CN115463153A - 一种MSCs微球滴眼液的制作方法 - Google Patents
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Abstract
本发明公开了一种MSCs微球滴眼液的制作方法,属于眼表疾病治疗药物技术领域,针对了目前MSCs应用于眼表的方式包括结膜下注射,泪腺注射,全身注射等,但有诸多弊端,如给药效率低、眼局部操作后疼痛难忍、眼周组织压力增高以及首过效应导致到达眼表效率低的问题,包括以下步骤:S1:MSCs的收集:取2‑5代的人脐带间充质干细胞,待细胞培养至60‑75%融合后,弃除上清,使用PBS清洗后,用胰酶将贴壁MSCs消化分散;本发明包裹MSCs的海藻酸钠微球在眼表缓慢降解,起到缓释作用,解决了MSCs在眼表穿透性差,代谢较快等问题,提高了MSCs在眼表的治疗效率,实现了微球中的MSCs长时间通过旁分泌发挥抗炎和免疫调节作用。
Description
技术领域
本发明属于眼表疾病治疗药物技术领域,具体涉及一种MSCs微球滴眼液的制作方法。
背景技术
间充质干细胞MSCs是一种存在于许多成人和胎儿组织中的多能干细胞,已被证实有高效的再生修复功能以及强大的免疫调节作用。近年来已有大量研究表明间充质干细胞(MSCs)具有治疗多种炎症性疾病的临床潜能,MSCs也逐渐被应用于眼表疾病治疗,据报道MSCs移植于眼部组织可改善干眼病、无菌性角膜溃疡、眼表烧伤等眼病的临床症状,降低眼表炎性水平。因此MSCs在眼表的应用方式对其疗效有极大的影响。
目前MSCs 应用于眼表的方式包括结膜下注射,泪腺注射,全身注射等,但有诸多弊端,如给药效率低、眼局部操作后疼痛难忍、眼周组织压力增高以及首过效应导致到达眼表效率低,滴眼剂是眼科常用的剂型,药物可直接接触角膜、结膜的病灶,高浓度快速起效,副作用少,因此MSC滴眼可能是一种较为理想的治疗方式,然而普通剂型滴眼液中的MSCs在眼表难以穿过泪膜屏障,不能在眼表稳定停留发挥抗炎作用,因此需要提高MSCs在眼表的生物利用度。
为此,我们提出了一种MSCs微球滴眼液的制作方法。
发明内容
本发明的目的在于提供一种MSCs微球滴眼液的制作方法,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:一种MSCs微球滴眼液的制作方法,包括以下步骤:
S1:MSCs的收集:取2-5代的人脐带间充质干细胞,待细胞培养至60-75%融合后,弃除上清,使用PBS清洗后,用胰酶将贴壁MSCs消化分散,使用含有9-12%胎牛血清和0.8-1.1%抗生素的F12/DMEM完全培养基终止消化后收集细胞悬液;
S2:负载MSCs微球海藻酸钠水凝胶的制备:将脐带间充质干细胞MSCs细胞悬液混合于0.9-1.2%w/v的海藻酸钠水凝胶海藻酸钠水凝胶中,再通过电喷微流控技术,在高电压的作用下将混合溶液喷射到氯化钙溶液中,获得包裹细胞的微凝胶液滴,形成MSCs微球海藻酸钠水凝胶;
S3:MSCs微球滴眼液的制备:将S2中所制MSCs微球海藻酸钠水凝胶使用PH调节剂将PH调至6.0-7.5,使用NaCl将渗透压调至300mOsm/L,形成MSCs微球滴眼液。
方案中需要说明的是:所述S1中采用1-4℃预冷的离心机进行离心操作弃除上清,其中离心力设置为500-800g,离心实际为5-9分钟。
作为一种优选的实施方式,所述S1中经过离心弃除上清后,获得的MSCs细胞沉淀使用PBS重悬,其中细胞密度为1-2*106个/ml。
作为一种优选的实施方式,所述S2中高电压得电压为3-10kV,其中S2中的电喷微流控技术流速为2-8ml/min。
作为一种优选的实施方式,所述S2中的微球海藻酸钠水凝胶的尺寸为140±10μm。
与现有技术相比,本发明提供的MSCs微球滴眼液的制作方法,至少包括如下有益效果:
(1)本发明为外用滴眼液,与现有的MSCs在眼部使用方式相比伤害小,安全性高,副作用小,使用方便;
(2)本发明包裹MSCs的海藻酸钠微球在眼表缓慢降解,起到缓释作用,解决了MSCs在眼表穿透性差,代谢较快等问题,提高了MSCs在眼表的治疗效率,实现了微球中的MSCs长时间通过旁分泌发挥抗炎和免疫调节作用;
(3)本发明选用海藻酸钠包裹MSCs制备微球,海藻酸钠是一种天然多糖,具有药物制剂辅料所需的稳定性、溶解性、粘性和安全性,它可以在体内完全代谢,无毒无害,且本产品PH与渗透压接近人体泪液,使用时无眼表不适及异物感,本发明不含防腐剂,长期使用对眼表组织也没有刺激伤害。
附图说明
图1为本发明的电镜下的MSCs微球示意图;
图2为本发明的光镜下的MSCs微球示意图;
图3为本发明的MSCs微球滴眼液可缓解干眼小鼠临床症状示意图;
图4为本发明的MSC微球滴眼治疗可降低眼周TH17相关的炎症水平示意图。
具体实施方式
下面结合实施例对本发明做进一步的描述。
为了使得本公开实施例的目的、技术方案和优点更加清楚,下面将结合本公开实施例的附图,对本公开实施例的技术方案进行清楚、完整地描述,显然,所描述的实施例是本公开的一部分实施例,而不是全部的实施例,基于所描述的本公开的实施例,本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例,都属于本公开保护的范围。
除非另外定义,本公开使用的技术术语或者科学术语应当为本公开所属领域内具有一般技能的人士所理解的通常意义,本公开中使用的“包括”或者“包含”等类似的词语意指出现该词前面的元件或者物件涵盖出现在该词后面列举的元件或者物件及其等同,而不排除其他元件或者物件,“连接”或者“相连”等类似的词语并非限定于物理的或者机械的连接,还可以包括电性的连接,不管是直接的还是间接的,“上”、“下”、“左”、“右”等仅用于表示相对位置关系,当被描述对象的绝对位置改变后,则该相对位置关系也可能相应地改变。
以下实施例用于说明本发明,但不能用来限制本发明的保护范围。实施例中的条件可以根据具体条件做进一步的调整,在本发明的构思前提下对本发明的方法简单改进都属于本发明要求保护的范围。
请参阅图1-4,本发明提供一种MSCs微球滴眼液的制作方法,包括以下步骤:
S1:MSCs的收集:取2-5代的人脐带间充质干细胞,待细胞培养至60-75%融合后,弃除上清,使用PBS清洗后,用胰酶将贴壁MSCs消化分散,使用含有9-12%胎牛血清和0.8-1.1%抗生素的F12/DMEM完全培养基终止消化后收集细胞悬液;
S2:负载MSCs微球海藻酸钠水凝胶的制备:将脐带间充质干细胞MSCs细胞悬液混合于0.9-1.2%w/v的海藻酸钠水凝胶海藻酸钠水凝胶中,再通过电喷微流控技术,在高电压的作用下将混合溶液喷射到氯化钙溶液中,获得包裹细胞的微凝胶液滴,形成MSCs微球海藻酸钠水凝胶;
S3:MSCs微球滴眼液的制备:将S2中所制MSCs微球海藻酸钠水凝胶使用PH调节剂将PH调至6.0-7.5,使用NaCl将渗透压调至300mOsm/L,形成MSCs微球滴眼液。
方案中需要说明的是:S1中采用1-4℃预冷的离心机进行离心操作弃除上清,其中离心力设置为500-800g,离心实际为5-9分钟,S1中经过离心弃除上清后,获得的MSCs细胞沉淀使用PBS重悬,其中细胞密度为1-2*106个/ml。
S2中高电压得电压为3-10kV,其中S2中的电喷微流控技术流速为2-8ml/min,S2中的微球海藻酸钠水凝胶的尺寸为140±10μm。
附图1是将MSCs混合于海藻酸钠水凝胶中,通过电喷微流控技术制备了海藻酸盐-MSCs微球,海藻酸钠微球尺寸为225±3.5μm的图像;
附图2是将MSCs混合于海藻酸钠水凝胶中,通过电喷微流控技术制备了海藻酸盐-MSCs微球,如图所示海藻酸钠微球中包裹着MSCs的图像;
附图3是将小鼠分为空白对照组、干眼组、MSC滴眼治疗组、PBS滴眼治疗组,治疗7天,分别在第4和第7天检测各组小鼠的临床指标A.各组小鼠基础泪液分泌的变化,可见MSC治疗组泪液水平回升接近正常水平B.各组小鼠眼表荧光素钠评分的变化,评分与眼表损伤程度正相关。可见MSC治疗组眼表FLS评分明显下降,眼表损伤减少。动物实验证明干眼小鼠使用外泌体凝胶后眼表炎症水平明显下降的图像;
附图4是将干眼病小鼠分为干眼病组、PBS滴眼治疗组、MSC滴眼治疗组,治疗7天后分别取角膜和引流淋巴结,消化成单细胞悬液后进行TH17的流式抗体染色,然后上机分析TH17细胞比例。A.各组角膜TH17流式代表图像B.各组淋巴结TH17流式的图像。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (5)
1.一种MSCs微球滴眼液的制作方法,其特征在于,包括以下步骤:
S1:MSCs的收集:取2-5代的人脐带间充质干细胞,待细胞培养至60-75%融合后,弃除上清,使用PBS清洗后,用胰酶将贴壁MSCs消化分散,使用含有9-12%胎牛血清和0.8-1.1%抗生素的F12/DMEM完全培养基终止消化后收集细胞悬液;
S2:负载MSCs微球海藻酸钠水凝胶的制备:将脐带间充质干细胞MSCs细胞悬液混合于0.9-1.2%w/v的海藻酸钠水凝胶海藻酸钠水凝胶中,再通过电喷微流控技术,在高电压的作用下将混合溶液喷射到氯化钙溶液中,获得包裹细胞的微凝胶液滴,形成MSCs微球海藻酸钠水凝胶;
S3:MSCs微球滴眼液的制备:将S2中所制MSCs微球海藻酸钠水凝胶使用PH调节剂将PH调至6.0-7.5,使用NaCl将渗透压调至300mOsm/L,形成MSCs微球滴眼液。
2.根据权利要求1所述的一种MSCs微球滴眼液的制作方法,其特征在于:所述S1中采用1-4℃预冷的离心机进行离心操作弃除上清,其中离心力设置为500-800g,离心实际为5-9分钟。
3.根据权利要求2所述的一种MSCs微球滴眼液的制作方法,其特征在于:所述S1中经过离心弃除上清后,获得的MSCs细胞沉淀使用PBS重悬,其中细胞密度为1-2*106个/ml。
4.根据权利要求3所述的一种MSCs微球滴眼液的制作方法,其特征在于:所述S2中高电压得电压为3-10kV,其中S2中的电喷微流控技术流速为2-8ml/min。
5.根据权利要求4所述的一种MSCs微球滴眼液的制作方法,其特征在于:所述S2中的微球海藻酸钠水凝胶的尺寸为140±10μm。
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