CN116474000B - 脐带间充质干细胞制剂、制备方法及其在治疗膝骨关节炎中的应用 - Google Patents
脐带间充质干细胞制剂、制备方法及其在治疗膝骨关节炎中的应用 Download PDFInfo
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Abstract
本发明公开了脐带间充质干细胞制剂、制备方法及其在治疗膝骨关节炎中的应用,属于生物医药技术领域。本发明通过优化脐带间充质干细胞的分离方法、传代培养基以及制剂的制备方法,得到的脐带间充质干细胞制剂的制备工艺成熟稳定,制剂稳定性好,可直接用于骨关节炎的临床使用,无需先冻存运输、临床使用时再现场复苏等操作。同时采用本发明的方法培养的脐带间充质干细胞可分泌细胞因子,有效改善骨关节炎的微环境,发挥免疫调节功能,有利于增强诱导软骨分化。
Description
技术领域
本发明涉及生物医药技术领域,尤其涉及一种脐带间充质干细胞制剂、制备方法及其在治疗膝骨关节炎中的应用。
背景技术
骨关节炎是老年人群中的高发疾病,近年来,利用干细胞再生医学治疗骨关节炎已经获得了很多的临床案例。干细胞能在体内微环境作用下主动迁移至受损部位进行软骨修复,促进退变软骨细胞的恢复和新生软骨细胞的生成。干细胞分泌的多种生物活性分子,具有调节免疫和抗炎作用,对于修复受损组织、缓解病痛、治疗膝关节炎,具有很好的效果。
骨关节炎(OA )是一种免疫介导的系统性炎症疾病,在患者的骨关节液中含有免疫细胞分泌的相关因子如白细胞介素 1β(IL-1β)、白介素细胞 6(IL-6)、肿瘤坏死因子α(INF-α),如调节这些因子的表达,可以抑制对软骨细胞的进一步损伤。而一些胰岛素样生长因子、转化生长因子等对软骨细胞增殖和基质的生成有调节作用。
当发生骨关节炎时,关节组织中高度浸润的 T 淋巴细胞释放大量炎性因子,这些炎性因子可增强微环境的免疫反应而促使软骨基质进一步破坏。在这种情况下,浸润性T淋巴细胞成为骨性关节炎发生发展的关键因素,促进软骨细胞的凋亡和软骨损伤,对骨关节产生实质性的损害。在 OA 中浸润性 T 淋巴细胞广泛分布于滑液和滑膜组织,浸润性T淋巴细胞在关节炎中特异表达骨保护素配体(Osteoprotegerin Ligand,OPGL),OPGL可调节淋巴结器官发生、淋巴细胞发育,通过与树突状细胞相互作用诱导T 淋巴细胞增殖。而软骨细胞表达OPGL受体,浸润性T淋巴细胞通过OPGL与软骨细胞膜上OPGL受体(RNAKL)引起骨丢失和关节损伤。持续免疫反应促使免疫不断増强,T细胞调节免疫平衡的作用受到抑制而促使OA不断恶化,这一过程受到分子信号通路的调控,软骨细胞膜上KL(Klotho蛋白)活化后,促进了IL-6和MMP-13(金属基质蛋白酶)的表达。近年研究发现,T淋巴细胞和干扰素调节因子-7(IRF-7)在某些与自身免疫相关的关节炎症疾病中表达上调,如类风湿性关节炎、银屑病和系统性红斑狼疮。因此,在OA进程中,浸润性T淋巴细胞与IRF-7可能密切相关,两者可能促使IL-6分泌增多,改变机体微环境从而导致OA恶化。在OA状态下,激活树突状细胞表面表达的TLRs(Toll样受体)与衔接分子髓系分化因子88(Myeloid differentiationfactor 88,MyD88)结合,活化关键调节因子IRF-7,并通过核因子NF-κB(Nuclear factor-kappa B,NF-kB)和AP-1(激活蛋白1)信号通路诱导IFN-α(α干扰素)和IFN-β(β干扰素)的表达。IRF-7在抗原诱导产生Ι型干扰素中起关键作用,是树突状细胞激活的关键调控子,IRF-7磷酸化后可活化NF-kB或PI3K(磷脂酰肌醇激酶)通路,促使IL-6表达上调,从而导致OA 治疗失败。
干细胞外泌体可有效消除自由基、改善关节内微环境、降低免疫反应,同时能够更好的促进间充质干细胞的增殖和向软骨干细胞的分化,进而实现快速修复软骨损伤。间充质干细胞可通过可溶性因子(如白细胞介素1、干扰素γ、肿瘤坏死因子α、吲哚胺2,3-双加氧酶、前列腺素E2、诱导型一氧化氮合酶、转化生长因子β、肝细胞生长因子、血管内皮生长因子、血小板源性生长因子、胰岛素样生长因子1、基质细胞衍生因子1、血管生成因子1、白细胞介素10、白细胞介素6和一氧化氮等)的分泌发挥免疫调节功能,或者通过细胞-细胞接触修复组织损伤,并能抑制不同细胞类型的先天免疫系统和适应性免疫系统。
由于炎症微环境中存在的炎细胞、炎性因子和白细胞代谢产物等各种炎性递质能对脐带间充质干细胞(UC-MSCs)产生持续的刺激,从而改变其增殖和分化能力。
炎性因子的分泌,可以通过多种复杂分析信号机制影响干细胞的生物学特性。白细胞介素1β和肿瘤坏死因子α是主要的促炎性因子,在炎症组织中高表达,而现在关于肿瘤坏死因子α对干细胞成骨分化的作用有不同的报道,有研究认为其抑制成骨,在炎症反应发生中,肿瘤坏死因子α可激活成骨细胞中的p38 MAPK(P38-分裂原激活的蛋白激酶信号通路),抑制成骨细胞的分化。也有研究表明肿瘤坏死因子α在干细胞成骨分化中起着积极的作用,通过激活核转录因子κB途径,提高成骨相关蛋白的表达,使基质矿化增多。
脐带来源间充质干细胞是从脐带中分离出来的一种成体干细胞,因其来源广泛、取材方便、免疫原性弱、増殖能力强的优势被广泛的应用于临床研究。同时有效的回避了胚胎干细胞涉及的伦理问题,也避免了类似诱导多能干细胞因复杂的诱导转分化机制所带来的安全性和风险问题。
现有技术中通常采用常规技术培养干细胞,即以收获一定数量的干细胞为主要目的,在培养过程中缺少针对骨关节炎的微环境改善及对软骨修复增强功能的考虑。
发明内容
为了解决现有技术中存在的问题,本发明提供了如下技术方案。
本发明第一方面提供了一种脐带间充质干细胞制剂的制备方法,包括:
从脐带中分离得到脐带间充质干细胞;
对脐带间充质干细胞进行传代培养,传代培养两次后得到种子细胞,传代培养四次后得到工作细胞;
对工作细胞进行复苏培养、消化和洗涤后,加入制剂缓冲液,形成用于临床使用的脐带间充质干细胞制剂;
其中,传代培养中用到的传代培养基包括DMEM培养基(Dulbecco's modifiedeagle medium,一种含各种氨基酸和葡萄糖的培养基)以及以DMEM培养基的用量为基准的:质量体积百分比为1~10%的UltraGROTM-Advanced(一种血清替代物,属于细胞培养补充剂),1~20mmol/L的谷胱苷肽,1~30nmol/L的胰岛素样生长因子,10~60ng/ml的重组人源血管内皮生长因子,10~500U/mL的血小板衍生生长因子,1~10μg/ml的纤连蛋白,10~50ng/mL的重组人碱性成纤维细胞生长因子,10~50ng/L的粒细胞-巨噬细胞集落刺激因子,1~5mmol/L的谷氨酰胺,10~60ng/ml的重组人表皮生长因子,4mg/L的L-抗坏血酸,5~100mg/L的β-巯基乙醇,0.5~5mg/L的乙醇胺,0.1~0.5g/L的丙二醇嵌段聚醚,10~1000mg/L的吐温80,2~50mg/L的胆固醇,0.2~5.0mg/L的腺嘌呤,2~10µg/mL的硫酸镁,质量体积百分比为0.5~1.5%的硫酸氨基葡萄糖,0.5~5µmol/L的甘油磷酸钠,100U/mL的青霉素,100mg/L的链霉素;所述传代培养基的pH为6.8-7.2。
优选地,采用组织块贴壁法或酶消化法从脐带中分离得到脐带间充质干细胞。
优选地,所述制剂缓冲液依次包括如下物质:低分子肝素钙注射液、右旋糖酐40注射液或氯化钠注射液、人血白蛋白、二甲基亚砜和复方电解质注射液,且各物质在所述制剂缓冲液中的体积百分比依次为:0.1%、10%、20%、2%、67.9%;其中,右旋糖酐40注射液的质量体积百分比为6%,氯化钠注射液的质量体积百分比为0.9%。
优选地,所述制备方法还包括:对脐带间充质干细胞进行传代培养后得到细胞上清液,所述细胞上清液中包括:9种促细胞迁移类细胞因子、9种受体类细胞因子、14种促细胞生长和分化类细胞因子、13种免疫调节类细胞因子和6种促血管生成类细胞因子。
优选地,所述制备方法还包括:对脐带间充质干细胞进行传代培养后得到细胞上清液,所述细胞上清液中包括肝细胞生长因子,且肝细胞生长因子的含量高于10000pg/mL。
优选地,所述制备方法还包括对脐带间充质干细胞进行传代培养后进行细胞冻存,所述细胞冻存的方法包括:取同一批培养至汇合度85%~95%的细胞,进行消化、离心、弃上清后使用冻存液按照5×106个细胞/mL~1×107个细胞/mL冻存密度重悬,得到细胞悬液,然后将所述细胞悬液分装至1.0mL冻存管中,经程序降温至-80℃后转入液氮保存,得到的冻存细胞稳定。
本发明第二方面提供了一种脐带间充质干细胞制剂,利用如第一方面所述的制备方法制备得到。
本发明第三方面提供了如第二方面所述的脐带间充质干细胞制剂在治疗膝骨关节炎中的应用。
本发明的有益效果是:本发明通过优化脐带间充质干细胞的分离方法、传代培养基以及制剂的制备方法,得到的脐带间充质干细胞制剂的制备工艺成熟稳定,制剂稳定性好,可直接用于骨关节炎的临床使用,无需冻存运输、临床使用时现场复苏等操作。同时采用本发明的方法培养的脐带间充质干细胞可分泌多种细胞因子,可有效改善骨关节炎的微环境,发挥免疫调节功能,有利于增强诱导软骨分化。
附图说明
图1为本发明实施例中组织贴壁法分离得到的脐带间充质干细胞P0代的细胞形态示意图;
图2为本发明实施例中酶消化法分离得到的脐带间充质干细胞P0代的细胞形态示意图;
图3为本发明实施例中组织贴壁法分离得到的脐带间充质干细胞P2代的细胞形态示意图;
图4为本发明实施例中酶消化法分离得到的脐带间充质干细胞P2代的细胞形态示意图;
图5为本发明实施例中不同分离方法的细胞生长曲线示意图;
图6为本发明实施例中HGF含量检测的标准曲线示意图;
图7为本发明实施例中P2代、P5代、P10代脐带间充质干细胞的形态特点及生长状态的图像示意图;
图8为本发明实施例中P2代细胞的分化成脂肪染色、成骨染色和成软骨染色的结果示意图;
图9为本发明实施例中P5代细胞的分化成脂肪染色、成骨染色和成软骨染色的结果示意图;
图10为本发明实施例中P10代细胞的分化成脂肪染色、成骨染色和成软骨染色的结果示意图;
图11为本发明实施例检测得到的57种细胞因子及其浓度分布示意图;
图12为本发明实施例检测得到的9种促细胞迁移类细胞因子及其浓度分布示意图;
图13为本发明实施例检测得到的9种受体类细胞因子及其浓度分布示意图;
图14为本发明实施例检测得到的14种促细胞生长和分化类细胞因子及其浓度分布示意图;
图15为本发明实施例检测得到的13种免疫调节类细胞因子及其浓度分布示意图;
图16为本发明实施例检测得到的6种促血管生成类细胞因子及其浓度分布示意图;
图17为本发明实施例中脐带间充质干细胞注射液对大鼠关节软骨损伤示意图;
图18为本发明实施例中脐带间充质干细胞注射液对大鼠膝关节HE染色检查示意图。
具体实施方式
为了更好地理解上述技术方案,下面将结合说明书附图以及具体的实施方式对上述技术方案做详细的说明。
实施例1 组织块贴壁及酶消化法分离脐带间充质干细胞
1.1贴壁法分离脐带间充质干细胞
将长20cm的脐带表面淤血清洗干净后,用镊子取出清洗后的脐带置于肾型盘中,将脐带纵向剪开,用镊子剥去脐带中的两根脐动脉和一根脐静脉,加入生理盐水冲洗,冲洗后将脐带剪成1.0-2.0mm3的脐带组织块,将脐带组织块平铺于T75培养瓶内,在培养瓶内加入含20%FBS(胎牛血清)的完全培养液3ml,缓慢摇匀使得脐带块均能接触到培养液,然后将培养瓶置于 5%CO2、37℃培养箱内静置2-3小时,待脐带组织块贴壁牢固后取出培养瓶,再向瓶中添加含20% FBS的完全培养液培养。置于5%CO2,37℃培养箱内培养,培养过程中培养液颜色变黄应换液,随培养时间延长可在脐带组织块周围观察到有细胞迁出,待贴壁细胞占培养瓶底总面积 85%以上时传代。
1.2 酶消化法分离脐带间充质干细胞:
脐带采集后在12h内进行处理,切除双侧夹痕及淤血的部分,用含双抗PBS缓冲液充分冲洗脐带外周以及脐静脉内腔,去除脐带上残留的淤血。将脐带剪开,剔除脐血管及脐带外膜,将剩余组织切成1.0-2.0mm3小块,放入50ml无菌离心管中,加入1g/L胶原酶Ⅱ,于37℃孵育16h,以PBS洗涤后加入25g/L胰酶于37℃孵育30min; 向离心管中加入FBS中止消化,然后以100µm的滤器过滤,收集细胞滤液;将细胞悬液离心弃上清,加入PBS洗涤后离心弃上清,反复洗涤数次至细胞悬液不再粘稠,离心管底部可见沉淀的细胞组织;以完全培养基重悬细胞,按1×104个细胞/cm2接种于T25培养瓶中,5%CO2,37℃培养箱培养,3d后全量换液,去除未贴壁细胞,待贴壁细胞汇合至70%-80%后传代。
1.3 UC-MSCs细胞生长情况的差异
1.3.1贴壁法:
(1)用5cm长的脐带,用贴壁法获得的组织,可以铺5个T75瓶进行原代细胞(P0代)的爬出培养。
(2)P0代细胞开始爬出组织块的时间约7天,利用P0代细胞进行四次传代培养,依次得到P1代、P2代、P3代、P4代细胞,在传代培养过程中,细胞汇合度达到70%-80%所需时间约7天。
1.3.2酶消化法:
(1)用5cm长的脐带,用酶消化法获得的组织,可以铺2个T75瓶进行原代细胞的爬出培养。
(2)利用P0代细胞进行四次传代培养,依次得到P1代、P2代、P3代、P4代细胞,在传代培养过程中,细胞汇合度达到70%-80%所需时间约4天。
组织贴壁法分离得到的脐带间充质干细胞P0代细胞形态与酶消化法分离得到的脐带间充质干细胞P0代细胞形态分别如图1和图2所示。从图1和图2可以看出,两种分离方法均有遮光性较强的长梭形单层P0代细胞爬出,细胞呈旋涡状排列。两种细胞形态差别不大。组织贴壁法分离得到的脐带间充质干细胞P2代细胞形态与酶消化法分离得到的脐带间充质干细胞P2代细胞形态分别如图3和图4所示。从图3和图4可以看出,两种分离方法获得的P2代UC-MSCs细胞均成长梭形单层细胞,细胞呈旋涡状排列。两种细胞形态差别不大。
1.4 UC-MSCs增殖能力的差异
通过 MTT法(四唑盐比色法)测定 P3代细胞增殖能力(以P3代的测定结果代表整个P0-P4代的培养周期的细胞增殖能力),结果如表1和图5所示。可以看出细胞接种后第 1天为潜伏期,第 2 天起细胞生长旺盛,开始进入指数生长期,3-5 天达到高峰后,进入平台期。由图5所示的生长曲线可见,不同分离方法得到的UC-MSCs增殖能力相似,贴壁法获得的UC-MSCs细胞增殖能力略高。
表1 不同分离方法得到的细胞增殖能力
1.5 UC-MSCs 表型的差异
由于UC-MSCs培养 P2代细胞中存在较多杂细胞,流式检测不准确,所以本发明实施例中按照企业标准将 P2 代细胞复苏至 P4 代,检测各组细胞表型。其中,企业标准要求:脐带间充质干细胞低表达(≤2%)CD34、CD11b、HLA-DR,高表达(≥95%)CD73、CD90、CD105。对贴壁法和酶消化法分离的两组UC-MSCs的P4代细胞进行细胞表型检测,各组的流式细胞检测结果见表2。
表2不同分离方法对P4代UC-MSCs表型的影响
1.6 结论
P0代次时,贴壁法在细胞爬出速率明显低于酶消化法,但是贴壁法操作程序相对简便,使用耗材、试剂的费用显著低于酶消化法。比较两者在P3代细胞的增殖速率,贴壁法获得的细胞增殖速率略高于酶消化法。比较P4代细胞的细胞形态和表面标记,贴壁法和酶消化法分离得到的脐带间充质干细胞在细胞形态、细胞表面标记等方面则没有太大的差别。经过多方面的比较,贴壁法具有费用低,操作简便,组织块损失少,获得的细胞数量多,后代细胞具有更快的生长速度等,遴选贴壁法用于后续工艺。
实施例2 脐带间充质干细胞分泌的肝细胞生长因子HGF的差异
2.1样本和试剂准备
2.1.1样本准备
本实施例选择的样本包括P2代、P4代和P5代细胞的培养上清,按照“序号-代次”的形式进行标记。比如 “M-2020042001-P2” 样本表示P2代序号为“M-2020042001”的细胞的培养上清。
标准品/样本稀释液(SR1)和待测样本按照体积比9:1配制待测样本稀释液。其中,标准品/样本稀释液(SR1)为做标准曲线时稀释标准品用的稀释液,不含HGF。待测样本为上述检测用的样本。
2.1.2试剂准备
(1)配制100mL洗涤液:用95mL双蒸水或去离子水将5mL20X浓缩洗涤液稀释成1倍应用液,未用完的浓缩洗涤液放入4℃冰箱保存。
(2)标准品梯度稀释:加入标准品/样本稀释液(SR1)1mL至冻干标准品中,静置15分钟待其完全溶解后轻轻混匀(浓度为8000pg/mL),然后在其余七管中各加入500μL标准品/样本稀释液(SR1),按照以下浓度进行2倍稀释:8000、4000、2000、1000、500、250、125、0pg/mL进行稀释。8000pg/mL为标准曲线最高点浓度,标准品/样本稀释液(SR1)作为标准曲线的零点(0pg/mL)。
2.2实验操作
(1)试剂回温:在实验前30min取出试剂盒,待测样本,恢复至室温。
(2)预洗板:标准品/样本加入前,加入洗涤液0.3mL/孔,洗板3次并甩干。
(3)加样并孵育:加入100μL标准品及检测样本至反应孔中,封板后于37℃孵箱孵育90min。配制2mL生物素化抗体工作液:吸取1.98mL检测稀释液(SR2)将20μL的100X抗体浓缩液稀释成1X应用工作液(稀释前充分混匀)。
(4)第一次洗板:加入洗涤液0.3mL/孔,洗板4次并甩干。
(5)加样生物素化抗体工作液并孵育:100μL/孔加入到反应孔中。封板后于37℃孵箱孵育60min。
(6)配制2mL酶结合物工作液:吸取1.98mL用酶结合物稀释液(SR3)将20μL的100X浓缩酶结合物稀释成1X应用工作液(稀释前离心)。
(7)第二次洗板:加入洗涤液0.3mL/孔,洗板4次并甩干。
(8)加样酶结合物工作液并孵育:100μL/孔加入到反应孔中。封板后于37℃孵箱孵育30min。
(9)第三次洗板:加入洗涤液0.3mL/孔,洗板5次并甩干。
(10)加样显色底物:加入100μL/孔至反应孔中,封板后于37℃避光显色15min。
(11)终止显色反应:加入50μL/孔终止液。
(12)测量OD值:加终止液5分钟内,酶标仪450nm、630nm波长下测OD值。
2.3 实验结果
标准曲线如图6所示,各样本对应的HGF含量如表3所示。图6的标准曲线为:y=0.0002396x+0.0007545,R²=0.9999。其中,x和y分别为图6中各点数据(x,y),即与坐标轴对应的数据。R2是标准曲线拟合的确定系数,表示拟合程度好坏。
表3 各样本HGF含量
其中,表3中的空白对照指的是不含细胞的传代培养基,其中不含HGF。
由表3可知,不同脐带间充质干细胞培养上清中HGF的含量不同,同一脐带的不同代次之间培养上清的HGF含量不同。但是,按照本发明实施例提供的方案得到的人脐带间充质干细胞培养上清中HGF含量均高于10000pg/mL。与现有技术相比,本发明实施例的培养上清中的HGF含量较高。
实施例3 细胞库冻存工艺
3.1 冻存密度
取同一批正常培养至汇合度85%~95%的 P4代细胞,消化、离心、弃上清后使用冻存液按照以下冻存密度重悬:5×106个细胞/mL、1×107个细胞/mL、1.5×107个细胞/mL、2×107个细胞/mL。将细胞悬液分装至1.0mL冻存管中,经程序降温至-80℃后转入液氮保存。保存4周后复苏细胞,取清洗液检测无菌,将冻存管内细胞悬液转移至含0.9%氯化钠注射液离心管中,离心重悬后测定细胞数量、细胞活率、支原体、细胞表型。结果如表4所示。
表4 不同冻存密度的检测结果
3.2 降温程序
取同一批正常培养的P4代细胞,消化、离心、弃上清后使用冻存液重悬,将密度调整至1.0×107/mL后,取1mL转入冻存管中,分别采用阶梯降温法(4℃ 30min、-18℃ 1~2h、-80℃ 12h、液氮)、程序降温法(1℃/min降温至-80℃、12~24h后液氮保存)、直接法(4℃直接-80℃保存12~24h后液氮保存)。液氮保存4周后复苏,检测细胞活率、细胞数量、无菌、支原体、细胞表型。结果如表5所示。
表5 细胞冻存降温程序的结果
实施例4 传代培养
4.1培养液体积
取同一批次工作细胞库(P4代)3支,复苏培养后,消化、离心、重悬、计数后分别取5.7×107细胞接种至3个细胞工厂,3个细胞工厂分别添加1050mL、1575mL、2100mL完全培养基。混合均匀后置于二氧化碳培养箱内(培养条件:37±1℃,5±0.5%CO2、饱和湿度)培养3~4天。
将培养液倒出细胞工厂,收集备用。每个细胞工厂加入360mL复方电解质注射液清洗后。每个细胞工厂加入经1:6(v:v)稀释的消化液(25g/L胰酶)280mL,于二氧化碳培养箱(37±1)℃环境中消化。消化结束后每个细胞工厂加入300mL上清液终止消化。使用400mL0.9%氯化钠注射液清洗每个细胞工厂,将清洗液和细胞悬液混合均匀,转移至50mL离心管中,离心弃上清,溶媒重悬细胞后进行计数。结果如表6所示。
表6 细胞工厂培养液体积结果
培养液体积 | 1050mL | 1575mL | 2100mL |
细胞数量 | 1.7×108 | 2.2×108 | 3.4×108 |
细胞活率 | 88.3% | 89.6% | 94.8% |
4.2 细胞传代接种量
取同一批次汇合度达到85%~95%的P4代细胞,经消化酶消化、收集细胞、细胞计数后,分别以10000/孔接种于24孔板,接种14孔,培养液体积1mL/孔。每隔24h,将一孔中培养液取出备用,加入1mL0.9%氯化钠注射液清洗后弃去,该孔加入0.2mL 1:6(v:v消化液:氯化钠注射液)稀释后的消化液,消化10min。消化后加入备用培养液终止消化,混合均匀后进行细胞数量检测。以后每天按照相同方法消化一孔,记录细胞数量。14天后以细胞数量为纵坐标,生长天数为横坐标,绘制生长曲线。
得到生长曲线后即可得到脐带间充质干细胞在该培养体系下的对数期范围2~4天、群体倍增时间21小时。根据汇合度研究结果,T175瓶达到99%汇合细胞量在1.3×107。反推每T175瓶接种量范围为:1.6×106~3.2×106。设置不同接种量进行传代工艺研究:
选取正常培养至汇合85%~95%的P3代细胞,消化酶消化为单细胞悬液,离心、弃上清,加入细胞培养液,将细胞悬液密度调至1×107/mL,设置3组(组一、组二、组三)不同接种密度接种至T175瓶中,摇晃均匀后置于细胞培养箱中(培养条件:37±1℃,5±0.5%CO2、饱和湿度)。观察每瓶细胞汇合度,消化、离心、重悬后检测细胞数量和细胞活率。结果如表7所示。
表7 细胞传代接种量研究结果
结果分析:接种量为1.0×106/T175瓶时细胞达不到最佳对数生长期,细胞数量及细胞活率均低于1.6×106/T175瓶,而接种量达到3.2×106/T175瓶时细胞生长速度过快而过于密集,导致细胞凋亡。
取同一批次工作细胞库(P4代)3支,经WCB细胞复苏、P5代细胞培养、P6代细胞培养等工序后,培养至细胞间汇合度达到85%~95%后,经消化、离心、重悬、计数后分别取4×107,6×107,8×107细胞量分别接种至3个细胞工厂,3个细胞工厂均添加以下2100mL完全培养液。混合均匀后置于二氧化碳培养箱内培养。消化酶消化,离心弃上清,细胞沉淀重悬,分别统计每个细胞工厂得到的细胞活率及细胞数量。结果如表8所示。
表8 细胞接种量研究结果
结果分析:接种4×107细胞明显未达到对数生长期,细胞数量及细胞活率均低于接种量6×107,而接种8×107细胞工厂第3天即可观察到部分细胞脱落,表明接种细胞量过多,细胞开始出现凋亡。结合细胞工厂中培养液量的研究结果,接种6×107的细胞量均能使细胞达到对数期。
实施例5 消化工艺
取同一批次细胞P0代细胞培养瓶4瓶,根据前述消化酶使用体积研究得到的消化液量最小体积,加入10mL消化酶(消化酶与0.9%氯化钠注射液按照体积比1:6的比例混合均匀)。细胞脱落后终止消化,离心、重悬细胞,进行细胞计数。
取同一批P2 代细胞培养瓶9瓶,每组3瓶,于二氧化碳培养箱中培养。分别添加消化液量7mL进行消化。显微镜下观察细胞消化情况。待观察到所有细胞均变圆终止消化,记录所有细胞变圆所消耗的时间(即为消化完成时间)后添加终止液终止消化,将消化液、终止液混合均匀后转移至离心管中。离心、重悬后,测定细胞活率和细胞数量。
取同一批次工作细胞库(P4代)4支,复苏后培养,经消化酶消化,离心得到P5细胞沉淀,完全培养液重悬细胞,计数后取6×107细胞量接种至4个细胞工厂。4个细胞工厂添加2100mL培养液,混合均匀后置于二氧化碳培养箱内。将培养液倒出细胞工厂,收集备用。每个细胞工厂加入360mL复方电解质清洗后。4个细胞工厂分别加入280mL经1:6(v:v)稀释的消化液。离心、重悬后,测定每个细胞工厂得到的细胞活率及细胞数量。
消化酶消化过程可使用培养上清液终止,也可以选择用血清终止,但血清存在动物源风险;上清液中过量的蛋白可与消化酶结合,竞争性抑制消化酶的作用,其次上清液中存在金属离子也可抑制消化酶的作用。
选取P2代冻存细胞,经复苏传代、培养后(P3代),收集每瓶上清液备用,吸取10mL0.9%氯化钠注射液清洗培养层细胞,清洗后弃去。加入7mL 1:6(v:v)稀释的消化酶。于培养箱中37±1℃消化10~15min,直至全部细胞脱落后,分别加入等体积胎牛血清、等体积上清液、双倍体积上清液终止消化,混合均匀后转移至离心管中。离心后弃上清。完全培养液重悬细胞,进行细胞计数。结果如表9所示。从结果可知,血清和上清液的终止效果无显著差异。
表9 消化酶终止方式研究结果
实施例6 细胞冻存稳定性
取P2、P4、P5代细胞,按照操作规范冻存细胞,液氮保存6月、12个月后复苏细胞,检测干细胞数量与活性、细胞表型,对其进行微生物和支原体进行检测,检测结果如表10所示。
表10 细胞冻存稳定性结果
实施例7 即用型制剂
7.1 方法:选取临床常用输注液:0.9%氯化钠注射液、5%葡萄糖注射液、复方电解质注射液细胞与制剂进行比较。实验温度设定为室温 25℃和恒温保存箱 4℃下进行对比。同源同批次间充质干细胞,分别置于 4℃和 25℃保存。在3个时间点(2h、4h、6h)检测各实验组细胞存活率,细胞增殖能力及倍增时间及UC-MSCs 表面特异性抗原,细胞贴壁率等。
7.2 检测结果
7.2.1 细胞活率检测
(1)细胞活率检测选用台盼蓝试剂。计数过程应控制在 3 分钟之内,避免染色过程时间过长细胞出现假阳性。
细胞活率(%)=活细胞数/细胞总数×100%。
(2)不同输注液对UC-MSCs活性的影响
不同温度和保存时间细胞活性如表11、表12所示。
表11 4℃条件下保存6hr的细胞活性
表12 25℃条件下保存6hr的细胞活性
(3)检测结果显示,1)制剂保存的UC-MSCs在不同温度条件下均能保持较高活性。2)总体来看,4℃条件下保存6hr的细胞活性高于23℃条件下保存6hr的细胞活性,说明温度对细胞活性有影响。4℃条件下更有利于细胞的保存。3)对比3种输注保存UC-MSCs的效果,在4℃或 23℃条件下各时间点,复方电解质注射液保存细胞活性最佳,0.9%氯化钠注射液次之,5%葡萄糖注射液保存的细胞活性最差。
7.2.2细胞贴壁率检测
间充质干细胞是贴壁细胞,细胞贴壁后才能增殖分裂,测定不同人输注液保持UC-MSCs 不同时间点的细胞贴壁率与细胞倍增时间,间接测定细胞增殖能力。
(1)抽取上述4℃或 23℃条件下保持 6 小时血袋中不同保存介质的细胞混悬液,置于 50mL离心管中,加入 30mL 完全培养液冲洗细胞后,400g 离心 5min,弃去上清。完全培养液重悬细胞,调节细胞浓度,按 1×104/孔接种 24 孔板。5% CO2、37℃培养箱内静置培养24小时,生理盐水洗涤 2 次,洗去未贴壁细胞后,用4%多聚甲醛固定。20min 后生理盐水洗涤 2 次,结晶紫染色 15min。生理盐水洗涤 3 次,去除背景色。倒置显微镜下观察计数:
细胞贴壁率(%)=贴壁细胞数/接种细胞数×100%。
(2)对 UC-MSCs 贴壁率的的影响
在不同温度和保存时间细胞贴壁率如表13、表14所示。
表13 4℃条件下细胞贴壁率与时间关系
表14 25℃条件下细胞贴壁率与时间关系
(3)检测结果显示:1)将UC-MSCs 保存于制剂,灌注在一次性使用塑料血袋中保存6hr后,细胞贴壁能力未受太大影响。但保存于复方电解质注射液、5%葡萄糖注射液、0.9%氯化钠注射液中的UC-MSCs 细胞贴壁率呈时间依赖性下降。复方电解质注射液和0.9%氯化钠注射液较5%葡萄糖注射液保存的细胞,UC-MSCs的贴壁率较好。
7.2.3脐带间充质干细胞制剂在4℃条件下的稳定性
脐带间充质干细胞制剂,4 ℃条件下保存,并于 0h、6h、12h、18h、24h、30h 抽样,对其进行相关指标的检测,检测结果如表15所示。从结果可知:4℃条件下干细胞保存18小时,细胞活率可维持在80%以上,质量稳定。
表15 4℃条件下细胞保存稳定性
实施例8 传代稳定性
8.1 细胞传代培养
主细胞库(利用P2代细胞组建):脐带间充质干细胞扩增至P2代后,可以有足够数量的细胞用于质量检测且能够为后续工作细胞库的构建留有足够数量的细胞,P2代细胞特性相对后续代次更接近最原始的P0代细胞。
工作细胞库(利用P4代细胞组建):脐带间充质干细胞在传至P10代后仍能保持与P2代一致的质量,按照使用代次=2/3研究最大代次的数值,选取P6代为使用代次,选择P4代细胞建立工作细胞库冻存,可保证P4代细胞复苏传代培养至P5代后再传至P6代时细胞功能完全恢复。传代培养至P10代,传代过程中记录相应代次细胞形态,自细胞收获得到的细胞悬液中取样检测,根据检测结果判断细胞的传代是否稳定。检测结果:细胞形态、细胞活率、细胞表型、分化能力等。
8.2检测结果
8.2.1不同代次干细胞形态、活率稳定性
应用倒置显微镜观察不同代次(P2代、P5代、P10代)脐带间充质干细胞的形态特点及生长状态,采集图像(如图7所示),可见细胞形态均为长梭形贴壁细胞,形态稳定。
重组胰酶消化后取部分细胞使用台盼蓝染液进行染色,显微镜下鉴定细胞活率。细胞活率:P2 、P5、P10传代活率均大于 80%。说明不同代次(P2、P5、P10)的脐带间充质干细胞形态稳定、活率稳定在80%以上。
8.2.2 不同代次干细胞流式标记稳定性
取培养的P2、P5、P10代细胞进行CD90、CD105、CD73、CD34、CD45、CD19、CD11b、HLA-DR检测。结果可如表16所示,从表16可以看出,不同代脐带间充质干细胞表型稳定。
表16 不同代脐带间充质干细胞表型稳定性检测结果
8.2.3不同代次的脐带间充质干细胞分化能力稳定性
取P2、P5、P10代细胞,按照脐带间充质干细胞分化鉴定标准操作规程进行鉴定,各代次细胞均具有分化成脂、成骨、成软骨的能力,分别如图8-10所示。从鉴定结果可知,不同代次的脐带干细胞均具有分化能力,可稳定保持干细胞分化特性。
实施例9 UC-MSCs分泌的细胞因子的检测
9.1实验步骤
9.1.1 样本准备
经传代培养基传代后,收集UC-MSCs上清液,4℃冰箱保存备用。传代培养基包括DMEM培养基以及以DMEM培养基的用量为基准的:质量体积百分比为5%的UltraGROTM-Advanced,10mmol/L的谷胱苷肽,1nmol/L的胰岛素样生长因子,10ng/ml的重组人源血管内皮生长因子,10U/mL的血小板衍生生长因子,5μg/ml的纤连蛋白,10ng/mL的重组人碱性成纤维细胞生长因子,10ng/L的粒细胞-巨噬细胞集落刺激因子,3mmol/L的谷氨酰胺,10ng/ml的重组人表皮生长因子,4mg/L的L-抗坏血酸,50mg/L的β-巯基乙醇,1mg/L的乙醇胺,0.1g/L的丙二醇嵌段聚醚,100mg/L的吐温80,20mg/L的胆固醇,0.2mg/L的腺嘌呤,2µg/mL的硫酸镁,质量体积百分比为1.0%的硫酸氨基葡萄糖,1µmol/L的甘油磷酸钠,100U/mL的青霉素,100mg/L的链霉素;所述传代培养基的pH为6.8-7.2。
9.1.2 样本检测
采用荧光标记的Streptavidin芯片检测方法检测样本中的UC-MSCs分泌的细胞因子。
9.2实验结果
共检测出57种细胞因子,其浓度分布可如图11所示。57种细胞因子包括:促细胞迁移类9种,其浓度分布可如图12所示,浓度较高的有MCP-1、GRO、MCP-3和GCP-2;受体类9种,其浓度分布可如图13所示,浓度较高的有Fas/TNFRSF6、IL-1R4/ST2和uPAR;促细胞生长和分化类14种,其浓度分布可如图14所示,浓度较高的有Osteo-protegerin、Activin A和IL-8;免疫调节类13种,其浓度分布可如图15所示,浓度较高的有IL-1β、TNF-β、IL-1α、IFN-γ和TNF-α;促血管生成类6种,其浓度分布可如图16所示,浓度较高的有:IL-6、VEGF。
从实验结果可知,本发明培养的UC-MSCs可分泌多种细胞因子,能够有效改善骨关节炎的微环境,发挥细胞因子的免疫调节功能,有利于增强诱导软骨分化。
实施例10 脐带间充质干细胞注射液(即用型)对大鼠膝骨关节炎的影响
研究方法:采用手术法诱导Lewis大鼠膝骨关节炎模型,右侧膝关节单侧造模,造模后进行行为学评价及X光检查并进行K-L评分,K-L评分1.0分及以上的为膝骨关节炎。观察培养的脐带间充质干细胞制剂对大鼠膝骨关节炎的影响。采用关节腔内注射给药,给药体积50 μL /关节腔(含1.5×106 个细胞)。研究指标包括:膝关节X光评分、关节软骨大体观察评分、血清指标检测和组织病理学HE染色检测。
研究结果:
1、膝关节X光K-L评分
对膝关节X光评分的影响结果如表17所示,表中数据为各组K-L评分均值±标准差,比如,未给药组在给药前的K-L评分均值±标准差为3.0±0.0。
给药前及给药后,与正常对照组相比,未给药组可见,X光K-L评分显著升高(P≤0.01)。与未给药组相比,给药组给药后可显著降低大鼠X光K-L评分(P≤0.05)。
表17 对膝关节X光K-L评分的影响(n=10)
注:1. 与正常对照组相比,△△ P≤0.01;与未给药组相比,* P≤0.05;2. n表示动物数。
2、关节软骨大体观察评分
对关节软骨大体评分的影响结果如表18所示,表中数据为各组关节软骨大体评分均值±标准差,比如,未给药组的关节软骨大体评分均值±标准差为2.7±0.2。
观察大鼠膝关节软骨损伤情况,如图17所示,其中,A图表示正常对照组,B图表示未给药组,C图表示给药组。从图中可看出正常对照组大鼠软骨关节面光滑,色泽红润;与正常对照组相比,未给药组大鼠关节软骨表面灰暗、粗糙、有小裂痕,部分关节软骨面出现溃疡、缺损,有关节腔积液。根据Pelletier评分标准,关节软骨大体评分显著升高(P≤0.01)。与未给药组相比,给药组可显著改善软骨面光泽度、表面粗糙、溃疡、裂纹等情况,降低大鼠关节软骨大体观察评分(P≤0.05)。
表18 对关节软骨大体评分的影响( n=10)
注:1. 与正常对照组相比,△△ P≤0.01;与未给药组相比,* P≤0.05;2. n:表示动物数
3、血清炎性因子及软骨代谢标志物检测
对血清炎性因子及软骨代谢标志物的影响结果如表19所示,表中数据为各检测结果均值±标准差,比如,未给药组的IL-1β的检测结果均值±标准差为47.2±1.8pg/mL。
如表19所示,与正常对照组相比,未给药组血清中白介素-1β(IL-1β)、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、II型胶原C端肽(CTX-II)、前列腺素 E2(PGE2)含量明显升高,转化生长因子-β(TGF-β)含量明显降低(P≤0.05),与未给药组相比,关节腔内注射给药后可显著降低血清中IL-1β、IL-6、CTX-Ⅱ、PGE2含量,升高TGF-β含量(P≤0.01)。
表19 对血清炎性因子及软骨代谢标志物的影响( n=10)
注:1. 与正常对照组相比,△△ P≤0.01;与未给药组相比,* P≤0.05, ** P≤0.01;2.n:表示动物数。
4.组织病理学检测(HE染色)
膝关节HE染色检查如图 18所示,其中,A图表示正常对照组,B图表示未给药组,C图表示给药组。从图中可以看出:正常对照组滑膜组织结构正常、无增生,关节软骨表面光滑平整,软骨层及软骨细胞、软骨下骨、骨小梁大小、排列正常,未见明显病理改变。未给药组滑膜组织增生,内膜下层纤维结缔组织增多,炎细胞浸润,毛细血管增多,增生的滑膜组织覆盖于关节软骨表面;软骨层被破坏,软骨细胞变性/坏死,伴纤维化变性;软骨下骨被破坏,部分被纤维结缔组织所覆盖,骨小梁间纤维组织增多。给药组膝关节滑膜组织增生,内膜下层纤维结缔组织增多,软骨层被破坏,软骨细胞变性/坏死,伴纤维化变性,软骨层增厚;软骨下骨被破坏,部分被纤维结缔组织所覆盖,骨小梁间纤维组织增多。
对HE染色评分的影响结果如表20所示,表中数据为各HE染色评分均值±标准差,比如,未给药组的滑膜的HE染色评分均值±标准差为3.2±0.2。与正常对照组相比,未给药组大鼠膝关节滑膜、软骨及软骨下骨的病理组织学评分显著升高(P≤0.01),与未给药组相比,给药组显著降低大鼠膝关节滑膜、软骨、软骨下骨的病理组织学评分(P≤0.05)。
表20 对HE染色评分的影响( n=10)
组别 | 滑膜 | 软骨 | 软骨下骨 |
正常对照组 | 0.0±0.0 | 0.0±0.0 | 0.0±0.0 |
未给药组 | 3.2±0.2△△ | 3.4±0.2△△ | 2.6±0.3△△ |
给药组 | 2.1±0.2* | 2.3±0.2* | 1.2±0.2* |
注:1. 与正常对照组相比,△△ P≤0.01;与未给药组相比,* P≤0.05;2. n表示动物数。
研究结论:关节腔内注射上述本发明提供的脐带干细胞制剂对大鼠膝骨关节炎有明显的治疗作用,可不同程度地改善大鼠膝关节X光评分、关节软骨大体观察评分、血清炎症因子及软骨代谢标志物含量、组织病理学评分等。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (6)
1.一种脐带间充质干细胞制剂的制备方法,其特征在于,包括:
从脐带中分离得到脐带间充质干细胞;
对脐带间充质干细胞进行传代培养,传代培养两次后得到种子细胞,传代培养四次后得到工作细胞库;
对工作细胞进行复苏培养、消化和洗涤后,加入制剂缓冲液,形成用于临床使用的脐带间充质干细胞制剂;
其中,传代培养中用到的传代培养基包括DMEM培养基以及以DMEM培养基的用量为基准的:质量体积百分比为1~10%的UltraGROTM-Advanced,1~20mmol/L的谷胱苷肽,1~30nmol/L的胰岛素样生长因子,10~60ng/ml的重组人源血管内皮生长因子,10~500U/mL的血小板衍生生长因子,1~10μg/ml的纤连蛋白,10~50ng/mL的重组人碱性成纤维细胞生长因子,10~50ng/L的粒细胞-巨噬细胞集落刺激因子,1~5mmol/L的谷氨酰胺,10~60ng/ml的重组人表皮生长因子,4mg/L的L-抗坏血酸,5~100mg/L的β-巯基乙醇,0.5~5mg/L的乙醇胺,0.1~0.5g/L的丙二醇嵌段聚醚,10~1000mg/L的吐温80,2~50mg/L的胆固醇,0.2~5.0mg/L的腺嘌呤,2~10µg/mL的硫酸镁,质量体积百分比为0.5~1.5%的硫酸氨基葡萄糖,0.5~5µmol/L的甘油磷酸钠,100U/mL的青霉素,100mg/L的链霉素;所述传代培养基的pH为6.8-7.2;
对脐带间充质干细胞进行传代培养后得到细胞上清液,所述细胞上清液中包括:9种促细胞迁移类细胞因子、9种受体类细胞因子、14种促细胞生长和分化类细胞因子、13种免疫调节类细胞因子和6种促血管生成类细胞因子;
对脐带间充质干细胞进行传代培养后得到细胞上清液,所述细胞上清液中包括肝细胞生长因子,且肝细胞生长因子的含量高于10000pg/mL;
4℃条件下脐带间充质干细胞制剂保存18小时,细胞活率维持在80%以上。
2.如权利要求1所述的制备方法,其特征在于,采用组织块贴壁法或酶消化法从脐带中分离得到脐带间充质干细胞。
3.如权利要求1所述的制备方法,其特征在于,所述制剂缓冲液包括如下物质:低分子肝素钙注射液、右旋糖酐40注射液、人血白蛋白、二甲基亚砜和复方电解质注射液,且各物质在所述制剂缓冲液中的体积百分比依次为:0.1%、10%、20%、2%、67.9%;其中,右旋糖酐40注射液的质量体积百分比为6%。
4.如权利要求1所述的制备方法,其特征在于,所述制剂缓冲液包括如下物质:低分子肝素钙注射液、氯化钠注射液、人血白蛋白、二甲基亚砜和复方电解质注射液,且各物质在所述制剂缓冲液中的体积百分比依次为:0.1%、10%、20%、2%、67.9%;其中,氯化钠注射液的质量体积百分比为0.9%。
5.如权利要求1所述的制备方法,其特征在于,所述制备方法还包括对脐带间充质干细胞进行传代培养后进行细胞冻存,所述细胞冻存的方法包括:取培养至汇合度85%~95%的细胞,进行消化、离心、弃上清后使用冻存液按照5×106个细胞/mL~1×107个细胞/mL冻存密度重悬,得到细胞悬液,然后将所述细胞悬液分装至1.0mL冻存管中,经程序降温至-80℃后转入液氮保存,得到的冻存细胞稳定。
6.一种脐带间充质干细胞制剂,其特征在于,利用如权利要求1-5任一项所述的制备方法制备得到。
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