CN106754640A - 一种表皮细胞培养基及其制备方法 - 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及其制备方法。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,飞燕草素,桑色素,儿茶素,表皮生长因子,糖皮质激素,转化生长因子,***钠,柠檬酸铁铵,硝酸铁,EDTA‑2Na。本发明表皮细胞培养基能使表皮细胞快速增殖,并意外发现本发明培养基中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。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及其制备方法。
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及其制备方法,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,其主要由以下成分及其浓度组成:基础培养基10-15g/L,胰岛素10-30mg/L,谷氨酰胺1-10mg/L,飞燕草素10-100μg/L,桑色素1-10ng/L,儿茶素45-65μM,表皮生长因子5-10μg/L,糖皮质激素50-300μg/L,转化生长因子-β1-3μg/L,***钠1-10μg/L,柠檬酸铁铵0.2-1.5mg/L,硝酸铁0.3-0.8mg/L和EDTA-2Na 4-10mg/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12g/L,胰岛素14.5mg/L,谷氨酰胺7.5mg/L,飞燕草素34μg/L,桑色素5.25ng/L,儿茶素63.5μM,表皮生长因子8.2μg/L,糖皮质激素150μg/L,转化生长因子-β2.6μg/L,***钠6μg/L,柠檬酸铁铵1.35mg/L,硝酸铁0.45mg/L和EDTA-2Na 6.25mg/L。
优选地,所述表皮细胞培养基中基础培养基为Ham’s F12或DMEM-L。
一种表皮细胞培养基的制备方法,由分为以下步骤:
(1)称取相应重量的基础培养基、飞燕草素、桑色素、儿茶素、***钠、柠檬酸铁铵、硝酸铁和EDTA-2Na,溶于35℃,800mL超纯水中,搅拌至完全溶解,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、表皮生长因子、糖皮质激素和转化生长因子,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滤膜正压过滤除菌,即得。
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。飞燕草素(C15H11ClO7,CAS NO.528530)、桑色素(C15Hl0O7·2H2O,CAS NO.480-16-0)属于生物类黄酮化合物,均具有很强的抗养化与抑菌作用,如桑色素对金黄色葡萄球菌、痢疾杆菌和伤寒杆菌有较强的抗菌作用。与此同时,飞燕草素对弹性蛋白酶和胶原蛋白酶具有抑制作用。儿茶素(C15H14O6,CAS NO.7295-85-4)又称儿茶精,茶单宁,为黄烷醇的衍生物,分子式C15H14O6。儿茶素最初由儿茶中提取出,为无色结晶形固体,能溶于水;儿茶素是天然的油脂抗氧化剂,并且可以清除人体产生的自由基,以保护细胞膜,可以抑制引起人类皮肤病的病菌,并且对治疗湿疹有很好的疗效。本发明培养基中加入飞燕草素、桑色素和儿茶素,不仅能够起到很好的抑菌效果,同时对表皮细胞的生长有极大地促进作用。
胰岛素可以促进RNA、蛋白质和脂肪酸的合成,抑制细胞凋亡,是重要的细胞存活因子。柠檬酸铁铵,硝酸铁和EDTA-2Na组合能够代替传统培养基中转铁蛋白的作用,能够促进铁的运输。表皮生长因子、转化生长因子-β和糖皮质激素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。
本发明培养基与传统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,并意外发现本发明培养基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。
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 F1212g/L,胰岛素14.5mg/L,谷氨酰胺7.5mg/L,飞燕草素34μg/L,桑色素5.25ng/L,儿茶素63.5μM,表皮生长因子8.2μg/L,糖皮质激素150μg/L,转化生长因子-β2.6μg/L,***钠6μg/L,柠檬酸铁铵1.35mg/L,硝酸铁0.45mg/L和EDTA-2Na 6.25mg/L。
制备方法:
(1)称取相应重量的基础培养基、飞燕草素、桑色素、儿茶素、***钠、柠檬酸铁铵、硝酸铁和EDTA-2Na,溶于35℃,800mL超纯水中,搅拌至完全溶解,得溶液I;
(2)将上述所得溶液I冷却至15℃,加入相应量的胰岛素、谷氨酰胺、表皮生长因子、糖皮质激素和转化生长因子,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30mg/L,谷氨酰胺10mg/L,飞燕草素100μg/L,桑色素10ng/L,儿茶素65μM,表皮生长因子10μg/L,糖皮质激素300μg/L,转化生长因子-β3μg/L,***钠10μg/L,柠檬酸铁铵1.5mg/L,硝酸铁0.8mg/L,EDTA-2Na 10mg/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培养基10g/L,胰岛素10mg/L,谷氨酰胺1mg/L,飞燕草素10μg/L,桑色素1ng/L,儿茶素45μM,表皮生长因子5μg/L,糖皮质激素50μg/L,转化生长因子-β1μg/L,***钠1μg/L,柠檬酸铁铵0.2mg/L,硝酸铁0.3mg/L,EDTA-2Na 4mg/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 F 12g/L,胰岛素14.5mg/L,谷氨酰胺7.5mg/L,飞燕草素97.5μg/L,桑色素5.25ng/L,表皮生长因子8.2μg/L,糖皮质激素150μg/L,转化生长因子-β2.6μg/L,***钠6μg/L,柠檬酸铁铵1.35mg/L,硝酸铁0.45mg/L和EDTA-2Na 6.25mg/L。
制备方法与实施例1类似。
与实施例1区别在于,不含有儿茶素而飞燕草素浓度提升至97.5μg/L。
试验例1细胞生长检测
取外科***手术切下的正常健康人的***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,凋亡速度减慢,因此本发明培养基对表皮细胞生长有极大地促进作用。
各培养基中表皮细胞生长趋势,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10-15g/L,胰岛素10-30mg/L,谷氨酰胺1-10mg/L,飞燕草素10-100μg/L,桑色素1-10ng/L,儿茶素45-65μM,表皮生长因子5-10μg/L,糖皮质激素50-300μg/L,转化生长因子-β1-3μg/L,***钠1-10μg/L,柠檬酸铁铵0.2-1.5mg/L,硝酸铁0.3-0.8mg/L和EDTA-2Na 4-10mg/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12g/L,胰岛素14.5mg/L,谷氨酰胺7.5mg/L,飞燕草素34μg/L,桑色素5.25ng/L,儿茶素63.5μM,表皮生长因子8.2μg/L,糖皮质激素150μg/L,转化生长因子-β2.6μg/L,***钠6μg/L,柠檬酸铁铵1.35mg/L,硝酸铁0.45mg/L和EDTA-2Na 6.25mg/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,由以下步骤组成:
(1)称取基础培养基、飞燕草素、桑色素、儿茶素、***钠、柠檬酸铁铵、硝酸铁和EDTA-2Na,溶于35℃,800mL超纯水中,搅拌至完全溶解,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、表皮生长因子、糖皮质激素和转化生长因子,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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CN105950542A (zh) * | 2016-06-24 | 2016-09-21 | 济南磐升生物技术有限公司 | 一种人皮肤表皮细胞培养基及其应用 |
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周燕等: "抗氧化剂对皮肤角质细胞体外寿命的影响", 《生物工程学报》 * |
蔄茂强等: "糖皮质激素对表皮功能的调节", 《临床皮肤科杂志》 * |
陈锋: "无血清细胞培养基的主要补充因子及研究进展", 《海峡药学》 * |
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CN107397710A (zh) * | 2017-07-31 | 2017-11-28 | 广州润虹医药科技股份有限公司 | 一种皮肤干细胞活性素的制备方法及其应用 |
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