Effect of Process Conditions on the Growth of Three-Dimensional Dermal-Equivalent Tissue Obtained by Microtissue Precursor Assembly

Abstract

Bottom-up approach is an appealing strategy to build complex three-dimensional (3D) viable tissues in vitro starting from microtissue precursors (μTP). In this work we biofabricated a thick dermal-like tissue by sequentially combining two steps: a μTPs production and assembly followed by tissue maturation in a purpose-built bioreactor. The μTPs were produced by first seeding bovine primary fibroblasts on gelatine microparticles and then cultivating them in stirring conditions until a thick layer of ~80 μm of de novo synthesized extracellular matrix uniformly covered the microparticle surface. The μTPs were then loaded into a cylindrical chamber (2 mm in depth and 35 mm in diameter) and let to maturate and assemble into a 3D viable biohybrid tissue under specific fluid flow conditions. Several combinations of perfusion and/or tangential fluid flow were applied and their effect on the tissue formation and maturation was assessed. Results show that structural composition and mechanical features of the final 3D bioengineered tissue are strongly affected by the hydrodynamic environment and demonstrate that by optimizing culture conditions a 3D viable tissue with properties similar to that of native derma could be produced.

Perfusion culture‐induced template‐assisted assembling of cell‐laden microcarriers is a promising route for fabricating macrotissues

“…Our previous reports had concerned the fabrication of microtissues and also demonstrated successful preparation of a macroscopic bone construct from hMSCs [8,14]. Among different strategies employed to assemble microtissues in literature [7,23,24], perfusion culture is able to promote interstitial flow and exert shearing force on cells and may favor cell growth and differentiation and tissue maturation [13,16,19,25]. Hence, in the present study, a systemic investigation on the process of assembling microtissues into macrotissues via perfusion culture was performed.…”

“…Twal et al reported that the mechanical property of a tubular construct made of cell-laden microcarriers was increased with culture time [18]. Urciuolo et al revealed that tissue properties largely relied on fluid flow conditions during the assembling of bovine fibroblast-laden gelatin microparticles [19]. Further, Imparato et al found that the microcarrier property could significantly influence the maturation of macrotissues [20].…”

Building a Tissue In Vitro from the Bottom Up: Implications in Regenerative Medicine

“…this micrometric tissue wrapped around and within the porosity of the scaffold constitutes micrometric tissue precursors (μtPs) for further large-tissue construction. 19,20 the process for μtP formation consisted of a dynamic cell seeding performed in a spinner flask bioreactor, which was loaded with cells and porous gelatine microbeads in the ratio of 50 cells per microbead and operated at 30 rpm. the disappearance of free cells from the inoculated spinner cultures was considered to indicate the attachment of cells to the microcarriers.…”

“…they can be assembled in an appropriate assembling chamber, and the tissue layers surrounding them allow their fusion through cell-cell and cell-matrix interactions. Following this strategy, a 3d functional dermal tissue equivalent has been created (Figure 1d, e), 20 and an assembling chamber able to (Figure 2a, B). it was observed that after 1 week in the maturation chamber under static conditions, the building blocks were able to assemble, leading to a compact tissue equivalent.…”

“…these building blocks can be created in a number of ways, such as self-assembled cell aggregates, [17][18] microfabrication of cell-laden microgel, 7 creation of cell sheet, 9 and microfabrication of cell seeded microbeads. [19][20] once obtained, these building blocks can be assembled in larger tissue through a number of methods including random packing, stacking of layers, or direct assembly. a bottom-up approach has been used by du et al 7 to direct the assembly of cell-laden microgels to generate 3-d tissue with tunable microarchitecture and complexity.…”