Three‐dimensional cell seeding and growth in radial‐flow perfusion bioreactor for in vitro tissue reconstruction

Abstract

Radial-flow perfusion bioreactor systems have been designed and evaluated to enable direct cell seeding into a three-dimensional (3-D) porous scaffold and subsequent cell culture for in vitro tissue reconstruction. However, one of the limitations of in vitro regeneration is the tissue necrosis that occurs at the central part of the 3-D scaffold. In the present study, tubular poly-L-lactic acid (PLLA) porous scaffolds with an optimized pore size and porosity were prepared by the lyophilization method, and the effect of different perfusion conditions on cell seeding and growth were compared with those of the conventional static culture. The medium flowed radially from the lumen toward the periphery of the tubular scaffolds. It was found that cell seeding under a radial-flow perfusion condition of 1.1 mL/cm2 x min was effective, and that the optimal flow rate for cell growth was 4.0 mL/cm2 x min. At this optimal rate, the increase in seeded cells in the perfusion culture over a period of 5 days was 7.3-fold greater than that by static culture over the same period. The perfusion cell seeding resulted in a uniform distribution of cells throughout the scaffold. Subsequently, the perfusion of medium and hence the provision of nutrients and oxygen permitted growth and maintenance of the tissue throughout the scaffold. The perfusion seeding/culture system was a much more effective strategy than the conventional system in which cells are seeded under a static condition and cultured in a bioreactor such as a spinner flask.

Effect of surface acoustic waves on the viability, proliferation and differentiation of primary osteoblast-like cells

“…As compared to the methods described in the literature, 4,[6][7][8][9][10][11][12] where the seeding process usually takes hours to days, the present SAW method significantly accelerates the cell seeding process. The fast seeding time also means that multiple drops containing the cells can be successively driven into the scaffold to achieve greater cell loading.…”

“…As we mentioned before, there are various cell seeding methods being developed and most of these studies investigate the cell viability and functionality. 4,[6][7][8][9][10][11][12] Undoubtedly, cell viability and functionality are critical to the usefulness of any new technique for tissue engineering. 5 Therefore, in addition to high cell viability, the proliferation and differentiation ability of the treated cells showed no difference from those of the untreated cells, indicating that the SAW has no deleterious effects on the cells' functionality.…”

“…However, low seeding efficiencies and nonuniform cell distributions have been reported with these methods as well. [3][4][5][6][7][8][9][10][11][12] Alvarez-Barreto and co-workers 10,11 developed a more refined approach from these ideas using flow perfusion; a cell suspension is first poured on top of the prewetted scaffold and then the flow is drawn into the scaffold by oscillatory pumping across it, driving the fluid with the suspended cells into the scaffold matrix. Compared to past methods, this technique has been reported to deliver superior cell adhesion and seeding efficiency.…”

Model-Based Optimization of Scaffold Geometry and Operating Conditions of Radial Flow Packed-Bed Bioreactors for Therapeutic Applications

“…In their paper, Olivier et al, raised an important practical issue influencing the decision as to whether an rPBB should be perfused with medium outwards or inwards. In fact, many cylindrical scaffolds have pores of increasing size towards their periphery [34], causing significant reduction of the local specific surface area. If cells attach weakly to the scaffold surface, for its chemical nature or for the little surface area available for adhesion, outward medium perfusion may exert high enough mechanical stresses to remove the adherent cells and wash them out.…”

“…Inward radial perfusion of 3D constructs in rPBBs has been shown to promote cell proliferation to a greater extent than static operation in the culture of NIH/3T3 cells in poly(L)lactic acid porous scaffolds [34], and of sheep mesenchymal stem cells [35] and MG63 osteoblast-like cells [36] in β-tricalcium phosphate (β-TCP) porous scaffolds. In the development of a bioartificial liver, inward radial perfusion culture in rPBBs has been reported to maintain in an active metabolic state high concentrations of human liver cancer cells FLC-7 [37], HepG2 cells [38], and porcine hepatocytes [39] in small-scale constructs, and of porcine primary hepatocytes in clinical-scale constructs [40,41].…”