Limited beneficial effects of perfluorocarbon emulsions on encapsulated cells in culture: experimental and modeling studies

Abstract

Due to the high solubility of oxygen in perfluorocarbons (PFCs), these compounds have been explored for improved cell and tissue oxygenation. The goal of this study is to investigate the effects of a PFC emulsion on cellular growth and function in a tissue engineered construct. A perfluorotributylamine (PFTBA) emulsion was co-encapsulated at 10 vol% with mouse βTC-tet insulinoma cells in calcium alginate beads and cultured under normoxic and severely hypoxic conditions. The number of metabolically active cells and the induced insulin secretion rate were measured over time for up to 16 days. Results showed no significant effect of PFTBA relative to the PFTBA-free control. The alginate-PFC-cell system was also modeled mathematically, and simulations tracked the number of viable cells over time under the same conditions used experimentally. Simulations revealed only a small, likely experimentally undetectable difference in cell density between the PFC-containing and PFC-free control beads. It is concluded that PFTBA up to 10 vol% has no significant effect on the growth and function of encapsulated βTC-tet cells under normoxic and hypoxic conditions.

Figure 1

Metabolic activity of encapsulated βTC-tet cells under normoxic conditions. The number of metabolically active cells was measured as a function of time by alamarBlue™ and normalized to Day 1. Measurements were performed with calcium alginate/ poly-L-lysine/ alginate (APA) beads of 1000 μm average diameter with no PFTBA, APA beads with 10 vol% lecithin, and APA beads with 10 vol% PFTBA (n = 3 each). The initial density of the encapsulated cells was 3.5×10⁷ cells/ml. * p<0.05 when compared to Day 1.

Figure 2

Stimulated insulin secretion rate (ISR) of βTC-tet cells under normoxic conditions. ISR was measured over 30 min of stimulation by 16 mM glucose, normalized to the initial number of encapsulated βTC-tet cells, and expressed on a per unit time basis. Measurements were carried out with 1000 μm average diameter APA beads with no PFTBA, APA beads with 10 vol% lecithin, and APA beads with 10 vol% PFTBA (n = 3 each). Each glucose stimulation episode followed 1 hour of exposure to basal, 0 mM glucose, medium. * p<0.05 when compared to Day 1.

Figure 3

Metabolic activity and % viability of encapsulated βTC-tet cells under severely hypoxic conditions (2% air saturation, or 0.004 mM). The number of metabolically active cells (A) and the % viability (B) were measured by alamarBlue™ and Trypan Blue, respectively, as a function of time and normalized to Day 1. Measurements were performed with calcium alginate beads of 500 μm average diameter with no PFTBA and 10 vol% PFTBA (n = 5 each). * p<0.05 when compared to Day 1.

Figure 4

Stimulated insulin secretion rate (ISR) of βTC-tet cells under severely hypoxic conditions. The encapsulated cells were cultured for 3 hours under hypoxic conditions, then subjected to a secretion test. The secretion episode consisted of cells exposed for 1 hour to basal, 0 mM glucose medium, followed by a 30 min of stimulation in 16 mM glucose medium, all under hypoxia. The ISR over the stimulation period was measured and is reported in the figure. The ISR under normoxic conditions is included for reference. Beads were of 500 μm average diameter.

Figure 5

Configuration considered to solve for the DO concentration profile. In solving for the total DO concentration (C_T, amount of DO in aqueous plus PFC phases divided by the compartment volume) at compartment ‘0’, diffusional transport occurs by the DO concentration differences in the aqueous phase (C_aq) between compartment ‘0’ and the neighboring compartments (‘-1’ & ‘+1’). Additionally, the DO concentrations in the PFC and aqueous phases, C_P and C_aq, respectively, are always in equilibrium according to the partition coefficient. Parameter S(r) is the oxygen consumption rate by the cells per unit volume of aqueous phase, as described in Equation (3).

Figure 6

Simulated temporal profiles under normoxic conditions (0.2 mM). The model equations were solved for 1000 μm average diameter beads to calculate the cell density (A) and the average intrabead DO concentration (AIDO) (B) in the aqueous phase in beads containing no PFC and 10% PFC. Simulated values are compared to the experimental data shown in Figure 1 obtained from APA beads under similar conditions. In (A), the scale of the graph makes the simulations for the 10% PFC and control beads essentially coincide.

Figure 7

Simulated factor increase in cell density vs. time resulting from the incorporation of 10% PFC relative to PFC-free control for 3 different oxygen consumption rates, v_max (mmol/(10⁹ cells·day)) and 2 different growth rates, μ_g,max (day^-1). Remaining model parameters are the same as those reported in Table 1.

Figure 8

Simulated temporal profiles under hypoxic conditions (0.004 mM). Model solutions for cell density (A) and average intrabead DO concentration (AIDO) (B) in the aqueous phase of 500 μm average diameter beads containing no PFC and 10% PFC. Simulated values are compared to the experimental data obtained from calcium alginate beads under similar conditions and reported in Figure 3. In (A), the scale of the graph makes the simulations for the 10% PFC and control beads essentially coincide. The inset in (B) shows the change in AIDO concentration for the initial 2 min.