An IB-LBM study of continuous cell sorting in deterministic lateral displacement arrays

The deterministic lateral displacement (DLD) is an important method used to sort particles and cells of different sizes. In this paper, the flexible cell sorting with the DLD method is studied by using a numerical model based on the immersed boundary-lattice Boltzmann method (IB-LBM). In this model, the fluid motion is solved by the LBM, and the cell membrane-fluid interaction is modeled with the LBM. The proposed model is validated by simulating the rigid particle sorted with the DLD method, and the results are found in good agreement with those measured in experiments. We first study the effect of flexibility on a single cell and multiple cells continuously going through a DLD device. It is found that the cell flexibility can significantly affect the cell path, which means the flexibility could have significant effects on the continuous cell sorting by the DLD method. The sorting characteristics of white blood cells and red blood cells are further studied by varying the spatial distribution of cylinder arrays and the initial cell-cell distance. The numerical results indicate that a well concentrated cell sorting can be obtained under a proper arrangement of cylinder arrays and a large enough initial cell-cell distance.

Microfabrication of Bubbular Cavities in PDMS for Cell Sorting and Microcell Culture Applications

We describe a novel technique, low surface energy Gas Expansion Molding （GEM）, to fabricate microbubble arrays in polydimethylsiloxane （PDMS） which are incorporated into parallel plate flow chambers and tested in cell sorting and microcell cuTture applications. This architecture confers several operational advantages that distinguish this technology approach from currently used methods. Herein we describe the GEM process and the parameters that are used to control microbubble formation and a Vacuum-Assisted Coating （VAC） process developed to selectively and spatially alter the PDMS surface chemistry in the wells and on the microchannel surface. We describe results from microflow image visualization studies conducted to investigate fluid streams above and within microbubble wells and conclude with a discussion of cell culture studies in PDMS.

An efficient method of sorting liver stem cells by using immuno-magnetic microbeads

瞄准：开发一个方法快并且高效地孤立肝干细胞。方法：胎儿的老鼠肝细胞被房间表面抗原(c 工具包和 CD45/TER119 ) 用流动血细胞计数描绘。候选人肝干细胞被使用免疫磁性的微祷告排序并且由形成克隆的文化， RT-PCR 和免疫荧光试金识别了。结果：c-Kit-(CD45/TER119 )- 有 97.9% 纯净的房间人口同时被免疫磁性的微祷告净化。这分离的收益是排序房间的大约 6% 总数，房间生存能力超过 98% 。什么时候这些房间有的有教养的在试管内高形成克隆、自我更新的能力和 hepatocytes 和胆汁管房间的表示标记。机能上地成熟的 hepatocytes 在文化的 21 d 以后被观察。结论：这个方法与高纯净和生存能力为肝干细胞的丰富提供一个优秀工具，它能被用于进一步的研究。它快、有效、简单、不昂贵。

The Cell Sorting Process of Xenopus Gastrula Cells Progresses in a Stepwise Fashion Involving Concentrification and Polarization

Animal pole cells (AC) and vegetal pole cells (VC) dissociated from early Xenopus gastrulae were intermingled, and the cell sorting process occurring within the aggregate was analyzed. The overall process of cell sorting was found to morphologically consist of two steps, “concentrification” and “polarization”, as designated here. First, AC and VC clusters emerged at random positions in the aggregate, and the individual clusters gradually assembled themselves by 5 hours in culture (5 hC), forming a concentric arrangement, in which the AC cluster was enveloped by the VC cluster. This concentrification step is essentially consistent with the descriptions in earlier studies. As the next step, the AC and VC clusters moved up and down from 7.5 to 12 hC, resulting in the vertical polarization, namely, a serial array just like in vivo. Immunohistochemical analyses showed that AC expressed both C- and E-cadherins, while VC only expressed C-cadherin, as in vivo, suggesting the normal participation of cadherin system. On the other hand, the actin localization showed that the actin bundles accumulated at the edge of the AC cluster until the concentrification was completed, and gradually decreased during the polarization step. Another important finding was that AC cluster could generate cartilage tissues during the long-term (7 days) culture, evidence for a healthy inductive interaction between the AC and VC. Taken together, the present experimental system allows the AC and VC to be viable and grow into an embryo-like organization.

Isolation and Characterization of Human CD34^+ Hematopoietic Progenitor Cells by High-gradient Magnetic Cell Sorting

Using a high-gradient magnetic cell sorting (MACS) system, CD34^+ cells were isolated from human bone marrow, cord blood, peripheral blood, and cultured in CFU system. The results showed that the excellent recovery (75%) and highest purity (95-99%) were acquired by using the MACS separation system, and the most CFCs were present in CD34^+ population, but not in CD34 fraction by clonogenic assays. The isolation of pure hematopoietic stem cells and progenitor cells is of experimental and clinical importance.

Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35

PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer＇s disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25-35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein.

应用流式细胞Index Sorting技术研究小鼠胚胎生血内皮细胞

目的:应用流式Index Sorting技术鉴定小鼠胚胎期d10(E10)的主动脉中生血内皮细胞(HEC)的表面分子表达特征,并结合体外孵育实验及免疫组化染色验证HEC的生血潜能。方法:已有研究显示细胞表面分子Kit和CD47均能标记造血相关群体。在此基础上,应用流式细胞Index Sorting技术分选单个分子表型为CD41^-CD43^-CD45^-CD31^+Kit^+的主动脉内皮细胞,并与基质细胞OP9-DL1共孵育诱导培养7 d,进而结合单个内皮细胞的流式分选特征参数,及诱导后生成CD45^+造血细胞和CD31^+内皮细胞的效率,综合回溯分析Kit和CD47富集HEC的效果。结果:具有生血潜能的HEC均富集在CD47^+内皮细胞群体中,CD47分子将HEC群体的精度提高1.5倍;CD47^+内皮群体中,仍有60%以上的内皮不具备生血能力,提示进一步探索寻找富集HEC表面标志的必要性。结论:应用流式细胞Index Sorting技术发现CD47分子能显著富集小鼠胚胎期HEC,为进一步精准富集并研究HEC提供了重要的技术手段。

基于直觉模糊n-cell数的多属性决策方法及应用

针对权重已知的直觉模糊多属性决策问题,提出了一种基于直觉模糊n-cell数的决策方法。定义了直觉模糊n-cell数的偏序和两种基于均值和离散度的弱序。研究了所提出序的理论性质,并进行比较分析。最后,将相关排序方法应用于一类人才选拔的问题之中,在解决问题的同时验证了方法的有效性。

Microfluidic platform for circulating tumor cells isolation and detection

Circulating tumor cells(CTCs)are essential biomarkers for liquid biopsies,which are important in the early screening,prognosis,and real-time monitoring of cancer.However,CTCs are less abundant in the peripheral blood of patients,therefore,their isolation is necessary.Recently,the use of microfluidics for CTC sorting has become a research hotspot owing to its low cost,ease of integration,low sample consumption,and unique advantages in the manipulation of micron-sized particles.Herein,we review the latest research on microfluidics-based CTC sorting.Specifically,we consider active sorting using external fields(electric,magnetic,acoustic,and optical tweezers)and passive sorting using the flow effects of cells in specific channel structures(microfiltration sorting,deterministic lateral displacement sorting,and inertial sorting).The advantages and limitations of each method and their recent applications are summarized here.To conclude,a forward-looking perspective is presented on future research on the microfluidic sorting of CTCs.

Multi-objective optimization of the cathode catalyst layer micro-composition of polymer electrolyte membrane fuel cells using a multi-scale,two-phase fuel cell model and data-driven surrogates

Polymer electrolyte membrane fuel cells(PEMFCs)are considered a promising alternative to internal combustion engines in the automotive sector.Their commercialization is mainly hindered due to the cost and effectiveness of using platinum(Pt)in them.The cathode catalyst layer(CL)is considered a core component in PEMFCs,and its composition often considerably affects the cell performance(V_(cell))also PEMFC fabrication and production(C_(stack))costs.In this study,a data-driven multi-objective optimization analysis is conducted to effectively evaluate the effects of various cathode CL compositions on Vcelland Cstack.Four essential cathode CL parameters,i.e.,platinum loading(L_(Pt)),weight ratio of ionomer to carbon(wt_(I/C)),weight ratio of Pt to carbon(wt_(Pt/c)),and porosity of cathode CL(ε_(cCL)),are considered as the design variables.The simulation results of a three-dimensional,multi-scale,two-phase comprehensive PEMFC model are used to train and test two famous surrogates:multi-layer perceptron(MLP)and response surface analysis(RSA).Their accuracies are verified using root mean square error and adjusted R^(2).MLP which outperforms RSA in terms of prediction capability is then linked to a multi-objective non-dominated sorting genetic algorithmⅡ.Compared to a typical PEMFC stack,the results of the optimal study show that the single-cell voltage,Vcellis improved by 28 m V for the same stack price and the stack cost evaluated through the U.S department of energy cost model is reduced by$5.86/k W for the same stack performance.

流式分选仪的创新调试及在初始T细胞纯化中的应用

针对敏感细胞样本如何调试流式分选仪以获得高纯度、高活性的分选产物是众多流式分选从业者关注的问题。该文以流式分选仪FACSAria Fusion为例,首创性地将工程师调试界面与用户调试界面相结合调整系统默认最低样品进样压力,并配合85μm喷嘴,按照用户常规操作流程,在流速1.0的情况下对小鼠原代初始T细胞进行纯化,并对产物的质量进行检测。结果证明阐述的调试方法可以成功地获得高纯度初始T细胞,并且诱导分化成的调节T细胞产生特异转录因子Foxp3的能力与未分选细胞相当。

流式细胞仪样品预处理系统的设计与实现

根据流式细胞仪对样品“均一化”的需求,研制了一套基于微流控细胞分选技术的流式细胞仪样品预处理系统,装置可以实现对粒径小于100µm细胞或微粒的驱动和分选.系统主要包含微流控分选芯片和样品驱动模块两部分,通过聚焦不同位置,实现了对不同粒径细胞/颗粒的有效分离.系统无需对细胞进行标记处理,经分选的细胞便于后续流式细胞仪检测.经验证,系统能够有效去除牡蛎血淋巴细胞样品中的大粒子杂质,提高细胞样品的稳定性和均一性,增加流式细胞仪检测结果的准确性.

深度学习驱动的液滴微流控单细胞分选系统

单细胞操作和分析对于研究许多基本生物学过程和揭示细胞异质性至关重要,并且在生物医学领域具有巨大的应用潜力。液滴微流控技术在单细胞分析方面具有显著优势。研制了一种深度学习驱动的液滴微流控单细胞分选系统,主要以液滴内所包含的生物样本种类以及数量作为标准分选目标液滴。根据实验需求制作好相应生物样本的数据集,在服务器上训练好对应的网络模型,并将该网络模型转移到NVIDIA Jetson TX2开发板上,利用该网络模型对实验过程中拍摄到的液滴图像进行实时检测判断,最后根据算法对包含特定物质的液滴进行分选,从而得到目标液滴。此方法能够有效地判断并分选出液滴内图像特征有差异的不同生物样本,可以实现对包含单个及2个细胞液滴的分选。该研究为液滴微流控单细胞分选技术在生物学和医学等领域的广泛应用提供了支撑。

Effects of brain-derived neurotrophic factor on induced differentiation of SH-SY5Y cells in vitro

BACKGROUND： Previous studies have demonstrated that brain-derived neurotrophic factor （BDNF） promotes neural differentiation. However, the mechanisms involved in cell cycle-related protein regulation, which highly correlates to neural proliferation and apoptosis, remain poorly understood. OBJECTIVE： To investigate the effects of various concentrations of BDNF on cycle-related protein mRNA expression in induce-differentiated SH-SY5Y cells in vitro prior to and following G2 phase, and to analyze the neuroprotective effects of BDNF. DESIGN, TIME AND SETTING： A comparison, observational study, based on cell biology, was performed at the Department of Biochemistry, Medical College of Tongji University, from March 2005 to October 2006. MATERIALS： SH-SY5Y cells were provided by Shanghai Institute of Cytology, Chinese Academy of Science; BDNF by Alomone Labs, Israel; all-trans retinoic acid （ATRA） by Sigma-Aldrich, USA. METHODS： SH-SY5Y cells were randomly divided into three groups： blank control [cells were treated in Insulin-Transferrin-Selenium （ITS） solution for 7 days], ATRA （cells were treated with ITS solution containing 10 μmol/L ATRA for 7 days）, and BDNF （cells were treated identical to the ATRA group for 5 days, and then respectively treated in ITS solution containing 1, 10, and 100 μg/L BDNF for 2 days）. The experiment was repeated three times for each group. MAIN OUTCOME MEASURES： mRNA expression levels of cyclin A1, B1, B2, cyclin-dependent kinase 1, and 5 were detected using quantitative real-time RT-PCR; percentage of cells in G1, S, and G2 phases were detected using fluorescence-activated cell sorting. RESULTS： mRNA expression levels of cyclin A1 in the high-dose BDNF group was significantly less than the ATRA group （P 〈 0.05）.mRNA expression levels of cyclin B1 was significantly less in the different BDNF concentration groups compared with the control and ATRA groups （P 〈 0.05 or P 〈 0.01）. mRNA expression levels of cyclin B2 and cyclin-dependent kinase 1 were significantly decreased in the high-dose BDNF group （P 〈 0.05 or P 〈 0.01）. Cyclin-dependent kinase 5 mRNA expression was significantly greater in the low-dose and moderate-dose BDNF groups compared with the ATRA group （P 〈 0.05）. The percentage of cells in G1 phase was significantly greater in the different BDNF concentration groups compared with the ATRA and control groups （P 〈 0.01）. Moreover, the percentage of cells in S phase was significantly less in the three BDNF groups compared with the ATRA group （P 〈 0.01）. However, the percentage of cells in S phase was significantly less in the low-dose and high-dose BDNF groups compared with the control group （P 〈 0.01）. CONCLUSION： BDNF enhanced the percentage of cells in G1 phase, but did not alter mRNA expression of cell cycle-related proteins prior to or following G2 phase. These results suggested that BDNF was not a risk factor for inducing apoptosis.

血清sMICA、PCNA、GASP-1、TIMP-1在非小细胞肺癌患者中的表达及相关性分析

目的探讨血清可溶性MHC-I类链相关蛋白A(sMICA)、增殖细胞核抗原(PCNA)、G蛋白偶联受体相关分选蛋白1(GASP-1)、组织金属蛋白酶抑制剂1(TIMP-1)在非小细胞肺癌(NSCLC)患者中的表达及与病理分型的相关性。方法2020年7月至2022年8月诊治的86例NSCLC患者作为研究对象,并设立为观察组,同期选取43例健康体检者设立为对照组;并根据不同病理分型将观察组分为腺癌组(n=33)和鳞癌组(n=53),对比血清sMICA、PCNA、GASP-1、TIMP-1;并采用Logistic回归模型分析sMICA、PCNA、GASP-1、TIMP-1对非小细胞肺癌的影响;采用ROC曲线模型分析sMICA、PCNA、GASP-1、TIMP-1诊断非小细胞肺癌的AUC、敏感度及特异度。结果观察组的sMICA、PCNA、GASP-1、TIMP-1均高于对照组(P<0.05)。腺癌组的sMICA、PCNA、GASP-1、TIMP-1均高于鳞癌组(P<0.05)。二元Logistic回归模型分析显示,sMICA、PCNA、GASP-1、TIMP-1高表达会对非小细胞肺癌的发生产生影响(P<0.05)。ROC曲线分析显示,sMICA、PCNA、GASP-1、TIMP-1及四项联合诊断NSCLC的AUC值分别为(0.750、0.654、0.819、0.788、0.843,P均<0.05),敏感度分别为57.00%、46.50%、67.40%、90.70%、79.10%;特异度分别为93.00%、93.00%、88.40%、58.10%、86.00%。结论sMICA、PCNA、GASP-1、TIMP-1在NSCLC患者中呈高表达趋势,其表达水平会随病理分型而升高。

磁性细胞分选技术的应用与生物学评价

磁性细胞分选技术是一种利用超顺磁性纳米复合材料进行细胞分选的细胞高度特异性快速分选技术,在免疫学、干细胞学、肿瘤学和临床医学等领域应用广泛。本文综合阐述了磁性细胞分选技术的分类和应用,讨论了近几年出现的几项基于磁性细胞分选的新技术和面临的挑战。重点分析了磁性细胞分选产品生物学评价的必要性,并提出了10项与磁性细胞分选产品相关的生物学评价技术参数:得率、纯度、无菌、细胞毒性、细胞形态、活率、细胞的光散射特性、细胞的荧光抗体标记能力、细胞活化、细胞增殖,该评价技术参数的提出对磁性细胞分选规范化应用具有重要的推动作用。

AI-assisted cell identification and optical sorting[Invited]

Cell identification and sorting have been hot topics recently.However,most conventional approaches can only predict the category of a single target,and lack the ability to perform multitarget tasks to provide coordinate information of the targets.This limits the development of high-throughput cell screening technologies.Fortunately,artificial intelligence(AI)systems based on deep-learning algorithms provide the possibility to extract hidden features of cells from original image information.Here,we demonstrate an AI-assisted multitarget processing system for cell identification and sorting.With this system,each target cell can be swiftly and accurately identified in a mixture by extracting cell morphological features,whereafter accurate cell sorting is achieved through noninvasive manipulation by optical tweezers.The AI-assisted model shows promise in guiding the precise manipulation and intelligent detection of high-flux cells,thereby realizing semiautomatic cell research.

微流控技术在过继细胞免疫治疗中的应用研究进展

过继细胞疗法(ACT)是一种个体化疗法,通过采集患者自身或供体的T细胞,在体外进行基因增强后重新引入患者体内,以对抗特定的癌细胞或感染因子。然而,ACT存在许多的局限性,如多步骤耗时且成本高昂等。微流控技术(MT)是以微观通道流动为基础,借助微加工技术制造的微流体芯片和微阀门系统进行分析和操作的技术。微流控平台具有集成度高、效率高、高通量、样品需求低等优点,能够有效简化ACT制备步骤,降低生产成本,探索药物开发过程中的多种因素,以及模拟肿瘤微环境。现对MT在ACT疗法中的细胞分选、细胞增殖和临床前测定及药物评估方面以及在其他肿瘤免疫治疗中的应用进行综述。

糖尿病肾病小鼠CD4^(+)T细胞的脂质组学研究

目的:初步探讨糖尿病肾病(DKD)小鼠CD4^(+)T免疫细胞在脂质组学方面的差异,筛选出具有生物学意义的差异代谢产物。方法:先采用CD4 (L3T4) MicroBeads免疫磁珠法分离BKS.Cg-Dock7^(m+/+)Lepr^(db)/J自发性DKD小鼠脾脏CD4^(+)T免疫细胞;流式细胞术鉴定CD4^(+)T免疫细胞纯度,LC-MS/MS技术检测CD4^(+)T免疫细胞非靶向脂质组学,对差异代谢产物进行分析。结果:LC-MS法检测出463个代谢产物;PCA和OPLS-DA分析显示代谢组分明显分离;筛选出24种差异代谢物。KEGG及富集分析可知差异代谢物涉及甘油磷脂代谢紊乱。结论:CD4^(+)T细胞磷脂类代谢与DKD的发生密切相关,靶向DKD CD4^(+)T细胞的磷脂类代谢可能是DKD治疗的新方向。

采用流式细胞分选术区分中枢神经系统内小胶质细胞和浸润巨噬细胞

目的建立通过流式细胞分选术区分中枢神经系统内小胶质细胞和浸润巨噬细胞的方法。方法用成年C57BL/6小鼠建立双侧颈总动脉狭窄模型,分别采用集落刺激因子1受体抑制剂PLX5622或氯膦酸盐脂质体处理。将分离、匀浆、重悬后的小鼠脑和脊髓组织进行Percoll密度梯度离心,得到单核细胞悬液。采用CD45、CD11b和淋巴细胞抗原6家族成员C(Ly6C)抗体进行流式分选,获得小胶质细胞(CD11b^(+)CD45^(low)Ly6C^(-)细胞)和浸润巨噬细胞(CD11b^(+)CD45^(high)Ly6C^(+)细胞),并验证PLX5622和氯膦酸盐脂质体2种给药范式获得的处理效果。结果通过CD45、CD11b和Ly6C抗体可以有效区分中枢神经系统中小胶质细胞和浸润巨噬细胞。与对照组比较,PLX5622处理后小胶质细胞数量减少(P=0.001),而氯膦酸盐脂质体处理后浸润巨噬细胞数量减少(P<0.001)。结论所建立的流式细胞分选方法可有效区分中枢神经系统中小胶质细胞和浸润巨噬细胞,PLX5622和氯膦酸盐脂质体2种给药范式可分别选择性清除中枢神经系统内的小胶质细胞和浸润巨噬细胞。