CN115595300A - 一种皮肤附属器的制备方法 - Google Patents
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Abstract
本发明公开了一种皮肤附属器的制备方法,涉及细胞3维培养技术领域,制备方法步骤如下:S1.用生理盐水洗涤脐带或羊膜并剪成段,去除小动脉和小静脉等小血管,撕取华通胶剪碎后分散至具有间充质干细胞培养基的培养瓶中静置培养,得到脐带或羊膜间充质干细胞;S2.2维扩增培养,传代达P3代后,得到2维扩增后的间充质干细胞的混悬液;S3.将2维的混悬液,离心,将细胞种到24孔板中,采用血管内皮细胞培养基或毛囊细胞培养基进行3维分化诱导培养,并进行鉴定,确认后进行混合培养至3维类器官形成,然后将凝胶共同取出,加入培养基离心,制得血管以及毛囊类器官,即所述的皮肤附属器。
Description
技术领域
本发明涉及细胞3维培养技术领域,具体涉及一种皮肤附属器,血管以及毛囊类器官的制备方法。
背景技术
越来越多的人受到脱发的困扰,其中病理性脱发的患者较难治愈。目前,治疗脱发的方法有药物治疗和毛囊移植技术。药物治疗对于脱发只能提供短期的改善作用,停止用药后,患者容易出现再次脱发的现象。自体毛囊移植技术见效快,但是自体毛囊细胞移植由自体皮肤经过培养移植,具有创伤,毛囊细胞数量来源有限,毛发的再生作用效果不完全能维持长久,其原因是供体患者本身的内分泌失调,头发的生长周期的衰退期和休止期毛囊细胞不能激活,故毛囊移植效果欠佳。因此,毛发移植也不能从根本上解决毛囊数量缺乏问题。
脱发和没有脱发的头皮组织中,毛囊干细胞(HFSCs)的数量都是相同的。而脱发头皮组织中的毛囊干细胞没有转化成生长头发的毛囊前体细胞。
目前,还没有一种方法可以将脐带或羊膜间充质干细胞等诱导分化为毛囊细胞、毛细血管以及周围成纤维细胞等皮肤附属器,制得的皮肤附属器,血管以及毛囊类器官,移植到皮肤缺损和无毛动物皮肤可以促进毛囊再生和改善脱发。
发明内容
针对现有技术中的缺陷,本发明提供一种皮肤附属器的制备方法。使用脐带或羊膜细胞种子细胞进行三维培养,获取方便,培养周期短,无排异。皮肤附属器血管以及毛囊类单体或混合体含有的毛细血管和毛囊类以及皮肤附属物的结构有利于增毛增发和创面的愈合。本发明使用的细胞培养方法合理,仿生态皮肤构造配方合理,满足了细胞生长所需要的局部循环和营养,并且可以有效的提高细胞的生长速度,缩短培养时间。本发明中提供的组织工程皮肤附属器中的血管能促进毛发毛囊的活性和促进长久的生发。也能够调节创面新陈代谢、更加有利于皮肤创口愈合不能自愈的严重烧伤,深度创伤创面的皮肤的修复和功能恢复。所含的毛囊、汗腺结构,使皮肤功能更完全。再经形成毛囊、汗腺结构的诱导培养,于其表面再接种羊膜上皮细胞和经向角质形成细胞诱导的羊膜上皮细胞,经组织工程双层皮肤的培养得到含有毛囊、汗腺结构的组织工程皮肤。
本发明的一个目的在于保护一种皮肤附属器的制备方法。所述的皮肤附属器的制备方法,步骤如下:
S1.采用脐带或羊膜培养脐带或羊膜间充质干细胞:使用生理盐水洗涤脐带或羊膜,剪成段,去除小动脉和小静脉,撕取华通胶或羊膜剪碎后分散至具有间充质干细胞培养基的培养瓶中静置培养,得到脐带或羊膜间充质干细胞;
S2.脐带或羊膜间充质干细胞传代采用2维扩增:进行2维扩增培养,传代达到所需P3代后,得到2维扩增后的间充质干细胞的混悬液;
S3.3维脐带或羊膜间充质干细胞分化诱导:将2维扩增后的间充质干细胞的混悬液,离心,将细胞种到24孔板中,采用血管内皮细胞培养基进行3维血管细胞分化诱导培养或采用毛囊细胞培养基的进行3维毛囊细胞分化诱导培养,培养两周后进行形态学的观察和免疫学的鉴定,确认后进行3维血管细胞和3维毛囊细胞的联合培养,至3维类器官形成,与凝胶共同取出,加入培养基离心,制得血管以及毛囊类器官,即制得所述的皮肤附属器。
优选地,步骤S1中,所述的撕取华通胶为撕取华通胶3-8cm。
优选地,步骤S1中,所述的间充质干细胞培养基的体积为25-40ml,所述的培养瓶的体积为175cm2。
优选地,步骤S1中,所述的静置培养为静置培养5-7天,进行第一换液,传代2次,再次换液。
优选地,步骤S2中,所述的2维扩增培养为置于5%CO2环境中37℃条件下培养14天,3-4天换液一次。
优选地,步骤S3中,所述的离心为以1500rpm/分的速率离心3-5min。
优选地,步骤S3中,所述的加入培养基离心为加入间充质干细胞培养基,以1500rpm/分的速率离心3-5min。
优选地,步骤S3中,所述的3维血管细胞分化诱导培养,进行3维培养的培养板的底层为胶原一型凝胶层;中间层为细胞层,细胞数为2-4×104细胞/孔;上层为I型胶原凝胶层,其上覆盖0.5-1ml的血管内皮细胞培养基;
所述的3维毛囊细胞分化诱导培养中,进行3维培养的培养板的底层为片状圆形羊膜组织,所述的片状圆形羊膜组织的面积等于24孔板的面积,上层为胶原一型凝胶层;次上层为细胞层,细胞数为2-4×104细胞/孔;其上覆盖0.5-1ml毛囊细胞培养基。
进一步优选地,所述的血管内皮细胞培养基为EGM-2大血管内皮细胞培养基LONZA;所述的毛囊细胞分化诱导培养基包括基础培养基、表皮生长因子5-20ng/mL、胰岛素2.0-6.0μg/mL、TGF-β0.5-4μg/mL、神经生长因子2-15ng/ml、胸腺素β2-10ng/ml的、氢化可的松2-9mmol/L、L-谷酰胺0.005-0.3μg/mL、维生素A 0.5-3μg/mL、维生素D2 0.5-3μg/mL、转铁蛋白0.5-3μg/mL、亚油酸50-200ng/mL、牛血清白蛋白10-40μg/mL、胎牛血清10-25%,或所述的毛囊细胞分化诱导培养基包括基础培养基、维生素C 20.0-40.0μg/mL、胰岛素2.0-6.0μg/mL、转铁蛋白1.0-5.0μg/mL、亚硒酸钠1.0-5.0ng/mL、成纤维细胞生长因子0.5-1.5ng/mL。
优选地,步骤S3中,所述的联合培养为采用体积比为1:1的血管内皮细胞培养基、毛囊细胞培养基进行联合培养;所述的联合培养的时间为7-10日。
本发明的有益效果体现在:
(1)本发明提供的方法可以将脐带或羊膜间充质干细胞等诱导分化为毛囊细胞、毛细血管以及周围成纤维细胞等皮肤附属器血管制得的皮肤附属器官移植到皮肤缺损动物皮肤可以促进皮肤修复,毛囊再生和改善脱发。
(2)本发明提供的方法通过制备皮肤附属器的血管,构建血管内皮细胞,毛囊类器官和成纤维细胞的生长的组织工程,全过程通过2维培养,扩增,再用3维培养,成目标的细胞与类器官毛囊类器官。本发明的方法首先使用2维细胞培养,目的是增殖,但是2维培养的细胞在形态发生、信号传导、细胞增殖、生长与分化与真实情况存在不同程度的差异。3维细胞培养优势明显,能够模拟体内环境,予细胞外环境进行实质性改善。血管培养进行夹层模拟培养,毛囊细胞增加底层生长因子的环境的模拟是本发明的技术关键。3维细胞培养基质用胶原支架为应用最广泛的3维细胞培养支架为天然的细胞外基质。3维细胞培养的功能比传统2维细胞培养更接近体内环境。更加接近细胞形态,生理状态,本发明模拟复杂的血管和毛囊的3维结构,也促使生长因子传递环境和体内生长环境。特别是细胞分化和诱导。在体外实验中模拟细胞在体内的行为,可以大量制备并取代2维细胞培养,取代了无法了解细胞在组织内的功能和应答反应,供生物体内等更真实的早期信息从而降低像市场推出细胞生物学的研究成本。
(3)毛囊类器官起重要生理作用,特别是对毛囊细胞起着修复、促进激活的重要作用。本发明提供的方法制得的皮肤附属器官,进行移植后,可以对非活化,休止和萎缩的毛囊细胞进行活化,既能有效改善头皮组织新陈代谢,也可阻止和逆转皮肤衰老也可以修复成纤维细胞损伤以促进毛囊细胞的再生,从而达到育发生发的目的。对于毛囊萎缩,坏死和对育发液长期无效的脱发可以采用本发明提供的方法制得的皮肤附属器血管以及毛囊类器官,进行毛囊移植。这种治疗方法不用脱发者的毛囊,可以用间充质干细胞进行量产。
(4)本发明提供的方法中采用的Ⅰ型胶原蛋白,其分子长度约300nm,直径约1.5nm,呈棒状,由三条肽链组成,其中有两条α(Ⅰ)链,一条α(Ⅱ)链,对机体功能作用最强。α(Ⅰ)链和α(Ⅱ)链之间的氨基酸序列只有微小的差异。且本发明采用的是不含端肽Ⅰ型胶原蛋白,免疫原性极低。
(5)本发明提供的方法采用的3维培养使用三维组织培养皿进行三维细胞培养在凝胶支架里自动三维培养,采用I型胶原包被表面、上层培养基,除了可诱导可分化成为不同类型细胞还可以模拟体内结构增加细胞基质的立体结构。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍。在所有附图中,类似的元件或部分一般由类似的附图标记标识。附图中,各元件或部分并不一定按照实际的比例绘制。
图1为间充质干细胞的3维血管细胞分化诱导培养4天的显微镜结果图;
图2为间充质干细胞的3维血管细胞分化诱导培养后进行CD31免疫荧光鉴定的显微镜结果图;
图3为间充质干细胞的3维血管细胞分化诱导培养2周后纵面电子扫描电镜的结果图;
图4为间充质干细胞的3维毛囊细胞分化诱导培养2周后的显微镜结果图;
图5为间充质干细胞的3维血管细胞分化诱导培养2周后的毛囊干细胞分子标记CK19免疫荧光鉴定的显微镜结果图;
图6为间充质干细胞的3维血管细胞分化诱导培养2周后的毛囊干细胞分子标记CK15免疫荧光鉴定的显微镜结果图;
图7为试验例3中试验组裸鼠一月后头部图;
图8为试验例3中对照组组裸鼠一月后头部图。
图9为试验例4移植后的皮肤毛囊结构中毛细血管分子标记CD31和毛囊干细胞分子标记CK15免疫染色结果;
图10为试验例5提供的皮肤缺损移植模型;
图11为试验例5的病理切片结果;
附图9中,a-CD31,b-CK15。
具体实施方式
下面将结合附图对本发明技术方案的实施例进行详细的描述。以下实施例仅用于更加清楚地说明本发明的技术方案,因此只作为示例,而不能以此来限制本发明的保护范围。
需要注意的是,除非另有说明,本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。
实施例
本实施例提供了一种皮肤附属器的制备方法,步骤如下:
S1.采用脐带或羊膜培养脐带或羊膜间充质干细胞:使用生理盐水洗涤脐带或羊膜,剪成段,去除小动脉和小静脉,撕取华通胶或羊膜剪碎后分散至具有间充质干细胞培养基的培养瓶中静置培养,得到脐带或羊膜间充质干细胞;
S2.脐带或羊膜间充质干细胞传代采用2维扩增:进行2维扩增培养,传代达到所需P3代后,得到2维扩增后的间充质干细胞的混悬液;
S3.3维脐带或羊膜间充质干细胞分化诱导:将2维扩增后的间充质干细胞的混悬液,离心,将细胞种到24孔板中,采用血管内皮细胞培养基进行3维血管细胞分化诱导培养或采用毛囊细胞培养基的进行3维毛囊细胞分化诱导培养,培养两周后进行形态学的观察和免疫学的鉴定,确认后进行3维血管细胞和3维毛囊细胞的联合培养,至3维类器官形成,与凝胶共同取出,加入培养基离心,制得血管以及毛囊类器官,即制得所述的皮肤附属器。
步骤S1中,所述的撕取华通胶为撕取华通胶3cm。
步骤S1中,所述的间充质干细胞培养基的体积为25ml,所述的培养瓶的体积为175cm2。
步骤S1中,所述的静置培养为静置培养7天,进行第一换液,传代2次,再次换液。
步骤S2中,所述的2维扩增培养为置于5%CO2环境中37℃条件下培养14天,4天换液一次。
步骤S3中,所述的离心为以1500rpm/分的速率离心5min。
步骤S3中,所述的加入培养基离心为加入间充质干细胞培养基,以1500rpm/分的速率离心5min。
步骤S3中,3维血管细胞分化诱导培养,进行3维培养的培养板的底层为胶原一型凝胶层;中间层为细胞层,细胞数为2-4×104细胞/孔;上层为I型胶原凝胶层,其上覆盖1ml的血管内皮细胞培养基;
3维毛囊细胞分化诱导培养中,进行3维培养的培养板的底层为片状圆形羊膜组织,片状圆形羊膜组织的面积等于24孔板的面积,上层为胶原一型凝胶层;次上层为细胞层,细胞数为2-4×104细胞/孔;其上覆盖1ml毛囊细胞培养基。
血管内皮细胞培养基为EGM-2大血管内皮细胞培养基LONZA;毛囊细胞分化诱导培养基包括基础培养基、表皮生长因子20ng/mL、胰岛素6.0μg/mL、TGF-β4μg/mL、神经生长因子15ng/ml、胸腺素β10ng/ml的、氢化可的松9mmol/L、L-谷酰胺0.3μg/mL、维生素A 3μg/mL、维生素D2 3μg/mL、转铁蛋白3μg/mL、亚油酸200ng/mL、牛血清白蛋白40μg/mL、胎牛血清25%,或所述的毛囊细胞分化诱导培养基包括基础培养基、维生素C 40.0μg/mL、胰岛素6.0μg/mL、转铁蛋白5.0μg/mL、亚硒酸钠5.0ng/mL、成纤维细胞生长因子1.5ng/mL。
步骤S3中,联合培养为采用体积比为1:1的血管内皮细胞培养基、毛囊细胞培养基进行联合培养;联合培养的时间为10日。
试验例1
检测实施例步骤S3中进行血管内皮细胞分化诱导,观察血管形成后,如图1,将水凝胶上面培养基移除,用戊二醛200ul/孔固定49小时后,PBS清洗三次。24孔板中的凝胶采用梯度酒精进行脱水处理,扫描电镜下拍照,和进行CD31免疫荧光染色检测、结果见图1-3。
从图1-3可知,间充质干细胞进行3维血管细胞分化诱导1天后,毛细血管细胞以及血管网形成。从图3可以清晰的分辨出Ⅰ型胶原蛋白和毛细血管断面和3个毛细血管管腔。
试验例2
检测实施例步骤S3中进行毛囊细胞分化诱导的效果
将实施例制得的皮肤附属器,血管以及毛囊类器官,的组织膜片进行用戊二醛200ul/孔固定48小时后,PBS清洗三次。24孔板中的凝胶采用梯度酒精进行脱水处理,以后进行CK19、CK15免疫荧光染色检测、显微镜检测,结果见图4-6。
从图4-6可知,间充质干细胞进行3维毛囊细胞分化诱导2周后,毛囊类器官形成。
试验例3
将实施例1制得的皮肤附属器血管以及毛囊类器官形成的组织膜片注射移植到裸鼠头部作为试验组,未移植细胞的仅注射生理盐水的裸鼠作为对照组;一个月后测试组裸鼠头部以及身体毛发生长的长度约3mm以上(见图7)与注射生理盐水的对照组(见图8)相比具有明显差异,对照组未见毛发。
试验例4
试验例3中试验组小鼠移植部位一个月后可以观察到正常毛囊的结构。将毛囊与周围组织取出用戊二醛200ul/孔固定48小时后,CD31和CK15双重染色,用共聚焦激光扫描显微镜olympus观测进行扫描,如图9所示,发现毛囊根部组织不仅可见毛细血管构造和CD31阳性也可见毛囊的正常结构和CK15阳性。
试验例5
测试方法:将雄性Wistar大鼠共8只,分为2组,每组4只。麻醉后,剃毛,在脊柱两侧各制造3个,共6个,直径为1.5厘米的同等大小的真皮层缺损,得到皮肤缺损模型,如图10所示;在试验组动物皮肤缺损处移植实施例制得的皮肤附属器血管以及毛囊类器官形成的组织膜片,对照组用生理盐水;术后试验动物背部敷创可贴。2周后解刨,将移植局部皮肤分离,用10%福尔马林中性固定后HE染色切片。
病理测试结果:病理切片在显微镜下结果如图11所示。可见试验组的皮肤缺损处皮肤修复,体外构建的表皮-真皮组织,新表皮以及新生毛囊构造,即皮肤附属物均可见。对照组皮肤溃疡处未见修复新生皮肤的修复和毛囊组织。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围,其均应涵盖在本发明的权利要求和说明书的范围当中。
Claims (10)
1.一种皮肤附属器的制备方法,其特征在于:所述的皮肤附属器的制备方法,步骤如下:
S1.采用脐带或羊膜培养脐带或羊膜间充质干细胞:使用生理盐水洗涤脐带或羊膜,剪成段,去除小动脉和小静脉,撕取华通胶或羊膜剪碎后分散至具有间充质干细胞培养基的培养瓶中静置培养,得到脐带或羊膜间充质干细胞;
S2.脐带或羊膜间充质干细胞传代采用2维扩增:进行2维扩增培养,传代达到所需P3代后,得到2维扩增后的间充质干细胞的混悬液;
S3.3维脐带或羊膜间充质干细胞分化诱导:将2维扩增后的间充质干细胞的混悬液,离心,将细胞种到24孔板中,采用血管内皮细胞培养基进行3维血管细胞分化诱导培养或采用毛囊细胞培养基进行3维毛囊细胞分化诱导培养,培养两周后进行形态学的观察和免疫学的鉴定,确认后进行3维血管细胞和3维毛囊细胞的联合培养,至3维类器官形成,与凝胶共同取出,加入培养基离心,制得血管以及毛囊类器官,即制得所述的皮肤附属器。
2.根据权利要求1所述的皮肤附属器的制备方法,其特征在于:步骤S1中,所述的撕取华通胶为撕取华通胶3-8cm。
3.根据权利要求1所述的皮肤附属器的制备方法,其特征在于:步骤S1中,所述的间充质干细胞培养基的体积为25-40ml,所述的培养瓶的体积为175cm2。
4.根据权利要求1所述的皮肤附属器的制备方法,其特征在于:步骤S1中,所述的静置培养为静置培养5-7天,进行第一换液,传代2次,再次换液。
5.根据权利要求1所述的皮肤附属器的制备方法,其特征在于:步骤S2中,所述的2维扩增培养为置于5%CO2环境中37℃条件下培养14天,3-4天换液一次。
6.根据权利要求1所述的皮肤附属器的制备方法,其特征在于:步骤S3中,所述的离心为以1500rpm/分的速率离心3-5min。
7.根据权利要求1所述的皮肤附属器的制备方法,其特征在于:步骤S3中,所述的加入培养基离心为加入间充质干细胞培养基,以1500rpm/分的速率离心3-5min。
8.根据权利要求1所述的皮肤附属器的制备方法,其特征在于:步骤S3中,所述的3维血管细胞分化诱导培养中,进行3维培养的培养板的底层为胶原一型凝胶层;中间层为细胞层,细胞数为2-4×104细胞/孔;上层为I型胶原凝胶层,其上覆盖0.5-1ml的血管内皮细胞培养基;
所述的3维毛囊细胞分化诱导培养中,进行3维培养的培养板的底层为片状圆形羊膜组织,所述的片状圆形羊膜组织的面积等于24孔板的面积,上层为胶原一型凝胶层;次上层为细胞层,细胞数为2-4×104细胞/孔;其上覆盖0.5-1ml毛囊细胞培养基。
9.根据权利要求8所述的皮肤附属器的制备方法,其特征在于:所述的血管内皮细胞培养基为EGM-2大血管内皮细胞培养基LONZA;所述的毛囊细胞分化诱导培养基包括基础培养基、表皮生长因子5-20ng/mL、胰岛素2.0-6.0μg/mL、TGF-β0.5-4μg/mL、神经生长因子2-15ng/ml、胸腺素β2-10ng/ml的、氢化可的松2-9mmol/L、L-谷酰胺0.005-0.3μg/mL、维生素A0.5-3μg/mL、维生素D2 0.5-3μg/mL、转铁蛋白0.5-3μg/mL、亚油酸50-200ng/mL、牛血清白蛋白10-40μg/mL、胎牛血清10-25%,或所述的毛囊细胞分化诱导培养基包括基础培养基、维生素C 20.0-40.0μg/mL、胰岛素2.0-6.0μg/mL、转铁蛋白1.0-5.0μg/mL、亚硒酸钠1.0-5.0ng/mL、成纤维细胞生长因子0.5-1.5ng/mL。
10.根据权利要求1所述的皮肤附属器的制备方法,其特征在于:步骤S3中,所述的联合培养为采用体积比为1:1的血管内皮细胞培养基、毛囊细胞培养基进行联合培养;所述的联合培养的时间为7-10日。
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