2.1 Isolation and Cell Culture of hASC.
This study was approved by the Research Ethics Committee, Faculty of Medicine, University of Chile. Adipose tissue was obtained from five donors aged between 30 and 45 years undergoing abdominal liposuction cosmetic surgery. Informed consent was obtained from all participants. hASC were obtained from lipoaspirated fat tissue, the tissue was digested by type I collagenase 0,2% p/v (Worthington, NJ, USA. Catalog number LS004196) and the stromal vascular fraction (SVF) was cultured in DMEM:F12 1:1 (GibcoTM, Dublin, Irlanda. Catalog number 12500062) medium supplemented with 10% fetal bovine serum (FBS), denominated supplemented medium, at 37°C and 5.0% CO2.
2.2 Differentiation to IPC from hASC in vitro.
hASC were differentiated in vitro to IPC using a two-stage protocol, [13, 14], with discrete modifications. Cells were stimulated for 7 days with DMEM high glucose (25 mM) (GibcoTM, Dublin, Irlanda. Catalog number 12100046); followed by 14 days with DMEM low glucose (5 mM) (GibcoTM, Dublin, Irlanda. Catalog number 31600034). Both media were supplemented with 2 nM activin A (Prospecbio, CA, USA. Catalog number CYT-145), 10 mM nicotinamide (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany. Catalog number N0636) and 10 nM glucagon like peptide (GLP-1) (Prospecbio, CA, USA. Catalog number HOR-284). However, we used 2% FBS instead of 10% as a supplement to differentiation medium during all differentiation, Figure 1A.
2.3 Differentiation to GPC from hASC in vitro.
GPC were obtained by the protocol reported for Rezania et al. (15) with modifications. The protocol consists in 6 stages: stage 1, incubated for 4 days with RPMI 1640 (GibcoTM. Catalog number 31800), bovine serum albumin (BSA) (Sigma-Aldrich, catalog number A9418) 2%, activin A 100 ng/mL (Prospecbio), Wnt3a 20 ng/mL (Sigma-Aldrich. Catalog number H17001), FGF2 8 ng/mL (Prospecbio. Catalog number CYT-085). Stage 2, incubated for 2 days with DMEM-F12 medium with BSA 2%, FGF7 50 ng/mL (Prospecbio. Catalog number CYT-303), Cyclopamine-KAAD 0,25 µM (SCBT, Dallas, Tx, USA. Catalog number sc-200929). Stage 3, incubation for 4 days with DMEM-F12 medium, Cyclopamin-KAAD 0,25 µM, retinoic acid (RA) 2 µM (Sigma-Aldrich. Catalog number R2625), FGF7 50 ng/mL (Prospecbio), Noggin 100 ng/mL (Prospecbio. Catalog number CYT-475). Stage 4, incubation for 3 days with DMEM:F12 medium, Inhibitor II ALK5 1 µM (SCBT. Catalog number sc-221234), Noggin 100 ng/mL, DAPT 1 µM (SCBT. Catalog number sc-201315). Stage 5, incubation for 7 days with DMEM/F12 medium, inhibitor II ALK5 1 µM. Stage 6, incubation for 7 days with DMEM F12 medium. However, we used 2% FBS instead of 1% B27 as a supplement to differentiation medium during stages 3 to 6, Figure 1B.
2.4 Formation of cell aggregates.
The formation of cells aggregates was induced applying a proprietary protocol that subjects cell to low adherence conditions and progressive clustering, as previously described . Briefly, differentiated IPC and GPC cells were incubated in a 4:1 ratio respectively, using 105 total cells, under conditions of non-adherence. This protocol combines 1) the use of bacteriological plates with low electrical charge, and 2) culturing in low Ca2+ and Mg2+ using a special formulation of Eagle Minimum Essential Medium, Joklik Modification (Sigma-Aldrich. Catalog number M0518) and 3) reduced concentration of FBS (2% v/v) for up to 7 days.
2.5 Immunofluorescent staining.
2.5.1 Immunofluorescence of adherent cells.
Undifferentiated and differentiated cells were grown in glass coverslips, and fixed for 10 min with 4% paraformaldehyde (PFA) (Sigma-Aldrich. Catalog number 158127) in phosphate buffer saline (PBS) at room temperature, washed, and then permeabilized with 0.3% triton X-100 (Sigma-Aldrich. Catalog number T8787) for 10 min and incubated with 3% BSA in PBS (to block non-specific binding) for 45 min. Cells were then incubated overnight at 4°C with Abcam primary antibodies (Abcam, Cambridge, UK) (Ms anti-C44 catalog number ab6124, Goat anti-Pdx1 catalog number ab47383, Rb anti-Ngn3 catalog number ab38548, GPig anti-Ins catalog number ab7842, Mouse anti-Gcg catalog number ab 10988, and Goat anti-Vim catalog number V-4630 (Sigma-Aldrich). Cells were then washed with PBS and incubated with corresponding secondary antibodies for 90 min at RT (Dnk anti-mouse catalog number ab96875, Dnk anti-goat catalog number ab96933, Dnk anti-rb catalog number ab96919, Dnk anti-GPig catalog number ab150185, Goat anti-mouse catalog number ab6787, Dnk anti-goat catalog number ab96931) (Abcam). Cells were washed with PBS and then incubated with Hoechst (Millipore, Massachusetts, USA) 1:500 for 5 min at RT. Covers were washed and mounted in slides with fluorescence mounting medium DAKO (Agilent, CA, USA. Catalog number S302380). The images were analyzed using confocal microscopy (LSM700, Zeiss) and the ImageJ public domain software.
2.5.2 Immunofluorescence of cell aggregates.
Prior to immunofluorescence, cell aggregates formed after 72 h were fixed in 4% PFA for 1 hour and added to 1% liquid low melting agarose (Sigma-Aldrich, catalog number A9414) in PBS (50°C). To obtain cell blocks, the agarose-cell mix was centrifuged at 1500 rpm for 5 min at RT. Cryostat sections of 6 µm were submitted to conventional IFI and visualized by confocal microscopy (LSM 700, Carl Zeiss).
2.6 ELISA assay.
To test if the hormonal release of IPC, GPC cells and aggregates was glucose-dependent, two glucose concentrations (2 mM and 25 mM) were assayed. After pre-incubation with Krebs-Ringer buffer (KRB) without glucose (120 mM NaCl, 5 mM KCl, 2,5 mM CaCl2, 1,1 mM MgCl2, 25 mM NaHCO3, 10 mM HEPES, 0.1 % BSA) at 37°C for 2 h, the cells were incubated with KRB containing 2 mM glucose at 37°C for 4 h. To induce insulin release, the cells were incubated with KRB containing 25 mM glucose for another 4 h. Then, the respective conditioned media were collected and tested for the content of released insulin with human insulin (Mercodia, Uppsala, Sweden. Catalog number 10-1132-01) and human glucagon (Cusabio, Houston, TX, USA. Catalog number CSB-E09207h) ELISA kits
2.7 Microencapsulation of cell aggregates.
Alginate microgels were automatically produced using the dripping technique. As a polymer, 1,5% (MW medium) sodium alginate (Sigma- Aldrich. Catalog number A2033) dissolved in 0,9% NaCl was used. The stabilizing solutions of calcium and barium chloride was maintained at pH 7.0 and was composed of (in mM): BaCl2 (20), NaCl (115), and histidine (50), or CaCl2 (40), NaCl (85), HEPES (10). First, 1 mL of sodium alginate solution (1,5% w/v) was mixed with 204 cellular aggregates previously filtered through 40 μm and 120 μm sieves . This anionic suspension was pumped using a Microencapsulator B-395Pro (Büchi, Flawil, Switzerland), equipped with a 150 µm nozzle over a stirring barium or calcium chloride solution for 30 seconds, with a dripping flow of 32 mL/min, 3000 Hz frequency and 1000 V voltage, as previously reported (27) (28). After its formation, the microgels were washed with 0.9% NaCl and incubated in DMEM / F12 medium supplemented with 10% FBS in cell culture conditions (37 °C, 5.0% CO2).
2.8 Statistical analysis.
For the experiments of cell aggregate formation and encapsulation of BSA or SpA proteins, statistical analysis was performed using one-way ANOVA followed by Student’s t test as post-hoc. Student t test was used in Ins or Gcg release tests. Differences were deemed statistically significant for p-values of less than 0.05.