Supporting Information
Microfluidic isolation of highly pure embryonic stem cells using feeder-separated co-culture system
Qiushui Chen, Jing Wu, Qichen Zhuang, Xuexia Lin, Jie Zhang and Jin-Ming Lin*
Department of Chemistry, Beijing Key Laboratory Microanalysis and Instrumentation, Tsinghua University, Beijing 100084, P.R. China
Corresponding to .
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1. Device fabrication
Fabrication and Assembly of PDMS porous membrane for 3D microdevice was conducted by using standard soft lithography and replica molding techniques.1 SU-8 posts were fabricated on a silicon wafer. After PDMS prepolymer was spin-coated on the wafer and cured at 65 ℃ for 3.0 min, the top layer with three parallel microchannes was aligned on the wafer and the residual surface was blocked by PDMS prepolymer for cleaning the wafer (Fig. S-1A). This process is critical to re-use the mold. After curing for 2 hours, the porous membrane was peeled with the top layer with microchannels. A bottom layer with 4 holes at the end of each microchannel was bonded with the top layer. The fabricated microdevice was put in the oven at 80 ℃ for 1 hour. Cut off the edges, the fabrication of PDMS porous membrane assembled 3D-device was finally finished (Fig. S-1B).
Figure S1. A) Photograph of the top PDMS layer alignment on a spin-coated PDMS prepolymer. B) Photograph of a PDMS porous membrane assembled 3D divice.
2. Microfluidic cell co-cultures
The mouse embryonic fibroblasts (mEFs) and mouse embryonic stem cells (mES cells) were separately co-cultured on the both sides of PDMS porous membrane in the 3D-device. First, 0.1% gelatin in H2O was introducing into microchannels for 30 min to keep the microchambers with good biocompatibility (Fig. S-2A). The microdevice was put on the UV irradiation for sterility conditions. Second, the DMEM media was injected into the microchambers to replace the 0.1% gelatin, resulting in cellular microenvironment (Fig. S-2B). After 30 min incubation, shutting down the inlet 1 using short PE tubing, a concentration of 107 cells ml-1 mEFs suspension was introduced into the bottom microchannels (inlet 2). The microchips were upturn to grow the mEFs onto PDMS porous membrane (Fig. S-2C). After culture 24 h, the mEFs was adherent to the PDMS porous membrane. Then, re-introducing the cell media in inlet 2 and shutting down inlet 2, the mES cells was injected into the top microchannels for co-cultures (Fig. S-2D). The media both in the two microchannels was supplied every 12 hours. And the co-culture of the mES cells and mEFs was observed under the microscope every day. Fig. S-2E illustrates the design of the microdevice with the top and bottom microchannels. The length is 10 mm and the width is 1.0 mm.
Figure S2. Illustration of microfluidic cell co-cultures of the mEFs and mES cells, and design of microdevice with the sizes of microchannels.
3. Stem cell co-culture and recovery
Traditionally, mouse embryonic stem (mES) cells are co-cultured with the feeder layers (mouse embryonic fibroblasts (mEFs)) to supply the essential intrinsic regulators and environment cues. In converntinal stem cells co-culture method (Fig. S-3A), the feeder layers are commonly needed to γ-irradiation or treatment of with mitomycin C before or after stem cells culture. A cells mixture will be recovered though the inactivated mEFs will be dead or apoptosis. Additional purification is commonly required before using the stem cells. In our method (Fig. S-3B), the co-culture of mEFs and mES cells were techniquely isolated by PDMS porous membrane in the microfabricated device, resulting in directly recovering pure stem cells, as well as allowing to use the normal mEFs without inactivation process. This simple and effective method is especially important to the human stem cells culture.
Figure S3. The illustration of conventional stem cells coculture on culture dish and microfabricated coculture platform on PDMS porous membrane-assembled 3D-microdevice.
4. Observation of RFP expressions on mEFs and pluripotent biomarker expressions
To observe the mitosis of normal mEFs on PDMS device, red fluorescence protein (RFP) expressing-mEFs were cultured on microchannels for several days. The growths of RFP-mEFs were observed and imaged under a fluorescence microscopy every day (Fig. S-4). We found that the mEFs still survived after 21 days. To compare the stem cell pluripotencies of the mES cells on feeder-separated system with that on direct contact co-cultures, the mES cells were directly co-cultured with the inactivated mEFs without PDMS porous membrane separation. After 5 days culture, the expressions of Oct-4 antibody and ALP positive level on mES cells were examined as a control (Fig. S-5). The quantification was conducted by multiple samples to control for differences in staining intensity. The same areas were used to calculate the staining intensities by control for the differences.
Figure S4. Photograph of mEFs on microfluidic chip from day 1 to day 7. The red fluorescence from RFP-mEFs was imaged under a fluorescence microscopy (Leica DMI 4000 B).
Figure S5. The expression of Oct-4 antibody (A) and ALP positive (B) on the mES cells by direct contact co-cultures.
Figure S6. Characterization of self-renewing pluripotency by Nanog-IgG/anti-IgG-FITC staining after 5 days culture on microfluidic system. Scale bar: 100 µm.
Reference
[1] Wei, H.et al. Particle sorting using a porous membrane in a microfluidic device. Lab Chip 11, 238-245 (2011).
Movie S1 shows 3D morphology of mES cells and mEFs co-cultures after 5 days by laser confocal fluorescence microscopy. The blue was the cells stained with Hoechst 33342, and the red was the expression of RFP from mEFs.