These days, it would not be enough to mimic the physical and biological properties of the original tissue, because different cells possess different physical properties such as stiffness or diffusion. This thesis indicates the development of three-dimensional microfluidic devices for cellular studies based on applications such as high throughput cell-encapsulated bead culture and cell-based assay. With microfluidics, we can generate cell-encapsulated microgel, which may be possible to observe cell morphology or drug testing due to high diffusion compared to bulk size hydrogel.
Microfluidics has a lot of advantages such as low cost, low consuming reagent, fast analysis and portable device and so on. I used this platform such as microfluidic chip to generate cell-encapsulated beads of uniform size. In Chapter III, it is Generated macrophage-encapsulated beads using microfluidic chip and observed cell morphology at different stiffness and stimulated protein such as LPS (lipopolysaccharide).
In chapter V is that the generation of breast cancer such as MDA-MB-231, MCF-7, SK-BR-3 has encapsulated beads and observed the morphology of cells from different stiffness and two types of chemotherapy such as paclitaxel and cisplatin. Finally, I presented hollow fiber to mimic blood vessels using endothelial cells and observed cell morphology and viability in Chapter VI.
General Introduction
Squeezing
Dripping
Jetting
Microfabrication of Microfluidic chip formed Double Flow focusing
- Mold Fabrication through MEMs Process
- Microchannel Fabrication using PDMS
Introduction
- ECM structure and Hydrogel Biomaterials
- ECM structure
- ECM property (Mechanotransduction)
- Hydrogel biomaterials
- Reference
- Introduction
- Materials and Methods
- Fabrication of PDMS microfluidic flow-focusing device
- Determination of droplet concentrations
- Synthesis of photo-crosslinkable gelatin
- Mechanical properties of hydrogels
- Fabrication of cell-laden microgels
- Fabrication of in vitro multi-tissue model
- In vitro evaluation
- Results and Discussion
- Physical properties of microgels
- Biocompatibility of macrophage-laden microgels
- Induction of macrophage differentiation in microgels
- In vitro multiplex tissue model
- Conclusion
- Reference
Microgel cell viability and proliferation in C2 (Aq1/Aq2=9%/12%) (a) Microscopic (top) and fluorescence (bottom) images of microgel-encapsulated macrophages taken at different times. Microscopic (top) and fluorescence (bottom) images of microgel-encapsulated macrophages taken at different times. Differentiation of macrophages in microgels. a) Microscopic images of macrophages in different microgels treated with LPS at day 7 (scale bar: 50 μm).
Microscopic images of macrophages encapsulated in MGel microgels at different concentrations (C1, C2 and C3) treated with LPS to induce Mϕ polarization (Scale bar: 50 μm). Microscopic images of macrophage cells cultured on the surfaces of MGel hydrogels at different concentrations (C1, C2 and C3) prepared separately to evaluate the effects of culture conditions (2D vs. 3D) on macrophage proliferation (scale bar: 200 μm). Macrophages were treated with LPS before embedding (untreated macrophages were used as control). d) Magnified view of macrophages in a fibroblast.
The macrophages were treated with LPS before recording (untreated macrophages were used as control). There was a significant decrease in the cell density of hepatocarcinoma surrounding the macrophage (highlighted area). c) A magnified view of the macrophages in the hepatocarcinoma tissue over time (scale bar: 50 µm).
Combined effects of co-culture and substrate mechanics on 3D
Fabrication of a microfluidic device
Immunocytochemistry
Determination of droplet concentrations
Synthesis of methacrylic gelatin (MGel)
Fabrication of cell-laden microgels
In vitro evaluation
- Viability and proliferation
- Immunostaining
Results and Discussion
- Effect of microgel mechanics on tumor spheroid formation
- Effect of co-culture on tumor spheroid formation
- Combined effect of co-culture and microgel mechanics on tumor spheroid formation
Conclusion
Cell subtype-dependent generation of breast tumor spheroids within
- Materials and Methods
- Microfluidic fabrication of cell-laden microgels
- In vitro evaluation of spheroids in microgels
- Chemotherapeutic screening
- Results and Discussion
- MDA-MB-231
- MCF-7 and SK-BR-3
- Chemotherapeutic response of spheroids in microgels
- Conclusion
- Reference
Microscopic images of MCF-7 cells cultured on MGel hydrogels with varying rigidity (scale bar: 200 um). Cytotoxicity of different concentrations of chemotherapeutic agents, paclitaxel and cisplatin, against 2D monolayer cultures of (a, d) MDA-MB-231, (b, e) MCF-7, and (c, f) SK-BR-3 cells, measured on day 1 and 3 of exposure. Solid and dashed lines represent the viability of monolayer cultures of MDA-MB-231 cells exposed to the same concentration on day 1 and 3, respectively.
Solid and dotted lines represent the viability of monolayer cultures of MCF-7 cells exposed to the same concentration on days 1 and 3, respectively. Fluorescent imaging of live (green) and dead (red) SK-BR-3 cells from spheroids in microgels with varying stiffness (C2-C5), taken after 1 day of exposure to different concentrations of (a) paclitaxel and (b) cisplatin (scale bar: 50 µm). Solid and dotted lines represent the viability of monolayer cultures of SK-BR-3 cells exposed to the same concentration on days 1 and 3, respectively.
Facile generation hollow fiber to mimic blood vessel using human
Materials and Methods
- Microgel using Semi IPN and IPN structure
- In vitro evaluation of Gels using Semi IPN and IPN structure
- Viability and proliferation
- Facile generation of cell-laden hollow fiber
Results and Discussion
- Physical properties of Microgel using Semi IPN and IPN structure
- Elastic modulus and swelling ratio
- SEM structure of IPN structure
- In vitro evaluation of Microgels using Semi IPN and IPN structure
- Viability and proliferation
- Effect of Sodium Citrate (Removing Alginate)
- Facile generation of cell-laden hollow fiber
This scheme represents overall experiments. a) Alginate and MGel mixed Ca2+ as a physical crosslink using Alginate (Semi-IPN) then chemical crosslink by UV exposure using MGel (IPN). In this study, we investigated several with stiffness gel and compared with semi-IPN or IPN. However, when UV is not exposed to the gel, which can maintain semi-IPN formation (Figure 1a).
Modulus of elasticity and swelling ratio. a) and (c) Moduli of elasticity (E) and swelling ratios (Q) of alginate 0.5% and MGel Semi-IPN and IPN mixtures (b) and (d) Moduli of elasticity (E) and swelling ratios (Q) of alginate 1 % and MGel mixed Semi-IPN and IPN. In Vitro Evaluation of Microgels Using Semi IPN and IPN Structure 6.3.3.1 Viability and Proliferation 6.3.3.1 Viability and Proliferation. The viability of 3T3 cells encapsulated in microgels with different stiffnesses was assessed at different time points (Figure 4a and 4b) with or without UV exposure to make a Semi-IPN or IPN structure.
At higher concentrations of MGel, cell viability increased regardless of Semi-IPN or IPN structure. Representative microscopic (left) and fluorescent (right) images of microgel-encapsulated 3T3 taken at different times up to 7 days. Cells were fluorescently stained to identify live (green) and dead (red) cells. a) Semi-IPN structure (b) IPN structure (c) and (d) Normalized number of living cells (Nt/N0) in the microgel was measured over time (Nt: number of living cells at time, t, N0: number of initial living cells cells) Semi-IPN and IPN. e) The graph in panels (c) and (d) was fitted with a power law model to obtain the spreading rate (kP).
Scheme of generation of hollow fiber using alginate and MGel with HUVECs outer phase and inner phase 0.05M calcium chloride, 10% FBS, 25mM HEPES. We performed two types of blood vessels such as Semi-IPN or IPN structure with or without UV exposure. Then we cultured for up to 7 days and observed cell viability by means of live/dead assay.
Cells were fluorescently stained to identify live (green) and dead (red) cells. a) Half-IPN structure (b) IPN structure. At low stiffness with hydrogels, low cell viability is observed regardless of the semi-IPN or IPN structure. Under all conditions, cell-encapsulated microgels did not propagate in semi-IPN due to the very low modulus of elasticity.
Conclusion
