Multi-functional Regulation of 3D Cell-laden Microspheres Culture on

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Multi-functional Regulation of 3D Cell-laden Microspheres Culture on an Integrated Microfluidic Device Yajing Zheng, Zengnan Wu, Mashooq Khan, Sifeng Mao, Kesavan Manibalan, Nan Li, Jin-Ming Lin, and Ling Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02434 • Publication Date (Web): 28 Aug 2019 Downloaded from pubs.acs.org on August 29, 2019

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Analytical Chemistry

Multi-functional Regulation of 3D Cell-laden Microspheres Culture on an Integrated Microfluidic Device Yajing Zheng, †, ¶ Zengnan Wu, ‡, ¶ Mashooq Khan, † Sifeng Mao, † Kesavan Manibalan, † Nan Li, † JinMing Lin,† and Ling Lin, ¶,* †

Beijing Key Laboratory of Microanalytical Methods and Instrumentation, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing, 100084, China. ‡ State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, 100029, China. ¶

CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China. E-mail: [email protected]. Phone: +86 10 82545621 ABSTRACT: Three-dimensional (3D) hydrogel microsphere has aroused increasing attention as an in-vitro cell culture model. Yet the preservation of cells’ original biological properties is overlooked during model construction. Here, we present an integrated microfluidic device to accomplish the overall process including cell-laden microsphere generation, on-line extraction and dynamicculture. The method extends the non-invasive and non-suppression capabilities of the droplet preparation system, provides constant microenvironment, which reduces the intracellular oxidative stress damage and the accumulation of mitochondria. Compared to conventional preparation method, the co-culture model of tumor-endothelial constructed on the integrated platform displays high-level angiogenic protein expression. We believe that this versatile and biocompatible platform will provide a more reliable analysis tool for tissue engineering and cancer therapy.

The research of interactions between neighboring cells1 and cell-matrix2 plays a pivotal role in regulating tumor growth, angiogenesis, and metastasis. In this contribution, the 3D extracorporeal cell culture models such as, spherical,3 fibroid,4 bone-shaped5 and more other sophisticated artificial tissue structures have been developed through biocompatible hydrogel scaffolds. The 3D architecuture reserves some specific phenotypes and functions of cells6 and rally the exploration complex cell culture study, such as organ/tumor models,7 8 anticarcinogen screening9 10and regenerative medicine.11,12 Hydrogel microspheres, the broadest and the most practicable 3D cell culture method, has been recognized as a promising tool for in-vitro culture.13 And the droplet microfluidic technique relying on flow-focusing mechanism is often the optimal production platform.14,15 Because it not only allows to produce plenty of monodisperse droplets in an automated fashion,16 but also has diverse selections of materials and gelation methods to meet different culture demands.17 However, in general, the offchip collection steps of cell-laden hydrogel microspheres are labor and tedious, including demulsification, washing and centrifugation. And the microspheres were incubated in petri dish in a single culture manner. The multi-step operations will destroy the spherical structure, harsh environment and oversimplified culture mode even influence the cell fate. Hence, it is urgent to develop a methodology to improve the overall quality of the bio-model system. (i.e. Considering both the prepreparation and post-treatment of cell-laden microsphere). Advanced microfluidic technology has shown great potential to overcome the above problems because of its powerful

integrated characteristics.18 It can not only customize microstructures flexibly such as dams,19 piles,20 and valves to extract hydrogel microspheres on-chip, but also manipulate fluid tightly to established some physiologically relevant regulation such as, gradient signal factor21, interstitial flow22. Yet few studies have developed such an integrated microfluidic platform with multi-functions. In this work, we engineered an integrated microfluidic device as a versatile and biocompatible platform for dynamic 3D cell culture. The device consisted of three modules. Firstly, the cellladen alginate microspheres generator, for the production of homogenous microspheres by utilizing water-soluble calciumethylenediaminetetraacetic acid (Ca-EDTA) complex as a crosslinking precursor.23 Secondly, microspheres extractor, for rapid and non-destructive purification and extraction of cellloaded microgel spheroids into the fresh culture medium. This module also retained the main channel for flow perfusion culture. Thirdly, the dynamic- culture created a lively and stable microenvironment for cell growth and also allowed monitoring of morphological and biochemical alteration of cells in-situ. The whole procedure was accomplished through the one-step method on a microfluidic device. The platform was compatible with physiological microenvironment and displayed high-level angiogenic protein expression in the extended tumorendothelial cell co-culture model.

EXPERIMENTAL SECTION Chemicals and Materials. SU-8 2050 Negative photoresist and developer were purchased from Microchem Corporation

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(Newton, MA, USA). Sylgard 184 poly(dimethylsiloxane) (PDMS) and initiators were purchased from Dow Corning (Midland, MI, USA). 1H,1H,2H,2H-perfluorooctyl trichlorosilane and 1H,1H,2H,2H-Perfluoro-1-octanol (PFO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aquapel were purchased from PPG industries (Pittsburgh, PA, USA). Pronova UP MVG Alginate was purchased from Novamatrix FMC Biopolymer (Ayrshire, GB). HFE-7500 was purchased from 3M Novec 7500 Engineered Fluid (3M, St. Paul, MN, USA). Fluorinated surfactant (Perfluorinated polyetherspolyethyleneglycol, PFPE-PEG) was purchased from RainDance Technologies (MA, USA), EDTA disodium salt solution was purchased from Macklin (Shanghai, China). Calcium chloride was purchased from Leagene (Beijing, China). 3.2 μm red fluorescent polystyrene nanomicrogels was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Black food dye was obtained from Kris Aromatics (Hertfordshire, UK). Oil-soluble dye was purchased from PCB creation (Benfeld, France). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin, penicillin and streptomycin were obtained from Gibco Corporation (NY, USA), Live/dead assay kit (CalceinAM/EthD-1) were obtained from Invitrogen. Cell tracker™ Deep Red、Cell Tracker™ Green CMFDA were obtained from Invitrogen. Mito-Tracker Green and Dihydroethidium dyes were purchased from Beyotime. EliKine ™ Human VEGF ELISA Kit was purchased from Abbkine (California, USA). Lactic Acid assay kit and Glucose Assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The fabrication of the integrated microfluidic device. The 1st PDMS microfluidic chip was prepared using soft lithography as previous work.24 SU-8 2050 photoresist was uniformly spun onto the 75 mm silicon wafer at a speed of 1500 rpm for 50 s in a spin-coater (KW-4A, Microelectronics Center, Chinese Academy of Sciences) to a thickness of 130 μm. When baking at 65 °C for 20 min, the silicon wafer was patterned by applying UV exposure using 1st transparency photomask (See Figure S1). Then the obtained pattern again rebaked at 65 °C for 20 min and developed with a SU-8 developer, right after it exist a clear microstructure. Then mold was treated with a silylation reagent (1H,1H,2H,2H-perfluorooctyl trichlorosilane) under vacuum condition for 4 h. Later on, degassed 10:1 mixture of PDMS and initiators was poured onto the mold, and cured at 65 °C for 1 h. Once the cured device was prepared, PDMS stamp was peeled off from mold pattern, cut, connection holes were punched, thereafter the gadget was tightly sealed to glass substrate resulting treatment in oxygen plasma (PDC-32G, Harrick Plasma, USA). Before using the chip, aquapel was anchored to generate the hydrophobic channels. Re-exposure technology was used to fabricate 2nd PDMS microfluidic chip fabrication.25 Briefly, the first layer patterns were constructed by using standard lithography method with a spin coating SU-8 2007 photoresist. Following this, it has generating a narrow and short connecting microchannels with the height of 20 μm by keep the speed at 1250 rpm for 30s. After baking and exposuring, the another layer of SU-8 2050 photoresist was coated at a speed of 1000 rpm for 30 s to obtain a 200 μm height of the main channel. After completing the second exposure step, the 1st PDMS microfluidic chip fabrication process (developing, silanization, pouring PDMS polymer and sealed to glass substrate) was then repeated again to generate the 2nd PDMS microfluidic chip.

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One-step generation and extraction of microsphere. For cell-laden microsphere, 4% (w/v) alginate was dissolved into DMEM cell culture medium instead of DI water and then mixed with 100 mM equal volume calcium-EDTA mixture. After the HUVEC cell was trypsinized, centrifuging at 1000 rpm for 1 min to obtain cell sediment. Next, the cell residue were resuspended into the 1 ml fresh alginate/calcium-EDTA solution to gain the desired cell concentration density at approximately 5×107 cell/ml. Thereafter, the fabrication of cellladen microsphere was carry out likewise one-step microsphere generation and extraction procedure. Once oil sucked out, we continuously injected medium for 5 min to wash and pure the cell-laden microspheres. Finally, we separated the 2nd microfluidic chip and placed it into incubator to culture the cellladen microspheres at 37 °C under 5 % CO2. Serve as dynamicculture state, we controled the perfusion rate of fresh medium at 5 μl/min by coupled the 2nd chip with the outer syringe pump. And as the static-culture state, we just set the 2nd chip in the incubator and replaced the medium every 24 h. The conventional centrifugation off-chip method was used to extract the cell-laden microsphere from oil phase into medium phase for culture. After microsphere generation, we collected the microsphere in a centrifuge tube containing 20% (v/v) PFO oil mixture and 7.5 mM CaCl2 solution. After removing PFO oil mixture out from centrifuge tube, we centrifuged CaCl2 supernatant and resuspended the cell-laden microsphere into fresh DMEM medium containing 7.5 mM CaCl2. Cell stain and analysed by confocal microscopy. Calcein AM/EthD-1 staining kit, Dihydroethium (DHE, 1 μM) and Mitotracker Green (1 μM) in phosphate buffer (PBS, 0.01 M, pH 7.4) were added to main channel of 2nd chip, respectively, and then incubated at 37 °C for 30 min. Finally, the channels were washed with PBS three times. For cell labeling, we used Cell tracker™ Deep Red (1 μM)、Cell Tracker™ Green CMFDA (1 μM) to separately stain U87-MG, HUVEC cells for 30 min. All the fluorescence images were observed a LSM780 inverted confocal laser scanning microscope (Zeiss, Germany). Study the co-culture model of U87-MG-HUVEC cell-laden microsphere. We increased to two dispersed phase channels to rebuild the 1st chip of microsphere generation module and introduced the alginate suspension containing U87-MG cells into the inner dispersed phase channel and the another alginate suspension containing HUVEC cells into the outer dispersed phase channel. The flow rate of the two phases were set as 50 µL/h and 100 µL/h, respectively. Thereafter, the other settings and operations were carry out likewise one-step microsphere generation and extraction procedure. After finished the on-line steps, we further set the heterogeneous cell-laden microspheres cultured under 5 μL/min medium flow condition. Measurement of VEGF, lactic acid secretion and glutamine consumption. We measured the concentrations of lactic acid, glutamine in the collected metabolic medium by commercial lactic acid assay kit, glutamine assay kit, respectively. Here, the culture medium of the two groups (the dynamic-culture group and the static-culture group) were separately collected every 12 h for 5 days. (Note: For staticculture group, the culture medium was changed every 24 h). We measured the concentration of VEGF by commercial EliKine™ Human VEGF ELISA Kit. Here, the culture medium


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Figure 1. A pictographic model of the multi-functional integrated microfluidic device. (A) A schematic representation of microfluidic chip which comprised two individual subdevices: Here the 1st PDMS device displays microgel droplets generation and 2nd PDMS device shows the oil extraction and culture cell-laden microgel on-chip under dynamic regulation (B) Schematic illustration of emulsification, cross-linking, demulsification and purification process of microsphere.

of the two groups (the integrated platform group and the control group) were separately collected every 24 h for 5 days. For the integrated platform group, we finished the preparation and collection of the cell-loaded microspheres on the integrated microfluidic device, and provided dynamic perfusion culture at 5 μL/min flow rate. For the control experiment group, we prepared the cell-loaded microspheres on the 1st chip and collected into a centrifuge tube. After centrifugation treatment, we transfered the microspheres into the 2nd chip for static culture. The culture medium was changed every 24 h.

RESULTS AND DISCUSSION Integrated Microfluidic Device Design and Performance. On the 1st microfluidic chip, flow-focusing microfluidic technique was used to produce cell-loaded alginate hydrogel spheroids (Figure 1A and Figure S1 a-c). The alginate containing Ca-EDTA complex was used to mix with cell suspensions as a dispersed phase, and the fluorocarbon oil containing 0.5 % perfluorinated polyetherspolyethyleneglycol (PFPE-PEG) surfactant and 0.15 % acetic acid was introduced as a continuous phase. The fluorocarbon oil containing 20 % 1H,1H,2H,2H-Perfluoro-1octanol (PFO) was utilized as a demulsifier phase. At the first cross-junction, Ca-Alginate gelation occurred because of rapid diffusion of H+ ion, yield premicrogel spheroids, which crosslinked in-situ inside the carrier oil shell. Then at the second cross-junction, introducing the PFO to dissociate the surfactant and break the stability of the oil-water interface (Figure 1B).26,27 After the oilwater interface was destabilized, the microspheres were immediately escaped from the outer oil shell, which facilitated to transfer the microspheres from oil to aqueous phase. The continuous operations of emulsification and demulsification were accomplished in a single microfluidic chip, the feature is crucial to avoid the long-term exposure of microspheres to harsh acidic oil solution and provide the encapsulated cells a biocompatible microenvironment. As verified by Figure 2A, the gelation and releasing of microspheres proceed simultaneously on the 1st microfluidic

chip. Subsequently, coupling the 1st microfluidic chip with the 2nd chip, which allowed hydrogel microspheres to be gently separated from immiscible oil solution. On either side of the main channel (width: 1.4 mm height: 170 μm, length: 7.8 mm) were provided two groups of microchannel arrays (width: 50 μm, height: 20 μm, length: 3 mm) (Figure S1 d-f) for Laplace resistance generation.28 When negative pressure was applied at the microchannel by a vacuum pump, the majority carrier oil solutions containing acid, PFPE-PEG and PFO were drawn off, while hydrogel microspheres were remained in the main channel due to deformation increased the pressure in the droplet. Besides, a line of micropillars was set in the terminal point of the main channel, the 50 μm space between adjacent pillars allow the liquid to pass through, yet the larger microspheres were trapped inside (Figure S1 g-h). Later, the low velocity of aqueous solution could wash out the residual oil around microspheres and achieve the medium transformation. During oil extraction, a density gradient distribution of microspheres had been observed in the main channel (Figure 2B). Then, an aqueous phase (e.g. CaCl2 solution, culture medium, or other stock solutions) was injected to the main channel through the side entrance to clean and resuspend microspheres. The morphology and diameter distribution of the microgel spheroids before and after treatment was obtained from bright field microscopy images (Figure 2C). About 9 % increase in microspheres diameters was observed which may be due to the affinity of carboxyl group of alginate to water molecules.29 Such on-line manipulation not only achieved rapid transformation from oil phase into aqueous phase, but also collected massive monodispersed microspheres as microspheres would not be stacked deformation in the oil phase. The size of the microgel spheroid was an essential factor for the long-term culture of the encapsulated cells. Broad size distributions (Figure 2D) and diverse flow patterns including dripping, injecting, and annular flow (Figure S2), related to the ratio of flow rate of the oil phase and alginate phase (Foil/Falg) were optimized to obtain the spheroid of the desired size. Thereafter, during cellencapsulation experiments, the microgel spheroids with core diameter size of 170 µm had been used,



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Figure 2. Simple one-step preparation, extraction of alginate microsphere from oil phase using integrated microfluidic device. Bright-field microscopy images depicting (A) microsphere generation process including emulsification and demulsification on 1st PDMS device (Here black food dye was mixed with alginate phase and oil-soluble dye was mixed with oil phase). (B) Distribution of the microspheres in the main channel during oil extraction process. (C) (a) Images of stable cell-laden microsphere without demulsification and oil extraction process. (c) Corresponding size distribution of the monodisperse microsphere in presence of oil and their average diameter to be 163.4 ± 8.1 µm. (b) Images of stable cell-laden microsphere immediately collected in medium solution after demulsification and oil extraction process. (d) Corresponding size distribution of the monodisperse microsphere in the absence of oil and their average diameter of 179.7 ± 10.0 μm. (Here 100 droplets were counted, which were used to calculate their average diameters), (D) Varying microsphere size as a function of the flow rate ratio of Foil/Falg, and corresponding confocal images of microsphere (To visualize microgel, 0.15 vol% red fluorescent polystyrene nanomicrogels was mixed with the alginate phase and green fluorescent dye of calcein-AM were used to stain the cells).

because it could encapsulate sufficient amounts of cells and allowed easy oxygen exchange.30 Cell Viability of Cell-Laden Microsphere on the Integrated Microfluidic Device. The cell viability encapsulated in hydrogel microspheres generated on the integrated microfluidic device was compared with the conventional centrifugation extraction method.23 A 9-fold increase in cell survival was observed through onchip extraction method (Figure 3A and B). The high cells viability was attributed to the gentle treatment of the integration design, which reduced the exposure time in harsh oil reagents and nullified high mechanical stress caused by centrifugation. Besides, the cell viability in mineral oil group was comparable with expensive biocompatible HFE-7500 oil group. Thus, this technique offers high cell viability and is compatible with diverse continuous phase systems, which has widespread applications in biomimetic cell culture engineering.

Evaluation of the Microenvironment and Cell Properties on The Integrated Microfluidic Device. Nutrients scarcity and metabolite accumulation in culture medium greatly affect the cellular functions.31-33 Therefore, we have reached the physiological dynamic state by supplying the culture medium to the cell-loaded hydrogel microspheres at flow rate of 5 μl/min.34 The flow penetrated the nutrients into cell-laden microgel spheroids and driven the metabolites into the medium. The medium passed from the main channel after interaction with cells was collected after every 12 h and analyzed for the amount of a nutrient, glutamine and a metabolite, lactic acid. As a comparison, the static-culture experimental group changed the medium every 24 h. In this microenvironment where there is no liquid circulation for a long time, nutrients gradually run out meanwhile metabolic waste accumulated. So the concentration of glutamine and lactic acid showed wide fluctuations in staticculture conditions, whereas the concentration of glutamine


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(2.95 mM) and lactic acid (0.74 mM) remained stable in dynamic-culture (Figure 4A and B). Furthermore, HUVEC

Figure 3. Fate of cells encapsulated in alginate microspheres. (A) Confocal fluorescent observation status of cell growth generated by (a) conventional centrifugation off-chip method in mineral oil continuous phase system, (b) extraction on the integrated microfluidic device in mineral oil continuous phase system, (c) conventional centrifugation off-chip method in HFE-7500 continuous phase system, (d) extraction on the integrated microfluidic device in HFE-7500 continuous phase system. Calcein AM/EthD-1 staining kit was used to stain the cells. (B) Quantitative analysis of cell viability rates of encapsulated HUVEC cells in microsphere among diverse experiment operations.

Figure 4. (A) The lactic acid secretion and (B) The glutamine consumption under static and dynamic-culture states. Statistical analysis of the amount of (C) GSSG and (D) mitochondria over 5 days under static and dynamic culture states. (E) The viability of various cells encapsulated in microgel was analysed by both in extraction on-chip method and dynamic modulation method (*P < 0.05, **P < 0.01).

Figure 5. The construction of U87-MG/HUVEC co-culture model on the integrated microfluidic chip. (A) Schematic illustration of the modified 1st PDMS module and their fabrication of speciallyassembled heterogeneous-cell-laden microgel. (Cell tracker™ Deep Red and Cell Tracker™ Green CMFDA were used to stain U87-MG, HUVEC cells, respectively). (B) Confocal fluorescent microscope images of in situ culturing heterogeneous -cell-laden microgel on-chip under dynamic-steady states. (The white arrow represents the medium flow direction and the cell viability was evaluated using Calcein AM/EthD-1 staining kit). (C). VEGF secretion of U87-MG/HUVEC co-culture was measured over 5 days based on our integrated platform. Here conventional centrifugation extraction and static culture data was used to compared (*P < 0.01).

cells were stained separately using dihydroethium (DHE) and Mitotracker green to visualize oxidized glutathione (GSSG) and mitochondria, respectively. The fluorescent intensities per unit area corresponds to the amount of stained intracellular content as a function of time were obtained from confocal microscopy images (Figure S3), using Image J software. Lower fluorescent intensities of DHE and MitoTracker were observed (Figure 4C and D), allude the less content of GSSG and mitochondria in cells. Contrary, high contents of GSSG were observed in cells cultured under the static state. The high level of GSSG in combination with high mass accumulation of mitochondria promotes cellular senescent and inhibit normal physiological metabolism.35,36 Similarly, uppsala 87 Malignant Glioma (U87MG) and Liver hepatocellular carcinoma (HepG2) cells showed the long-term activity (Figure 4E). These results imply that the dynamic microfluidic device offered a non-suppressed environment for cell-growth and ensured the normal cellular behavior. Fabrication and Evaluation of the Co-culture Model of U87-MG-HUVEC Cell-Laden Microspheres. Furthermore, the architecture of the first module was slightly modified to provide two alginate disperse phases, which displayed stable laminar flow in the micron scale microchannels. When mixing with HUVEC and U87-MG cells, respectively, we obtained a tumor-endothelial co-culture model with a well-assembled 3D core-shell structure. The rapid alginate-calcium ionic gelation rendered two types of cells stay in their respective spatial regions without obvious external mixing (Figure 5A and Figure S4). The cell-laden microgel spheroids were then gently



transformed to the main channel and cultured in a fresh cell medium. The high cell viability remained for one week inside the stable-structure hydrogel spheroid under dynamic-culture condition (Figure 5B). In comparison to conventional centrifugal extraction and static culture (see Surpporting Information), the experimental group based on the integrated microfluidic platform released more VEGF (Figure 5C). This data revealed significant differences (*p < 0.01) from the conventional co-culture model. The high VEGF release was also in agreement with low GSSG level and lower mitochondrial activity. These findings verified the validity of microfluidic dynamic platform for cells co-culture, which could be significant to mimic the physiological tumor- endothelia coculture model for cancer therapy and tissue engineering.

CONCLUSIONS In summary, an efficient integrated microfluidic platform was developed for the generation and culture of cell-laden 3D alginate microspheres. The platform allowed the on-line extraction of cell-laden microspheres into the main microchannel. The main channel provided the continuous supply of fresh cell medium to the cells and washed out the metabolite, mimicked a more biomimetic physiological environment. As verified by constructing a simple endothelialtumor co-culture model using the presented microfluidic approach, high-level secretion of angiogenic stimulator VEGF was observed. In addition, the built-in cells can be easily replaced with other cells to construct diverse artificial models and study cell-cell communications. Furthermore, the platform has potential applications to couple with mass spectrometry to provide qualitative and quantitative analysis of cellular behavior.37,38

ASSOCIATED CONTENT Supporting Information Four figures showing the design and detail of microfluidic chip, the flow patterns of droplet generation, the confocal fluorescent images of GSSG and mitochondria under dynamic-culture and staticculture states and the assembly process of U87-MG cells and HUVEC cells in the core-shell microsphere.

AUTHOR INFORMATION Corresponding Author *Phone: +86 10 82545621. E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (Nos. 21727814, 21435002, 21621003) and National Key R&D Program of China (2017YFC0906800).

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