Construction of a chondrocyte cell sheet using temperature-responsive poly(N-isopropylacrylamide)-co-acrylamide

Abstract

In this study, a novel temperature-responsive poly(N-isopropylacrylamide)-co-acrylamide was used to prepare a chondrocyte cell sheet. Chondrocytes were isolated from human articular cartilage and plated on the copolymer film grafted tissue culture plates. The cell attachment on the copolymer film was shown to be similar to that of the ungrafted surface. To harvest a cell sheet, the incubation temperature was reduced to 10°C for 30 minutes to allow the polymer chain to fully extend, changing the copolymer's phase from hydrophobicity to hydrophilicity. Additional incubation at 20°C for 60 minutes was necessary to activate the cellular metabolism required for cytoskeletal organization and cell detachment. A complete cell sheet recovery was achieved when a PVDF membrane was used as a cell sheet carrier. Unfortunately, the shrinkage of the cell sheet was observed. Nonetheless, the harvested cell sheet was shown to be viable and healthy.

Quantitative relation between thickness and grafting density of temperature‐responsive poly(N‐isopropylacrylamide‐co‐acrylamide) thin film using synchrotron‐source ATR‐FTIR and spectroscopic ellipsometry

“…Adhesion forces between the cell and the surface are lost upon polymer hydration. However, the cell and extracellular matrix (ECM) protein interactions are still active 4,11,15 …”

“…6 In an aqueous environment, PNIAM shows a hydrophilic-hydrophobic phase transition at its lower critical solution temperature (LCST) of about 32 C. [7][8][9][10] When the temperature is above the LCST, cells adhere to the PNIAM surface as the polymer chains collapse, allowing proteins to adsorb to the surface. [11][12][13] In contrast, the adsorption of protein and cell attachment is negligible below the LCST due to extended chain conformation. As a result, cultured cells along with their adhesion proteins can be recovered as a monolayer cell sheet when the temperature is below the LCST.…”

“…However, the cell and extracellular matrix (ECM) protein interactions are still active. 4,11,15 The amount of the PNIAM grafted layer is believed to influence protein adhesion and cell attachment. [16][17][18] When the amount of polymer grafted on the tissue culture polystyrene surface (TCPS) is low, the polymer chain hydration is restricted.…”

Improving Cell Detachment from Temperature-Responsive Poly(N-isopropylacrylamide-co-acrylamide)-Grafted Culture Surfaces by Spin Coating

“…Recent studies have focused on attempts to fabricate a temperature-sensitive surface, which can be used for detachment of different cell types. ,,− The use of ultraviolet (UV) is a more economical alternative, compared to EB irradiation. , In addition, it can be used in conjunction with other techniques, such as surface patterning and photolithography techniques, to graft polymers onto TCPS. , Our previous studies showed that temperature-sensitive Poly­( N -isopropylacrylamide- co -acrylamide) (PNIAM- co -AM) can be grafted onto TCPS by UV irradiation. , When harvesting cell sheets, the cell monolayers had to be agitated with the culture media. This, however, resulted in 85% detachment of mouse preosteoblast MC3T3-E1 cell sheets.…”