CN114836370B - 一种用于细胞三维培养的壳聚糖微球压片 - Google Patents
一种用于细胞三维培养的壳聚糖微球压片 Download PDFInfo
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Abstract
本发明提供了一种用于细胞三维培养的壳聚糖微球压片及其制备方法。本发明的壳聚糖微球压片是由无交联剂残留的壳聚糖微球与崩解剂压制成片制备，在溶液中具有良好的分散性，经分散后的壳聚糖微球可实现大量细胞的三维培养。在溶菌酶的作用下，可以实现壳聚糖微球的降解，进而收获细胞。本发明避免了壳聚糖微球在储存和运输时损耗大、灭菌时操作困难、定量不精确等问题，且利用所述壳聚糖微球压片进行三维培养时细胞具有良好的粘附、生长和增殖性能，可进行大规模培养扩增获取高质量细胞。
Description
技术领域
本发明属于生物材料科学技术领域。具体涉及使用一种壳聚糖微球压片在培养基中分散后进行大量细胞的三维培养。
背景技术
细胞的三维培养是实现大量细胞培养、药物开发筛选、肿瘤耐药性检测等的重要手段。目前细胞的三维培养主要方法有悬滴法、悬浮法、微孔阵列培养法、微型支架法和微流控培养法，其中微型支架由于具有较大比表面积利于细胞粘附受到广泛的关注。目前细胞的三维培养所用的微型支架主要是具有微米级尺寸且生物相容性良好的具有一定强度的高分子材料，如聚乳酸-羟基乙酸共聚物、海藻酸盐、透明质酸和壳聚糖等。然而这类粉末状的微型高分子支架由于支架材料之间静电作用使得在保存、运输、称取和灭菌过程中存在损耗大、定量困难和操作繁琐等问题。因此，提供一种保存运输简易、取用快捷、定量准确的用于大量细胞三维培养的材料具有重要的应用前景。
发明内容
本发明的目的旨在提供一种用于大量细胞三维培养的壳聚糖微球压片及其制备方法，避免壳聚糖微球运输困难、取用繁琐等问题。
本发明的技术方案如下：
一种用于细胞三维培养的壳聚糖微球压片，原料包括壳聚糖微球与崩解剂，所述的崩解剂与所述的壳聚糖微球的质量比例为1:8-12；所述的壳聚糖微球粒径在80-120μm，在直径为 8mm厚为2mm的每压片体积内包含2000-8000个壳聚糖微球。
优选地，崩解剂包括淀粉、交联聚乙烯吡咯烷酮、泡腾片(主要成分为柠檬酸和碳酸氢钠)和交联羧甲基纤维素钠中的一种。
优选地，所述的壳聚糖微球原料压片所用的压制力为2-5kN。
优选地，所述的壳聚糖的分子量范围为2万-30万。
优选地，壳聚糖微球的制备方法为，
称取壳聚糖、冰醋酸溶于纯水或超纯水中配置成溶液A，其中，壳聚糖与水的比例为 0.05-0.5g/10ml，冰醋酸与水的体积比为0.005-0.05:1；
称取司班80、吐温60溶于石油醚中配置成溶液B,其中，司班80与石油醚的比例为1-5g/50ml,吐温60与石油醚的比例为0.1-0.5g/50ml；
称取氢氧化钠、超纯水溶于无水乙醇中配置成溶液C，其中，氢氧化钠与无水乙醇的比例为0.5-5g/50ml，水与无水乙醇的体积比为1:10-15；
将溶液A缓慢滴加入溶液B，A与B体积比为1:4-6；并在30-50℃，500-2000rpm下搅拌1-10h，形成溶液D；
溶液D经液氮冷却后加入溶液C，溶液C与溶液D的体积比为1:1-2；并在室温，100-1000 rpm下搅拌1-3h获得壳聚糖微球。
本发明还提供所述的壳聚糖微球压片在细胞三维培养用的应用。
本发明还提供所述的壳聚糖微球压片的使用方法，将其放入培养基后快速分散成壳聚糖微球。
优选地，细胞培养之后，加入溶菌酶降解壳聚糖微球。
本发明所述的壳聚糖微球压片是由壳聚糖微球与崩解剂在压片机下压制而成。所述的壳聚糖微球压片优选的直径为8mm，厚为2mm，每片包含2000-8000个壳聚糖微球。所述的壳聚糖微球压片的成型压力范围为2-5kN，在本发明的一个实施例中，成型压力选用2.5kN。
所述的壳聚糖微球压片放入培养基后可快速分散成壳聚糖微球，其粒径与制备压片前的壳聚糖微球无异。在加入溶菌酶后，壳聚糖微球可实现降解。
本发明所述的壳聚糖微球无交联剂残留。所述壳聚糖微球可通过溶菌酶实现降解。
所述壳聚糖微球中可培养的细胞密度为20-80万个细胞/mg壳聚糖微球；
所述的壳聚糖微球压片为直径8mm厚2mm的薄片；在运输和储存过程中片型完好不松散；当投放在培养基后可快速分散成大量壳聚糖微球。
本发明的优点如下：
在本发明中，所述的壳聚糖微球压片在进行大量细胞三维培养时，细胞在壳聚糖微球上具有良好的增殖情况，可用于细胞的大量快速培养和三维肿瘤模型的构建。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1是典型的壳聚糖微球扫描电镜图。
图2是壳聚糖微球压片实物图。
图3是壳聚糖微球压片在细胞培养基中的崩解及分散过程。
图4是典型的壳聚糖微球压片中的微球扫描电镜图。
图5是壳聚糖微球压片培养CT26细胞增殖情况。
图6是壳聚糖微球压片培养CT26细胞粘附情况。
图7是CT26细胞在壳聚糖微球压片上细胞荧光分布图。
图8是鼠源CEC原代细胞在壳聚糖微球压片上细胞荧光分布图
图9是壳聚糖微球压片在溶菌酶的作用下的降解情况。
具体实施方式
为了更好地理解本发明，下面结合附图、实施例进一步阐明本发明的内容，但本发明的内容不仅仅局限于下面的实例。
以下实施例中，所用的壳聚糖的分子量为200000，产自上海麦克林生化科技有限公司；所用司班80纯度为化学纯，产自广东汕头西陇化工厂有限公司；所用吐温60纯度为化学纯，产自广东汕头西陇化工厂有限公司；所用氢氧化钠浓度为分析纯，产自国药集团；所用无水乙醇浓度为分析纯，产自国药集团；溶菌酶浓度为20000U/mg，产自上海源叶生物科技有限公司。
实施例1壳聚糖微球的制备
准确称取0.1g壳聚糖、100μL冰醋酸溶于10mL超纯水中配置成溶液A。
准确称取1.52g司班80、0.13g吐温60溶于50mL石油醚中配置成溶液B。
准确称取1g氢氧化钠、4mL超纯水溶于46mL无水乙醇中配置成溶液C。
将溶液A缓慢滴加入溶液B，并在40℃，1200rpm下搅拌3h，形成溶液D。
溶液D经液氮冷却15min后加入溶液C，并在室温，500rpm下搅拌2h获得壳聚糖微球。
所得到的壳聚糖微球经无水乙醇清洗3次后，冻干备用。所得到的壳聚糖微球的形貌和粒径大小如图1所示。
实施例2壳聚糖微球的制备
准确称取0.05g壳聚糖、100μL冰醋酸溶于10mL超纯水中配置成溶液A。
准确称取1g司班80、0.1g吐温60溶于50mL石油醚中配置成溶液B。
准确称取1g氢氧化钠、4mL超纯水溶于46mL无水乙醇中配置成溶液C。
将溶液A缓慢滴加入溶液B，并在40℃，1200rpm下搅拌3h，形成溶液D。
溶液D经液氮冷却15min后加入溶液C，并在室温，500rpm下搅拌2h获得壳聚糖微球。所得到的壳聚糖微球经无水乙醇清洗3次后，冻干备用。
实施例3壳聚糖微球的制备
准确称取0.2g壳聚糖、100μL冰醋酸溶于10mL超纯水中配置成溶液A。
准确称取3g司班80、0.2g吐温60溶于50mL石油醚中配置成溶液B。
准确称取1g氢氧化钠、4mL超纯水溶于46mL无水乙醇中配置成溶液C。
将溶液A缓慢滴加入溶液B，并在40℃，1200rpm下搅拌3h，形成溶液D。
溶液D经液氮冷却15min后加入溶液C，并在室温，500rpm下搅拌2h获得壳聚糖微球。所得到的壳聚糖微球经无水乙醇清洗3次后，冻干备用。
实施例4壳聚糖微球的制备
准确称取0.3g壳聚糖、100μL冰醋酸溶于10mL超纯水中配置成溶液A。
准确称取4g司班80、0.3g吐温60溶于50mL石油醚中配置成溶液B。
准确称取1g氢氧化钠、4mL超纯水溶于46mL无水乙醇中配置成溶液C。
将溶液A缓慢滴加入溶液B，并在40℃，1200rpm下搅拌3h，形成溶液D。
溶液D经液氮冷却15min后加入溶液C，并在室温，500rpm下搅拌2h获得壳聚糖微球。所得到的壳聚糖微球经无水乙醇清洗3次后，冻干备用。
实施例5壳聚糖微球压片的制备
称取实施例1中得到的壳聚糖微球50mg、交联聚乙烯吡咯烷酮5mg，混合均匀后，使用压片机在2.5kN压力下进行冲压成型，所得到的壳聚糖微球压片如图2所示。
取壳聚糖微球压片1片加入4mL溶液中，浸泡1min，超声分散5min，观察并记录壳聚糖微球压片的分散情况(图3)。将壳聚糖微球压片在溶液中分散的壳聚糖微球取出进行形貌观察(图4)。壳聚糖微球压片在溶液中分散的壳聚糖微球保持原本的球形外观不变，粒径为 80-120μm。
实施例6壳聚糖微球压片用于鼠源结直肠癌细胞CT26的三维培养
取实施例5中得到的壳聚糖微球压片经过高压120℃灭菌1h。取1片壳聚糖微球压片在 5mL 1640细胞培养基中进行分散，并接种1mL细胞密度为105个/mL的鼠源结直肠癌细胞 (CT26)悬液。将培养皿置于37℃，5％CO2进行培养。取实施例1中得到的壳聚糖微球50mg进行相同条件培养作为对照组，传统二维细胞贴壁培养作为空白组。所用到的1640细胞培养基以胎牛血清(FBS)、青霉素-链霉素(Penicillin-Streptomycin)和RPMI-1640细胞培养基按10:1:89体积比配置。
在培养12、24、36、48和60h后，使用CCK-8试剂对细胞活力进行检测。如图5所示，壳聚糖微球压片培养的细胞与壳聚糖微球培养的细胞的细胞增殖能力相近，且均高与传统二维贴壁培养的细胞。培养4天后，经乙醇梯度脱水后，使用扫描电镜观察细胞粘附情况，如图6所示，使用壳聚糖微球压片培养的细胞与壳聚糖微球培养的细胞均粘附在微球上表面，连接紧密，聚团生长。通过钙黄绿素和碘化丙啶对细胞进行染色可以看到，如图7所示，使用壳聚糖微球压片培养的细胞在微球上三维粘附且生长情况良好。
实施例7：壳聚糖微球压片用于大鼠角膜上皮原代细胞CEC的三维培养
取实施例5中得到的壳聚糖微球压片经过高压120℃灭菌1h。将1片壳聚糖微球压片在 5mL DMEM细胞培养基中进行分散，并接种1mL细胞密度为105mL-1的GFP大鼠角膜上皮原代细胞(CEC)悬液。置于37℃，5％CO2进行培养7天后直接在荧光显微镜下观察细胞分布。所用到的DMEM细胞培养基以胎牛血清、青霉素-链霉素、氢化可的松、细胞培养添加物(ITS)、二甲基亚砜、小鼠表皮生长因子(m-EGF)、DMEM/F12细胞培养基按 6:1:0.02:1:0.5:0.1：91.38体积比配置。
如图8所示，CEC-GFP细胞本身在传统二维贴壁培养中增殖缓慢且不易培养，但在壳聚糖微球压片上生长情况良好，具备优异的增殖性能，且微球上细胞连接紧密，有聚集生长趋势，证明壳聚糖微球压片为细胞提供了良好的增殖环境，有利于CEC-GFP细胞的增殖。
实施例8：壳聚糖微球压片的降解性能
按实施例6构建CT26细胞三维培养模型，经培养4天后加入100U/mL的溶菌酶，继续培养24h后，观察壳聚糖降解情况并收集细胞。
如图9显示，在加入溶菌酶后，壳聚糖微球在24h内被逐渐降解，原本粘附生长在壳聚糖微球上的细胞团散落贴附在培养皿上。该结果表面壳聚糖微球压片在可利用溶菌酶进行降解，并收获细胞，避免了传统二维贴壁培养中胰蛋白酶的使用对细胞造成的损伤。
Claims (7)
1.一种用于细胞三维培养的壳聚糖微球压片，其特征在于：原料包括壳聚糖微球与崩解剂，所述的崩解剂与所述的壳聚糖微球的质量比例为1:8-12；所述的壳聚糖微球粒径在80-120 μm，在直径为8 mm厚为2 mm的每压片体积内包含2000-8000个壳聚糖微球；
壳聚糖微球的制备方法包括如下步骤：
称取壳聚糖、冰醋酸溶于纯水或超纯水中配置成溶液A，其中，壳聚糖与水的比例为0.05-0.5g/10ml，冰醋酸与水的体积比为0.005-0.05:1；
称取司班80、吐温60溶于石油醚中配置成溶液B, 其中，司班80与石油醚的比例为1-5g/50ml, 吐温60与石油醚的比例为0.1-0.5g/50ml；
称取氢氧化钠、超纯水溶于无水乙醇中配置成溶液C，其中，氢氧化钠与无水乙醇的比例为0.5-5g/50ml，水与无水乙醇的体积比为1:10-15；
将溶液A缓慢滴加入溶液B，A与B体积比为1:4-6；并在30-50 ℃，500-2000 rpm下搅拌1-10 h，形成溶液D；
溶液D经液氮冷却后加入溶液C，溶液C与溶液D的体积比为1:1-2； 并在室温，100-1000rpm下搅拌1-3 h获得壳聚糖微球。
2.根据权利要求1所述的壳聚糖微球压片，其特征在于：崩解剂包括淀粉、交联聚乙烯吡咯烷酮、泡腾片和交联羧甲基纤维素钠中的至少一种。
3. 根据权利要求1所述的壳聚糖微球压片，其特征在于：所述的壳聚糖微球原料压片所用的压制力为2-5 kN。
4.根据权利要求1所述的壳聚糖微球压片，其特征在于：所述的壳聚糖的分子量范围为2万-30万。
5.如权利要求1-4任一项所述的壳聚糖微球压片在细胞三维培养中的应用。
6.如权利要求1-4任一项所述的壳聚糖微球压片的使用方法，将其放入培养基后快速分散成壳聚糖微球。
7.如权利要求6所述的壳聚糖微球压片的使用方法，细胞培养之后，加入溶菌酶降解壳聚糖微球。
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