微流控芯片技术用于精子与上皮细胞分离的研究

为研究建立使用微流控芯片技术分离精子与阴道上皮细胞的方法,选用制作工艺简单的玻璃-PDMS芯片,对混合样本进行分离。加样前分别在进、出口池中加入7此和lO此缓冲液,然后在进样口加入2此混合样本。至少静置8nlin后,从出口池中取出3此形成重力驱动的微流体后开始分离,每隔5min在进样口补加1此缓冲液。达到理想分离效果时,用移液器从出口池取出分离出的精子,核酸酶去除游离DNA,经过提取、扩增和电泳分离脸测等步骤得到精子的分型结果。结果显示,使用基于重力驱动微流体原理的微流控芯片可在30min内分离出精子,不会有上皮细胞进入分离通道;通过核酸酶对分离出的精子液的去游DNA处理,可得到单一、完整的精子分型。与传统的差异裂解法相比,这种方法在很大程度上节省了检验时间,在性侵案件中具有一定的法医物证分析价值。     

Enrichment using antibody-coated microfluidic chambers in shear flow: model mixtures of human lymphocytes

Isolation of phenotypically-pure cell subpopulations from heterogeneous cell mixtures such as blood is a difficult yet fundamentally important task. Current techniques such as fluorescent activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) require pre-incubation with antibodies which lead to processing times of at least 15-60 min. In this study, we explored the use of antibody-coated microfluidic chambers to negative deplete undesired cell types, thus obtaining an enriched cell subpopulation at the outlet. We used human lymphocyte cell lines, MOLT-3 and Raji, as a model system to examine the dynamic cell binding behavior on antibody coated surfaces under shear flow. Shear stress ranging between 0.75 and 1.0 dyn/cm2 was found to provide most efficient separation. Cell adhesion was shown to follow pseudo-first order kinetics, and an anti-CD19 coated (Raji-depletion) device with approximately 2.6 min residence time was demonstrated to produce 100% pure MOLT-3 cells from 50-50 MOLT-3/Raji mixture. We have developed a mathematical model of the separation device based on the experimentally determined kinetic parameters that can be extended to design future separation modules for other cell mixtures. We conclude that we can design microfluidic devices that exploits the kinetics of dynamic cell adhesion to antibody coated surfaces to provide enriched cell subpopulations within minutes of total processing time.     

High-gradient magnetic affinity separation of trypsin from porcine pancreatin

We introduce a robust and scale-flexible approach to macromolecule purification employing tailor-made magnetic adsorbents and high-gradient magnetic separation technology adapted from the mineral processing industries. Detailed procedures for the synthesis of large quantities of low-cost defined submicron-sized magnetic supports are presented. These support materials exhibit unique features, which facilitate their large-scale processing using high magnetic field gradients, namely sufficiently high magnetization, a relatively narrow particle size distribution and ideal superparamagnetism. Following systematic optimization with respect to activation chemistry, spacer length and ligand density, conditions for preparation of effective high capacity (Q(max) = 120 mg g(-1)) strongly interacting (Kd < 0.3 microm) trypsin-binding adsorbents based on immobilized benzamidine were established. In small-scale studies approximately 95% of the endogenous trypsin present in a crude porcine pancreatin feedstock was recovered with a purification factor of approximately 4.1 at the expense of only a 4% loss in alpha-amylase activity. Efficient recovery of trypsin from the same feedstock was demonstrated at a vastly increased scale using a high-gradient magnetic separation system to capture loaded benzamidine-linked adsorbents following batch adsorption. With the aid of a simple recycle loop over 80% of the initially adsorbed trypsin was recovered in-line with an overall purification factor of approximately 3.5.     

Semi‐continuous in situ magnetic separation for enhanced extracellular protease production—modeling and experimental validation

In modern biotechnology proteases play a major role as detergent ingredients. Especially the production of extracellular protease by Bacillus species facilitates downstream processing because the protease can be directly harvested from the biosuspension. In situ magnetic separation (ISMS) constitutes an excellent adsorptive method for efficient extracellular protease removal during cultivation. In this work, the impact of semi‐continuous ISMS on the overall protease yield has been investigated. Results reveal significant removal of the protease from Bacillus licheniformis cultivations. Bacitracin‐functionalized magnetic particles were successfully applied, regenerated and reused up to 30 times. Immediate reproduction of the protease after ISMS proved the biocompatibility of this integrated approach. Six subsequent ISMS steps significantly increased the overall protease yield up to 98% because proteolytic degradation and potential inhibition of the protease in the medium could be minimized. Furthermore, integration of semi‐continuous ISMS increased the overall process efficiency due to reduction of the medium consumption. Process simulation revealed a deeper insight into protease production, and was used to optimize ISMS steps to obtain the maximum overall protease yield. Biotechnol. Bioeng. 2013; 110: 2161–2172. © 2013 Wiley Periodicals, Inc.     

Stem cell separation: A bottleneck in stem cell therapy

The substantial progress in embryonic stem cell (ESC) research could lead to new possibilities in the treatment of various diseases. Currently, applications of ESC for cell therapy are impeded by the presence of potentially teratoma-forming undifferentiated ESC. Thus, a selective and quantitative removal of undifferentiated ESC from a pool of differentiated and undifferentiated cells is essential before cell therapy. We evaluated the highly selective magnetic activated cell sorting (MACS) method for the quantitative removal of undifferentiated ESC. We found that the clearance rates for undifferentiated ESC decreased with decreasing amount of undifferentiated ESC in the cell pool. Using a simplified model calculation we could predict that, assuming an initial purity of 60%, an estimated 31 steps are required to achieve less than 10^–1 cell per 10⁹ cells. Thus, a log clearance rate of 10, which would be necessary for a therapeutically application, is hard to achieve. Our work clearly indicates that the current MACS technology is insufficient to meet the purification needs for cell therapy.     

Avidity‐mediated virus separation using a hyperthermophilic affinity ligand

Immunoaffinity separation of large multivalent species such as viruses is limited by the stringent elution conditions necessary to overcome their strong and highly avid interaction with immobilized affinity ligands on the capture surface. Here we present an alternate strategy that harnesses the avidity effect to overcome this limitation. Red clover necrotic mosaic virus (RCNMV), a plant virus relevant to drug delivery applications, was chosen as a model target for this study. An RCNMV binding protein (RBP) with modest binding affinity (K_D ～100 nM) was generated through mutagenesis of the Sso7d protein from Sulfolobus solfataricus and used as the affinity ligand. In our separation scheme, RCNMV is captured by a highly avid interaction with RBP immobilized on a nickel surface through a hexahistidine (6xHis) tag. Subsequently, disruption of the multivalent interaction and release of RCNMV is achieved by elution of RBP from the nickel surface. Finally, RCNMV is separated from RBP by exploiting the large difference in their molecular weights (～8 MDa vs. ～10 kDa). Our strategy not only eliminates the need for harsh elution conditions, but also bypasses chemical conjugation of the affinity ligand to the capture surface. Stable non‐antibody affinity ligands to a wide spectrum of targets can be generated through mutagenesis of Sso7d and other hyperthermophilic proteins. Therefore, our approach may be broadly relevant to cases where capture of large multivalent species from complex mixtures and subsequent release without the use of harsh elution conditions is necessary. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Antibody‐immobilized column for quick cell separation based on cell rolling

Cell separation using methodological standards that ensure high purity is a very important step in cell transplantation for regenerative medicine and for stem cell research. A separation protocol using magnetic beads has been widely used for cell separation to isolate negative and positive cells. However, not only the surface marker pattern, e.g., negative or positive, but also the density of a cell depends on its developmental stage and differentiation ability. Rapid and label‐free separation procedures based on surface marker density are the focus of our interest. In this study, we have successfully developed an antiCD34 antibody‐immobilized cell‐rolling column, that can separate cells depending on the CD34 density of the cell surfaces. Various conditions for the cell‐rolling column were optimized including graft copolymerization, and adjustment of the column tilt angle, and medium flow rate. Using CD34‐positive and ‐negative cell lines, the cell separation potential of the column was established. We observed a difference in the rolling velocities between CD34‐positive and CD34‐negative cells on antibody‐immobilized microfluidic device. Cell separation was achieved by tilting the surface 20 degrees and the increasing medium flow. Surface marker characteristics of the isolated cells in each fraction were analyzed using a cell‐sorting system, and it was found that populations containing high density of CD34 were eluted in the delayed fractions. These results demonstrate that cells with a given surface marker density can be continuously separated using the cell rolling column. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Bacteriocidal effects and inhibition of cell separation of cinnamic aldehyde on Bacillus cereus

AIMS: In this study, bacteriocidal effects of cinnamic aldehyde on Bacillus cereus were investigated. METHODS: The bacterial culture or cell suspension in 0.85% NaCl was treated with cinnamic aldehyde at a concentration of 0.3 ml l(-1). Viable cells were counted on a nutrient agar plate. Protein leakage from the cell was determined using a protein dye. Cell morphology was observed using a scanning electron microscope. RESULTS: Bacillus cereus cells were the most sensitive to cinnamic aldehyde among four different food-borne pathogens. When the cells were treated with 0.3 ml l(-1) of cinnamic aldehyde, the viable counts decreased about 6 log cycles after 6 h of incubation. The bacterial cells remained unlysed although they were killed by cinnamic aldehyde. Treatment of cinnamic aldehyde to the exponential phase cells resulted in no significant protein leakage but strong inhibition of cell separation. CONCLUSIONS: The present findings suggest that cinnamic aldehyde exhibits bacteriocidal effects and inhibition of cell separation on B. cereus. SIGNIFICANCE AND IMPACT OF THE STUDY: These data represent an interesting background for a possible mechanism for antibacterial effects of cinnamic aldehyde.     

Investigation of a two‐step device implementing magnetophoresis and dielectrophoresis for separation of circulating tumor cells from blood cells

Identifying tumor cells from a pool of other cells has always been an appealing topic for different purposes. The objective of this study is to discriminate circulating tumor cells (CTCs) from blood cells for diagnostic purposes in a novel microfluidic device using two active methods: magnetophoresis and dielectrophoresis. The most specific feature of this device is the differentiation of CTCs without labeling them in order to achieve a more reliable and less complicated method. This device was analyzed and evaluated using finite element method. Four cell lines are separated in this device containing red blood cells, platelets, white blood cells, and CTCs. Primarily, red blood cells and platelets, which constitute the largest part of a blood sample, are removed in the magnetophoresis section. Remaining cells enter the dielectrophoresis part and based on their inherent dielectric properties and diameters, final separation occurs. In each step, different parameters are examined to obtain the maximum purification. The results demonstrate the potential of different CTCs separation by changing the effective parameters in the designed device based on the inherent properties of the cells.     

A metal‐chelate affinity reverse micellar system for protein extraction

A new nonionic reverse micellar system is developed by blending two nonionic surfactants, Triton X‐45 and Span 80. At total surfactant concentrations lower than 60 mmol/L and molar fractions of Triton X‐45 less than 0.6, thermodynamically stable reverse micelles of water content (W₀) up to 30 are formed. Di(2‐ethylhexyl) phosphoric acid (HDEHP; 1–2 mmol/L) is introduced into the system for chelating transition metal ions that have binding affinity for histidine‐rich proteins. HDEHP exists in a dimeric form in organic solvents and a dimer associated with one transition metal ion, including copper, zinc, and nickel. The copper‐chelate reverse micelles (Cu‐RM) are characterized for their W₀, hydrodynamic radius (R_h), and aggregation number (N_ag). Similar with reverse micelles of bis‐2‐ethylhexyl sodium sulfosuccinate (AOT), R_h of the Cu‐RM is also linearly related to W₀. However, N_ag is determined to be 30–90 at W₀ of 5–30, only quarter to half of the AOT reverse micelles. Then, selective metal‐chelate extraction of histidine‐rich protein (myoglobin) by the Cu‐RM is successfully performed with pure and mixed protein systems (myoglobin and lysozyme). The solubilized protein can be recovered by stripping with imidazole or ethylinediaminetetraacetic acid (EDTA) solution. Because various transition metal ions can be chelated to the reverse micelles, it is convinced that the system would be useful for application in protein purification as well as simultaneous isolation and refolding of recombinant histidine‐tagged proteins expressed as inclusion bodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A uniform‐shear rate microfluidic bioreactor for real‐time study of proplatelet formation and rapidly‐released platelets

Platelet transfusions, with profound clinical importance in blood clotting and wound healing, are entirely derived from human volunteer donors. Hospitals rely on a steady supply of donations, but these methods are limited by a 5‐day shelf life, the potential risk of contamination, and differences in donor/recipient histocompatibility. These challenges invite the opportunity to generate platelets ex vivo. Although much progress has been made in generating large numbers of culture‐derived megakaryocytes (Mks, the precursor cells to platelets), stimulating a high percentage of Mks to undergo platelet release remains a major challenge. Recent studies have demonstrated the utility of shear forces to enhance platelet release from cultured Mks. In this study, we performed a computational fluid dynamics (CFD) analysis of several published platelet microbioreactor systems, and used the results to develop a new 7‐µm slit bioreactor—with well‐defined flow patterns and uniform shear profiles. This uniform‐shear‐rate bioreactor (USRB‐7µm) permits real‐time visualization of the proplatelet (proPLT) formation process and the rapid‐release of individual platelet‐like‐particles (PLPs), which has been observed in vivo, but not previously reported for platelet bioreactors. We showed that modulating shear forces and flow patterns had an immediate and significant impact on PLP generation. Surprisingly, using a single flow instead of dual flows led to an unexpected six‐fold increase in PLP production. By identifying particularly effective operating conditions within a physiologically relevant environment, this USRB‐7µm will be a useful tool for the study and analysis of proPLT/PLP formation that will further understanding of how to increase ex vivo platelet release. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1614–1629, 2017     

Daughter cell separation is controlled by cytokinetic ring‐activated cell wall hydrolysis

During bacterial cytokinesis, hydrolytic enzymes are used to split wall material shared by adjacent daughter cells to promote their separation. Precise control over these enzymes is critical to prevent breaches in wall integrity that can cause cell lysis. How these potentially lethal hydrolases are regulated has remained unknown. Here, we investigate the regulation of cell wall turnover at the Escherichia coli division site. We show that two components of the division machinery with LytM domains (EnvC and NlpD) are direct regulators of the cell wall hydrolases (amidases) responsible for cell separation (AmiA, AmiB and AmiC). Using in vitro cell wall cleavage assays, we show that EnvC activates AmiA and AmiB, whereas NlpD activates AmiC. Consistent with these findings, we show that an unregulated EnvC mutant requires functional AmiA or AmiB but not AmiC to induce cell lysis, and that the loss of NlpD phenocopies an AmiC^? defect. Overall, our results suggest that cellular amidase activity is regulated spatially and temporally by coupling their activation to the assembly of the cytokinetic ring.     

Exploring additivity effects of double mutations on the binding affinity of protein‐protein complexes

Additivity in binding affinity of protein‐protein complexes refers to the change in free energy of binding (ΔΔG_bind) for double (or multiple) mutations which is approximately equal to the sum of their corresponding single mutation ΔΔG_bind values. In this study, we have explored the additivity effect of double mutants, which shows a linear relationship between the binding affinity of double and sum of single mutants with a correlation of 0.90. However, the comparison of ΔΔG_bind values showed a mean absolute deviation of 0.86 kcal/mol, and 25.6% of the double mutants show a deviation of more than 1 kcal/mol, which are identified as non‐additive. The additivity effects have been analyzed based on the influence of structural features such as accessible surface area, long range order, binding propensity change, surrounding hydrophobicity, flexibility, atomic contacts between the mutations and distance between the 2 mutations. We found that non‐additive mutations tend to be closer to each other and have more contacts. We have also used machine learning methods to discriminate additive and non‐additive mutations using structure‐based features, which showed the accuracies in the range of 0.77–0.92 for protein‐protein complexes belonging to different functions. Further, we have compared the additivity effects of protein stability along with binding affinity and explored the similarities and differences between them. The results obtained in this study provide insights into the effects of various structural features on binding affinity of double mutants, and will aid the development of accurate methods to predict the binding affinity of double mutants.     

High‐efficiency affinity precipitation of multiple industrial mAbs and Fc‐fusion proteins from cell culture harvests using Z‐ELP‐E2 nanocages

Affinity precipitation using Z‐elastin‐like polypeptide‐functionalized E2 protein nanocages has been shown to be a promising alternative to Protein A chromatography for monoclonal antibody (mAb) purification. We have previously described a high‐yielding, affinity precipitation process capable of rapidly capturing mAbs from cell culture through spontaneous, multivalent crosslinking into large aggregates. To challenge the capabilities of this technology, nanocage affinity precipitation was investigated using four industrial mAbs (mAbs A–D) and one Fc fusion protein (Fc A) with diverse molecular properties. A molar binding ratio of 3:1 Z:mAb was sufficient to precipitate >95% mAb in solution for all molecules evaluated at ambient temperature without added salt. The effect of solution pH on aggregation kinetics was studied using a simplified two‐step model to investigate the protein interactions that occur during mAb–nanocage crosslinking and to determine the optimal solution pH for precipitation. After centrifugation, the pelleted mAb–nanocage complex remained insoluble and was capable of being washed at pH ≥ 5 and eluted with at pH < 4 with >90% mAb recovery for all molecules. The four mAbs and one Fc fusion were purified from cell culture using optimal process conditions, and >94% yield and >97% monomer content were obtained. mAb A–D purification resulted in a 99.9% reduction in host cell protein and >99.99% reduction in DNA from the cell culture fluids. Nanocage affinity precipitation was equivalent to or exceeded expected Protein A chromatography performance. This study highlights the benefits of nanoparticle crosslinking for enhanced affinity capture and presents a robust platform that can be applied to any target mAb or Fc‐containing proteins with minimal optimization of process parameters.     

The alpha-glucanase Agn1p is required for cell separation in Schizosaccharomyces pombe

BACKGROUND INFORMATION: In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast, ring constriction is followed by deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomyosin ring and septum assembly, little is known about the later steps involving the cleavage of the cell wall. RESULTS: We identified a novel gene in Schizosaccharomyces pombe, namely the agn1(+) gene that has homology to fungal 1,3-alpha-glucanases (mutanases). Disruption of the agn1(+) gene is not lethal to the cells, but does interfere with their separation, whereas overexpression of Agn1p is toxic and causes cell lysis. Agn1p levels reach a peak during septation and the protein localizes to the septum region before cell separation. Moreover, agn1(+) is responsible for the 1,3-alpha-glucanase activity, which shows a maximum at the end of septation. CONCLUSIONS: Our results clearly suggest the existence of a relationship between agn1(+), 1,3-alpha-glucanase activity and the completion of septation in S. pombe. Agn1p could be involved in the cleavage of the cylinder of the old wall that surrounds the primary septum, a region rich in alpha-glucans.     

Quantification of chemical–polymer surface interactions in microfluidic cell culture devices

Microfluidic cell culture devices have been used for drug development, chemical analysis, and environmental pollutant detection. Because of the decreased fluid volume and increased surface area to volume ratio, interactions between device surfaces and the fluid is a key element that affects the performance and detection accuracy of microfluidic devices, particularly if fluid is recirculated by a peristaltic pump. However, this issue has not been studied in detail in a microfluidic cell culture environment. In this study, chemical loss and contaminant leakage from various polymer surfaces in a microfluidic setup were characterized. The effects of hydrophilic coating with Poly (vinyl alcohol), Pluronic® F‐68, and multi‐layer ionic coating were measured. We observed significant surface adsorption of estradiol, doxorubicin, and verapamil with PharMed® BPT tubing, whereas PTFE/BPT and stainless steel/BPT hybrid tubing caused less chemical loss in proportion to the fraction of BPT tubing in the hybrid system. Contaminants leaching out of the BPT tubing were found to be estrogen receptor agonists as determined by estrogen‐induced green fluorescence expression in an estrogen responsive Ishikawa cell line and also caused interference with an estradiol enzyme‐linked immunosorbent assay (ELISA) assay. Stainless steel/BPT hybrid tubing caused the least interference with ELISA. In summary, polymer surface and chemical interactions inside microfluidic systems should not be neglected and require careful investigations when results from a microfluidic system are compared with results from a macroscale cell culture setup. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Quantification of non‐specific binding of magnetic micro‐ and nanoparticles using cell tracking velocimetry: Implication for magnetic cell separation and detection

The maturation of magnetic cell separation technology places increasing demands on magnetic cell separation performance. While a number of factors can cause sub‐optimal performance, one of the major challenges can be non‐specific binding of magnetic nano‐ or microparticles to non‐targeted cells. Depending on the type of separation, this non‐specific binding can have a negative effect on the final purity, the recovery of the targeted cells, or both. In this work, we quantitatively demonstrate that non‐specific binding of magnetic nanoparticles can impart a magnetization to cells such that these cells can be retained in a separation column and thus negatively impact the purity of the final product and the recovery of the desired cells. Through experimental data and theoretical arguments, we demonstrate that the number of MACS magnetic particles needed to impart a magnetization that is sufficient to cause non‐targeted cells to be retained in the column to be on the order of 500–1,000 nanoparticles. This number of non‐specifically bound particles was demonstrated experimentally with an instrument, cell tracking velocimeter, CTV, and it is demonstrated that the sensitivity of the CTV instrument for Fe atoms contained in magnetic nanoparticles on the order of 1 × 10^?15 g/mL of Fe. Biotechnol. Bioeng. 2010;105: 1078–1093. © 2009 Wiley Periodicals, Inc.