CN110699322B - 一种肿瘤细胞三维培养基及其制备方法 - Google Patents
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Abstract
一种肿瘤细胞三维培养基,其制备方法包括以下步骤:将改性壳聚糖溶于30‑35℃的蒸馏水,调节pH到7‑8,经超声处理10‑24h,然后按比例加入交联剂、引发剂、乙醇搅拌反应30‑60min,停止搅拌后在50‑60℃下继续反应2‑5h,得到培养基;所述改性壳聚糖是将壳聚糖溶液与N‑异丙基丙烯酰胺或N,N‑亚甲基双丙烯酰胺溶液按照1.5‑5:1的摩尔比混合,调节pH值调节到3‑6,在25‑35℃下反应1‑10h得到改性壳聚糖溶液,改性壳聚糖溶液在60‑80℃下经真空脱水干燥,再经去离子水清洗得到改性壳聚糖。该培养基具有良好的生物相容性,对细胞无毒副作用,机械性能好,便于进行动物细胞实验异种移植,便于对细胞进行后续的分析。
Description
技术领域
本发明属于生物医学材料领域,具体涉及一种肿瘤细胞三维培养基及其制备方法。
背景技术
肿瘤从产生到发展到转移的过程到目前为止仍然有大量的关键环节未被揭示,由于这个过程难以直观的被观察记录,因此在动物模型以外,传统体外细胞培养模型在肿瘤研究中有着广泛的应用。在传统二维培养中的肿瘤细胞可以被视为单细胞的集合,细胞之间的相互作用较弱,不能进一步形成接近组织的二级结构。而在三维细胞培养中,多细胞球中的细胞之间有较强的相互作用,同时形成的球形结构中的细胞存在养分,细胞周期,代谢产物,氧气及凋亡等一系列的梯度差异,这种差异为进一步研究肿瘤细胞的增殖,活性,能量代谢过程,转移侵袭行为,细胞间相互作用等提供了新的平台。
此外,肿瘤药物在体外的评价对于肿瘤治疗有着至关重要的意义,传统二维细胞培养为药物筛选提供了大量的有效反馈,但很多药物被用于动物模型或者临床实验后发现效果并不理想。近年来,针对癌细胞的体外三维培养成为研究癌细胞分子生物学生理病理学以及药物评价和治疗等工作提供一个新的平台。虽然动物模型可以更好的模拟肿瘤在体内的生长条件,但是由于异源性等因素造成在动物模型中研究获得结果与应用到临床后存在一定差异,同时难以进行实时观察等问题也给研究造成一定的负面影响。
体外三维培养模型在癌细胞方面的应用具有良好的前景。Bisell等人通过构建三维细胞培养微环境使得在二维培养中获得的人乳腺癌细胞正常化等一系列研究都表明有必要建立三维培养基质来作为二维传统培养的补充,而单纯构建一个仅具有三维结构的惰性支架研究细胞基因表达与功能的联系而不研究支架本身与细胞之间作用的做法正在逐渐被淘汰,通过模拟细胞微环境设计材料,使细胞的各种信号通路在时间和空间维度上保持完整,从而使细胞表型更接近体内正成为研究的主流方向之一。
较低的细胞毒性和较好的细胞相容性是材料用于细胞培养的先决条件,而材料的化学特性对这两方面起决定性作用。细胞生长需要合适的pH值,电性及离子浓度等,当这些指标偏离正常范围,细胞可能会启动程序性死亡或凋亡;材料的亲疏水性影响细胞在其表面的粘附,不少研究表明太过亲水或疏水的材料都不适合细胞生长。
在体外三维细胞培养模型上获得的信息对化疗,放疗,基于药物/细胞的免疫治疗,基因治疗和光动力治疗的指导作用受到越来越多的重视,其在生物医学方面主要应用分为两个方面:第一,研究肿瘤的产生发展机制,为肿瘤治疗寻找新的靶点,为新的肿瘤诊断和治疗方法提供设计思路;第二,为肿瘤治疗手段改进提供另一个视角的反馈,与高通量药物筛选结合,缩短药物及其他治疗手段的研发应用周期。
水凝胶可以作为体外细胞培养基质或者组织工程植入材料。作为细胞培养或组织工程支架的水凝胶可分为来源于天然材料的水凝胶和源于人工合成材料的水凝胶。用于细胞培养的来源于天然材料的水凝胶通常有胶原蛋白I型(collagentype I)、纤连蛋白(fibronectin)、层粘连蛋白(laminin)及透明质酸(hyaluronic acid)等,由于这些水凝胶来源于生物体,因此具有和细胞相互作用力强、具有一定的被细胞重构能力,生长在其中的细胞可以进行迁移,但同时由于这些材料的提取方法所限,导致来源于天然材料的水凝胶其力学性能(弹性模量)较差,虽然可以通过物理、化学或生物的方法进行改善,但是仍然存在局限性,不菲的价格、批次间的差异、较难进一步改性等也限制了源于天然材料水凝胶的应用。
CN102206583 A公开了一种细胞共培养用芯片及共培养方法;该芯片由2~99个相同的细胞培养结构构成,这些培养结构之间通过隔板结构相互分隔;其细胞共培养方法为将细胞悬液加入细胞培养结构中,放入细胞培养箱中培养,然后将芯片置于显微镜下观察细胞的贴壁及生长情况;所述芯片利用了隔板的物理阻挡作用,形成不同的细胞培养区域,只需在不同的培养区域接种不同的细胞即可实现细胞的共培养,操作简便,实验重复性高;该芯片可用于检测细胞共培养后的多种指标。
CN102399693 A公开了一种仿真三维细胞培养器。由两侧紧密嵌套的内容器和外容器组成基本结构单元,所述内容器和外容器由用于细胞培养的有机材料制成;内容器和外容器呈圆柱状,内容器的底部由渗透支持膜构成且与外容器的底部不连接;所述外容器的底部设有一排液口;上述基本结构单元的单个或多个组成仿真三维细胞培养器。该培养器可以模拟体内肿瘤生理环境,可用来检测筛选新型抗肿瘤药物的效果或检测肿瘤细胞对不同抗肿瘤药物敏感性,考虑了肿瘤细胞的营养供应、代谢废物排除、血管及血管内皮细胞的因素,接近于体内肿瘤的生长环境。
CN102898660 A公开了一种用于肿瘤细胞三维培养的水凝胶,应用聚甲基乙烯基醚马来酸酐水解产物、甲基乙烯基醚马来酸共聚物和交联剂聚乙二醇、聚乙交酯丙交酯-聚乙二醇-聚乙交酯丙交酯三嵌段共聚物、聚-ε-己内酯丙交酯-聚乙二醇-聚-ε-己内酯丙交酯三嵌段共聚物或葡聚糖进行交联反应,通过调整甲基乙烯基醚/马来酸共聚物中羧基和交联剂中羟基的比例,获得不同交联密度的凝胶,交联后凝胶中多余的羧基再用聚乙二醇单甲醚进行酯化,最后将获得的凝胶配成5%的水溶液,再用弱碱,如饱和碳酸氢钠水溶液进行中和,至pH=7.0,制得可适合用作肿瘤细胞三维培养基质的水凝胶。
CN106148285 A公布了一种新型的用于肿瘤细胞三维培养的基质,该种基质是通过海藻酸钠溶液、透明质酸钠溶液及细胞外培养基质Matrigel以适当的比例混合,然后包裹细胞。所述基质具有较好的机械性能,同时有效促进体外培养细胞组织结构的形成,减少甚至替代Matrigel的使用,大大降低了肿瘤细胞三维培养的成本,便于对三维细胞进行异种移植和动物实验,且制作方法简单,实用性强。
CN107794243 A公开了一种体外培养或扩增人上皮肿瘤细胞的方法及培养基,其包含:(a)饲养细胞;和(b)确定成分的细胞培养基,其包含至少一种Rho相关的含螺旋线圈的蛋白激酶(ROCK)抑制剂,并且不含或基本不含骨形态生成蛋白(BMP)抑制剂和Wnt/β-连环蛋白信号通路激动剂。该培养方法及培养基能够富集上皮肿瘤细胞,降低基质细胞及正常上皮细胞生长,并且维持原始肿瘤样品的克隆多样性。
发明内容
基于现有技术的不足,本发明提供过一种具有广泛细胞适应性,高强度,对温度和pH敏感的三维细胞培养用水凝胶。本申请通过对壳聚糖的预处理,使得壳聚糖分子中嵌入具有一定给交联活性的N-异丙基丙烯酰胺或N,N-亚甲基双丙烯酰胺分子,然后再与低分子量的聚甲基丙烯酸混合,在引发剂添加下,发生较为复杂的交联反应,生成一种复合结构的水凝胶,在交联的过程中,加入一定量的乙醇,可以中和未反应的羧酸分子,并加强水凝胶的骨架强度,提高其机械性能。
所述壳聚糖的改性处理是将壳聚糖中的氨基部分质子化,然后用改性剂使得壳聚糖中的氨基部分与N-异丙基丙烯酰胺之间形成弱化学键,使得壳聚糖分子中出现更多可以进行反应的活性基团,进一步提高壳聚糖的交联活性。聚壳聚糖上氨基带有正电荷,聚甲基丙烯酸上的羧基带有负电荷,壳聚糖和丙烯酸通过静电相互作用连接在一起。引发剂产生自由基,引发N-异丙基丙烯酰胺和甲基丙烯酸酸上的双键发生共同聚合,从而缠绕生成水凝胶的三维网络结构。
一种肿瘤细胞三维培养基,其制备方法包括以下步骤:将改性壳聚糖溶于30-35℃的蒸馏水,调节pH到7-8,经超声处理10-24h,然后按比例加入交联剂、引发剂、乙醇搅拌反应30-60min,停止搅拌后在50-60℃下继续反应2-5h,得到培养基;所述改性壳聚糖是将壳聚糖溶液与N-异丙基丙烯酰胺或N,N-亚甲基双丙烯酰胺溶液按照1.5-5:1的摩尔比混合,调节pH值调节到3-6,在25-35℃下反应1-10h得到改性壳聚糖溶液,改性壳聚糖溶液在60-80℃下经真空脱水干燥,再经去离子水清洗得到改性壳聚糖。
所述交联剂是聚甲基丙烯酸溶液,优选是浓度为0.1-1wt%的聚甲基丙烯酸溶液;所述交联剂用量是壳聚糖质量的10-30%,所述引发剂是过硫化物,优选是过硫酸钾、过硫酸铵或过硫酸钠;所述引发剂是改性壳聚糖质量的0.5-2%;所述乙醇的加入量是聚甲基丙烯酸质量的10-50%。
所述聚甲基丙烯酸是低分子量聚甲基丙烯酸,其重均分子量为1000-5000,优选是1000-3000。
所述培养基中还包含用于细胞培养的细胞外基质、生长因子和其它营养成分。
本发明的另一方面,还公开了所述水凝胶用于肿瘤细胞培养的用途。
本发明的有益效果:(1)具有良好的生物相容性,对细胞无毒副作用,环境友好,价格较低,降低了细胞三维培养的成本;(2)机械性能好,便于进行动物细胞实验异种移植,便于对细胞进行后续的分析;(3)具有广泛的细胞适应性,适应各种上皮细胞肿瘤细胞的培养。
以下结合实施例对本发明作出进一步描述。
实施例1
所述改性壳聚糖是将壳聚糖溶液与N-异丙基丙烯酰胺溶液按照1.5:1的摩尔比混合,调节pH值调节到3,在25℃下反应10h得到改性壳聚糖溶液,改性壳聚糖溶液在80℃下经真空脱水干燥,再经去离子水清洗得到改性壳聚糖。
肿瘤细胞三维培养基的制备,包括以下步骤:将改性壳聚糖溶于30℃的蒸馏水,调节pH到8,经超声处理10h,然后按比例加入交联剂、引发剂、乙醇,搅拌反应30min,停止搅拌后在60℃下继续反应5h,得到培养基;所述交联剂是1wt%的聚甲基丙烯酸溶液;所述交联剂用量是壳聚糖质量的20%,所述引发剂是过硫酸钾;所述引发剂是改性壳聚糖质量的0.5%,所述乙醇用量是聚甲基丙烯酸质量的20%。
实施例2
所述改性壳聚糖是将壳聚糖溶液与N-异丙基丙烯酰胺或N,N-亚甲基双丙烯酰胺溶液按照3:1的摩尔比混合,调节pH值调节到6,在35℃下反应10h得到改性壳聚糖溶液,改性壳聚糖溶液在60-80℃下经真空脱水干燥,再经去离子水清洗得到改性壳聚糖。
肿瘤细胞三维培养基的制备:包括以下步骤:将改性壳聚糖溶于30℃的蒸馏水,调节pH到7,经超声处理24h,然后按比例加入交联剂、引发剂、乙醇,搅拌反应30min,停止搅拌后在60℃下继续反应5h,得到培养基;
所述交联剂是所述交联剂是0.5wt%的聚甲基丙烯酸溶液;所述交联剂用量是壳聚糖质量的30%,所述引发剂是过硫酸铵;所述引发剂是改性壳聚糖质量的2%,所述乙醇用量是聚甲基丙烯酸质量的30%。
实施例3
所述改性壳聚糖是将壳聚糖溶液与N-异丙基丙烯酰胺溶液按照5:1的摩尔比混合,调节pH值调节到3,在25℃下反应8h得到改性壳聚糖溶液,改性壳聚糖溶液在60℃下经真空脱水干燥,再经去离子水清洗得到改性壳聚糖。
肿瘤细胞三维培养基,其制备方法包括以下步骤:将改性壳聚糖溶于33℃的蒸馏水,调节pH到7,经超声处理18h,然后按比例加入交联剂、引发剂、乙醇,搅拌反应60min,停止搅拌后在50℃下继续反应5h,得到培养基;
所述交联剂是所述交联剂是0.2wt%的聚甲基丙烯酸溶液;所述交联剂用量是壳聚糖质量的20%,所述引发剂是过硫酸钠;所述引发剂是改性壳聚糖质量的1%,所述乙醇用量是聚甲基丙烯酸质量的50%。
对比例1
不对壳聚糖进行改性,其他步骤与实施例1相同。
肿瘤细胞三维培养基,其制备方法包括以下步骤:将改性壳聚糖溶于33℃的蒸馏水,调节pH到7,经超声处理18h,然后按比例加入交联剂、引发剂、乙醇,搅拌反应60min,停止搅拌后在50℃下继续反应5h,得到培养基。
对比例2
采用常规的甲基丙烯酸作为交联剂,其他步骤与实施例1相同。
所述改性壳聚糖是将壳聚糖溶液与N-异丙基丙烯酰胺溶液按照1.5:1的摩尔比混合,调节pH值调节到3,在25℃下反应10h得到改性壳聚糖溶液,改性壳聚糖溶液在80℃下经真空脱水干燥,再经去离子水清洗得到改性壳聚糖。
肿瘤细胞三维培养基的制备,包括以下步骤:将改性壳聚糖溶于30℃的蒸馏水,调节pH到8,经超声处理10h,然后按比例加入交联剂、引发剂,搅拌反应30min,停止搅拌后在60℃下继续反应5h,得到培养基;所述交联剂是1wt%的聚甲基丙烯酸溶液;所述交联剂用量是壳聚糖质量的20%,所述引发剂是过硫酸钾;所述引发剂是改性壳聚糖质量的0.5%。
对比例3
不加入乙醇,其他步骤与对比例1相同。
所述改性壳聚糖是将壳聚糖溶液与N-异丙基丙烯酰胺溶液按照1.5:1的摩尔比混合,调节pH值调节到3,在25℃下反应10h得到改性壳聚糖溶液,改性壳聚糖溶液在80℃下经真空脱水干燥,再经去离子水清洗得到改性壳聚糖。
肿瘤细胞三维培养基的制备,包括以下步骤:将改性壳聚糖溶于30℃的蒸馏水,调节pH到8,经超声处理10h,然后按比例加入交联剂、引发剂,搅拌反应30min,停止搅拌后在60℃下继续反应5h,得到培养基;所述交联剂是1wt%的聚甲基丙烯酸溶液;所述交联剂用量是壳聚糖质量的20%,所述引发剂是过硫酸钾;所述引发剂是改性壳聚糖质量的0.5%。
对实施例1-3和对比例1-3得到的水凝胶进行测试,结果如下表所示:
最大拉伸断裂强度(KPa) | 断裂应变(%) | 肝细胞球成活率(%) | 肺细胞球成活率(%) | |
实施例1 | 1260 | 1625 | 95 | 97 |
实施例2 | 1272 | 1561 | 96 | 97 |
实施例3 | 1269 | 1606 | 95 | 95 |
对比例1 | 1013 | 1192 | 82 | 80 |
对比例2 | 582 | 711 | 75 | 78 |
对比例3 | 821 | 1235 | 69 | 78 |
所述细胞球成活率实验是将分离出的肿瘤组织上皮细胞铺于已准备好的滋养层细胞上,用水凝胶培养基在35℃-38℃,3%-10%CO2条件下培养7天后进行观察。
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 (6)
1.一种肿瘤细胞三维培养基,其制备方法包括以下步骤:将改性壳聚糖溶于30-35℃的蒸馏水,调节pH到7-8,经超声处理10-24h,然后按比例加入交联剂、引发剂、乙醇搅拌反应30-60min,停止搅拌后在50-60℃下继续反应2-5h,得到培养基;所述改性壳聚糖是将壳聚糖溶液与N-异丙基丙烯酰胺或N,N-亚甲基双丙烯酰胺溶液按照1.5-5:1的摩尔比混合,调节pH值调节到3-6,在25-35℃下反应1-10h得到改性壳聚糖溶液,改性壳聚糖溶液在60-80℃下经真空脱水干燥,再经去离子水清洗得到改性壳聚糖;所述交联剂是浓度为0.1-1wt%的聚甲基丙烯酸溶液;所述交联剂用量是壳聚糖质量的10-30%;所述引发剂是过硫化物,所述引发剂是改性壳聚糖质量的0.5-2%;所述乙醇的加入量是聚甲基丙烯酸质量的10-50%。
2.根据权利要求1所述的培养基,其特征在于所述引发剂是过硫酸钾、过硫酸铵或过硫酸钠。
3.根据权利要求1所述的培养基,其特征在于所述聚甲基丙烯酸是低分子量聚甲基丙烯酸,其重均分子量为1000-5000。
4.根据权利要求3所述的培养基,其特征在于所述聚甲基丙烯酸的重均分子量为1000-3000。
5.根据权利要求1所述的培养基,其特征在于还包含用于细胞培养的细胞外基质、生长因子和其它营养成分。
6.权利要求1-5中任一项所述培养基用于肿瘤细胞培养的用途。
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