A novel fragment of antigen binding (Fab) surface display platform using glycoengineered Pichia pastoris

A fragment of antigen binding (Fab) surface display system was developed using a glycoengineered Pichia pastoris host strain genetically modified to secrete glycoproteins with mammalian mannose-type Man(5)GlcNAc(2) N-linked glycans. The surface display method described here takes advantage of a pair of coiled-coil peptides as the linker while using the Saccharomyces cerevisiae Sed1p GPI-anchored cell surface protein as an anchoring domain. Several Fabs were successfully displayed on the cell surface using this system and the expression level of the displayed Fabs was correlated to that of secreted Fabs from the same glycoengineered host in the absence of the cell wall anchor. Strains displaying different model Fabs were mixed and, through cell sorting, the strain displaying more expressed Fab molecule or the strain displaying the Fab with higher affinity for an antigen was effectively enriched by FACS. This novel yeast surface display system provides a general platform for the display of Fab libraries for affinity and/or expression maturation using glycoengineered Pichia.     

A microfluidic cell culture platform for real-time cellular imaging

This study reports a new microfluidic cell culture platform for real-time, in vitro microscopic observation and evaluation of cellular functions. Microheaters, a micro temperature sensor, and micropumps are integrated into the system to achieve a self-contained, perfusion-based, cell culture microenvironment. The key feature of the platform includes a unique, ultra-thin, culture chamber with a depth of 180 μm, allowing for real-time, high-resolution cellular imaging by combining bright field and fluorescent optics to visualize nanoparticle-cell/organelle interactions. The cell plating, culturing, harvesting and replenishing processes are performed automatically. The developed platform also enables drug screening and real-time, in situ investigation of the cellular and sub-cellular delivery process of nano vectors. The mitotic activity and the interaction between cells and the nano drug carriers (conjugated quantum dots-epirubicin) are successfully monitored in this device. This developed system could be a promising platform for a wide variety of applications such as high-throughput, cell-based studies and as a diagnostic cellular imaging system.     

A microfluidic platform for 3-dimensional cell culture and cell-based assays

This paper reports a novel microfluidic platform introducing peptide hydrogel to make biocompatible microenvironment as well as realizing in situ cell-based assays. Collagen composite, OPLA and Puramatrix scaffolds are compared to select good environment for human hepatocellular carcinoma cells (HepG2) by albumin measurement. The selected biocompatible self-assembling peptide hydrogel, Puramatrix, is hydrodynamically focused in the middle of main channel of a microfluidic device, and at the same time the cells are 3-dimensionally immobilized and encapsulated without any additional surface treatment. HepG2 cells have been 3-dimensionally cultured in a poly(dimethylsiloxane) (PDMS) microfluidic device for 4 days. The cells cultured in micro peptide scaffold are compared with those cultured by conventional petri dish in morphology and the rate of albumin secretion. By injection of different reagents into either side of the peptide scaffold, the microfluidic device also forms a linear concentration gradient profile across the peptide scaffold due to molecular diffusion. Based on this characteristic, toxicity tests are performed by Triton X-100. As the higher toxicant concentration gradient forms, the wider dead zone of cells in the peptide scaffold represents. This microfluidic platform facilitates in vivo-like 3-dimensional microenvironment, and have a potential for the applications of reliable cell-based screening and assays including cytotoxicity test, real-time cell viability monitoring, and continuous dose-response assay.     

Hot embossing for fabrication of a microfluidic 3D cell culture platform

Clinically relevant studies of cell function in vitro require a physiologically-representative microenvironment possessing aspects such as a 3D extracellular matrix (ECM) and controlled biochemical and biophysical parameters. A polydimethylsiloxane (PDMS) microfluidic system with a 3D collagen gel has previously served for analysis of factors inducing different responses of cells in a 3D microenvironment under controlled biochemical and biophysical parameters. In the present study, applying the known commercially-viable manufacturing methods to a cyclic olefin copolymer (COC) material resulted in a microfluidic device with enhanced 3D gel capabilities, controlled surface properties, and improved potential to serve high-volume applications. Hot embossing and roller lamination molded and sealed the microfluidic device. A combination of oxygen plasma and thermal treatments enhanced the sealing, ensured proper placement of the 3D gel, and created controlled and stable surface properties within the device. Culture of cells in the new device indicated no adverse effects of the COC material or processing as compared to previous PDMS devices. The results demonstrate a methodology to transition microfludic devices for 3D cell culture from scientific research to high-volume applications with broad clinical impact.     

A versatile valve-enabled microfluidic cell co-culture platform and demonstration of its applications to neurobiology and cancer biology

A versatile microfluidic platform allowing co-culture of multiple cell populations in close proximity with separate control of their microenvironments would be extremely valuable for many biological applications. Here, we report a simple and compact microfluidic platform that has these desirable features and allows for real-time, live-cell imaging of cell-cell interactions. Using a pneumatically/hydraulically controlled poly(dimethylsiloxane) (PDMS) valve barrier, distinct cell types can be cultured in side-by-side microfluidic chambers with their optimum culture media and treated separately without affecting the other cell population. The platform is capable of both two-dimensional and three-dimensional cell co-culture and through variations of the valve barrier design, the platform allows for cell-cell interactions through either direct cell contact or soluble factors alone. The platform has been used to perform dynamic imaging of synapse formation in hippocampal neurons by separate transfection of two groups of neurons with fluorescent pre- and post-synaptic protein markers. In addition, cross-migration of 4T1 tumor cells and endothelial cells has been studied under normoxic and hypoxic conditions, which revealed different migration patterns, suggesting the importance of the microenvironments in cell-cell interactions and biological activities.     

A microfluidic cell co-culture platform with a liquid fluorocarbon separator

A microfluidic cell co-culture platform that uses a liquid fluorocarbon oil barrier to separate cells into different culture chambers has been developed. Characterization indicates that the oil barrier could be effective for multiple days, and a maximum pressure difference between the oil barrier and aqueous media in the cell culture chamber could be as large as ~3.43 kPa before the oil barrier fails. Biological applications have been demonstrated with the separate transfection of two groups of primary hippocampal neurons with two different fluorescent proteins and subsequent observation of synaptic contacts between the neurons. In addition, the quality of the fluidic seal provided by the oil barrier is shown to be greater than that of an alternative solid-PDMS valve barrier design by testing the ability of each device to block low molecular weight CellTracker dyes used to stain cells in the culture chambers.     

The culture and differentiation of amniotic stem cells using a microfluidic system

Human mesenchymal stem cells (MSCs) have the potential to differentiate into multiple tissue lineages for cell therapy and, therefore, have attracted considerable interest recently. In this study, a new microfluidic system is presented which can culture and differentiate MSCs in situ. It is composed of several components, including stem cell culture areas, micropumps, microgates, seeding reservoirs, waste reservoirs and fluid microchannels; all fabricated by using micro-electro-mechanical-systems (MEMS) technology. The developed automated system allows for the long-term culture and differentiation of MSCs. Three methods, including Oil Red O staining for adipogenic cells, alkaline phosphatase staining and immunofluorescence staining are used to assess the differentiation of MSCs. Experimental results clearly demonstrate that the MSCs can be cultured for proliferation and different types of differentiation are possible in this microfluidic system, which can maintain a suitable and stable pH value over long time periods. This prototype microfluidic system has great potential as a powerful tool for future MSC studies.     

Quantitative proteomic profiling of breast cancers using a multiplexed microfluidic platform for immunohistochemistry and immunocytochemistry

This paper describes a multiplexed microfluidic immunohistochemistry (IHC)/immunocytochemistry (ICC) platform for quantitative proteomic profiling in breast cancer samples. Proteomic profiling via ICC was examined for four breast cancer cell lines (AU-565, HCC70, MCF-7, and SK-BR-3). The microfluidic device enabled 20 ICC assays on a biological specimen at the same time and a 16-fold decrease in time consumption, and could be used to quantitatively compare the expression level of each biomarker. The immunohistochemical staining from the microfluidic system showed an accurate localization of protein and comparable quality to that of the conventional IHC method. Although AU-565 and SK-BR-3 cell lines were classified by luminal subtype and adenocarcinomas and were derived from the same patient, weak p63 expression was seen only in SK-BR-3. The HCC70 cell line showed a triple-negative (estrogen receptor-negative/progesterone receptor-negative/human epidermal growth factor receptor 2-negative) phenotype and showed only cytokeratin 5 expression, a representative basal/myoepithelial cell marker. To demonstrate the applicability of the system to clinical samples for proteomic profiling, we were also able to apply this platform to human breast cancer tissue. This result indicates that the microfluidic IHC/ICC platform is useful for accurate histopathological diagnoses using numerous specific biomarkers simultaneously, facilitating the individualization of cancer therapy.     

An agarose-based microfluidic platform with a gradient buffer for 3D chemotaxis studies

The current state-of-art in 3D microfluidic chemotaxis device (μFCD) is limited by the inherent coupling of the fluid flow and chemical concentration gradients. Here, we present an agarose-based 3D μFCD that decouples these two important parameters, in that the flow control channels are separated from the cell compartment by an agarose gel wall. This decoupling is enabled by the transport property of the agarose gel, which—in contrast to the conventional microfabrication material such as polydimethylsiloxane (PDMS)—provides an adequate physical barrier for convective fluid flow while at the same time readily allowing protein diffusion. We demonstrate that in this device, a gradient can be pre-established in an agarose layer above the cell compartment (a gradient buffer) before adding the 3D cell-containing matrix, and the dextran (10 kDa) concentration gradients can be re-established within 10 min across the cell-containing matrix and remain stable indefinitely. We successfully quantified the chemotactic response of murine dendritic cells to a gradient of CCL19, an 8.8 kDa lymphoid chemokine, within a type I collagen matrix. This model system is easy to set up, highly reproducible, and will benefit research on 3D chemoinvasion studies, for example with cancer cells or immune cells. Because of its gradient buffering capacity, it is particularly suitable for studying rapidly migrating cells like mature dendritic cells and neutrophils. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Ulrike Haessler and Yevgeniy Kalinin have equal contribution.     

巴斯德毕赤酵母表达系统表达重组蛋白的影响因素及优化

巴斯德毕赤酵母表达系统是一种优秀的重组蛋白表达系统,近年来被广泛应用于重组蛋白的表达。本文介绍了巴斯德毕赤酵母表达系统的特点及其用于外源蛋白表达的基本策略。随后重点讨论了发酵过程中影响外源蛋白表达的主要环境因素、影响机制及控制策略,这些影响因素包括诱导温度、甲醇浓度、溶解氧、氮源浓度和类型及诱导pH。它们影响重组蛋白的表达量和品质,并且这几种因素的作用是相互影响和制约的,因此需要系统优化。     

Development of an indirect ELISA for serological detection of reticuloendotheliosis virus using the gp90 protein expressed in Pichia pastoris

The present study was undertaken to express the gp90 protein of reticuloendotheliosis virus (REV) in Pichia pastoris and evaluate its potential use as a diagnostic antigen in ELISA. The full-length gp90 gene of REV was cloned into pPIC9k vector and then integrated into the chromosome of P. pastoris for induced expression. SDS-PAGE and western blot assay demonstrated that gp90 protein was expressed and secreted into the culture medium at about 100mg/L of culture under optimized condition. An indirect ELISA was then established by using the recombinant gp90 protein as the coating antigen. The optimal concentration of coated antigen was 0.1 μg/well at a serum dilution of 1:200 and the optimal positive threshold value of the assay was 0.409. Cross-reactivity assay showed that this antigen was REV specific. The reproducibility experiment displayed good consistency. Furthermore, the gp90 protein based indirect ELISA showed good correlation rates of 96.3% and 97.5% with virus neutralization test and a commercially whole virus based indirect ELISA, respectively. This study demonstrates the efficacy of recombinant gp90 protein as an antigen in ELISA for seroepidemiological study of REV infection on a large scale.     

A self-contained, programmable microfluidic cell culture system with real-time microscopy access

Utilizing microfluidics is a promising way for increasing the throughput and automation of cell biology research. We present a complete self-contained system for automated cell culture and experiments with real-time optical read-out. The system offers a high degree of user-friendliness, stability due to simple construction principles and compactness for integration with standard instruments. Furthermore, the self-contained system is highly portable enabling transfer between work stations such as laminar flow benches, incubators and microscopes. Accommodation of 24 individual inlet channels enables the system to perform parallel, programmable and multiconditional assays on a single chip. A modular approach provides system versatility and allows many different chips to be used dependent upon application. We validate the system’s performance by demonstrating on-chip passive switching and mixing by peristaltically driven flows. Applicability for biological assays is demonstrated by on-chip cell culture including on-chip transfection and temporally programmable gene expression.     

Invasion-Inhibitory Antibodies Elicited by Immunization with Plasmodium vivax Apical Membrane Antigen-1 Expressed in Pichia pastoris Yeast

In a recent vaccine trial performed with African children, immunization with a recombinant protein based on Plasmodium falciparum apical membrane antigen 1 (AMA-1) conferred a significant degree of strain-specific resistance against malaria. To contribute to the efforts of generating a vaccine against Plasmodium vivax malaria, we expressed the ectodomain of P. vivax AMA-1 (PvAMA-1) as a secreted soluble protein in the methylotrophic yeast Pichia pastoris. Recognized by a high percentage of sera from individuals infected by P. vivax, this recombinant protein was found to have maintained its antigenicity. The immunogenicity of this protein was evaluated in mice using immunization protocols that included homologous and heterologous prime-boost strategies with plasmid DNA and recombinant protein. We used the following formulations containing different adjuvants: aluminum salts (Alum), Bordetella pertussis monophosphoryl lipid A (MPLA), flagellin FliC from Salmonella enterica serovar Typhimurium, saponin Quil A, or incomplete Freund''s adjuvant (IFA). The formulations containing the adjuvants Quil A or IFA elicited the highest IgG antibody titers. Significant antibody titers were also obtained using a formulation developed for human use containing MPLA or Alum plus MPLA. Recombinant PvAMA-1 produced under “conditions of good laboratory practice” provided a good yield, high purity, low endotoxin levels, and no microbial contaminants and reproduced the experimental immunizations. Most relevant for vaccine development was the fact that immunization with PvAMA-1 elicited invasion-inhibitory antibodies against different Asian isolates of P. vivax. Our results show that AMA-1 expressed in P. pastoris is a promising antigen for use in future preclinical and clinical studies.     

Theoretical development and critical analysis of burst frequency equations for passive valves on centrifugal microfluidic platforms

This paper presents a theoretical development and critical analysis of the burst frequency equations for capillary valves on a microfluidic compact disc (CD) platform. This analysis includes background on passive capillary valves and the governing models/equations that have been developed to date. The implicit assumptions and limitations of these models are discussed. The fluid meniscus dynamics before bursting is broken up into a multi-stage model and a more accurate version of the burst frequency equation for the capillary valves is proposed. The modified equations are used to evaluate the effects of various CD design parameters such as the hydraulic diameter, the height to width aspect ratio, and the opening wedge angle of the channel on the burst pressure.     

重组酵母鸡α干扰素的研制及其抗病毒活性测定

目的通过酵母真核表达系统获得鸡α干扰素,并鉴定其抗病毒生物活性.方法以Con A刺激4～10小时后的鸡全血淋巴细胞提取的总RNA为模板,通过RT-PCR方法克隆出鸡α干扰素成熟蛋白基因,通过EcoR I 和Xba I两个酶切位点使该基因插入酵母表达载体(pPICZa-A),并把线性化该重组酵母鸡α干扰素载体,通过电转化使鸡α干扰素基因整和到酵母(X33)基因组上,利用微量病变抑制法测定表达的鸡α干扰素的抗病毒活性.结果实验结果表明重组酵母鸡α干扰素活性单位为10×48U/ml,具有较好的抗病毒效果.结论实验研究结果表明重组酵母鸡α干扰素具有较好的抗病毒能力.     

A microfluidic platform to isolate avian erythrocytes infected with Plasmodium gallinaceum malaria parasites based on surface morphological changes

This paper reports on a microfluidic platform to isolate and study avian red blood cells (RBCs) infected to various degrees by the malaria parasite Plasmodium gallinaceum. The experimental findings point to the feasibility of using the morphological changes on the surface of the malaria infected avian RBC (miaRBCs) as biomarkers for diagnosis. A glass substrate with a controlled surface roughness was used as part of a polydimethylsiloxane (PDMS) microfluidic channels. When whole-blood samples were introduced into the channels, the miaRBCs would be preferentially slowed and eventually become immobilized on the roughened surface. The surface lesions and furrow-like structures on the miaRBC surfaces offered a markedly higher probability to interact with the roughened substrate and allowed the cells to become imobilized on the surface. The captured miaRBCs were from blood samples at various degrees of infection at 3.2%, 3.9%, 9.1%, 13.4%, 20.1%, 28%, and 37%. It was observed that the miaRBCs could be selectively captured under a wall shear rate between 2.1 to 3.2 s^?1, which was directly proportional to the flow rate through the channels. This capture rate could be improved by increasing the channel length and finer flow control. It was also found that a roughened glass substrate with ten-point-height larger than the depth of surface lesions and furrow-like structures of miaRBCs showed a substantial enhancement on the number of immobilized infected RBCs. These findings indicated that surface morphologies, including surface lesions and furrow-like structures, can serve as an alternative biomarker for malaria diagnosis.     

Genetic Engineering of Native Chain Combinations of B-Cell Repertoires on the Surface of Methylotrophic Yeasts Pichia pastoris

Bulletin of Experimental Biology and Medicine - We designed genetic constructs for exposing Fab-fragment library of natively paired single cell B-cell receptors on the surface of Pichia pastoris...     

Prevention of air bubble formation in a microfluidic perfusion cell culture system using a microscale bubble trap

Formation of air bubbles is a serious obstacle to a successful operation of a long-term microfluidic systems using cell culture. We developed a microscale bubble trap that can be integrated with a microfluidic device to prevent air bubbles from entering the device. It consists of two PDMS (polydimethyldisiloxane) layers, a top layer providing barriers for blocking bubbles and a bottom layer providing alternative fluidic paths. Rather than relying solely on the buoyancy of air bubbles, bubbles are physically trapped and prevented from entering a microfluidic device. Two different modes of a bubble trap were fabricated, an independent module that is connected to the main microfluidic system by tubes, and a bubble trap integrated with a main system. The bubble trap was tested for the efficiency of bubble capture, and for potential effects a bubble trap may have on fluid flow pattern. The bubble trap was able to efficiently trap air bubbles of up to 10 μl volume, and the presence of captured air bubbles did not cause alterations in the flow pattern. The performance of the bubble trap in a long-term cell culture with medium recirculation was examined by culturing a hepatoma cell line in a microfluidic cell culture device. This bubble trap can be useful for enhancing the consistency of microfluidic perfusion cell culture operation.     

Cell culture studies with a cytopathic bovine rotavirus

Summary The growth characteristics in MDBK cells of a calf rotavirus isolated in Northern Ireland are described. Of a range of cell cultures tested, the virus grew in secondary bovine kidney and MDBK cells, but consistently produced a CPE only in MDBK cells. The CPE consisted of cytoplasmic vacuolation, development of eosinophilic intracytoplasmic inclusions, and degeneration and detachment of cells from the monolayer. The onset of CPE was more rapid and its effects more severe in rolled cultures than stationary cultures. Immunofluorescent staining showed that the rotavirus was antigenically unrelated to avian and mammalian reoviruses. There was no significant difference in virus growth over the pH range 6.5 to 8.4.With 3 Figures