The device was assembled in five layers (Fig.?1) consisting of a lower layer of a culture substrate, on top of an intermediate layer formed by two patterned glass and two patterned polydimethylsiloxane (PDMS) membranes (Sylgard 184; DowCorning, Midland, MI, USA), with a top layer of polymethyl methacrylate (PMMA), including three adaptors for producing the vacuum, medium inlet, and store. uneven flow profile in a circle cultural chamber. The dimension and parameters of flow field were based on a previous study . (DOCX 979 kb) 13287_2016_371_MOESM1_ESM.docx (979K) GUID:?878AC417-4BF5-47E4-A24B-DB69F7EB9CFC Additional file 2: Is Video 1 showing the movie of air bubble removal from the cell culture chamber of the microfluidic device. polydimethylsiloxane, polymethyl methacrylate The microfluidic device was designed to have a culture chamber dimension of 10?mm??40?mm??350?m (width??length??height), with a culture area of 400?mm2. The device was assembled in five layers (Fig.?1) consisting of a lower layer of a culture substrate, on top of an intermediate layer formed by two patterned glass and two patterned polydimethylsiloxane (PDMS) membranes (Sylgard 184; DowCorning, Midland, MI, USA), with a top layer of polymethyl methacrylate (PMMA), including three adaptors for producing the vacuum, medium inlet, and store. The PDMS membranes were prepared and fabricated according to the manufacturers instructions. These PDMS membranes were patterned by a CO2 laser machine and the glass was patterned by an ultrasonic drilling machine (LUD-1200; Lapidary & Sonic Enterprises, Taipei, Taiwan). The substrate was made from a polystyrene plate (PS) (25?mm??75?mm) cut from a culture dish using a CO2 laser. Finally, the patterned glass and PDMS were bonded together by a plasma treatment system (PX-250; Nordson, Westlake, OH, USA) and stuck to the PMMA adaptor with double-sided tape to completely assemble the microfluidic device. The microfluidic device, which included a cell culture chamber, GS-9901 a vacuum, and air bubble trap regions, was placed on top of the PS culture substrate. The function of the vacuum region was to seal the culture substrates within the microfluidic device by unfavorable pressure. The pressure applied for sealing is about 85?mmHg. For future large-scale studies, the culture chamber can be further scaled up (up to now, its maximal culture area is usually 32,400?mm2, as shown in Additional file 1: Determine S1). In addition, the device was sterilized by -ray radiation before the experiments. The assembled microfluidic culture system included the actual microfluidic device with a thermal sensor and regulator, a syringe pump, an inlet connecting the syringe for culture medium injection, a separate outlet connected to the waste GS-9901 tube, and a vacuum (Fig.?2a, ?,b).b). The device was connected to a time-lapse microscope for real-time observation, attributed to the transparency of the device chamber. The heat controller ensures a stable heat of the culture chamber. The syringe pump supplied new medium into the system, and the time-lapse microscope allowed real-time observation of the cellular morphology of MSCs during hepatic differentiation. Open in a separate window Fig. 2 Assemblage of the complete microfluidic system for cell culture and time-lapse observation of MSC hepatic differentiation. a Actual microfluidic system for cell culture. shows the presence of a thermal sensor attached to the microfluidic device for heat regulation. b Developed microfluidic system. The culture system GS-9901 including the designed microfluidic device consists of a temporal sensor, a syringe pump, a heat controller, one inlet connecting the syringe unto the device, one outlet connecting waste tube, and a vacuum. polydimethylsiloxane Cultivation of MSCs MSCs were harvested from the bone marrow of postnatal 7-week-old C57BL/6?J mice (National Laboratory Animal Center, Taipei, Taiwan). Approval for the experiment was obtained from the Taipei Veterans CACNB4 General Hospital Institutional Animal Care and Use Committee (IACUC) regarding the use of animals prior to commencement of the experiments. For maintenance and culture expansion, MSCs were maintained in Dulbeccos altered Eagles medium with 1000?mg/L glucose (LG-DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10?% fetal bovine serum (FBS; Gibco Invitrogen, Carlsbad, CA, USA), 100 models/ml penicillin, 100?g/ml streptomycin, 2?mM?l-glutamine (Gibco Invitrogen), 10?ng/ml basic fibroblast growth factor (bFGF; Sigma-Aldrich), and 10?ng/ml epidermal growth factor (EGF; R&D Systems, Minneapolis, MN, USA). Cells were seeded at a density of 3??103 cells/cm2 (30C40?% confluence). They were subcultured and expanded when reaching 80C90?% confluence. Confluent cells were detached with 0.1?% trypsin-EDTA (Gibco Invitrogen), rinsed twice with PBS, and centrifuged at 200??for 5?minutes. Cell pellets were rinsed twice with PBS and resuspended in culture medium. The cells were re-seeded at a density of 8??103 cells/cm2 prior to hepatic differentiation under the same culture conditions. The culture medium was replaced three times a week. All cultures were maintained at 37?C in a humidified atmosphere containing 5?% CO2. Proliferation and hepatic differentiation of MSCs around the microfluidic device The procedures for proliferation and hepatic differentiation of MSCs.