CN112553159A - 模拟骨髓环境培养造血干细胞的3d模型及其制备方法和应用 - Google Patents
模拟骨髓环境培养造血干细胞的3d模型及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了模拟骨髓环境培养造血干细胞的3D模型及其制备方法和应用，所述3D模型包括细胞培养基和胶原蛋白溶液；其中，细胞培养基包括：v/v为1％的青霉素/链霉素无血清干细胞基础培养基、100ng/mL重组人干细胞因子、50ng/mL重组人血小板生成素、50ng/mL重组人FMS样酪氨酸激酶3和50ng/mL白介素‑3。与现有技术相比，本发明具有以下优点：(1)所述3D模型首先将细胞加入胶原蛋白中促使细胞聚合，最大程度地模拟了造血干细胞在细胞外基质内的环境；(2)所述3D模型后续将细胞培养基加入含有干细胞的胶原蛋白中，能够很好地渗入胶原蛋白为细胞增殖提供营养；(3)所述3D模型保留了造血干细胞的多向分化能力，因此为造血干细胞的体外扩增提供了良好的应用前景。
Description
技术领域
本发明属于细胞体外培养技术领域，涉及一种模拟体内的复杂环境培养造血干细胞的方法，具体为模拟骨髓环境培养造血干细胞的3D模型及其制备方法和应用。
背景技术
在骨髓中，造血干细胞具有无限自我更新的能力，产生与原始细胞相同的后代。它们通常处于细胞周期的G0期，并可以分化为具有特定功能的细胞。由于其具有自我更新和多向分化的能力，造血干细胞在治疗血液系统恶性和非恶性疾病中有着广泛应用，而研发出一种高效的体外扩增技术也显得越来越重要。
传统的2D细胞培养技术是将造血干细胞培养于培养瓶或者培养皿中，在其中加入血清替代物和多种生长因子。但是,这种培养模式没有考虑到细胞在生理环境中与微环境和其他细胞之间的相互作用。对造血干细胞自我更新和分化的调控取决于局部和全身细胞因子的分泌作用，并且骨髓内复杂的微环境和细胞间的相互作用在体外很难完全复制。多项研究表明，造血干细胞与基质细胞之间的接触对于维持其功能很重要。来自骨髓的基质细胞包括成骨细胞，巨噬细胞，内皮细胞和间充质细胞。间充质细胞是多能祖细胞，具有分化为成骨细胞、脂肪细胞、软骨细胞、神经元细胞等的潜力。这些细胞与造血细胞相互作用，支持造血功能的发展并充当免疫调节剂。此外，最近的研究表明，其他分子，例如N6-甲基腺苷(M6A)，通过在mRNA水平上调节一组YTHDF2基因的表达，是造血干细胞自我更新的重要调节剂。
目前已经有一些技术用于构建模拟骨髓环境，用于在3D环境中培养造血干细胞。但现有3D模型存在一定的限制，如培养成本较高、批量生产较难等。开发一种简单、高效、低成本的3D培养技术具有很高的应用价值。
发明内容
解决的技术问题：为了克服现有技术的不足，获得一种能够模拟造血干细胞在细胞外基质内的环境，同时使得造血干细胞较好的聚合并获取营养，从而保留造血干细胞的多向分化能力的体外培养模型，本发明提供了模拟骨髓环境培养造血干细胞的3D模型及其制备方法和应用。
技术方案：模拟骨髓环境培养造血干细胞的3D模型，所述3D模型包括细胞培养基和胶原蛋白凝胶，培养基与胶原蛋白凝胶的体积比为3:1-2:1；其中，细胞培养基包括：v/v为1％的青霉素/链霉素无血清干细胞基础培养基、100ng/mL重组人干细胞因子、50ng/mL重组人血小板生成素、50ng/mL重组人FMS样酪氨酸激酶3和50ng/mL白介素-3。
优选的，胶原蛋白凝胶的配方为100μL的10×PBS，10μL的1mol/L NaOH，250μL水和640μL 3.2mg/mL胶原蛋白。
以上任一所述的模拟骨髓环境培养造血干细胞的3D模型的制备方法，所述方法为首先在胶原蛋白溶液中接种待培养的造血干细胞，然后将含有造血干细胞的胶原蛋白溶液置于37℃条件下孵育，待胶原蛋白聚合后加入细胞培养基，从而制备获得模拟骨髓环境培养造血干细胞的3D模型。
优选的，胶原蛋白溶液的浓度为2mg/mL。
优选的，造血干细胞在胶原蛋白溶液中的接种量为105个/mL。
优选的，所述细胞培养基每天更换。
以上任一所述的模拟骨髓环境培养造血干细胞的3D模型在制备造血干细胞体外扩增试剂盒中的应用。
所述模拟骨髓环境培养造血干细胞的3D模型的工作原理在于：一方面，胶原蛋白促使细胞聚合，模拟造血干细胞在细胞外基质内的环境；另一方面，胶原蛋白能使干细胞很好的渗入其中，并为细胞增殖提供营养。
有益效果：(1)本发明所述3D模型首先将细胞加入胶原蛋白中促使细胞聚合，最大程度地模拟了造血干细胞在细胞外基质内的环境；(2)所述3D模型后续将细胞培养基加入含有干细胞的胶原蛋白中，能够很好地渗入胶原蛋白为细胞增殖提供营养；(3)所述3D模型保留了造血干细胞的多向分化能力，因此为造血干细胞的体外扩增提供了良好的应用前景。
附图说明
图1为本发明所述3D模型制备流程示意图；
图2为本发明中3D模型(A)和2D模型(B)培养造血干细胞的照片；
图3为本发明中3D模型和2D模型干细胞含量和活性检测结果图；
图4为本发明中3D模型和2D模型培养的造血干细胞移植免疫缺陷NOD-SCID小鼠4周后，外周血中人源CD45+细胞量检测。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改和替换，均属于本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1
模拟骨髓环境培养造血干细胞的3D模型，所述3D模型包括细胞培养基和胶原蛋白溶液；其中，细胞培养基包括：v/v为1％的青霉素/链霉素无血清干细胞基础培养基、100ng/mL重组人干细胞因子、50ng/mL重组人血小板生成素、50ng/mL重组人FMS样酪氨酸激酶3和50ng/mL白介素-3。
胶原蛋白溶液的配方为100μL的10×PBS，10μL的1mol/L NaOH，250μL水和640μL3.2mg/mL胶原蛋白。
以上所述的模拟骨髓环境培养造血干细胞的3D模型的制备方法，所述方法为首先在胶原蛋白溶液中接种待培养的造血干细胞，然后将含有造血干细胞的胶原蛋白溶液置于37℃条件下孵育，待胶原蛋白聚合后加入细胞培养基，从而制备获得模拟骨髓环境培养造血干细胞的3D模型。
其中，胶原蛋白溶液的浓度为2mg/mL；造血干细胞在胶原蛋白溶液中的接种量为105个/mL；所述细胞培养基每天更换。
实施例2
采用实施例1所述模拟骨髓环境培养造血干细胞的3D模型检测干细胞活性及含量，以CD34+细胞为例：
脐带血采集及CD34+细胞分离纯化：采集新鲜脐带血，将未分离的脐带血细胞用羟乙基淀粉溶液以1:6稀释，用CD34+磁珠分离CD34+细胞。
将105个CD34+细胞接种至胶原蛋白溶液中，先将24孔板预热，然后将含有造血干细胞的胶原蛋白溶液加入24孔板，并置于37℃培养箱中进行孵育，待胶原蛋白聚合后加入细胞培养基，每天更换培养基。
并以2D细胞培养模型作为对照：将105个造血干细胞直接加入含有上述培养基的24孔板中，放入37℃培养箱中培养，每天更换培养基。
细胞活性及CD34+细胞含量检测：培养7天后，取3D细胞培养组的胶原蛋白球，将其小心剪成小块后用酶进行消化，离心，取出细胞并进行计数以及细胞活性检测；对2D培养组的细胞可以直接进行细胞计数及细胞活性检测。如图3所示，用流式细胞仪对两组的CD34+细胞含量进行检测，3D培养的细胞活力大于2D培养。
实施例3
利用免疫缺陷NOD-SCID小鼠观察实施例2的3D模型和2D模型培养的细胞的造血能力：培养10天后，将两组细胞分别移植入NOD-SCID小鼠内，每只小鼠移植600个细胞，4周后检测小鼠外周血中人源CD45+细胞的量。如图4所示，3D模型培养的细胞检测到的数量远高于2D培养的细胞。
Claims (7)
1.模拟骨髓环境培养造血干细胞的3D模型，其特征在于，所述3D模型包括细胞培养基和胶原蛋白凝胶，培养基与胶原蛋白凝胶的体积比为3:1-2:1；其中，细胞培养基包括：v/v为1％的青霉素/链霉素无血清干细胞基础培养基、100ng/mL重组人干细胞因子、50ng/mL重组人血小板生成素、50ng/mL重组人FMS样酪氨酸激酶3和50ng/mL白介素-3。
2.根据权利要求1所述的模拟骨髓环境培养造血干细胞的3D模型，其特征在于，胶原蛋白凝胶的配方为100μL的10×PBS，10μL的1mol/L NaOH，250μL水和640μL 3.2mg/mL胶原蛋白。
3.权利要求1或2所述的模拟骨髓环境培养造血干细胞的3D模型的制备方法，其特征在于，所述方法为首先在胶原蛋白溶液中接种待培养的造血干细胞，然后将含有造血干细胞的胶原蛋白溶液置于37℃条件下孵育，待胶原蛋白聚合后加入细胞培养基，从而制备获得模拟骨髓环境培养造血干细胞的3D模型。
4.根据权利要求3所述的模拟骨髓环境培养造血干细胞的3D模型的制备方法，其特征在于，胶原蛋白溶液的浓度为2mg/mL。
5.根据权利要求3所述的模拟骨髓环境培养造血干细胞的3D模型的制备方法，其特征在于，造血干细胞在胶原蛋白溶液中的接种量为105个/mL。
6.根据权利要求3所述的模拟骨髓环境培养造血干细胞的3D模型的制备方法，其特征在于，所述细胞培养基每天更换。
7.权利要求1或2所述的模拟骨髓环境培养造血干细胞的3D模型在制备造血干细胞体外扩增试剂盒中的应用。
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