RESUMO
Over the past two decades, poly(N-isopropyl acrylamide) (pNIPAM) has become widely used for bioengineering applications. In particular, pNIPAM substrates have been used for the nondestructive release of biological cells and proteins. In this feature article, we review the applications for which pNIPAM substrates have been used to release biological cells, including for the study of the extracellular matrix (ECM), for cell sheet engineering and tissue transplantation, the formation of tumorlike spheroids, the study of bioadhesion and bioadsorption, and the manipulation or deformation of individual cells. The articles reviewed include submissions from our own group as well as from those performing research in the field worldwide.
Assuntos
Acrilamidas/química , Adesão Celular , Matriz Extracelular/química , Polímeros/química , Proteínas/química , Resinas Acrílicas , Adsorção , Animais , Células CultivadasRESUMO
Poly(N-isopropyl acrylamide) (pNIPAM) is a stimulus-responsive polymer that has been of great interest to the bioengineering community. When the temperature is lowered below its lower critical solution temperature (â¼32 °C), pNIPAM rapidly hydrates, and adherent cells detach as intact cell sheets. This cell-releasing behavior in a physiologically relevant temperature range has led to NIPAM's use for engineered tissues and other devices. In a previous study, however, the authors found that although most techniques used to polymerize NIPAM yield biocompatible films, some formulations from commercially-available NIPAM (cpNIPAM) can be cytotoxic. In this work, the authors investigate the reasons underlying this anomaly. The authors evaluated the response of a variety of cell types (e.g., bovine aortic endothelial cells, BAECs; monkey kidney epithelial cells, Vero cells; and mouse embryonic fibroblasts, 3T3s) after culture on substrates spin-coated with sol-gel (spNIPAM) and commercially-prepared (cpNIPAM). The relative biocompatibility of each cell type was evaluated using observations of its cell morphology and function (e.g., XTT and Live/Dead assays) after 48 and 96 h in culture. In addition, the substrates themselves were analyzed using NMR, goniometry, and XPS. The authors find that all the cell types were compromised by 96 h in culture with cpNIPAM, although the manner in which the cells are compromised differs; in particular, while Vero and 3T3 cells appear to be undergoing cytotoxic death, BAECs undergo apoptic death. The authors believe that this result is due to a combination of factors, including the presence of short chain oligomers of NIPAM in the commercially-available preparation. This work will provide valuable insights into the cytotoxicity of commercially-prepared polymer substrates for this type of bioengineering work and therefore into the applicability of cells grown on such surfaces for human subjects.
Assuntos
Resinas Acrílicas/toxicidade , Células Endoteliais/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Resinas Acrílicas/química , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Células Endoteliais/fisiologia , Células Epiteliais/fisiologia , Fibroblastos/fisiologia , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Espectroscopia Fotoeletrônica , Fatores de TempoRESUMO
Although there is a great deal of research focused on cell sheet engineering from polymers such as poly(N-isopropyl acrylamide) (pNIPAM), the biocompatibility of pNIPAM surfaces and the nature of cellular detachment from this polymer is still unclear. The most extensive study of the mechanism of detachment proposed a two-step process, with a first (passive) phase involving hydration of pNIPAM chains, and the second (active) phase involving cellular metabolism. However, a number of studies performed successful cell sheet detachment from pNIPAM-grafted surfaces at low temperatures which calls this hypothesis into question. Furthermore, although it has been demonstrated that low-temperature cell sheet detachment using pNIPAM-grafted surfaces is less destructive than other methods of detachment, it has not been investigated if cell sheet detachment removes a portion of pNIPAM from the surfaces as well. It is essential to know if any fragments of the polymer are removed along with the cells, as small polymer fragments could have cytotoxic effects on the cells. This is especially important if these cells are used for the generation of tissues used for transplantation. In this work, the mechanism of cell detachment from pNIPAM coated surfaces is investigated by testing how temperature and presence of an adenosine triphosephase inhibitor affect cellular detachment. Surface initiated atom transfer polymerization (ATRP) was utilized to synthesize thermoresponsive atrpNIPAM surfaces. pNIPAM surfaces were labeled to assess whether cell sheet detachment from pNIPAM is accompanied by the removal of pNIPAM from the substrate itself. Using a semipermeable superstrate, cell sheets were transferred to a secondary culture dish to assess whether cell detachment resulted in any pNIPAM removal. In addition, the function of the transplanted bovine aortic endothelial cells was assessed by determining whether they would proliferate and grow on a new secondary substrate.
Assuntos
Resinas Acrílicas/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Células Endoteliais/citologia , Animais , Aorta/citologia , Bovinos , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Microscopia de Fluorescência , Azida Sódica/farmacologia , Propriedades de Superfície , TemperaturaRESUMO
Although there are many stimulus-responsive polymers, poly(N-isopropyl acrylamide) (pNIPAM) is of special interest due to the phase change it undergoes in a physiologically relevant temperature range that leads to the release of cells and proteins. The nondestructive release of cells opens up a wide range of applications, including the use of pNIPAM for cell sheet and tissue engineering. In this work, pNIPAM surfaces were generated that can be distinguished from the extracellular matrix. A polymerization technique was adapted that was previously used by Mendez, and the existing protocol was optimized for the culture of mammalian cells. The resulting surfaces were characterized with X-ray photoelectron spectroscopy and goniometry. The developed pNIPAM surfaces were further adapted by incorporation of 5-acrylamidofluorescein to generate fluorescent pNIPAM-coated surfaces. Both types of surfaces (fluorescent and nonfluorescent) sustained cellular attachment and produced cellular detachment of â¼90%, and are therefore suitable for the generation of cell sheets for engineered tissues and other purposes. These surfaces will be useful tools for experiments investigating cellular detachment from pNIPAM and the pNIPAM/cell interface.
Assuntos
Resinas Acrílicas/síntese química , Adesão Celular , Técnicas de Cultura de Células/métodos , Engenharia Tecidual/métodos , Animais , Linhagem Celular , Humanos , Mamíferos , Espectroscopia Fotoeletrônica , Propriedades de SuperfícieRESUMO
Poly(N-isopropyl acrylamide) (pNIPAM) is one of the most popular stimulus-responsive polymers for research. It is especially of great interest in the field of tissue engineering. While it is known that the NIPAM monomer is toxic, there is little conclusive research on the cytotoxicity of the polymer. In this work, the relative biocompatibility of the NIPAM monomer, pNIPAM, and pNIPAM-coated substrates prepared using different polymerization (free radical and plasma polymerization) and deposition (spin coating and plasma polymerization) techniques was evaluated using appropriate cytotoxicity tests (MTS, Live/Dead, plating efficiency). Four different mammalian cell types (endothelial, epithelial, smooth muscle, and fibroblasts) were used for the cytotoxicity testing. The pNIPAM-coated surfaces were evaluated for their thermoresponse and surface chemistry using X-ray photoelectron spectroscopy and goniometry. We found that while cell viability on pNIPAM surfaces decreases when compared to controls, the viability also seems to be deposition type dependent, with sol-gel based pNIPAM surfaces being the least biocompatible. Long term experiments proved that all pNIPAM-coated surfaces were not cytotoxic to the four cell types evaluated in a direct contact test. Plating efficiency experiments did not show cytotoxicity. Cellular sensitivity to pNIPAM and to the NIPAM monomer varied depending on cell type. Endothelial cells consistently showed decreased viability after 48 hours of exposure to pNIPAM extracts and were more sensitive than the other cell lines to impurities in the polymer.