Fabrication of Functional Cell Sheets with Human Thyrocytes from Non-Tumorous Thyroid Tissue

Abstract

BACKGROUND: Engineered cell sheet transplantation has been considered an alternative physiological therapy for endocrine disorders. In this study, we attempted to fabricate functional human thyroid cell sheets using the engineering technology by culturing primary thyrocytes in free-feeder monolayers and assessed their proliferation and function in two different media. METHODS: The non-tumorous tissues (approximately 2 g) were dissected during surgery. Primary human thyroid cells were isolated by mechanical dispersion and treatment with isolation solution. The cells were cultured on tissue culture dishes or temperature-responsive culture dishes to induce the formation of detached cell sheets. RESULTS: Primary thyroid cells isolated from nine patients were positive for thyroid transcription factor 1, thyroglobulin (TG) and cytokeratin 7. Cell sheets with follicles were fabricated by cells incubated in both Dulbecco's Modified Eagle Medium (DMEM) and hepatocyte-defined medium (HDM) culture medium. The diameter and thickness of sheets fabricated in HDM were larger and thicker than those fabricated from DMEM. Furthermore, the cells incubated in HDM secreted higher levels of fT3 and fT4 than those incubated in DMEM. The thyroid peroxidase and TG mRNA of cells maintained in HDM were higher than those in cells maintained in DMEM. CONCLUSION: HDM appears suitable as a culture medium for maintaining primary thyrocytes and fabricating functional cell sheets. These in vitro findings may contribute to the development of appropriate culture conditions for human thyrocytes as well as engineered functional cell sheets.

Regenerative medicine for the hepatobiliary system: A review

“…68 This technology enables preparation of transplantable tissue constructs preserving intact cell-cell contacts and extracellular matrices. 67 In the authors' laboratory, these cell sheets were successfully generated: hepatic cell, 69 thyrocyte, 70 myoblast, 71 adiposederived stem cell, 72 oral epithelial cell, 73 islet cell, 74 fibroblast, 75 and epidermal cell 76 sheets.…”

The bioengineering of perfusable endocrine tissue with anastomosable blood vessels

“…Techniques based on cell sheets [3] and cell aggregates [4] can improve engraftment after transplantation. Several studies have demonstrated hormone secretion by bioengineered endocrine tissue, including islet β-cell sheets [5], islet β-cell spheroids [6] and islet-like organoids [4,7] in models of diabetes mellitus, and thyroid cell sheets [8,9] and thyroid gland organoids [10] in models of hypothyroidism.…”

Alternative method for trypsin-based cell dissociation using poly (amino ester) coating and pH 6.0 PBS

“…Several researches have used a strategy based on culturing cells on tissue culture plates (TCPs) with unique polymer coating. [4][5][6][7][8] For example, poly(N-isopropylacrylamide) (PNIPAAm) has been applied to the surface of TCP for detaching cells since PNIPAAm can change its hydrophilicity in temperature-dependent manner. 5 However, PNIPAAm has several disadvantages.…”