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

Sakulaue, Phongphot; Swe, Aye Yu Yu; Benchaprathanphorn, Kanokaon; Lertvanithphol, Tossaporn; Viravaidya-Pasuwat, Kwanchanok; Siriwatwechakul, Wanwipa

doi:10.1021/acsomega.8b02514

Cited by 22 publications

(32 citation statements)

References 48 publications

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“…To confirm the relationships obtained from the prediction model, random samples prepared with different UV irradiation times and spin‐coating speeds were measured for their grafting density and thickness. Our previous study showed that by increasing the UV exposure time, the grafting thickness increased, and by increasing the spin‐coating speed, the consistency of the film thickness was improved 39 . The grafting density–thickness model was used to predict the grafting density of the samples, prepared with six different conditions with known thickness.…”

Section: Resultsmentioning

confidence: 99%

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

Sakulaue

Lertvanithphol

Eiamchai

et al. 2020

Surface & Interface Analysis

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Poly(N‐isopropylacrylamide‐co‐acrylamide) (PNIAM‐co‐AM) grafted onto tissue culture polystyrene surfaces (TCPSs) with different thicknesses and grafting densities were prepared by varying the spin‐coating speed and UV irradiation time for use in cell sheet engineering. The thickness of the PNIAM grafted surface, less than 30 nm, was believed to influence protein adhesion and cell attachment. However, methods for determining the thicknesses of a thin surface (nanometer scale) are still a challenge. In this study, a model was developed to relate the thickness of the PNIAM‐co‐AM film to its grafting density. Spectroscopic ellipsometry (SE) and attenuated total reflection‐Fourier transform infrared spectroscopy (ATR‐FTIR) with synchrotron radiation were used to quantify the thickness and the grafting density, respectively. The amount of the grafted copolymer was determined from quantitative ATR‐FTIR by relating the signals at wavenumber 1,654 to 1,600 cm−1 (A1,654/A1,600) to the known grafting density. A combination of SE and ATR‐FTIR was used to construct a linear model (h = 6.29σ + 2.24), to obtain the grafting density (σ) from the film thickness (h) with R2 = 0.93. To validate the model, random samples were prepared with different thicknesses and grafting densities. The linear model was used to predict the grafting density, which was nearly equal to the measured values.

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Section: Resultsmentioning

confidence: 99%

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

Sakulaue

Lertvanithphol

Eiamchai

et al. 2020

Surface & Interface Analysis

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“…Biocompatibility of the copolymer is the most important feature to be used as a cell attachment substrate. Thus, the potential toxicity of PNIPAm-co -AAc-b -PS and PNIPAm-co -AAc-b -PS copolymers have been evaluated on RAW264.7 cell lines using in vitro MTT dye reduction assay (Fentahun Darge et al, 2021;Sakulaue et al, 2018). The toxicity of the copolymers was performed by quantifying the viability of cells treated with copolymers (PNIPAm-co -AAc-b -PS and PNIPAm-co -AAc-b -PS) for 24 h at a concentration range of 0.0005 to 5 mg/mL.…”

Section: Cytotoxicity Test (Mtt Assay)mentioning

confidence: 99%

“…Therefore, unlike the bare cytodex 3, thermosensitive coated cytodex 3 microcarriers showed spontaneous cell detachments with reducing temperature lower than LCST. This temperature induced cell detachment strategy might be essential to harvest adequate viable cells with preserved cell features and functionality that are mostly lost during enzymatic cell dissociation (Kim et al, 2020;Sakulaue et al, 2018).…”

Section: Characterization Of Pnipam-co-aam-b-ps Coated Microcarriersmentioning

confidence: 99%

“…Because temperature triggered reversible hydrophobic to hydrophilic phase transition of the thermosensitive copolymers (PNIPAm-co -AAc-b -PS and PNIPAm-co -AAm-b -PS) promote cell adhesion and detachment, respectively (Kumashiro, Yamato, & Okano, 2010;N. Li et al, 2021;Sakulaue et al, 2018). The presence of repeating hydrophilic amide and hydrophobic isopropyl groups in PNIPAm chains allowed the copolymers to undergo reversible conformational change with temperature alterations in aqueous environments.…”

Section: Introductionmentioning

confidence: 99%

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Preparation of thermosensitive PNIPAm-based copolymer coated cytodex 3 microcarriers for efficient non-enzymatic cell harvesting during 3D culturing

Darge

Chuang

Lai

et al. 2021

Preprint

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Enzymatic detachment of cells might damage important features of cells and could affect subsequent function of cells in various applications. Therefore, non-enzymatic cell detachment using thermosensitive polymer matrix is necessary for maintaining cell quality after harvesting. In this study, we synthesized thermosensitive PNIPAm-co-AAc-b-PS and PNIPAm-co-AAm-b-PS copolymers and LCST was tuned near to body temperature. Then, polymer solutions (5% w/v, 10% w/v, and 20% w/v) were spin coated to prepare films for cell adhesion and thermal-induced cell detachment. The apha-step analysis and SEM image of the films suggested that the thickness of the films depends on the molecular weight and concentration which ranged from 206 nm to 1330 nm for PNIPAm-co-AAc-b-PS and 97.5 nm to 497 nm for PNIPAm-co-AAm-b-PS. The contact angles of the films verified that the polymer surface was moderately hydrophilic at 37°C. From cell attachment and detachment studies, RAW264.7 cells, were convincingly proliferated on the films to a confluent of >80 % within 48 days. However, relatively more cells were grown on PNIPAm-co-AAm-b-PS (5%w/v) films and thermal-induced cell detachment was more abundant in this formulation. As a result, commercial cytodex 3 microcarrier was coated with PNIPAm-co-AAm-b-PS (5%w/v) and interestingly enhanced cell detachment with preserved potential of recovery was observed at low temperature during 3D culturing. Thus, surface modification of microcarriers with PNIPAm-co-AAm-b-PS could be vital strategy for non-enzymatic cell dissociation and able to achieve adequate number of cells with maximum cell viability, and functionality for various cell-based applications. Keywords: surface coated microcarriers; thermosensitive polymer; non-enzymatic cell detachment

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“…Nakayama et al reported that a polyNIPAm-coated petri dish was used successfully to culture endothelial cells while the cells were able to detach from the polyNIPAm-coated petri dish when the culture temperature was lowered from 37 °C to 20 °C [ 10 ]. Similarly, Sakulaue et al found that mouse preosteoblast MC3T-E1 cells were able to detach from a poly( N -isopropylacrymide- co -acrylamide)-grafted culture surface upon reducing the culture temperature to 20 °C [ 11 ]. Furthermore, in a study by Capella et al [ 12 ], polyNIPAm films were shown to be biocompatible and non-cytotoxic to mouse predipocytes cells, human embryonic kidney cells (HEK293) and human lung carcinoma epithelial cells (A549).…”

Section: Introductionmentioning

confidence: 99%

Development of Thermally Responsive PolyNIPAm Microcarrier for Application of Cell Culturing—Part I: A Feasibility Study

et al. 2021

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Poly(N-isopropylacrylamide) (polyNIPAm) microspheres were synthesized via the suspension polymerization technique. Thermal and redox initiators were compared for the polymerization, in order to study the effect of initiator type on the surface charge and particle size of polyNIPAm microspheres. The successful polymerization of NIPAm was confirmed by FTIR analysis. Microspheres of diameter >50 µm were synthesized when a pair of ammonium persulfate (APS) and N,N,N’,N’-tetramethylene-diamine (TEMED) redox initiators was used, whilst relatively small microspheres of ~1 µm diameter were produced using an Azobis-isobutyronitrile (AIBN) thermal initiator. Hence, suspension polymerization using a redox initiator pair was found to be more appropriate for the synthesis of polyNIPAm microspheres of a size suitable for human embryonic kidney (HEK) cell culturing. However, the zeta potential of polyNIPAm microspheres prepared using an APS/TEMED redox initiator was significantly more negative than AIBN thermal initiator prepared microspheres and acted to inhibit cell attachment. Conversely, strong cell attachment was observed in the case of polyNIPAm microspheres of diameter ~90 µm, prepared using an APS/TEMED redox initiator in the presence of a cetyl trimethyl ammonium bromide (CTAB) cationic surfactant; demonstrating that surface charge modified polyNIPAm microspheres have great potential for use in cell culturing.

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

Cited by 22 publications

References 48 publications

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

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

Preparation of thermosensitive PNIPAm-based copolymer coated cytodex 3 microcarriers for efficient non-enzymatic cell harvesting during 3D culturing

Development of Thermally Responsive PolyNIPAm Microcarrier for Application of Cell Culturing—Part I: A Feasibility Study

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