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Keywords:

  • embryoid bodies;
  • concave microwells;
  • surface tension;
  • homogeneous-size;
  • mass production

Abstract

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results
  6. 4. Discussion
  7. 5. Conclusion
  8. Supporting Information
  9. Acknowledgements
  10. Supporting Information

Embryonic stem cells (ESCs) are pluripotent and capable of self-renewal. ESC aggregates, termed embryoid bodies (EBs), have been widely adopted as an in vitro differentiation model. However, the mass production of uniform size and shaped EBs has been challenging. Herein is described the development of a culture plate containing a large number of concave microwells with minimal use of tools, labor, skill, and cost, enabling the production of a large number of homogeneous EBs simultaneously using the culture plate. The large number of concave well structures is self-constructed through the surface tension of the viscoelastic PDMS prepolymer. Murine ESCs (mESCs) are then seeded onto the concave wells for mass production of monodisperse EBs. It is observed that the EBs produced over a large area are uniform in shape and size regardless of microwell position and differences in cell seeding densities, and whether their phenotype is maintained. The capability to differentiate into adult cells (neuron and endothelial cells) from EBs is also evaluated and the neural spikes from differentiated neuron cells are measured to observe their function. Uniform size and shape EBs are successfully generated in large scale and their pluripotency is maintained similar to other methods.


1. Introduction

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results
  6. 4. Discussion
  7. 5. Conclusion
  8. Supporting Information
  9. Acknowledgements
  10. Supporting Information

Embryonic stem cells (ESCs) are pluripotent, and can differentiate into specific cell lineages in response to spatio-temporally regulated intrinsic and extrinsic biochemical signals1–4 and mechanical forces.5–7 These differentiated cells can ultimately grow to form an organ or a tissue and have been extensively studied for applications in tissue engineering,8–10 regenerative medicine,4 and cell-based therapy.3, 11 The principle step in ESC differentiation is formation of an embryoid body (EB), which recapitulates features of early embryogenesis.12, 13 Considerable research effort has been devoted to obtain well-organized EBs, but prevailing methods, like scraping techniques and suspension culture methods,14–16 produce a polydisperse EB size distribution that prevents the homogeneous differentiation necessary to generate large amounts of specific lineages.11, 12, 17, 18 Recent progress in microtechnology has led to the development of diverse microwells for the formation of uniform-sized EBs19–24 as well as cell behavior researches.25, 26 Polyurethane microwells, including self-assembled monolayers27, 28 and non-adhesive polyethylene glycol microwell arrays,29 have also been used to control the homogeneity of EB size and shape, but difficulties in seeding of uniform number of cells, retrieving of EBs and obtaining larger EBs have been encountered. Recently, we reported the formation of homogeneous EBs in a concave-microwell having neither edge nor corner structure thus, reducing cells adhesion to microwell surfaces array by replicating the deflection of a thin elastic membrane,20, 30 but, with this method, the large-scale fabrication of densely packed concave structures remain a challenge. Despite such difficulties, the production of homogeneous EBs in large amounts is critical for generating large numbers of the desired cell type with high purity.17, 31, 32 Even though it is possible to construct large scale-microwell arrays, it remains unclear whether EBs produced in large scale maintain their shape and size uniformly because the number of cells seeded into each microwell is not even over large scale-microwell arrays.

In this paper, we present a simple and novel method for fabricating a culture plate containing a large number of concave microwells with minimum tools, labor, skill, and cost, and we produced a large number of homogeneous EBs simultaneously using this plate. The concave well structures were self-constructed through the surface tension of the PDMS prepolymer based on the cylindrical polydimethylsiloxane (PDMS) microwell arrays that have been prepared using a conventional soft lithography method. This procedure is simple enough to be accessible to any laboratory, including a general biology or material laboratory. Throughout this process, 10,900 concave microwell structures can be produced simultaneously in a single plate. Murine ESCs (mESCs) were then seeded onto the concave wells for mass production of monodisperse EBs. Here, we focused on whether the EBs produced over a large area are uniform in shape and size, regardless of microwell position and differences in cell seeding densities, and whether their phenotype is maintained. The capability to differentiate to adult cells (neuron and endothelial cells) from EBs was also evaluated and the neural spikes from differentiated neuron cells were measured to observe their function. The proposed approach will reduce the scale gap between production of thousands or millions of cells at the laboratory bench and the much larger number of cells required per patient for clinical application.

2. Experimental Section

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results
  6. 4. Discussion
  7. 5. Conclusion
  8. Supporting Information
  9. Acknowledgements
  10. Supporting Information

2.1. Creation of Concave-Microwell Arrays

The large-scale, concave-microwell array structure was fabricated by exploiting the surface tension of the PDMS prepolymer. A PDMS microwell plate (70 × 70 × 5 mm) containing 10,900 arrayed cylindrical well structures was prepared using a standard soft lithography process. Onto this plate, we poured a PDMS prepolymer consisting of a 10:1 mixture of silicon elastomer (Sylgard 184) and curing agent, and allowed it to completely fill all cylindrical microwells (Figure 1a & b). Using a slide glass (76 × 52 × 1.2 mm), we raked out the PDMS prepolymer, applying slight pressure to the soft PDMS microwell plate (Figure 1a & b). The softness of the PDMS plate resulted in approximately half of the PDMS prepolymer being removed by wiping, with the remaining half of the PDMS prepolymer filling the cylindrical microwells. The surface tension of the PDMS prepolymer caused a meniscus to be self-organized in the cylindrical microwells (Figure 1a & d; white dotted line indicates cylindrical microwells); the contact angle between the prepolymer meniscus and the PDMS sidewall was determined to be approximately 20°.33, 34 The PDMS prepolymer in each well was polymerized by thermal curing on a hot plate (80 °C for 1 hour), forming the final concave structure. This fabrication procedure produced a large number of concave wells simultaneously (Figure 1c and inset in Figure 1d) without using any specialized tool or complicated procedure. Figure 1d shows scanning electron microscopy (SEM) images, demonstrating the hemispherical structure of the microwells. The PDMS plate containing a large number of concave wells was replicated with SU-8 (MicroChem, MA, USA) was replicated by pouring SU-8 (MicroChem, MA, USA) onto the concave array and exposed to UV, creating a convex master mold (Figure S1). The PDMS concave microwell arrays were finally fabricated using the SU-8 replica.

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Figure 1. Fabrication of a concave microwell-array PDMS plate. (a) A schematic diagram of concave-microwell arrays made using the meniscus of the PDMS prepolymer. The PDMS prepolymer was poured onto the prepared base-plate containing cylindrical microwells. The contact angle between the meniscus of the PDMS prepolymer and the cylindrical microwell formed was approximately 20°. (b) The PDMS prepolymer on the cylindrical microwell arrays was raked out using a flat plate (e.g., glass plate). (c) A picture of a microwell- plate fabricated to contain a large numbers of concave microwells. (d) SEM image of the meniscus-induced concave microwell. The figure is a side view of a 300-μm device. The white dotted lines indicate the cylindrical well before pouring the prepolymer. After pouring and raking out the prepolymer, deep concave wells are formed by a meniscus effect. Scale bar; 300 μm.

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2.2. Preparation of mESCs

The R1 ESC line (American Type Culture Collection, USA) used in these experiments is a sub-line of J1 ESCs established by Dr. Rudolph Jaenisch (Whitehead Institute for Biomedical Research, Cambridge, MA, USA). The prepared ESCs were cultured on gelatin-coated petri dishes in Knockout-Dulbecco's Modified Eagle Medium (Knockout-DMEM; Invitrogen, USA) containing 15% ES-qualified fetal bovine serum (FBS; Invitrogen) and 1400 Unit/ml leukemia inhibitory factor (LIF; Millipore, USA). Cells were passaged every 3 days by dissociating the cells into small colonies using 0.25% trypsin (Invitrogen) and replating on a 0.1% gelatin-coated dish at a subculture ratio of 1:6.

2.3. Seeding mESCs into Concave Microwells

For seeding cells into concave microwells, suspensions of cells at different densities (6.0 × 106, 9.0 × 106, and 12.0 × 106 cells/ml) were prepared and the affects of cell density on the size and shape of EBs were investigated. A 2-ml suspension of mESCs was directly seeded on top of the concave microwell-arrayed PDMS plate with gentle shaking, and the cells were allowed to become trapped within concave microwells (Figure S3a). Thirty minutes after seeding, when mESCs were settled within concave microwells, a flow of culture medium was gently applied to remove cells that did not settle within the microwells (Figure S3b).

2.4. Cell Viability Test

Cell viability was assayed by adding 5 ml of a phosphate-buffered saline (PBS) solution containing 2 ml of calcein AM solution and 10 ml of ethidium homodimer-1 solution to EBs and then by incubating at 37 °C in a 5% CO2 incubator for 40 minutes. Stained mESCs were observed under an inverted fluorescence microscope (EVOS; AMG, USA). Live and dead cells were indicated by calcein–AM (green) and ethidium homodimer-1 (red) staining, respectively.

2.5. Three Germ-Layer Differentiation of EBs and Gene Analysis

mESCs were cultured within concave microwells for 3 days, after which mESC agglomerations were retrieved and cultured for an additional 3, 6, or 9 days in a petri dish in a static suspension state to allow EB development (Figure S3c). EBs were gently retrieved and subsequently resuspended in Knockout-DMEM containing 15% FBS, non-essential amino acids, 2 mM L-Glutamine, β-mercaptoethanol (Invitrogen), and penicillin-streptomycin. Cell density effects were assessed in mESC agglomerations in concave microwells (diameter, 500 μm) from three different seeding densities (6.0, 9.0, and 12.0 × 106 cells/ml). For gene-profile tests, EBs were digested in TRIzol Reagent (Invitrogen) followed by chloroform extraction and precipitation of total RNA with isopropyl alcohol. cDNA was synthesized from total RNA using reverse transcriptase (TAKARA, Japan) as described by manufacturer.

2.6. Immunocytochemistry

EBs were fixed with 4% paraformaldehyde in PBS at 4 °C for 60 minutes, washed gently three times with PBS, equilibrated in 20% sucrose overnight at 4 °C, then frozen in O.C.T compound (Sakura Finetek Japan, Tokyo) and kept at -80 °C until use. Frozen EBs were sectioned (∼15-μm-thick slices) using a cryostat and placed on Histobond adhesive glass slides (Marienfeld, Germany). Thereafter, glass slides containing samples were dried for 30 minutes and subsequently washed three times with PBS (5 minutes each). Sectioned EBs were incubated with primary antibodies overnight at 4 °C, then washed with PBS/0.05% Tween (PBST), and incubated with appropriate secondary antibodies (Jackson ImmunoResearch Laboratories or Molecular Probes, USA) in PBS/0.1% BSA at room temperature for 1.5 hours. After counterstaining with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, Invitrogen, CA), stained mESCs were observed under an inverted fluorescence microscope (EVOS; AMG, USA)

2.7. mESC-Derived Neuronal and Endothelial Cell Differentiation

The differentiation capability of EBs to each neuronal (from ectoderm) and endothelial (from mesoderm) cell was evaluated. EBs cultured in concave microwells for 3 days were retrieved, and transferred to a petri dish for additional culture in a suspension state for 6 days. For the neuronal cell differentiation, EBs were transferred to tissue culture dishes, and cultured in neuronal differentiation medium supplemented with insulin/transferrin/selenium/fibronectin (ITSFn) medium (Invitrogen) for 10 days. For the endothelial cell differentiation, EBs were transferred to a Matrigel (BD Biosciences, USA)-coated dish and cultured in endothelial cell growth medium (EGM-2; Lonza, USA) containing a full complement of supplements (EGM-2 bullet kit: 2% FBS, 0.4% hFGF-2, 0.1% VEGF, 0.1% R3-IGF-1, 0.1% hEGF, 0.04% hydrocortisone, 0.1% ascorbic acid, 0.1% heparin, and 0.1%-GA-100) for an additional 4 days. Culture media were replaced every other day. Neuroepithelial progenitor cells that differentiated from EBs were observed after culturing for 10 days. The lengths of neurofilament branches were measured using Image J software (http://rsbweb.nih.gov/ij/). For immunostaining, differentiated cells were fixed for 20 minutes with 4% formaldehyde at 4 °C. Cells were permeabilized using 0.1% Triton-X100 in 0.1% PBS for 20 minutes at room temperature, blocked with 3% BSA in PBS for 30 minutes, and then incubated with primary antibody overnight at 4 °C. Primary antibodies (Stemcell Technologies, Canada) against neurofilament (1:1000) and nestin (1:100) were used to observe various cell types. After incubating overnight, cells were washed with PBST for 5 min. Cells were incubated with secondary antibodies (1:1000 dilution; Invitrogen) for 1.5 hours at room temperature and counterstained with DAPI. Endothelial cell differentiation was evaluated by staining EBs with primary anti-mouse CD31 antibody (BD Sciences) and secondary phycoerythrin (PE)-conjugated antibodies (Santa Cruz Biotechnology, USA). Stained cells were observed by fluorescence microscopy (EVOS; AMG).

2.8. Neuro-Spike Signal Analysis

For monitoring electrical activity of differentiated neuronal cells, EBs were cultured in a Petri dish for an additional 6 days, and then transferred to a commercial multi-electrode array (MEA) chip (Multi Channel Systems, Germany). Neuronal differentiation was induced by applying neuronal differentiation medium supplemented with ITSFn medium (Invitrogen) for an additional 20 days, at which time (total, 29 days) electric signals were recorded using a multi-channel data acquisition system (MC_Rack; Multi Channel Systems, Germany).

2.9. Scanning Electron Microscopy

Field emission scanning electron microscopy (FE-SEM; JEOL 4701F, JAPAN) was used to analyze the shape of deep concave wells and EBs in concave wells. The morphology of EBs cultured in concave wells and in the suspension state was observed by fixing EBs in the well with 2.5% glutaraldehyde in deionized water for 1 hour, and then gently washing with deionized water. As a secondary fixation step, EBs were incubated in 1% osmium tetroxide in deionized water for 1 hour. Fixed EBs were dehydrated using a series of graded ethanol (25%, 50%, 75%, 95%, and 100%). After dehydration, EBs were washed three times (30 minutes) in tetra butyl alcohol at room temperature and frozen at -70 °C. EBs were freeze-dried until the tetra butyl alcohol had evaporated, mounted on a specimen stub with graphite paste, coated with palladium alloy, and observed under a scanning electron microscope (JEOL Ltd., Japan).

2.10. Statistics and Image Analysis

All quantitative data were expressed as means ± standard errors of the mean. All collected data were analyzed using unpaired t-tests or one-way analyses of variance (ANOVA). P-values < 0.05 were considered statistically significant.

3. Results

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results
  6. 4. Discussion
  7. 5. Conclusion
  8. Supporting Information
  9. Acknowledgements
  10. Supporting Information

3.1. Large-Scale, Concave-Microwell Arrays Induced by Surface Tension of PDMS

A large number of concave microwell arrays (10,900 wells) were simultaneously fabricated on a 70-mm circular PDMS plate by exploiting the surface tension of the PDMS solution (Figure 1c). Figure 1d shows a lateral SEM image of concave wells and a top view of microwell arrays (inset). As predicted, the shape and size of microwells were nearly uniform, despite using a manual process to rake out the PDMS prepolymer to create the structure. Diverse-sized concave-microwell arrays, with diameters of 300, 500 and 700 μm, were also successfully produced, as shown in Figure S2 (a, b, and c).

3.2. Mass Production of EBs and Evaluation of their Size and Shape

We produced EBs on a large scale and investigated their size and shape. mESCs were seeded onto microwell arrays and cells outside of the concave microwells were washed out to prevent undesired mESC aggregation and proliferation (Figure S3a & b). mESCs were settled into each microwell within 30 minutes after seeding. A microscopic image of the EB formation process over time in 500-μm concave-microwell array is shown in Figure 2a. One day after seeding, ESCs started to self-aggregate and form spherical shapes at the center of concave wells, and EB agglomerations became tight at day 2. At day 3, EBs formed agglomerations that occupied approximately half the diameter of the concave microwell. An SEM image of EB agglomerations in concave wells showed that their shape was almost spherical (Figure 2b). A Live/Dead assay showed that the viability of EBs in concave wells was greater than 95% (Figure 2c, calcein AM-stained [white]; white dotted lines indicate the edge of concave microwells), indicating that the mass production method did not cause serious cell damage or central necrosis. Figure 2d shows EBs harvested from 500-μm concave microwells; they were easily retrieved from the concave microwells by gently shaking or tapping the microwell plate. The harvested EBs were homogeneous in shape and size (Figure 2d), indicating that the harvesting process does not damage EBs. The homogeneity of the shape of EBs was quantified by calculating EB circularity, defined as 4π × [Area/Perimeter2]. The circularity denotes the ratio of irregular EB clusters to regular spherical EB clusters32 and was calculated using Image J software. The circularity value in 500-μm microwells was 0.984 ± 0.021 (n = 20), indicating that EBs harvested from concave microwells have a highly homogeneous circular shape.

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Figure 2. EB formation in concave microwell-arrays used for mass production. (a) Variation in EBs from day 0 to day 3. (b) An SEM image showing formation of EBs in microwells. (c) Results of LIVE/DEAD assays at day 3 showing that most mESCs were viable. (d) EBs harvested from the concave wells after culturing for 3 days, demonstrating that mass production is possible in concave-microwell arrays.

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To investigate the relationship between cell density and EB size, we seeded concave-microwell arrays with three different densities of mESCs (6.0, 9.0, and 12.0 × 106 cells/ml), cultured them for 3 days, and determined the size of the resulting EBs (Figure 3a and Figure S5). As predicted, the diameter of mESCs agglomerations 30 minutes after cell seeding increased as the cell density increased. However, as time passed, these density-dependent differences in EB size decreased, and at day 3 (Figure 3a & b), the size of EBs seeded at different cell densities became similar (Figure 3a and Figure S5). A calculation of the ratio of EB size to microwell diameter at 3 days showed that the rate of reduction in size of EB differed slightly according to the size of the concave well (Figure 3c). Such a reduction in EB size minimizes the contact area between the EB and the concave well, and enables easy retrieval of EBs from the well without causing damage.

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Figure 3. Size-tunable EB formation in concave microwells. (a) The size of EBs approached half the size of well diameters. Scale bar, 500 μm. (b) The diameter of ESC aggregates seeded at different cell densities and cultured in different-sized concave microwells (diameter: 300, 500, and 700 μm) decreased over time. (c) ESC aggregates cultured in concave microwells were tuned to half the size of the well diameter at day 3.

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3.3. Differentiation Characteristics of EBs

EB sections frozen at days 9 and 12 were immunostained for Sox17, brachyury, and nestin expression to assess endodermal, mesodermal and ectodermal differentiation, respectively. The immunostained image (Figure 4a) shows that all three embryonic germ layers were generated in all cases and cell density is not a critical determinant of differentiation. However, the lineage-specificity of differentiation differed somewhat over time. Strong expression of brachyury was observed in both 9- and 12-day EBs independent of our cell density conditions. In contrast, expression of Sox17 at day 12 was much stronger than that at day 9. Nestin expression at day 12 was slightly less than that on day 9.

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Figure 4. Time-course of the expression of markers for the three germ layers in EBs and evaluation of neuronal differentiation in harvested EBs. (a) Fluorescence images of cryo-sectioned EBs immunostained for Sox17 (endoderm), brachyury (mesoderm), and nestin at days 9 and 12. (b) Top: Representative figure showing the expression of mRNA for the three germ-layer markers, AFP (endoderm), BMP-4 (mesoderm) and nestin (ectoderm), and the pluripotency marker, Oct-04, in ESCs cultured in concave microwells for 3 to 12 days in vitro. Bottom: Quantification of the relative expression levels of the markers shown above. The error bars indicate standard deviation (*p < 0.05, **p < 0.01 compared to 300-μm EBs; n = 4; Student's t-test).

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In addition to immunostaining, RT-PCR was used to analyze quantitatively the expression of several markers, including Oct-4, BMP−4, AFP and nestin, to investigate the trends in differentiation and pluripotency according to culture time (3, 6, 9 and 12 days) and cell density (6.0, 9.0, and 12.0 × 106 cells/ml). The results of these analyses are presented in Figure 4b. Similar to the results obtained by immunostaining, our cell density conditions did not significantly affect gene expression; in contrast, culture time had a critical effect on differentiation toward specific lineages. We found no evidence for expression of the endodermal differentiation marker, AFP, in any of the microwells at day 3 or 6; however, AFP was strongly expressed after day 9. In contrast, expression of the mesodermal differentiation marker, BMP-4, did not significantly differ according to time or our cell density conditions. Expression of the ectodermal differentiation marker, nestin, increased during the first 6 days after seeding and decreased after day 6. Oct-4, a marker for undifferentiated cells, was highly expressed at days 3 and 6, and decreased rapidly after day 6 (Figure 4a).

To investigate whether the EBs differentiated to mature cell lineages, we transferred harvested EBs from 500-μm concave wells to a cell culture dish and cultured them in differentiation medium for an additional 10 days to allow differentiation into neurons (Figure 5a). The differentiation into endothelial cell was observed after 14 days culture in endothelial cell inducing media (Figure S7). Figure 5a demonstrates sprouting of cells differentiating into neurons and neurofilaments (green) from EBs; nestin staining (red) indicates the appearance of neural progenitor cells. The average length of sprouted filaments was greater than 1.0 mm. To evaluate neuronal functions, we measured the neuronal spikes of neurons differentiated from EBs by culturing EBs on an MEA chip (Figure S8, red dotted line). The spike signal, plotted in Figure 5b, confirmed that the waveform of signals evoked from differentiated EBs was similar to normal neuronal signals, but the duration of the spikes was longer than neuronal signals from a normal rat35 (Figure 5b). To evaluate endothelial cell differentiation, we immunostained cells with CD31 (PECAM) and smooth muscle actin (SMA); both CD31 and SMA were strongly expressed (Figure S7). Collectively, these results indicate that EBs are capable of differentiating into diverse-lineage adult cells.

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Figure 5. (a) Illustration of neuronal differentiation of an EB cultured for 10 days; the figure was generated from 26 fluorescent images (10x). The average lengths of neurites exceeded 1 mm. Scale bar, 500 μm. (b) Neuronal spikes in differentiated neuron cells from EBs were measured in MEA-patterned culture dishes.

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4. Discussion

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results
  6. 4. Discussion
  7. 5. Conclusion
  8. Supporting Information
  9. Acknowledgements
  10. Supporting Information

The viscoelastic properties and surface tension of the PDMS prepolymer enabled the construction of concave 10,900-microwell arrays with uniform shape and size using a simple raking-out process. Once the cylindrical microwell arrays have been prepared using conventional soft lithography, the process of constructing concave microwells is straightforward and fast (<1 hour), even in a biology or material laboratory setting. Little labor, and no specialized skills, devices, or facilities are required contrary to conventional MEMS technology.

It is generally recognized that differentiation of ESCs is affected by the size of the EB, and precise control of EB size is a critical issue.19, 20, 36 In our platform, the size of EBs is determined just by changing the diameter of the concave well, and the size of EBs created in same-sized microwells was almost uniform. In principle, it is difficult to seed a uniform number of mESCs into each concave microwell, however, the size of EBs 3 days after cell seeding were almost similar. We hypotheses the reason for such size uniformity may be from the proliferation of cells during the EB-formation process. And the concave microwell may inhibit the further growth of EBs. To test this assumption, we seeded concave-microwell arrays with three different densities of ESC suspensions (6.0, 9.0, and 12.0 × 106 cells/ml) and cultured them. As predicted, initial differences in EB size at different cell-seeding densities decreased over time such that by day 3, EB sizes had become similar. This experimental result explains why EB size becomes uniform, despite differences in the number of cells trapped in each microwell. Once ESCs are aggregated, forming a spheroid shape, cells may proliferate until EBs reach a certain size. Although further studies are required to confirm this, such a property could be very useful in the large-scale production of uniform EBs. For mass production of EBs, a large area (7-cm diameter) PDMS plate containing 10,900 microwells was used, raising questions about possible location-specific differences in size. To test whether the size of EBs was uniform over all microwells regardless of position, we measured the size of EBs at five locations, defined in Figure S4a, after culturing for 3 days (Figure S4c.) The differences in EBs sizes were within ±10%, indicating that the size of EBs could be controlled, even when generated on a large scale on large-area plates.

The gene profile of RT-PCR and immunostaining analyses were consistent with each other The endodermal differentiation marker, AFP, was more strongly expressed at day 12 than at day 9, a result identical to that obtained by immunostaining for the endodermal marker, Sox17. The mesodermal markers, brachyury and BMP-4, were expressed similarly in both analyses.

After 3 days, EBs were cultured in suspension in culture dishes, and grew in size, forming cystic EBs; tight cell-cell interactions on the EB surface were observed after 8–10 days, as reported previously.13, 37 Figure S6 shows an SEM image of growing EBs over time, revealing the cystic structure (Figure 4a, white arrowheads) at day 9 (total, 12 days) after suspension culture. The differentiation of EBs into neurons (from ectoderm) and endothelial cells (from mesoderm) indicate the capacity of EBs to differentiate into specific-lineage adult cells. Measurements of spike signals using an MEA system indicated that neurons differentiated from EBs perform the basic function of transmitting neural signals, indicating that the proposed method for mass producing EBs is useful for the large-scale differentiation of mature cells. Successful clinical application of these differentiated cells will require bridging the scale gap between production of thousands or millions of cells at the laboratory bench and the much larger numbers of cells required per patient for clinical protocols. The proposed method, which is cost-effective and capable of producing a large number of uniform EBs, will decrease this gap and may enable extensive clinical trials to proceed much more rapidly.

5. Conclusion

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results
  6. 4. Discussion
  7. 5. Conclusion
  8. Supporting Information
  9. Acknowledgements
  10. Supporting Information

We established a method for simultaneously fabricating concave-microwell arrays on a large scale using an extremely simple and cost-effective method that exploits the surface tension of a PDMS solution. This device allowed us to rapidly produce EBs with ultra-high throughput without the requirement for additional processing during cell seeding or EB retrieval. The size of EBs was tunable by simply changing the size of microwells, and the sizes of EBs from wells of the same size were uniform, regardless of our cell-seeding density conditions and location of microwell. The proposed EB production method may enable the large-scale expansion of specific-lineage cells, and has potentially extensive applications in drug discovery, bio-artificial organ development, and regenerative medicine.

Supporting Information

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results
  6. 4. Discussion
  7. 5. Conclusion
  8. Supporting Information
  9. Acknowledgements
  10. Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results
  6. 4. Discussion
  7. 5. Conclusion
  8. Supporting Information
  9. Acknowledgements
  10. Supporting Information

This study was supported by a grant from the NRL (National Research Lab) program, the Korea Science and Engineering Foundation (KOSEF), Republic of Korea (No.20110020455).

Supporting Information

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results
  6. 4. Discussion
  7. 5. Conclusion
  8. Supporting Information
  9. Acknowledgements
  10. Supporting Information

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