Ahmadi Seyed Esmaeil, Shabestari Rima Manafi, Kojabad Amir Asri, Safa Majid
Department of Hematology and Blood Banking, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.
Biotechnol Rep (Amst). 2023 Mar 12;38:e00792. doi: 10.1016/j.btre.2023.e00792. eCollection 2023 Jun.
HIV-1-derived lentiviral vectors (LVs) are capable of transducing human cells by integrating the transgene into the host genome. In order to do that, LVs should have enough time and space to interact with the surface of the target cells. Herein, we used a microfluidic system to facilitate the transduction of BCP-ALL cells.
We used a SU-8 mold to fabricate a PDMS microfluidic chip containing three channels with a 50 μm height and a surface matching 96-well plates. In order to produce LVs, we used HEK293T cells to package the second generation of LVs. First, we evaluated the cell recovery from the microfluidic chip. Cell recovery assessment showcased that 3 h and 6 h of incubation in microfluidic channels containing 100,000 NALM-6 (BCP-ALL) cells with 2μL of culture media yielded 87±7.2% and 80.6 ± 10% of cell recovery, respectively. Afterward, the effects of LV-induced toxicity were evaluated using 10-30% LV concentrations in time frames ranging from 3 h to 24 h. In 96-well plates, it took 12-24 h for the viruses with 20% and 30% concentrations to affect the cell survival significantly. These effects were intensified in the microfluidic system implying that microfluidic is capable of enhancing LV transduction. Based on the evidence of cell recovery and cell survival we chose 6 h of incubation with 20% LV.
The results from EGFP expression showcased that a microfluidic system could increase the LV transduction in BCP-ALL cells by almost 9-folds. All in all, the microfluidic system seems to be a great armamentarium in optimizing LV-based transduction.
源自HIV-1的慢病毒载体（LVs）能够通过将转基因整合到宿主基因组中来转导人类细胞。为了实现这一点，LVs应有足够的时间和空间与靶细胞表面相互作用。在此，我们使用微流控系统来促进BCP-ALL细胞的转导。
我们使用SU-8模具制造了一个PDMS微流控芯片，该芯片包含三个高度为50μm且表面与96孔板匹配的通道。为了生产LVs，我们使用HEK293T细胞包装第二代LVs。首先，我们评估了从微流控芯片中回收的细胞。细胞回收评估表明，在含有100,000个NALM-6（BCP-ALL）细胞和2μL培养基的微流控通道中孵育3小时和6小时，细胞回收率分别为87±7.2%和80.6±10%。随后，在3小时至24小时的时间范围内，使用10%-30%的LV浓度评估LV诱导的毒性作用。在96孔板中，浓度为20%和30%的病毒需要12-24小时才能显著影响细胞存活。这些作用在微流控系统中增强，这意味着微流控能够增强LV转导。基于细胞回收和细胞存活的证据，我们选择了用20%的LV孵育6小时。
EGFP表达结果表明，微流控系统可使BCP-ALL细胞中的LV转导增加近9倍。总而言之，微流控系统似乎是优化基于LV的转导的有力工具。
