Construction of novel temperature-responsive hydrogel culture system based on the biomimetic method for stem cell sheet harvest

Abstract

Temperature-responsive hydrogel culture system is considered as an ideal platform for cell sheet harvest, but its complex preparation methods and harsh reaction conditions limit its application. Inspired by the marine mussels, a biomimetic method presented here is to construct a novel temperature-responsive hydrogel culture system for stem cell sheet harvest. The tissue culture polystyrene is first modified with polydopamine coating, and then amine-terminated poly(N-isopropylacrylamide) is grafted onto the coating via the Schiff base or Michael addition reaction to construct the temperature-sensitive hydrogel culture system. Then, bone marrow stromal cells are cultured on the culture system to construct cell sheets. The prepared culture system shows significant temperature-sensitive property with the grafted concentrations of poly(N-isopropylacrylamide) ranging from 0.5 to 1 g/L. Meanwhile, the constructed culture system has low cytotoxicity and facilitates the stem cell adhesion, proliferation, and cell sheet formation at 37°C. When the culture system is placed in a 20°C environment, the cell sheet can be completely detached from the surface of tissue culture polystyrene without being treated with any enzymes. More importantly, the cell morphology, cell sheet thickness, and the fibril structure of the associated proteins are similar to the cells cultured on the tissue culture polystyrene without modification. The biomimetic, simple, inexpensive, and environmentally friendly preparation of the culture system enables it to be used for the harvest of cell sheet and even applied to tissue engineering for tissue regeneration.

Adhesion of Epithelial Cells to PNIPAm Treated Surfaces for Temperature-Controlled Cell-Sheet Harvesting

“…As we have a three layer system (PMS–ECM–cell), another main contribution to the reduction of cell adhesion upon temperature decrease might be ECM dehydration. Previous literature has also shown destabilization of collagen 1 and fibronectin fibers on PNIPAm substrates . This ECM dehydration could be induced by water uptake through the swelling PNIPAm, and ECM adsorption on the PMS or the detachment of the cell layer would then be based on the stability of each ECM component against loss of water.…”

“…Previous literature has also shown destabilization of collagen 1 and fibronectin fibers on PNIPAm substrates. 28 This ECM dehydration could be induced by water uptake through the swelling PNIPAm, and ECM adsorption on the PMS or the detachment of the cell layer would then be based on the stability of each ECM component against loss of water. This should be less influential for fibrillar components than gel-like matrices.…”

“… 27 More recently, inspired by marine mussels, a polystyrene surface, layer-by-layer coated with a polydopamine and PNIPAm, has been constructed to culture bone marrow stromal cells. 28 …”

“…For instance, Yoon et al developed an elastic piezoelectric substrate based on PNIPAm in order to apply both electrical and mechanical stimuli to skeletal muscle cell sheets . More recently, inspired by marine mussels, a polystyrene surface, layer-by-layer coated with a polydopamine and PNIPAm, has been constructed to culture bone marrow stromal cells …”