CN106591219A - 一种表皮细胞培养基 - Google Patents
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Abstract
本发明属于细胞培养基技术领域,具体涉及一种表皮细胞培养基。本发明培养基主要由以下成分组成:基础培养基,胰岛素,谷氨酰胺,辅酶Q10,燕麦β‑葡聚糖,维生素A,维生素E,维生素C,表皮生长因子,纤维母细胞生长因子,转化生长因子‑β,亚硒酸钠,柠檬酸铁,硝酸铁,EDTA‑2Na,硫酸锌和硫酸钼。本发明培养基对表皮细胞生长有极大地促进作用,能使表皮细胞的凋亡速度减慢。本发明培养基成分简单、成本低廉,适合用于表皮细胞的大规模培养。
Description
技术领域
本发明属于细胞培养基技术领域,具体涉及一种表皮细胞培养基。
背景技术
人表皮细胞在创伤愈合中起至关重要的作用,尤其是严重烧伤病人表皮细胞的匮乏与其病程长短、并发症的发生、后期瘢痕的形成均有密切关系,因此应用组织工程技术在体外进行表皮细胞的分离、培养与保存并应用于临床始终都是烧伤工作者的一个研究热点。
传统的表皮培养基不仅需要昂贵的添加因子,而且其培养的表皮细胞扩增速度缓慢,在时间上不能满足烧烫伤治疗的需求,这个因素严重束缚了组织工程表皮用于急性皮肤创伤的治疗的临床应用。
中国专利申请(CN105950542A)提供了一种人皮肤表皮细胞培养基及其应用,本发明培养基将K-SFM及DMEM和F12培养基按照2:1:1混合,添加BPE、EGF、SCGF、FGF、TGF-β、VEGF、CaCl2、谷氨酰胺、双抗。此本发明培养基不含血清,可用于人皮肤表皮细胞原代及传代培养,但培养基成本昂贵。
因此,目前亟需一种能够促进表皮细胞快速扩增、成本低廉的表皮细胞培养基。
发明内容
为解决上述问题,本发明提供了一种表皮细胞培养基,其具有快速促进表皮细胞生长,成分简单,成本低廉的优点。
本发明通过以下技术方案实现:
一种表皮细胞培养基,主要由以下成分及其浓度组成:基础培养基15-20g/L,胰岛素20-30mg/L,谷氨酰胺5-10mg/L,辅酶Q1010-30μg/L,燕麦β-葡聚糖1-10mg/L,维生素A10-20mg/L,维生素E7-8mg/L,维生素C 1-3mg/L,表皮生长因子10-13μg/L,纤维母细胞生长因子50-100μg/L,转化生长因子-β1.5-5μg/L,亚硒酸钠1-10μg/L,柠檬酸铁1-1.5mg/L,硝酸铁0.5-0.8mg/L,EDTA-2Na5-8mg/L,硫酸锌1-3mg/L和硫酸钼0.1-0.5mg/L。
优选地,所述表皮细胞培养基由以下成分及其浓度组成:基础培养基18g/L,胰岛素25mg/L,谷氨酰胺7.5mg/L,辅酶Q1023μg/L,燕麦β-葡聚糖6.5mg/L,维生素A 13.5mg/L,维生素E 7.5mg/L,维生素C 2.5mg/L,表皮生长因子12.5μg/L,纤维母细胞生长因子75μg/L,转化生长因子-β4.5μg/L,亚硒酸钠8μg/L,柠檬酸铁1.35mg/L,硝酸铁0.75mg/L,EDTA-2Na 6.8mg/L,硫酸锌1.75mg/L和硫酸钼0.34mg/L。
优选地,所述基础培养基为Ham’s F12或DMEM-L。
本发明培养基成分及其浓度是经过大量实验筛选得到的,是一种组分配比合理、科学、有效的表皮细胞培养基。辅酶Q10(C59H90O4,CAS NO.303-98-0)是组成细胞线粒体呼吸链的成分之一,是传递电子、质子的递氢体,能激活细胞呼吸,加速产生ATP。燕麦β-葡聚糖是一种水溶性膳食纤维,有助于提高表皮活性细胞和促进表皮细胞的新陈代谢。维生素A是脂溶性的醇类物质,能够增强表皮细胞的新陈代谢和有丝分裂。维生素E是脂溶性维生素,为细胞膜上的重要组成成分,亦是细胞膜上的主要抗氧化剂,防止因细胞的损伤或酶失活而引起的脂肪酸氧化物的伤害。维生素C是一种抗氧化剂,因为它能够保护细胞免于氧化剂的威胁。表皮生长因子、转化生长因子-β和纤维母细胞生长因子分别是重要的促生长因子和激素,对表皮细胞生长具有促进作用。柠檬酸铁,硝酸铁和EDTA-2Na复合可以作为转铁蛋白的替代物,能够为细胞获取生长所需的铁。
表皮细胞培养基的制备方法,分为以下步骤:
(1)准确称取基础培养基、燕麦β-葡聚糖、亚硒酸钠,柠檬酸铁,硝酸铁,EDTA-2Na,硫酸锌,硫酸钼,溶于35℃,800mL超纯水中,搅拌均匀,得溶液I;
(2)将上述所得溶液I冷却至15-16℃,加入相应量的胰岛素,谷氨酰胺,辅酶Q10,维生素A,维生素E,维生素C,表皮生长因子,纤维母细胞生长因子,转化生长因子-β,加入200mL超纯水轻微搅拌至均匀,得溶液II;
(3)用1mol/L氢氧化钠溶液或1mol/L盐酸溶液调溶液II的pH至7.2,得溶液III;将溶液III用0.22μm无菌滤膜正压过滤除菌,即得。
本发明培养基与现有表皮细胞培养基相比,具有以下优点:
(1)本发明表皮细胞培养基能使表皮细胞快速增殖,并且表皮细胞的凋亡速度减慢,因而本发明培养基对表皮细胞生长有极大地促进作用;
(2)本发明培养基成分简单、成本低廉,适合用于表皮细胞的大规模培养。
附图说明
图1不同培养基中表皮细胞生长曲线:培养基分别为实施例1-3及对比例1制备得到的培养基。
具体实施方式
下面结合附图和实施例对本发明做进一步详细说明,但这并非是对本发明的限制,本领域技术人员根据本发明的基本思想,可以做出各种修改或改进,但是只要不脱离本发明的基本思想,均在本发明的范围之内。
燕麦β-葡聚糖购于陕西慈缘生物技术有限公司;其它均为常规药品。
实施例1一种表皮细胞培养基
一种表皮细胞培养基,由以下成分及其浓度组成:Ham’s F1218g/L,胰岛素25mg/L,谷氨酰胺7.5mg/L,辅酶Q1023μg/L,燕麦β-葡聚糖6.5mg/L,维生素A 13.5mg/L,维生素E7.5mg/L,维生素C 2.5mg/L,表皮生长因子12.5μg/L,纤维母细胞生长因子75μg/L,转化生长因子-β4.5μg/L,亚硒酸钠8μg/L,柠檬酸铁1.35mg/L,硝酸铁0.75mg/L,EDTA-2Na 6.8mg/L,硫酸锌1.75mg/L和硫酸钼0.34mg/L。
制备方法,分为以下步骤:
(1)称取基础培养基、燕麦β-葡聚糖、亚硒酸钠,柠檬酸铁,硝酸铁,EDTA-2Na,硫酸锌,硫酸钼,溶于35℃,800mL超纯水中,搅拌均匀,得溶液I;
(2)将上述所得溶液I冷却至15-16℃,加入相应量的胰岛素,谷氨酰胺,辅酶Q10,维生素A,维生素E,维生素C,表皮生长因子,纤维母细胞生长因子,转化生长因子-β,加入200mL超纯水轻微搅拌至均匀,得溶液II;
(3)用1mol/L氢氧化钠溶液或1mol/L盐酸溶液调溶液II的pH至7.2,得溶液III;将溶液III用0.22μm无菌滤膜正压过滤除菌,即得。
实施例2一种表皮细胞培养基
一种表皮细胞培养基,由以下成分及其浓度组成:DMEM-L 15g/L,胰岛素20mg/L,谷氨酰胺5mg/L,辅酶Q1010μg/L,燕麦β-葡聚糖1mg/L,维生素A10mg/L,维生素E 7mg/L,维生素C 1mg/L,表皮生长因子10μg/L,纤维母细胞生长因子50μg/L,转化生长因子-β1.5μg/L,亚硒酸钠1μg/L,柠檬酸铁1mg/L,硝酸铁0.5mg/L,EDTA-2Na 5mg/L,硫酸锌1mg/L和硫酸钼0.1mg/L。
制备方法与实施例1类似。
实施例3一种表皮细胞培养基
一种表皮细胞培养基,由以下成分及其浓度组成:DMEM-L 20g/L,胰岛素30mg/L,谷氨酰胺10mg/L,辅酶Q1030μg/L,燕麦β-葡聚糖10mg/L,维生素A20mg/L,维生素E 8mg/L,维生素C 3mg/L,表皮生长因子13μg/L,纤维母细胞生长因子100μg/L,转化生长因子-β5μg/L,亚硒酸钠10μg/L,柠檬酸铁1.5mg/L,硝酸铁0.8mg/L,EDTA-2Na 8mg/L,硫酸锌3mg/L和硫酸钼0.5mg/L。
制备方法与实施例1类似。
对比例1一种表皮细胞培养基
一种表皮细胞培养基,由以下成分及其浓度组成:Ham’s F1218g/L,胰岛素25mg/L,谷氨酰胺7.5mg/L,辅酶Q1023μg/L,葡萄糖6.5mg/L,维生素A13.5mg/L,维生素E 7.5mg/L,维生素C 2.5mg/L,表皮生长因子12.5μg/L,纤维母细胞生长因子75μg/L,转化生长因子-β4.5μg/L,亚硒酸钠8μg/L,柠檬酸铁1.35mg/L,硝酸铁0.75mg/L,EDTA-2Na 6.8mg/L,硫酸锌1.75mg/L和硫酸钼0.34mg/L。
制备方法与实施例1类似。
与实施例1区别在于,将燕麦β-葡聚糖替换为葡萄糖。
试验例1细胞分离与培养
取外科包皮手术切下的正常健康人的包皮组织加入含500U/mL双抗的MEM取样培养基中,尽快返回细胞室。剪除脂肪组织,将皮片剪成2mm×5mm的狭长条,再在100U/mL的双抗D-hanks液中浸泡3次,每次3min。用0.2%的胰酶冷消化过夜,然后在平皿内分离表皮和真皮,尽量将皮片剪碎,37℃热消化30min,加等量MEM终止消化,过铜网离心弃上清,分别加入3mL实施例1-3及对比例1培养液,均匀接种在无菌培养瓶内,置37℃CO2培养箱中培养。
利用MTT法检测检测细胞生长状况,如表1所示。
表1不同培养基中表皮细胞生长状况
由表1可知,实施例培养基中表皮细胞生长状况明显优于对比例,实施例细胞培养基中表皮细胞生长迅速,能够快速达到平顶期,其中实施例1中细胞最大生物量是对比例1最大生物量的1.3倍;并且并且实施例培养基中表皮细胞平顶期为3天,在培养8天后才开始衰减,对比例1培养基中表皮细胞平顶期为2天,在培养7天后开始出现明显衰减,由此说明实施例培养基能够使表皮细胞凋亡速度减慢,因此本发明培养基有利于表皮细胞生长。
各培养基中表皮细胞生长具体趋势,如图1所示。
Claims (4)
1.一种表皮细胞培养基,其特征在于,主要由以下成分及其浓度组成:基础培养基15-20g/L,胰岛素20-30mg/L,谷氨酰胺5-10mg/L,辅酶Q1010-30μg/L,燕麦β-葡聚糖1-10mg/L,维生素A 10-20mg/L,维生素E 7-8mg/L,维生素C 1-3mg/L,表皮生长因子10-13μg/L,纤维母细胞生长因子50-100μg/L,转化生长因子-β1.5-5μg/L,亚硒酸钠1-10μg/L,柠檬酸铁1-1.5mg/L,硝酸铁0.5-0.8mg/L,EDTA-2Na 5-8mg/L,硫酸锌1-3mg/L和硫酸钼0.1-0.5mg/L。
2.根据权利要求1所述表皮细胞培养基,其特征在于,由以下成分及其浓度组成:基础培养基18g/L,胰岛素25mg/L,谷氨酰胺7.5mg/L,辅酶Q1023μg/L,燕麦β-葡聚糖6.5mg/L,维生素A 13.5mg/L,维生素E 7.5mg/L,维生素C 2.5mg/L,表皮生长因子12.5μg/L,纤维母细胞生长因子75μg/L,转化生长因子-β4.5μg/L,亚硒酸钠8μg/L,柠檬酸铁1.35mg/L,硝酸铁0.75mg/L,EDTA-2Na 6.8mg/L,硫酸锌1.75mg/L和硫酸钼0.34mg/L。
3.根据权利要求1或2所述表皮细胞培养基,其特征在于,所述基础培养基为Ham’s F12或DMEM-L。
4.根据权利要求1-3任一所述的表皮细胞培养基的制备方法,其特征在于,分为以下步骤:
(1)准确称取基础培养基、燕麦β-葡聚糖、亚硒酸钠,柠檬酸铁,硝酸铁,EDTA-2Na,硫酸锌,硫酸钼,溶于35℃,800mL超纯水中,搅拌均匀,得溶液I;
(2)将上述所得溶液I冷却至15-16℃,加入相应量的胰岛素,谷氨酰胺,辅酶Q10,维生素A,维生素E,维生素C,表皮生长因子,纤维母细胞生长因子,转化生长因子-β,加入200mL超纯水轻微搅拌至均匀,得溶液II;
(3)用1mol/L氢氧化钠溶液或1mol/L盐酸溶液调溶液II的pH至7.2,得溶液III;
(4)将溶液III用0.22μm无菌滤膜正压过滤除菌,即得。
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