Cell Therapy Production Unit, Department of Cerebrovascular Disease - IRCCS Neurologic Institute C. Besta Foundation, Milano, Italy.
Cell Therapy Production Unit, Department of Cerebrovascular Disease - IRCCS Neurologic Institute C. Besta Foundation, Milano, Italy.
Curr Res Transl Med. 2019 Feb;67(1):20-27. doi: 10.1016/j.retram.2018.06.002. Epub 2018 Aug 10.
Mesenchymal stromal cells (MSCs) are considered a promising tool for cell therapy approaches. The translation of research-based cell culture protocols into procedures that comply with Good Manufacturing Practice (GMP) is critical. The aim of this study was to design a new method for the expansion of MSCs from Adipose Tissue (AT-MSCs) in compliance with GMP, without enzymatic tissue digestion and without the use of animal proteins as source of growth factors.
MSCs were expanded from 10 periumbilical biopsies. Our new isolation approach is based on: (1) disruption of AT with an automated, closed system; (2) use of GMP-grade medium without the addition of fetal bovine serum or platelet lysate; (3) use of human recombinant Trypsin. AT-MSCs cultured in α-MEM and minced by scalpel were used as control.
It was possible to expand MSCs from all the AT-samples for at least eight passages. MSCs displayed the typical spindle-shape morphology, a high viability, multilineage differentiation potential and high expression levels of the typical MSC-specific surface antigens and genes. Compared to standard method, MSCs obtained with the new method showed higher yield, up to passage 6, and higher purity in terms of percentage of CD34 and CD45 markers. All AT-MSCs exhibit in vitro immunosuppressive capacity and possess a normal karyotype.
Our data clearly demonstrate that our new approach permits to generate AT-MSCs fully compliant for therapeutic use and better at least in terms of quantity and purity than those obtained with the standard method.
间充质基质细胞（MSCs）被认为是细胞治疗方法的有前途的工具。将基于研究的细胞培养方案转化为符合良好生产规范（GMP）的程序至关重要。本研究的目的是设计一种新的方法，从脂肪组织（AT-MSCs）中扩张 MSCs，符合 GMP 要求，无需酶组织消化，也不使用动物蛋白作为生长因子的来源。
从 10 个脐周活检中扩增 MSC。我们的新分离方法基于：（1）使用自动化、封闭系统破坏 AT；（2）使用不含胎牛血清或血小板裂解液的 GMP 级培养基；（3）使用人重组胰蛋白酶。α-MEM 中培养的 AT-MSCs 和手术刀切碎的 AT-MSCs 用作对照。
从所有 AT 样本中至少可以扩增 8 代 MSC。MSCs 呈现典型的纺锤形形态，高活力，多谱系分化潜能，以及高表达典型的 MSC 特异性表面抗原和基因。与标准方法相比，新方法获得的 MSC 在前 6 代时产量更高，CD34 和 CD45 标志物的纯度更高。所有 AT-MSCs 均表现出体外免疫抑制能力，并具有正常核型。
我们的数据清楚地表明，我们的新方法可以生成完全符合治疗用途的 AT-MSCs，至少在数量和纯度方面优于标准方法获得的 MSC。
