An integrated microfluidic bubble pocket for long-term perfused three-dimensional intestine-on-a-chip model

Perfused three-dimensional (3D) cultures enable long-term in situ growth and monitoring of 3D organoids making them well-suited for investigating organoid development, growth, and function. One of the limitations of this long-term on-chip perfused 3D culture is unintended and disruptive air bubbles. To overcome this obstacle, we invented an imaging platform that integrates an innovative microfluidic bubble pocket for long-term perfused 3D culture of gastrointestinal (GI) organoids. We successfully applied 3D printing technology to create polymer molds that cast polydimethylsiloxane (PDMS) culture chambers in addition to bubble pockets. Our developed platform traps unintended, or induced, air bubbles in an integrated PDMS pocket chamber, where the bubbles diffuse out across the gas permeable PDMS or an outlet tube. We demonstrated that our robust platform integrated with the novel bubble pocket effectively circumvents the development of bubbles into human and mouse GI organoid cultures during long-term perfused time-course imaging. Our platform with the innovative integrated bubble pocket is ideally suited for studies requiring long-term perfusion monitoring of organ growth and morphogenesis as well as function.     

Traceable clonal culture and chemodrug assay of heterogeneous prostate carcinoma PC3 cells in microfluidic single cell array chips

Cancer heterogeneity has received considerable attention for its role in tumor initiation and progression, and its implication for diagnostics and therapeutics in the clinic. To facilitate a cellular heterogeneity study in a low cost and highly efficient manner, we present a microfluidic platform that allows traceable clonal culture and characterization. The platform captures single cells into a microwell array and cultures them for clonal expansion, subsequently allowing on-chip characterization of clonal phenotype and response against drug treatments. Using a heterogeneous prostate cancer model, the PC3 cell line, we verified our prototype, identifying three different sub-phenotypes and correlating their clonal drug responsiveness to cell phenotype.     

A pump-free membrane-controlled perfusion microfluidic platform

In this article, we present a microfluidic platform for passive fluid pumping for pump-free perfusion cell culture, cell-based assay, and chemical applications. By adapting the passive membrane-controlled pumping principle from the previously developed perfusion microplate, which utilizes a combination of hydrostatic pressure generated by different liquid levels in the wells and fluid wicking through narrow strips of a porous membrane connecting the wells to generate fluid flow, a series of pump-free membrane-controlled perfusion microfluidic devices was developed and their use for pump-free perfusion cell culture and cell-based assays was demonstrated. Each pump-free membrane-controlled perfusion microfluidic device comprises at least three basic components: an open well for generating fluid flow, a micron-sized deep chamber/channel for cell culture or for fluid connection, and a wettable porous membrane for controlling the fluid flow. Each component is fluidically connected either by the porous membrane or by the micron-sized deep chamber/channel. By adapting and incorporating the passive membrane-controlled pumping principle into microfluidic devices, all the benefits of microfluidic technologies, such as small sample volumes, fast and efficient fluid exchanges, and fluid properties at the micro-scale, can be fully taken advantage of with this pump-free membrane-controlled perfusion microfluidic platform.     

Treatment of streptozotocin induced diabetes in male rats by immunoisolated transplantation of islet cells

Insulin injection is the main way to combat against insulin-dependent diabetes mellitus effects. Today in some laboratories in the world, the investigators are trying to find some treatments for this disease with insulin-secreting pancreatic islet cells transplantation. Donor tissue in each step of work was prepared from 36 adult male wistar Rats weighted 250–300 grams (75–90 days). Transplantation was done in rats after 2–4 weeks induction of diabetes with 60mg/kg of streptozotocin injection by intravenous method. Encapsulation of pancreatic islet cells allows for transplantation in the absence of immunosuppression. This technique that is called “immunoisolation” is based on the principle that transplanted tissue is protected for the host immune system by an artificial or natural membrane. In this study, the levels of insulin, C-peptide and glucose in diabetic rats have been reached to normal range as compared to un-diabetic rats in 20 days after transplantation of islet cells, so that testis is immunoisolated place for islet cells transplantation. Inside the testis subcutaneously and intrapretoneally implantation of pure islet cells graft, that is a natural immunoisolation method, rapidly and permanently normalized the diabetic state of streptozocin-administered animals.     

Isolation and purification of rat islet cells by flow cytometry

Flow cytometry has been employed as a method to study homogeneity of isolated islet subpopulations. After collagenase digestion of rat pancreas and elutriation of tissue fragments, islets were isolated and dissociated, and cells were analyzed and sorted according to their low forward angle light scattering properties by using automated flow cytometry. A standardized procedure was developed for the preparation of rat islet cell grafts for purification of islet cells. In this process, after collagenase digestion of pancreas, islets were isolated, dissociated, identification by dithizone method and then with enzymatic procedure by DNase and trypsin, the islet cells changed into single cells and beta cells were identified by immunofluorescence method and then assayed by flow cytometry. Methods have been developed for the preparation of suspension of viable rat pancreatic islet cells and their analysis and sorting in the fluorescence activated cell sorter (FACC IV, Becton Dickinson, Sunnyvale, Ca). Flow cytometry of these cells indicated that there were 91% of beta cells in cell suspension. Most of the exocrine particles were lost during digestion. Purified endocrine islet cell grafts were prepared by pure beta-cells, without endocrine non-beta cells. The purified aggregates were devoid of endocrine non-beta cells and damaged cells.     

A programmable microvalve-based microfluidic array for characterization of neurotoxin-induced responses of individual C. elegans

The soil dwelling nematode Caenorhabditis elegans (C. elegans) is an excellent model organism for the study of numerous disease including neurodegenerative disease. In this study, a programmable microvalve-based microfluidic array for real-time and long-term monitoring of the neurotoxin-induced responses of the individual C. elegans was developed. The device consisted of a flow layer and a control layer, which were used for worm manipulation. By activating the programmable microvalves in the control layer, mutiple worms could be individually captured and intermittently immobilized in parallel channels. Thus the mobility behavior, together with the corresponding dopaminergic neuron features of the worms in response to neurotoxin, could be investigated simultaneously. It was found that the neurotoxin MPP+ enabled to induce mobility defects and dopaminergic neurons loss in worms. The established system is easy and fast to operate, which offers not only the controllable microenvironment for analyzing the individual worms in parallel, monitoring the same worm over time, but also the capability to characterize the mobility behavior and neuron features in response to stimuli simultaneously. In addition, the device enabled to sustain the worm culture over most of their adult lifespan without any harm to worm, providing a potential platform for lifespan and aging research.     

Vascular smooth muscle cell culture in microfluidic devices

This paper presents a microfluidic device enabling culture of vascular smooth muscle cells (VSMCs) where extracellular matrix coating, VSMC seeding, culture, and immunostaining are demonstrated in a tubing-free manner. By optimizing droplet volume differences between inlets and outlets of micro channels, VSMCs were evenly seeded into microfluidic devices. Furthermore, the effects of extracellular matrix (e.g., collagen, poly-l-Lysine (PLL), and fibronectin) on VSMC proliferation and phenotype expression were explored. As a platform technology, this microfluidic device may function as a new VSMC culture model enabling VSMC studies.     

High yield fabrication of multilayer polydimethylsiloxane devices with freestanding micropillar arrays

A versatile method to fabricate a multilayer polydimethylsiloxane (PDMS) device with micropillar arrays within the inner layer is reported. The method includes an inexpensive but repeatable approach for PDMS lamination at high compressive force to achieve high yield of pillar molding and transfer to a temporary carrier. The process also enables micropillar-containing thin films to be used as the inner layer of PDMS devices integrated with polymer membranes. A microfluidic cell culture device was demonstrated which included multiple vertically stacked flow channels and a pillar array serving as a cage for a collagen hydrogel. The functionality of the multilayer device was demonstrated by culturing collagen-embedded fibroblasts under interstitial flow through the three-dimensional scaffold. The fabrication methods described in this paper can find applications in a variety of devices, particularly for organ-on-chip applications.     

Osteocyte culture in microfluidic devices

This paper presents a microfluidic device (poly-dimethylsiloxane micro channels bonded with glass slides) enabling culture of MLO-Y4 osteocyte like cells. In this study, on-chip collagen coating, cell seeding and culture, as well as staining were demonstrated in a tubing-free manner where gravity was used as the driving force for liquid transportation. MLO-Y4 cells were cultured in microfluidic channels with and without collagen coating where cellular images in a time sequence were taken and analyzed, confirming the positive effect of collagen coating on phenotype maintaining of MLO-Y4 cells. The proliferating cell nuclear antigen based proliferation assay was used to study cellular proliferation, revealing a higher proliferation rate of MLO-Y4 cells seeded in microfluidic channels without collagen coating compared to the substrates coated with collagen. Furthermore, the effects of channel dimensions (variations in width and height) on the viability of MLO-Y4 cells were explored based on the Calcein-AM and propidium iodide based live/dead assay and the Hoechst 33258 based apoptosis assay, locating the correlation between the decrease in channel width or height and the decrease in cell viability. As a platform technology, this microfluidic device may function as a new cell culture model enabling studies of osteocytes.     

Sustained release of hydrophobic drugs by the microfluidic assembly of multistage microgel/poly (lactic-co-glycolic acid) nanoparticle composites

The poor solubility of many newly discovered drugs has resulted in numerous challenges for the time-controlled release of therapeutics. In this study, an advanced drug delivery platform to encapsulate and deliver hydrophobic drugs, consisting of poly (lactic-co-glycolic acid) (PLGA) nanoparticles incorporated within poly (ethylene glycol) (PEG) microgels, was developed. PLGA nanoparticles were used as the hydrophobic drug carrier, while the PEG matrix functioned to slow down the drug release. Encapsulation of the hydrophobic agents was characterized by fluorescence detection of the hydrophobic dye Nile Red within the microgels. In addition, the microcomposites prepared via the droplet-based microfluidic technology showed size tunability and a monodisperse size distribution, along with improved release kinetics of the loaded cargo compared with bare PLGA nanoparticles. This composite system has potential as a universal delivery platform for a variety of hydrophobic molecules.     

A microfluidic platform for real-time and in situ monitoring of virus infection process

Microfluidic chip is a promising platform for studying virus behaviors at the cell level. However, only a few chip-based studies on virus infection have been reported. Here, a three-layer microfluidic chip with low shear stress was designed to monitor the infection process of a recombinant Pseudorabies virus (GFP-PrV) in real time and in situ, which could express green fluorescent protein during the genome replication. The infection and proliferation characteristics of GFP-PrV were measured by monitoring the fluorescence intensity of GFP and determining the one-step growth curve. It was found that the infection behaviors of GFP-PrV in the host cells could hardly be influenced by the microenvironment in the microfluidic chip. Furthermore, the results of drug inhibition assays on the microfluidic chip with a tree-like concentration gradient generator showed that one of the infection pathways of GFP-PrV in the host cells was microtubule-dependent. This work established a promising microfluidic platform for the research on virus infection.     

Immunoisolated Transplantation of Purified Langerhans Islet Cells in Testis Cortex of Male Rats for Treatment of Streptozotocin Induced Diabetes Mellitus

The objective of this study is to induce experimental diabetes mellitus by streptozotocin in normal adult Wistar rats via comparison of changes in body weight, consumption of food, volume of water, urine and levels of glucose, insulin and C-peptide in serum, between normal and diabetic rats. Intra-venous injection of 60 mg/kg dose of streptozotocin in 250–300 g (75–90 days) adult Wistar rats makes pancreas swell and causes degeneration in Langerhans islet β-cells and induces experimental diabetes mellitus in 2–4 days. For a microscopic study of degeneration of Langerhans islet β-cells of diabetic rats, biopsy from pancreas tissue of diabetic and normal rats, staining and comparison between them, were done. In this process, after collagenase digestion of pancreas, islets were isolated, dissociated and identified by dithizone method and then with enzymatic procedure by DNase and trypsin, the islet cells changed into single cells and β-cells were identified by immune fluorescence method and then assayed by flow-cytometer. Donor tissue in each step of work was prepared from 38 adult male Wistar rats weighted 250–300 g (75–90 days). Transplantation was performed in rats after 2–4 weeks of diabetes induction. In this study, the levels of insulin, C-peptide and glucose in diabetic rats reached to normal range as compared to un-diabetic rats in 20 days after transplantation of islet cells. Transplantation was performed under the cortex of testis as immunoisolated place for islet cells transplantation.     

High-throughput study of alpha-synuclein expression in yeast using microfluidics for control of local cellular microenvironment

Microfluidics is an emerging technology which allows the miniaturization, integration, and automation of fluid handling processes. Microfluidic systems offer low sample consumption, significantly reduced processing time, and the prospect of massive parallelization. A microfluidic platform was developed for the control of the soluble cellular microenvironment of Saccharomyces cerevisiae cells, which enabled high-throughput monitoring of the controlled expression of alpha-synuclein (aSyn), a protein involved in Parkinson's disease. Y-shaped structures were fabricated using particle desorption mass spectrometry-based soft-lithography techniques to generate biomolecular gradients along a microchannel. Cell traps integrated along the microchannel allowed the positioning and monitoring of cells in precise locations, where different, well-controlled chemical environments were established. S. cerevisiae cells genetically engineered to encode the fusion protein aSyn-GFP (green fluorescent protein) under the control of GAL1, a galactose inducible promoter, were loaded in the microfluidic structure. A galactose concentration gradient was established in the channel and a time-dependent aSyn-GFP expression was obtained as a function of the positioning of cells along the galactose gradient. Our results demonstrate the applicability of this microfluidic platform to the spatiotemporal control of cellular microenvironment and open a range of possibilities for the study of cellular processes based on single-cell analysis.     

Stem cells in microfluidics

Microfluidic techniques have been recently developed for cell-based assays. In microfluidic systems, the objective is for these microenvironments to mimic in vivo surroundings. With advantageous characteristics such as optical transparency and the capability for automating protocols, different types of cells can be cultured, screened, and monitored in real time to systematically investigate their morphology and functions under well-controlled microenvironments in response to various stimuli. Recently, the study of stem cells using microfluidic platforms has attracted considerable interest. Even though stem cells have been studied extensively using bench-top systems, an understanding of their behavior in in vivo-like microenvironments which stimulate cell proliferation and differentiation is still lacking. In this paper, recent cell studies using microfluidic systems are first introduced. The various miniature systems for cell culture, sorting and isolation, and stimulation are then systematically reviewed. The main focus of this review is on papers published in recent years studying stem cells by using microfluidic technology. This review aims to provide experts in microfluidics an overview of various microfluidic systems for stem cell research.     

Microfluidic-driven viral infection on cell cultures: Theoretical and experimental study

Advanced cell culture systems creating a controlled and predictable microenvironment together with computational modeling may be useful tools to optimize the efficiency of cell infections. In this paper, we will present a phenomenological study of a virus-host infection system, and the development of a multilayered microfluidic platform used to accurately tune the virus delivery from a diffusive-limited regime to a convective-dominated regime. Mathematical models predicted the convective-diffusive regimes developed within the system itself and determined the dominating mass transport phenomena. Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) transgene were used at different multiplicities of infection (MOI) to infect multiple cell types, both in standard static and in perfused conditions. Our results validate the mathematical models and demonstrate how the infection processes through perfusion via microfluidic platform led to an enhancement of adenoviral infection efficiency even at low MOIs. This was particularly evident at the longer time points, since the establishment of steady-state condition guaranteed a constant viral concentration close to cells, thus strengthening the efficiency of infection. Finally, we introduced the concept of effective MOI, a more appropriate variable for microfluidic infections that considers the number of adenoviruses in solution per cell at a certain time.     

Polyester μ-assay chip for stem cell studies

The application of microfluidic technologies to stem cell research is of great interest to biologists and bioengineers. This is chiefly due to the intricate ability to control the cellular environment, the reduction of reagent volume, experimentation time and cost, and the high-throughput screening capabilities of microscale devices. Despite this importance, a simple-to-use microfluidic platform for studying the effects of growth factors on stem cell differentiation has not yet emerged. With this consideration, we have designed and characterized a microfluidic device that is easy to fabricate and operate, yet contains several functional elements. Our device is a simple polyester-based microfluidic chip capable of simultaneously screening multiple independent stem cell culture conditions. Generated by laser ablation and stacking of multiple layers of polyester film, this device integrates a 10 × 10 microwell array for cell culture with a continuous perfusion system and a non-linear concentration gradient generator. We performed numerical calculations to predict the gradient formation and calculate the shear stress acting on the cells inside the device. The device operation was validated by culturing murine embryonic stem cells inside the microwells for 5 days. Furthermore, we showed the ability to maintain the pluripotency of stem cell aggregates in response to concentrations of leukemia inhibitory factor ranging from 0 to ∼1000 U/ml. Given its simplicity, fast manufacturing method, scalability, and the cell-compatible nature of the device, it may be a useful platform for long-term stem cell culture and studies.     

Parallel generation of uniform fine droplets at hundreds of kilohertz in a flow-focusing module

Droplet-based microfluidic systems enable a variety of biomedical applications from point-of-care diagnostics with third world implications, to targeted therapeutics alongside medical ultrasound, to molecular screening and genetic testing. Though these systems maintain the key advantage of precise control of the size and composition of the droplet as compared to conventional methods of production, the low rates at which droplets are produced limits translation beyond the laboratory setting. As well, previous attempts to scale up shear-based microfluidic systems focused on increasing the volumetric throughput and formed large droplets, negating many practical applications of emulsions such as site-specific therapeutics. We present the operation of a parallel module with eight flow-focusing orifices in the dripping regime of droplet formation for the generation of uniform fine droplets at rates in the hundreds of kilohertz. Elevating the capillary number to access dripping, generation of monodisperse droplets of liquid perfluoropentane in the parallel module exceeded 3.69 × 10⁵ droplets per second, or 1.33 × 10⁹ droplets per hour, at a mean diameter of 9.8 μm. Our microfluidic method offers a novel means to amass uniform fine droplets in practical amounts, for instance, to satisfy clinical needs, with the potential for modification to form massive amounts of more complex droplets.     

基于扎根理论的平台就业用户能力信息识别与匹配研究

【目的/意义】平台就业用户的服务能力是平台经济发展的基础,识别平台就业用户的能力信息构成要素, 为平台经济数据信息匹配及监管提供参考。【方法/过程】运用扎根理论,以平台就业用户为研究对象,提炼其能力信 息构成要素,明确各信息要素之间的作用关系。适用量质协调的评价方法,构建平台就业用户信息匹配评价体系。 【结果/结论】平台就业用户能力信息要素主要体现在四个维度,包括基础信息、服务信息、协作信息、发展信息,基于 平台就业用户的能力信息匹配监测体系,可监测平台就业用户的信息匹配质量。【创新/局限】通过扎根理论针对平 台就业用户的能力信息进行萃取,并探讨了能力信息匹配监测的理论路径,有待进一步获取平台就业用户数据进 行实践检验。     

Continuously perfused microbubble array for 3D tumor spheroid model

Multi-cellular tumor spheroids (MCTSs) have been established as a 3D physiologically relevant tumor model for drug testing in cancer research. However, it is difficult to control the MCTS testing parameters and the entire process is time-consuming and expensive. To overcome these limitations, we developed a simple microfluidic system using polydimethylsiloxane (PDMS) microbubbles to culture tumor spheroids under physiological flow. The flow characteristics such as streamline directions, shear stress profile, and velocity profile inside the microfluidic system were first examined computationally using a COMSOL simulation. Colo205 tumor spheroids were created by a modified hanging drop method and maintained inside PDMS microbubble cavities in perfusion culture. Cell viability inside the microbubbles was examined by live cell staining and confocal imaging. E-selectin mediated cell sorting of Colo205 and MDA-MB-231 cell lines on functionalized microbubble and PDMS surfaces was achieved. Finally, to validate this microfluidic system for drug screening purposes, the toxicity of the anti-cancer drug, doxorubicin, on Colo205 cells in spheroids was tested and compared to cells in 2D culture. Colo205 spheroids cultured in flow showed a threefold increase in resistance to doxorubicin compared to Colo205 monolayer cells cultured under static conditions, consistent with the resistance observed previously in other MCTS models. The advantages presented by our microfluidic system, such as the ability to control the size uniformity of the spheroids and to perform real-time imaging on cells in the growth platform, show potential for high throughput drug screening development.