CN114214278B - 一种神经元细胞体外培养方法 - Google Patents
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Abstract
本发明提供了一种神经元细胞体外培养方法,包括:S1制备贮存液;S2制备灭菌液;S3制备培养液。本发明通过向培养液中加入特定的蛋白因子,使得所述神经元细胞体外培养生存率显著提升,对于科研和批量利用神经元细胞具有十分积极的意义。
Description
技术领域
本发明涉及医药技术领域,具体涉及一种神经元细胞体外培养方法。
背景技术
细胞的体外培养技术为研究人员能够更为全面、深入地研究细胞形态和功能提供了便利,同时也排除了其他类型细胞的干扰,其中培养液的配制对于细胞体外培养效果至关重要。
不可逆致盲性眼病包括青光眼、黄斑变性等,是临床医学领域亟待攻克的难题。视网膜神经元的病变是上述所有不可逆致盲性眼病共同且关键的病理生理基础,因此保护视网膜神经元的相关科学研究对于防盲、治盲具有重要价值。视网膜神经元无法自身***增殖,且受损后无法再生,这使得该细胞的体外培养极具挑战。现有技术中的体外培养液培养的视网膜神经元存活率及存活时间仍然有限,难以适用于诸多对细胞存活率要求较高的科学研究,也难以进行诸多对细胞数要求较高的检测,如Western Blot、各类测序检测等。因此,提高视网膜神经元体外培养的存活率依然是亟待解决的问题,对科学研究具有重要价值。
发明内容
本发明采用如下技术方案:
一种神经元细胞的体外培养液的制备方法,包括以下步骤:
S1制备贮存液:
S11制备100X佐藤贮存液:取一定量的杜氏磷酸盐缓冲液(DPBS),在其中加入转铁蛋白、牛血清白蛋白(bovine serum albumin,BSA)、***、腐胺以及***钠后,经滤网滤过后灭菌,制成100X佐藤贮存液,并在-30~-10℃下冷冻保存,所述100X佐藤贮存液中,转铁蛋白、牛血清白蛋白、***、腐胺以及***钠的浓度分别为5~15mg/mL、5~15mg/mL、0.04~0.08mg/mL、1~2mg/mL、0.05~0.15mg/mL;优选的,所述100X佐藤贮存液中,转铁蛋白、牛血清白蛋白、***、腐胺以及***钠的浓度分别约为10mg/mL、10mg/mL、0.0625mg/mL、1.6mg/mL、0.1mg/mL;优选的,所述冷冻保存温度为-20℃;
S12制备100X三碘甲状腺氨酸贮存液:将三碘甲状腺氨酸溶于一定体积、浓度为1mol/L氢氧化钠溶液内,加入氢氧化钠溶液75倍体积的DPBS,过滤灭菌,取续滤液,制成100X三碘甲状腺氨酸贮存液,并在-30~-10℃下冷冻保存,所述100X三碘甲状腺氨酸贮存液中,三碘甲状腺氨酸的浓度约为0.04~0.5mg/mL;更进一步的,将0.1~0.5mg三碘甲状腺氨酸溶于50~1000μl、浓度为1mol/L氢氧化钠溶液内,加入氢氧化钠溶液75倍体积的DPBS,过滤灭菌,取续滤液,制成100X三碘甲状腺氨酸贮存液,并在-30~-10℃下冷冻保存,所述100X三碘甲状腺氨酸贮存液中,三碘甲状腺氨酸的浓度为0.04~0.08mg/mL;更进一步的,所述100X三碘甲状腺氨酸贮存液中,三碘甲状腺氨酸的浓度约为0.066mg/mL;更进一步优选的,将0.2mg三碘甲状腺氨酸溶于400μl、1mol/L氢氧化钠溶液内,加入氢氧化钠溶液75倍体积的DPBS,过滤灭菌,取续滤液,制成100X三碘甲状腺氨酸贮存液,并在-20℃冷冻保存;
S13制备100X N-乙酰-L-半胱氨酸贮存液:取一定量的N-乙酰-L-半胱氨酸粉末,溶于Neurobasal培养液中,滤过灭菌,制成100X N-乙酰-L-半胱氨酸贮存液,并在-30~-10℃下冷冻保存,其中,所述100X N-乙酰-L-半胱氨酸贮存液中N-乙酰-L-半胱氨酸浓度为3~10mg/mL;优选的,所述100X N-乙酰-L-半胱氨酸贮存液中N-乙酰-L-半胱氨酸浓度约为5mg/mL;优选的,所述冷冻保存温度为-20℃;
S14制备1000X毛喉素贮存液:取一定量的毛喉素,溶于二甲基亚砜中,滤过灭菌,制成1000X毛喉素贮存液贮存液,并在-30~-10℃下冷冻保存,其中,所述1000X毛喉素贮存液中毛喉素浓度为1~5mg/mL;优选的,所述100X N-乙酰-L-半胱氨酸贮存液中毛喉素浓度约为2.08mg/mL;优选的,所述冷冻保存温度为-20℃;
S15制备1000X BDNF贮存液:取一定量的BDNF冻干粉,溶于含有质量百分含量0.5~5%BSA的杜氏磷酸盐缓冲液(DPBS)中,滤过灭菌,制成1000XBDNF贮存液中,并在-100~-70℃下冷冻保存,其中,所述1000X BDNF贮存液中BDNF浓度为0.01~0.1mg/mL;优选的,所述杜氏磷酸盐缓冲液(DPBS)中BSA的质量百分含量为1%;优选的,所述1000X BDNF贮存液中BDNF浓度约为0.02mg/mL;优选的,所述冷冻保存温度为-80℃;
S16制备1000X CNTF贮存液:取一定量的CNTF冻干粉,溶于含有质量百分含量0.5~5%BSA的杜氏磷酸盐缓冲液(DPBS)中,滤过灭菌,制成1000XCNTF贮存液中,并在-100~-70℃下冷冻保存,其中,所述1000XCNTF贮存液中CNTF浓度为0.01~0.1mg/mL;优选的,所述1000X CNTF贮存液中CNTF浓度约为0.02mg/mL;优选的,所述冷冻保存温度为-80℃;
S17制备500X FGF贮存液:取一定量的FGF冻干粉,溶于含有质量百分含量0.5~5%BSA的杜氏磷酸盐缓冲液(DPBS)中,滤过灭菌,制成1000X FGF贮存液中,并在-100~-70℃下冷冻保存,其中,所述1000X FGF贮存液中FGF浓度为0.01~0.1mg/mL;优选的,所述1000X FGF贮存液中FGF浓度约为0.02mg/mL;优选的,所述冷冻保存温度为-80℃;
S18制备1000X GDNF贮存液:取一定量的GDNF冻干粉,溶于含有质量百分含量0.5~5%BSA的杜氏磷酸盐缓冲液(DPBS)中,滤过灭菌,制成1000XGDNF贮存液中,并在-100~-70℃下冷冻保存,其中,所述1000X GDNF贮存液中GDNF浓度为0.01~0.1mg/mL;优选的,所述1000X GDNF贮存液中FGF浓度约为0.02mg/mL;优选的,所述冷冻保存温度为-80℃;
S19制备1000X SCGF-β贮存液:取一定量的SCGF-β冻干粉,溶于含有质量百分含量0.5~5%BSA的杜氏磷酸盐缓冲液(DPBS)中,滤过灭菌,制成1000XSCGF-β贮存液中,并在-100~-70℃下冷冻保存,其中,所述1000X SCGF-β贮存液中SCGF-β浓度为0.005~0.05mg/mL;优选的,所述1000X SCGF-β贮存液中FGF浓度约为0.01mg/mL;优选的,所述冷冻保存温度为-80℃;
S20制备100X胰岛素贮存液:取一定量的胰岛素粉末,溶于蒸馏水中,再加入适量1.0mol/L的盐酸,滤过灭菌,制成100X的胰岛素贮存液中,并在-30~-10℃下冷冻保存,其中,所述100X胰岛素贮存液中胰岛素浓度为0.1~5mg/mL;优选的,所述100X胰岛素贮存液中胰岛素浓度约为0.5mg/mL;优选的,所述冷冻保存温度为-20℃;
S2制备灭菌液:取10~30ml Neurobasal培养液,分别加入上述S1步骤制备的100~300μL的100X佐藤贮存液、浓度为100~300mmol/L左旋谷氨酰胺贮存液、100X三碘甲状腺氨酸贮存液、100X胰岛素贮存液,加入300~500μl的B27Plus溶液,10~30μL的100X N-乙酰-L-半胱氨酸贮存液、50~200mmol/L的丙酮酸钠溶液以及3000~6000U/mL的青霉素/链霉素双抗溶液,然后过滤灭菌,获得灭菌液;优选的,加入Neurobasal培养液20mL;优选的,所述100X佐藤贮存液、左旋谷氨酰胺贮存液、100X三碘甲状腺氨酸贮存液、100X胰岛素贮存液均为200μL;优选的,所述B27 Plus溶液的体积为400μL;所述100X N-乙酰-L-半胱氨酸贮存液、丙酮酸钠溶液以及青霉素/链霉素双抗溶液均为20μl;优选的,的,左旋谷氨酰胺贮存液的浓度为200mmol/L;优选的,丙酮酸钠溶液的浓度为100mmol/L;优选的,所述青霉素/链霉素双抗溶液的浓度为5000U/mL的;
S3制备培养液:取10~30ml上述灭菌液,再分别加入10~50μL的1000X毛喉素贮存液、1000X CNTF贮存液、1000X BDNF贮存液、1000X GDNF贮存液、1000X SCGF-β贮存液,以及30~100μl的500X FGF贮存液,获得培养液;优选的,所述灭菌液为20ml;优选的,所述1000X毛喉素贮存液、1000X CNTF贮存液、1000X BDNF贮存液、1000X GDNF贮存液、1000X SCGF-β贮存液分别为20μL;优选的,所述500X FGF贮存液为40μL;
进一步的,所述神经元细胞为视网膜神经元细胞;
附图说明
图1:不同视网膜神经节细胞培养液体外培养神经节细胞存活率比较
有益效果
本方法制备的神经元细胞体外培养液能够显著延长神经元细胞特别是视网膜神经节细胞在体外的存活率;本发明通过实验证实,在所述培养液中加入特定的蛋白因子能够显著延长神经元细胞在体外的存活率。
具体实施方式
下面结合附图和具体实施方式对本发明作进一步详细的说明。
本发明具体实施方式中的试剂和实验材料均为市售获得。
实施例1:制备神经元细胞的体外培养液
一种神经元细胞的体外培养液的制备方法,包括以下步骤:
S1制备贮存液:
S11制备100X佐藤贮存液:取40ml的杜氏磷酸盐缓冲液(DPBS),在其中加入400mg转铁蛋白、400mg牛血清白蛋白(bovine serum albumin,BSA)、2.5mg***、64mg腐胺以及4mg***钠后,经孔径为0.22μm的滤网滤过后灭菌,制成贮存液,-20℃冷冻保存;
S12制备100X三碘甲状腺氨酸贮存液:将0.2mg三碘甲状腺氨酸溶于400μl、1mol/L氢氧化钠溶液内,加入30ml的DPBS,使用孔径为0.22μm的滤网过滤灭菌(弃去最先滤出的10mL),制成贮存液,-20℃冷冻保存;
S13制备100X N-乙酰-L-半胱氨酸贮存液:取50mg N-乙酰-L-半胱氨酸粉末,溶于10ml Neurobasal培养液(来自ThermFisher scientific,https://www.thermofisher.cn/ cn/zh/home/technical-resources/media-formulation.251.html)中,滤网滤过灭菌,制成贮存液,-20℃冷冻保存;
S14制备1000X毛喉素贮存液:将10mg毛喉素溶于4.8ml二甲基亚砜(DimethylSulphoxide,DMSO)中,然后经孔径为0.22μm的滤网过滤灭菌,制成贮存液,-20℃冷冻保存;
S15制备1000X BDNF贮存液:将100μg人源性BDNF冻干粉溶于5mL含有1%BSA的杜氏磷酸盐缓冲液DPBS中,然后经孔径为0.22μm的滤网过滤灭菌,制成贮存液,-80℃冷冻保存;
S16制备1000X CNTF贮存液:将100μg人源性CNTF冻干粉溶于5mL含有1%BSA的DPBS中,制成贮存液,-80℃冷冻保存;
S17制备500X FGF贮存液:将100μg人源性FGF冻干粉溶于5mL含有1%BSA的Dulbecco磷酸盐缓冲液(Dulbecco phosphate-buffered saline,DPBS)中,然后经孔径为0.22μm的滤网过滤灭菌,制成贮存液,-80℃冷冻保存;
S18制备1000X GDNF贮存液:将100μg人源性GDNF冻干粉溶于5mL含有1%BSA的DPBS中,然后经孔径为0.22μm的滤网过滤灭菌,制成贮存液,-80℃冷冻保存。
S19制备1000X SCGF-β贮存液:将50μg人源性SCGF-β冻干粉溶于5mL含有1%BSA的DPBS中,然后经孔径为0.22μm的滤网过滤灭菌,制成贮存液,-80℃冷冻保存。
S20制备100X的胰岛素贮存液:将10mg的胰岛素粉末加入20mL蒸馏水中,再加入100μl 1.0N的盐酸,然后经孔径为0.22μm的滤网过滤灭菌,制成贮存液,-20℃冷冻保存。
S2制备灭菌液:取20ml Neurobasal培养液(来自Gibco Inc.),分别加入上述S1步骤制备的200μl的100X佐藤贮存液、200mmol/L左旋谷氨酰胺贮存液(来自Gibco Inc.)、100X三碘甲状腺氨酸贮存液、100X胰岛素贮存液,400μl的B27 Plus溶液(来自GibcoInc.),20μl的100X N-乙酰-L-半胱氨酸贮存液、100mmol/L的丙酮酸钠溶液(来自GibcoInc.)以及5000U/mL的青霉素/链霉素双抗溶液(来自Gibco Inc.),然后经孔径为0.22μm的滤网过滤灭菌,获得灭菌液;
S3制备培养液:取20ml上述灭菌液,再分别加入20μl的1000X毛喉素贮存液、1000XCNTF贮存液、1000X BDNF贮存液、1000X GDNF贮存液、1000X SCGF-β贮存液以及40μl的500XFGF贮存液,获得培养液。
该培养液可在4℃下贮存3天。
实施例2:培养板包被
S1:配制100X多聚赖氨酸(poly-D-lysine,PDL)贮存液:在每5mL蒸馏水中加入5mgPDL冻干粉,每200μl分装,滤网滤过灭菌,-20℃冷冻保存;
S2:配制层粘连蛋白(laminin)溶液:在每5mL Neurobasal培养液中加入20μl层粘连蛋白(Sigma L-6274),即得;
S3配制PDL工作液:每20mL无菌蒸馏水中加入200μl 100X PDL贮存液,即得PDL工作液;
S4培养板包被:取无菌24孔细胞培养板,每孔加入350μl PDL工作液,室温静置30分钟后弃去该液,使用无菌蒸馏水洗涤2次。此后每孔加入350μl层粘连蛋白溶液,37℃、10%v/v CO2环境下放置1小时以上即包被完成,使用前弃去层粘连蛋白溶液即可。
对比例1:
其余步骤不变,将实施例1步骤S3S3制备培养液改为:取20ml上述灭菌液,再分别加入20μl的1000X毛喉素贮存液、1000X CNTF贮存液以及1000XBDNF贮存液,获得培养液。
对比例2:
其余步骤不变,将实施例1步骤S3S3制备培养液改为:取20ml上述灭菌液,再分别加入20μl的1000X毛喉素贮存液、1000X CNTF贮存液、1000X BDNF贮存液以及1000X GDNF贮存液,获得培养液。
对比例3:
取20ml上述灭菌液,再分别加入20μl的1000X毛喉素贮存液、1000X CNTF贮存液、1000X BDNF贮存液、1000X GDNF贮存液以及500X FGF贮存液,获得培养液。
实验例:视网膜神经节细胞培养
将一定量(约40000个)的视网膜神经节细胞分别重悬于实施例1(OriginalMedium+GNDF+FGF+SCGF-β)、对比例1(Original Medium)、对比例2(Original Medium+GNDF)以及对比例3(Original Medium+GNDF+FGF)制备的培养液中,再加入实施例2包被的培养孔中,每孔培养液体积约400μl,置于37℃、10%v/vCO2环境下培养,每隔一日将孔内一半(200μL)培养液更换成新鲜培养液;将该实验重复3次。实验结果如图1所示,分别为对比例1、对比例2、对比例3以及实施例1培养液对视网膜神经节细胞进行培养后,视网膜神经节细胞4天后的细胞存活率,由图1可以得出,本发明制备的视网膜神经节细胞培养液具有良好的技术效果,能够显著延长神经节细胞的体外存活率。
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术人员无需创造性劳动就可以根据本发明的构思做出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
Claims (29)
1.一种视网膜神经元细胞体外培养液的制备方法,其特征在于,包括以下步骤:
S1制备贮存液:
S11制备100X佐藤贮存液:取一定量的DPBS,在其中加入转铁蛋白、牛血清白蛋白、***、腐胺以及***钠后,经滤网过滤后灭菌,制成100X佐藤贮存液,并在-30~-10℃下冷冻保存,所述100X佐藤贮存液中,转铁蛋白、牛血清白蛋白、***、腐胺以及***钠的浓度分别为5~15mg/mL、5~15mg/mL、0.04~0.08mg/mL、1~2mg/mL、0.05~0.15mg/mL;
S12制备100X三碘甲状腺氨酸贮存液:将0.1~0.5mg三碘甲状腺氨酸溶于50~1000μl、浓度为1mol/L氢氧化钠溶液内,加入氢氧化钠溶液75倍体积的DPBS,过滤灭菌,取续滤液,制成100X三碘甲状腺氨酸贮存液,并在-30~-10℃下冷冻保存,所述100X三碘甲状腺氨酸贮存液中,三碘甲状腺氨酸的浓度为0.04~0.08mg/mL;
S13制备100X N-乙酰-L-半胱氨酸贮存液:取一定量的N-乙酰-L-半胱氨酸粉末,溶于Neurobasal培养液中,过滤灭菌,制成100X N-乙酰-L-半胱氨酸贮存液,并在-30~-10℃下冷冻保存,其中,所述100XN-乙酰-L-半胱氨酸贮存液中N-乙酰-L-半胱氨酸浓度为3~10mg/mL;
S14制备1000X毛喉素贮存液:取一定量的毛喉素,溶于二甲基亚砜中,过滤灭菌,制成1000X毛喉素贮存液贮存液,并在-30~-10℃下冷冻保存,其中,所述1000X毛喉素贮存液中毛喉素浓度为1~5mg/mL;
S15制备1000X BDNF贮存液:取一定量的BDNF冻干粉,溶于含有质量百分含量0.5~5%BSA的DPBS中,过滤灭菌,制成1000XBDNF贮存液中,并在-100~-70℃下冷冻保存,其中,所述1000XBDNF贮存液中BDNF浓度为0.01~0.1mg/mL;
S16制备1000X CNTF贮存液:取一定量的CNTF冻干粉,溶于含有质量百分含量0.5~5%BSA的DPBS中,过滤灭菌,制成1000XCNTF贮存液中,并在-100~-70℃下冷冻保存,其中,所述1000XCNTF贮存液中CNTF浓度为0.01~0.1mg/mL;
S17制备500X FGF贮存液:取一定量的FGF冻干粉,溶于含有质量百分含量0.5~5%BSA的DPBS中,过滤灭菌,制成500X FGF贮存液中,并在-100~-70℃下冷冻保存,其中,所述500X FGF贮存液中FGF浓度为0.01~0.1mg/mL;
S18制备1000X GDNF贮存液:取一定量的GDNF冻干粉,溶于含有质量百分含量0.5~5%BSA的DPBS中,过滤灭菌,制成1000XGDNF贮存液中,并在-100~-70℃下冷冻保存,其中,所述1000XGDNF贮存液中GDNF浓度为0.01~0.1mg/mL;
S19制备1000X SCGF-β贮存液:取一定量的SCGF-β冻干粉,溶于含有质量百分含量0.5~5%BSA的DPBS中,过滤灭菌,制成1000X SCGF-β贮存液中,并在-100~-70℃下冷冻保存,其中,所述1000X SCGF-β贮存液中SCGF-β浓度为0.005~0.05mg/mL;
S20制备100X胰岛素贮存液:取一定量的胰岛素粉末,溶于蒸馏水中,再加入适量1.0mol/L的盐酸,过滤灭菌,制成100X的胰岛素贮存液中,并在-30~-10℃下冷冻保存,其中,所述100X胰岛素贮存液中胰岛素浓度为0.1~5mg/mL;
S2制备灭菌液:取10~30ml Neurobasal培养液,分别加入上述S1步骤制备的100~300μL的100X佐藤贮存液、浓度为100~300mmol/L左旋谷氨酰胺贮存液、100X三碘甲状腺氨酸贮存液、100X胰岛素贮存液,加入300~500μl的B27 Plus溶液,10~30μL的100X N-乙酰-L-半胱氨酸贮存液、50~200mmol/L的丙酮酸钠溶液以及3000~6000U/mL的青霉素/链霉素双抗溶液,然后过滤灭菌,获得灭菌液;
S3制备培养液:取10~30ml上述灭菌液,再分别加入10~50μL的1000X毛喉素贮存液、1000X CNTF贮存液、1000X BDNF贮存液、1000X GDNF贮存液、1000X SCGF-β贮存液,以及30~100μl的500XFGF贮存液,获得培养液;
2.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S11中,所述100X佐藤贮存液中,转铁蛋白、牛血清白蛋白、***、腐胺以及***钠的浓度分别约为10mg/mL、10mg/mL、0.0625mg/mL、1.6mg/mL、0.1mg/mL。
3.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S11中,所述冷冻保存温度为-20℃。
4.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S12中,所述100X三碘甲状腺氨酸贮存液中,三碘甲状腺氨酸的浓度约为0.066mg/mL。
5.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S12中,将0.2mg三碘甲状腺氨酸溶于400μl、1mol/L氢氧化钠溶液内,加入氢氧化钠溶液75倍体积的DPBS,过滤灭菌,取续滤液,制成100X三碘甲状腺氨酸贮存液,并在-20℃冷冻保存。
6.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S13中,所述100X N-乙酰-L-半胱氨酸贮存液中N-乙酰-L-半胱氨酸浓度约为5mg/mL。
7.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S13中,所述冷冻保存温度为-20℃。
8.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S14中,所述1000X毛喉素贮存液中毛喉素浓度约为2.08mg/mL。
9.根据权利要求8所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S14中,所述冷冻保存温度为-20℃。
10.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S15中,所述DPBS中BSA的质量百分含量为1%。
11.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S15中,所述1000X BDNF贮存液中BDNF浓度约为0.02mg/mL。
12.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S15中,所述冷冻保存温度为-80℃。
13.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S16中,所述1000X CNTF贮存液中CNTF浓度约为0.02mg/mL。
14.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S16中,所述冷冻保存温度为-80℃。
15.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S18中,所述1000X GDNF贮存液中GDNF浓度约为0.02mg/mL。
16.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S18中,所述冷冻保存温度为-80℃。
17.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S19中,所述1000X SCGF-β贮存液中SCGF-β浓度约为0.01mg/mL。
18.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S19中,所述冷冻保存温度为-80℃。
19.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S20中,所述100X胰岛素贮存液中胰岛素浓度约为0.5mg/mL。
20.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S20中,所述冷冻保存温度为-20℃。
21.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S2中,加入Neurobasal培养液20mL。
22.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S2中,所述100X佐藤贮存液、左旋谷氨酰胺贮存液、100X三碘甲状腺氨酸贮存液、100X胰岛素贮存液均为200μL。
23.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S2中,所述B27 Plus溶液的体积为400μL。
24.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S2中,所述100X N-乙酰-L-半胱氨酸贮存液、丙酮酸钠溶液以及青霉素/链霉素双抗溶液均为20μl。
25.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S2中,左旋谷氨酰胺贮存液的浓度为200mmol/L。
26.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S2中,丙酮酸钠溶液的浓度为100mmol/L。
27.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S2中,所述青霉素/链霉素双抗溶液的浓度为5000U/mL的。
28.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S3中,所述灭菌液为20ml。
29.根据权利要求1所述视网膜神经元细胞体外培养液的制备方法,其特征在于,S3中,所述1000X毛喉素贮存液、1000X CNTF贮存液、1000X BDNF贮存液、1000X GDNF贮存液、1000X SCGF-β贮存液分别为20μL,且所述500X FGF贮存液为40μL。
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