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Materials and methods
Cells from an established cell line of T-47D mammary carcinoma cells1 cultured in monolayer using RPMI supplemented with 10% foetal bovine serum was entrapped in alginate gels of approx. 1.0 mL. Gel formulations were prepared by quickly mixing the contents of two syringes containing sodium alginate solution (PRONOVA SLG-20 or SLM-20, FMC BioPolymer, Norway) and calcium alginate (PRONOVA
CaM, FMC BioPolymer) suspension with added cells, respectively, using a three way connector. The final alginate concentration was 2% w/w. The cell-containing alginate mixture was emptied into a small tube with 10 mm inner diameter.
A computer-connected optical oxygen sensor (NTH, flat-broken tip, Ø=140 μm, PreSens GmbH, Regensburg, Germany) was embedded in the gelling alginate mixture before it set, which occurred within minutes after initial mixing (fig 1). Growth medium was added in the tube above the gel. The oxygen
tension gradient in the gel and medium was also measured and logged for ~24 h using a Clarke-type micro-sensor (Ø = 8-12 μm) and profiling system with a micromanipulator, both computer-controlled (Unisense, Århus, Denmark)(fig. 2). Viability of the entrapped cells was registered by 3-D confocal microscopy on live/dead–stained (Calcein/Propidium iodide), vibratome sectioned gel slices. The parameters tested were alginate type and amount of oxygen initially present in the formulations.
Results and discussion
The oxygen level in the gels decreased rapidly after entrapment, depending on cell density. Data from three different gels (n = 3 in each set) with a starting cell concentration of 3,7·105 cells/mL are presented in fig. 1. The data indicate relatively constant oxygen consumption per cell with time until hypoxic conditions are reached. Oxygen concentration profiles taken in the vertical dimension of the system showed that oxygen concentration was homogeneous within the gel and increased linearly in the medium layer,
implying quasi-steady state diffusion (fig. 3). The tested T-47D cells were found to have an apparently constant oxygen consumption rate of 37±5 fmol/h/cell in mannuronate-rich gels, 60±10 fmol/h/cell in guluronate-rich gels and 100±20 fmol/h/cell in mannuronate-rich gels with suspended air-bubbles. This is significantly less than published data for T-47D cells in exponential growth.Confocal imaging showed that no proliferation had occurred at neither 3 nor 7 days. The plating efficiency was estimated to 56±8% for mannuronate-rich gels with or without suspended air bubbles, and 60±10% for guluronate-rich gels. Most studies on alginate gels report unimpaired diffusion of small molecules, compared to water, although macromolecular substances show lower diffusivity. As guluronate-rich gels have higher porosity than mannuronate-rich gels, the relative amount of guluronate in the gel, and the effect this has on macromolecular diffusion is a possible explanatory factor. The inclusion of air bubbles greatly increased the time needed to deplete oxygen stores in the system (fig.4). In this case, the oxygen tension as a function of time was non-linear and consistent with depletion of an additional oxygen compartment
in the system.
Conclusions
Alginate entrapped T-47D cells depleted the oxygen stores in the alginate gel structures as a function of time. Comparing guluronate- and mannuronate-rich gels demonstrated a significantly higher oxygen consumption rate by cells entrapped in the former gel type, although there was no significant difference between the plating efficiencies obtained. Adding an additional oxygen reservoir into the gel system increased the time-span during which oxygen was available in the matrix. No proliferation was registered in the examined system.
