**3. Electrical stimulation and gene expression analysis with cell-adhesive microcavity-array electrode substrate**

As described in section 2, it was difficult to stimulate a large number of EBs, over a hundred numbers, when using the cell-nonadhesive electrode substrate with SU-8 negative thick photoresist. This was because that the SU-8 structures were strictly integrated with ITO substrate and thus all surface of the electrode substrate became cell-nonadhesive after the MPC polymer coating of microcavity-array region (Fig. 5-a). Although the cell-nonadhesive surface was advantageous to handle EBs, maintain suspension culture conditions and trap EBs into the microcavities temporally, it was disadvantageous to fix EBs to the bottom surfaces of the microcavities not to remove away by mechanical perturbations. Based on these problems, we attempted to propose an alternative method to stimulate a large number of EBs simultaneously. We fabricated cell-adhesive electrode substrate by attaching a PDMS sheet, featuring microcavity-array patterns, onto an ITO substrate (Fig. 5-b). PDMS is well used elastomeric material in cell engineering research because of its capability of easy processing, biocompatibility and cell-nonadhesiveness (Mata et al, 2005). PDMS is also an insulative material (Nam et al., 2006) and thus useful for insulator as the SU-8 structures used in section 2. By using the cell-adhesive electrode substrate with the PDMS sheet and plating P19 cells onto it, we attempted to obtain a large number of EBs and stimulate them. We also attempted to analyse gene expression changes after ensemble electrical stimulation of EBs by polymerase chain reaction (PCR) and electrophoresis Techniques.

The experimental procedures were described as follows. The microcavity-array pattern was fabricated in PDMS using soft lithography technique. First, column-array structures were fabricated as a master mold onto a standard glass substrate using SU-8 photoresist. SU-8 3050 was spin-coated onto a glass substrate (76 52 mm, Matsunami Glass) for 60 s at 1000

(a) Cell-nonadhesive electrode substrate using SU-8 photoresist.

(b) Cell-adhesive electrode substrate using PDMS elastomer.

40 Advances in Unconventional Lithography

It is widely recognized that intracellular calcium dynamics play important roles in regulation of cell differentiation processes (Dolmetsch et al., 1998; Yamada et al., 2007). Particularly, Spitzer et al. reported that spatio-temporal patterns of intracellular calcium transients regulated neuronal differentiation processes, such as extension of neurite and selection of neurotransmitter (Spitzer et al., 2004). Thus, inducing and controlling intracellular calcium transients in undifferentiated stem cells by applying electrical stimulation, which parameters are flexibly determined, is promising method to affect cell differentiation processes artificially. Optimizing parameters of electrical stimulation to induce desirable spatio-temporal patterns of intracellular calcium transients will be next

There are several problems, however, with the cell-nonadhesive electrode substrate using SU-8 thick photoresist. In the experiments, we manually transferred EBs one by one from 96 well culture plates into each microcavity using micropipettes. Furthermore, due to the cellnonadhesiveness of the electrode substrate by the MPC polymer coating, mild mechanical perturbations by moving the electrode substrate easily remove EBs from the microcavities. These problems make it difficult to stimulate more than 20 EBs simultaneously with the SU-8-based cell non-adhesive electrode substrates and to perform subsequent gene expression analysis and culture test after stimulation. On the basis of these disadvantages, we attempt to fabricate PDMS (poly(dimethylsiloxane))-based cell-adhesive electrode substrate to stimulate more number of EBs. The experimental procedures and present results for the cell-

**3. Electrical stimulation and gene expression analysis with cell-adhesive** 

of EBs by polymerase chain reaction (PCR) and electrophoresis Techniques.

The experimental procedures were described as follows. The microcavity-array pattern was fabricated in PDMS using soft lithography technique. First, column-array structures were fabricated as a master mold onto a standard glass substrate using SU-8 photoresist. SU-8 3050 was spin-coated onto a glass substrate (76 52 mm, Matsunami Glass) for 60 s at 1000

As described in section 2, it was difficult to stimulate a large number of EBs, over a hundred numbers, when using the cell-nonadhesive electrode substrate with SU-8 negative thick photoresist. This was because that the SU-8 structures were strictly integrated with ITO substrate and thus all surface of the electrode substrate became cell-nonadhesive after the MPC polymer coating of microcavity-array region (Fig. 5-a). Although the cell-nonadhesive surface was advantageous to handle EBs, maintain suspension culture conditions and trap EBs into the microcavities temporally, it was disadvantageous to fix EBs to the bottom surfaces of the microcavities not to remove away by mechanical perturbations. Based on these problems, we attempted to propose an alternative method to stimulate a large number of EBs simultaneously. We fabricated cell-adhesive electrode substrate by attaching a PDMS sheet, featuring microcavity-array patterns, onto an ITO substrate (Fig. 5-b). PDMS is well used elastomeric material in cell engineering research because of its capability of easy processing, biocompatibility and cell-nonadhesiveness (Mata et al, 2005). PDMS is also an insulative material (Nam et al., 2006) and thus useful for insulator as the SU-8 structures used in section 2. By using the cell-adhesive electrode substrate with the PDMS sheet and plating P19 cells onto it, we attempted to obtain a large number of EBs and stimulate them. We also attempted to analyse gene expression changes after ensemble electrical stimulation

step for controlling cell differentiation processes.

**microcavity-array electrode substrate** 

adhesive electrode substrate are summarized in a next section.

Fig. 5. Structures of the electrode sustrates for stimulating EBs

rpm with a thickness of 100 m. After exposure and development with the photomasks which had inverted patterns as used in section 2, column-array structures were formed on a glass substrate. In this experiment, 289 (17 17 matrix pattern) and 64 columns (8 8 matrix pattern) were fabricated for 200 and 500 m diameter microcavities, respectively. Before pouring PDMS, a thin layer of photoresist (OFPR-800, positive photoresist, Tokyo Ohka) with a thickness of 4 m was formed onto the substrate by spin-coating for 40 s at 2000 rpm and then baking for 30 min at 80 oC. The thin photoresist layer worked as a sacrifice layer to release a cured PDMS sheet from the substrate (Nam et al., 2006). Then, the mixture of PDMS pre-polymer and catalyst (10:1 ratio, Silpot 184, Dow Corning) was spin-coated on the substrate for 80 s at 2500 rpm and 40 s at 2000 rpm, for 200 and 500 m diameter microcavites, respectively. The PDMS coated substrate was put on a hotplate and then a premanufactured thick PDMS annulus (20 mm square and 3 mm thickness) which had a 15 mm diameter hole was immediately attached on the substrate. The PDMS layer and annulus

Toward the Precise Control of Cell Differentiation Processes by Using Micro and Soft Lithography 43

stimulation, a stranded cupper wire was attached to the substrate as described in section 2. Electrical pulses were chronically applied to the EBs with culturing them in an incubator after 1 day of cell plating (EB forming). In this experiment, a single biphasic pulse, same

To analyse the effects of chronic electrical stimulation on cell differentiation processes, reverse transcriptase-PCR (RT-PCR) analysis was carried out after electrical stimulation. First, extraction and purification of RNA were carried out by using a commercial RNA extraction kit (NucleoSpin RNA II kit, Nippon Genetics) from the EBs after applying electrical stimulation for 2 days. RNA of the EBs after induction of neuronal differentiation with retinoic acid was also extracted as the control sample. A 250 ng of total RNA was used for synthesis of first strand complementary DNA (cDNA) by reverse transcription for 80 min at 42 oC using a commercial kit (iScript Select cDNA Synthesis kit, Bio-Rad). Then, amplification of cDNA was carried out with 0.5 l of synthesized cDNA for 30 s at 94 oC and 1 min at 74 oC, for denaturation and primer extension, respectively. The primer information used in this experiment and primer annealing conditions were summarized in Table 1. The PCR products were analysed using an Experion automated electrophoresis system (Bio-Rad). Temperature controls for reverse transcription and PCR reactions were carried out

The Oct 3/4 gene is thought to play important roles in maintaining undifferentiated conditions and thus is widely used as a marker for undifferentiated stem cells (Ronser et al., 1990). The BMP-4 gene is mainly expressed in ectoderm, meanwhile it inhibit the neuronal differentiation. Thus, an expression level of the BMP-4 is transiently increased and then gradually decreased during neuronal differentiation (Wilson and Hemmti-Brivanlou, 1995). The Mash-1 gene is mainly expressed in neuronal progenitor cells and thus is used as a neuronal marker (Lo et al., 1991). The Wnt-1 gene is also used as a neuronal marker, particularly it inhibit glial differentiation and promote neuronal differentiation (Tang et al., 2002). The GAPDH (glyceraldehyde-3-phosphatedehydrogenase) gene is a widely-used housekeeping gene. These genes are also expressed and regulated in neuronal

Figure 7 showed a photograph of the cell-adhesive electrode substrate using a PDMS sheet and a phase-contrast image of the microcavity-array region (diameters of 200 m in this figure). SEM (scanning electron microscope) images of the SU-8 column-array mold and the corresponding PDMS microcavity-array structures were also shown in Fig. 8. The roundshape microcavity-array patterns as in section 2 were fabricated with fine reproducibility. The depth of the microcavities depended on the height of the column mold (100 m in this

experiment) and could be regulated by changing the experimental parameters.

conditions as in section 2, was applied at intervals of 30 min for 2 days.

using a C1000 thermal cycler (Bio-Rad).

Table 1. Primer sequences and reaction parameters for RT-PCR

differentiation processes of P19 cells (Bain et al., 1994).

were baked for an hour at 80 oC. After curing, the PDMS sheet was cut along the thick annulus using forceps and then the substrate was soaked in an acetone to solve the photoresist layer. The released PDMS sheet was rinsed with IPA and distilled water. The microcavity-array pattern electrode substrate was formed by attaching the PDMS sheet onto an ITO substrate (50 50 mm). The protocols for fabricating the microcavity-array electrode substrate using PDMS were shown in Fig. 6.

Fig. 6. Schematic of procedures for fabricating the microcavity-array electrode substrate using PDMS elastomer.

Methods for subculture of P19 cells were same as described in section 2. Proliferated P19 cells were collected and then replated onto the microcavity-array electrode substrates. Due to its cell-nonadhesive property of PDMS, P19 cells adhered only to the bottom surfaces of the microcavities. After several hours of plating, non-adhered cells were removed by washing the substrates with culture medium or PBS. P19 cells were proliferated within the microcavities and EBs were self-organizingly formed after 1 day in culture. For electrical

were baked for an hour at 80 oC. After curing, the PDMS sheet was cut along the thick annulus using forceps and then the substrate was soaked in an acetone to solve the photoresist layer. The released PDMS sheet was rinsed with IPA and distilled water. The microcavity-array pattern electrode substrate was formed by attaching the PDMS sheet onto an ITO substrate (50 50 mm). The protocols for fabricating the microcavity-array electrode

Fig. 6. Schematic of procedures for fabricating the microcavity-array electrode substrate

Methods for subculture of P19 cells were same as described in section 2. Proliferated P19 cells were collected and then replated onto the microcavity-array electrode substrates. Due to its cell-nonadhesive property of PDMS, P19 cells adhered only to the bottom surfaces of the microcavities. After several hours of plating, non-adhered cells were removed by washing the substrates with culture medium or PBS. P19 cells were proliferated within the microcavities and EBs were self-organizingly formed after 1 day in culture. For electrical

substrate using PDMS were shown in Fig. 6.

using PDMS elastomer.

stimulation, a stranded cupper wire was attached to the substrate as described in section 2. Electrical pulses were chronically applied to the EBs with culturing them in an incubator after 1 day of cell plating (EB forming). In this experiment, a single biphasic pulse, same conditions as in section 2, was applied at intervals of 30 min for 2 days.

To analyse the effects of chronic electrical stimulation on cell differentiation processes, reverse transcriptase-PCR (RT-PCR) analysis was carried out after electrical stimulation. First, extraction and purification of RNA were carried out by using a commercial RNA extraction kit (NucleoSpin RNA II kit, Nippon Genetics) from the EBs after applying electrical stimulation for 2 days. RNA of the EBs after induction of neuronal differentiation with retinoic acid was also extracted as the control sample. A 250 ng of total RNA was used for synthesis of first strand complementary DNA (cDNA) by reverse transcription for 80 min at 42 oC using a commercial kit (iScript Select cDNA Synthesis kit, Bio-Rad). Then, amplification of cDNA was carried out with 0.5 l of synthesized cDNA for 30 s at 94 oC and 1 min at 74 oC, for denaturation and primer extension, respectively. The primer information used in this experiment and primer annealing conditions were summarized in Table 1. The PCR products were analysed using an Experion automated electrophoresis system (Bio-Rad). Temperature controls for reverse transcription and PCR reactions were carried out using a C1000 thermal cycler (Bio-Rad).


Table 1. Primer sequences and reaction parameters for RT-PCR

The Oct 3/4 gene is thought to play important roles in maintaining undifferentiated conditions and thus is widely used as a marker for undifferentiated stem cells (Ronser et al., 1990). The BMP-4 gene is mainly expressed in ectoderm, meanwhile it inhibit the neuronal differentiation. Thus, an expression level of the BMP-4 is transiently increased and then gradually decreased during neuronal differentiation (Wilson and Hemmti-Brivanlou, 1995). The Mash-1 gene is mainly expressed in neuronal progenitor cells and thus is used as a neuronal marker (Lo et al., 1991). The Wnt-1 gene is also used as a neuronal marker, particularly it inhibit glial differentiation and promote neuronal differentiation (Tang et al., 2002). The GAPDH (glyceraldehyde-3-phosphatedehydrogenase) gene is a widely-used housekeeping gene. These genes are also expressed and regulated in neuronal differentiation processes of P19 cells (Bain et al., 1994).

Figure 7 showed a photograph of the cell-adhesive electrode substrate using a PDMS sheet and a phase-contrast image of the microcavity-array region (diameters of 200 m in this figure). SEM (scanning electron microscope) images of the SU-8 column-array mold and the corresponding PDMS microcavity-array structures were also shown in Fig. 8. The roundshape microcavity-array patterns as in section 2 were fabricated with fine reproducibility. The depth of the microcavities depended on the height of the column mold (100 m in this experiment) and could be regulated by changing the experimental parameters.

Toward the Precise Control of Cell Differentiation Processes by Using Micro and Soft Lithography 45

microcavities. Thus, the EBs were not easily remove away by mechanical perturbations. We confirmed that applying electrical pulses evoked intracellular calcium transients in the EBs as those in the experiments of section 2. We concluded that we could obtain a large number of size-controlled P19 EBs and stimulate them simultaneously by using the cell-adhesive

Fig. 9. Self-formed EBs within the microcavity-array substrate using PDMS. (a) the 200 m

After ensemble electrical stimulation of P19 EBs for 2 days using the cell-adhesive electrode substrate, we attempted to analyse the effects of electrical stimulation on cell differentiation processes of P19 cells by gene expression analysis using a RT-PCR method. Chronic electrical stimulation to P19 EBs was started to be applied after 1 day of cell plating and continued for 2 days with an interval of 30 min. The experimental setup for chronic electrical stimulation of EBs was shown in Fig. 10. A platinum wire was used as a

diamter pattern sample. (b) the 500 m diamter pattern sample.

counter electrode as in section 2.

microcavity-array electrode substrate.

Fig. 7. The cell-adhesive electrode substrate using PDMS (a) the outview of the subsrate. (b) the phase-contrast image of the microcavity-array region.

Fig. 8. SEM images of the microcavity-array structures. The diameter of each SU-8 column and microcavity is 200 m. (a) The SU-8 column-array mold for the microcavites. (b) The corresponding microcavity-array of PDMS.

Then, we plated P19 cells onto the electrode substrate and culture them. The results were shown in Fig. 9. The figure showed the microcavity-array regions of the 200 m diameter sample (Fig. 9-a) and the 500 m diameter sample (Fig. 9-b) after 2 days in culture. There were no adhesion and proliferation of P19 cells on the PDMS region. The P19 cells adhered to the bottom surfaces of the microcavities proliferated, aggregated within the microcavities and formed EBs. The characteristic feature of these EBs, different from the EBs in the experiments of section 2, was that they were tightly adhered to the bottom surfaces of the

Fig. 7. The cell-adhesive electrode substrate using PDMS (a) the outview of the subsrate. (b)

Fig. 8. SEM images of the microcavity-array structures. The diameter of each SU-8 column and microcavity is 200 m. (a) The SU-8 column-array mold for the microcavites. (b) The

Then, we plated P19 cells onto the electrode substrate and culture them. The results were shown in Fig. 9. The figure showed the microcavity-array regions of the 200 m diameter sample (Fig. 9-a) and the 500 m diameter sample (Fig. 9-b) after 2 days in culture. There were no adhesion and proliferation of P19 cells on the PDMS region. The P19 cells adhered to the bottom surfaces of the microcavities proliferated, aggregated within the microcavities and formed EBs. The characteristic feature of these EBs, different from the EBs in the experiments of section 2, was that they were tightly adhered to the bottom surfaces of the

the phase-contrast image of the microcavity-array region.

corresponding microcavity-array of PDMS.

microcavities. Thus, the EBs were not easily remove away by mechanical perturbations. We confirmed that applying electrical pulses evoked intracellular calcium transients in the EBs as those in the experiments of section 2. We concluded that we could obtain a large number of size-controlled P19 EBs and stimulate them simultaneously by using the cell-adhesive microcavity-array electrode substrate.

Fig. 9. Self-formed EBs within the microcavity-array substrate using PDMS. (a) the 200 m diamter pattern sample. (b) the 500 m diamter pattern sample.

After ensemble electrical stimulation of P19 EBs for 2 days using the cell-adhesive electrode substrate, we attempted to analyse the effects of electrical stimulation on cell differentiation processes of P19 cells by gene expression analysis using a RT-PCR method. Chronic electrical stimulation to P19 EBs was started to be applied after 1 day of cell plating and continued for 2 days with an interval of 30 min. The experimental setup for chronic electrical stimulation of EBs was shown in Fig. 10. A platinum wire was used as a counter electrode as in section 2.

Toward the Precise Control of Cell Differentiation Processes by Using Micro and Soft Lithography 47

genes during the differentiation processes of P19 cells following the application of retinoic

Then, the results for gene expression analysis of P19 EBs after application of chronic electrical stimulation using the microcavity-array electrode substrate were shown in Fig. 12. The figure showed the results of two samples for each 200 and 500 m diameter pattern.

Fig. 11. The gene expression patterns in P19 cells induced by retinoic acid under suspension cultures. U; undifferentiated cells. Agg; EBs in suspension culture. Numbers indicate culture

In the four samples used in this experiment, the expression of the Oct 3/4 gene was maintained and there was no distinct expression of the BMP-4, Mash-1 and Wnt-1 genes after electrical stimulation. Thus, in the present situation, we could not induce distinct differentiation processes of P19 cells from its undifferentiated state by electrical stimulation

In this section, we proposed an alternative microcavity-array electrode substrate with PDMS elastomer using micro and soft lithography technique. We could obtain hundreds of sizecontrolled EBs. The EBs were tightly adhered to the bottom surfaces of the microcavities, thus subsequent electrical stimulation and gene expression analysis could be stably carried out. However, applying electrical stimulation of EBs for 2 days could not induce changes in the gene expression patterns of P19 cells. One of the possible reasons was that an excessive

acid.

days.

and EBs size treatment.

Fig. 10. Chronic electrical stimulation of P19 EBs with the microcavity-array electrode substrate using a PDMS sheet. Electrical pulses were applied for 2 days with an interval of 30 min.

First, we analysed the gene expression of P19 cells induced with retinoic acid under suspension culture conditions for setting a criteria of the gene expression level of neuronal differentiation pathways in P19 cells. To initiate neuronal differentiation, P19 cells were plated into bacteria culture dishes ( 100 mm; Fisherbrand) and were allowed to aggregate with 2 106 cells/dish. During the induction period, -MEM containing 5 % FBS and 1 10-6 M all-trans-retinoic acid (Sigma-Aldrich) was used as the culture medium.The expression levels of 5 genes described above were shown in Fig. 11. The figure showed the gene expression patterns under five conditions of P19 cells; undifferentiated cells, EBs under suspension culture conditions with retinoic acid (each of 1 ~ 4 days in culture). The presence of white band indicated the expression of the relevant genes.

The expression of the GAPDH gene was confirmed in all conditions, indicating that PCR reactions in the experiment worked well. In undifferentiated P19 cells, distinct expression of the Oct 3/4 was confirmed, while there were no expression of the BMP-4, Mash-1 and Wnt-1 genes. Following the application of retinoic acid under suspension cultures, the expression of the Oct 3/4 rapidly decreased and disappeared. Alternatively, the BMP-4, Mash-1 and Wnt-1 genes showed elevated expression levels in the EBs. We confirmed the inhibition of undifferentiated associated gene and the expression of neuronal differentiation associated

Fig. 10. Chronic electrical stimulation of P19 EBs with the microcavity-array electrode substrate using a PDMS sheet. Electrical pulses were applied for 2 days with an interval of

of white band indicated the expression of the relevant genes.

First, we analysed the gene expression of P19 cells induced with retinoic acid under suspension culture conditions for setting a criteria of the gene expression level of neuronal differentiation pathways in P19 cells. To initiate neuronal differentiation, P19 cells were plated into bacteria culture dishes ( 100 mm; Fisherbrand) and were allowed to aggregate with 2 106 cells/dish. During the induction period, -MEM containing 5 % FBS and 1 10-6 M all-trans-retinoic acid (Sigma-Aldrich) was used as the culture medium.The expression levels of 5 genes described above were shown in Fig. 11. The figure showed the gene expression patterns under five conditions of P19 cells; undifferentiated cells, EBs under suspension culture conditions with retinoic acid (each of 1 ~ 4 days in culture). The presence

The expression of the GAPDH gene was confirmed in all conditions, indicating that PCR reactions in the experiment worked well. In undifferentiated P19 cells, distinct expression of the Oct 3/4 was confirmed, while there were no expression of the BMP-4, Mash-1 and Wnt-1 genes. Following the application of retinoic acid under suspension cultures, the expression of the Oct 3/4 rapidly decreased and disappeared. Alternatively, the BMP-4, Mash-1 and Wnt-1 genes showed elevated expression levels in the EBs. We confirmed the inhibition of undifferentiated associated gene and the expression of neuronal differentiation associated

30 min.

genes during the differentiation processes of P19 cells following the application of retinoic acid.

Then, the results for gene expression analysis of P19 EBs after application of chronic electrical stimulation using the microcavity-array electrode substrate were shown in Fig. 12. The figure showed the results of two samples for each 200 and 500 m diameter pattern.

Fig. 11. The gene expression patterns in P19 cells induced by retinoic acid under suspension cultures. U; undifferentiated cells. Agg; EBs in suspension culture. Numbers indicate culture days.

In the four samples used in this experiment, the expression of the Oct 3/4 gene was maintained and there was no distinct expression of the BMP-4, Mash-1 and Wnt-1 genes after electrical stimulation. Thus, in the present situation, we could not induce distinct differentiation processes of P19 cells from its undifferentiated state by electrical stimulation and EBs size treatment.

In this section, we proposed an alternative microcavity-array electrode substrate with PDMS elastomer using micro and soft lithography technique. We could obtain hundreds of sizecontrolled EBs. The EBs were tightly adhered to the bottom surfaces of the microcavities, thus subsequent electrical stimulation and gene expression analysis could be stably carried out. However, applying electrical stimulation of EBs for 2 days could not induce changes in the gene expression patterns of P19 cells. One of the possible reasons was that an excessive

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Fig. 12. The gene expression patterns in P19 cells after electrical stimulation for 2 days. #200 indicate the results in 200m diamter samples and #500 in 500 m diamter samples.
