Using living radical polymerization to enable facile incorporation of materials in microfluidic cell culture devices

High throughput screening tools are expediting cell culture studies with applications in drug discovery and tissue engineering. This contribution demonstrates a method to incorporate 3D cell culture sites into microfluidic devices and enables the fabrication of high throughput screening tools with uniquely addressable culture environments. Contact lithographic photopolymerization (CLiPP) was used to fabricate microfluidic devices with two types of 3D culture sites: macroporous rigid polymer cell scaffolds and poly(ethylene glycol) (PEG) encapsulated cell matrices. Cells were cultured on-device with both types of culture sites, demonstrating material cytocompatibility. Multilayer microfluidic devices were fabricated with channels passing the top and bottom sides of a series of rigid porous polymer scaffolds. Cells were seeded and cultured on device, demonstrating the ability to deliver cells and culture cells on multiple scaffolds along the length of a single channel. Flow control through these rigid porous polymer scaffolds was demonstrated. Finally, devices were modified by grafting of PEG methacrylate from surfaces to prevent non-specific protein adsorption and ultimately cell adhesion to channel surfaces. The living radical component of this CLiPP device fabrication platform enables facile incorporation of 3D culture sites into microfluidic cell culture devices, which can be utilized for high throughput screening of cell-material interactions.     

Fabrication of phospholipid–xanthan microcapsules by combining microfluidics with self-assembly

We report the synthesis of an amphiphilic polysaccharide, a phospholipid (1,2-dioleoyl-sn-glycero-phosphoetilamine, DOPE) conjugated with the anionic xanthan gum, and its ability to spontaneously self-assemble under mild aqueous conditions. This work also aimed to apply a microfluidic platform that can precisely fabricate microsized and monodispersed capsules for cell encapsulation. Stable hollow capsular structures were obtained by the generation of homogeneous spherical droplets of the self-assembled polymer in the microfluidic device through the formation of a water-in-oil emulsion, followed by the stabilization of the polymer aggregates in a separate collection vessel containing phosphate-buffered saline (physiological ionic strength and pH). The properties (size, morphology, permeability) and performance (stability) of the obtained microcapsules were studied, as well their ability to support the viability, function and proliferation of encapsulated cells. ATDC5 cells were encapsulated within the capsules and shown to remain viable, evidencing increased cellular metabolic activity over 21 days of in vitro culture. By combining microfluidic droplet generation and self-assembly of xanthan–DOPE, we were able to fabricate microcapsules that provided an adequate environment for cells to survive and proliferate.     

A self-contained microfluidic cell culture system

Conventional in vitro cell culture that utilizes culture dishes or microtiter plates is labor-intensive and time-consuming, and requires technical expertise and specific facilities to handle cell harvesting, media exchange and cell subculturing procedures. A microfluidic array platform with eight microsieves in each cell culture chamber is presented for continuous cell culture. With the help of the microsieves, uniform cell loading and distribution can be obtained. Within the arrays, cells grown to the point of confluency can be trypsinized and recovered from the device. Cells trapped in the microsieves after trypsinization function to seed the chambers for subsequent on-chip culturing, creating a sustainable platform for multiple cycles. The capability of the microfluidic array platform was demonstrated with a BALB/3T3 (murine embryonic fibroblast) cell line. The present microfluidic cell culture platform has potential to develop into a fully automated cell culture system integrated with temperature control, fluidic control, and micropumps, maximizing cell culture health with minimal intervention.     

Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device: effect of cytostatic drugs

Breast cancer is the leading cause of cancer deaths among non-smoking women worldwide. At the moment the treatment regime is such that patients receive different chemotherapeutic and/or hormonal treatments dependent on the hormone receptor status, the menopausal status and age. However, in vitro sensitivity testing of tumor biopsies could rationalize and improve the choice of chemo- and hormone therapy. Lab-on-a-Chip devices, using microfluidic techniques, make detailed cellular analysis possible using fewer cells, enabling working with a patients' own cells and performing chemo- and hormone sensitivity testing in an ex vivo setting. This article describes the development of two microfluidic devices made in poly(dimethylsiloxane) (PDMS) to validate the cell culture properties and analyze the chemosensitivity of MCF-7 cells (estrogen receptor positive human breast cancer cells) in response to the drug staurosporine (SSP). In both cases, cell viability was assessed using the life-stain Calcein-AM (CAAM) and the death dye propidium iodide (PI). MCF-7 cells could be statically cultured for up to 7 days in the microfluidic chip. A 30 min flow with SSP and a subsequent 24 h static incubation in the incubator induced apoptosis in MCF-7 cells, as shown by a disappearance of the aggregate-like morphology, a decrease in CAAM staining and an increase in PI staining. This work provides valuable leads to develop a microfluidic chip to test the chemosensitivity of tumor cells in response to therapeutics and in this way improve cancer treatment towards personalized medicine.     

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.     

Nanoporous membrane-sealed microfluidic devices for improved cell viability

Cell-laden microfluidic devices have broad potential in various biomedical applications, including tissue engineering and drug discovery. However, multiple difficulties encountered while culturing cells within devices affecting cell viability, proliferation, and behavior has complicated their use. While active perfusion systems have been used to overcome the diffusive limitations associated with nutrient delivery into microchannels to support longer culture times, these systems can result in non-uniform oxygen and nutrient delivery and subject cells to shear stresses, which can affect cell behavior. Additionally, histological analysis of cell cultures within devices is generally laborious and yields inconsistent results due to difficulties in delivering labeling agents in microchannels. Herein, we describe a simple, cost-effective approach to preserve cell viability and simplify labeling within microfluidic networks without the need for active perfusion. Instead of bonding a microfluidic network to glass, PDMS, or other solid substrate, the network is bonded to a semi-permeable nanoporous membrane. The membrane-sealed devices allow free exchange of proteins, nutrients, buffers, and labeling reagents between the microfluidic channels and culture media in static culture plates under sterile conditions. The use of the semi-permeable membrane dramatically simplifies microniche cell culturing while avoiding many of the complications which arise from perfusion systems.     

Microfluidic environment for high density hepatocyte culture

We present a microfluidic bioreactor for culturing high-density arrays of hepatocytes in a tissue-like micro-architecture. The microfluidic environment mimicked physiological liver mass transport, enabling sustained culture of high density cells (>2,000 cells/mm²) without nutrient limitation for over 1 week. The key feature of this design was a microporous microfluidic barrier that formed a sieved-pocket to concentrate cells during loading. Nutrient depletion within the cell mass was avoided by maintaining a continuous flow of medium (10 μl/day) that diffused across the porous barrier. Human hepatoma cells (HepG2/C3A) remained viable and functional as demonstrated by fluorescent viability assays and secretion of albumin for the one-week culture period.     

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.     

Development of a novel microfluidic device for long-term in situ monitoring of live cells in 3-dimensional matrices

Using the latest innovations in microfabrication technology, 3-dimensional microfluidic cell culture systems have been developed as an attractive alternative to traditional 2-dimensional culturing systems as a model for long-term microscale cell-based research. Most microfluidic systems are based on the embedding of cells in hydrogels. However, physiologically realistic conditions based on hydrogels are difficult to obtain and the systems are often too complicated. We have developed a microfluidic cell culture device that incorporates a biodegradable rigid 3D polymer scaffold using standard soft lithography methods. The device permits repeated high-resolution fluorescent imaging of live cell populations within the matrix over a 4?week period. It was also possible to track cell development at the same spatial location throughout this time. In addition, human primary periodontal ligament cells were induced to produce quantifiable calcium deposits within the system. This simple and versatile device should be readily applicable for cell-based studies that require long-term culture and high-resolution bioimaging.     

Microfabricated scaffold-guided endothelial morphogenesis in three-dimensional culture

Morphogenesis is a fundamental process by which new blood vessels are formed during angiogenesis. The ability to control angiogenesis would lead to improvements in tissue engineering constructions; indeed, the study of angiogenesis has numerous clinical applications, for example, in the investigation of metastatic cancer, peripheral and coronary vascular disease, and wound healing. Conventional in vitro organotypic cell culture approaches to these studies are limited primarily by their reliance on microvascular vessel formation through a random process of morphogenesis that lacks the spatial reproducibility and orientation needed for high-throughput drug testing. We have developed a bioreactor system for scaffold-guided tubulogenesis coupled with 3-D organotypic culture to spatially control vessel formation and its orientation. To create microchannels to guide microvessel formation, we fabricated rigid scaffolds using photolithography and light curing epoxy, and soft scaffolds formed by a polydimethylsiloxane (PDMS) stamp directly into collagen. Scaffolds seeded with dermal microvascular endothelial cells were placed between gelled layers of collagen containing dermal fibroblasts within a Transwell filter system and cultured for up to 2 weeks to allow for vessel maturation. Morphological analysis of thin tissue sections following standard histology and immunohistochemical detection of endothelial cells, fibroblasts, and basement membrane confirmed vessel formation along the microchannel walls with either scaffold. This system may also provide a means to explore revascularization within decellularized extracellular matrices, the culture of microvessel networks with controlled geometries, and possibly the spatial guidance of angiogenesis for interfacing with an external microfluidic supply network. As a new tool for guided angiogenesis, our approach introduces new possibilities for identification of anti-angiogenic therapeutics.     

An open-access microfluidic model for lung-specific functional studies at an air-liquid interface

In an effort to improve the physiologic relevance of existing in vitro models for alveolar cells, we present a microfluidic platform which provides an air-interface in a dynamic system combining microfluidic and suspended membrane culture systems. Such a system provides the ability to manipulate multiple parameters on a single platform along with ease in cell seeding and manipulation. The current study presents a comparison of the efficacy of the hybrid system with conventional platforms using assays analyzing the maintenance of function and integrity of A549 alveolar epithelial cell monolayer cultures. The hybrid system incorporates bio-mimetic nourishment on the basal side of the epithelial cells along with an open system on the apical side of the cells exposed to air allowing for easy access for assays.     

Microfluidic array for three-dimensional perfusion culture of human mammary epithelial cells

The ability to culture cells in three dimensional extracellular matrix (3D ECM) has proven to be an important tool for laboratory biology. Here, we demonstrate a microfluidic perfusion array on a 96-well plate format capable of long term 3D ECM culture within biomimetic microchambers. The array consists of 32 independent flow units, each with a 4 μl open-top culture chamber, and 350 μl inlet and outlet wells. Perfusion is generated using gravity and surface tension forces, allowing the array to be operated without any external pumps. MCF-10A mammary epithelial cells cultured in Matrigel in the microfluidic array exhibit acinus morphology over 9 days consistent with previous literature. We further demonstrated the application of the microfluidic array for in vitro anti-cancer drug screening.     

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 cell culturing system that integrates the cell loading function on a single platform and evaluation of the pulsatile pumping effect on cells

In this paper, we present a novel microfluidic system with pulsatile cell storing, cell-delivering and cell culturing functions on a single PDMS platform. For this purpose, we have integrated two reservoirs, a pulsatile pumping system containing two soft check valves, which were fabricated by in situ photopolymerization, six switch valves, and three cell culture chambers all developed through a simple and rapid fabrication process. The sample volume delivered per stroke was 120 nl and the transported volume was linearly related to the pumping frequency. We have investigated the effect of the pulsatile pneumatic micropumping on the cells during transport. For this purpose, we pumped two types of cell suspensions, one containing human breast adenocarcinoma cells (MCF-7) and the other mesenchymal stem cells (hMSCs) derived from bone marrow. The effect of pulsatile pumping on both cell types was examined by short and long-term culture experiments. Our results showed that the characteristics of both cells were maintained; they were not damaged by the pumping system. Evaluations were carried out by morphological inspection, viability assay and immunophenotyping analysis. The delivered MCF-7 cells and hMSCs spread and proliferated onto the gelatin coated cell culture chamber. This total micro cell culture system can be applied to cell-based high throughput screening and for co-culture of different cells with different volume.     

Characterization of encapsulated cells within hyaluronic acid and alginate microcapsules produced via horseradish peroxidase-catalyzed crosslinking

Hydrogel microcapsules having the ability to promote cell adhesion and proliferation are a useful tool for fabricating tissue in vitro. The present study explored the effects of two anionic polysaccharide hydrogel membranes which have an impact on the adhesiveness, morphology and growth of cells. Microcapsules were made by coating a cell-laden gelatin microparticle with a hydrogel membrane produced from modified hyaluronic acid or alginate possessing phenolic hydroxyl moieties (HA-Ph or Alg-Ph respectively) via a horseradish peroxidase-catalyzed crosslinking reaction. Some gelatin was retained within the microcapsules to support the attachment and growth of encapsulated cells. The morphological and functional characteristics of encapsulated HeLa and 10T1/2 cells were evaluated. The HA-Ph hydrogel, which exhibited greater retention of gelatin, showed a higher degree of cytocompatibility with respect to cell adhesion, spreading and proliferation compared with the Alg-Ph hydrogel membrane. These findings indicate that HA-Ph microcapsules synthesized around a temporary gelatin microparticles are a promising cell vehicle for tissue engineering applications.     

A gel-free 3D microfluidic cell culture system

3D microfluidic cell culture systems offer a biologically relevant model to conduct micro-scale mammalian cell-based research and applications. Various natural and synthetic hydrogels have been successfully incorporated into microfluidic systems to support mammalian cells in 3D. However, embedment of cells in hydrogels introduces operational complexity, potentially hinders mass transfer, and is not suitable for establishing cell-dense, ECM-poor constructs. We present here a gel-free method for seeding and culturing mammalian cells three-dimensionally in a microfluidic channel. A combination of transient inter-cellular polymeric linker and micro-fabricated pillar arrays was used for the in situ formation and immobilization of 3D multi-cellular aggregates in a microfluidic channel. 3D cellular constructs formed this way are relieved of hydrogel embedment for cellular support. Two mammalian cell lines (A549 and C3A) and a primary mammalian cell (bone marrow mesenchymal stem cells) were cultured in the gel-free 3D microfluidic cell culture system. The cells displayed 3D cellular morphology, cellular functions and differentiation capability, affirming the versatility of the system as a 3D cell perfusion culture platform for anchorage-dependent mammalian cells.     

Microfluidic system for formation of PC-3 prostate cancer co-culture spheroids

The niche microenvironment in which cancer cells reside plays a prominent role in the growth of cancer. It is therefore imperative to mimic the in vivo tumor niche in vitro to better understand cancer and enhance development of therapeutics. Here, we engineer a 3D metastatic prostate cancer model that includes the types of surrounding cells in the bone microenvironment that the metastatic prostate cancer cells reside in. Specifically, we used a two-layer microfluidic system to culture 3D multi-cell type spheroids of fluorescently labeled metastatic prostate cancer cells (PC-3 cell line), osteoblasts and endothelial cells. This method ensures uniform incorporation of all co-culture cell types into each spheroid and keeps the spheroids stationary for easy tracking of individual spheroids and the PC-3's residing inside them over the course of at least a week. This culture system greatly decreased the proliferation rate of PC-3 cells without reducing viability and may more faithfully recapitulate the in vivo growth behavior of malignant cancer cells within the bone metastatic prostate cancer microenvironment.     

Microfabricated polymeric vessel mimetics for 3-D cancer cell culture

Modeling tumor growth in vitro is essential for cost-effective testing of hypotheses in preclinical cancer research. 3-D cell culture offers an improvement over monolayer culture for studying cellular processes in cancer biology because of the preservation of cell–cell and cell-ECM interactions. Oxygen transport poses a major barrier to mimicking in vivo environments and is not replicated in conventional cell culture systems. We hypothesized that we can better mimic the tumor microenvironment using a bioreactor system for controlling gas exchange in cancer cell cultures with silicone hydrogel synthetic vessels. Soft-lithography techniques were used to fabricate oxygen-permeable silicone hydrogel membranes containing arrays of micropillars. These membranes were inserted into a bioreactor and surrounded by basement membrane extract (BME) within which fluorescent ovarian cancer (OVCAR8) cells were cultured. Cell clusters oxygenated by synthetic vessels showed a ∼100μm drop-off to anoxia, consistent with in vivo studies of tumor nodules fed by the microvasculature. Oxygen transport in the bioreactor system was characterized by experimental testing with a dissolved oxygen probe and finite element modeling of convective flow. Our study demonstrates differing growth patterns associated with controlling gas distributions to better mimic in vivo conditions.     

Rheologically biomimetic cell suspensions for decreased cell settling in microfluidic devices

Many microfluidic devices operate with cells suspended in buffer solutions. Researchers who work with large cell types in such devices often run into problems with gravitational cell settling in the injection equipment and in the device itself. A method for reducing this problematic settling is discussed in this paper using tumor cell lines as an example. Microfluidic circulating tumor cell (CTC) isolation devices (MCIDs) are benchmarked using buffer solutions spiked with in-vitro tumor cell lines prior to validation with clinical samples (i.e. whole blood). However, buffer solutions have different rheological properties than whole blood. Here we describe the use of alginate in PBS buffer solutions to mimic blood rheology and reduce cell settling during preliminary validation experiments. Because alginate increases the viscosity of a solution, it helps to maintain cells in suspension. We report that vertical equipment configurations are important to further mitigate the effects of cell settling for MDA-MB-468 carcinoma cells. We also report that alginate does not disrupt the specific binding interactions that are the basis of carcinoma cell capture in MCIDs. These results indicate that vertical equipment configurations and the addition of alginates can be used to reduce cell settling in buffer based MCID testing and other applications involving large cells suspended in buffer solution.     

Embryonic body culturing in an all-glass microfluidic device with laser-processed 4 μm thick ultra-thin glass sheet filter

In this paper, we report the development and demonstration of a method to fabricate an all-glass microfluidic cell culturing device without circulation flow. On-chip microfluidic cell culturing is an indispensable technique for cellular replacement therapies and experimental cell biology. Polydimethylsiloxane (PDMS) have become a popular material for fabricating microfluidic cell culture devices because it is a transparent, biocompatible, deformable, easy-to-mold, and gas-permeable. However, PDMS is also a chemically and physically unstable material. For example, PDMS undergoes aging easily even in room temperature conditions. Therefore, it is difficult to control long term experimental culturing conditions. On the other hand, glass is expected to be stable not only in physically but also chemically even in the presence of organic solvents. However, cell culturing still requires substance exchanges such as gases and nutrients, and so on, which cannot be done in a closed space of a glass device without circulation flow that may influence cell behavior. Thus, we introduce a filter structure with micropores onto a glass device to improve permeability to the cell culture space. Normally, it is extremely difficult to fabricate a filter structure on a normal glass plate by using a conventional fabrication method. Here, we demonstrated a method for fabricating an all-glass microfluidic cell culturing device having filters structure. The function of this all-glass culturing device was confirmed by culturing HeLa, fibroblast and ES cells. Compared with the closed glass devices without a filter structure, the numbers of cells in our device increased and embryonic bodies (EBs) were formed. This method offers a new tool in microfluidic cell culture technology for biological analysis and it expands the field of microfluidic cell culture.