Comparison of substance supply in static and perfusion cultures based on mass transport phenomena

Mass transport phenomena in cell culture can be formulated by using classical reaction-diffusion equations; however, in practice, it is difficult to solve these equations analytically. Here, we used computer simulation to solve these equations, and compared the time-dependent concentration profile of substances in conventional static culture with that in perfusion culture. The simulated glucose consumption in static culture agreed with that of actual culture conditions used in a general cell culture experiment. The simulation of perfusion culture revealed that the geometry of the chamber and its operating parameters are critical to obtaining a sufficient supply of substances. We also found that the previously reported perfusion chambers are well designed and operate under adequate conditions. Our kinetic model of time-dependent concentration profiles for general substances based on mass transport phenomena could, therefore, be used for the optimal design of a microfluidic perfusion culture chip.     

Applying microfluidics to electrophysiology

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities. Specifically, the motivation for the microfluidic brain slice device is discussed including how the device docks to standard perfusion chambers and the technique of passive pumping which is used to deliver boluses of neuromodulators to the brain slice. By simplifying the device design, we are able to achieve a practical solution to the current unmet electrophysiology need of applying multiple neuromodulators across multiple regions of the brain slice. This is achieved by substituting the standard coverglass substrate of the perfusion chamber with a thin microfluidic device bonded to the coverglass substrate. This was then attached to the perfusion chamber and small holes connect the open-well of the perfusion chamber to the microfluidic channels buried within the microfluidic substrate. These microfluidic channels are interfaced with ports drilled into the edge of the perfusion chamber to access and deliver stimulants. This project represents how the field of microfluidics is transitioning away from proof-of concept device demonstrations and into practical solutions for unmet experimental and clinical needs.     

Microfluidic components and bio-reactors for miniaturized bio-chip applications

In this paper miniaturized disposable micro/nanofluidic components applicable to bio chip, chemical analyzer and biomedical monitoring system, such as blood analysis, micro dosing system and cell experiment, etc are reported. This system includes various microfluidic components including a micropump, micromixer, DNA purification chip and single-cell assay chip. For low voltage and low power operation, a surface tension-driven micropump is presented, as well as a micromixer, which was implemented using MEMS technology, for efficient liquid mixing is also introduced. As bio-reactors, DNA purification and single-cell assay devices, for the extraction of pure DNA from liquid mixture or blood and for cellular engineering or high-throughput screening, respectively, are presented.     

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane technology. Understanding the fouling process could lead to optimization and higher efficiency of membrane based filtration. Here we show the design and fabrication of an automated three-dimensionally (3-D) printed microfluidic cross-flow filtration system that can test up to 4 membranes in parallel. The microfluidic cells were printed using multi-material photopolymer 3-D printing technology, which used a transparent hard polymer for the microfluidic cell body and incorporated a thin rubber-like polymer layer, which prevents leakages during operation. The performance of ultrafiltration (UF), and nanofiltration (NF) membranes were tested and membrane fouling could be observed with a model foulant bovine serum albumin (BSA). Feed solutions containing BSA showed flux decline of the membrane. This protocol may be extended to measure fouling or biofouling with many other organic, inorganic or microbial containing solutions. The microfluidic design is especially advantageous for testing materials that are costly or only available in small quantities, for example polysaccharides, proteins, or lipids due to the small surface area of the membrane being tested. This modular system may also be easily expanded for high throughput testing of membranes.      

An integrated microfluidic device for single cell trapping and osmotic behavior investigation of mouse oocytes

Transport properties of oocytes play an important role in the optimization of their cryopreservation. However, there are still no systematical investigations on oocyte transport properties from the viewpoint of single-cell trapping and high precision perfusion, especially with the powerful microfluidic approach. To this end, we developed an easy-to-fabricate and easy-to-use microfluidic chip along with automatic single cell trapping capability to investigate the oocyte membrane transport properties. The experimental results indicate that the device is available and reliable. We further performed a comparative study of the oocyte membrane transport properties between single and multi-step CPA addition protocols and confirmed that the transport property parameters measured by single-step osmotic shift could not be used for prediction of the osmotic responses of oocytes in multi-step CPA addition. This study provides a powerful tool for investigation of oocyte osmotic responses.     

高通量灌流培养模型在生物工艺开发中的应用研究

近年来,连续型细胞培养由于其高单位体积产量、稳定的产品质量属性以及潜在的成本节约效应正成为生物大分子制药生产的工艺焦点。相比传统的流加培养模式,灌流培养因培养的连续性、操作的复杂性,致使其反应器规模培养需消耗大量培养基,产生更高人力成本,不能满足当今加速化高效化的工艺开发需求。为获得稳健的灌流培养工艺并控制较低成本,高通量灌流培养模型被用于批量化的小规模灌流培养,进行灌流培养前期的克隆筛选、培养基筛选及工艺参数优化等工作,为后期大规模培养提供实用性数据支持,同时也被用于预测大规模培养的细胞表型和产品质量属性。重点介绍了当前高通量系统包括摇瓶/摇管系统、多平行自动化系统以及微流控体系用作灌流培养的特征、具体应用及比较,同时论述当前高通量灌流培养系统在生物工艺领域发展所面临的机遇及挑战,并展望其应用前景。     

Fluorescence optical detection in situ for real‐time monitoring of cytochrome P450 enzymatic activity of liver cells in multiple microfluidic devices

We describe an in situ fluorescence optical detection system to demonstrate real‐time and non‐invasive detection of reaction products in a microfluidic device while under perfusion within a standard incubator. The detection system is designed to be compact and robust for operation inside a mammalian cell culture incubator for quantitative detection of fluorescent signal from microfluidic devices. When compared to a standard plate reader, both systems showed similar biphasic response curves with two linear regions. Such a detection system allows real‐time measurements in microfluidic devices with cells without perturbing the culture environment. In a proof‐of‐concept experiment, the cytochrome P450 1A1/1A2 activity of a hepatoma cell line (HepG2/C3A) was monitored by measuring the enzymatic conversion of ethoxyresorufin to resorufin. The hepatoma cell line was embedded in Matrigel^TM construct and cultured in a microfluidic device with medium perfusion. The response of the cells, in terms of P450 1A1/1A2 activity, was significantly different in a plate well system and the microfluidic device. Uninduced cells showed almost no activity in the plate assay, while uninduced cells in Matrigel^TM with perfusion in a microfluidic device showed high activity. Cells in the plate assay showed a significant response to induction with 3‐Methylcholanthrene while cells in the microfluidic device did not respond to the inducer. These results demonstrate that the system is a potentially useful method to measure cell response in a microfluidic system. Biotechnol. Bioeng. 2009; 104: 516–525 © 2009 Wiley Periodicals, Inc.     

A new tool for probing of cell–cell communication: human embryonic germ cells inducing apoptosis of SKOV3 ovarian cancer cells on a microfluidic chip

A microfluidic device with unidirectional perfusion has been developed to observe the effect of human embryonic germ (hEG) cells on SKOV3 cells. The hEG and SKOV3 cells were seeded in the inlet and the outlet reservoirs separately, and co-cultured for 2 days. The medium was perfused unidirectionally from the inlet to the outlet. The growth inhibition of SKOV3 cells was monitored online and the apoptosis signals in SKOV3 culture area decreased along the flow of the medium. In conclusion, microfluidic chip is a potentially useful tool to investigate the effect of stem cells on cancer cells with intuitionistic cell-based screens.     

Brain slice stimulation using a microfluidic network and standard perfusion chamber

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The two-level microfluidic device is fabricated using a two-step standard negative resist lithography process. The first level contains microchannels with inlet and outlet ports at each end. The second level contains microscale circular holes located midway of the channel length and centered along with channel width. Passive pumping method is used to pump fluids from the inlet port to the outlet port. The microfluidic device is integrated with off-the-shelf perfusion chambers and allows seamless integration with the electrophysiology setup. The fluids introduced at the inlet ports flow through the microchannels towards the outlet ports and also escape through the circular openings located on top of the microchannels into the bath of the perfusion. Thus the bottom surface of the brain slice placed in the perfusion chamber bath and above the microfluidic device can be exposed with different neurotransmitters. The microscale thickness of the microfluidic device and the transparent nature of the materials [glass coverslip and PDMS (polydimethylsiloxane)] used to make the microfluidic device allow microscopy of the brain slice. The microfluidic device allows modulation (both spatial and temporal) of the chemical stimuli introduced to the brain slice microenvironments.     

Bioprocess iterative batch-to-batch optimization based on hybrid parametric/nonparametric models

This paper presents a novel method for iterative batch-to-batch dynamic optimization of bioprocesses. The relationship between process performance and control inputs is established by means of hybrid grey-box models combining parametric and nonparametric structures. The bioreactor dynamics are defined by material balance equations, whereas the cell population subsystem is represented by an adjustable mixture of nonparametric and parametric models. Thus optimizations are possible without detailed mechanistic knowledge concerning the biological system. A clustering technique is used to supervise the reliability of the nonparametric subsystem during the optimization. Whenever the nonparametric outputs are unreliable, the objective function is penalized. The technique was evaluated with three simulation case studies. The overall results suggest that the convergence to the optimal process performance may be achieved after a small number of batches. The model unreliability risk constraint along with sampling scheduling are crucial to minimize the experimental effort required to attain a given process performance. In general terms, it may be concluded that the proposed method broadens the application of the hybrid parametric/nonparametric modeling technique to "newer" processes with higher potential for optimization.     

Magnetoresistive immunosensor for the detection of Escherichia coli O157:H7 including a microfluidic network

A hand held device has been designed for the immunomagnetic detection and quantification of the pathogen Escherichia coli O157:H7 in food and clinical samples. In this work, a technology to manufacture a Lab on a Chip that integrates a 3D microfluidic network with a microfabricated biosensor has been developed. With this aim, the sensing film optimization, the design of the microfluidic circuitry, the development of the biological protocols involved in the measurements and, finally, the packaging needed to carry out the assays in a safe and straightforward way have been completed. The biosensor is designed to be capable to detect and quantify small magnetic field variations caused by the presence of superparamagnetic beads bound to the antigens previously immobilized on the sensor surface via an antibody-antigen reaction. The giant magnetoresistive multilayer structure implemented as sensing film consists of 20[Cu(5.10nm)/Co(2.47 nm)] with a magnetoresistance of 3.20% at 235Oe and a sensitivity up to 0.06 Omega/Oe between 150Oe and 230Oe. Silicon nitride has been selected as optimum sensor surface coating due to its suitability for antibody immobilization. In order to guide the biological samples towards the sensing area, a microfluidic network made of SU-8 photoresist has been included. Finally, a novel packaging design has been fabricated employing 3D stereolithographic techniques. The microchannels are connected to the outside using standard tubing. Hence, this packaging allows an easy replacement of the used devices.     

Microfluidic blood–brain barrier model provides in vivo‐like barrier properties for drug permeability screening

Efficient delivery of therapeutics across the neuroprotective blood–brain barrier (BBB) remains a formidable challenge for central nervous system drug development. High‐fidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study, we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues, allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing, meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo‐like barrier properties in a microfluidic BBB model. This BBB‐on‐a‐chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo‐like values of trans‐endothelial electrical resistance (TEER). The TEER levels peaked above 4000 Ω · cm² on day 3 on chip and were sustained above 2000 Ω · cm² up to 10 days, which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC‐dextrans) and model drugs (caffeine, cimetidine, and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB‐on‐a‐chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time‐based design of a microfluidic platform will enable integration with other organ modules to simulate multi‐organ interactions on drug response. Biotechnol. Bioeng. 2017;114: 184–194. © 2016 Wiley Periodicals, Inc.     

Pumpless,unidirectional microphysiological system for testing metabolism-dependent chemotherapeutic toxicity

Drug development is often hindered by the failure of preclinical models to accurately assess and predict the efficacy and safety of drug candidates. Body-on-a-chip (BOC) microfluidic devices, a subset of microphysiological systems (MPS), are being created to better predict human responses to drugs. Each BOC is designed with separate organ chambers interconnected with microfluidic channels mimicking blood recirculation. Here, we describe the design of the first pumpless, unidirectional, multiorgan system and apply this design concept for testing anticancer drug treatments. HCT-116 colon cancer spheroids, HepG2/C3A hepatocytes, and HL-60 promyeloblasts were embedded in collagen hydrogels and cultured within compartments representing “colon tumor”, “liver,” and “bone marrow” tissue, respectively. Operating on a pumpless platform, the microfluidic channel design provides unidirectional perfusion at physiologically realistic ratios to multiple channels simultaneously. The metabolism-dependent toxic effect of Tegafur, an oral prodrug of 5-fluorouracil, combined with uracil was examined in each cell type. Tegafur-uracil treatment induced substantial cell death in HCT-116 cells and this cytotoxic response was reduced for multicellular spheroids compared to single cells, likely due to diffusion-limited drug penetration. Additionally, off-target toxicity was detected by HL-60 cells, which demonstrate that such systems can provide useful information on dose-limiting side effects. Collectively, this microscale cell culture analog is a valuable physiologically-based pharmacokinetic drug screening platform that may be used to support cancer drug development.     

A hybrid microfluidic-vacuum device for direct interfacing with conventional cell culture methods

Background

Microfluidics is an enabling technology with a number of advantages over traditional tissue culture methods when precise control of cellular microenvironment is required. However, there are a number of practical and technical limitations that impede wider implementation in routine biomedical research. Specialized equipment and protocols required for fabrication and setting up microfluidic experiments present hurdles for routine use by most biology laboratories.

Results

We have developed and validated a novel microfluidic device that can directly interface with conventional tissue culture methods to generate and maintain controlled soluble environments in a Petri dish. It incorporates separate sets of fluidic channels and vacuum networks on a single device that allows reversible application of microfluidic gradients onto wet cell culture surfaces. Stable, precise concentration gradients of soluble factors were generated using simple microfluidic channels that were attached to a perfusion system. We successfully demonstrated real-time optical live/dead cell imaging of neural stem cells exposed to a hydrogen peroxide gradient and chemotaxis of metastatic breast cancer cells in a growth factor gradient.

Conclusion

This paper describes the design and application of a versatile microfluidic device that can directly interface with conventional cell culture methods. This platform provides a simple yet versatile tool for incorporating the advantages of a microfluidic approach to biological assays without changing established tissue culture protocols.     

Applications of nonequilibrium response spectroscopy to the study of channel gating. Experimental design and optimization

A novel experimental technique known as non-equilibrium response spectroscopy (NRS) based on ion channel responses to rapidly fluctuating voltage waveforms was recently described (Millonas & Hanck, 1998a). It was demonstrated that such responses can be affected by subtle details of the kinetics that are otherwise invisible when conventional stepped pulses are applied. As a consequence, the kinetics can be probed in a much more sensitive way by supplementing conventional techniques with measurements of the responses to more complex voltage waveforms. In this paper we provide an analysis of the problem of the design and optimization of such waveforms. We introduce some methods for determination of the parametric uncertainty of a class of kinetic models for a particular data set. The parametric uncertainty allows for a characterization of the amount of kinetic information acquired through a set of experiments which can in turn be used to design new experiments that increase this information. We revisit the application of dichotomous noise (Millonas & Hanck, 1998a, b), and further consider applications of a more general class of continuous wavelet -based waveforms. A controlled illustration of these methods is provided by making use of a simplified "toy" model for the potassium channel kinetics.     

CFD-aided cell settler design optimization and scale-up: effect of geometric design and operational variables on separation performance

The inclined multiplate (lamella) gravity settler has proven to be an effective cell retention device in industrial perfusion cell culture applications. Investigations on the effects of geometric design and operational variables of the cell settler are crucial to understanding how to best improve the settler performance. Maximizing the harvest/perfusion flow rate while minimizing viable cell loss out of the harvest is the primary challenge for optimization of the settler design. This study demonstrated that computational fluid dynamics (CFD) can be utilized to accurately model and evaluate the settler separation performance for near-monodisperse suspensions and therefore aid in the design optimization of the settler under these baseline conditions. With the preferred geometric features that were identified from CFD modeling results, we proposed design guidelines for the scale-up of these multiplate settler systems. With these guidelines and performance verification using the CFD model, a new large-scale settler was designed and fabricated for a perfusion cell culture process using a minimally aggregating production cell line. Perfusion cell culture runs with this particular cell line were performed with this settler, and the CFD model was able to predict the initial ramp-up performance, proving it to be a valuable scale-up design tool for this production process.     

外置式与内置式秸秆生物反应堆对番茄生长及光合性能的影响

以“魁冠108”番茄为试验材料,对比研究了外置式和内置式秸秆生物反应堆在秋延后番茄生产过程中对日光温室内CO₂浓度、空气相对湿度、空气饱和水汽压差以及番茄生长和光合性能的影响.结果表明:与对照相比,晴天9:30-11:30和14:30-15:00 外置式秸秆生物反应堆温室内CO₂浓度平均提高了207.3和103 μmol·mol^-1,差异显著;晴天9:30-11:30内置式秸秆生物反应堆温室内CO₂浓度平均提高了19.0 μmol·mol^-1;外置式和内置式秸秆生物反应堆促进了番茄株高生长,使植株提早开花,显著提高了番茄净光合速率、单株产量及单位面积产量,显著降低了营养生长期和结果期的蒸腾速率.与内置式相比,外置式秸秆生物反应堆更适合在日光温室秋延后生产中推广应用.