A novel recovery system for cultured cells using plasma‐treated polystyrene dishes grafted with poly(N‐isopropylacrylamide)

Abstract

Poly(N-isopropyl acrylamide) (PIPAAm) demonstrated a fully expanded chain conformation below 32 degrees C and a collapsed, compact conformation at high temperatures. This unique temperature responsive polymer was grafted onto surfaces of commercial polystyrene dishes and used as temperature switches for creating hydrophilic surfaces below 32 degrees C and hydrophobic surfaces above 32 degrees C. Cell attachment and the growth of bovine endothelial cells and rat hepatocytes on PIPAAm-grafted surfaces at 37 degrees C demonstrated similar behavior to the commercialized culture dishes. Both cell types were observed to detach from the PIPAAm-grafted surface simply by reducing the temperature below the polymer transition temperature (collapse). Cells recovered by this method maintained substrate adhesivity, growth, and secretion activities nearly identical to those found in primary cultured cells in contrast to the compromised function found in cultured cells damaged by trypsinization. These results provide strong evidence that PIPAAm-grafted surfaces, as thermal switches are very effective for reversing cell attachment and detachment without cell damage. Properties of cell culture surfaces can be readily transformed by this technique reversibly into hydrophilic and hydrophobic coatings of PIPAAm-grafted polymers.

Scaffolding in tissue engineering: general approaches and tissue-specific considerations

“…Such approach can be repeated to laminate multiple single cell layers to form thicker matrix. This technology is pioneered by Japanese group [84,85,110] and has been systemically applied to a number of applications such as cornea [82] in clinical trials and myocardium [106] in preclinical trials. The same approach has been modified and improved to produce patterned substrates for thicker tissue fabrication and for injectable applications [57].…”

Regenerative medicine of cornea by cell sheet engineering using temperature-responsive culture surfaces

“…PIPAAm shows a hydrophobic character above its lower critical solution temperature (LCST: 32°C), but reversely obtains a hydrophilic character at temperatures lower than 32°C through spontaneous conformational changes ( Figure 2(a)). Therefore, the culture dishes on which PIPAAm is covalently immobilized at nanometer level thickness enable the control of cell adhesion only by temperature alteration [24]. In fact, under general conditions of cell culture (37°C: above LCST), the temperature-responsive surface shows an appropriate hydrophobic status, allowing various types of cells to adhere, spread, and proliferate, similar to cells cultured on normal polystyrene culture dishes (Figure 2(b)).…”

“…In fact, under general conditions of cell culture (37°C: above LCST), the temperature-responsive surface shows an appropriate hydrophobic status, allowing various types of cells to adhere, spread, and proliferate, similar to cells cultured on normal polystyrene culture dishes (Figure 2(b)). In contrast, a reduced temperature below the LCST lets the culture dish surfaces change to a hydrophilic character, resulting in the detachment of cells by the formation of a hydration layer between the culture dish surface and the attached cells (Figure 2(b)) [24]. Therefore, following the confluent conditions of cultured cells on this intelligent surface, low-temperature treatment allows us to obtain sheet-like tissues, "cell sheets," that exhibit high cell density without the need of any scaffolds and proteases ( Figure 3) [23].…”

“…Previously, we established a novel method to prepare bioengineered tissues without the use of any carriers or scaffolds made from biological materials as well as protease treatments, through intelligently modifying the surface of culture dishes [23,24]. Our technology addresses the issues that arose from the use of biologic materials or protease treatments.…”

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“…The grafted polymer reacts due to temperature variations and changes to its molecular form affecting cell adhesion on the surface of the culture dish in vitro. [1] The dish surface is hydrophobic and kept at 37 °C in order to optimize cultured cell's ability to adhere onto the surface. On the other hand, the grafted polymer takes a hydrophilic form at 20 °C, and the cultured cells easily detach from the surface of the dish without degradation of intercellular adhesion molecules [2] or extracellular matrix (ECM).…”