CN111375361B - 一种用于大规模干细胞培养的纳米海藻糖3d微胶囊 - Google Patents
一种用于大规模干细胞培养的纳米海藻糖3d微胶囊 Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种用于大规模干细胞培养的纳米海藻糖3D微胶囊,该3D微胶囊的制备方法包括以下步骤:(1)将重量百分比为1‑6wt%的海藻酸钠溶液与重量百分比为0.1‑5wt%的明胶溶液以0.1‑2:0.1‑2的比例混合均匀,制得第一混合溶液;(2)在第一混合溶液中加入起泡剂混合均匀,起泡剂在第一混合溶液中的浓度为5‑100g/L,制得第二混合溶液;(3)在第二混合溶液中滴加CaCl2溶液,CaCl2溶液与第一混合溶液的体积比为1‑2:1‑2,制得微胶囊溶液;(4)将微胶囊溶液用生理盐水洗涤3‑6次,再进行真空干燥,制得干燥微胶囊;(5)将干燥微胶囊压扁、灭菌,即得3D微胶囊;本发明的3D微胶囊使用方便、解囊简单,在5天可使细胞增殖500倍左右,7天可使细胞增殖1000倍左右,收获时细胞活率能达到90%以上。
Description
技术领域
本发明属于干细胞技术领域,特别涉及一种用于大规模干细胞培养的纳米海藻糖3D微胶囊。
背景技术
干细胞是一类具有多向分化潜能和自我复制能力的原始的未分化细胞,是形成哺乳类动物的各组织器官的原始细胞,干细胞的用途非常广泛,涉及到医学的多个领域,人体的衰老、皱纹的出现,究其根源实质上都是细胞的衰老和减少,而细胞的衰老和减少则是由干细胞老化引起的,干细胞是各种组织细胞更新换代的种子细胞,是人体细胞的生产厂,干细胞族群的老化严重减弱了其增殖和分化的能力,新生的细胞补充不足,衰老细胞不能及时被替代,全身各系统功能下降,让人一天天老去,而皮肤也因为皮肤干细胞的衰老而无法及时更新,衰老的皮肤得不到修复,所以,有了皱纹,失去了青春容颜,干细胞美容原理是通过输注特定的多种细胞(包括各种干细胞和免疫细胞),激活人体自身的“自愈功能”,对病变的细胞进行补充与调控,激活细胞功能,增加正常细胞的数量,提高细胞的活性,改善细胞的质量,防止和延缓细胞的病变,恢复细胞的正常生理功能,从而达到疾病康复、对抗衰老的目的,但干细胞移植面临两大难题:干细胞移植成活率低和干细胞有效分化率低,这严重阻碍了干细胞治疗的进行。
目前国内外存在着一种微胶囊技术,可以使干细胞存活增殖,微胶囊技术很早就被应用在了细胞培养和移植领域,相较于传统培养方式,微胶囊所构建的三维微环境和真实机体环境更加相似,大量的研究表明这种体系可以为胚胎干细胞、骨髓间充质干细胞和造血干细胞等细胞类型提供良好的体外扩增环境。
微胶囊是以天然或合成高分子为材料制作的球形小囊,直径依不同需求可做成几到几千微米,微胶囊可以包封气体、固体或液体物质,可以使物质与环境隔绝而减少来自环境的影响,起到屏蔽物质颜色、味道和气味或控制释放活性物质等功能,构成微胶囊的材料被称为壁材,内部包囊的物质被称为芯材,起隔离作用的壁材依据其通透性可分为不透微胶囊和半透性微胶囊,不透微胶囊一般用于非生物领域,壁材厚,芯材与外部环境完全隔离,需要时往往通过机械或化学的方法使微胶囊破裂,芯材被释放,与环境发生作用,在生物医学领域则主要应用半透性微胶囊,半透性微胶囊一方面要保持其隔离性能,起到储库的作用,一些大分子不允许通过,又要保证一些小分子物质可以任意进出,微胶囊的膜厚度、表面结构、多孔性和芯材分布等物理性质是决定芯材缓释性能的主要因素。
随着微胶囊研究的不断发展,为了满足不同应用场合下的性能要求,人们开发了越来越多的成囊技术,更多的新材料也被陆续用于微胶囊的制备,目前生物医学领域研究最深入的材料为海藻酸钠、聚氨基酸和壳聚糖。
海藻酸钠被作为微胶囊制备材料而被广泛应用是因为它有一些独特性质,在水溶液状态下,单纯的海藻酸钠以溶胶的形式存在,而当其遇到Ca2+、Ba2+等二价阳离子时,原本与海藻酸根结合的Na+会被逐渐置换出来,形成一种既有一定强度又有一定弹性的凝胶结构。
根据专利CN201510920872,大连化物所曾研究过海藻酸盐—壳聚糖酰基衍生物微胶囊,这个胶囊的特点是强度较好,生物相容性好,但是通透性和降解性较差;主要问题是:(1)细胞很快在囊内死亡;(2)微胶囊降解困难。
发明内容
为了解决以上技术问题,本发明提供了一种用于大规模干细胞培养的纳米海藻糖3D微胶囊。
本发明具体技术方案如下:
本发明提供了一种用于大规模干细胞培养的纳米海藻糖3D微胶囊,该3D微胶囊的制备方法包括以下步骤:
(1)将重量百分比为1-6wt%的海藻酸钠溶液与重量百分比为0.1-5wt%的明胶溶液以0.1-2:0.1-2的比例混合均匀,制得第一混合溶液;
(2)在第一混合溶液中加入起泡剂混合均匀,起泡剂在第一混合溶液中的浓度为5-100g/L,制得第二混合溶液;
(3)在第二混合溶液中滴加CaCl2溶液,CaCl2溶液与第一混合溶液的体积比为1-2:1-2,制得微胶囊溶液;
(4)将微胶囊溶液用生理盐水洗涤3-6次,再进行真空干燥,制得干燥微胶囊;
(5)将干燥微胶囊压扁、灭菌,即得3D微胶囊。
其中,以上方法中的步骤(2)加入起泡剂后会形成具有丰富气泡结构的溶液;最终生成的微胶囊中富含气泡,本发明提供的微胶囊是直径大小为0.5-10毫米的蜂巢结构,微胶囊内部的孔径为10nm到10000nm,本发明提供的3D微胶囊支持多种贴附依赖性细胞(贴壁细胞)、兼性贴壁细胞等绝大多数细胞类型的高效生产和收获,包括:成纤维型细胞(内皮细胞、间充质细胞、成骨细胞、心肌细胞、软骨细胞等)、上皮型细胞(皮肤细胞、表皮衍生物、消化管上皮细胞等)、游走型细胞(巨噬细胞、肿瘤细胞等)、多形性型细胞(神经元细胞、神经胶质细胞等)等;本发明提供的3D微胶囊的使用方法如下:首先将微胶囊称重,按照1g对应10ml培养基的比例加入DMEM/F12培养基中静置4h完全溶胀。溶胀后将微胶囊转移到含10%FBS的DMEM/F12培养基中,按照5000-10000个细胞/ml的密度接种,采取波动培养的方式经行接种及培养,放置在37℃二氧化碳培养箱中培养,补充培养基,培养完成后,用溶解剂解囊,溶解剂为氨基酸+柠檬酸+柠檬酸钠+EDTA钠盐,其中,氨基酸包括甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、精氨酸和组氨酸中的任意一种,氨基酸、柠檬酸、柠檬酸钠、EDTA钠盐的比例:0.1-1.0:0.1-2.0:0.1-1.0:0.1-2.0,收获细胞,计数、对比,本发明提供的3D微胶囊可实现干细胞三维大规模增殖,生产收获过程无酶化、快速便捷,对干细胞基本没有伤害,如图1所示,先在3D微胶囊上接种干细胞,然后干细胞在3D微胶囊中大规模增殖,培养完成后进行解囊,收集干细胞即可。
进一步地,步骤(5)的将干燥微胶囊压扁是通过机械压扁的方法将干燥微胶囊压扁。
进一步地,步骤(5)的将干燥微胶囊压扁、灭菌是采用放射性同位素放射的γ射线杀灭微生物和芽孢,其中,辐射灭菌剂量为25000Gy。
进一步地,起泡剂包括氨基酸起泡剂、椰油起泡剂、柠檬酸、柠檬酸钠、碳酸钠和碳酸氢钠中的一种。
进一步地,步骤(3)真空干燥是将洗涤后的微胶囊用真空干燥机进行干燥。
进一步地,步骤(3)的CaCl2溶液的滴加速度为5-10s/d。
进一步地,步骤(3)的CaCl2溶液滴加前保持第二混合溶液的气泡大小稳定。
进一步地,CaCl2溶液的浓度为0.1-0.6mol/L。
本发明还提供了一种3D微胶囊的用途,用于三维培养大规模的干细胞。
本发明提供的3D微胶囊是一种水凝胶,能允许水和小分子通过,但孔径不足以让细胞通过,具有光学透明、直径0.5-10毫米的特性,使用本发明提供的3D微胶囊培养细胞,在5天可使细胞增殖500倍左右,7天可使细胞增殖1000倍左右,收获时细胞活率能达到90%以上;且溶胀时可直接用培养基进行溶胀,操作简单,收获后细胞的贴壁良好,细胞培养完成后,使用溶解剂即可进行解囊,操作简单、方便。
附图说明
图1.为3D微胶囊的培养细胞的流程图;
图2.为用TFF分离设备收集细胞的流程图;
图3.为实施例2的3D微胶囊溶胀后的显微镜下观察图;
图4.为实施例2的3D微胶囊溶胀后的显微镜下观察图;
图5.为使用实施例1的3D微胶囊培养细胞时细胞的增殖曲线;
图6.为使用实施例1的3D微胶囊培养细胞时细胞的增殖曲线;
图7.为使用实施例1的3D微胶囊培养细胞时细胞的增殖曲线;
图8.为使用新加坡ESCO商品化纸片载体培养细胞时细胞的增殖曲线;
图9.为使用实施例2的3D微胶囊培养细胞时细胞贴壁状态图;
图10.为使用新加坡ESCO商品化纸片载体培养细胞时细胞的贴壁状态图;
图11.为实施例2的3D微胶囊的实物图。
具体实施方式
实施例1
本实施例提供了一种用于大规模干细胞培养的纳米海藻糖3D微胶囊,该3D微胶囊的制备方法包括以下步骤:
(1)将10ml重量百分比为1wt%的海藻酸钠溶液与200ml重量百分比为0.1wt%的明胶溶液混合均匀,制得第一混合溶液;
(2)在第一混合溶液中加入1.05g椰油起泡剂混合均匀,制得第二混合溶液;
(3)待第二混合溶液的气泡大小稳定后,在第二混合溶液中滴加420ml浓度为0.1mol/L的CaCl2溶液,滴加速度为5s/d,制得微胶囊溶液;
(4)将微胶囊溶液用生理盐水洗涤3次,再进行真空干燥,制得干燥微胶囊;
(5)用机械压扁的方法将干燥微胶囊压扁,再采用放射性同位素放射的γ射线对干燥微胶囊进行灭菌,即得3D微胶囊;
其中,辐射灭菌剂量为25000Gy;
实施例1的微胶囊溶胀后的溶胀图见图3和图4。
实施例2
本实施例提供了一种用于大规模干细胞培养的纳米海藻糖3D微胶囊,该3D微胶囊的制备方法包括以下步骤:
(1)将100ml重量百分比为3wt%的海藻酸钠溶液与100ml重量百分比为2wt%的明胶溶液混合均匀,制得第一混合溶液;
(2)在第一混合溶液中加入10g氨基酸起泡剂混合均匀,制得第二混合溶液;
(3)待第二混合溶液的气泡大小稳定后,在第二混合溶液中滴加200ml浓度为0.35mol/L的CaCl2溶液,滴加速度为8s/d,制得微胶囊溶液;
(4)将微胶囊溶液用生理盐水洗涤4次,再进行真空干燥,制得干燥微胶囊;
(5)用机械压扁的方法将干燥微胶囊压扁,再采用放射性同位素放射的γ射线对干燥微胶囊进行灭菌,即得3D微胶囊;
其中,辐射灭菌剂量为25000Gy;
实施例2的微胶囊的实物图见图11。
实施例3
本实施例提供了一种用于大规模干细胞培养的纳米海藻糖3D微胶囊,该3D微胶囊的制备方法包括以下步骤:
(1)将200ml重量百分比为6wt%的海藻酸钠溶液与10ml重量百分比为5wt%的明胶溶液混合均匀,制得第一混合溶液;
(2)在第一混合溶液中加入21g氨基酸起泡剂混合均匀,制得第二混合溶液;
(3)待第二混合溶液的气泡大小稳定后,在第二混合溶液中滴加105ml浓度为0.6mol/L的CaCl2溶液,滴加速度为10s/d,制得微胶囊溶液;
(4)将微胶囊溶液用生理盐水洗涤6次,再进行真空干燥,制得干燥微胶囊;
(5)用机械压扁的方法将干燥微胶囊压扁,再采用放射性同位素放射的γ射线对干燥微胶囊进行灭菌,即得3D微胶囊;
其中,辐射灭菌剂量为25000Gy。
试验例1
试验方法:在无菌条件下,分别取实施例1-3制得的3D微胶囊各5g倒入玻璃瓶中,加入50mlDMEM/F12培养基搅拌均匀,静置4小时,将溶胀后的微胶囊转移到50ml含10%FBS的DMEM/F12培养基中,按照8000个细胞/ml的密度接种间充质干细胞,将其放置于37℃二氧化碳培养箱中培养,培养条件为,每2-3天补液一次,去除50%DMEM/F12培养基后补充等体积新鲜DMEM/F12培养基,当细胞在可溶性微胶囊上达到约90%汇合度时,去除培养基,用与溶胀后的微胶囊等体积的溶解剂溶解载体(质量比为1:2:1:2的赖氨酸、柠檬酸、柠檬酸钠和EDTA钠盐)解囊15min,溶解后用TFF分离设备收集细胞,如图2所示,TFF是切向流过滤,它是一种压力驱动的,根据分子尺寸的膜分离过程,流体通过多次再循环的方式,切向通过膜的表面,降低初始样本在膜表面的积累,比膜截留分子量大的目标分子得到了保留,然而小分子和缓冲液通过了膜;吹打混匀细胞悬液,取样进行显微观察和细胞计数,并计算最终的细胞产量、存活率和增值率;取新加坡ESCO商品化纸片载体,按照说明进行细胞培养,测定其增值率,其中,细胞增值率可采用台盼蓝计数法进行测定,具体方法为:将负载细胞的微胶囊裂解后定容,取0.5ml细胞悬液放入小试管里,再加0.4%台酚蓝染液0.5ml,用吸管轻轻吹打混匀,染色3分钟,然后把悬液摇匀,吸取悬液滴在血球计数板上,蓝色细胞为死细胞;按以下公式计算细胞存活率,细胞活率=(细胞总数-死细胞数)/细胞总数。
实验结果:实施例1的3D微胶囊溶胀后的显微镜下观察图见图3,由实施例1-3的3D微胶囊培养细胞后的细胞增殖率见图5-7,使用新加坡商品化纸片载体培养细胞后细胞的增值率见图8,由图5-8可知,使用实施例1-3的3D微胶囊培养的细胞,增殖率在第6天达到1000倍以上,增殖率远远高于使用新加坡ESCO商品化纸片载体培养细胞的增值率,经测定,实施例1-3的3D微胶囊培养细胞后的细胞活性分别为94.8%、95.0%和96.7%;将实施例2的3D微胶囊解囊后,将细胞收集后重新贴壁,结果见图9,细胞贴壁状态良好;将新加坡ESCO商品化纸片载体进行贴壁,结果见图10。
综上,仅为本发明之较佳实施例,不以此限定本发明的保护范围,凡依本发明专利范围及说明书内容所作的等效变化与修饰,皆为本发明专利涵盖的范围之内。
Claims (10)
1.一种用于大规模干细胞培养的纳米海藻糖3D微胶囊,其特征在于,所述3D微胶囊的制备方法包括以下步骤:
(1)将重量百分比为1-6wt%的海藻酸钠溶液与重量百分比为0.1-5wt%的明胶溶液以0.1-2:0.1-2的比例混合均匀,制得第一混合溶液;
(2)在所述第一混合溶液中加入起泡剂混合均匀,所述起泡剂在所述第一混合溶液中的浓度为5-100g/L,制得第二混合溶液;
(3)在所述第二混合溶液中滴加CaCl2溶液,所述CaCl2溶液与所述第一混合溶液的体积比为1-2:1-2,制得微胶囊溶液;
(4)将所述微胶囊溶液用生理盐水洗涤3-6次,再进行真空干燥,制得干燥微胶囊;
(5)将所述干燥微胶囊压扁、灭菌,即得3D微胶囊。
2.如权利要求1所述的用于大规模干细胞培养的纳米海藻糖3D微胶囊,其特征在于,步骤(5)所述的将所述干燥微胶囊压扁是通过机械压扁的方法将所述干燥微胶囊压扁。
3.如权利要求1所述的用于大规模干细胞培养的纳米海藻糖3D微胶囊,其特征在于,步骤(5)所述的将所述干燥微胶囊压扁、灭菌是采用放射性同位素放射的γ射线杀灭微生物和芽孢,其中,辐射灭菌剂量为25000Gy。
4.如权利要求1所述的用于大规模干细胞培养的纳米海藻糖3D微胶囊,其特征在于,所述起泡剂包括氨基酸起泡剂、椰油起泡剂、柠檬酸、柠檬酸钠、碳酸钠和碳酸氢钠中的一种。
5.如权利要求1所述的用于大规模干细胞培养的纳米海藻糖3D微胶囊,其特征在于,步骤(3)的CaCl2溶液的滴加速度为5-10s/d。
6.如权利要求1所述的用于大规模干细胞培养的纳米海藻糖3D微胶囊,其特征在于,步骤(3)的CaCl2溶液的滴加速度为10s/d。
7.如权利要求1所述的用于大规模干细胞培养的纳米海藻糖3D微胶囊,其特征在于,步骤(3)的CaCl2溶液滴加前保持第二混合溶液的气泡大小稳定。
8.如权利要求1所述的用于大规模干细胞培养的纳米海藻糖3D微胶囊,其特征在于,所述CaCl2溶液的浓度为0.1-0.6mol/L。
9.一种应用于权利要求1所述的3D微胶囊的解囊剂,其特征在于,所述解囊剂包括质量比为0.1-1.0:0.1-2.0:0.1-1.0:0.1-2.0的氨基酸+柠檬酸+柠檬酸钠+EDTA钠盐,所述氨基酸包括甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、精氨酸和组氨酸中的任意一种。
10.一种权利要求1所述的用于大规模干细胞培养的纳米海藻糖3D微胶囊的应用,其特征在于,所述3D微胶囊用于三维培养大规模的干细胞。
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