Photothermally Induced Local Dissociation of Collagens for Harvesting of Cell Sheets

Abstract

The local heating of poly(3,4-ethylenedioxythiophene) (PEDOT) by ap hotothermal effect directed by nearinfrared (NIR) light induces unfolding of absorbed collagen triple helices,yielding soluble collagen single-helical structures. This dissociation of collagens allowed the harvesting of aliving idiomorphic cell sheet, achieved upon irradiation with NIR light (l = 808 nm). The PEDOTlayer was patterned and cells were successfully cultured on the patterned substrate.C ell sheets of various shapes mirroring the PEDOTp attern could be detached after af ew minutes of irradiation with NIR light. The PEDOTp atterns guided not only the entire shape of the cell sheets but also the spreading direction of the cells in the sheets.T his photothermally induced dissociation of collagen provided af ast non-invasive harvesting method and tailormade cell-sheet patterns.Collagen, which has at riple-helical structure,i st he one of the most important proteins in cell science. [1][2][3][4] Each triple helixisstabilized by numerous hydrogen bonds and supports cell binding.H owever,t he triple helix of collagens unfolds upon heating. [5] Therefore,c ontrolling the dissociation of collagen could be highly important for cell-to-surface interactions and eventually for the detachment of cells.Most celldetachment studies are undertaken using temperatureresponsive culture dishes on which thermoresponsive polymers,s uch as poly(N-isopropylacrylamide) (pNIPAAm), are covalently grafted. [6][7][8][9][10] Although this is one of the most promising methods and is well-established, spatial control of dissociation is particularly difficult using these thermoresponsive substrates.N ear-IR (NIR) laser irradiation is "biologically friendly" and can be employed to provide au nique method to spatially control cell behavior. [11][12][13][14] By spatially dissociating collagens through the photothermal effect, we now describe,for the first time,tunable cell-sheet detachment and harvesting from ap oly(3,4-ethylenedioxythiophene) (PEDOT) substrate without achange in the cell morphology.PEDOTwas directly coated onto polystyrene by polymer solution casting to form SP-PEDOT (PEDOT made by solution casting polymerization (SP)). Then collagen type I (0.3 wt %) was dropcast onto the SP-PEDOT substrate.T he thickness of the collagen layer was 14-18 mm, as determined using an Alpha-Step surface profiler.Fibroblasts were seeded on top of ac ollagen-coated PEDOT substrate (CSP-PEDOT) at ac oncentration of 310 5 cells/dish and were cultured for 3days.T he CSP-PEDOTw as irradiated with aN IR diode laser (l = 808 nm), with an input power density (I pw )between 1.9 Wcm À2 and 2.5 Wcm À2 (see Table S1 in the Supporting Information). When exposed to NIR light, the temperature of the CSP-PEDOTw as increased to 41.4 8 8C ( Figure S1). After 5min of irradiation, the cell sheets were detached from the substrate (Figure 1a). Theh arvested cell sheets showed positive E-cadherin expression (Figure 1b), verifying that cell-cell interactions are maintained in the harvested cell sheet...

Protein-Engineered Large Area Adipose-derived Stem Cell Sheets for Wound Healing

“…To harvest the cell sheets, CPP-PEDOT substrates covered with confluent hADSCs were irradiated from the bottom face of TCPS with a coherent NIR laser (λ = 808 nm, I pw = 2.7 W cm −2 ). The temperature at the CPP-PEDOT, increased up to 43 °C which is above the temperature of dissociation of collagen 25 , 26 leading cell sheet detachment as shown in Fig. 1g .…”

“…A photothermal polymer film (PP-PEDOT) was prepared on a tissue culture polystyrene dish (TCPS) through a solution casting polymerization (SCP) process using a solution containing EDOT, pyridine, a polymeric surfactant (PEPG), and Fe(III) p-tosylate, similar to the polymer film (SP-PEDOT) for the formation of a human dermal fibroblast sheet 25 . The prepared PEDOT-coated substrate went through 5 times of washing step (dipped in fresh ethanol for 2 h and washed out) to remove any residual impurities.…”

“…We have previously shown that collagen-coated PEDOT substrates are effective for the preparation of a human dermal fibroblast sheet 25 . Contrary to the human dermal fibroblasts cells, which were adhered and were homogeneously distributed on the collagens coated PEDOT surfaces, the hADSCs were not evenly adhered onto the CPP-PEDOT surfaces and left a partially aggregated mass when the hADSCs were seeded at 4 × 10 5 cells/dish.…”

“…1c ) and collagen layer is in-between cells and PEDOT film, cells are protected from direct exposure to a NIR source. As the collagens of triple helix structure in the film become unfolded into single strands at the light exposed area (43 °C), they are dissolved into solution media and the CS interactions are lost 25 , 26 . On the contrary, the CC interactions would stay intact to maintain as a sheet form during the harvesting of sheets due to CAM (Fig.…”

“…As observed before, the temperature of the cell medium at the same condition was 37~38 °C that could not damage cells within short NIR irradiation time (<10 min). For this reason, the harvested cell sheets showed a high viability after harvesting by photothermal method 25 , 26 .…”