3.3. Macroporous Scaffolds
Macroporous scaffolds may allow larger constructs as the mass transfer of nutrients, oxygen and waste removal will be enhanced [100]. The structure does also have predefined dimensions and they can better withstand deformation compared to compact hydrogels [101]. Also, the pore size and porosity can be tuned and optimized for the specific application and cell type [102,103,104]. However, a major challenge with such scaffolds may be cell seeding efficiency and cell distribution, as the pores are often either too small to let cells in or too large to retain cells inside. A variety of approaches have been investigated to overcome this challenge utilizing, for example, cell seeding devices [105], bioreactors [106], centrifugal force [107] and vacuum [108,109].
Two alginate based macroporous systems for cell culture are available, AlgiMatrix® and NovaMatrix®-3D from Thermo Fisher Scientific/Life Technologies (Carlsbad, CA, USA) and FMC BioPolymer/NovaMatrix (Sandvika, Norway), respectively. Both systems are based on ionically gelled and dried macroporous scaffolds and are available as sterile discs pre-filled in different formats of standard cell culture well plates [77,110]. Both scaffolds turn into hydrogels upon rehydration following cell seeding. Further, cells can be stained and microscopically evaluated while entrapped, the gels can be dissolved for easy retrieval of cells and multicellular structures and their gel elasticity is tunable. The main differences between these sponge‑like matrices are the seeding technique and ability to control cell to matrix interactions. A concentrated suspension of cells in culture media is absorbed by the dry AlgiMatrix® scaffold as it is applied on the top surface and cells will be entrapped inside the porous structure. Centrifugation may be utilized to ensure a more homogeneous distribution of cells throughout the scaffold. For NovaMatrix®-3D, cells are applied on top of the scaffold suspended in an alginate solution dissolved in cell culture media. As shown in Figure 10, the foam structure is utilized as a scaffold and source of gel forming ions to induce gelation of the applied alginate solution. When the pores are filled with the alginate solution, a hydrogel is subsequently formed in situ. This technique ensures a cell seeding efficiency of about 100% and an even distribution of cells throughout the thickness of the gel. NovaMatrix®-3D cell culture kits are also available with RGD-coupled alginate which is added to the dry sponge together with the cells. See Section 3.2 for examples of cellular responses with or without RGD-coupled alginate.
Figure 10  Schematic presentation of the steps for in situ gelation in macroporous alginate scaffolds. (A) Am alginate solution with cells is applied on top of a dry scaffold containing calcium ions, (B) rehydration of the scaffold by the alginate solution filling its pores and diffusion of calcium ions from the foam lamellas to the absorbed alginate, and (C) formation of a calcium cross-linked alginate hydrogel inside the pores of the foam. From reference [77].

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