CN112755250A - 一种组织工程化周围神经组织及其制备方法 - Google Patents
一种组织工程化周围神经组织及其制备方法 Download PDFInfo
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- CN112755250A CN112755250A CN202011620688.5A CN202011620688A CN112755250A CN 112755250 A CN112755250 A CN 112755250A CN 202011620688 A CN202011620688 A CN 202011620688A CN 112755250 A CN112755250 A CN 112755250A
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Abstract
本发明公开了一种组织工程化周围神经组织及其制备方法,属于组织工程技术领域,制备方法如下:将种子细胞分离和培养、纯化和扩增后,体外诱导种子细胞分化成雪旺细胞,然后制备干细胞聚合体,再将干细胞聚合体植入丝素蛋白导管支架材料,形成组织工程化周围神经组织。采用上述方法制备的组织工程化周围神经组织可用于面神经及三叉神经分支等周围神经组织缺损再生修复,与目前存在的其他类型组织工程化神经相比,具有种子细胞获取容易,创伤小,体外诱导分化雪旺细胞分泌成神经相关因子提高神经再生效率,干细胞聚合体内包含充足的细胞量和丰富的细胞外基质,同时具有更好的生物活性和良好的生物安全性,并可显著缩短神经修复时间。
Description
技术领域
本发明涉及组织工程技术领域,具体涉及一种组织工程化周围神经组织及其制备方法。
背景技术
因创伤、肿瘤侵袭或手术等原因造成的神经阻滞破坏是常见的临床问题。而神经组织损伤后伴随着神经功能障碍,如面神经分支损伤引起的面部表情肌运动障碍,这将给患者及其家人的身心带来巨大伤害和痛苦。因此,探索神经组织缺损修复的方法,再生具有生物活性的神经组织对患者组织功能的恢复和生活质量的提高具有重大的临床意义。
目前临床修复周围神经组织缺损的方法主要采用自体神经移植,这种方法也取得了良好的疗效。然而,自体神经移植也存在着明显的缺点:(1)获取的自体神经的长度和直径受限,造成因部分自体神经与受区神经不匹配而影响神经修复疗效;(2)自体神经获取不可避免的造成供区因手术导致的周围组织损伤、畸形和神经功能的丧失。此外,采用神经诱导再生材料(如:聚乙醇酸(PGA)神经桥接管、纤维蛋白胶胶连等)修复神经缺损,主要起到引导和支持作用(CN106730010A公开的《去细胞神经水凝胶用于制备周围神经损伤修复组合物的用途》和CN106729980B公开的《一种用于外周神经修复的仿生神经移植物及其制备方法》),但神经再生的长度受到了限制。因此,目前临床上缺乏一种有效周围神经组织缺损再生修复的方法。
随着组织工程和再生医学的飞速发展,越来越多的研究者利用组织工程技术开展组织、器官再生研究,这为临床上各种组织、器官缺损的治疗提供了新的方向。多项发明基于神经组织发育结合组织工程与再生医学技术,设计了种子细胞/支架材料复合体用于神经组织再生修复的策略,但目前这种策略存在一些不足:(1)多数采用早期外胚间充质干细胞或神经元细胞等作为种子细胞,存在临床获取困难且细胞量不足;(2)种子细胞需要采用生物胶等支架材料支持、传递和固定细胞,这对细胞生物学活性有一定影响。
综上,目前采用周围神经再生的大部分组织工程化周围神经组织,虽然可促进神经组织再生,但仅通过神经断端残留的少量细胞增殖并形成髓鞘,髓鞘化不全很难获得良好的支撑和引导神经再生。因此,目前需研制一种具有种子细胞临床获取容易、创伤小、细胞量充足,且包含丰富细胞外基质特点的,可在体内显著加速神经纤维平行性定向生长,释放成神经相关因子促进髓鞘形成的神经支架导管结构供受区神经缺损处移植,更有效地再生修复神经缺损的组织工程化周围神经组织。
发明内容
本发明为了解决上述的技术问题,而提供一种组织工程化周围神经组织及其制备方法。
本发明是按照以下技术方案实现的:
一种组织工程化周围神经组织的制备方法,将种子细胞分离和培养、纯化和扩增后,体外诱导种子细胞分化成雪旺细胞,然后制备干细胞聚合体,再将干细胞聚合体植入丝素蛋白导管支架材料,形成组织工程化周围神经组织。
进一步的,所述种子细胞包括去分化脂肪细胞、脂肪干细胞或骨髓间充质干细胞。
进一步的,所述去分化脂肪细胞的分离和培养、纯化和扩增的步骤如下:
a. 自口腔颊脂垫、大腿或腹部皮下脂肪组织处抽取3-10 mL脂肪组织,放入含青、链霉素50-150U/mL的PBS于培养皿中浸泡、清洗3次,每次3-5min,将脂肪组织剪碎至1-3mm3,加入脂肪组织2-4倍体积的0.1-0.4% I型胶原酶,震荡,37℃消化0.5-1.5h,加干细胞培养基终止消化;用200μm、100μm滤网依次过滤,离心后收集上层脂肪细胞;PBS洗3次,离心后弃上清液,加干细胞培养基重悬,并接种于25cm2培养瓶中,再加满干细胞培养基,培养瓶翻转180°,置于5%CO2、37℃孵箱内,7天后去培养基,PBS洗2次,加干细胞培养基并翻转180°培养瓶培养,每2-3天换液一次,待细胞生长达60-80%时传代,并标记第一代去分化脂肪细胞;
b.取第一代去分化脂肪细胞以100~200个/mL的细胞密度接种,置于5%CO2、37℃孵箱内培养,每隔1天换液1次,待细胞生长到60%~80%时传代,标记筛选后的种子细胞。
所述干细胞培养基为10%FBS和100U/mL青、链霉素的商用a-MEM培养液。
进一步的,所述脂肪干细胞的分离和培养、纯化和扩增的步骤如下:
a. 自口腔颊脂垫、大腿或腹部皮下脂肪组织处抽取3-10 mL脂肪组织,放入含青、链霉素50-150U/mL的PBS于培养皿中浸泡、清洗3次,每次3-5min,将脂肪组织剪碎至1-3mm3,加入脂肪组织2-4倍体积的0.1-0.4% I型胶原酶,震荡,37℃消化0.5-1.5h,加干细胞培养基终止消化;用200μm、100μm滤网依次过滤,离心收集下层基质血管细胞混合物,PBS重悬,70μm滤网过滤,离心后弃上清液,加干细胞培养基重悬,接种于25cm2培养瓶中,并置于5%CO2、37℃孵箱内培养,每2-3天换液一次,待细胞生长达60-80%时传代,并标记第一代脂肪干细胞;
b. 取第一代脂肪干细胞以100~200个/mL的细胞密度接种,置于5%CO2、37℃孵箱内培养,每隔1天换液1次,待细胞生长到60%~80%时传代,标记筛选后的种子细胞。
所述干细胞培养基为10%FBS和100U/mL青、链霉素的商用a-MEM培养液。
进一步的,所述骨髓间充质干细胞的分离和培养、纯化和扩增的步骤如下:
a.自髂骨处抽取3mL骨髓,加入到含800~1200 U/mL肝素钠的生理盐水中,摇晃后再加入1~1.2 g/mL的淋巴细胞分离液,离心后移除单核细胞白色膜状层,PBS洗涤,再离心后弃上清,加入干细胞培养基重悬,置于5%CO2、37℃条件下进行培养,每隔1天换液1次,待细胞生长到75%~85%时传代,标记第一代骨髓间充质干细胞;
b.取第一代骨髓间充质干细胞以100~200个/mL的细胞密度接种,置于5%CO2、37℃孵箱内培养,每隔1天换液1次,待细胞生长到60%~80%时传代,标记筛选后的种子细胞。
进一步的,体外诱导种子细胞分化成雪旺细胞的步骤如下:将筛选后的种子细胞密度调整为1×105~5×105个/mL后接种于培养皿中,24~48小时细胞贴壁后,PBS洗2次,加入2-4mL成神经预诱导液A,并置于5%CO2、37℃孵箱内培养24小时,弃培养基,PBS洗2次,再加入2-4mL成神经预诱导液B,继续培养72小时,弃培养基后,PBS洗2次,最后加入2-4mL成神经诱导液,每2-3天换液一次,持续培养1~2周,即完成雪旺细胞诱导分化。
通过采用上述技术方案,由于雪旺细胞一方面可分化形成髓鞘组织,保护受损轴突和引导轴突生长方向,另一方面分泌NGF、GDF及FGF等成神经相关因子诱导轴突生长,因此,通过体外获取大量雪旺细胞可显著提高神经再生效率。
进一步的,所述成神经预诱导液A为含1mol/L β-ME的10% FBS DMEM培养基;成神经预诱导液B为含35ng/mL A-TRA的10% FBS DMEM培养基;成神经诱导液为含14µmol/LFSK、10ng/mL bFGF、5ng/mL PDGF-AA和200ng/mL HRG-β的10% FBS DMEM培养基。
进一步的,所述干细胞聚合体的制备步骤如下:
a. 弃诱导分化为雪旺细胞的培养皿中神经预诱导培养基,PBS洗2次,加入干细胞聚合体培养基并置于5%CO2、37℃孵箱内培养,每2~3天换液一次,持续培养1~2周,可在培养皿底部形成白色绒状聚合物,即干细胞膜片;
b. 将2~8个干细胞膜片折叠、塑性,加入干细胞聚合体培养基,并置于5%CO2、37℃孵箱内培养,每天半量换液,持续培养3~5天,可见培养皿内形成粉红色绒状聚合物,即干细胞聚合体。
所述干细胞聚合体培养基为加入50-100μg/mL抗坏血酸的干细胞培养基。
通过采用上述技术方案,制备具有可塑性、充足种子细胞、丰富细胞外基质的干细胞聚合体,细胞外基质可保证种子细胞营养和信号的传递,其内含有I型胶原蛋白和生长因子可促进种子细胞神经向分化。
进一步的,所述组织工程化周围神经组织的制备步骤如下:植入干细胞聚合体于丝素蛋白导管支架材料内,并置于37℃ 孵箱孵育30分钟,即获得组织工程化周围神经组织。
所述丝素蛋白导管支架材料为商业丝素蛋白膜并根据受损周围神经直径所制备。
一种上述组织工程化周围神经组织的制备方法制备得到的组织工程化周围神经组织。
本发明具有的优点和有益效果是:
(1)本发明的组织工程化周围神经组织具有与天然神经组织相似的成分与结构,因此组织工程化周围神经组织移植入机体后可发挥神经组织的正常功能,其内已分化的雪旺细胞可持续分泌神经营养因子,促进具有正常生物学活性的神经组织再生,最终修复周围神经缺损;
(2)本发明以去分化脂肪细胞和脂肪干细胞为种子细胞,便于从临床获取且细胞量足够,经体外诱导可分化雪旺细胞;干细胞聚合体的培养技术的使用,突破了传统组织工程中种子细胞、支架材料和微环境的理念,干细胞聚合体内不仅保证了足够的种子细胞,还包含丰富的细胞外基质,可体内显著加速神经纤维平行性定向生长,释放成神经相关因子促进髓鞘形成,更有效地再生修复神经缺损。间充质干细胞所自分泌的成神经相关生长因子和I型胶原等与细胞外基质蛋白结合储存在细胞外基质内,并且细胞外基质保证了细胞之间传递信号和营养的作用,提供干细胞增殖和分化的微环境。此外,使用的丝素蛋白导管支架材料具有良好的物理特性,无生物安全隐患,拥有良好的抑菌作用;
(3)本发明的组织工程化周围神经组织的制备方法具有操作简便、强度适宜、可塑性强、良好的细胞密度和细胞分布、丰富的细胞外基质、避免细胞接种材料时细胞流失等优点。
附图说明
图1是本发明去分化脂肪细胞、脂肪干细胞及骨髓间充质干细胞的培养图以及体外诱导分化的雪旺细胞图;
图2是本发明细胞聚合体的制备流程及组织学观察图;
图3是本发明组织工程化周围神经组织移植于大鼠面神经颊支10mm缺损处8周后的组织学检测结果图。
具体实施方式
下面结合附图及实施例对本发明进行详细的说明。
实施例1
本实施例制备的组织工程化周围神经组织采用去分化脂肪细胞为种子细胞,经分离、培养、扩增并体外诱导为雪旺细胞,再制备去分化脂肪细胞聚合体,最后将细胞聚合体与丝素蛋白导管支架材料复合,形成组织工程化周围神经组织,其制备具体步骤如下:
(1)自口腔颊脂垫、大腿或腹部皮下脂肪组织处抽取8 mL脂肪组织,放入含青、链霉素100U/mL的PBS于培养皿中浸泡、清洗3次,每次4min,将脂肪组织剪碎至1mm3,加入脂肪组织3倍体积的0.25% I型胶原酶,震荡,37℃消化1h,加干细胞培养基终止消化。用200μm、100μm滤网依次过滤,离心后收集上层脂肪细胞。PBS洗3次,离心后弃上清液,加干细胞培养基重悬,并接种于25cm2培养瓶中,再加满干细胞培养基,培养瓶翻转180°,置于5%CO2、37℃孵箱内,7天后去培养基,PBS洗2次,加干细胞培养基并翻转180°培养瓶培养,每2天换液一次,待细胞生长达80%时传代,并标记第一代去分化脂肪细胞;
所述干细胞培养基:10%FBS和100U/mL青、链霉素的商用a-MEM培养液;
(2)去分化脂肪细胞筛选:取第一代去分化脂肪细胞,以200个/mL的细胞密度接种,置于5%CO2、37℃条件下进行培养,每隔1天换液1次,待细胞生长到80%时传代,标记筛选后的去分化脂肪细胞;
(3)将筛选后的去分化脂肪细胞密度调整为3×105个/mL后接种于培养皿中,24小时细胞贴壁后,PBS洗2次,加入4mL成神经预诱导液A,并置于5%CO2、37℃孵箱内培养24小时,弃培养基,PBS洗2次,再加入4mL成神经预诱导液B,继续培养72小时,弃培养基后,PBS洗2次,最后加入4mL成神经诱导液,每2天换液一次,持续培养10天,即完成雪旺细胞诱导分化;
所述成神经预诱导液A为含1mol/L β-ME的10% FBS DMEM培养基;成神经预诱导液B为含35ng/mL A-TRA的10% FBS DMEM培养基;成神经诱导液为含14µmol/L FSK、10ng/mLbFGF、5ng/mL PDGF-AA和200ng/mL HRG-β的10% FBS DMEM培养基。
(4)弃诱导分化为雪旺细胞的培养皿中神经预诱导培养基,PBS洗2次,加入含100μg/mL抗坏血酸干细胞培养基并置于5%CO2、37℃孵箱内培养,每3天换液一次,持续培养2周,可在培养皿底部形成白色绒状聚合物,即去分化脂肪细胞膜片;将3个细胞膜片折叠、塑性,加入细胞聚合体培养基,并置于5%CO2、37℃孵箱内培养,每天半量换液,持续培养5天,可见培养皿内形成粉红色绒状聚合物,即去分化脂肪细胞聚合体。
(5)根据受区周围神经缺损处直径,采用丝素蛋白导管支架材料包裹去分化脂肪细胞聚合体进行体外复合,加入100 μg/mL抗坏血酸干细胞培养基,并置于5%CO2、37℃孵箱内培养,每天半量换液,持续培养3天,完成去分化脂肪细胞聚合体与丝素蛋白导管支架材料复合,即获得组织工程化周围神经组织。
所述丝素蛋白导管支架材料是将丝素蛋白膜根据受损周围神经直径制备而成,其中丝素蛋白膜购自苏州丝美特生物技术有限公司。
本实施例制备的组织工程周围神经组织,如图1 A是倒置显微镜下原代的去分化脂肪细胞,图1 D是倒置显微镜下体外诱导分化的雪旺细胞。
实施例2
本实施例制备的组织工程化周围神经组织采用骨髓间充质干细胞为种子细胞,经分离、培养、扩增并体外诱导为雪旺细胞,再制备骨髓间充质干细胞聚合体,最后将骨髓间充质干细胞聚合体与支架材料体外复合,形成组织工程化周围神经组织,其制备具体步骤如下:
(1)骨髓间充质干细胞分离、培养及筛选:自髂骨处抽取3mL,注入含0.3mL肝素钠生理盐水(1000 U/mL)的离心管内,摇晃后再加入6mL淋巴细胞分离液(1.2 g/mL),离心后移除单核细胞白色膜状层,PBS洗2次,再离心后弃上清,加入干细胞培养基重悬,置于5%CO2、37℃条件下进行培养,每隔1天换液1次,待细胞生长到80%时传代,标记第一代骨髓间充质干细胞。
所述干细胞培养基:10%FBS和100U/mL青、链霉素的商用a-MEM培养液。
(2)骨髓基质干细胞筛选:取第一代骨髓间充质干细胞,以100个/mL的细胞密度接种,置于5%CO2、37℃条件下进行培养,每隔1天换液1次,待细胞生长到80%时传代,标记筛选后的骨髓间充质干细胞。
(3)将筛选后的骨髓间充质干细胞密度调整为5×105个/mL后接种于培养皿中,24小时细胞贴壁后,PBS洗2次,加入4mL成神经预诱导液A,并置于5%CO2、37℃孵箱内培养24小时,弃培养基,PBS洗2次,再加入4mL成神经预诱导液B,继续培养72小时,弃培养基后,PBS洗2次,最后加入4mL成神经诱导液,每3天换液一次,持续培养12天,即完成雪旺细胞诱导分化;
所述成神经预诱导液A为含1mol/L β-ME的10% FBS DMEM培养基;成神经预诱导液B为含35ng/mL A-TRA的10% FBS DMEM培养基;成神经诱导液为含14µmol/L FSK、10ng/mLbFGF、5ng/mL PDGF-AA和200ng/mL HRG-β的10% FBS DMEM培养基。
(4)弃诱导分化为雪旺细胞的培养皿中神经预诱导培养基,PBS洗2次,加入含50μg/mL抗坏血酸干细胞培养基并置于5%CO2、37℃孵箱内培养,每3天换液一次,持续培养2周,可在培养皿底部形成白色绒状聚合物,即干细胞膜片;将3个干细胞膜片折叠、塑性,加入干细胞聚合体培养基,并置于5%CO2、37℃孵箱内培养,每天半量换液,持续培养5天,可见培养皿内形成粉红色绒状聚合物,即骨髓间充质干细胞聚合体。
(5)根据受区周围神经缺损处直径,采用丝素蛋白导管支架材料包裹骨髓间充质干细胞聚合体进行体外复合,加入50 μg/mL抗坏血酸干细胞培养基,并置于5%CO2、37℃孵箱内培养,每天半量换液,持续培养3天,完成骨髓间充质干细胞聚合体与丝素蛋白导管支架材料复合,即获得组织工程化周围神经组织。
所述丝素蛋白导管支架材料是将丝素蛋白膜根据受损周围神经直径制备而成,其中丝素蛋白膜购自苏州丝美特生物技术有限公司。
本实施例制备的组织工程化周围神经组织,如图1 C是倒置显微镜下原代的骨髓间充质干细胞;图2 A是体式显微镜下的间充质干细胞膜片,图2 B是间充质干细胞膜片聚合的干细胞聚合体,图2 C是扫描电镜下间充质干细胞聚合体表面结构,图2 D & E是HE、Masson染色观察间充质干细胞聚合体结构,图2 F是免疫组织化学染色观察间充质干细胞聚合体I型胶原蛋白的表达。
实施例3
本实施例制备的组织工程化周围神经组织采用脂肪干细胞为种子细胞,经分离、培养、扩增并体外诱导为雪旺细胞,再制备脂肪干细胞聚合体,最后将脂肪干细胞聚合体与支架材料体外复合,形成组织工程化周围神经组织,其制备具体步骤如下:
(1)自口腔颊脂垫、大腿或腹部皮下脂肪组织处抽取6mL脂肪组织,放入含青、链霉素100U/mL的PBS于培养皿中浸泡、清洗3次,每次3min,将脂肪组织剪碎至1mm3,加入脂肪组织3倍体积的0.3% I型胶原酶,震荡,37℃消化1h,加干细胞培养基终止消化。用200μm、100μm滤网依次过滤,离心收集下层基质血管细胞混合物(SVF),PBS重悬,70μm滤网过滤,离心后弃上清液,加干细胞培养基重悬,接种于25cm2培养瓶中,并置于5%CO2、37℃孵箱内培养,每2天换液一次,待细胞生长达75%时传代,并标记第一代脂肪干细胞
所述干细胞培养基:10%FBS和100U/mL青、链霉素的商用a-MEM培养液。
(2)脂肪干细胞筛选:取第一代脂肪干细胞,以200个/mL的细胞密度接种,置于5%CO2、37℃条件下进行培养,每隔1天换液1次,待细胞生长到75%时传代,标记筛选后的脂肪干细胞。
(3)将筛选后的脂肪干细胞密度调整为4×105个/mL后接种于培养皿中,24小时细胞贴壁后,PBS洗2次,加入4mL成神经预诱导液A,并置于5%CO2、37℃孵箱内培养24小时,弃培养基,PBS洗2次,再加入4mL成神经预诱导液B,继续培养72小时,弃培养基后,PBS洗2次,最后加入4mL成神经诱导液,每2天换液一次,持续培养10天,即完成雪旺细胞诱导分化;
所述成神经预诱导液A为含1mol/L β-ME的10% FBS DMEM培养基;成神经预诱导液B为含35ng/mL A-TRA的10% FBS DMEM培养基;成神经诱导液为含14µmol/L FSK、10ng/mLbFGF、5ng/mL PDGF-AA和200ng/mL HRG-β的10% FBS DMEM培养基。
(4)弃诱导分化为雪旺细胞的培养皿中神经预诱导培养基,PBS洗2次,加入含100μg/mL抗坏血酸干细胞培养基并置于5%CO2、37℃孵箱内培养,每3天换液一次,持续培养2周,可在培养皿底部形成白色绒状聚合物,即脂肪干细胞膜片;将3个干细胞膜片折叠、塑性,加入细胞聚合体培养基,并置于5%CO2、37℃孵箱内培养,每天半量换液,持续培养5天,可见培养皿内形成粉红色绒状聚合物,即脂肪干细胞聚合体。
(5)根据受区周围神经缺损处直径,采用丝素蛋白导管支架材料包裹脂肪干细胞聚合体进行体外复合,加入100 μg/mL抗坏血酸干细胞培养基,并置于5%CO2、37℃孵箱内培养,每天半量换液,持续培养3天,完成脂肪干细胞聚合体与丝素蛋白导管支架材料复合,即获得组织工程化周围神经组织。
所述丝素蛋白导管支架材料是将丝素蛋白膜根据受损周围神经直径制备而成,其中丝素蛋白膜购自苏州丝美特生物技术有限公司。
本实施例制备的组织工程化周围神经组织,如图 1B是倒置显微镜下原代的脂肪干细胞;图 3A是组织工程化周围神经组织移植于大鼠面神经颊支10mm缺损;图 3B-D是8周后组织工程化周围神经组织学检查,可见组织工程化周围神经组织的生理结构与天然周围神经组织十分接近,具有典型的髓鞘、神经外膜和轴突等结构。
图1 A是倒置显微镜下的原代去分化脂肪细胞;图1 B是倒置显微镜下的原代脂肪干细胞;图1 C是倒置显微镜下的原代骨髓间充质干细胞;图1 D是倒置显微镜下体外诱导后的雪旺细胞。
图2 A是体式显微镜下细胞膜片的聚合过程,图2 B是大体观察的细胞聚合体聚合过程,图2 C是扫描电镜下的细胞聚合体表面,图2 D、E是HE和Masson三色染色观察细胞聚合体内部细胞分布和细胞外基质量,图2 F是免疫组化染色观察I型胶原在细胞聚合中的表达情况。
图3A是组织工程化周围神经组织移植修复大鼠长约10mm面神经颊支 缺损;图3B是大鼠面神经颊支修复重建8周后,组织工程化周围神经组织 甲苯胺蓝染色,并观察到其髓鞘数目、排列和形态均与天然周围神经组织相 似;图3C1-3是NF-200免疫荧光染色,观察组织工程化周围神经组织神经 纤维的排列和走形,其与天然周围神经组织相似;图3D1-4是GFAP(绿色) 和β-Tubulin(红色)免疫荧光染色组织工程化周围神经组织,观察到绿色的神经外膜和紫色的神经轴突结构与天然周围神经组织相似。综上,组织工程 化周围神经组织与天然周围神经组织生理结构十分接近,具有典型的髓鞘、 神经外膜和轴突等结构。
Claims (12)
1.一种组织工程化周围神经组织的制备方法,其特征在于:将种子细胞分离和培养、纯化和扩增后,体外诱导种子细胞分化成雪旺细胞,然后制备干细胞聚合体,再将干细胞聚合体植入丝素蛋白导管支架材料,形成组织工程化周围神经组织。
2.根据权利要求1所述的一种组织工程化周围神经组织的制备方法,其特征在于:所述种子细胞包括去分化脂肪细胞、脂肪干细胞或骨髓间充质干细胞。
3.根据权利要求2所述的一种组织工程化周围神经组织的制备方法,其特征在于:所述去分化脂肪细胞的分离和培养、纯化和扩增的步骤如下:
a.自口腔颊脂垫、大腿或腹部皮下脂肪组织处抽取脂肪组织,放入含青、链霉素50-150U/mL的PBS于培养皿中浸泡、清洗3次,每次3-5min,将脂肪组织剪碎至1-3mm3,加入脂肪组织2-4倍体积的0.1-0.4% I型胶原酶,震荡,37℃消化0.5-1.5h,加干细胞培养基终止消化;用200μm、100μm滤网依次过滤,离心后收集上层脂肪细胞;PBS洗3次,离心后弃上清液,加干细胞培养基重悬,并接种于培养瓶中,再加满干细胞培养基,培养瓶翻转180°,置于5%CO2、37℃孵箱内,7天后去培养基,PBS洗2次,加干细胞培养基并翻转180°培养瓶培养,每2-3天换液一次,待细胞生长达60-80%时传代,并标记第一代去分化脂肪细胞;
b.取第一代去分化脂肪细胞以100~200个/mL的细胞密度接种,置于5%CO2、37℃孵箱内培养,每隔1天换液1次,待细胞生长到60%~80%时传代,标记筛选后的种子细胞。
4.根据权利要求2所述的一种组织工程化周围神经组织的制备方法,其特征在于:所述脂肪干细胞的分离和培养、纯化和扩增的步骤如下:
a.自口腔颊脂垫、大腿或腹部皮下脂肪组织处抽取脂肪组织,放入含青、链霉素50-150U/mL的PBS于培养皿中浸泡、清洗3次,每次3-5min,将脂肪组织剪碎至1-3mm3,加入脂肪组织2-4倍体积的0.1-0.4% I型胶原酶,震荡,37℃消化0.5-1.5h,加干细胞培养基终止消化;用200μm、100μm滤网依次过滤,离心收集下层基质血管细胞混合物,PBS重悬,70μm滤网过滤,离心后弃上清液,加干细胞培养基重悬,接种于培养瓶中,并置于5%CO2、37℃孵箱内培养,每2-3天换液一次,待细胞生长达60-80%时传代,并标记第一代脂肪干细胞;
b. 取第一代脂肪干细胞以100~200个/mL的细胞密度接种,置于5%CO2、37℃孵箱内培养,每隔1天换液1次,待细胞生长到60%~80%时传代,标记筛选后的种子细胞。
5.根据权利要求2所述的一种组织工程化周围神经组织的制备方法,其特征在于:所述骨髓间充质干细胞的分离和培养、纯化和扩增的步骤如下:
a.自髂骨处抽取骨髓,加入到含800~1200 U/mL肝素钠的生理盐水中,摇晃后再加入1~1.2 g/mL的淋巴细胞分离液,离心后移除单核细胞白色膜状层,PBS洗涤,再离心后弃上清,加入干细胞培养基重悬,置于5%CO2、37℃条件下进行培养,每隔1天换液1次,待细胞生长到75%~85%时传代,标记第一代骨髓间充质干细胞;
b.取第一代骨髓间充质干细胞以100~200个/mL的细胞密度接种,置于5%CO2、37℃孵箱内培养,每隔1天换液1次,待细胞生长到60%~80%时传代,标记筛选后的种子细胞。
6.根据权利要求1所述的一种组织工程化周围神经组织的制备方法,其特征在于:体外诱导种子细胞分化成雪旺细胞的步骤如下:将筛选后的种子细胞密度调整为1×105~5×105个/mL后接种于培养皿中,24~48小时细胞贴壁后,PBS洗2次,加入成神经预诱导液A,并置于5%CO2、37℃孵箱内培养24小时,弃培养基,PBS洗2次,再加入成神经预诱导液B,继续培养72小时,弃培养基后,PBS洗2次,最后加入成神经诱导液,每2-3天换液一次,持续培养1~2周,即完成雪旺细胞诱导分化。
7.根据权利要求6所述的一种组织工程化周围神经组织的制备方法,其特征在于:所述成神经预诱导液A为含1mol/L β-ME的10% FBS DMEM培养基;成神经预诱导液B为含35ng/mLA-TRA的10% FBS DMEM培养基;成神经诱导液为含14µmol/L FSK、10ng/mL bFGF、5ng/mLPDGF-AA和200ng/mL HRG-β的10% FBS DMEM培养基。
8.根据权利要求1所述的一种组织工程化周围神经组织的制备方法,其特征在于:所述干细胞聚合体的制备步骤如下:
a. 弃诱导分化为雪旺细胞的培养皿中神经预诱导培养基,PBS洗2次,加入干细胞聚合体培养基并置于5%CO2、37℃孵箱内培养,每2~3天换液一次,持续培养1~2周,可在培养皿底部形成白色绒状聚合物,即干细胞膜片;
b. 将2~8个干细胞膜片折叠、塑性,加入干细胞聚合体培养基,并置于5%CO2、37℃孵箱内培养,每天半量换液,持续培养3~5天,可见培养皿内形成粉红色绒状聚合物,即干细胞聚合体。
9.根据权利要求1所述的一种组织工程化周围神经组织的制备方法,其特征在于:所述组织工程化周围神经组织的制备步骤如下:植入干细胞聚合体于丝素蛋白导管支架材料内,并置于37℃ 孵箱孵育30分钟,即获得组织工程化周围神经组织。
10.根据权利要求3-5任一所述的一种组织工程化周围神经组织的制备方法,其特征在于:所述干细胞培养基为10%FBS和含100U/mL青、链霉素的a-MEM培养液。
11.根据权利要求8所述的一种组织工程化周围神经组织的制备方法,其特征在于:所述干细胞聚合体培养基为加入50-100μg/mL抗坏血酸的干细胞培养基。
12.一种权利要求1-11任一所述的组织工程化周围神经组织的制备方法制备得到的组织工程化周围神经组织。
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