Supplementary MaterialsAdditional file 1: Is supplementary materials and methods: Table S1 presenting the sequence of quantitative PCR primers for MSCs, Figure S1 showing the microfluidic device with larger culture chamber used for the study, Figure S2 showing the processes of proliferation (A) and hepatic differentiation (B) of MSCs in the culture dish and microfluidic device, Figure S3 showing the growth curve of human MSCs cultured in the microfluidic device and culture dish from 0 to 9?days, Figure S4 showing the comparative expression of surface markers in mouse MSCs cultured in static culture dish and microfluidic device at day 0?and day time 3, and Shape S5 displaying the simulation of tradition medium diffusion inside a group cultural chamber. demonstrated an uneven movement profile inside a group cultural chamber. The parameters and dimension of flow field were predicated on a previous study . (DOCX 979 kb) 13287_2016_371_MOESM1_ESM.docx (979K) GUID:?878AC417-4BF5-47E4-A24B-DB69F7EB9CFC Extra file 2: Is Video 1 showing the movie of air bubble removal through the cell culture chamber from the microfluidic device. polydimethylsiloxane, polymethyl methacrylate The microfluidic gadget was made to possess a tradition chamber sizing of 10?mm??40?mm??350?m (width??size??height), having a tradition part of 400?mm2. These devices was constructed in five levels (Fig.?1) comprising a lower coating of a tradition substrate, together with an intermediate coating formed by two patterned cup and two patterned polydimethylsiloxane (PDMS) membranes (Sylgard 184; DowCorning, Midland, MI, USA), with a high coating of polymethyl methacrylate (PMMA), including three adaptors for creating the vacuum, moderate inlet, and wall socket. The PDMS membranes were fabricated and prepared based on the producers instructions. These PDMS membranes had been patterned with a CO2 laser beam machine as well as the cup was patterned by an ultrasonic drilling machine (LUD-1200; Lapidary & Sonic Corporations, Taipei, Taiwan). The substrate was created from a polystyrene dish (PS) (25?mm??75?mm) lower from a tradition dish utilizing a CO2 laser beam. Finally, the patterned glass and PDMS were bonded together by a plasma treatment Y15 system (PX-250; Nordson, Westlake, OH, USA) and stuck to the PMMA Y15 adaptor with double-sided tape to completely assemble the microfluidic device. The microfluidic device, which included a cell culture chamber, 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 negative 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 32,400?mm2, as shown in Additional file 1: Figure 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, another outlet linked to the waste materials tube, and vacuum pressure (Fig.?2a, ?,b).b). These devices was linked to a time-lapse microscope for Y15 real-time observation, related to the transparency of these devices chamber. The temperatures controller Y15 ensures a well balanced temperatures of the lifestyle chamber. The syringe pump provided clean moderate in to the functional program, as well as the time-lapse microscope allowed real-time observation from the mobile morphology of MSCs during hepatic differentiation. Open up in another window Fig. 2 Assemblage of the entire microfluidic program for cell time-lapse and lifestyle observation of MSC hepatic differentiation. a Real microfluidic program for cell lifestyle. shows the current presence of a thermal sensor mounted on the microfluidic gadget for temperatures legislation. b Developed microfluidic program. The lifestyle system 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 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). These were Rabbit Polyclonal to Catenin-beta expanded and subcultured when reaching 80C90?% confluence. Confluent cells had been detached with 0.1?% trypsin-EDTA (Gibco Invitrogen), rinsed with PBS twice, and centrifuged at 200??for 5?a few minutes. Cell pellets were rinsed with PBS and resuspended in lifestyle moderate double. The cells had been re-seeded at a thickness of 8??103 cells/cm2 to hepatic differentiation beneath the same culture conditions preceding. The culture medium was replaced 3 x a complete week. All cultures had been preserved at 37?C within a humidified atmosphere containing 5?% CO2. Proliferation and hepatic differentiation of MSCs in the microfluidic gadget The techniques for proliferation and hepatic differentiation of MSCs in the lifestyle dish as well as the microfluidic gadget are defined in the supplementary materials (Additional document 1: Body S2). Hepatic differentiation was initiated using the two-step process we reported  previously. Mouse MSCs.