In situ gelation for cell immobilization and culture in alginate foam scaffolds

Abstract

Essential cellular functions are often lost under culture in traditional two-dimensional (2D) systems. Therefore, biologically more realistic three-dimensional (3D) cell culture systems are needed that provide mechanical and biochemical cues which may otherwise be unavailable in 2D. For the present study, an alginate-based hydrogel system was used in which cells in an alginate solution were seeded onto dried alginate foams. A uniform distribution of NIH:3T3 and NHIK 3025 cells entrapped within the foam was achieved by in situ gelation induced by calcium ions integrated in the foam. The seeding efficiency of the cells was about 100% for cells added in a seeding solution containing 0.1-1.0% alginate compared with 18% when seeded without alginate. The NHIK 3025 cells were allowed to proliferate and form multi-cellular structures inside the transparent gel that were later vital stained and evaluated by confocal microscopy. Gels were de-gelled at different time points to isolate the multi-cellular structures and to determine the spheroid growth rate. It was also demonstrated that the mechanical properties of the gel could largely be varied through selection of type and concentration of the applied alginate and by immersing the already gelled disks in solutions providing additional gel-forming ions. Cells can efficiently be incorporated into the gel, and single cells and multi-cellular structures that may be formed inside can be retrieved without influencing cell viability or contaminating the sample with enzymes. The data show that the current system may overcome some limitations of current 3D scaffolds such as cell retrieval and in situ cell staining and imaging.

FIG. 1.

Schematic presentation of the steps of in situ gelation including (A) application of sodium alginate solution on top of a dry calcium alginate foam; (B) Rehydration of the foam by the alginate solution filling its pores, and diffusion of calcium ions from the foam lamellas to the absorbed alginate; (C) Formation of a calcium alginate hydrogel inside the pores of the foam. The alginate solution may contain cells or other materials that will be entrapped in the hydrogel that is formed.

FIG. 2.

Gelation kinetics described by oscillatory rheometry after addition of 0.5% alginate solutions to dry alginate foam disks shown as storage modulus, G′ (thick black), loss modulus, G′′ (thick gray), and phase angle, δ (thin black). The gelation properties of PRONOVA UP LVG (solid line), PRONOVA UP MVG (dashed line), and PRONOVA UP LVM (dotted line) alginates in alginate foams are shown. The relative SD in the measurements was between 2–20%.

FIG. 3.

Young's modulus of gel disks as a function of the applied LVG alginate concentration (A). The effect on Young's modulus of gel disks added 1.0% or 0.5% LVG alginate followed by immersion in isotonic gelling solutions of either SrCl₂ or CaCl₂ (B). Young's modulus over time for gel disks-added 0.5% LVG alginate and kept in Dulbecco's-modified Eagle's medium (DMEM), which was replaced thrice per week (C). Characterization of gel disks in A, B, and C (Day 0) was accomplished on disks kept in DMEM for 2 h. Significance is considered between different treatments for the same concentration of added alginate. *p<0.05, **p<0.01, and ***p<0.001. Error bars present SD.

FIG. 4.

Distribution of NIH:3T3 cells in gel disks and cell seeding efficiency influenced by the alginate concentration in the cell seeding solution 2 h after seeding. Cell localization throughout the thickness of the gel indicated by colors when cells were suspended in cell seeding solutions containing alginate concentrations of 0% (A), 0.1% (B), 0.5% (C), and 1.0% (D). Cell distribution in gels showed for xy- and xz-directions (E). Percent cell seeding efficiency as a function of alginate concentration in the cell seeding solution (F). Magnification: ×40. Scale bars: 100 μm, Color bars: foam thickness 0–400 μm (A), 0–700 μm (B, D) and 0–600 μm (C). The error bars depict SD.

FIG. 5.

Proliferation of NHIK 3025 cells normalized to initial cell seeding when cultured in alginate gel disks as a function of time (A). An approximate exponential increase in cell number was found with a cell doubling time of 3.0±0.3 days. Volume of spheroids as a function of time, with an almost exponential increase between days 0 and 16 before it levels off (B). The calculated time to obtain double volume was based on the exponential fit of data between days 0 and 16 and was found to be 1.43±0.05 days. The images show distribution of calcein-stained cells throughout the thickness of the gel at the day of seeding (C), inside the gel after 6 days of culture (D), isolated after 16 days of culture (E), and inside the gel after 23 days of culture (F). Magnification: ×40 (C–E) and ×100 (F). Scale bars: 100 μm. Error bars present SD.