用于肝病研究的外周血人肝球
Summary
在这里，我们提出了一种使用从稳态外周血分离的单核细胞产生人类自体肝球的非遗传方法。
Abstract
人肝细胞可以形成三维（3D）结构，能够在培养物中生长数周，保持其功能能力。由于它们在具有低粘附特性或无粘附特性的培养皿中聚集的性质，它们形成多个肝细胞的聚集体，称为人肝球体。3D肝球体的形成依赖于肝细胞在没有粘性基质的情况下聚集的自然趋势。这些3D结构比更接近 体内 环境的细胞具有更好的生理反应。与传统的二维（2D）培养物相比，使用3D肝细胞培养物具有许多优势，包括生物学相关性更高的微环境，重组天然器官的结构形态以及更好地预测疾病状态和 体内对药物的反应。各种来源可用于生成球状体，如原代肝组织或永生化细胞系。3D肝组织也可以通过使用人类胚胎干细胞（hESC）或诱导多能干细胞（hiPSC）来衍生肝细胞来设计。我们使用血液来源的多能干细胞（BD-PSC）获得了人肝球体，该干细胞通过激活人膜结合的GPI连接蛋白由未经操纵的外周血产生，并分化为人肝细胞。采用人肝细胞标志物进行光学显微镜和免疫分型分析BD-PSCs来源的人肝细胞和人肝球。
Introduction
近年来，三维（3D）球状体培养系统已成为研究癌症研究，药物发现和毒理学各个领域的重要工具。这种培养物引起了极大的兴趣，因为它们弥合了二维（2D）细胞培养单层和复杂器官之间的差距1。
在没有粘合表面的情况下，与2D细胞培养物相比，球状体的形成基于这些细胞以3D形式聚集的天然亲和力。这些细胞将自己组织成由一种或多种成熟细胞组成的组。这些细胞没有异物，像在原始微环境中一样相互作用。3D培养中的细胞更近，彼此之间具有适当的方向，比2D培养物具有更高的细胞外基质产生，并构成接近自然的环境 2。
动物模型长期以来一直用于研究人类生物学和疾病3.在这方面，人类和动物之间存在内在差异，这使得这些模型并不完全适合外推研究。3D培养球体和类器官代表了一种有前途的工具，用于研究 体内 发生的不同细胞类型之间的组织样结构，相互作用和串扰，并有助于减少甚至取代动物模型。它们对研究肝病的发病机制以及药物筛选平台特别感兴趣4.
3D球状体培养对于癌症研究尤为重要，因为它可以通过减少制备用于2D培养的肿瘤细胞单层所需的胰蛋白酶消化或胶原酶处理的需要来消除细胞与其环境之间的不连续性。肿瘤球状体能够研究正常细胞与恶性细胞如何接收和响应来自周围环境的信号5 ，并且是肿瘤生物学研究的重要组成部分。
与单层相比，由各种细胞类型组成的3D培养物在结构和功能特性上类似于肿瘤组织，因此适用于研究肿瘤细胞的转移和侵袭。这就是为什么这种球体模型有助于加速癌症研究6。
球状体也有助于开发制造人类类器官的技术，因为组织和器官生物学的研究非常具有挑战性，特别是在人类中。干细胞培养的进步使得开发3D培养成为可能，例如由干细胞和组织祖细胞组成的类器官，以及来自具有某些功能特征的器官的不同类型的成熟（组织）细胞，例如可用于模拟器官发育，疾病的真实器官，但它们也可以被认为在再生医学中有用7。
原代人肝细胞通常用于研究人肝细胞的 体外 生物学、肝功能和药物诱导的毒性。人肝细胞培养有两个主要缺点，首先，原代组织（如人肝细胞）的可用性有限，其次，肝细胞在2D培养中倾向于快速去分化，从而失去其特定的肝细胞功能8。3D肝培养物在这方面具有优越性，最近由分化的人胚胎干细胞（hESC）或诱导多能干细胞（hiPSC）制成9。生物工程肝3D球体对研究肝脏的发展，毒性，遗传和传染病以及用于治疗肝脏疾病的药物发现特别感兴趣10。最后，它们也有潜力用于临床，知道急性肝病的死亡率接近80%，生物人工肝和/或肝球可以通过提供部分肝功能来拯救这些患者，直到找到合适的供体11。
我们已经建立了一个使用血液来源的多能干细胞（BD-PSC）生成人肝球的方案，以制备含有4000至1 x 106 细胞的不同大小的球体，并通过光学显微镜和免疫荧光对其进行分析。我们还测试了肝细胞特异性功能的能力，评估了属于细胞色素P450家族的细胞色素P450 3A4（CYP3A4）和2E1（CYP2E1）酶的表达，这些酶通过解毒过程在细胞和药物代谢中起重要作用12。
Protocol
Representative Results
Discussion
肝脏是人体的主要器官，具有许多基本的生物学功能，例如代谢物的解毒。由于肝硬化和/或病毒性肝炎等严重肝功能衰竭，全世界每年有近200万人死亡。肝移植在全球实体器官移植中排名第二，但目前的需求仅得到满足约10%22。
原代人肝细胞（PHH）通常用于研究肝毒性。这些细胞可以在培养物中维持短时间，保留其特定功能。此外，来自单个供体的细胞数?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者特别感谢Oksana和John Greenacre提供的技术援助。这项工作得到了德国海德堡ACA CELL Biotech GmbH的支持。
Materials
| Albumin antibody | Sigma-Aldrich | SAB3500217 | produced in chicken |
| Albumin Fraction V | Carl Roth GmbH+Co. KG | T8444.4 | |
| Alpha-1 Fetoprotein | Proteintech Germany GmbH | 14550-1-AP | rabbit polyclonal IgG |
| Biolaminin 111 LN | BioLamina | LN111-02 | human recombinant |
| CD45 MicroBeads | Miltenyi | 130-045-801 | nano-sized magnetic beads |
| Cell Strainer | pluriSelect | 43-10040-40 | |
| CellSens | Olympus | imaging software | |
| Centrifuge tubes 50 mL | Greiner Bio-One | 210270 | |
| CEROplate 96 well | OLS OMNI Life Science | 2800-109-96 | |
| CKX53 | Olympus | ||
| Commercially available detergent | Procter & Gamble | nonionic detergent | |
| CYP2E1-specific antibody | Proteintech Germany GmbH | 19937-1-AP | rabbit polyclonal antibody IgG |
| CYP3A4 | Proteintech Germany GmbH | 67110-1-lg | mouse monoclonal antibody IgG1 |
| Cytokeratin 18 | DakoCytomation | M7010 | mouse monoclonal antibody IgG1 |
| DMSO | Sigma-Aldrich | D8418-50ML | |
| DPBS | Thermo Fisher Scientific | 14040091 | |
| FBS | Merck Millipore | S0115/1030B | Discontinued. Available under: TMS-013-B |
| Glass cover slips 14 mm | R. Langenbrinck | 01-0014/1 | |
| GlutaMax 100x Gibco | Thermo Fisher Scientific | 35050038 | L-glutamine |
| Glutaraldehyde 25% | Sigma-Aldrich | G588.2-50ML | |
| Goat anti-mouse IgG Cy3 | Antibodies online | ABIN1673767 | polyclonal |
| Goat anti-mouse IgG DyLight 488 | Antibodies online | ABIN1889284 | polyclonal |
| Goat anti-rabbit IgG Alexa Fluor 488 | Life Technologies | A-11008 | |
| HCl | Sigma-Aldrich | 30721-1LGL | |
| HepatoZYME-SFM | Thermo Fisher Scientific | 17705021 | hepatocyte maturation medium |
| HGF | Thermo Fisher Scientific | PHG0324 | human recombinant |
| HNF4α antibody | Sigma-Aldrich | ZRB1457-25UL | clone 4C19 ZooMAb Rbmono |
| Hydrocortisone 21-hemisuccinate (sodium salt) | Biomol | Cay18226-100 | |
| Knock out Serum Replacement – Multi Species Gibco | Fisher Scientific | A3181501 | KSR |
| KnockOut DMEM/F-12 | Thermo Fisher Scientific | 12660012 | Discontinued. Available under Catalog No. 10-828-010 |
| MACS Buffer | Miltenyi | 130-091-221 | |
| MACS MultiStand | Miltenyi | 130-042-303 | magnetic stand |
| MEM NEAA 100x Gibco | Thermo Fisher Scientific | 11140035 | |
| Mercaptoethanol | Thermo Fisher Scientific | 31350010 | 50mM |
| MiniMACS columns | Miltenyi | 130-042-201 | |
| Nunclon Multidishes | Sigma-Aldrich | D6789 | 4 well plates |
| Oncostatin M | Thermo Fisher Scientific | PHC5015 | human recombinant |
| Paraformaldehyde | Sigma-Aldrich | 158127 | |
| PBS sterile | Carl Roth GmbH+Co. KG | 9143.2 | |
| Penicillin/Streptomycin | Biochrom GmbH | A2213 | 10000 U/ml |
| PS 15ml tubes sterile | Greiner Bio-One | 188171 | |
| Rabbit anti-chicken IgG Texas red | Antibodies online | ABIN637943 | |
| Roti Cell Iscoves MDM | Carl Roth GmbH+Co. KG | 9033.1 | |
| Roti Mount FluorCare DAPI | Carl Roth GmbH+Co. KG | HP20.1 | |
| Roti Sep 1077 human | Carl Roth GmbH+Co. KG | 0642.2 | |
| Transthyretin antibody | Sigma-Aldrich | SAB3500378 | produced in chicken |
| Triton X-100 | Thermo Fisher Scientific | HFH10 | 1% |
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