Thorbow Jan, Strauch Andrea, Pfening Viktoria, Klee Jan-Philip, Brücher Patricia, Boshof Björn, Petry Florian, Czermak Peter, Herrera Sanchez Maria Beatriz, Salzig Denise
Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, 35390 Giessen, Germany.
Faculty of Biology and Chemistry, University of Giessen, 35392 Giessen, Germany.
Bioengineering (Basel). 2024 Jul 9;11(7):692. doi: 10.3390/bioengineering11070692.
The assessment of human liver stem cells (HLSCs) as cell therapeutics requires scalable, controlled expansion processes. We first focused on defining appropriate process parameters for HLSC expansion such as seeding density, use of antibiotics, optimal cell age and critical metabolite concentrations in conventional 2D culture systems. For scale-up, we transferred HLSC expansion to multi-plate and stirred-tank bioreactor systems to determine their limitations. A seeding density of 4000 cells cm was needed for efficient expansion. Although growth was not significantly affected by antibiotics, the concentrations of lactate and ammonia were important. A maximum expansion capacity of at least 20 cumulative population doublings (cPDs) was observed, confirming HLSC growth, identity and functionality. For the expansion of HLSCs in the multi-plate bioreactor system Xpansion (XPN), the oxygen supply strategy was optimized due to a low of 0.076 h. The XPN bioreactor yielded a final mean cell density of 94 ± 8 × 10 cells cm, more than double that of the standard process in T-flasks. However, in the larger XPN50 device, HLSC density reached only 28 ± 0.9 × 10 cells cm, while the glucose consumption rate increased 8-fold. In a fully-controlled 2 L stirred-tank bioreactor (STR), HLSCs expanded at a comparable rate to the T-flask and XPN50 processes in a homogeneous microenvironment using advanced process analytical technology. Ultimately, the scale-up of HLSCs was successful using two different bioreactor systems, resulting in sufficient numbers of viable, functional and undifferentiated HLSCs for therapeutic applications.
将人肝干细胞（HLSCs）用作细胞疗法需要可扩展、可控的扩增过程。我们首先专注于确定HLSC扩增的合适工艺参数，如接种密度、抗生素的使用、最佳细胞年龄以及传统二维培养系统中关键代谢物的浓度。为了扩大规模，我们将HLSC扩增转移到多板和搅拌罐生物反应器系统中，以确定其局限性。高效扩增需要4000个细胞/cm²的接种密度。虽然抗生素对生长没有显著影响，但乳酸和氨的浓度很重要。观察到最大扩增能力至少为20次累积群体倍增（cPDs），证实了HLSC的生长、特性和功能。对于在多板生物反应器系统Xpansion（XPN）中扩增HLSCs，由于低的0.076 h⁻¹的氧气供应策略进行了优化。XPN生物反应器产生的最终平均细胞密度为94±8×10⁶个细胞/cm²，是T型烧瓶中标准工艺的两倍多。然而，在更大的XPN50装置中，HLSC密度仅达到28±0.9×10⁶个细胞/cm²，而葡萄糖消耗率增加了8倍。在一个完全受控的2 L搅拌罐生物反应器（STR）中，使用先进的过程分析技术，HLSCs在均匀的微环境中以与T型烧瓶和XPN50工艺相当的速率扩增。最终，使用两种不同的生物反应器系统成功扩大了HLSCs的规模，从而获得了足够数量的有活力、功能正常且未分化的HLSCs用于治疗应用。
