Transparent bioreactor based on nanoparticle-coated liquid marble for

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Biological and Medical Applications of Materials and Interfaces

Transparent bioreactor based on nanoparticle-coated liquid marble for in-situ observation of suspending embryonic body formation and differentiation Kejun Lin, Ruoyang Chen, Liyuan Zhang, Duyang Zang, Xingguo Geng, and Wei Shen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20169 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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ACS Applied Materials & Interfaces

Transparent bioreactor based on nanoparticle-coated liquid marble for insitu observation of suspending embryonic body formation and differentiation Kejun Lin,a,b,c Ruoyang Chen,a,b Liyuan Zhang,a,b,d,* Duyang Zang,c Xingguo Geng,c Wei Shena,b,* a

Department of Chemical Engineering, Monash University, Wellington Road,

Clayton Campus, Victoria 3800, Australia b

Monash Institute of Medical Engineering, Monash University, Wellington

Road, Clayton Campus, Victoria 3800, Australia c

Functional Soft Matter & Materials Group, Key Laboratory of Space Applied

Physics and Chemistry of Ministry of Education, School of Science, Northwestern Polytechnical University, Xi’an, 710129, China d

Science and Technology Institute, Wuhan Textile University, Jiangxia, Hubei

430200, China *Corresponding authors: Wei Shen; Liyuan Zhang Tel: +61399053447 E-mail: [email protected]; [email protected] 1 ACS Paragon Plus Environment

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KEYWORDS: transparent liquid marble; embryonic stem cells (ESCs); embryonic bodies (EBs), “one-pot” differentiation, nanoparticle-liquid interface ABSTRACT Transparent liquid marbles coated with hydrophobic silica nanoparticles were used as micro bioreactors for embryonic stem cell (ESC) culturing. The high transparency of silica liquid marbles enables the real-time and in situ monitoring of the embryonic body (EB) formation and differentiation. The experimental result shows that ESCs can aggregate one another close to the bottom of the liquid marble and form EBs while remaining suspended in the culture media. The differentiation of the suspending EBs into the contractile cardiomyocytes has been demonstrated inside the transparent liquid marbles, which enables the in situ microscopic observation. It was also found, through comparison, that ESCs in a bare sessile drop placed on a superhydrophobic substrate tend to anchor on the substrate and then differentiate following the normal way of cell spreading, i.e., withdrawal from the cell cycle, fusion with nascent myotubes, and final differentiation into cardiomyocytes. In contrast, liquid marble particle shell weakens the adhesion of spherical EBs to the substrate, encouraging them to differentiate in suspension into cardiomyocytes, without anchoring. The results of this study highlight the promising performance of liquid marble as a “one-pot” microbioreactor for EB formation and differentiation.

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1. INTRODUCTION A liquid marble, characterized by a liquid core covered by hydrophobic powder particles, was first reported by Aussillous and Quéré1 in 2000. The hydrophobic particle shell gives rise to the non-stick property of the liquid marble and provides a confined micro-environment for the core liquid inside the marble. Because of the mechanical robustness and elasticity of the particle shell,2-4 liquid marbles have been explored as a soft micro-reactor to control a wide range of chemical and biological processes.5-11 Our group has conducted a series of experimental trails to achieve the bioreactor function of the liquid marble.5, 6, 12, 13 Arbatan et al. explored the use of liquid marble as a micro bioreactor to culture tumour spheroids.6 This work demonstrated the feasibility of the liquid marble to be used for cell culture applications for the first time. Sarvi et al. carried out experiments to indicate that liquid marbles could serve as a platform for culturing the embryonic bodies (EBs) from embryonic stem cells (ESCs) and to demonstrate that EBs in liquid marble made from 35 m PTFE particles has higher efficiency and lower cell adhesion than that in liquid marble made from 100 m PTFE particles.12 Tian et al. used the liquid marble to cultivate microorganisms by taking advantage of the gas permeability of the liquid marble.5 Li et al. investigated the transfer of the mass and electric charge, showing the material mixing condition inside the liquid marble.13 Oliveira et al. showed that liquid marbles could be used as cell culture reactors for highthroughput drug screening by culturing the anchorage-dependent cells.14 Recently, transparent liquid marbles containing tumour cells were prepared to produce threedimensional (3D) tumour spheroids with high efficiency (50 - 200 spheroids in a liquid marble micro reactor) and apply the tumour spheroids to the high-throughput drug resistance test.15 Raja K. Vadivelu et al. also employed liquid marble as a simple 3D micro-reactors to study cell-cell interactions.16 Other works also reported the applicability and advantages of using liquid marbles to culture cells, with the ability to form 3D aggregates inside the marble, such as olfactory ensheathing cells.17 Pluripotent stem cells, including ESCs and induced pluripotent stem cells, have the ability to differentiate into a variety of specific lineages18,

19,

such as

cardiomyocytes20-22 and neurons23-25. When being cultured in suspension without any anti-differentiation factor, such as leukemia inhibitory factor (LIF) and feeder layer cells, ESCs can form 3D aggregates, or EBs, and spontaneously differentiate.26 It was previously shown that the quality of EBs, such as size, shape, and uniformity, played 3 ACS Paragon Plus Environment


an important role in the subsequent differentiation of ESCs into a variety of cell lineages in vitro.27 Various methods have been developed to form EBs, however, it is still challenging to obtain EBs with a controlled size and a high level of uniformity in an efficient manner. Current micro-scaled methods, such as the hanging-drop26, microwell28, 29 and microfluidic devices30, are typically involving two steps: the aggregation of ESCs in hanging drops or wells and the maturation of cell aggregates to EBs in suspension; these culturing methods relies on the use of the low adherent bacterial petri dishes. The common limitations of these methods are the significant loss of EBs during transfer of EBs from step one to step two; the attachment of premature EBs on to the petri dish presents a high risk for EBs to be damaged, despite that low adherent petri dishes are used. However, transfer of EBs from step one to step two has been thought to be necessary; the subsequent differentiation has been believed to occur after the progenitor cells anchor to a substrate. Given the limitations of the micro-scaled methods, the less laborious methods, e.g., the stirred-suspension culture method31, have been proposed. These methods, however, have a high potential to form the non-uniform EBs and cause the damage of the fragile cells, which affects the subsequent differentiation outcome. Using liquid marbles as micro bioreactors for the stem cell differentiation was first demonstrated by Sarvi et al.; these authors showed that EBs could be formed from ESCs in liquid marbles at high efficiency and quality.32 In particular, dozens of uniformity EBs could be formed in a single liquid marble, which requires fewer steps of preparation. In addition, the hydrophobic particle shell of the liquid marble is porous, stretchable, and air-permeable, which is expected to promote the multilayer cell-cell interactions within the particle-liquid system.33 Although previous research has compared the “bare” drop micro bioreactor (hanged on a substrate) and the liquid marble and found that the latter had higher efficiency to achieve the EBs with higher uniformity12, it was still not clear why the liquid marble bioreactor could facilitate the formation of the uniform EBs at such a high efficiency than a “bare” drop bioreactor. To find an answer to this question and to address the limitations of the existing methods for EB culturing, a transparent liquid marble bioreactor was designed in this work to generate the cardiac lineage from ESCs. Our aim is to develop an effective “one-pot” method for the generation and differentiation of EBs, i.e., using a liquid marble bioreactor to generate EBs from ESCs 4 ACS Paragon Plus Environment

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and to trigger the cell differentiation in suspension in the same reactor without transfer. The transparent liquid marble enabled the in situ observation of the culturing process, therefore, provided a capable tool to clarify the role of the hydrophobic shell of the liquid marble that makes it such a highly efficient bioreactor than a “bare” drop for cell cultivation. The novel “one-pot” method can avoid the transportation of the EBs to a separate substrate, which has been an essential step of all traditional methods, thus enhancing the efficiency of the cell generation and differentiation in the liquid marble bioreactor. 2. RESULTS AND DISCUSSION 2.1 In-situ observation of suspending EB formation To monitor the EB formation process, transparent liquid marbles were used as micro bioreactors to culture the Oct4B2-ESCs. The transparent liquid marble was fabricated by using the shaking-transfer approach34, which resulted in the coating of a monolayer of nanoparticles at the drop surface. Because of the low roughness of this nanoparticle monolayer, the liquid marble surface exhibits excellent transparency34, 35, as shown in Fig. 1(a). Due to the sufficient mechanical robustness of the nanoparticle shell36, the liquid marble containing cell culture media can be conveniently transport in the petri dish. The volume of the culture media was 300 µL, the nutrients of which were reported to be sufficient to keep the ESCs alive and active inside the liquid marble12. The capillary length (𝑙𝑐) can be calculated from2: 𝑙𝑐 = 𝛾𝑒𝑓𝑓 𝜌𝑔

(1)

where 𝜌 is the density of the culture media, 𝑔 is the gravitational acceleration, and 𝛾𝑒𝑓𝑓 is the effective surface tension of the liquid marble shell, which is ~84 mN/m37. This capillary length predicts that the liquid marble formed for this study has a puddle shape (Fig. 1(b)), since the size of the liquid marble was much larger than its capillary length. As illustrated in Fig. 1c, the cells aggregated to one another, forming the smallsized cell-clusters at the bottom of the liquid marble; this is due to the gravity and internal water flow.4, 38 During the culturing process, the high transparency of the silica nanoparticle shell enables the direct monitoring of the EB development and the conditions of the culturing media inside the liquid marble bioreactor; the colour of the media offers a convenient indication for evaluation of the nutrient replenishment through media change. After the incubation of 60 h, the old media within the marbles 5 ACS Paragon Plus Environment


was gently removed and replaced by the same amount of fresh media using a micropipette (Fig. 1c). Due to the internal water flow and gas permeability of the liquid marble, the culture medium and oxygen could be allowed to transport into the bottom of the liquid marble.15 For the initial period of culturing, Oct4B2-ESCs tended to form cell aggregates (Fig. 1d-f). After 30 h, the spherical EBs began to form and grew through further ESC aggregation and proliferation (Fig. 1g-i). From the morphology of EBs where no branches could be observed; it can be concluded that the EBs were remained suspending in the culture media without adhering to the particle shell of the liquid marble.

Figure 1. EB formation in a micro bioreactor (300 L). (a) and (b) Transparent liquid marble containing water (Top view) and culture media (Side view), respectively. (c) A 6 ACS Paragon Plus Environment

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schematic of the EB formation in a liquid marble. (d-i) Phase contrast images showing the formation process of an EB in a liquid marble. Scale bars = 100 µm. 2.2 “One-pot” differentiation of suspending EBs By utilizing the mechanical stability and volume-variability of silica liquid marbles, the ESCs could always be maintained in one liquid marble without being taken out from the microreactor. Therefore, the approach reported in this work was referred to the “one-pot” differentiation. Typically, about 6.5% of suspending EBs exhibited spontaneous contraction on day 9, as shown in Fig. 2. The percentage of contractile EBs increased over time, reaching a maximum of 75% on day 17; this is 10% higher than the previous study conducted using liquid marble micro bio reactor, and 45% higher than the result obtained using hanging drops.32 Sarvi et al.12 reported an observation result that the cardiomyocyte-like cells were formed through differentiation in suspension inside a liquid marble and the number of the contractile cells in suspension was about 4.5%; whereas, the contraction of these cells only lasted for a few hours. Their research showed the possibility of the development of a “one-pot” method for cell culturing using a liquid marble. The percentage of contractile EBs obtained by our “one-pot” method (Figure 2) is higher than that obtained using the two-step approach reported by Sarvi et al, which involved EB formation in liquid marble, harvesting and then plating.32 After 18 days, the contractile EBs reduced gradually due to the decline of the EB activity, and only 3.5% of suspending EBs still contracted on day 25. Previous literature reported that adhesion signalling of EB with gelatin-coated plates, e.g., Src family kinases and focal adhesion kinase, had significant inhibitory effect on subsequent differentiation on day 4 or earlier.39, 40 However, our experimental data indicated that the EBs inside the liquid marble remain suspending without the adhesion signalling and present similar contractile percentage during subsequent differentiation. These results demonstrated that liquid marble can be explored as a micro bioreactor to firstly realize “one-pot” EB formation and differentiation to create beating cardiac cells.



Figure 2. The percentage of the contractile EBs at different culture time. In previously reported studies, researchers focused only on the EB formation in the liquid marble microreactor rather than its differentiation because EB differentiation required extra complex operations, including media change, EB harvest and plating.20, 29

The operations may introduce the external force which may damage the EBs, thus

lowering the efficiency of the subsequent differentiation. Here, by utilizing the transparency and mechanically robustness of the liquid marble, the EBs could remain suspending and subsequently differentiate to contractile cardiac cells in just one liquid marble. This “one-pot” method for culturing the suspending EBs provides a unique approach to achieve the low-cost and high-effective differentiation of stem cells. 2.3 Influence of nanoparticle shell on cell viability To investigate the influence of silica nanoparticles on the ESCs and EBs, the cell viability was evaluated by using the live/dead assay. The newly-formed cell aggregates, i.e., an EB, in the liquid marble were labelled by fluorochromes on day 2, day 5 and day 7, respectively. As shown in Fig. 3a, most cells were alive (green) and only a few cells were dead (red) throughout the period of culturing. However, it is difficult to quantify the viability of cells by using such a fluorescent label method. To precisely analyse the viability of the cells, the MTT (3-(4,5-dimethylthiazol-2-y-l)-2,5diphenyltetrazolium bromide) assay was employed, as illustrated in Fig. 3d. In this case, the ESCs cultured in hanging drops (without exposure to nanoparticles) were also subject to the same treatment process as the positive control. Within 24 h of culturing, the viability of cells in the liquid marble covered by the silica nanoparticle shell decreased from 100% to 90%  2%, which suggests that the presence of the silica nanoparticle shell has no inhibition for the cell viability in liquid marble. We think that, 8 ACS Paragon Plus Environment

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in the liquid marble environment, suspended cells may not be in contact with silica nanoparticles, which are deposited on the marble shell. This reduces the change for silica nanoparticles being ingested by cells. After 48 h of culturing, the viability of cells in liquid marble decreased to 67% ± 5%, whereas the viability for cells in hanging drops decreased to 47% ± 2% (Fig. 3d). Comparing to cell viability in hanging drops, the liquid marble covered by the silica nanoparticle shell is beneficial to the cell viability. These results also suggest that the presence of silica nanoparticle shell could sustain a sufficient level of mass transfer between the culture media in liquid marble and the surrounding environment and consequently enhance the viability of cultured cells.

Figure 3. Cell viability of EBs cultured for different time in liquid marble was analyzed by using a live/dead assay: a) 2 days, b) 5 days and c) 7 days. The live cells stained by Calcein AM show the green and the dead cells stained by ethidium homodimer show the red. d) MTT assay after cells cultured in hanging drop and liquid marble. 2.4 Comparison between bare sessile drop and liquid marble bioreactors Literature information shows that works on cultivating EBs by using a bare sessile drop reactor placed on a superhydrophobic substrate, have been unsuccessful. This suggests that the particle-liquid interface of a liquid marble may have a desired function that could influence the behaviour of the cells inside a liquid marble micro bioreactor. It is expected that a comparison of the cell behaviour in a bare sessile drop and a transparent liquid marble would reveal the difference of the cell behaviour in the two 9 ACS Paragon Plus Environment


types of bioreactors, and aid our understanding of the critical advantage of a liquid marble over a bare liquid drop in cell cultivation. A sessile drop of culturing media (~300 µL) containing 2  104 ESCs was placed on a superhydrophobic paper with the contact angle of 162.4  3.3 and its hysteresis of less than 5,41 to form a bare sessile drop micro bioreactor and subjected to the same process of culturing as that for the liquid marble. This superhydrophobic paper was fabricated by our group; the superhydrophobic property was decorated onto the surface of a piece of Whatman #4 filter paper via dip-coating. Calcium carbonate, montmorillonite pigments and cellulose nanofibres were used to construct a dual-scale roughness and alkyl ketene dimer to provide hydrophobicity.41 Sectional areas of the suspending EBs in the bare drop and liquid marble reactors were measured using the ImageJ software to characterize the EB development process by randomly selecting EBs in both micro reactors. As illustrated in Fig. 4a, the sectioned area (𝑆) of suspending EBs in both liquid marbles increased exponentially with culture time (𝑡), which can be fitted with 𝑆 = 1600𝑒0.037𝑡. This relates to the logarithmic growth phase of the cell, indicating the EB with high-activity. Interestingly, however, EBs formed in the bare sessile drop could remain suspending only for ~24 h. After that, EBs began to adhere to the substrate and developed branches stemming out from the edge of the cells (Fig. 4b).

In addition, the similar experimental result was also presented for the bare

sessile drops on a silica superhydrophobic substrate. By controlling the process of the media transfer (not allow the drop to roll on the substrate), it can be deduced that EBs have adhered to the superhydrophobic substrate, leaving adhered EB residual on the substrate (characterized in Fig. S1, Supporting Information). The number of suspending EBs in the bare sessile drop decreased after 36 h of culturing. These results suggest that EBs in bare sessile drop on superhydrophobic substrates have a tendency to anchor on the substrate and then differentiate following the normal way of cell spreading in a short culturing period (~24 h), i.e., withdrawal from the cell cycle, fusion with nascent myotubes, and final differentiation into cardiomyocytes, therefore making EBs difficult to harvest and transfer for further investigation.


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Figure 4. Comparison of the size of the suspending EBs in liquid marble and bare sessile drop. (a) Sectioned areas (𝑆) of suspending EBs plotted as a function of culture time (𝑡), which can be well fitted with the trend curve 𝑆 = 1600𝑒0.037𝑡. (b) The morphology of EBs after culturing for 36 h in bare sessile drop, the arrows indicate the branches stemming out from the edge of adherent cells. Scale bar is 100 µm. It still remains to be answered as to why EBs in a liquid marble could remain suspended for a long time, while they could only remain suspended in a bare sessile drop on a superhydrophobic substrate for less than 24 hours. Previous studies have shown that the cell fate and lineage commitment are significantly influenced by the mechanical properties of the microenvironment, such as the mechanical stiffness and elasticity. For instance, stiff substrates that mimic the bone-like property could induce the differentiation of the mesenchymal stem cells to osteogenic42; cell spreading and self-renewal were inhibited on soft substrates43. In the present study, the factor that makes the liquid marble different from the bare sessile drop on a superhydrophobic substrate could most likely be the presence of the shell of nanoparticle at the air-liquid interface of the liquid marble. It has been extensively reported that a particle layer on the particle/liquid interface possesses a unique mechanical property, namely viscoelasticity,44, 45 which makes it different to the traditional solid substrate, although the former was also comprised by nano- or micro-scale solid particles. Furthermore, the presence of the nanoparticle shell may result in a special solute distribution on the liquid marble due to the Laplace pressure arising from the meniscus around a particle in the shell46, which may facilitate the mass transport and favour the EBs culturing and suspending in the media. However, the detail mechanism responsible for the nonsticking property of the liquid marble is not clear yet. Observations made in the present study have improved our understanding to the uniqueness of the liquid marble bioreactor, which still requires further investigation. 11 ACS Paragon Plus Environment


To further characterize the effect of the particle shell on the EB differentiation, the EB was kept in the bare sessile drop to observe the contraction of contractile EBs. Figure 5 shows the mean intensity and contractile frequency obtained from representative areas of contractile EBs on day 10 (Video S1 and S2, Supporting Information). The mean intensity at the point of the contractile EB presents the rhythmic change, and yet there is no underlying biological meaning therein. As illustrated in Fig. 5a, the contractile rhythmic is similar in two bioreactors and the value change of the mean intensity is insignificant. On day 10, the adhered EBs exhibited spontaneous contraction, thus, it was confirmed that EBs can form and differentiate into cardiac cells when adhere to the surface of the supporting substrate in a bare sessile drop micro bioreactor. As shown in Fig. 5b, the contractile frequency was 0.34 Hz for the adhered EB in bare sessile drop and 0.35 Hz for the suspended EB in liquid marble, respectively. Compared with the suspended EBs generated in a liquid marble, the adhered EBs in a bare sessile drop did not show difference in contractile property. The critical advantage of the liquid marble micro bioreactor over the bare sessile drop is that the former enables EBs to suspend inside the liquid marble and proliferate and differentiate; this makes the harvest of the differentiated cardiac cells easier and with higher viability. Liquid marble therefore provides a micro bioreactor capable of achieving the “one-pot” suspending EB formation and differentiation into cardiac lineages.

Figure 5. Comparison of contractile EBs after culturing for 10 days in liquid marble and bare sessile drop micro bioreactors. (a) Mean intensity patterns and (b) the contractile frequency obtained from the representative area. 3. CONCLUSIONS In this work, transparent liquid marbles made of silica nanoparticles were used as micro bioreactors to generate EBs from ESCs. The high transparency of the liquid marble shell enables the observation of the entire process of EB formation and 12 ACS Paragon Plus Environment

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differentiation on a continuous basis. The aggregation of ESCs and formation of EBs were observed in suspension at the bottom of the liquid marble micro bioreactor. The formed EBs remained suspended in the liquid marble for the entire duration of cultivation (~ 25 days), whereas the EBs in a bare sessile drop on a superhydrophobic substrate remained suspended only for ~24 h and then adhered to the substrate. Therefore, the suspending EBs and the differentiated cardiac cells in liquid marble can be harvested and transferred conveniently for further steps of cultivation. The transparent liquid marble enables the observation of the differences in cell behaviour in a liquid marble and in a bare sessile drop. This study demonstrated that the liquid marble micro bioreactor is able to provide a suitable micro environment for ESCs to differentiate into cardiac lineage; therefore the liquid marble micro bioreactor can be used as a “one pot” reactor to achieve the formation and differentiation of EBs. This work presented a unique technological advance in establishing a simple and costeffective method for cardiac research, although the effect of the particle shell on mass transfer between the culture media and the surrounding environment deserves further study. 4. EXPERIMENTAL SECTION 4.1 Murine ES cell culture Undifferentiated mES cells (Oct4B2-ESCs) were cultured in DMEM (cat#10569-010; Gibco) supplemented with 10% FBS (JRH Biosciences, Australia), 1% non-essential amino acids solution (cat#11140-050, Life Technologies), 0.5% penicillinstreptomycin (cat#15070-063, Invitrogen), 0.1  10−3 M β-mercaptoethanol (cat#21985-023; Sigma), and 1000 U mL−1 ESGRO leukemia inhibitory factor (LIF: ESGRO, ESG1106; Sigma). Cells were passaged every 2-3 day by dissociating the cells into small colonies using Tryple express (Gibco, Life Technologies) and replating on a 0.1% gelatin-coated dish at a subculture ratio of 1:10. 4.2 Cell culture in liquid marble Our previous work has indicated that the micro-bioreactor containing 2  104 ESCs in 300 µL culture media was the most appropriate reactor because of its the high efficiency in the production of EBs.12 These cells were suspended in DMEM (cat#10569-010; Gibco) supplemented with 10% FBS (JRH Biosciences, Australia), 1% non-essential amino acids solution (cat#11140-050, life technologies), 0.5% penicillin-streptomycin (cat#15070-063, Invitrogen), 0.1  10−3 M β-mercaptoethanol (cat#21985-023; Sigma), 13 ACS Paragon Plus Environment


and without mLIF. The suspension drop (300 µL) was coated with hydrophobic silica nanoparticles through a shaking-transferring approach to form a transparent liquid marble.34, 37, 47 The petri dish containing the liquid marble micro reactor was then placed inside a capped Petri dish (100 mm diameter) containing sterile water to reduce evaporation from the liquid marble. The set of dishes was always incubated at 37C in a humidified air atmosphere of 5% CO2. Besides that, the old media within the marbles was replaced by the fresh media every 3-5 days, so the weak evaporation within 3-5 days has little effect on the nutrients concentration. 4.3 EB morphology analysis The suspension of ESCs was seeded inside the liquid marble micro bioreactor, allowing cells to aggregate one another and form EBs. The cells were incubated in micro bioreactors for different culture time (1 h–120 h). To capture the phase-contrast images of EB morphology, an optical imaging system (Olympus BX60 microscope) was used. The sectional area of EBs was measured from the collected micrographs using ImageJ software (NIH, USA) to characterize the process of EB formation. 4.4 “One-pot” EB formation and differentiation For cardiac differentiation, EBs were suspended inside liquid marbles culturing in differentiation media for another 15-20 days. Every 2-3 days, the old media within the marbles was gently removed and replaced by the same amount of fresh media using a micropipette, as illustrated in Fig. 1a. The suspending EBs were examined daily for contractile activity based on videos captured at 24 fps using a camera (SpeedXT core CCD) through an optical microscope. 4.5 Live and dead assay A live/dead staining was used to evaluate the cell viability in the liquid marble micro reactor according to the manufacturer’s instructions. After the incubation in different time, cells in each micro bioreactor were transferred into each well of 96-well plates, following the EB in micro bioreactors was stained by the live/dead assay. Briefly, samples were gently washed in warmed phosphate-buffered saline (PBS) for twice. 1 µM Calcein AM (cat#C1430, Life Technologies) and 2 µM ethidium homodimer-1 (cat#E1169, Life Technologies) were used to stain live cells (green) and dead cells (red) and then incubated at 37 C in a 5% CO2 incubator for 40 min. The stained EBs were analysed by using an inverted fluorescence microscope (Olympus IX71 microscope). 4.6 MTT assay 14 ACS Paragon Plus Environment

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ESCs were seeded at a density of 2  104 ES cells in 300 µL liquid marble. Cells seeded in bare sessile drop without nanoparticles shell were used as control group. After incubating for 24 h and 48 h, cells in each micro bioreactor were added into a microwell, following the previously reported procedure; cell viability was then assessed by the MTT assay. Briefly, 10 μL of MTT solution in phosphate buffered saline (5 mg mL−1) was added to each well and the sample plate was incubated in 37 C for 4 h. 100 μL of dimethyl sulfoxide (DMSO) was added into each well to dissolve the purple formazan crystals. The absorbance at 490 nm of each well was measured by a microplate reader. The liquid marble and bare sessile drop micro reactors without cells were prepared to remove the media interference on MTT measurement. At least three individual measurements were performed. 4.7 Image analysis After culturing for 9-10 days, suspending or adherent EBs began to show the contractile activity. Videos of the contractile activity were captured using the microscope camera system; these videos were converted into image sequences with gray scale by Matlab. As the EB underwent rhythmic contraction, the cell cluster colour changed from dark to light. Ten different areas were selected from each image sequence, the contracting activity through changes in gray scale intensity in each area was analyzed using ImageJ software (NIH, USA), and the resulting mean intensity was plotted against time. ASSOCIATED CONTENT Supporting Information Figure S1. Cell viability obtained from MTT assay after cells cultured in hanging drop and liquid marble. Figure S2. The morphology of cell aggregates after culturing for 48 h in bare sessile drop: (a) before and (b) after sucking the media. Scale bars are 200 µm. Video S1. Beating of the suspending EB after culturing for 10 days in a liquid marble micro bioreactor. Video S2. Contracting foci of adhered EB after culturing for 10 days in a bare sessile drop micro bioreactor. CONFLICTS OF INTEREST There are no conflicts to declare. ACKNOWLEDGEMENTS



The authors thank Karla Contreras for the technical training on cell culture. Kejun Lin acknowledges the China Scholarship Council for scholarships, and Xiaoguang Li for the technical discussion. Ruoyang Chen acknowledges the Monash Graduate Education and the Faculty of Engineering for the scholarships. Duyang Zang thanks the National Natural Science Foundation of China (U1732129). Liyuan Zhang and Wei Shen acknowledges the Monash Institute of Medical Engineering (MIME) for research funds, the Australian Research Council for the funds support through Research Hub for Energy efficient Separation (IH170100009) Project, and the Department of Industry, Innovation and Science (DIIS) and MyHealthTest Pty Ltd for the research funds support through Innovation Connections Grant (ICG000457). REFERENCES [1] Quéré, D.; Aussillous, P. Liquid Marbles, Nature 2001, 411, 924-927. [2] Zang, D.; Chen, Z.; Zhang, Y.; Lin, K.; Geng, X.; Binks, B. Effect of Particle Hydrophobicity on the Properties of Liquid Water Marbles, Soft Matter 2013, 9, 50675073. [3] Rendos, A.; Alsharif, N.; Kima, B.; Brown, K. Elasticity and Failure of Liquid Marbles: Influence of Particle Coating and Marble Volume, Soft Matter 2017, 13, 89038909. [4] Bormashenko, E. Liquid Marbles, Elastic Nonstick Droplets: from Minireactors to Self-Propulsion, Langmuir 2017, 33, 663-669. [5] Tian, J.; Fu, N.; Chen, X.; Shen, W. Respirable Liquid Marble for the Cultivation of Microorganisms, Colloids Surf., B 2013, 106, 187-190. [6] Arbatan, T.;

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Graphic Abstract 116x78mm (600 x 600 DPI)

ACS Paragon Plus Environment

Transparent bioreactor based on nanoparticle-coated liquid marble for

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