结构,粗双期染色体缩短变粗)及各阶段特征性基因的表达可以判断该类器官中存在细线期、偶线期、粗线期、双线期的精母细胞。
表4 HEB的成分及含量
组分 体积
7.25mL
600mM Tris Hcl 500uL
500mM蔗糖 1mL
170mM柠檬酸 1mL
500mM EDTA 100uL
500mM DTT 50uL
100mM PMSF 100uL
3.鉴定睾丸类器官中体细胞类型
将实施例1得到的睾丸类器官(聚合球)用4%多聚甲醛固定,然后进行石蜡包埋切片,对组织切片进行免疫荧光染色,具体为:添加一抗:anti-SOX9(ab13840,abcam)与anti-A CTA2(sc-53142,Santacruz),anti-3βHSD(sc-515120,Santacruz)与anti-SOX9(ab13840,abcam),抗体稀释比为1:400,放置于4℃冰箱过夜;添加二抗:Alexa Fluor 488(115-545-003,Jackson),Alexa Fluor 594(111-585-003,Jackson),抗体稀释比1:400,室温静置1h;Hoechst33342染核,稀释比l:1000,室温染10min;观察,结果如图4所示:SOX9阳性细胞表示睾丸中的支持细胞,是形成曲细精管结构的主要体细胞类型,可以看到SOX9阳性细胞在类器官中形成曲细精管结构,ACTA2是肌样细胞特征性基因,主要分布在曲细精管外层,3βHSD表示的是Leydig细胞,主要分布在间质区域;以上表明体外的得到的睾丸类器官体细胞类型及分布与体内比较一致。
4.鉴定睾丸类器官精子细胞
将实施例1得到的睾丸类器官(聚合球)用4%多聚甲醛固定,然后进行石蜡包埋切片,对组织切片进行免疫荧光染色,具体为:添加一抗(anti-DDX4(ab13840,abcam)),抗体稀释比为1:400,放置于4℃冰箱过夜;添加二抗:PNA(L32460,Thermo Fisher)),AlexaFluor 488(115-545-003,Jackson),抗体稀释比1:400,室温静置1h;Hoechst33342(H3570,Thermo Fisher)染核,稀释比l:1000,室温染10min;观察,结果如图5所示:类器官中存在精子细胞,精子细胞可特异性在细胞质中表达DDX4,并且通过PNA染色可以看到精子顶体呈半月牙状。
实施例3小鼠睾丸类器官中精子细胞功能的验证
1.流式分选睾丸类器官中的精子细胞
用1mL枪头吸取实施例1得到的睾丸类器官(聚合球)至国产3.5cm皿中,PBS洗涤一次,用1mL一次性注射器将睾丸类器官切成小块,加入2mL胶原酶IV(4mg/ml),消化10min,中途用移液枪吹打,然后加入4mL DMEM+10%FBS终止消化,将细胞消化得到的细胞悬液转移至15mL离心管中,1300rpm离心3min,弃上清,得到细胞沉淀(实验组)。
同时,取6周成年鼠(ICR小鼠,购于广东省医学实验动物中心)脱颈处死,取一侧睾丸放入PBS中,去除白膜后清洗3次,用0.25%胰酶消化15分钟后,将细胞悬液转移到15ml离心管中,1300rpm离心3min,弃上清,得到细胞沉淀(对照组)。
将实验组和对照组的细胞沉淀分别加入5mLDMEM+10%FBS重悬,再加入2uL Hoechst(H3570,Thermo Fisher),Hoechst的终浓度为4ug/mL,混匀,放入37℃水浴锅染色15min,1300rpm离心3min,弃上清,加入500uL流式液(PBS+2%FBS),枪头轻轻吹打混匀,过筛(40μm)至流式管中,然后进行流式分选(流式分选仪为RD公司),流式分选图如图6中A、B所示:对照组中单倍体(1N),二倍体(2N)及四倍体(4N)分群明显;同时实验组中单倍体,二倍体和四倍体群分群也明显,与体内比较一致,并且实验组中单倍体效率约为2.3%;将这些单倍体细胞分选下来后,实验组分选得到的单倍体白光图这些细胞具有典型的小鼠圆形精子的形态特征:直径约为8~9μm,细胞小且圆,能明显看到核区有突出的染色质区域,且细胞核中央有明显的染色质;实验组分选得到的单倍体白光图如图6中C所示。
2.卵母细胞胞浆内圆形精子注射(ROSI)
用激活液(M2(M7167,Sigma)、细胞松弛素(C6762,Sigma))处理卵母细胞(来源于C57和DBA背景的雌鼠,雌鼠购于广东省医学实验动物中心)10min,将卵母细胞激活,然后将步骤1实验组、对照组分选得到的精子细胞注射入激活的卵母细胞中,在有激活液的培养液中37℃培养6小时,然后更换正常培养液M16(M7292,Sigma),继续培养,结果如图7所示:24小时发育成2细胞(如图7中A所示)、48小时发育成4细胞(如图7中B所示)、72小时发育成桑葚(如图7中C所示)、96小时发育成囊胚(如图7中D所示)。
3.健康子代的获得
将步骤2ROSI后形成的胚胎移植到假孕母鼠的输卵管中,19.5天,将移植的母鼠脱颈处死,快速取出胎儿,其中睾丸类器官来源的精子细胞得到两只健康子代(均为一雄一雌)如图8所示。5周后将ROSI来源的雌雄鼠合拢交配,成功产下可育后代,因睾丸类器官来源的小鼠是白色毛发,因此在F2代出现毛色性状分离如图9所示。说明通过该方法得到的单倍体具有发育成个体的潜能。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。

Claims (3)

1.一种小鼠睾丸类器官的构建方法,其特征在于,包括如下步骤:
(1)用聚合球培养液重悬睾丸细胞,得到的睾丸细胞重悬液置于37℃下培养48h,得到聚合睾丸细胞的聚合球;
(2)将步骤(1)得到的聚合球置于琼脂块上,加入精原干细胞培养液,37℃下培养4天;
(3)除去精原干细胞培养液,加入睾丸分化液,34℃下培养18天;
步骤(1)中所述聚合球培养液包含以下成分:MEMα、KOSR、L-谷氨酰胺、非必需氨基酸、丙酮酸钠、β-巯基乙醇和GDNF,其中,所述MEMα与所述KOSR的体积比为9:1、所述L-谷氨酰胺的终浓度为2mM、所述非必需氨基酸的终浓度为1mM、所述丙酮酸钠的终浓度为1mM、所述β-巯基乙醇的终浓度为5×10-5M、所述GDNF的终浓度为20ng/mL;
步骤(1)中所述睾丸细胞重悬液中睾丸细胞的浓度为700个细胞/μL;
步骤(2)中所述精原干细胞培养液包含以下组分:StemPro34基础培养液45mL、非必需氨基酸1mM、丙酮酸钠1mM、L-谷氨酰胺2mM、β-巯基乙醇5×10-5M、stemPro34 supplement1.3mL、bFGF 10ng/mL、LIF 1000U/mL、EGF 10ng/mL、GDNF 10ng/mL;
步骤(2)中所述精原干细胞培养液的加入量为到达琼脂块高度的90%;
步骤(3)中所述睾丸分化液包含以下组分:MEMα 45mL、KOSR 5mL、L-谷氨酰胺2mM、非必需氨基酸1mM、丙酮酸钠1mM、β-巯基乙醇5×10-5M;
步骤(3)中所述睾丸分化液的加入量为到达琼脂块高度的90%。
2.根据权利要求1所述的构建方法,其特征在于:步骤(2)中所述聚合球置于琼脂块上的密度为2~5个/cm2
3.根据权利要求1所述的构建方法,其特征在于:步骤(1)中所述睾丸细胞采用两步酶消化法得到。
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