Figure 1.
(A) Layout of the integrated microfluidic device mainly composed of an upstream CGG and a downstream cell culture module. (B) The microfluidic channels in the upstream dilution module generate several concentrations of the stimulus by continuous flow diffusive mixing of adjacent laminar flow streams in the serpentine channels. (C) Three chambers were connected to every channel of downstream culture module. Solutions would diffuse into the chambers by osmosis till the concentration of the mixture in the chambers is uniform to the connected channel. (D) Microfluidic device operation. A syringe pump was connected to a microfluidic chip by teflon tubes. It drived the solutions into the chip at a flow speed of 0.1 ml/min continuously in laminar mode, meanwhile, waste liquid was collected.
Figure 2.
FACS analysis for BMSCs surface markers.
The cells were indicated positive for CD29 (99.17%) and CD90 (98.95%), but negative for CD45 (0.76%) after P3.
Figure 3.
Multilineage potential and morphological change of BMSCs after differentiation.
BMSCs (P3) differentiated into chondrocytes (Toluidine blue staining: A), osteocytes (Alizarin red staining: B) and adipocytes (Oil Red staining: C). (D) Undifferentiated BMSCs showed a flat, fibroblast-like morphology. (E) After differentiating into SC-like cells, the cells changed to a bipolar, spindle-like shape. Scale bar: 50 µm.
Figure 4.
Immunocytochemistry for S100β immunopositive cells on-chip.
(A) The cells in every chamber were stained with S100β antibody (green) and counterstained with DAPI (blue). Scale bar: 50 µm. (B) Among the combinations of the induce factors, the 6th group (4.00 µM FSK and 250.00 ng/ml HRG) indicated the largest number of S100β immunopositive cells. Control vs. the 5th group (5.00 µM FSK and 200.00 ng/ml HRG): *p<0.05. (C) The average expression of S100β in per immunopositive cell reflected by normalized fluorescent intensities was at the highest level in the same group. Control vs. the 5th group: *p<0.05. The normalized fluorescent intensities per cell were determined by the total fluorescent intensity in a field divided by the number of immunopositive cells in that field. All the experiments were repeated at least three times.
Figure 5.
Immunocytochemistry for p75NTR immunopositive cells on-chip.
(A) Cells cultured in chambers were stained with p75NTR antibody (red) and counterstained with DAPI (blue). Scale bar: 50 µm. (B) In consistent with S100β expression, cells cultured in the same combination of induces factors showed the largest number of p75NTR immunopositive cells. Control vs. the 5th group: *p<0.05. (C) The normalized fluorescent intensity per immunopositive cell was at the maximal gray scale in the same group. Control vs. the 5th group: *p<0.05. All the experiments were repeated at least three times.
Figure 6.
DRG neurons co-cultured with cells of the four groups.
(A–E) Immunocytochemical staining for βIII-tubulin (FITC) to show neurites sprouting from DRG neurons after co-culture for 24 h with basal media (control, A), undifferentiated BMSCs (B), SC-like cells cultured in conventional differentiate media (C), SC-like cells cultured in optimal differentiate media (D) and Schwann cells (E). Scale bar: 100 µm. (F–H) Three independent parameters measured: percentage of DRG neurons sprouting neurites (F), length of longest neurite (G) and total neurite density (H). Data were listed as mean ± S.E.M. Control vs. group A and basal media: *p<0.05, **p<0.01; Control vs. group B: #p<0.05, ##p<0.01.
Figure 7.
Assessment of neurotrophins production.
ELISA quantification of BDNF (A), NGF (B) and NT-3 (C) secreted by the cells of the four groups. Data were listed as mean ± S.E.M. Control vs. group A: *p<0.05, **p<0.01; Control vs. group B: #p<0.05, ##p<0.01.
Table 1.
The concentrations between FSK and HRG in the cell chambers integrated with ten channels.