An integrated microfluidic culture device to regulate endothelial cell differentiation from embryonic stem cells

We developed an integrated microfluidic culture device to regulate embryonic stem (ES) cell fate. The integrated microfluidic culture device consists of an air control channel and a fluidic channel with 4×4 micropillar arrays. We hypothesized that the microscale posts within the micropillar arrays would enable the control of uniform cell docking and shear stress profiles. We demonstrated that ES cells cultured for 6 days in the integrated microfluidic culture device differentiated into endothelial cells. Therefore, our integrated microfluidic culture device is a potentially powerful tool for directing ES cell fate.     

Microfabricated embryonic stem cell divider for large-scale propagation of human embryonic stem cells

We have developed a novel method for fabricating an embryonic stem cell divider (ESCD) constructed from a poly(dimethylsiloxane) (PDMS) replica with a square or hexagonal pattern, and have proposed a new dissociation method for human embryonic stem cells (ESCs). An aspect ratio of the device as high as 2 was perfectly replicated in the cutting line. Using the ESCD, human ESC colonies can be easily and efficiently dissociated into regular-sized ESC clumps without enzymatic treatment. The regularity of the ESC clumps dissociated by the ESCD was compared to that dissociated by a conventional mechanical method. Its quality and reliability were confirmed by maintaining undifferentiated ESCs up to the 15th passage. The ESCD will contribute to the advance quality control of in vitro ESC cultures and allow large-scale production of qualified ESCs with tremendous time- and work-saving.     

Microfluidic chemostat and turbidostat with flow rate, oxygen, and temperature control for dynamic continuous culture

This work reports on an instrument capable of supporting automated microscale continuous culture experiments. The instrument consists of a plastic-PDMS device capable of continuous flow without volume drift or evaporation. We apply direct computer controlled machining and chemical bonding fabrication for production of fluidic devices with a 1 mL working volume, high oxygen transfer rate (k(L)a≈0.025 s(-1)), fast mixing (2 s), accurate flow control (±18 nL), and closed loop control over temperature, cell density, dissolved oxygen, and pH. Integrated peristaltic pumps and valves provide control over input concentrations and allow the system to perform different types of cell culture on a single device, such as batch, chemostat, and turbidostat continuous cultures. Continuous cultures are demonstrated without contamination for 3 weeks in a single device and both steady state and dynamically controlled conditions are possible.     

Controlling size, shape and homogeneity of embryoid bodies using poly(ethylene glycol) microwells

Directed differentiation of embryonic stem (ES) cells is useful for creating models of human disease and could potentially generate a wide array of functional cell types for therapeutic applications. Methods to differentiate ES cells often involve the formation of cell aggregates called embryoid bodies (EBs), which recapitulate early stages of embryonic development. EBs are typically made from suspension cultures, resulting in heterogeneous structures with a wide range of sizes and shapes, which may influence differentiation. Here, we use microfabricated cell-repellant poly(ethylene glycol) (PEG) wells as templates to initiate the formation of homogenous EBs. ES cell aggregates were formed with controlled sizes and shapes defined by the geometry of the microwells. EBs generated in this manner remained viable and maintained their size and shape within the microwells relative to their suspension counterparts. Intact EBs could be easily retrieved from the microwells with high viability (>95%). These results suggest that the microwell technique could be a useful approach for in vitro studies involving ES cells and, more specifically, for initiating the differentiation of EBs of greater uniformity based on controlled microenvironments.     

Three-Dimensional Collagen Gel Networks for Neural Stem Cell-Based Neural Tissue Engineering

Stem and progenitor cells isolated from the embryonic rat cerebral cortex were immobilized by matrix entrapment in three-dimensional (3D) Type I collagen gels, and cultured in serum-free medium containing basic fibroblast growth factor. The cells trapped within the collagen networks actively proliferated and formed clone-like aggregates. Neurons were the first differentiated cells to appear within the aggregates, followed by generation of astrocytes and oligodendrocytes. In addition, necrotic cores were developed as the aggregate diameter increased and cell viability declined significantly after 3 weeks in culture. To overcome these problems, the cell-collagen constructs were transferred to Rotary Wall Vessel bioreactors for up to 10 weeks. In the rotary culture, the collagen gels compacted 3-4 folds and a long-term growth and differentiation of neural stem and progenitor cells was dynamically maintained. Remarkably, the cell-collagen constructs formed a complex two-layered structure that superficially emulated to a certain extent the cerebral cortex of the embryonic brain in architecture and functionality. The engineered 3D tissue-like constructs displaying characteristic properties of neuronal circuits may have potential use in tissue replacement therapy for injured brain and spinal cord.     

Bioprocessing technology for plant cell suspension cultures

Considering various forms of in vitro plant tissue cultures, cell suspension culture is most amenable to large-scale production of natural compounds, owing primarily to its superior culture homogeneity. This fact has already been demonstrated in several largescale applications, including the commercial shikonin process. The scope of this work is to review the state of the art in bioprocessing technologies pertinent to the secondary metabolite production from suspension cultures of callus cells. In the first part of the review, plant cell physiology relevant to bioprocess design is considered. This is followed by an in-depth discussion on the bioreactor design and operation and its effect on plant cell suspension cultures. Finally, recent commercial exploitation and development are summarized. Following the review, related patents and literature are listed.     

Microfabrication-based modulation of embryonic stem cell differentiation

Embryonic stem (ES) cells form spontaneous aggregates during differentiation, and cell-cell communication in the aggregates plays an important role in differentiation. The development of a controlled differentiation scheme for ES cells has been hindered by the lack of a reliable method to produce uniform aggregate sizes. Conventional techniques, such as hanging drop and suspension cultures, do not allow precise control over size of ES cell aggregates. To surmount this problem, we microfabricated adhesive stencils to make mouse ES (mES) cell aggregates of specific sizes ranging from 100 microm to 500 microm in diameter. With this technique, we studied the effect of the initial aggregate size on ES cell differentiation. After 20 days of induction of differentiation, we analyzed the stem cell populations using gene and protein expression assays as well as biochemical functions. Notably, we found that germ layer differentiation depends on the initial size of the ES cell aggregate. Among the ES cell aggregate sizes tested, the aggregates with 300 microm diameter showed similar differentiation profiles of three germ layers as embryoid bodies made using the "hanging drop" technique. The smaller (100 microm) aggregates showed the increased expression of ectodermal markers compared to the larger (500 microm) aggregates, while the 500 microm aggregates showed the increased expression of mesodermal and endodermal markers compared to the 100 microm aggregates. These results indicate that the initial size of the aggregate is an important factor for ES cell differentiation, and can affect germ layer selection as well as the extent of differentiation.     

Growth Characteristics of Human Adipose-Derived Stem Cells During Long Time Culture Regulated by Cyclin A and Cyclin D1

Abundant and less passaged cells are highly expected in clinical application since repeated subculture reduces stem cell characteristics. Long time culture of stem cells without passage is therefore needed. The growth and cell viability of human adipose-derived stem cells (hADSCs) were investigated by live/dead staining, cck-8 kits, and hemocytometer every day in 30?days of culture. The stem cell characteristics of hADSCs at the beginning and the end of culture were detected by flow cytometry and histochemical staining. hADSCs can be cultured up to the 30th day in one passage while maintaining high level cell viability and their stem cell characteristics. In addition, the cells displayed two plateau phases and three logarithmic phases during 1?month of culture. Increasing expression of cyclin A at protein level resulted in an increase in the percentage of hADSCs in the S and G₂/M phases, while decreasing protein level of cyclin D1 induced a decline in the proportion of hADSCs in the G₀/G₁ phase, regulating cells to move into rapid proliferation. This study demonstrates that a great quantity of hADSCs can be obtained in vitro by prolonging the culture time of each passage. And cyclin A and cyclin D1 affect the distribution of cell cycle and regulate the growth of hADSCs.     

Microfabricated polyester conical microwells for cell culture applications

Over the past few years there has been a great deal of interest in reducing experimental systems to a lab-on-a-chip scale. There has been particular interest in conducting high-throughput screening studies using microscale devices, for example in stem cell research. Microwells have emerged as the structure of choice for such tests. Most manufacturing approaches for microwell fabrication are based on photolithography, soft lithography, and etching. However, some of these approaches require extensive equipment, lengthy fabrication process, and modifications to the existing microwell patterns are costly. Here we show a convenient, fast, and low-cost method for fabricating microwells for cell culture applications by laser ablation of a polyester film coated with silicone glue. Microwell diameter was controlled by adjusting the laser power and speed, and the well depth by stacking several layers of film. By using this setup, a device containing hundreds of microwells can be fabricated in a few minutes to analyze cell behavior. Murine embryonic stem cells and human hepatoblastoma cells were seeded in polyester microwells of different sizes and showed that after 9 days in culture cell aggregates were formed without a noticeable deleterious effect of the polyester film and glue. These results show that the polyester microwell platform may be useful for cell culture applications. The ease of fabrication adds to the appeal of this device as minimal technological skill and equipment is required.     

Microfluidic arrays for logarithmically perfused embryonic stem cell culture

We present a microfluidic device for culturing adherent cells over a logarithmic range of flow rates. The device sets flow rates through four separate cell-culture chambers using syringe-driven flow and a network of fluidic resistances. The design is easy to fabricate with no on-chip valves and is scalable both in the number of culture chambers as well as in the range of applied flow rates. Using particle velocimetry, we have characterized the flow-rate range. We have also demonstrated an extension of the design that combines the logarithmic flow-rate functionality with a logarithmic concentration gradient across the array. Using fluorescence measurements we have verified that a logarithmic concentration gradient was established in the extended device. Compared with static cell culture, both devices enable greater control over the soluble microenvironment by controlling the transport of molecules to and away from the cells. This approach is particularly relevant for cell types such as embryonic stem cells (ESCs) which are especially sensitive to the microenvironment. We have demonstrated for the first time culture of murine ESCs (mESCs) in continuous, logarithmically scaled perfusion for 4 days, with flow rates varying >300x across the array. Cells grown in the slowest flow rate did not proliferate, while colonies grown in higher flow rates exhibited healthy round morphology. We have also demonstrated logarithmically scaled continuous perfusion culture of 3T3 fibroblasts for 3 days, with proliferation at all flow rates except the slowest rate.     

A Facile Method for the Preparation of Monodisperse Beads with Uniform Pore Sizes for Cell Culture

This paper describes a facile method for the preparation of porous gelatin beads with uniform pore sizes using a simple fluidic device and their application as supporting materials for cell culture. An aqueous gelatin droplet containing many uniform toluene droplets, produced in the fluidic device, is dropped into liquid nitrogen for instant freezing and the small toluene droplets evolve into pores in the gelatin beads after removal of toluene and then freeze‐drying. The porous gelatin beads exhibit a uniform pore size and monodisperse diameter as well as large open pores at the surface. Fluorescence microscopy images of fibroblast‐loaded gelatin beads confirm the attachment and proliferation of the cells throughout the porous gelatin beads.     

Patterned cell culture inside microfluidic devices

This paper describes a simple plasma-based dry etching method that enables patterned cell culture inside microfluidic devices by allowing patterning, fluidic bonding and sterilization steps to be carried out in a single step. This plasma-based dry etching method was used to pattern cell-adhesive and non-adhesive areas on the glass and polystyrene substrates. The patterned substrate was used for selective attachment and growth of human umbilical vein endothelial cells, MDA-MB-231 human breast cancer cells, NIH 3T3 mouse fibroblasts, and primary rat cortical neurons. Finally, we have successfully combined the dry-patterned substrate with a microfluidic device. Patterned primary rat neurons were maintained for up to 6 days inside the microfluidic devices and the neurons' somas and processes were confined to the cell-adhesive region. The method developed in this work offers a convenient way of micropatterning biomaterials for selective attachment of cells on the substrates, and enables culturing of patterned cells inside microfluidic devices for a number of biological research applications where cells need to be exposed to well-controlled fluidic microenvironment.     

Enrichment of putative stem cells from adipose tissue using dielectrophoretic field-flow fractionation

We have applied the microfluidic cell separation method of dielectrophoretic field-flow fractionation (DEP-FFF) to the enrichment of a putative stem cell population from an enzyme-digested adipose tissue derived cell suspension. A DEP-FFF separator device was constructed using a novel microfluidic-microelectronic hybrid flex-circuit fabrication approach that is scaleable and anticipates future low-cost volume manufacturing. We report the separation of a nucleated cell fraction from cell debris and the bulk of the erythrocyte population, with the relatively rare (<2% starting concentration) NG2-positive cell population (pericytes and/or putative progenitor cells) being enriched up to 14-fold. This work demonstrates a potential clinical application for DEP-FFF and further establishes the utility of the method for achieving label-free fractionation of cell subpopulations.     

Gravitational field-flow fractionation of human hemopoietic stem cells

New cell sorting methodologies, which are simple, fast, non-invasive, and able to isolate homogeneous cell populations, are needed for applications ranging from gene expression analysis to cell-based therapy. In particular, in the forefront of stem cell isolation, progenitor cells have to be separated under mild experimental conditions from complex heterogeneous mixtures prepared from human tissues. Most of the methodologies now employed make use of immunological markers. However, it is widely acknowledged that specific markers for pluripotent stem cells are not as yet available, and cell labelling may interfere with the differentiation process. This work presents for the first time gravitational field-flow fractionation (GrFFF), as a tool for tag-less, direct selection of human hematopoietic stem and progenitor cells from cell samples obtained by peripheral blood aphaeresis. These cells are responsible to repopulate the hemopoietic system and they are used in transplantation therapies. Blood aphaeresis sample were injected into a GrFFF system and collected fractions were characterized by flow cytometry for CD34 and CD45 expression, and then tested for viability and multi-differentiation potential. The developed GrFFF method allowed obtaining high enrichment levels of viable, multi-potent hematopoietic stem cells in specific fraction and it showed to fulfil major requirements of analytical performance, such as selectivity and reproducibility of the fractionation process and high sample recovery.     

Dual Elicitation for Improved Production of Withaferin A by Cell Suspension Cultures of Withania somnifera

High yielding transformed callus culture of W. somnifera was established by infecting hypocotyls with Agrobacterium tumefaciens MTCC-2250. Maximum withaferin A content of 0.0875 mg/g dry cell weight and transformation efficiency of 80% were obtained. Confirmation of transformation was done on the basis of the presence of the ags gene by using polymerase chain reaction. Various abiotic elicitors (arachidonic acid, methyl jasmonate, calcium chloride, and copper sulfate) and biotic elicitors (cell extracts and culture filtrates of Alternia alternata, Fusarium solani, and Verticilium dahaliae) were tested at different concentrations to enhance withaferin A production in suspension culture of transformed cells. Maximum enhancements of 5.4 times and 9.7 times, respectively, were obtained when copper sulfate (100 microM) and the cell extract of V. dahaliae (5% v/v) were added separately to suspension cultures. The dual elicitation strategy by the combined addition of these two elicitors resulted in 13.8-fold enhancement of withaferin A content in comparison to control cultures (2.65 mg/L). The present study indicates the potential of this biotechnology-based methodology for the large-scale production of withaferin A.     

Microfluidic‐Printed Microcarrier for In Vitro Expansion of Adherent Stem Cells in 3D Culture Platform

Microcarrier‐based stem cell expansion cultures can increase the dimensions of in vitro stem cell cultures from 2D to 3D. The culture handling process then becomes more efficient compared with conventional 2D cultures. However, the use of spherical plastic microcarriers complicates the monitoring of cell culture. To facilitate monitoring, transparent disc‐shaped microcarriers are manufactured using a light‐initiated microfluidic printing system and the obtained microcarriers are named as 2.5D microcarrier. The 2.5D microcarriers (diameter/height ≈ 5) enable us to use conventional monitoring tools in 2D‐based platform during the in vitro expansion on a 3D culture platform. Surface modification via a 1 h‐long poly‐dopamine (PDA) reaction can maintain the transparent nature of the microcarriers while optimizing the cell attachment. The surface marker expression and differentiation potential of the 2.5D microcarrier‐expanded stem cells reveal that the characteristics and functionalities preserved during expansion. The 2.5D microcarrier is readily integrated into an on‐bead assay to conserve reagents and permit a high number (n = 9) of repeated measurements with reliable results. These results demonstrate that the 2.5D microcarrier‐based scale‐up culture provides a valuable tool for the in vitro expansion of adherent stem cells, especially if repetitive monitoring is required.     

Human neural stem cell growth and differentiation in a gradient-generating microfluidic device

This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell-based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.     

Ex Vitro Expansion of Human Placenta-Derived Mesenchymal Stem Cells in Stirred Bioreactor

The high demand of human placenta-derived mesenchymal stem cells (hPDMSCs) for therapeutic applications requires reproducible production of large numbers of well-characterized cells under well-controlled conditions. However, no method for fast hPDMSCs proliferation has yet been reported. In the present study, the feasibility of using a stirred bioreactor system to expand hPDMSCs was examined. hPDMSCs were cultured either in stirred bioreactors or in tissue culture flasks (T-flasks) for 5 days. Total cell density and several parameters of physical microenvironments were monitored in the two culture systems every 24 h. The maintenance of the antigenic phenotype of hPDMSCs before and after culturing in the stirred bioreactor system was cytometrically assessed. Data suggested that the physical microenvironment in the stirred bioreactors was much more favorable than that of the T-flasks. At the end of 144 h culturing, the total cell number was increased 1.73 times from the T-flasks to the stirred bioreactors. In addition, hPDMSCs could maintain their antigenic phenotype when cultured in stirred bioreactors. These results provide the initial assessment for large-scale hPDMSCs production using suspension culture bioreactors.     

A phenotypic small-molecule screen identifies an orphan ligand-receptor pair that regulates neural stem cell differentiation

High-throughput identification of small molecules that selectively modulate molecular, cellular, or systems-level properties of the mammalian brain is a significant challenge. Here we report the chemical genetic identification of the orphan ligand phosphoserine (P-Ser) as an enhancer of neurogenesis. P-Ser inhibits neural stem cell/progenitor proliferation and self-renewal, enhances neurogenic fate commitment, and improves neuronal survival. We further demonstrate that the effects of P-Ser are mediated by the group III metabotropic glutamate receptor 4 (mGluR4). siRNA-mediated knockdown of mGluR4 abolished the effects of P-Ser and increased neurosphere proliferation, at least in part through upregulation of mTOR pathway activity. We also found that P-Ser increases neurogenesis in human embryonic stem cell-derived neural progenitors. This work highlights the tremendous potential of developing effective small-molecule drugs for use in regenerative medicine or transplantation therapy.     

Mass cultivation of catharanthus roseus cells using a nonmechanically agitated bioreactor

Batch suspension cultures ofCatharanthus roseus G. Don were grown in a 5 L LKB Ultraferm fermenter, converted to operate as an airlift bioreactor, to test the suitability of such a system for the mass culture of plant cells. Results show that the airlift system has considerable merits as a culture vessel for such a purpose, including: conversion rates of carbohydrate substrate to cell mass equivalent to > 50% under optimum conditions. (Operating under these conditions, growth rates of approximately 0.4 d^-1 are typical). In the absence of the mechanical shear normally associated with mechanically driven bioreactors, the gently agitated environment of the airlift vessel proves to be an ideal system for the growth of fragile plant cells. Use of a nozzle sparger reduces the possibility of a high mass transfer coefficient, except at very high gassing rates, thereby eliminating any interference with the growth rate caused by high rates of gaseous exchange.