Research ArticleCELL BIOLOGY

In vivo–mimicking microfluidic perfusion culture of pancreatic islet spheroids

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Science Advances  27 Nov 2019:
Vol. 5, no. 11, eaax4520
DOI: 10.1126/sciadv.aax4520
  • Fig. 1 Spheroid-based microfluidic perfusion culture of pancreatic islets to mimic the in vivo environment.

    (A) Clusters of endocrine cells dispersed throughout the exocrine acini in native pancreas form islets of Langerhans. A dense vascular network exists within islets and facilitates efficient nutrient supply and adequate responses to glucose stimulation by diffusion through extracellular spaces with interstitial level of flow. Insulin-producing β cells are tightly connected via adherent junctions to coordinate hormone release and maximize their function. (B) Microchip-based engineering of islet spheroids with perfusion system is reconstituted to mimic in vivo environment. Microwell arrays are homogeneously shaped and distributed, facilitating the formation of uniform-sized spheroids and enhancing cell-cell interactions. The flow chip provides continuous supply of nutrient and oxygen and removal of metabolic waste products with interstitial levels of slow flow. (C to E) Schematics of experimental setup. (C) Isolated intact islets from Sprague-Dawley rats were dispersed into single islet cells and seeded to the inlet of PDMS-based microfluidic chip. The outlet was connected to the coiled tube of an osmotic micropump. (D) The osmotic pump was dipped into the polyethylene glycol (PEG) solution to generate the main driving power of the system. D.W., distilled water. (E) The dissociated cells including islet cells and iECs aggregate and form compact spheroids in concave microwells. (F) Pancreatic islet cells were cultured in microfluidic chips under three different conditions: (i) static condition without micropump, (ii) dynamic I condition with a flow rate of 8 μl/hour, and (iii) dynamic II condition with a flow rate of 25 μl/hour. Both fluidic conditions (dynamics I and II) are in the range of in vivo interstitial velocities.

  • Fig. 2 Survival of iECs from islets under dynamic culture.

    (A) Morphology of islet spheroids within concave microwells and survival of iECs over time when cultured in static or dynamic culture conditions. Scale bars, 100 μm. (B and C) Detection of endothelial cell–specific markers in iECs under dynamic culture. (B) XZ image of a spheroid within a well containing expanded iECs. Staining for islet endocrine cells (insulin; red), endothelial cells (vWF; green), and cell nuclei [4′,6-diamidino-2-phenylindole (DAPI); blue] is shown. (C) XY, YZ, and XZ projection images of expanded iECs (vWF; green) on chips and XY images with Z-scan series. (D) Shear-activated expansion of iECs on flat channels. Morphology of iECs in different culture groups (static and dynamics I and II), immunofluorescent images (insets; insulin, red; vWF, green; DAPI, blue), and quantification of cell area on flat channels for 14 days are shown. Scale bar, 200 μm. The data are expressed as the mean ± SD (n = 12; ***P < 0.001 versus other groups at the same time points). (E) Survival of iECs within islet spheroids under diffusion-dominant microenvironment. Immunostaining of cross-sectioned islet spheroids cultured for 14 days under different culture conditions (vWF, green; DAPI, blue) is shown. Scale bar, 100 μm. The ratio of vWF+ cells to nuclei of sectioned spheroids in static and dynamic I and II groups is shown. The data are expressed as the mean ± SD (n = 12; ***P < 0.001 versus dynamic groups). n.s., not significant.

  • Fig. 3 Improved viability and function of islet spheroids in dynamic culture compared with those in static culture.

    (A and B) Cell viability in islet spheroids under static and dynamic (I and II) conditions on days 7 and 14. (A) LIVE/DEAD assay showing live cells in green and dead cells in red. Scale bars, 100 μm. (B) Quantification of LIVE/DEAD assay results. The data are expressed as the mean ± SD (n = 17; **P < 0.005 and ***P < 0.001 versus dynamic groups). (C) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of proapoptotic genes, Fas and Bax, and an antiapoptotic gene, Bcl-2, in intact islets and islet spheroids under static and dynamic conditions in the devices over 14 days of culture. Gene expression in each group was calculated relative to the 18S rRNA expression and normalized to levels from intact islets at day 7. The data are expressed as the mean ± SD (n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001 versus other groups at the same time point). (D) Mathematical simulation of glucose concentration consumed by islet spheroids in microfluidic devices under static and dynamic I and II conditions. It was assumed that the initial concentration of glucose in the medium is 11.1 mol/m3 with a diffusion coefficient of 580 μm2/s and a consumption rate of 0.267 mol/m3 per second. (E and F) Immunofluorescent analysis of islet spheroids for insulin and E-cadherin on days 7 and 14. (E) Confocal z-stacked and cross-sectioned images of islet spheroids in different culture conditions (insulin, red; E-cadherin, green; DAPI, blue). Scale bars, 50 μm. (F) Ratio of insulin or E-cadherin to nuclei in three groups (left, z-stacked; right, sectioned). The data are expressed as the mean ± SD (n = 12; **P < 0.005 and ***P < 0.001 versus dynamic groups).

  • Fig. 4 Abundant microvilli and tight junctions in islet spheroids in controlled dynamic flow conditions.

    (A, C, and E) Scanning electron microscopy images of both exterior and interior of islet spheroids under static or dynamic conditions. Scale bars, 10 μm. (B and D) Total length of microvilli per field (625 μm2) was measured in each group, as described in Materials and Methods. The data are expressed as the mean ± SD (n = 8; **P = 0.0035; ***P < 0.001 versus dynamic group). (A to D) Magnified surface images show that only flow-exposed islet spheroids maintained abundant microvilli (arrowheads) on day 7. After 2 weeks, tight cell-cell junctions existed only in spheroids under dynamic I condition, whereas those under dynamic II condition lost tight connections. Both loss of tight junctions and microvilli are observed under fast fluid flow of 200 μl/hour in the device. (E) Inner structures of spheroids under static or dynamic conditions on day 7.

  • Fig. 5 Improved glucose responsiveness of islet spheroids in controlled dynamic flow conditions.

    (A) GSIS assay at low (2.8 mM) and high (16.7 mM) glucose and (B) stimulation index (SI) values. The amount of insulin secreted from islet spheroids in response to different glucose concentrations was measured after incubation for 1 hour with either low or high glucose. SI was calculated by dividing insulin concentrations at high and low glucose. The data are expressed as the mean ± SD (n = 5 for static and dynamics I and II and n = 3 for fast flow; *P < 0.05, **P < 0.005, and ***P < 0.001 versus dynamic groups at the same time point).

  • Fig. 6 Long-term maintenance of islet characteristics and application to in vitro drug testing.

    (A) Comparison of islet culture conditions; conventional suspension culture of isolated intact islets and microfluidic perfusion culture of reconstituted islet spheroids. For the following experiments, intact islets with the conventional method and islet spheroids with static and dynamics I and II were retrieved after culturing for 7 and 14 days. One-day cultured fresh intact islets were used as a reference. (B to E) qRT-PCR analysis showed higher expression of islet-specific genes in dynamic culture. Gene expression of (B) β cells (insulin, Pdx1, and Glut2), (C) α cells (glucagon), (D) nonislet cells including endothelial (Pecam) and neural (Tubb3) cells, and (E) islet ECM proteins (collagen IV and I). Gene expression in each group was calculated relative to the 18S rRNA expression and normalized to levels from intact islets at day 7. The data are expressed as the mean ± SD (n = 3; *P < 0.01, **P < 0.005, and ***P < 0.001 versus other groups at the same time point). (F and G) Drug efficacy testing on islet spheroids at day 7 using (F) tolbutamide and (G) GLP-1 with different dosages. The data are expressed as the mean ± SD (n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001, significantly above baseline of each group). (H and I) Drug toxicity was evaluated on islet spheroids at day 7 by culturing for additional 4 days under static or dynamic conditions with culture medium containing rapamycin at different concentrations (0, 200, and 400 nM). (H) Islet viability (live cells, green; dead cells, red) and (I) glucose responsiveness upon drug exposure for 4 days. Quantified viability results are represented as line plots. Scale bars, 100 μm. The data are expressed as the mean ± SD (n = 3 for SI and n = 15 for viability; *P < 0.05 and ***P < 0.001, significantly below baseline of each group).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaax4520/DC1

    Fig. S1. Size distribution and DNA analysis of pancreatic islet spheroids over 4 weeks of culture.

    Fig. S2. Immunofluorescent detection of iECs on flow chips.

    Fig. S3. Simulation of flow velocity and shear stress on channel bottom and spheroid surface.

    Fig. S4. Interstitial flow effect on expansion of iECs cultured in conditioned media.

    Fig. S5. Simulation of the diffusion and consumption of nutrients introduced into the culture systems.

    Fig. S6. Fast flow condition beyond interstitial levels resulted in decreased viability of islet spheroids.

    Fig. S7. Simulation of localized accumulation of secreted soluble factors from islet spheroids under two different dynamic conditions.

    Fig. S8. Long-term culture (4 weeks) of islet spheroids in microfluidic chips.

    Table S1. Primer design for qRT-PCR.

    References (4151)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Size distribution and DNA analysis of pancreatic islet spheroids over 4 weeks of culture.
    • Fig. S2. Immunofluorescent detection of iECs on flow chips.
    • Fig. S3. Simulation of flow velocity and shear stress on channel bottom and spheroid surface.
    • Fig. S4. Interstitial flow effect on expansion of iECs cultured in conditioned media.
    • Fig. S5. Simulation of the diffusion and consumption of nutrients introduced into the culture systems.
    • Fig. S6. Fast flow condition beyond interstitial levels resulted in decreased viability of islet spheroids.
    • Fig. S7. Simulation of localized accumulation of secreted soluble factors from islet spheroids under two different dynamic conditions.
    • Fig. S8. Long-term culture (4 weeks) of islet spheroids in microfluidic chips.
    • Table S1. Primer design for qRT-PCR.
    • References (4151)

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