Nanofabrication for micropatterned cell arrays by combining electron beam-irradiated polymer grafting and localized laser ablation.

Most methods reported for cell-surface patterning are generally based on photolithography and use of silicon or glass substrates with processing analogous to semiconductor manufacturing. Herein, we report a novel method to prepare patterned plastic surfaces to achieve cell arrays by combining homogeneous polymer grafting by electron beam irradiation and localized laser ablation of the grafted polymer. Poly(N-isopropylacrylamide) (PIPAAm) was covalently grafted to surfaces of tissue culture-grade polystyrene dishes. Subsequent ultraviolet ArF excimer laser exposure to limited square areas (sides of 30 or 50 microm) produced patterned ablative photodecomposition of only the surface region (approximately 100-nm depth). Three-dimensional surface profiles showed that these ablated surfaces were as smooth and flat as the original tissue culture-grade polystyrene surfaces. Time-of-flight secondary ion mass spectrometry analysis revealed that the ablated domains exposed basal polystyrene and were surrounded with PIPAAm-grafted chemistry. Before cell seeding, fibronectin was adsorbed selectively onto ablated domains at 20 degrees C, a condition in which the non-ablated grafted PIPAAm matrix remains highly hydrated. Hepatocytes seeded specifically adhered onto the ablated domains adsorbed with fibronectin. Because PIPAAm, inhibits cell adhesion and migration even at 37 degrees C when the grafted density is > 3 microg/cm2, all the cells were confined within the ablated domains. A 100-cell domain array was achieved by this method. This surface modification technique can be utilized for fabrication of cell-based biosensors as well as tissue-engineered constructs.

Thermally responsive polymer-grafted surfaces facilitate patterned cell seeding and co-culture.

Tissue engineering constructs that effectively duplicate natural tissue function must also maintain tissue architectural and organization features, particularly the integration of multiple cell types preserving distinct, integrated phenotypes. Cell-cell communication and biochemical cross-talk have been shown to be essential for the maintenance of differentiated cell functions in tissues and organs. Current limitations of cell-culture hinder progress in understanding the features and dynamics of heterotypic cell communication pathways critical to developing more sophisticated or effective tissue-engineered devices. We describe a method to conveniently electron-beam pattern cell culture surfaces with thermo-responsive polymer chemistry that exploits changes in cell-polymer adhesive interactions over a temperature window amenable for high-throughput cell culture. Cells seeded on these patterned surfaces at 20 degrees C adhere only to surface areas lacking thermo-responsive grafting chemistry; grafted domains at 20 degrees C are hydrophilic and non-cell adhesive. The culture temperature is then increased to 37 degrees C collapsing the hydrated grafted chemistry. A second cell type is added to the culture and adheres only to these exposed relatively hydrophobic grafted patterns. Both cell types can then be effectively co-cultured at 37 degrees C under multiple conditions. Long-term cell pattern fidelity and differentiated cell functions characteristic of each co-planar cell type are observed. This method is simple and has few limitations, compared with other existing co-culture methods.

Two-dimensional cell sheet manipulation of heterotypically co-cultured lung cells utilizing temperature-responsive culture dishes results in long-term maintenance of differentiated epithelial cell functions.

Here we report two-dimensional cell sheet manipulation (2D CSM) of heterotypically co-cultured lung cell sheets and the maintenance of differentiated phenotypes of lung epithelial cells over prolonged periods of up to 70 days. This was facilitated by poly(N-isopropylacrylamide) (PIPAAm)-grafted tissue culture dishes. PIPAAm-grafted dishes are responsive to temperature changes and offer a unique surface on which cells adhere and multiply like on ordinary tissue culture dishes under the permissive temperature of 37 degrees C, but on lowering of temperature resulting in changes in hydration of the polymer the cells spontaneously detach from the surface without use of enzymes like trypsin which is the common procedure. It has been well documented that type II pneumocytes of the lung lose many of their special features rapidly in culture. The culture system detailed here comprises random co-culture of epithelial and mesenchymal cells of lung. The heterotypic cell culture system promotes cell-cell interactions maintaining a harmonized physiology. When this heterotypic monolayer on PIPAAm-grafted dishes was subjected to lower temperature of 20 degrees C and 2D CSM we were able to transfer the monolayer as a single contiguous sheet with cell-cell connections intact to other surfaces. This non-invasive transfer of cell sheet resulted in shrinkage of the monolayer, enabling the type II cells to regain their cuboidal morphology and specialized characters like Maclura pomifera lectin binding and surfactant protein A (SP-A) expression. The active dome formation also observed subsequent to transfer reaffirms the uniqueness of the culture conditions and 2D CSM in future for developing tissue like architecture in vitro.