CN114196632A - 一种高效体外培养造血干细胞的培养基 - Google Patents
一种高效体外培养造血干细胞的培养基 Download PDFInfo
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Abstract
本申请涉及一种高效体外培养造血干细胞的培养基,属于细胞体外培养技术领域。本申请的一种高效体外培养造血干细胞的培养基,含有基础培养基和添加因子;所述基础培养基为无血清培养基;重组人干细胞因子:50‑200ng/ml、重组人血小板生成素:20‑100ng/ml、重组人FMS样酪氨酸激酶3配体:100‑200ng/ml、白介素‑3:30‑60ng/ml、聚乙烯醇:1‑10mg/ml、胰岛素‑转铁蛋白‑硒‑乙醇胺添加剂:1‑2:100ml/ml、吲哚类似物:10‑200ng/ml、氮杂环化合物:10‑200ng/ml。解决了现有技术中存在的造血干细胞扩增率低、易分化等缺陷;将造血干细胞增殖效率提高了50‑100倍,得到的造血干细胞数量更多。
Description
技术领域
本申请涉及一种高效体外培养造血干细胞的培养基,属于细胞体外培养技术领域。
背景技术
造血干细胞移植技术是临床上治疗白血病、淋巴瘤、再生障碍性贫血、地中海贫血等多种血液类疾病和免疫系统疾病的常用且有效的治疗手段。造血干细胞通常有三个来源:骨髓、外周血和脐带血。与骨髓和外周血造血干细胞相比,脐带血造血干细胞获取方便、来源丰富、对供者无损伤无副作用,因此成为造血干细胞移植供体的一大重要来源。
目前,脐带血造血干细胞移植技术的瓶颈在于脐带血中的造血干细胞含量少,一份脐带血中所含的造血干细胞及祖细胞数量不足以快速恢复一位成人患者的免疫系统,而造成机会性感染致死率的增高。目前暂行的策略是双份脐带血移植,即一位患者清髓后先后接受两份脐带血的移植,但这增加了供体的HLA配型匹配难度,因此,亟需扩增脐带血造血干细胞的方法,以获得足量的可供移植的造血干细胞。
人们对于造血干细胞的体外扩增进行了大量尝试,但都没有取得理想效果。早期人们利用血液中的细胞因子来培养造血干细胞,结果导致细胞分化,而移植功能减弱。后来,人们发现骨髓造血干细胞微环境中的Wnt(分泌型糖蛋白)信号分子、Notch配体、视黄酸拮抗因子等能够有效扩增CD34+造血干/祖细胞。利用CHIR99021(化学式:C22H18Cl2N8)或者BIO(化学式C16H10BrN3O2)激活Wnt信号通路维持体外培养的造血干细胞的移植能力;而在造血干细胞的培养体系中添加DLL1抗体(Notch信号通路Delta样配体1抗体),DSL1(Notch信号通路 Delta样配体1的衍生多肽)等,能够通过激活Notch信号而适度扩增造血干细胞。另有研究发现,骨髓内皮基质细胞分泌的PTN(多效生长因子)也能够轻微扩增造血干细胞。生理状态下造血干细胞处在低氧条件下,而体外培养产生的氧胁迫会通过增高ROS(细胞内活性氧)水平损害造血干细胞的自我更新和移植功能;人们发现,抗氧化剂的添加以及mTOR(雷帕霉素靶蛋白)的抑制能够抵消这些损害。然而,上述技术并没能够显著扩增脐带血造血干细胞。偶然的发现,铜离子螯合剂TEPA(膦酰基乙酸三乙酯),SIRT(沉默调节蛋白)抑制剂 Nicotinamide(烟酰胺)能够显著提高造血干细胞移植水平,且在临床实验中显示初步疗效,但扩增后的细胞体内存活时间不够长,且分化谱系不够完整。总的来说,造血干细胞最佳的体外扩增条件至今尚没有明确的共识。
发明内容
为了解决现有体外培养造血干细胞技术存在的增加造血干细胞数量少、同时造血干细胞CFU集落形成能力差的技术问题,本申请提供了一种高效体外培养造血干细胞的培养基。该培养基能增加造血干细胞数量同时又提高造血干细胞CFU 集落形成能力的效果,使得造血干细胞能够处于增殖不分化的状态,进而达到临床移植需求。
本发明的技术方案如下:
一种高效体外培养造血干细胞的培养基,由基础培养基和添加因子组成;
所述基础培养基为无血清培养基;
所述添加因子及其相对于基础培养基的含量如下:
重组人干细胞因子:50-200ng/ml、
重组人血小板生成素:20-100ng/ml、
重组人FMS样酪氨酸激酶3配体:100-200ng/ml、
白介素-3:30-60ng/ml、
聚乙烯醇:1-10mg/ml、
胰岛素-转铁蛋白-硒-乙醇胺添加剂:1-2:100ml/ml、
吲哚类似物:10-200ng/ml、
氮杂环化合物:10-200ng/ml。
本发明通过在无血清培养基的基础上,添加了特定量的重组人干细胞因子(SCF)、重组人血小板生成素、重组人FMS样酪氨酸激酶3配体rhFLT3L、白介素-3(IL-3)、聚乙烯醇、胰岛素-转铁蛋白-硒-乙醇胺、UM171和SR1。其中,各添加因子在本发明培养基中的具体作用为:
干细胞因子(Stem Cell Factor,SCF)是重要的造血细胞因子,它是酪氨酸激酶受体c-Kit的配体。SCF主要作用于早期造血干细胞、原始造血祖细胞,并且诱导这些细胞的存活、增殖和分化。SCF单独使用生物学作用低,但它与其它细胞因子具有强的协同作用。重组人干细胞因子(Recombinant Human Stem Cell Factor,rhSCF),是指采用基因工程方法,使用大肠杆菌等表达的人的干细胞因子。
重组人血小板生成素(TPO)可促进造血干细胞的增殖。
重组人FMS样酪氨酸激酶3配体rhFLT3L可促进造血干细胞增殖。
白介素-3主要刺激髓系细胞扩增分化的多系细胞因子,促进造血干细胞增殖和集落形成。
重组人干细胞因子、重组人血小板生成素、重组人FMS样酪氨酸激酶3配体和白介素-3协同作用可促进造血干细胞的增殖和维持造血干细胞的自我更新。
聚乙烯醇是极安全的高分子有机物,可为造血干细胞体外培养的基底材料,为细胞生长提供合适的场所。
胰岛素-转铁蛋白-硒-乙醇胺作为一种细胞培养生长添加剂可降低细胞维持和低密度贴壁所需的胎牛血清浓度,其中胰岛素促进葡萄糖和氨基酸的吸收,转铁蛋白促进铁离子转运,硒维持谷胱甘肽的还原性,乙醇胺是磷脂的前体。
吲哚类似物和氮杂环化合物这两个小分子被认为是较为成熟稳定的造血干细胞体外扩增剂,且经一段时间的体外扩增培养后的细胞仍具有较强干性。
实验研究证明,氮杂环化合物SR1和吲哚类似物UM171能够更有效的扩增具备长期移植能力的造血干细胞。临床实验表明,SR1扩增的造血干细胞具备重建患者免疫系统的能力,但其依然没有摆脱对双份脐带血移植的依赖。但是,在本申请中,氮杂环化合物SR1和吲哚类似物UM171与其他添加因子之间具有协同作用;各添加因子之间协同作用提高造血干细胞的增殖速度及造血干细胞的干性。
上述本申请的一种高效体外培养造血干细胞的培养基,可选的,
所述添加因子及其相对于基础培养基的用量如下:
重组人干细胞因子:50-100ng/ml、
重组人血小板生成素:20-50ng/ml、
重组人FMS样酪氨酸激酶3配体:100-150ng/ml、
白介素-3:30-50ng/ml、
聚乙烯醇:1-6mg/ml、
胰岛素-转铁蛋白-硒-乙醇胺添加剂:2:100ml/ml、
吲哚类似物:10-100ng/ml、
氮杂环化合物:10-100ng/ml;
具体的,所述添加因子及其相对于基础培养基的用量如下:
重组人干细胞因子:100ng/ml、
重组人血小板生成素:50ng/ml、
重组人FMS样酪氨酸激酶3配体:150ng/ml、
白介素-3:50ng/ml、
聚乙烯醇:6mg/ml、
胰岛素-转铁蛋白-硒-乙醇胺添加剂:2:100ml/ml、
吲哚类似物:100ng/ml、
氮杂环化合物:100ng/ml。
本申请中,
所述重组人干细胞因子rhSCF可以根据现有技术自行制备,也可以从现有商品中选择,具体的可以选择Peprotech AF-250-03;
所述重组人血小板生成素rhTPO可以根据现有技术自行制备,也可以从现有商品中选择,具体的可以选择Peprotech AF-315-14;
所述重组人FMS样酪氨酸激酶3配体rhFLT3L可以根据现有技术自行制备,也可以从现有商品中选择,具体的可以选择R&D Bio-Techne 308E-GMP。
所述重组人白介素-3可以根据现有技术自行制备,也可以从现有商品中选择,具体的可以选择Peprotech 200-03;
所述聚乙烯醇是指聚乙烯醇(PVA);可以根据现有技术自行制备,也可以从现有商品中选择,具体的可以选择上海阿拉丁生化科技股份有限公司,品牌:阿拉丁,醇解度:72.5-74.5mol%,黏度:4.2-5.0mPa.s,CAS编号9002-89-5;
所述胰岛素-转铁蛋白-硒-乙醇胺添加剂(ITS-X)可以根据现有技术自行制备,也可以从现有商品中选择,具体的可以选择Gibco 51500-056。
所述的述吲哚类似物(UM171)可以从现有商品中选择,具体的可以选择MedChemExpress HY-12878。
所述的氮杂环化合物(SR1)可以从现有商品中选择,具体的可以选择MedChemExpressHY-15001。
有益效果
1、解决了现有技术中存在的造血干细胞扩增率低、易分化等缺陷;将造血干细胞增殖效率提高了50-100倍(相对于SFI和SFTI组合的培养基);得到的造血干细胞数量更多;
2、可维持造血干细胞干性,培养后细胞可分化为血液系统的多种类型细胞;
3、本培养基的操作简便,成本低廉。
具体实施方式
下面对本发明的实施例作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
本申请中,所述重组人干细胞因子简称rhSCF;
所述重组人血小板生成素简称rhTPO;
所述重组人FMS样酪氨酸激酶3配体简称rhFLT3L;
所述胰岛素-转铁蛋白-硒-乙醇胺添加剂简称ITSE;
所述吲哚类似物,简称UM171,其CAS号为1448724-09-1;
所述氮杂环化合物,简称SR1,其CAS号为1227633-49-9。
实施例1CD34+细胞的磁珠分选
选用德国美天旎生物技术有限公司CD34+细胞的磁珠分选培养基
免疫磁珠细胞分离(MACS)培养基(美国Miltenyi Biotec公司,包括试剂A1、 A2、B);细胞过滤网(Becton Dickinson公司,孔径70μm)
用磷酸缓冲盐溶液(PBS)制成细胞悬液(密度1×108/ml),同时加入试剂 A1和A2,4℃冰箱孵育15min,用PBS洗1次,加入试剂B,4℃冰箱孵育15min, 离心后再次制成单个核细胞悬液(密度1×108/ml)。具体操作按MACS培养基说明书进行。
细胞悬液用细胞过滤网过滤后,将细胞加入固定于高强度磁场的分选柱中, 利用重力使未与磁珠结合的细胞随缓冲液流出,与磁珠结合的CD34+细胞保留在柱中,加PBS洗2次,将分选柱移出磁场,用PBS将吸附于柱上的CD34+细胞快速洗脱出来,并将其收集待用。精提:将上述获得的CD34+细胞再次加入分选柱中,重复分选1次,即可得到高纯度的CD34+细胞。
实施例2造血干细胞培养基的配置
将聚乙烯醇(PVA)储存在无菌去离子水中,置于一个耐热玻璃瓶中,高压灭菌,得聚乙烯醇水溶液2.5%w/w。于4℃温度条件下储存。
按照表1的用量将重组人干细胞因子rhSCF、重组人血小板生成素rhTPO、重组人FMS样酪氨酸激酶3配体rhFLT3L、IL-3、聚乙烯醇水溶液和胰岛素-转铁蛋白-硒-乙醇胺添加剂、UM171、SR1添加至500ml无血清培养基中;分别得造血干细胞培养基T1-T8、C1-C3(作为对照);
其中,所述重组人干细胞因子rhSCF为Peprotech AF-250-03,所述重组人血小板生成素rhTPO为Peprotech AF-315-14;所述重组人FMS样酪氨酸激酶3 配体rhFLT3L为R&DBio-Techne 308E-GMP;白介素-3(IL-3)为 Peprotech-200-03,所述聚乙烯醇为阿拉丁,9002-89-5;所述胰岛素-转铁蛋白 -硒-乙醇胺添加剂(ITS-X)为Gibco 51500-056;UM171为MedChemExpress HY-12878;SR1为MedChemExpressHY-15001。
表1
实施例3
采用实施例2制备的造血干细胞培养基体外培养CD34+细胞,并进行CFU培养,选出最佳比例。具体操作如下:
1.1、将实施例1中得到的CD34+用生理盐水洗涤3次后计数,分别用实施例2中的T1-T8、C1-C3培养基调整细胞浓度,配成2×105/ml细胞悬液各6ml,将细胞悬液铺到六孔细胞培养板中,每孔2ml。
1.2、将六孔细胞培养板置于质量浓度为5%CO2、相对湿度100%、37℃的 CO2细胞培养箱中,培养24h;然后进行换液,继续培养12h后收获计数,采用终浓度(质量浓度)为0.04%的台昐蓝染色法检测CD34+细胞数目及活率。流式细胞术检测各组细胞中CD34+细胞百分比。各培养基条件下造血干细胞的细胞总数、细胞活率和CD34+细胞比例结果如表2所示。
2.1、待细胞生长至融合度80%时,用胰酶消化收获,用RPMI1640培养液洗涤3次后计数,以RPMI1640培养液调整细胞浓度,配成1-2×107/ml细胞悬液。
2.2、将步骤2.1分离的细胞悬液培养于α-MEM-甲基纤维素培养基,接种 35mm培养皿中,每皿1ml,重复3个皿。轻轻摇匀,观察CD34+细胞分布情况, CD34+细胞应均匀分布,不能聚集成团,于质量浓度为5%CO2、相对湿度100%、 37℃的CO2培养箱中,培养14天后倒置显微镜下观察结果。
2.3、以3个皿的均数做为结果,平皿内细胞团≥40个细胞的计为集落,记录集落数量,见表3。
表2
表3
培养基 | 集落总数 |
T1 | 197.33 |
T2 | 214.33 |
T3 | 187.67 |
T4 | 179.67 |
T5 | 154.00 |
T6 | 146.67 |
T7 | 139.33 |
T8 | 1.79 |
C1 | 72 |
C2 | 98.67 |
C3 | 134.33 |
实施例4
收集各组培养基(T1-T8、C1-C3)培养的CD34+细胞,并取3×109个CD34+ 细胞经尾静脉注射至NSI鼠体内,观察CD34+细胞增殖分化情况。
具体操作如下:
1、收集各组培养基(T1-T8、C1-C3)培养的CD34+细胞,每组分8份,每份经尾静脉注射至免疫缺陷小鼠体内,(每组被注射的8只小鼠中雌雄各半)。注射1个月后观察CD34+细胞植入情况。
2、移植后受体小鼠定期尾静脉取血,用血细胞分析仪检测血常规(白细胞、红细胞比容、血小板),观察移植后受体鼠的存活时间及移植后7天、14天、30天的存活情况。具体结果如表4.1、4.2、4.3所示。
表4.1移植后7天
表4.2移植后14d
分组 | 白细胞(10<sup>9</sup>/L) | 红细胞比容(%) | 血小板(10<sup>9</sup>/L) |
T1 | 1.12 | 24.13 | 51.27 |
T2 | 1.37 | 31.24 | 79.52 |
T3 | 1.16 | 25.37 | 60.45 |
T4 | 1.23 | 26.71 | 57.24 |
T5 | 1.27 | 19.28 | 59.37 |
T6 | 1.29 | 25.19 | 58.16 |
T7 | 1.17 | 26.34 | 54.93 |
T8 | 1.15 | 28.97 | 61.25 |
C1 | 0.42 | 16.57 | 35.14 |
C2 | 0.95 | 27.81 | 49.78 |
C3 | 0.91 | 30.57 | 57.69 |
表4.3移植后30天
表4.1-4.3中,“分组”这一列中的“T1-T8,C1-C3”是指给小鼠注射的 CD34+细胞分别是由表1中的“T1-T8,C1-C3”培养基培养的。以注射了表1中“T1-T8”培养基培养的CD34+细胞的小鼠作为实验组1-8,以注射了表1中“C1-C3”培养基培养的CD34+细胞的小鼠作为对照组1-3。
结果分析:
(1)外周血白细胞的变化:实验组小鼠的外周血白细胞明显高于对照组;除对照组1以外的其他实验组和对照组的小鼠外周血白细胞均有不同程度的升高;实验组2的小鼠外周血白细胞第30天时达到正常范围;对照组1小鼠外周血白细胞一直下降,对照组1小鼠全部死亡。
(2)外周血红细胞比容变化:对照组小鼠红细胞比容水平逐渐下降;实验组小鼠红细胞比容明显高于对照组,实验组小鼠移植后30天检测时其红细胞比容仍持续升高直至正常范围。
(3)外周血血小板变化:对照组7天左右小鼠血小板迅速下降,实验组小鼠血小板明显高于对照组。实验组小鼠血小板持续升高,30d时恢复至正常范围。
通过比较:实验组2各类细胞升高速度最快,最先达到正常水平;实验组1、 3-8次之,添加了UM171和SR1因子的对照组1-3再次之。结果显示造血干细胞培养体系中UM171和SR1有非常重要的作用,而聚乙烯醇可以更好的维持造血干细胞的活性并提高增殖速度。
Claims (10)
1.一种高效体外培养造血干细胞的培养基,其特征在于,由基础培养基和添加因子组成;
所述基础培养基为无血清培养基;
所述添加因子及其相对于基础培养基的含量如下:
重组人干细胞因子:50-200ng/ml、
重组人血小板生成素:20-100ng/ml、
重组人FMS样酪氨酸激酶3配体:100-200ng/ml、
白介素-3:30-60ng/ml、
聚乙烯醇:1-10mg/ml、
胰岛素-转铁蛋白-硒-乙醇胺添加剂:1-2:100ml/ml、
吲哚类似物:10-200ng/ml、
氮杂环化合物:10-200ng/ml。
2.根据权利要求1所述的培养基,其特征在于,重组人干细胞因子:50-100ng/ml。
3.根据权利要求1所述的培养基,其特征在于,重组人血小板生成素:20-50ng/ml。
4.根据权利要求1所述的培养基,其特征在于,重组人FMS样酪氨酸激酶3配体:100-150ng/ml。
5.根据权利要求1所述的培养基,其特征在于,白介素-3:30-50ng/ml。
6.根据权利要求1所述的培养基,其特征在于,聚乙烯醇:1-6mg/ml。
7.根据权利要求1所述的培养基,其特征在于,胰岛素-转铁蛋白-硒-乙醇胺添加剂:2:100ml/ml。
8.根据权利要求1所述的培养基,其特征在于,吲哚类似物:10-100ng/ml。
9.根据权利要求1所述的培养基,其特征在于,氮杂环化合物:10-100ng/ml。
10.根据权利要求1所述的培养基,其特征在于,
重组人干细胞因子:100ng/ml、
重组人血小板生成素:50ng/ml、
重组人FMS样酪氨酸激酶3配体:150ng/ml、
白介素-3:50ng/ml、
聚乙烯醇:6mg/ml、
胰岛素-转铁蛋白-硒-乙醇胺添加剂:2:100ml/ml、
吲哚类似物:100ng/ml、
氮杂环化合物:100ng/ml。
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