Bioprinting of a functional vascularized mouse thyroid gland construct

Abstract

Bioprinting can be defined as additive biofabrication of three-dimensional (3D) tissues and organ constructs using tissue spheroids, capable of self-assembly, as building blocks. The thyroid gland, a relatively simple endocrine organ, is suitable for testing the proposed bioprinting technology. Here we report the bioprinting of a functional vascularized mouse thyroid gland construct from embryonic tissue spheroids as a proof of concept. Based on the self-assembly principle, we generated thyroid tissue starting from thyroid spheroids (TS) and allantoic spheroids (AS) as a source of thyrocytes and endothelial cells (EC), respectively. Inspired by mathematical modeling of spheroid fusion, we used an original 3D bioprinter to print TS in close association with AS within a collagen hydrogel. During the culture, closely placed embryonic tissue spheroids fused into a single integral construct, EC from AS invaded and vascularized TS, and epithelial cells from the TS progressively formed follicles. In this experimental setting, we observed formation of a capillary network around follicular cells, as observed during in utero thyroid development when thyroid epithelium controls the recruitment, invasion and expansion of EC around follicles. To prove that EC from AS are responsible for vascularization of the thyroid gland construct, we depleted endogenous EC from TS before bioprinting. EC from AS completely revascularized depleted thyroid tissue. The cultured bioprinted construct was functional as it could normalize blood thyroxine levels and body temperature after grafting under the kidney capsule of hypothyroid mice. Bioprinting of functional vascularized mouse thyroid gland construct represents a further advance in bioprinting technology, exploring the self-assembling properties of tissue spheroids.

From Shape to Function: The Next Step in Bioprinting

“…One successful example relates to biofabricated ovaries, obtained by including ovary follicle cells in printed gelatin‐based scaffolds with cell‐instructive architectures that were shown to rescue reproductive capacity, enabling sterilized mice to obtain healthy offspring from natural mating . An additional example of a functional bioprinted structure are the printed spheroids of vascular and thyroid gland cells that were capable of replacing the bioactivity of native thyroid, including its thermoregulatory ability and thyroxine hormone secretion into the systemic circulation, when implanted in mice with a surgically induced hypothyroidism . Also, bioprinted muscle cell‐laden hydrogel fibers were demonstrated to mature into homogenously shaped bundles of myofibers, which exhibited contractile function, and could be matured upon ectopic implantation in vivo, showing a more pronounced organization and alignment compared to nonprinted controls …”

“…These structures that only encompass an intermediate complexity could then be bioprinted into more complex tissue and organ progenitors with geometries and patterns designed to instruct the formation of functional tissue . The fusion capability of multicellular spheroids representing such intermediate tissue modules has been characterized in detail and utilized in proof‐of‐principle studies for tissue generation . The bioprinting of such spheroids or microtissues into either 3D plotted or MEW‐generated thermoplastic polymer scaffolds has recently been proposed and demonstrated, facilitating the assembly into larger tissue units …”

“…Importantly, while our ability to precisely mimic architectural facets of living tissues increases, the extent of resolution that is required in order to achieve fully functional biofabricated constructs, remains unknown. Salient advances in the creation of tissues that exerted native functions in vivo have been achieved with precisely patterned, yet relatively simple architectures . At the same time, an increasing number of studies is emerging, suggesting improved maturation of biofabricated tissues mimicking native structures, particularly when multiple cell types are distributed in precise areas of the produced constructs .…”

Engineered 3D Polymer and Hydrogel Microenvironments for Cell Culture Applications

“…Highly crosslinked collagen fabricated using the crosslinking agent genipin allowed osteoblast-like cells and human adipose stem cells to be viable and to proliferate [295]. Tissue spheroids encapsulated in collagen [296], keratinocytes and fibroblasts encapsulated in collagen [297], as well as primary mesenchymal stem cells [298], were all bioprinted for applications in thyroid gland engineering, human skin engineering, and human meniscus engineering, respectively.…”

“…Inclusion of genipin, feature size 400 µm, compressive E: 17 kPa to 1.4 MPa [295] Indirect coating of collagen onto TPP scaffold, feature size 30 µm [290] Osteoblast cells, human adipose stem cells [295]; tissue spheroids [296]; keratinocytes and fibroblasts [297], hMSCs [298]; human corneal epithelial cells [257]; osteocytes [300][301][302]; 3D liver microenvironments [209] Fibrin (4.4.2) Mixed with PVA, feature size 100 µm [303] Indirect methods of coating [306], micro-molding with feature size 20 µm [61] Bone marrow stromal cells [307]; neural tissue [308]; dental pulp stem cells [309]; Schwann cells [219,303]; human umbilical vein endothelial cells, hMSCs [310] Gelatin (4.4.3)…”