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Biomaterials 34 (2013) 11e18

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Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

Light-induced cell detachment for cell sheet technology

Yi Hong a,1, Mengfei Yu b,1, Wenjian Weng a,c,1, Kui Cheng a,*, Huiming Wang b, Jun Lin b

aDepartment of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Zhejiang University, No. 38, Zheda Road, Hangzhou 310027, Chinab The First Affiliated Hospital of Medical College, Zhejiang University, Hangzhou 310003, Chinac The Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, China

a r t i c l e i n f o

Article history:Received 24 August 2012Accepted 20 September 2012Available online 12 October 2012

Keywords:Cell detachmentLight-inducedTitanium dioxideNanodots film

* Corresponding author. Tel.: þ86 571 87953945; faE-mail address: chengkui@zju.edu.cn (K. Cheng).

1 Contributed equally.

0142-9612/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2012.09.043

a b s t r a c t

The phenomenon of light-induced cell detachment is reported. Mouse calvaria-derived, pre-osteoblastic(MC3T3-E1) cells were cultured on a TiO2 nanodot-coated quartz substrate. After 20 min of UV365illumination, over 90% of the cells would detach from the surface. Moreover, intact cell sheets could beobtained in the same way. It was found that the as-obtained cells showed good viability, and could beused for further culture processes and other applications. Also, biocompatibility and safety character-izations indicated that the use of TiO2 nanodots and UV365 illumination was safe for such cell detach-ment. It is suggested that adsorbed extracellular matrix proteins play key roles in developing cell sheetsand ensuring biocompatibility. The present light-induced cell detachment method demonstratesa promising way for rapid cell/cell sheet harvesting.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The cell is a key object in biological, medical, pharmaceuticalsciences andmany other scientific areas. To date, in vitro cell cultureremains one of the most common ways to obtain various types ofcells for research. Because numerous cultivatable cells areanchorage-dependent, which means that the cells only survivewhen attached to the culture surface mediated by pre-adsorbedextracellular matrix (ECM) proteins [1,2], the harvesting ofcultured cells becomes a key issue. In typical cell culture protocols,enzymatic treatment is inevitable, so that ECM proteins are diges-ted and cultured cells can be released. This treatment is invasivebecause cell surface proteins, such as ion channel proteins, receptorproteins, and cell-to-cell junction proteins, are also digested.

As an alternative, a temperature-induced cell harvestingmethod, based on the mechanism that extracellular matrix gener-ally adheres to a hydrophobic surface rather than highly hydro-philic surfaces [3,4], has been developed. Through a thermallyresponsive poly(N-isopropylacrylamide)-coated culture surface[5e8], a temperature-induced hydrophobicehydrophilic transitionoccurs, leading to cells being harvested as single cells or even in anintact layer of confluent cells (i.e., a cell sheet). Due to the preser-vation of ECM and cell junctions, this temperature-induced cellharvesting is less invasive and thus significant. In fact, the

x: þ86 571 87953787.

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acquisition of intact cell sheets from this method has resulted ina new direction in tissue engineering strategies, referred to as cellsheet technology. Indeed, this technology is rapidly becoming a hotarea of research because of the directly transplantable cell sheets orthree-dimensional layers, composed of different individual cellsheets, that are possible [5,9,10].

Although other still less invasive cell harvesting methods wouldbe useful for cell sheet technology, only a few other methods havebeen developed, such as electricity-induced [11], magnetism-induced [12], and pH change-induced [13,14] methods. In theelectricity-induced method, cells are cultured on an electricity-responsive thiol layer and detached through detachment of thethiol layer. In the magnetic method, a magnet is placed under theculture dish to immobilize magnetic nanoparticles on which toform a surface layer. Then, the cells are cultured on this layer anddetached upon removal of the magnet. In both methods, however,detached cells or cell sheets may contain residual materials fromthe layer beneath. In the pH change-induced method, cells arecultured on a pH-responsive culture surface. With pH variation, thesurface changes from electropositive to electronegative, while ECMproteins remain negatively charged at such physiological pH values[15]. As a result, ECM proteins and cells release from the surfacebecause of charge repulsion. However, regulation of pH value isusually realized through diffusion of protons or hydroxyl groups,which may easily result in local deviations from the physiologicalpH values and eventually harmful to cells.

To date, most of these less invasive cell harvestingmethods haveused surface property variations to induce cell detachment. Amongthem, changes in wettability and charge are considered to be more

Y. Hong et al. / Biomaterials 34 (2013) 11e1812

favorable for cells. In view of these surface properties, light illu-mination is another way to induce a transition in surface wetta-bility of certain materials [16,17]. Thus, light-induced surfaceproperty changes could potentially provide a more convenient andeffective approach for cell harvesting.

Some points must be considered if a light-induced approach isto be adopted. First is biocompatibility and second is wavelength.The wavelength should be safe for the cells and it should not bepossible to induce cell detachment during the normal culturingprocess. Based on these considerations, titanium dioxide (TiO2) isbelieved to be one of the best candidates because it has both goodbiocompatibility [18] and light-induced wettability variationunder illumination below 380 nm [16,17], although several othermaterials have been reported to have light-induced super-hydrophilicity. It has been demonstrated that 365 nm ultraviolet(UV) illumination is safe for cells, even up to doses of thousands ofmJ/cm2 [19e21]. Moreover, nanoscale surface topography hasbeen reported to greatly influence surface wettability [22,23],improve lightesurface interaction through incident light scat-tering [24], and improve cell adhesion [25]. From these consider-ations, a nanostructured TiO2 surface and 365 nm UV may bea reasonable combination to investigate for light-induced celldetachment.

In this study, a new light-induced cell detachment approachwasinvestigated. TiO2 nanodot films developed in our previous work[26e28] were chosen for the culture surface. Cell detachmentbehavior under 365 nm UV (UV365) illumination and the viabilityof the detached cells and cell sheets were investigated. Also, to gaininsights into the light-induced interface response, the proteinadsorption behavior of the TiO2 nanodot surface under UV365illumination was also examined.

2. Materials and methods

2.1. Preparation and characterization of TiO2 nanodot film

A TiO2 nanodot film (TN) was prepared on a quartz substrate through phaseseparation-induced self-assembly [24]. Briefly, a precursor sol containing titaniumtetrabutoxide (TBOT, Sinopharm Chemical Reagent, CP, >98%), acetylacetone (AcAc,Lingfeng Chemical Reagent, AR, >99%) and polyvinyl pyrrolidone (PVP, K30, Sino-pharm Chemical Reagent, AR, >99%), was spin-coated on the surface and allowed tophase-separate after further heat treatment at 500 �C. A TiO2 nanodot film wasobtained. The diameter of the nanodots ranged from 30 to 110 nm, and the dotdensity was about 5.6 � 1010/cm2. The dots were polycrystalline. The nanodots werecharacterized by transmission electron microscopy (TEM; FEI, F-20).

2.2. Cell culture

Mouse calvaria-derived, pre-osteoblastic MC3T3-E1 cells (CRL-2594, ATCC)were used as model cells. Subconfluent MC3T3-E1 on polystyrene (PS) dishes weretrypsinized with 0.25% trypsin/1 mM EDTA (Gibco) and were subcultured on TN orTN-free quartz or on PS with alpha-modified Minimum Essential Medium (MEMAlpha, Gibco), supplemented with 10% fetal bovine serum (FBS, PAA, Australia), 1%sodium pyruvate (Gibco), 1% antibiotic solution containing 10,000 units/mL peni-cillin and 10,000 mg/mL streptomycin (Gibco), 1% MEM non-essential amino acids(Gibco) in a humidified atmosphere of 5% CO2 at 37 �C.

2.3. Cell attachment and detachment assay

2.3.1. UV resources and illumination methodA cold LED UV light was used to eliminate any interference by heat. A UV light

with a wavelength of 365 nm and a power of 2.0 mW/cm2 was used. The trans-mittance power was measured to be 1.4 mW/cm2. As calculated following thelongest illumination time in this work (40 min), the total energy was about 3360mJ/cm2, much lower than the reported safe value (7500 mJ/cm2).

2.3.2. Single cell attachment and detachment assayMC3T3-E1 cells were seeded on TN-coated quartz (1 � 1 cm2) and PS at a final

cell density of 5 � 104 cells/cm2 and cultured for 24 h. Before UV365 illumination,the samples were rinsed gently with PBS three times. After the intended time ofUV365 illumination, samples were rinsed gently again before observation witha phase contact microscope (CKX41, Olympus, Tokyo). The MTS assay was used to

evaluate residual cells on the surface [29], so that the detachment ratio could becalculated. Cells detached from TN after 20 min of UV365 illumination were seededon TN again to evaluate cell reattachability. Themorphology of reseeded cells after 1,3, and 5 days was also observedwith a phase-contrast microscope. As a control, cellsharvested with 0.25% trypsin/1 mM EDTA treatment were also tested forreattachability.

2.3.3. Cell sheet and detachment assayMC3T3-E1 were seeded on TN and TN-free quartz (1 � 1 cm2) in tissue culture

dishes at a final cell density of 1 � 105 cells/cm2 and cultured for 5 days to obtainconfluent cell sheets. Both the TN and TN-free samples were illuminated by UV365for 20 min. Then, cell sheets on TN were acquired readily. This operationwas carriedout gently and quickly to avoid damage to the cell sheets. The detachment processwas monitored using a digital camera (Nikon 3100, Tokyo).

2.4. Cell viability and proliferation

2.4.1. Viability by flow cytometry measurementsMC3T3-E1 cells were seeded on quartz (diameter: 4.5 cm) with TN and PS at

a final cell density of 1 � 105 cells/cm2. After 24 h, the samples were transferred tonew tissue culture dishes for 20, 40, or 60 min of UV365 illumination. Then, allsamples were kept in culture in the incubator for another 24 h. Annexin V-fluo-rescein isothiocyanate (FITC, 0.1 mg/mL) was used to assess phosphatidylserine (amarker of cellsinitiating apoptosis) and propidium iodide (PI; 0.5 mg/mL) was usedfor cell viability analysis. Cell death and apoptosis were measured using a FACSCantoII flowcytometer (Becton Dickinson, Mountain View, CA). Compensationwas used asnecessary.

2.4.2. Integrity of DNAThe integrity of DNAwas evaluated through oxidative damage. An 8-hydroxy- 2-

deoxyguanosine (8-OH-dG) EIA kit (SKT-120-96, StressMarq) was used to determinethe amount of 8-OH-dG, an established marker of oxidative stress [30e33]. MC3T3-E1 cells were seeded on TN-coated quartz (1 � 1 cm2) and PS in culture dishes ata final cell density of 5 � 104 cells/cm2 and cultured for 24 h. The culture mediumfrom each sample was collected after adding 500 mL serum-free medium to eachwell and 20, 40, or 60 min of UV365 illumination. Then, the collected culture mediawas characterized for the relative amounts of 8-OH-dG following standard proce-dures [34e36].

2.4.3. Live-dead staining of harvested cell sheetsThe cell sheet viability was assessed using a live/dead viability/cytotoxicity assay

kit (Invitrogen), based on a simultaneous determination of living and dead cells withtwo probes, calcein-AM for intracellular esterase activity and ethidium homodimer-1 for plasma membrane integrity [37]. Cell sheets were first rinsed three times withPBS and then incubated with a mixture of 1 mM calcein-AM and 2 mg/mL ethidiumhomodimer-1 for 30 min [14]. After rinsing three times with PBS, cell sheets wereexamined using a fluorescence microscope (IX81, Olympus, Tokyo, Japan), andanalyzed with the Image-pro plus 6.0 software. As a negative control, a piece of cellsheet was also tested following the same procedure after 3 min cytocidal treatmentin ethanol.

2.4.4. Alkaline phosphatase expressionThe cell sheets obtained from TN after 20 min UV365 illumination were

treated with cell lysis buffer (9803, Cell Signaling) and used to assess alkalinephosphatase (ALP) expression. Cells cultured in a standard PS dish following thenormal protocol were also processed as a control. Proteins were separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 5%TriseHCl reducing gels, ALP (ab65834, Abcam) expression was assessed byWestern blotting with HRP-anti-Rb antibody (Lot no. 050884, KPL). Band densitieson the Western blots were assessed using the Quantity One software (VersaDoc5000, BioRad).

2.5. Protein adsorption and influence of adsorbed proteins

2.5.1. Surface composition analysisX-ray photoelectron spectroscopy (XPS, Kratos AXIS Ultra DLD) was used to

characterize the surface composition of TN and PATN samples with an Al Ka source(1486.6 eV). A detailed scan for N and Ti was carried out with a step of 0.1 eV. Ti 2 p,at 458.0 eV, was used for calibration.

2.5.2. Wettability measurementThe water contact angle (WCA) was determined using a sessile drop method

with a contact angle meter (Dataphysics, OCA20). Three replicate measure-ments were recorded for each value point. Before measurement, the sampleswere kept in the dark and under vacuum (1 Pa) for 8 h, and then exposed toUV365 for 10e60 min in air; WCA was then measured after the designated timeintervals. Also, some TN samples were soaked in culture medium for 24 h forproteins to adsorb (PATN), and were then used for WCA measurements in thesame manner.

Y. Hong et al. / Biomaterials 34 (2013) 11e18 13

2.5.3. Effects of adsorbed proteins on photochemical reactionThe effects of protein adsorption on free radical production were evaluated

through electron spin resonance (ESR, JEOL JES FA200) at room temperature. A flatcell, modulation amplitude of 1 G, microwave power of 1 mW, and a microwavefrequency of 9.4 GHz were used for characterization. Samples for ESR studies weresoaked in solutions containing the spin-trapping reagent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO; 3 mM). The mixture was illuminated with UV light (l ¼ 365 nm,P ¼ 2.0 mW/cm2) for 30 min and transferred to the ESR measurement cells.

A disinfection assay was used to evaluate the effects of protein adsorption.S. aureus bacteria (ATCC 25293) were suspended in LB Broth to provide a finaldensity of 1 � 105 cells/mL. Then, 100 mL of the S. aureus bacterial suspensions wasseparately dripped onto TN, PATN, and PS. After 60 min, the solutions were dilutedappropriately with NaCl solution (0.9%) and plated on glass Petri dishes using platecount agar. After incubation for 24 h at 37 �C, CFUs were then counted. When theS. aureus number was <5 CFU/mL, it was considered that no CFUs were observed.

2.6. Statistical analysis

All values are expressed asmeans� standard deviation. Statistical analyses werecarried out by a one-way analysis of variance (one-way ANOVA) and Scheffe’s posthoc test with the SPSS software for multiple comparison tests, or using Student’s t-test. Differences were considered statistically significant when p < 0.05.

3. Results and discussion

3.1. Light-induced cell detachment

3.1.1. Efficiency of light-induced cell detachmentAfter a 24 h culture at 37 �C, cells attached and spread well on

both substrates (Fig. 1A) and the number of attached cells on TN

Fig. 1. (A) Morphology of MC3T3-E1 cells after 24 h in culture and different UV365 illumbehavior from TN and PS with differing UV365 illumination times. The closed red circlesinterpretation of the references to colour in this figure legend, the reader is referred to the

was similar to that on PS (Fig. 1B). Cell detachment behaviors onthese surfaces with differing illumination times were furtherinvestigated after culturing cells for 24 h (Fig. 1). With increasedUV365 illumination time, more and more cells contracted theirfilopodia, became spherical, and detached from TN(Fig. 1A).However, the cells on PS still spread well, and no signifi-cant change was found even after 40 min of UV365 illumination.The detachment ratio of cells on TN after 20 min of UV365 illumi-nation was 90.5%. This detachment rate is much higher than otherreported harvesting methods [6,13]. With increased UV365 illu-mination time beyond 20 min, the detachment ratio did not changesignificantly (Fig. 1B). The detachment ratio with 20 min of UV365illumination is actually comparable to trypsin treatment, whichusually gives a level of 85%.

Similarly, a MC3T3-E1 cell sheet cultured on TN surfacedetached as a continuous and intact cell sheet after 20 min ofUV365 illumination. That means TN films did response to UV365light and led the cell sheet to detach. If trypsin treatment is used,cell-to-cell junction proteins are readily digested, and eventuallylead single cells, not cell sheets, to detach from the culture surface.The harvesting of the sheets with the present method indicatedthat the light-induced effects had little or no influence on cell-to-cell junction proteins.

Compared with other cell sheet detachment methods, light-induced cell detachment exhibits advantages on no materialsresidual and no local environmental changes. That makes it suitable

ination times on TN and PS, Scale bar ¼ 100 mm. (B) Time-course of cell detachmentrepresent the number of attaching cells on PS; the closed black diamonds, TN. (Forweb version of this article.)

Fig. 2. (A) Cell viability of samples examined by flow cytometry. (B) Proliferation of reseeded cells examined by MTS (C) Cell morphology of reseeded cells detached using trypsintreatment and 20 min of UV365 illumination from TN. Scale bar ¼ 100 mm. (D) Oxidative damage of DNA assessed using 8-OH-dG. All data in these experiments were determinedfrom three independent cultures and are expressed as means � standard deviation. No significant difference was found.

Y. Hong et al. / Biomaterials 34 (2013) 11e1814

Y. Hong et al. / Biomaterials 34 (2013) 11e18 15

for cell sheet acquirement. Moreover, the easy regulation of lightwavelength, illumination intensity and time makes it possible toaccurately control cell sheet detachment.

3.1.2. Viability of light-induced detached cellsThe survival of MC3T3-E1 cells detached using UV365 illumi-

nationwas assessed using flow cytometry. All of the detached cells,with differing times of illumination, showed normal cells above97%, death of around 0.6%, and apoptosis of less than 2.0% (Fig. 2A).Compared with trypsin treatment, no significant difference wasfound. This result demonstrates that cells harvested by the light-induced detachment method had viability as good as with trypsintreatment.

The reattachment ability of detached cells is another importantindicator of activity, because cells need to be harvested and thenreattach repeatedly in the cell passage process. The reattachmentbehavior of MC3T3-E1 cells on TN is shown in Fig. 2B and C. Thelight-detached cells could reattach on TN, and showed similar cellmorphology and proliferation to cells harvested by trypsin treat-ment. This indicates that, morphologically, light-detached cellsshow good reattachability.

A more detailed characterization was carried out at a molecularbiology level on DNA integrity. As shown in Fig. 2D, no significantdifference in 8-hydroxy-2-deoxy guanosine (8-OH-dG) concentra-tion was found for different illumination times in comparison withthe control group. Because 8-OH-dG is considered a marker for theoxidative damage of DNA [32,33], this result shows that the cellsobtained with the present light-induced detachment method havegood viability without any significant DNA oxidative damage.

Fig. 3. (A) Viability of MC3T3-E1 cell sheet demonstrated by live-dead staining with calceinEthanol treatment is the negative control. (B) and (C) Western blot analysis of ALP expremeans � standard deviation. Asterisks (*) indicate significant differences (p < 0.01) compapretation of the references to colour in this figure legend, the reader is referred to the web

Also, the survival of a detached cell sheet was tested immedi-ately by live-dead staining. As shown in Fig. 3A, live-dead stainingperformed after detachment demonstrated that cell survival wasnot significantly compromised. Western blot analysis (Fig. 3B, C)also showed that alkaline phosphatase (ALP) expression of cellsobtained from cell sheets was much higher than cells on PS afterthe same time in culture. This high expression of ALP could beattributable to the nanostructured TN promoting cell proliferationand differentiation [25]. Additionally, ALP is an early mineralizationmarker, which also reveals cell type-specific trends in osteoblasticcommitment [38]. This result demonstrates that cell sheets har-vested by the light-induced method had good activity and werewell functionalized, and can be used in tissue engineering.

3.2. Analyses of cell-free TN surfaces under UV light illumination

3.2.1. Evolution of adsorbed protein with UV light illuminationDuring cell adhesion, proteins from the culture medium first

adsorb on the surface and then mediate the adhesion and detach-ment behaviors of cells [2]. As shown in Fig. 4A, an obvious N1ssignal at 399.6 eV was observed, indicating the existence ofproteins on the soaked TN samples (protein-adsorbed TN, PATN).With increased illumination time, the N1s signal decreased gradu-ally, while the Ti2p signal increased accordingly. After 60 min ofUV365 illumination, the N1s signal was still obvious and the Ti2psignal, although it had increased significantly, was still weaker thanthat without adsorption.

HRTEM images showed that the proteins formed a compactlayer on the surface of individual nanodots (Fig. 4C) in comparison

(green), specific for live cells, and ethidium homodimer (red), specific for dead cells.ssion. Data were determined from three independent cultures and are expressed asred with PS, calculated using one-way ANOVA followed by a post hoc test. (For inter-version of this article.)

Fig. 4. (A) XPS characterization of differently treated samples. TEM image of nanodots from (B) non-treated TN, (C) PATN, (D) 20-min UV365 illuminated PATN (E) 60-min UV365illuminated PATN (F) cell sheet-detached TN. (Arrows indicate the protein adsorption).

Fig. 5. Time-course of water contact angles with differing UV365 illumination times.The closed red squares indicate PATN, the closed black diamonds, TN. (For interpre-tation of the references to colour in this figure legend, the reader is referred to the webversion of this article.)

Y. Hong et al. / Biomaterials 34 (2013) 11e1816

with that without adsorption (Fig. 4B). After UV365 illumination(Fig. 4D and E), PATN still showed the presence of proteins, whilethe thickness of the protein layer on PATN became thinner as illu-mination time increased. Both the HR TEM and XPS results indicatethat some of the adsorbed proteins were released from theadsorption layer on UV illumination, while others remained. Asshown in Fig. 4F, a more “real” situation observed on the nanodotsscratched from a post-detached TN surface further supported thatsome proteins remained on the nanodots surface.

Obviously, proteins adsorbed rapidly on the surface of TiO2nanodots during in vitro cell culture, and were released graduallyduring UV365 illumination. Such behavior may, in turn, influencethe surface properties of the TiO2 nanodots and eventually influ-ence cell attachment on the surface.

3.2.2. Response of PATN under UV light illuminationSurface property variations resulting from protein release were

further investigated through water contact angle evaluations. TheWCA of non-adsorbed TN dropped rapidly with illumination time(Fig. 5), and the surface turned into superhydrophilic after 20 minUV365 illumination. It is noteworthy that the WCA of PATN drop-ped much more slowly than non-adsorbed TN. The WCA of PATN atthe time of cell sheets detachment was around 60 �, that is in wellagreement with the reported threshold WCA of cells attachmentand detachment on poly(N-isopropylacrylamide) [6,39]. Theseresults indicate that the surface wettability and protein releaseprovide a basis for meeting the requirement of cell detachmentfrom the culture substrate surface. The protein release during thetransition from hydrophobicity to hydrophilicity is possibly themain reason for the light-induced cell detachment.

It is well-known that TiO2 inwater with UV illumination leads tothe generation of light-induced hydroxyl radicals. It has been re-ported that bare TiO2 with UVA has strong germicidal effectsbecause of oxidative damage caused by hydroxyl radicals [40]. TN atthe very beginning (5 min) of the UV365 illumination had anobviously strong hydroxyl radical signal (Fig. 6A), and behavednormally. However, with PATN, no hydroxyl radical signal wasobserved even after 30 min of illumination. This result

demonstrates that the conventional hydroxyl radical-productionreaction does not happen in a protein-adsorbed system. Rajen-dran’s study suggested that protein-conjugated CdS particleseffectively suppressed the production of radical species [41]. That isconsistent with our result, indicating that the reaction in protein-coated TiO2 differs from the conventional photochemical reaction.

The disinfection assay was designed to examine the effects ofthe adsorbed protein layer at a biological level. The nucleoid ofbacteria is more sensitive to oxidative damage because of the lackof protection by a nuclear membrane. The disinfection resultdemonstrated that bare TiO2 (TN) did kill the bacteria effectivelyafter 60 min of UV365 illumination; indeed, only about 20% of thebacteria survived. However, the protein-adsorbed TiO2 (PATN)showed no disinfection effect after 60 min of UV365 illumination

Fig. 6. (A) ESR analysis of TN and PATN after differing UV365 illumination times. (B) Disinfection assay of TN and PATN with 60 min of UV365 illumination. Data were determinedfrom three independent cultures and are expressed as means � standard deviation. Asterisks (*) denote significant differences (p < 0.01) compared with PS, calculated using a one-way ANOVA followed by a post hoc test.

Y. Hong et al. / Biomaterials 34 (2013) 11e18 17

(Fig. 6B). This result further illustrates that the reaction happeningon the protein-adsorbed TiO2 behaves in a different way than onbare TiO2 under UVA illumination. It also implies that such PATNhas no oxidative damage to cells, i.e., the light-induced celldetachment is safe for cells.

In general, this light-induced cell detachment is most probablycaused by adsorbed protein release on the basis of Fig. 4, and therelease of adsorbed protein is triggered by the light-induced tran-sition from hydrophobicity to hydrophilicity on the basis of Fig. 5.However, the exact process and mechanism of the release ofadsorbed proteins remain unclear. Further study is ongoing toinvestigate the interaction between TiO2 and the protein layer.

4. Conclusions

This work demonstrated that a light-sensitive TiO2 nanodot filmshowed advantages in the light-induced detachment of culturedcells on easy operation and accurate control. Cells/cell sheets can beharvested effectively with good cell viability comparable to enzy-matic method. Our findings suggest that light-induced celldetachment is closely related to protein adsorption and releasingbehavior. Further studies should be carried out to clarify the exact

mechanism reason of cell detachment. Moreover, such light-induced cell detachment was shown to be safe and efficient.Thus, a new harvesting process for in vitro cultured cells may bedeveloped based on this phenomenon, and it has a promisingfuture in tissue engineering applications.

Acknowledgments

This work is financially supported by National Natural ScienceFoundation of China (30870627, 81071258, 81171003, 51072178),National Basic Research Program of China (973 Program,2012CB933601) and Science & Technology Department of ZhejiangProvince of China (2010C33088).

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