Figure 1.
Flow chart for production of alginate bilayers with and without BaSO4 crystals.
Figure 2.
Phase contrast microscopy of alginate layers.
A–C: Alginate bilayers after 10 minutes incubation in 0.3% alginate solution; the varied crystal density due to the different precipitation procedures causes a change in resulting mean layer thickness; A: crystal free; B: precipitation after washing with NaCl; C: precipitation before washing with NaCl; D: One core capsule with 9 step by step absorbed alginate layer (0.7%). Note a slight compression of the inner core capsule, which was possibly due to syneresis. The final size of this capsule system was about 2 mm.
Figure 3.
Mean alginate layer thickness.
A: Thickness of alginate bilayer increases with incubation time in alginate. Results for incubation in 0.7 % alginate solution are shown; CG-: crystal gun core capsule without BaSO4 precipitation; CG+: crystal gun core capsule with BaSO4 precipitation; Std-: core capsule without crystal gun, without BaSO4 precipitation; Std+: core capsule without crystal gun, with BaSO4 precipitation B: The layer thickness varies if the capsules were coated directly after production (t = 0) or previously stored in isotonic NaCl. Mean layer thickness decreases slightly within the first two hours of storage, but stays subsequently stable up to 21 days (data not shown). Core capsules were produced out of 0.65% alginate solution without the crystal gun approach but with BaSO4 precipitation.
Figure 4.
Swelling behavior of core capsules and alginate layers in DMEM based culture medium (containing 10% FBS); core capsules were produced without the crystal gun approach using 0.65% alginate (dashed lines); double layer capsules were produced as described using alginate solution with 0.7% (solid lines); capsules were fixated by poly-L-lysine coated 24 well plate to reduce movement during long term observation.
Figure 5.
Examples of encapsulated RIN-m spheroids after 10 days of cultivation; Top row: transmission microscopy; Bottom row: fluorescence microscopy via live-dead assay (fluorescein diacetate and ethidium bromide); A/B: free RIN-m spheroid; C/D: standard encapsulated RIN-m spheroid; E/F:RIN-m spheroid encapsulated using the bilayer approach without BaSO4 precipitation; G/H:RIN-m spheroid encapsulated using the bilayer approach with BaSO4 precipitation.
Due to cell growth spheroids grew out of core capsules and grew into the interface between the core capsules and the adsorbed alginate layers; I: early stage of interface growth of RIN-m spheroid; J: advanced stadium of interface growth after 14 days of cell culture the shape of the cell mass growing around the core capsule is clearly visible.
Figure 6.
Encapsulated Langerhans’ islets.
A: Insulin release of encapsulated islets with and without double layer expressed as stimulation index, p=0.16 (no significant difference), B: FDA/EB viability staining of encapsulated islet (green = vital), C: microscopic image of encapsulated islets with alginate double layer.