Liquid Marbles As Bioreactors for the Study of Three-Dimensional Cell Interactions

Biomed Microdevices (2017) 19:31 DOI 10.1007/s10544-017-0171-6

Liquid marbles as bioreactors for the study of three-dimensional cell interactions

Raja K. Vadivelu1 & Harshad Kamble2 & Ahmed Munaz2 & Nam-Trung Nguyen2

# Springer Science+Business Media New York 2017

Abstract Liquid marble as a bioreactor platform for cell- developing bioassays that could substantially contribute to based studies has received significant attention, especially therapeutic applications. Especially, insights for improving for developing 3D cell-based assays. This platform is partic- the survival and adherence of transplanted cells. ularly suitable for 3D in-vitro modeling of cell-cell interactions. For the first time, we demonstrated the interaction of Keywords Liquid marble . Bioreactor . Spinal cord injury . olfactory ensheathing cells (OECs) with nerve debris and Post-injury model . Post-transplantation model . Digital meningeal fibroblast using liquid marbles. As the transplanta- microfluidics . 3D cell interaction tion of OECs can be used for repairing nerve injury, degenerating cell debris within the transplantation site can adversely affect the survival of transplanted OECs. In this 1 Introduction paper, we used liquid marbles to mimic the hostile 3D environment to analyze the functional behavior of the cells and to Repairing injured tissues using cellular therapy is attractive. form the basis for cell-based therapy. We show that OECs Recently, this approach has been increasingly adopted in clin- interact with debris and enhanced cellular aggregation to form ical practice. Over the past two decades, cell-based therapy has a larger 3D spheroidal tissue. However, these spheroids indi- been improving significantly, especially in post-transplantation cated limitation in biological functions such as the inability of survival of cells in the host niche (Wan et al. 2015). Despite the cells within the spheroids to migrate out and adherence to impressive potential, cell-based therapy still encounters several neighboring tissue by fusion. The coalescence of two liquid obstacles. The transplanted cells face several challenges to marbles allows for analyzing the interaction between two dis- maintain its viability and functional physiology (Terrovitis tinct cell types and their respective environment. We created a et al. 2010). Eventually, the transplanted cells are subjected to microenvironment consisting of 3D fibroblast spheroids and death due to oxidative stress and inflammation. The functional nerve debris and let it interact with OECs. We found that properties of transplanted cells in the host environment could OECs initiate adherence with nerve debris in this 3D environ- not be successfully addressed by in-vivo assessment. Thus, ment. The results suggest that liquid marbles are ideal for novel approaches to mimic cell-cell interaction in a 3D cell culture model are urgently needed. This in vitro model is especially important to provide new insights into the effectiveness * Raja K. Vadivelu of cell-based therapies for tissue repair. [email protected] Bioassays enabling 3D cell cultures can be utilized to mim- * Nam-Trung Nguyen ic the environment of recipient body or injury milieu. The [email protected] investigation of the cellular physiology of transplants with respect to their ability to interact adhere and survive is espe- 1 School of Natural Sciences, Nathan Campus, Griffith University, 170 cially helpful. Another important aspect of a 3D cell culture is Kessels Road, Brisbane QLD 4111, Australia modeling an in-vivo environment for testing and developing 2 QLD Micro- and Nanotechnology Centre, Nathan Campus, Griffith suitable therapeutics. This approach requires a multipurpose University, 170 Kessels Road, Brisbane QLD 4111, Australia 3D platform to produce a well-controlled microenvironment 31 Page 2 of 9 Biomed Microdevices (2017) 19:31 to meet various experimental conditions. Therefore the com- as a repair strategy has shown significant neurological improve- bination of various cell types and therapeutics in this platform ment in SCI patient (Tabakow et al. 2014). OECs exhibit axonal is crucial. A major bottle neck of existing in vitro scaffold- growth promoting properties and display neuronal survival and based platforms is the reproducibility (Rimann and Graf- axonal extension (Raisman et al. 2012). However, this therapeutic Hausner 2012). Scaffold-free 3D platforms have been used, effect is still limited due to the insulating features of the post- mainly because they are readily available, flexible to generate injury site. After SCI, the post-injury site often becomes hostile and reproducible (Duraine et al. 2015). Thus, the scaffold-free for cell growth. The main cause is the abundant axonal debris, approach is more suitable to promote intracellular interaction originating from dead or damaged neurons. At the same time, and assembly of cells (Rogozhnikov et al. 2016). A scaffold- fibroblast cells invade into this hostile environment and cooperate free 3D platform allows for optimal cell-cell interactions and with the astrocytes to create tissue scarring (Khankan et al. 2015). the formation of heterogeneous 3D cell structures. The scar tissue impedes the function of transplanted OECs Recognizing these needs, we aim to establish a simple and (Afshari et al. 2009). OECs post-implantation appears to clear robust lab-in-a-droplet method to allow for the study of cell-cell the abundant cell debris and provides a permissive substrate for interactions. Recently, we have demonstrated a reliable method axonal regeneration (He et al. 2014). Despite this advantage, the for 3D cell culture using a floating liquid marble (Vadivelu et al. interaction of OECs with meningeal fibroblast is still unclear. 2015). A liquid marble (LM) is a liquid droplet coated with hy- Moreover, there are almost no reports on the prevalence of fibro- drophobic particles (Aussillous and Quéré 2001). A liquid marble blast in the presence of cell debris. can serve as a suitable platform as a scaffold-free approach for Here, we aim to employ floating LM as a platform to create cells compartmentalization and growing in 3D environments a simple 3D hostile environment to study OECs interactions in (Sarvi et al. 2015), and requires only a small quantity of liquid. 3D configuration with nerve debris and meningeal fibroblast. LM functions as a distinct micro-bioreactor for 3D cell culture. Besides facilitating spheroid growth, LM also serves as a dig- The hydrophobic coating is non-adhesive, thus providing a con- ital microfluidic platform with the ability to mix solutes and fined microenvironment to maximize cell-cell interactions. These cells. This microfluidic function is achieved by merging two interactions can promote the self-assembly or aggregation of LMs with distinct cellular and biochemical contents. cells. Therefore, the effect of a drug, therapeutics or of a par- Previously, the coalescence between two LMs were achieved ticular environment mimicking in-vivo niche over cellular inter- with external mechanical force (Castro et al. 2016), or elec- actions or behavior can be analyzed. As mentioned above, we trostatic force (Liu et al. 2016). In this study, we triggered the have previously developed a new platform by floating the LM coalescence with collision and impact. Coalesce occurred af- on a carrier liquid to generate spheroids of olfactory ter the momentum is transferred from a falling LM towards ensheathing cells (OECs) with a high yield (Vadivelu et al. another stationary LM. For the first time, we demonstrate the 2015). Additionally, we have also grown different glial cell coalescence of LMs for studying cell-cell interactions between types with OECs to understand the complex cell interactions OECs, meningeal fibroblast and nerve debris. The insights (Vadivelu et al. 2015). Several physical properties of a floating into cell-cell interaction will potentially allow for the signifi- LM have been reported and ultimately proved to be supportive cant enhancement of therapeutic benefit of OEC implantation. of cell interaction. Upon floating, the buoyancy and surface tension create an internal flow within the marble (Ooi et al. 2015a). The floating LM behaves as a Leidenfrost drop 2 Material and methods (Bormashenko 2017) and can easily move on the liquid surface. During floating, the marble is not in direct contact with 2.1 Cell cultures the liquid surface (Ooi et al. 2016). The movement and internal flow minimizes sedimentation due to gravity. As result, a We used green fluorescent protein (GFP) expressing immortalized floating LM enhances random cell distribution and associa- mouse OECs which were previously generated (Moreno-Flores tion. Floating LM can move by self-propelling effect (Ooi et al. 2003, 2006). Cells were cultured in DMEM/F12 (Life et al. 2015b). Furthermore, a LM is scalable with magnetic Technologies) supplemented with 10% FBS (vol/vol), 2 μM actuation as a digital microfluidic platform (Khaw et al. 2016). forskolin (Sigma), 20 μgml−1 pituitary extract (Gibco), 10 ng In the context of cell imaging, a floating LM is accessible for ml−1 FGF-2 (PeproTech), 10 ng ml−1 EGF (PeproTech) and real-time analysis or time-lapse microscopy. However, it 0.5% (vol/vol) gentamicin (Life Technologies). We used should be highlighted that floating LM is only suitable for DMEM (Gibco) with 10% FBS as the standard medium for rou- imaging cells labeled with florescent or florescent protein ex- tine culture of meningeal fibroblast cells. After cells growth was pressing cells. sub-confluent in the T25, they were washed twice with HBSS In this study, we aim to explore the behavior of olfactory (Life Technologies) and detached with TrypLE Express (Life ensheathing cells (OECs) in a 3D in vitro model, which resembles Technologies) for 5 min at 37 °C. The enzymatic reaction was in vivo spinal cord injury (SCI) features. Transplantation of OECs stopped by adding 2 mL 10% FBS media followed by Biomed Microdevices (2017) 19:31 Page 3 of 9 31 centrifugation at 1000 rpm for 5 min. The culture medium was that was modified by trimming the tip edge to the approximate changed every 2 days. The both cells types were grown in hu- diameter of the LM. This allows sucking the LM inside the tip midified atmosphere with 5% CO2 in air at 37 °C. andgripbyfictionfit.TheLMwasdispensedonto96wellplates out from the tip by pressure displacement. The wells are filled 2.2 Preparation of cellular debris with the liquid medium by slowly pipetting 100 μLmediaonthe wall of the wells. This ensures the liquid medium flowing under- Cellular debris was generated by dissecting out the nerve fiber neath the LM and gently carries it. It is critical to avoid direct layer of the olfactory bulb and the olfactory mucosa lining of the exposure of the medium to the LM because wetting can break the nasal septum from olfactory marker protein (OMP)-ZsGreen LM. For incubation, the 96-well plate containing floating-liquid mice (Windus et al. 2007). The dissected tissue was prepared marble gently into the incubator in incubation condition at 5% by partially digesting it in TrypLE Express (Life Technologies) CO2 in air at 37 °C for 24 h. To examine the cell growth and 1 for 15 min and followed by triturated using a syringe with a 18 /2 spheroid formation, the content of LM was exanimated and im- gauge needle. The reaction was stopped by addition of fetal aged by during incubation by using an optical–fluorescent mi- bovine serum (Bovogen) and sieved through 40 μm nylon mesh croscope (Olympus IX70). Puncturing with a needle broke the (Falcon™ Cell Strainers). The cellular debris was then centri- floating LMs. The marble collapses and deposits its content on fuged at 10,000 rpm for 10 min and the pellet was weighed the bottom of the wells ready for the examination of morphology and suspended in 1 mL of media to determine the concentration and dimension of the spheroids. Microscopic examination was weight/volume and stored at −20 °C until used. carried after spheroids were settled in the wells. The spheroids can be collected by multiple wells by centrifugation for 5 min at 2.3 Assessment of the effect of cellular debris 1000 rpm. on the viability of OECs 2.5 Spheroid migration assay OECs were seeded at a density of 3 × 103 cells/100 μL in each well of a 96-well plate (Nunc TM). After 24 h incubation at 37 °C The spheroids were collected and transferred to a 15 ml tube in a humidified incubator under 5% CO2, the cells were treated containing fresh media. The spheroids were suspended at the with cellular debris for 24 h. The stock of cellular debris was then density of 10 spheroids per mL. Then, 100 μL of the suspension diluted with fresh medium in series of concentration ranging be- was seeded onto a flat bottom of 96 well plates. We expect to tween (0.0097–10 mg/mL). The viability of the OECs was tested obtain a single spheroid in each well. The plate was incubated by adding 20 μL MTS solution into each well containing 100 μL under standard cell culture conditions for 24 h to evaluate the media and then incubated at 37 °C for 2 houres in darkness. migration capacity and then imaged with a fluorescence micro- Absorbance was measured using a microplate reader at 490 nm. scope (Olympus IX70) and imaging software (SPOT). The mi- Cells grown without debris served as the comparison control gration of the spheroid was measured by subtraction of the spher- group and resulted in 100% cell viability. Wells with the medium oid radius at t = 0 from the farthest distance of migrated cells. but without cells provided the background reading. Cell viability was determined as a percentage of the control group cells without 2.6 Spheroid fusion debris. Statistical differences between groups were analyzed with on-way Analysis of Variance (ANOVA) using the statistical soft- Spheroid fusion was carried out by using a polydimethylsilox- ware Prism 5. The viability results were expressed as percentages ane (PDMS) microfluidic device which was designed with [mean (± standard deviation)]. microchannel arrays to trap spheroids and at the same time serve as a culture chamber (Munaz et al. 2016). Spheroids 2.4 Generation of floating liquid marble for spheroid with an average diameters ranging between 80 and 120 μm culture were harvested from a number of liquid marbles (between 10 and15).WeestimatethateachLMcontaining15–25 spher- Liquid marbles were created by dropping a droplet containing oids. A clean stock of spheroid without debris and powder 5×103 cells onto polytetrafluoroethylene (PTFE) (Sigma- particles was obtained by centrifugation 500 rpm for 5 min. Aldrich, product number 430935) powder bed with particle sizes Slow centrifugation is critical to avoid damages to the spher- of 1 μm inside a 6-well plate. For co-culture spheroids, OECs at a oid structure. Before injecting the spheroids, we wash the density of 5 × 103 cells/mL were suspended with nerve debris at device by flushing the chamber with DI water. Then, approx- a density of 300 μg/mL in a total volume of 1Ml. A 10-μL imately 100 spheroids in 30 μL volume were injected into the droplet was subsequently dispensed on the powder bed, followed inlet of the device. The spheroid injection was carried out by gentle shaking in a circular motion for 3 min. The droplet was using a 10-μL pipette. After injection, the device was imme- entirely encapsulated by the hydrophobic particles resulting in a diately placed vertically to ensure deposition of spheroids on LM. For floating, the LM was picked by using a p1000 pipette the filtering microchannel array by gravitation force. After 31 Page 4 of 9 Biomed Microdevices (2017) 19:31

0.85 Figure 2 schematically depicts the experimental setup for 0.8 LM 1 0.75 LM 2 μ Surface volumetric fraction of fluid the LM. The LM was dispensed using a 1000- L pipette, 0.7 Fig. 2a. We cut the edge of the 1000-μL pipette tips with 0.65 0.6 = 1 approximately the same diameter of the LM, Fig. 2b. The tip 0.55 will accommodate the LM after sucking it up. The tip creates a 0.5 LM 1 0.45 = 0 press fit to hold the LM in place. To coalesce, the LM was 0.4 = 0.5 dispensed to impact on another sessile LM, Fig. 2c.Thispro- 0.35 0.3 LM 2 cess requires accurate positioning of the pipette. We assumed 0.25 = 0 that the dropping was due to gravitational acceleration, and the 0.2 Volume fraction of fluid (1) 0.15 t= 0 t= 0.25 t= 0.5 t=1 t=2 t=3 t=4 pipetting force was negligible. The kinetic energy overcomes 0.1 the surface tension and causes coalescence, Fig. 2d. If the 0.05 impact is not strong enough, the LM bounces after contact 0 0 0.5 11.52 2.5 3 3.5 4 or deform into a non-spherical shape. After the coalescence, Time (s) the merged LM is picked by pipetting and subsequently Fig. 1 Representative coalescence process of two liquid marbles. The mixing the fluid content by rolling them vertically inside the results for change in the surface volumetric fraction of LM 1 and LM 2 over time (Inset: Initial conditions for FEA model and change in the tip with low suspension force, Fig. 2e. Finally, the merged surface volumetric fraction of LMs over time) LMs were transferred onto the good plate and floated by dis- pensing liquid medium as described in Section 2.4,Fig.2f. trapping the spheroids, the device was placed under cell culture condition and supplied with fresh media every 4 h through an integrated inlet reservoir, which directly connects the microchannel arrays. Images were taken every 6 h using 3 Results and discussion the fluorescent microscope to evaluate the fusion process. 3.1 Effect of cellular debris on OECs viability

2.7 Coalescence of liquid marble OECs were incubated with different concentrations of axonal debris (0.0097–10 mg/mL) for 24 h to determine the surviv- For the first time, we attempted to demonstrate coalescence of ability of OEC in the presence of nerve debris. The purpose of LMs for studying cell-cell interactions. Before encapsulating this experiment is the determination of the amount of nerve the cells inside a LM, we simulated the coalescence of two debris that can potentially exert cytotoxicity to the OECs. No LMs to prove the concept. A finite element analysis (FEA) significant changes in the percentage of cell viability were model in COMSOL Multiphysics 5.2 was used Fig. 1.The observed at low concentrations (0.0097 to 2.5 mg/mL). LMs are modeled as liquid droplets with the same apparent Debris at higher concentrations between 5 to 10 mg signifi- surface tension of the real liquid marbles. The simulation cantly reduced cell viability (p < 0.001), Fig. 3. We estimated shows that coalescence of the LMs by gravity can occur with- that 1 mg /mL is the optimal concentration for the following in seconds. experiment.

Fig. 2 Step-by-step illustration of the coalescence process: a First (a) (b) (c) LM (Yellow); b The first LM is picked by a 1000 μLpipette (white arrow indicates the cut p1000 pipette); c The first LM (yellow) is dropped onto the second marble (green); d The merged LM (grey); e The merged LM is picked up and transferred to a liquid surface; f The floating (d) (e) (f) merged LM. The scale bar is 1000 μm Biomed Microdevices (2017) 19:31 Page 5 of 9 31

Figure 5 indicates that compared to culture condition without debris (Fig.5a) LM with nerve debris produces larger multiple co- cultured spheroids (Fig. 5d). We next assessed the number and dimension of the spheroids by breaking the LM. We examined its content after spheroids settled on the bottom of the 96 well plates. We observed that co-cultured spheroids formed in LM containing debris are larger but less abundant compared to the condition without debris. (Fig. b, e). For detail examination of the spheroid composition, we compared the appearance of the debris by 20× magnification. The debris inside the co-cultured spheroid appears as tiny dots (Fig. 5f) and is easily distinguishable when compared with normal spheroids, Fig. 5c. The source of the debris is from Fig. 3 Effect of nerve debris on OECs viability. No significant reduction ZS green fluorescent expressing olfactory neurons (Ekberg et al. in cell viability was observed when debris was added at low concentrations. However, debris at a higher concentration (5 to 10 mg/ 2011). Nakamura et al. 2013 reported that the ZsGreen1 protein is ml) significantly decreases OECs cell viability. Values are the expressed expressed better than GFP protein in yeast cells and mammalian as the mean and s.e. (n =3).(*p < 0.001) cell lines (Harrell et al. 2007). It was reported that the ZsGreen protein is expressed better than GFP protein in yeast cells 3.2 Interaction of nerve debris with OECs (Nakamura et al. 2013) and mammalian cell lines (Harrell et al. 2007). Thus, the fluorescent intensity of ZsGreen1 protein is The interaction between OECs and nerve debris inside liquid brighter compared to GFP. It is not clear whether the aggregates marbles was subsequently investigated. We tested the interac- are composed of ingested debris after OECs phagocytes team, or tion at different intervals. A floating LM allows cells to freely the debris is trapped within cells. However, the composition of interact with debris to form cell aggregates containing debris. debris inside the OECs cytoplasm was noticed in 2D assays (Data Further, LM permits visualization at different time points. We not shown). One-way ANOVA indicated in the presence of de- created LMs with the 10-μL droplet containing cells at a con- bris, cells aggregate to form significantly larger spheroids. The μ centration of 5 × 103 cells/μL and nerve debris at a concen- spheroid size ranges between 91 and 120 m(Fig.2b,p<0.05). tration of 1 mg/mL. Fluorescent microscopy was used to view the interaction between cells and nerve debris which showed 3.3 Spheroid spreading OECs came into contact with debris to form aggregates. An increase of aggregation was seen from 6 h to 24 h inside the Our next question is whether cells within a spheroid consist of LM (Fig. 4a–d) and aggregates from broken LM (Fig.4e–h). debris are viable and feature both motility and dissemination. It is The cellular aggregates changed shape to the spheroidal struc- well established that healthy cells migrate out of spheres with a ture at 24 h. higher migration rate. Thus, we used the migration test to

6 hours 12 hours 18 hours 24 hours (a) (b) (c) (d) Inside LM

(e) (f) (g) (h) Broken LM

Fig. 4 The interaction of OECs with nerve debris between 6 h to 24 h. a– are observed (a and e). After 12 h, cells aggregate with debris (b and f). d Merged bright -field and fluorescent microscopic images show the The formation of spheroids like can be observed at 18 h (c and g)and interaction of OECs with nerve debris spheroids inside the liquid marble. complete spheroid morphology was seen at 24 h (d and h). Scale bar in e–h The florescent images of broken LM indicate that OECs interact with (a–d)and(e–h) are 500 μm and 200 μm, respectively nerve debris and forms spheroids. At 6 h, loose dispersion debris and cells 31 Page 6 of 9 Biomed Microdevices (2017) 19:31

(a) (b) (c)

(d) (e) (f)

(g)

Fig. 5 The effect of nerve debris on OECs aggregation. Spheroid of spheroids and the number of spheroids within the indicated diameter formation inside the marbles without debris (a) and marble with debris ranges in condition without debris (red) and in the presence of debris (d) at the seeding densities of 5 × 103 cells/mL. The floating marbles were (green). The co-cultured of OECs and debris produces significantly larger broken and spheroids are allowed to settle to the bottom of the well; spheroids. The distribution of spheroids size indicates more spheroids in panels show spheroids from a single broken marble with debris (b)and the diameter range 90–151 μm compared to control. (*** p <0.001). without debris (d). The morphology of single spheroid was imaged at 20× However, significantly less number of co-cultured spheroids are formed magnification. c The representative of a single spheroid with debris and when compared with control. (### p < 0.001). Data were analyzed using (f) show brighter green dots of debris, which express ZS green protein. one-way ANOVA followed by post hoc Bonferroni’stest.All Scale bar in (a and d), (b and e)and(c and f) are 500 μm, 200 μmand experiments were repeated three times. Bars represent the mean; error 100 μm, respectively. g Quantification of spheroid size. The total number bars represent the standard error of the mean characterize the functional behavior of OECs in contact with (p < 0.001) of OECs from spheroids consist of debris. In contrast, debris. To assess the migration of cells out of a spheroid, we OECs from spheroids without debris migrating out over an area harvested the spheroids by breaking the LM, selected and seeded 2–3 times larger (Fig. 6c). spheroids with diameters between 90 and 120 μmatt =0hina 96-well plate. The OECs migrated out of the spheroids onto the 3.4 Spheroid fusion surrounding surface. The spheroid spreading parameters are clearly visible. The changes in the OECs spreading area are Tissue fusion plays a central role in regeneration and repair. illustrated in the respective florescent oimages (Fig. 6a,b).The Moreover, it is critical to accelerating the cohesiveness of ANOVA showed that there was significantly less migration cellular aggregates to fuse and form larger functional Biomed Microdevices (2017) 19:31 Page 7 of 9 31

s (a) bri e d hout hout t i W

(b) is r b hde it W

Fig. 6 Migration of cells out of spheroids grown with nerve debris. Co- migrated significantly further than cells from spheroids containing debris spheroids with OECs and nerve debris were removed from marbles and (**p <0.01;n = 30 spheroids). Data were analyzed using Student’st-test. plated and cultured for 24 h. Cells migrated further from spheroids grown Bars represent the mean; error bars represent the standard error of the without debris (a)thanwithdebris(b) at the same cell-seeding densities. mean The scale bar is 100 μm. c Cells from spheroids grown without debris structure. The viability and function of multicellular tissue 12 h. Figure 7a, d depicts the placement of trapped spheroids with debris are critical for the fusion process. Thus, we sought inside the chamber after 1 h. In general, tissue spheroids with- to understand whether spheroid/debris complex initiates fu- out debris were more cohesive. The spheroids without debris sion upon contact. Two sets of spheroid conditions were gen- form better connection and initiate fusion mechanism. As fu- erated: OECs spheroid with debris and without debris. To sion process begins, the area of aggregates decreases and grad- investigate the fusion of multicellular spheroids we developed ually forms a tissue consist of compact spheroids after 6 h, a microfluidic chamber to trap the spheroids (Munaz et al. Fig. 7b. The length of the fused spheroids further decreased 2016). When the suspension of spheroids (approximately and formed a non-spheroidal like tissue at 12 h, Fig. 7c.In n = 100) was injected into the device, spheroids disperse and contrast, we observed that spheroid with debris possess weak- captured onto the trap. Only 50% of the spheroids tend to er connection after 1 h, Fig. 7d. The fusion rate was slower and settle in linear array of two or more to establish a connection narcosis of cells commenced at the outer layer between 6 to as clusters of spheroids along the trap. For measuring the 12 h, Fig. 7e–f. The change of morphogenetic shape during fusion process we select clusters of spheroid that lined up fusion is dependent on the abundance of viable cells at the closely. Images of optical fluorescence microscopy were used outer layer of the spheroids. The active contact of viable cells to illustrate the morphological changes during the fusion pro- may accelerate the migration of cells within the spheroids and cess.Imageswerecapturedevery6hoveratimeperiodof enhances cell adherence. Subsequent cell-cell and cell-matrix

1 hour 6 hour 12 hour (a) (b) (c) Without debris (d) (e) (f) With debris

Fig. 7 Effect of nerve debris on spheroids fusion inside the microfluidic structures were observed (c). In contrast, spheroid with debris showed chamber. The spheroid fusion mechanism was assessed from images imperfect contact at 1 h (d). After 6 h, necrotic-like morphology was seen taken at an interval of 6. Spheroids grown without debris forms contact in the outer region of spheroids and exhibit limited fusion. (e). Necrosis in the microfluidic trap device at 1 h (a) and fusion were seen after 6 h (b). accelerates after 12 h and tissue size decreases (f). The white arrows After 12 h, individual spheroids were no longer identified, and tissue-like indicate the necrotic regions. The scale bar is 200 μm 31 Page 8 of 9 Biomed Microdevices (2017) 19:31 interaction occurs and contributes to tissue contraction. In Interestingly, with nerve debris OECs remain in its position, general, OECs mediates phagocytic clearance of apoptotic and only fibroblasts migrated out, Fig. 8f. cells and cellular debris (He et al. 2014;Suetal.2013). However, it is unknown whether OECs sustains cell viability and preserves the cellular function after phagocytosis process. 4 Conclusion

3.5 Coalescence of liquid marble to study cell interaction This paper presents a new method for recapitulating the behavior of transplanted cells in a host tissue. We simulated the Next, we demonstrated the coalescence of LMs for studying hostile environment of the injury by encapsulating nerve de- cell-cell interactions. In the experiment, we generated the first bris with OECs inside the LM. The simplicity and reproduc- LM encapsulated with meningeal fibroblast and nerve debris ibility of LMs fulfils the requirement of a bioassay for study- to serve as the hostile post-Ninjury site. We then floated and ing the interaction between OECs and nerve debris. The abil- incubated the first LM for 24 h to produce spheroids of ity to manipulate the coalescence of two liquid marbles as fibroblast/debris complex. The nerve debris was seen associ- compartmentalized bioassays with a small volume has many ated with the outer layer of fibroblast spheroid (Fig. 8d). Then, practical applications in biotherapeutics development. This the first LM coalesced with a second LM carrying GFP- paper demonstrates the complete models of injury sites expressing OECs in suspension to serve as the transplanted and post-transplantation site to gain preliminary under- therapeutic cells. After the coalescence, the merged LM standing on the ability of OECs to interact with nerve debris served as a 3D post-transplantation model. Over 24 h of incu- and meningeal fibroblast. Clearly, LM is a paradigm-shifting bation, OECs eventually interact with fibroblast/debris spher- and versatile tool for studying 3D in vitro post-injury models. oid and fused to form a single aggregate. We found that OECs The method can use to distinguish the differences in the be- exhibit invasion-like behavior and became concentrated in the havior between distinct cell types or interaction of cells with center of the aggregate, Fig. 8e. In contrast, in the absence of other substances. The presented method minimizes animal debris, fibroblast spheroids fused to form larger aggregates. models for evaluating the efficiency of cell-based therapy. OECs encapsulated of the aggregates, Fig. 8b.Wethenplaced Furthermore, the LMs and the coalescence method reported the aggregates on a flat surface to investigate the cell spread- here potentially can serve a platform technology to expedite ing behavior. In the absence of nerve debris, noticeably both the interaction of cells and biomaterial for testing biocompat- OECs and fibroblast possess migration capacity, Fig. 8c. ibility. The present study, of course, still has some limitations.

(a) (b) (c)

(d) (e) (f)

Fig. 8 Interaction of meningeal fibroblast with OECs and cell debris: a surrounding of meningeal fibroblast spheroid (white arrow); e OECs Spheroids of meningeal fibroblast; b Co-culture of OECs (green) and promote fusion of fibroblast/debris complex and exhibit invasion like fibroblast. OECs promotes fusion of meningeal fibroblast spheroids and behavior with debris; f The fibroblast migrated out of (fibroblast/debris became localized to the exterior of the fused spheroids and did not form a complex + OECs) but not OECs. OECs remain localized in the interior heterogeneous cell population; c Meningeal fibroblast and OECs migrate core of the spheroid. The scale bar is 100 μm out of their co-cultured spheroid; d Nerve debris layered at the outer Biomed Microdevices (2017) 19:31 Page 9 of 9 31

The experiments are based on microscopic observations and M. T. Moreno-Flores, E. J. Bradbury, M. J. Martin-Bermejo, M. Agudo, are not associated with investigations at a molecular level. F. Lim, E. Pastrana, J. Avila, J. Diaz-Nido, S. B. McMahon, F. Wandosell, A clonal cell line from immortalized olfactory However, we provided here the fundamental basis for the ensheathing glia promotes functional recovery in the injured spinal analysis of complex biological interactions, which can involve cord. Mol. Ther. 13,598–608 (2006) florescent-labeled molecules or protein. Overall, LM reveals a A. Munaz, R.K. Vadivelu, J.A. St John, N.-T. Nguyen, A lab-on-a-chip promising milestone as a digital platform for large-scale drug device for investigating the fusion process of olfactory ensheathing cell spheroids. Lab Chip 16,2946–2954 (2016) screening at a higher throughput. The liquid marble platform Y. Nakamura, J. Ishii, A. Kondo, Bright fluorescence monitoring system would serve as a powerful tool to further expand the pipeline utilizing Zoanthus sp. green fluorescent protein (ZsGreen) for hu- of therapeutic interventions. man G-protein-coupled receptor signaling in microbial yeast cells. PLoS One 8, e82237 (2013) C.H. Ooi, R.K. Vadivelu, J. St John, D.V. Dao, N.T. Nguyen, Acknowledgement We acknowledge the funding support of the Deformation of a floating liquid marble. Soft Matter 11, 4576– Australian Research Council for the discovery grant DP170100277. We 4583 (2015a) thank the members of Griffith Institute for Drug Discovery, especially to C.H. Ooi, A. Van Nguyen, G.M. Evans, O. Gendelman, E. Bormashenko, Dr. James St. Johns and his group for providing the cell lines and cell N.-T. Nguyen, A floating self-propelling liquid marble containing culture facilities. aqueous ethanol solutions. RSC Adv. 5, 101006–101012 (2015b) C.H. Ooi, C. Plackowski, A.V.Nguyen, R.K. Vadivelu, J.A. St John, D.V. Dao, N.T. 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