CN116376818A - 一种干细胞三维培养生物支架的制备方法 - Google Patents
一种干细胞三维培养生物支架的制备方法 Download PDFInfo
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Abstract
本发明公开了一种干细胞三维培养生物支架的制备方法,该水凝胶为没食子酸修饰的羟丁基壳聚糖,具备良好的生物相容性和可降解性能。其具体实施过程如下:分离并培养来自人体的胎盘、脐带、牙髓、脂肪及成纤维组织等的干细胞以获得合格的干细胞来源,低温下将其均匀分散于所制备的温敏型壳聚糖水溶液中,在37℃形成温敏型壳聚糖水凝胶干细胞三维培养生物支架。在体外三维培养时可显著提高干细胞的增殖水平和生长状态,再现人体内细胞生长的真实立体微环境。注射到皮肤局部组织后,可显著提高移植干细胞在皮肤区域的保留时间和存活率,大大提高干细胞在体内可发挥的功能,进一步推进间充质干细胞细胞制剂的发展。
Description
技术领域:
本发明涉及生物医药材料技术领域,具体地说,涉及一种干细胞三维培养生物支架的制备方法。
背景技术:
人体的皮肤等组织可能伤口、组织切割或断裂以及烧伤而受损,并且皮肤老化是一个自然发生的过程,同时环境污染、紫外线照射、饮酒、吸烟和营养不良都会加剧皮肤老化,在影响美观的同时还会带来多种皮肤疾病,所以当前社会对于治疗皮肤损伤和逆转皮肤老化的需求量十分巨大。干细胞是形成哺乳类动物的各组织器官的原始细胞,具有多向分化潜能、造血支持和自我复制能力,已经被临床应用于解决多种心血管疾病、肝硬化、神经系统疾病、膝关节半月板损伤修复和自身免疫性疾病,挽救了许多病患的生命。
尽管将干细胞从实验室转移到临床使用是理论可行的,但在许多早期或晚期的临床实验中都出现了重大失败,导致大量的干细胞治疗产品不被批准,其主要失败因素包括但不限于干细胞在免疫相容性、稳定性、异质性、分化和迁移能力方面的质量控制不佳和特征不一致。当前干细胞培养最主要的方法仍是二维培养,仅支持干细胞在一个平面上生长,无法再现生物体内细胞真实的生长状况。二维培养环境下干细胞的生物活性、培养结构和营养物质的释放等很多方面均远不及三维培养,使原本的干细胞逐渐丧失其原有的性状、形态、结构和功能,导致其研究结果与体内试验结果经常不一致,实验参考价值和准确性较低。二维培养所提供的环境与生物体内微环境差距甚大,必然对干细胞的增殖分化产生负面作用,同时二维培养的低增殖效率也无法满足临床干细胞大剂量应用的需求。
除此之外,单纯的二维干细胞移植发挥的作用也极为有限,主要是由于移植的干细胞进入人体之后很快会发生大批量死亡。首先是细胞在移植过程中,由于从贴壁生长状态转变为悬浮状态,会启动一种名为失巢凋亡的细胞死亡途径;随后注射的干细胞会倾向于形成各种大小不一的团块,团块在形成血管之前扩散是唯一的营养来源,导致干细胞由于营养不足发生死亡;最后是植入部位的炎症环境对干细胞的杀伤导致其存活率进一步降低。
因此,研究人员一直在致力开发更为有效的体内外干细胞培养和使用方法。生物支架可以模拟体内细胞生长环境实现细胞与培养环境之间的相互作用,并有助于细胞黏附、增殖和分化,实现对干细胞的三维培养。在体外进行干细胞三维培养既能保留体内细胞微环境的物质及结构基础,又能展现细胞培养的直观性及条件可控性的优势。当前用于干细胞培养的生物支架,如胶原海绵和脱细胞真皮基质,但是这种支架需要额外的细胞接种程序,不能很好地适应不同的移植部位情况。因此,具有溶胶-凝胶转变特性且易于细胞包封和填充不规则的植入部位的水凝胶生物支架是用于干细胞三维培养的更好选择。
发明内容:
本发明的目的是克服现有技术的不足,提供一种可显著提高干细胞的增殖水平和生长状态,能模拟人体内细胞生长真实立体微环境的干细胞三维培养生物支架的制备方法。
为解决上述技术问题,本发明采用如下技术方案:
一种干细胞三维培养生物支架的制备方法,将人源干细胞悬液和温敏型壳聚糖溶液在0~4℃混合均匀,形成干细胞浓度为1×102~1×108cells/mL的温敏型壳聚糖水溶液,然后置于37℃的培养箱中,混合液迅速形成凝胶,形成干细胞三维培养生物支架。
作为优选的,在上述的制备方法中,所述人源干细胞来源于人体的胎盘、脐带、牙髓、脂肪或成纤维组织。
作为优选的,在上述的制备方法中,所述人源干细胞是指符合细胞库要求的第2~5代干细胞。
作为优选的,在上述的制备方法中,所述人源干细胞悬液的制备方法为:将人源干细胞用胰蛋白酶消化并离心,用高糖无血清DMEM培养基重悬细胞,轻轻吹打均匀制备成细胞悬液。
作为优选的,在上述的制备方法中,所述温敏型壳聚糖溶液的制备方法包括如下步骤:
(1)将壳聚糖溶于质量分数1~10%盐酸溶液,加入1~5mol/L的NaOH溶液得沉淀,过滤得纯化的壳聚糖;
(2)将步骤(1)所得纯化壳聚糖在质量分数50%NaOH溶液中碱化之后,加入1,2-环氧丁烷在40-50℃反应24-48h,透析冻干后得到羟丁基壳聚糖(HBC);
(3)将步骤(2)所得羟丁基壳聚糖(HBC)与被1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)活化之后的没食子酸(GA)在50-60℃反应24-48h反应,透析冻干后得到羟丁基没食子酸壳聚糖(HBC-GA);
(4)将步骤(3)所得羟丁基没食子酸壳聚糖(HBC-GA)在0~4℃溶于超纯水水,配置成质量分数为1%-5%的温敏型壳聚糖溶液。
作为优选的,在上述的制备方法中,所述干细胞浓度为1×103~1×108cells/mL。
与现有技术相比,本发明具有如下有益效果:
(1)本发明的三维培养生物支架采用的温敏型壳聚糖水凝胶为羟丁基和多酚改性的壳聚糖,为天然高分子改性,具备更好的组织黏附能力和抗氧化能的同时生物相容性和生物降解性良好,在低温下水溶液呈流动可注射状态,温度升至体温附近时,即可形成水凝胶,并且具备可逆性温敏能力,该特性为医疗操作、细胞培养和组织工程提供了极大的便利。
(2)本发明制备的温敏型壳聚糖水凝胶干细胞三维培养生物支架可以模拟体内细胞生长环境,实现细胞与培养环境之间的相互作用,并有助于细胞黏附、增殖和分化,实现对干细胞的三维培养。在体外进行干细胞三维培养既能保留体内细胞微环境的物质及结构基础,又能展现细胞培养的直观性及条件可控性的优势。
(3)本发明制备的温敏型壳聚糖水凝胶干细胞三维培养生物支架在注射到皮肤局部组织后,原位形成干细胞三维培养三维支架,可显著提高移植干细胞在机体内的的保留时间和存活率,大大提高干细胞在体内可发挥的功能,进一步推进干细胞细胞制剂的发展和在毛囊、牙髓、皮肤及机体各部位的靶向应用。
(4)本发明制备的温敏型壳聚糖水凝胶干细胞三维培养生物支架在毛囊、牙髓、皮肤及机体各部位的靶向应用前景广阔,可用于机体各部位的器官修复或再生。
温敏型壳聚糖水凝胶干细胞三维培养生物支架在在包载干细胞后注射到体内中的应用。通过注射器均匀注射到体内使其通过温敏性原位形成干细胞三维培养生物支架,分析干细胞存活和保留情况。检测内容包括对细胞形态、细胞分化能力、细胞表明标志物和细胞表达因子的检测以及对温敏型壳聚糖水凝胶生物相容性和对细胞生长状态促进作用的研究。
附图说明:
图1为温敏型羟丁基和没食子酸改性壳聚糖的制备过程图;
图2为温敏型壳聚糖水凝胶在不同温度间的形态转换图;
图3为温敏型壳聚糖水凝胶干细胞三维培养生物支架的制备;
图4为脐带干细胞表面抗原分析;
图5为脐带干细胞三维培养生物支架中干细胞的细胞形态分析测试图。
具体实施方式:
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:干细胞的分离和培养
分离提取来源于人体的胎盘、脐带、牙髓、脂肪及成纤维组织的干细胞后,传代培养后建立细胞库并进行细胞库检测获取合格的干细胞,采用符合细胞库要求的第2~5代干细胞用于开发干细胞三维培养生物支架。对干细胞三维培养生物支架进行检测,所述检测内容包括对细胞形态、细胞分化能力、细胞表明标志物和细胞表达因子的检测以及对温敏型壳聚糖水凝胶生物相容性和对细胞生长状态促进作用的研究(如图3)。
实施例2:脐带干细胞合格性分析
分离提取来源于人体脐带的干细胞后,传代培养至2代的干细胞通过表面抗体免疫荧光标记后进行流式细胞分析。脐带干细胞表面的CD73,CD90和CD105等抗原显示阳性,而CD34,CD45和HLA-DR等抗原显示阴性,说明所分离提取的脐带干细胞可用于建立稳定的细胞库用于后续实验(如图4)。
实施例3:温敏型壳聚糖水凝胶干细胞三维培养生物支架的制备
1.温敏型壳聚糖水凝胶的制备:
(1)将壳聚糖溶于1%盐酸溶液,加入1mol/L的NaOH溶液得沉淀,过滤得纯化的壳聚糖;
(2)将步骤(1)所得纯化壳聚糖在50%NaOH溶液中碱化之后,加入1,2-环氧丁烷在50℃反应24h,透析冻干后得到羟丁基壳聚糖HBC;
(3)将步骤(2)所得羟丁基壳聚糖HBC与被EDC和NHS活化之后得没食子酸GA在60℃反应24h反应,透析冻干后得到羟丁基没食子酸壳聚糖HBC-GA;
(4)将步骤(3)所得HBC-GA在0℃溶于超纯水水,配置成3%的温敏型壳聚糖HBC-GA水凝胶溶液;制备过程如图1所示。
2.干细胞三维培养生物支架的制备:
分离并培养来自人体脐带干细胞以获得合格的干细胞来源,建立稳定的干细胞库后,将培养至2代的干细胞在0℃下均匀分散于所制备的温敏型壳聚糖水溶液中,在37℃形成温敏型壳聚糖水凝胶三维间充质干细胞培养生物支架,于三维间充质干细胞培养生物支架中的细胞浓度为106cells/mL。
实施例4:温敏型壳聚糖水凝胶干细胞三维培养生物支架的制备
1.温敏型壳聚糖水凝胶的制备:
(1)将壳聚糖溶于2%盐酸溶液,加入5mol/L的NaOH溶液得沉淀,过滤得纯化的壳聚糖;
(2)将步骤(1)所得纯化壳聚糖在50%NaOH溶液中碱化之后,加入1,2-环氧丁烷在50℃反应24h,透析冻干后得到羟丁基壳聚糖HBC;
(3)将步骤(2)所得羟丁基壳聚糖HBC与被EDC和NHS活化之后得没食子酸GA在60℃反应48h反应,透析冻干后得到羟丁基没食子酸壳聚糖HBC-GA;
(4)将步骤(3)所得HBC-GA在4℃溶于超纯水水,配置成5%的温敏型壳聚糖HBC-GA水凝胶溶液;制备过程如图1所示。
2.干细胞三维培养生物支架的制备:
分离并培养来自人体牙髓的干细胞以获得合格的干细胞来源,建立稳定的干细胞库后,将培养至2代的干细胞在4℃下均匀分散于所制备的温敏型壳聚糖水溶液中,在37℃形成温敏型壳聚糖水凝胶三维间充质干细胞培养生物支架,于三维间充质干细胞培养生物支架中的细胞浓度为107cells/mL。
实施例5:温敏型壳聚糖水凝胶在不同温度间的形态转换
取实施例3所得HBC-GA在4℃溶于水,配置成3%的HBC-GA水溶液,将该溶液多次至于4℃和37℃环境中,观察其性状。实验结果如图2所示,该溶液在4℃为透明溶液,37℃下成透明凝胶,且该转变随着温度变化具有可逆性,说明该羟丁基和多酚改性壳聚糖HBC-GA的水溶液具备温度响应性成胶能力。
实施例6:骨髓间充质干细胞三维培养生物支架的制备
(1)取实施例3的HBC-GA在4℃溶于水,配置成1%的HBC-GA水溶液;
(2)将培养的骨髓间充质干细胞用胰蛋白酶消化并离心,用高糖无血清DMEM培养基重悬细胞,轻轻吹打均匀,并调整合适细胞密度待用;
(3)将准备好的骨髓间充质干细胞细胞悬液与壳聚糖水溶液混合,使干细胞的细胞浓度为106cells/mL,制成的细胞-壳聚糖溶液在冰浴上静置约10分钟,滴加60μL至96孔板中,立即将孔板置于37℃、饱和湿度、5%CO2的培养箱中孵育20min,一旦干细胞三维生物支架形成,每孔补加100μL的培养基,继续放入孵箱内培养。
实施例7:骨髓间充质干细胞在三维生物支架内的增殖能力检测
(1)负载有干细胞的壳聚糖水凝胶固化成型后,转移至48孔板中,加入适宜体积的基础培养基后,将孔板置于37℃、饱和湿度、5%CO2的培养箱中孵育;
(2)每天更换一次培养基,并在培养板的每个孔中加入CCK-8,培养箱中再孵育4小时。最后,立即使用酶标仪测量450纳米处的吸光度。
实施例8:脂肪干细胞三维培养生物支架的制备
(1)取实施例3的HBC-GA在4℃溶于水,配置成2%的HBC-GA水溶液;
(2)将培养的脂肪干细胞用胰蛋白酶消化并离心,用高糖无血清DMEM培养基重悬细胞,轻轻吹打均匀,并调整合适细胞密度待用;
(3)将准备好的脂肪干细胞悬液与壳聚糖水溶液混合,使干细胞的细胞浓度为106cells/mL,制成的细胞-壳聚糖溶液在冰浴上静置约10分钟,滴加60μL至96孔板中,立即将孔板置于37℃、饱和湿度、5%CO2的培养箱中孵育20min,一旦干细胞三维生物支架形成,每孔补加100μL的培养基,继续放入孵箱内培养。
实施例9:脂肪干细胞在三维生物支架内的存活能力检测
(1)负载有脂肪干细胞的壳聚糖水凝胶固化成型后,转移至48孔板中,加入适宜体积的基础培养基后,将孔板置于37℃、饱和湿度、5%CO2的培养箱中孵育。
(2)分别在第1,3,5天吸去脂肪干细胞三维培养生物支架的培养基上清,利用PBS缓冲液润洗3次,再加入calcein AM/PI染液,再将孔板置于孵箱内孵育15min,最后利用新鲜PBS润洗3次,在倒置相差显微镜下观察水凝胶中脂肪干细胞的荧光染色情况。
实施例10:脐带干细胞三维培养生物支架的制备
(1)取实施例3的HBC-GA在4℃溶于水,配置成3%的HBC-GA水溶液;
(2)将培养的脐带干细胞用胰蛋白酶消化并离心,用高糖无血清DMEM培养基重悬细胞,轻轻吹打均匀,并调整合适细胞密度待用;
(3)将准备好的脐带干细胞悬液与壳聚糖水溶液混合,使干细胞的细胞浓度为106cells/mL,制成的细胞-壳聚糖溶液在冰浴上静置约10分钟,滴加60μL至96孔板中,立即将孔板置于37℃、饱和湿度、5%CO2的培养箱中孵育20min,一旦干细胞三维生物支架形成,每孔补加100μL的培养基,继续放入孵箱内培养。
实施例11:脐带干细胞在三维生物支架内的形态分析
(1)负载有脐带干细胞的壳聚糖水凝胶固化成型后,转移至共聚焦培养皿中,加入适宜体积的基础培养基后,将孔板置于37℃、饱和湿度、5%CO2的培养箱中孵育。
(2)培养5天之后,为了评估水凝胶内脐带干细胞的细胞骨架结构,将凝胶构建体在 37°C预热的0.5%多聚甲醛中固定30分钟,PBS洗涤3次,每次3min;
(3)配制0.1%triton-X 100的PBS溶液,加入共聚焦培养皿孵育室温2min,PBS洗涤3次,每次3min;
(4)配制165 nM荧光素-鬼笔环肽溶液,加入脐带干细胞三维培养生物支架中,避光孵育30min,PBS洗涤3次,每次3min;
(5)配制0.1μg/mL的DAPI溶液,加入脐带干细胞三维培养生物支架中,避光孵育10min,PBS洗涤3次,每次3min;
(6)共聚焦显微镜下观察细胞形态,肌动蛋白细胞骨架呈绿色,细胞核呈蓝色。
结果如图5所示,在壳聚糖水凝胶内部的多孔结构中,脐带间充质干细胞整体铺展,细胞呈现出纤维拉伸结构,细胞骨架和细胞核的形态结构十分完整,表明干细胞生长状态良好,并且细胞形态接近正常脐带组织中的脐带间充质干细胞形态。说明所制备的壳聚糖水凝胶可以完全满足三维条件下间充质干细胞的培养,维持其更为优秀的细胞形态,促进干细胞的功能,有应用于体内外培养干细胞的潜力。
实施例12:牙髓干细胞三维培养生物支架的制备
(1)取实施例3的HBC-GA在4℃溶于水,配置成5%的HBC-GA水溶液;
(2)将培养的牙髓干细胞用胰蛋白酶消化并离心,用高糖无血清DMEM培养基重悬细胞,轻轻吹打均匀,并调整合适细胞密度待用;
(3)将准备好的细胞悬液与壳聚糖水溶液混合,使牙髓干细胞的细胞浓度为106cells/mL,制成的细胞-壳聚糖溶液在冰浴上静置约10分钟,滴加60μL至96孔板中,立即将孔板置于37℃、饱和湿度、5%CO2的培养箱中孵育20min,一旦干细胞三维生物支架形成,每孔补加100μL的培养基,继续放入孵箱内培养。
总的来说,本发明制备的温敏型壳聚糖水凝胶干细胞三维培养生物支架可以模拟体内细胞生长环境实现细胞与培养环境之间的相互作用,可供培养的细胞类型有骨髓间充质干细胞、脂肪干细胞、脐带干细胞和牙髓干细胞等,并有助于细胞黏附、增殖和分化,实现对干细胞的三维培养。在体外进行干细胞三维培养既能保留体内细胞微环境的物质及结构基础,又能展现细胞培养的直观性及条件可控性的优势。并且可以原位形成干细胞再提水凝胶,可显著提高移植干细胞在皮肤区域的保留时间和存活率,大大提高干细胞在体内可发挥的功能,进一步推进间充质干细胞细胞制剂的发展。
Claims (7)
1.一种干细胞三维培养生物支架的制备方法,其特征在于:将人源干细胞悬液和温敏型壳聚糖溶液在0~4℃混合均匀,形成干细胞浓度为1×102~1×108cells/mL的温敏型壳聚糖水溶液,然后置于37℃的培养箱中,混合液迅速形成凝胶,形成干细胞三维培养生物支架。
2.根据权利要求1的所述的制备方法,其特征在于所述人源干细胞来源于人体的胎盘、脐带、牙髓、脂肪或成纤维组织。
3.根据权利要求1的所述的制备方法,其特征在于所述人源干细胞是指符合细胞库要求的第2~5代干细胞。
4.根据权利要求1的所述的制备方法,其特征在于所述人源干细胞悬液的制备方法为:将人源干细胞用胰蛋白酶消化并离心,用高糖无血清DMEM培养基重悬细胞,轻轻吹打均匀制备成细胞悬液。
5.根据权利要求1的所述的制备方法,其特征在于所述温敏型壳聚糖溶液的制备方法包括如下步骤:
(1)将壳聚糖溶于质量分数1~10%盐酸溶液,加入1~5mol/L的NaOH溶液得沉淀,过滤得纯化的壳聚糖;
(2)将步骤(1)所得纯化壳聚糖在质量分数50%NaOH溶液中碱化之后,加入1,2-环氧丁烷在40-50℃反应24-48h,透析冻干后得到羟丁基壳聚糖;
(3)将步骤(2)所得羟丁基壳聚糖与被1-(3-二甲氨基丙基)-3-乙基碳二亚胺和N-羟基琥珀酰亚胺活化之后的没食子酸在50-60℃反应24-48h反应,透析冻干后得到羟丁基没食子酸壳聚糖;
(4)将步骤(3)所得羟丁基没食子酸壳聚糖在0~4℃溶于超纯水水,配置成质量分数为1%-5%的温敏型壳聚糖溶液。
6.根据权利要求1的所述的制备方法,其特征在于所述干细胞浓度为1×103~1×108cells/mL。
7.一种干细胞三维培养生物支架,其特征在于由权利要求1-6任一项的制备方法制成。
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