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A Decellularized Matrix Produced by Mesenchymal Stem Cells Modulates Growth and Metabolic Activity of Hepatic Cell Cluster Jooyeon Park, Joyeon Kim, Kathryn Michele Sullivan, Seungyun Baik, Eunkyung Ko, Myung-Joo Kim, Young Jun Kim, and Hyunjoon Kong ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00494 • Publication Date (Web): 10 Nov 2017 Downloaded from http://pubs.acs.org on November 13, 2017

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ACS Biomaterials Science & Engineering

A Decellularized Matrix Produced by Mesenchymal Stem Cells Modulates Growth and Metabolic Activity of Hepatic Cell Cluster Jooyeon Park,1 Joyeon Kim,2 Kathryn Michele Sullivan,3 Seungyun Baik,4 Eunkyung Ko,3 Myung-Joo Kim,5 Young Jun Kim,4 Hyunjoon Kong*,1,3, 6 1

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-

Champaign, Urbana, IL 61801, USA 2

Department of Materials Engineering, University of Illinois at Urbana-Champaign, Urbana,

IL 61801, USA 3

Department of Bioengineering, Institute for Genomic Biology, University of Illinois at

Urbana-Champaign, Urbana, IL 61801, USA 4

Environmental Safety Group, Korea Institute of Science and Technology (KIST Europe),

Campus E 7.1, Universitaet des Saarlandes, Saarbrucken 66123, Germany 5

Department of Prosthodontics and Dental Research Institute, School of Dentistry, Seoul

National University, Seoul 110-749, Korea 6

Carl L. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign,

Urbana, IL 61801, USA

KEYWORDS: Liver organoid, cell spheroid, stroma, P450 cytochrome detoxification, vascular endothelial growth factor

ACS Paragon Plus Environment


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ABSTRACT Miniature organ-like three-dimensional cell clusters often called organoids have emerged as a useful tool for both fundamental and applied bioscience studies. However, there is still a great need to improve the quality of organoids to a level where they exhibit similar biological functionality to an organ. To this end, we hypothesized that a decellularized matrix derived from mesenchymal stem cell (MSC) could regulate the phenotypic and metabolic activity of organoids. This hypothesis was examined by culturing cells of interest in the decellularized matrix of MSCs cultured on a 2D substrate at confluency or in the form of spheroids. The decellularized matrix prepared with MSC spheroids showed a 3D porous structure with a higher content of extracellular matrix molecules than the decellularized matrix derived from MSCs cultured on a 2D substrate. HepG2 hepatocarcinoma cells, which retain the metabolic activity of hepatocytes, were cultured in these decellularized matrices. Interestingly, the decellularized matrix from the MSC spheroids served to develop the hepatic cell clusters with higher levels of E-cadherin-mediated cell-cell adhesion and detoxification activity than the decellularized matrix from the MSCs cultured on a 2D substrate. Overall, the results of this study are useful in improving biological functionality of a wide array of organoids.


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INTRODUCTION A small cell cluster organized to present a similar architecture of normal or pathologic tissues, often called as an "organoid," has emerged as a vital tool to fundamental and applied bioscience studies.2-4 Organoids are engineered by assembling lineage-committed cells or stem cells with pluripotency or multipotency into a cluster form and activating cell differentiation.5-6 These organoids present great potential that would allow one to better understand development and regeneration processes of tissues and organs of interest. Organoids would be useful to study disease-related tissue remodeling and evaluate the therapeutic efficacy of newly developed drug molecules before in vivo evaluation.7-8 Also, these cell clusters can be transplanted to treat various tissue defects, acute injury, and chronic diseases.9-10 The organoids are prepared by culturing cells of interests in the MatrigelTM supplemented with a series of peptides, growth factors, and cytokines.11-13 These bioactive molecules reconstitute soluble signals provided by the extracellular microenvironment in vivo and, in turn, stimulate proliferation and differentiation of cells in a 3D matrix. In contrast, little attention was paid to study whether a matrix that surrounds cell clusters can act as an insoluble signal that influences the growth and metabolic activity of cell clusters. In general, cells that constitute tissues and organs are surrounded by connective tissue termed as stroma.14 It is well agreed that the chemical and mechanical properties of the stroma significantly affect the growth and metabolic activities of cells and subsequently, homeostasis of tissues and organs.15-17 Also, abrupt or gradual changes in structure and properties of the stroma due to injury or pathogenesis result in aberrant biological activities of cells and finally, dysfunctional tissues and organs.18



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As a way to recapitulate insoluble signals to cell clusters in vitro, this study hypothesized that a decellularized matrix produced by mesenchymal stem cells (MSCs) would regulate both morphology and phenotypic activity of cell clusters.19-21 To examine this hypothesis, MSCs were cultured in a spheroidal form followed by decellularization with a surfactant.22 This spheroid-derived matrix fabrication is different from traditional methods used to prepare the decellularized matrix. In the past, the decellularized matrix was prepared by removing cells in a primary tissue.23-25 Alternatively, the decellularized matrix was prepared by sequentially culturing cells on a 2D substrate to confluency, detaching them from the substrate, and incubating them with surfactant. In this study, human liver cancer cell line (HepG2) cells were cultured in the decellularized matrices to form hepatic cell clusters. Although the HepG2 cells present tumorigenicity, these cells can organize in the form of hepatic lobules and retain some of the physiological function of non-cancerous, normal hepatocytes such as detoxification activity.26-28 These cells were also cultured on agarose gel or in a matrix assembled by decellularizing a cell sheet formed on a 2D substrate. We examined the architecture and density of the decellularized matrix by using electron microscopy and mass spectroscopy, respectively. We further studied the effects of these decellularized matrices on regulating cellular adhesion protein expression, growth, hepatocyte-specific metabolic activities, and angiogenic growth factor secretion of resulting hepatic cell clusters. MATERIALS AND METHODS Fabrication of decellularized matrices. Decellularized matrices were fabricated by using mouse mesenchymal stem cells (MSCs) (D1 cells, ATCC). MSCs were cultured in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco). Once cells reach confluence, they were


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harvested and used to form decellularized matrices. First, MSCs were harvested from the cell culture flask by using the trypsin-disodium ethy le-nediaminetetraacetic acid (EDTA) dissolved in neutral phosphate buffer saline. Then, MSCs were suspended in the decellularizing solution and incubated overnight at room temperature. The decellularizing solution was prepared by adding 20 mM ammonium hydroxide (Sigma) and 0.5% (v/v) Triton-X to the phosphate buffered saline (PBS). Alternatively, the decellularized matrix was derived from the MSC spheroids. The spheroids were prepared by the hanging-drop method.1 First, MSCs were diluted at a concentration of 1 × 106 cells/mL media, and 20 µL of cells were dropped on the lid of the cell culture plate. The plate was flipped to an up-side down position to form droplets hanging from the top. Then, the drops of cells were cultured for three days until they aggregate and form cell spheroids. The MSC spheroids were then harvested and incubated in the decellularizing solution. These decellularized matrices were washed with PBS and remaining deoxyribonucleic acids were removed by using 0.1 M deoxyribonuclease (Sigma). Morphological characterization of decellularized matrices. Scanning electron microscope (SEM) was used to examine the morphology of decellularized matrices. The gels were fixed with 3.7% paraformaldehyde for 4 h, and then gently removed from the 96 well plate and added to a petri dish. The gels were gradually dehydrated by incubating in 30%, 50%, 70%, and 100% ethanol. Each ethanol solution was added for at least 60 min. The gels were then briefly incubated in sieve ethanol, and then thoroughly dried using an automated critical point dryer (Tousimis). Then, the gels were immediately coated with a 6-8 nm layer of gold (EMITECH 575). Scanning electron microscope images (Hitachi S4700) were taken with at an accelerating voltage of 5 to 10 kV, and a working distance of around 4-8 mm. The



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emission current was adjusted as needed to reduce sample damage. LC-MS analysis of decellularized matrices. To minimize ionization suppression, polyethylene glycol (PEG) in samples was separated and removed by extracting through SPE cartridges. 6 mL Oasis MCX (150 mg) was used to separate PEG by the cation exchange function and target compounds by hydrophilic-lipophilic balance function of the cartridges. Oasis MCX (Mixed Cation Exchange) cartridges for solid phase extraction (SPE) of samples were obtained from Waters (Eschborn, Germany). The cartridges were conditioned with 100% methanol followed by water with 1% formic acid. 100 µL of each sample was taken and diluted by water with 1% formic acid up to 10 mL. This diluted and acidified samples were then loaded to the cartridges followed by washing with 1 mL of water with 1% formic acid. 4 mL of 100% methanol was eluted to separate PEG. The final elution of samples was then processed with 8 mL of 100% methanol with 5% ammonium hydroxide to elute basic residues in samples. These two separated elution parts for each sample were then evaporated by nitrogen gas blowing until dryness. 200 µL of water with 20% acetonitrile was added to each sample to reconstitute it. The concentrations of extracellular matrix molecules in decellularized matrices were subsequently determined by BCA protein assay kit (PIERCE, CA, USA). The final concentrations of 1 µg/ml decellularized matrix samples were used for proteome analysis with mass spectrometer. Samples prepared at a concentration of 1 µg/ml by SPE extraction were analyzed on Agilent 6460 Jet Stream electrospray ionization (ESI) triple quadrupole (QQQ) mass spectrometer (MS) coupled with Agilent 1290 LC system (Agilent Technology, CA, USA). Separation of compounds in samples was performed on Thermo Scientific BetaBasic C-18 column (2.1 mm × 150 mm, 3.0 µm particle size) equipped with a guard column with the same material (Thermo Fisher Scientific, CA, USA). A 10 µL was applied as an injection


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volume. Gradient elution at a flow rate of 2.0 mL/min was used with two mobile phases, water with 0.1 % formic acid (A) and acetonitrile (B). As the gradient profile used for separation, the initial conditions were 95% (A) and 5% (B), and after keeping the same condition for 5 minutes, (B) was increased to 95% in 25 minutes followed by holding the same conditions over 10 more minutes. These mobile phases were then moved back to the initial conditions within 0.5 minutes, followed by keeping it for 4.5 minutes more. The equilibration time was following for 5 more minutes, which resulted in a total run time of 50 minutes including a post-run protocol. Mass spectrometric conditions include: gas temperature of 300 °C, gas flow of 10 L/min, nebulizer gas of 45 psi, capillary voltage of 3500 V, and source temperature set to 350 °C. Nitrogen gas supplied by the nitrogen generator (Agilent Technology, CA, USA) was used as a drying and a nebulizing gas. The MS analysis in Full scan mode was carried out in a positive ionization mode with fragmentor of 110V and cell accelerator voltage of 7 eV. All data were collected and processed using Agilent Technologies MassHunter Software (version B). Assembly of HepG2 cell clusters. HepG2 cells (ATCC) were cultured in DMEM with 10% FBS and 1% penicillin-streptomycin (Gibco). When cells were confluent, they were harvested. 1 × 105 cells were plated on 1% (w/v) agar coated 12-well plates with or without decellularized matrices obtained from 1 × 106 MSCs. The clustered HepG2 cells were harvested for evaluation at Day 3. The relative number of metabolically active cells was measured by using the CCK-8 kit (Sigma). Evaluation of the morphology and phenotype of HepG2 cell clusters. The morphology and phenotype of HepG2 cell clusters were examined by immunostaining the HepG2 cells. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton-X, and blocked with 1% (w/v) bovine serum albumin. For intracellular staining of F-actin, cells were



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incubated with phalloidin-Alexa 488 (Invitrogen). For staining of the E-cadherin, cells were incubated sequentially with the monoclonal E-cadherin primary antibody (Cell Signaling Technologies) and the Alexa Fluor 568 secondary antibody (Invitrogen). For staining of integrin β1, cells were incubated with mouse monoclonal β1-integrin primary antibody (Abcam, ab15148) and Alexa Fluor 488 secondary antibody (Invitrogen). 4’-6-diamidino-2phenylindole (Sigma) was used to stain the nucleus of cells. After staining with the antibodies, the samples were washed with phosphate buffered saline. Then, cells were imaged with the multi-photon confocal microscope (Zeiss LSM 710) using the 2-photon mode for phalloidin and the single-photon mode for integrin β1, E-cadherin and cell nucleus imaging. Analysis of angiogenesis activities of HepG2 cell clusters. Three days after seeding HepG2 cells into decellularized matrices, the medium was changed to DMEM without FBS for 24 h. The concentration of VEGF in the supernatant was then measured by using the mouse VEGF enzyme-linked immunosorbent assay (ELISA) kit (n = 3 per group, R&D Systems). The measured VEGF amount was normalized to the initial cell density (i.e., 105 cells/condition). Analysis

of

hepatocyte-specific

metabolic

activities.

7-ethoxy-resorufin

O-

deethylation (EROD) assay was performed to analyze the cytochrome P450 activity of HepG2 cells. HepG2 cells were incubated in DMEM containing 20 µM 7-ethoxyresorufin, 25 µM dicumarol, and 2 mM probenecid for 5 min. The fluorescence from the cells was captured by using confocal microscopy (Zeiss LSM 710). Cells were excited at the laser wavelength of 510 nm. The resulting emission above 570 nm was collected. Pinhole size was set at 100 µm. The same settings were used to image and process all samples. The fluorescence intensity was converted to 256 pseudo-color map by using ImageJ. Statistical analysis. The quantitative data were expressed as the means ± standard deviations. The statistical analysis was performed by using the one-way analysis of variance (ANOVA)


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combined with the Tukey significant difference post hoc test. The GraphPad Prism 6 software was used for this analysis. A value of p < 0.05 was considered to denote statistical significance. RESULTS AND DISCUSSION 1. Assembly and analysis of decellularized matrices The decellularized matrix was prepared either by incubating MSCs cultured on a cell culture flask (Figure 1a) or MSC spheroids formed via a hanging drop method in a Triton-X surfactant-water mixture (Figure 1b). This surfactant-water mixture lyses the cell and in turn, enables the removal of cells and soluble factors associated with the cell membrane. According to the scanning electron microscopy (SEM) images, the decellularized matrix obtained from MSCs cultured on a 2D substrate was in a powder form (Figure 2). In contrast, the decellularized matrix derived from MSC spheroids showed a 3D porous structure with scattered particles and fibers.

Figure 1. Schematic description of the processes to prepare decellularized matrices of mesenchymal stem cells (MSCs). (a) After culture on a cell culture flask at confluency over 3 days, MSCs were detached and incubated in the water-surfactant mixture for decellularization. (b) MSCs spheroids assembled via the Hanging drop method were incubated in the watersurfactant mixture for decellularization.



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Figure 2. Scanning electron microscopy images of decellularized matrices. (a) Decellularized matrix formed by MSCs cultured on the 2D substrate (b) Decellularized matrix of MSC spheroids. In (b), red dotted line represents the boundary of a pore and white arrows represent the fibrous matrix. Decellularized matrix molecules were compared qualitatively and quantitatively from chromatograms obtained with the liquid chromatography-mass spectroscopy (LC-MS).29-30 For the qualitative comparison of samples, most of the components in decellularized matrices were detected within 20 minutes of separation (Figure 3a). In particular, the chromatogram displayed two characteristic major peaks which were found around 3 (Peak 1) and 16 (Peak 2) minutes of retention time. It is suggested that these peaks represent decellularized matrix molecules because no peaks were found from blank media. In contrast, the peaks appearing after 20 minutes of separation (peaks after peak 3) were obtained from all samples including the blank media. Therefore, these peaks were not taken into consideration.


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Figure 3. Chromatograms for samples run with LC-MS full scan mode; (a) Base peak chromatograms (BPC) in 40 min of running. (b) Extracted ion chromatograms (EIC) from the major peak (1). (c) Extracted ion chromatograms (EIC) from the major peak (2). For all chromatograms, black, blue and red lines indicated medium blank, the decellularized matrix of single MSCs and the decellularized matrix of MSC spheroids, respectively. Ranges beyond retention time selected (3), retention time between 20 and 40 minutes, was neglected for EIC due to existence of same peaks from medium blank. Heights of peaks 1 and 2 were compared for relatively quantitative comparison of the mass of decellularized matrix molecules. The height of peak 1 obtained with the



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decellularized matrix from MSC spheroids was 2.5 times higher than that obtained with the decellularized matrix from MSCs cultured on a 2D substrate (Figure 3b). The height of peak 2 appeared on 12.7 minutes of separation was also higher with the decellularized matrix from MSC spheroids than the decellularized matrix from MSCs cultured on a 2D substrate (Figure 3c). These results address that both MSC spheroids and MSCs grown on a 2D substrate produce the same extracellular matrix molecules. However, MSC spheroids produce a larger mass of extracellular matrix molecules than those cultured on a 2D substrate. Taken together, we propose that the decellularized process of the MSCs cultured on a 2D substrate negatively affects the association between extracellular matrix molecules. Therefore, the resulting decellularized matrix is in a fragmented form. In contrast, it is likely that the larger number of extracellular matrix molecules produced by MSC spheroids retains structural integrity during the cell lysis. 2. Morphological analysis of HepG2 cell cluster cultured in decellularized matrices Effects of decellularized matrices on the formation and phenotypic activities of hepatic cell clusters were examined by culturing HepG2 cells within decellularized matrices. Cells were introduced into the matrices by rehydrating the lyophilized decellularized matrices with the suspension of HepG2 cells. As a control, HepG2 cell clusters were prepared by culturing 1 × 105 HepG2 cells on 1% (w/v) agar-coated 12-well plates (Figure 4a).31-32 Agar prevented cell adhesion to the plates and promoted the formation of 3D cell clusters.


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Figure 4. Immunohistochemical analysis of HepG2 cell clusters formed by three different processes. (a) Schematic description of the processes to prepare HepG2 cell clusters by culturing cells on the non-adherent agarose gel. In (b)-(d), Condition I represents the HepG2 cell clusters formed on the non-adherent agarose gel. Condition II represents the HepG2 cell clusters formed in the decellularized matrix of MSCs cultured on the 2D substrate. Condition III represents the HepG2 cell clusters formed in the decellularized matrix of MSC spheroids. (b) Immunostained images of F-actin (green color) and DAPI (blue color) of HepG2 cell clusters. (c) The relative number of metabolically active cells in each condition. The number of cells was normalized to the number of metabolically active cells in Condition I. Three samples were used for analyses. The difference of values for each condition was statistically significant (*p < 0.05). (d) Staining of E-cadherin (red color) and Integrin β1 (green color) to examine the cellular adhesion to neighboring cells and the decellularized matrix. Scale bars in (b) and (d) represent 20 µm. According to images of filamentous intracellular actin (F-actin), the HepG2 cells cultured on the agarose gel formed a thin cell sheet with about 1 µm thickness (Figure 4b-I). In contrast, HepG2 cells grown in the decellularized matrices formed cell clusters. In particular, the decellularized matrix of MSC spheroids led HepG2 cells to cluster into a larger tissue mass than the matrix of MSCs cultured on a 2D substrate (Figure 4b-II & III). The viability of HepG2 cells in each condition was evaluated by using the cell counting kit-8.33 This kit marks the metabolically active cells with yellow colored formazan dye, a



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reduced product of tetrazolium salt by dehydrogenase. HepG2 cells cultured without decellularized matrices had the least number of live cells. Also, the HepG2 cell clusters formed within the MSC spheroid-derived matrix presented the larger number of viable cells than those prepared within the matrix of MSCs cultured on a 2D substrate (Figure 4c). As such, the analysis of total proteins collected from lysates of HepG2 cell clusters displayed that the matrix derived from MSC spheroids supports the continuous growth of HepG2 cells (Supplemental Figure S1). The interactions between cells in the clusters were examined by staining E-cadherin and integrin β1 of the cells. The E-cadherin represents the cellular adhesion to neighboring cells,34 while the integrin β1 represents the cellular adhesion to an extracellular matrix,35-36 The malignant liver cancer cells display the increased expression of integrin β1 due to active matrix invasion, while normal hepatocytes exhibit the increased expression of E-cadherin.18, 26

The HepG2 cell sheets formed by culturing cells on the agarose gel showed lower E-

cadherin and higher integrin β1 expressions than cell clusters formed in the decellularized matrix derived from MSC spheroids (Figure 4d). Therefore, these results indicate that the MSC spheroid-derived matrix suppresses malignant activity of HepG2 cells. 3. Physiological analysis of HepG2 cell cluster cultured in decellularized matrices The activity of HepG2 cell clusters to secrete vascular endothelial growth factor (VEGF) was evaluated by measuring the concentration of VEGF in the cell culture media. It is well agreed that malignant cancer cells overproduce VEGF and, in turn, stimulate angiogenesis.3739

The resulting microvasculature supplies oxygen and plasma to cancer cells and also

facilitates metastasis.40-42 The amount of VEGF secreted by cells was measured after 3 days of cell culture because there was a noticeable difference of cell growth rate between conditions. HepG2 cells cultured on the agarose gel exhibited the highest level of VEGF


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secretion (Figure 5a). In contrast, HepG2 cells cultured in the decellularized matrix of MSC spheroids displayed the lowest level of VEGF secretion, similar to normal hepatocytes.

Figure 5. Evaluation of the metabolic activity of HepG2 cell clusters. (a) Quantification of the VEGF secretion demonstrated that the HepG2 cell clusters formed on the agarose gel (Condition I) present the largest secretion activity. The measured VEGF concentration was normalized to the total protein concentration from HepG2 cell lysates measured on Day 3. Conditions II and III represent HepG2 cell clusters formed in the decellularized matrix of MSCs and those formed in the decellularized matrix of MSC spheroids. Four samples were used for analyses. The difference of the values between each condition was statistically significant (*p < 0.05). (b) Cytochrome P450 detoxification assay to examine the hepatocytespecific metabolic activity. HepG2 cell clusters formed in the decellularized matrix of MSC spheroids (Condition III) compared with HepG2 cell clusters formed on the agarose gel (Condition I) and HepG2 cell clusters formed in the decellularized matrix of MSCs cultured on the 2D substrate (Condition II). The scale bar represents 100 µm. The hepatocyte-specific metabolic activities of HepG2 cells were evaluated by using the ethoxy resorufin-O-deethylase (EROD) assay that measures the cytochrome P450 detoxification level.43 The cytochrome P450 enzyme can convert ethoxy resorufin into fluorescent resorufin through deethylation. Therefore, by measuring the intensity of fluorescence from resorufin, it is possible to evaluate the cytochrome P450 activity. Interestingly, the HepG2 cells cultured on the agarose gel showed the lowest level of P450 detoxification. In contrast, HepG2 cells cultured in the MSC spheroid-derived matrix showed the most intense fluorescence signals (Figure 5b). Again, this result demonstrates that the



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decellularized matrix of MSC spheroids plays a significant role in retaining the hepatic function of HepG2 cells.44 These results highlight the important role of decellularized matrices in regulating the secretion and metabolic activities of HepG2 cells. We propose that a thick and porous decellularized matrix produced by MSC spheroids, as shown in figure 2, provide HepG2 cells with a three-dimensional environment, similar to that of a 3D liver organ. As such, HepG2 cells could retain the detoxification function more actively than those cultured on a fragmented decellularized matrix of MSCs. There may also be additional biochemical and biomechanical effects on the HepG2 cell function, which will be examined systematically in future studies. CONCLUSIONS This study demonstrates that the microstructure of decellularized matrices regulates the secretion and detoxification activities of hepatic cell clusters. In particular, MSC spheroids produced a porous decellularized matrix while MSCs cultured on the 2D substrate produced a thin, fragmented decellularized matrix. The amount of matrix molecule was also higher with the matrix of MSC spheroids than with the matrix of MSCs. The decellularization of MSC spheroids was also advantageous to retain hepatocyte-function of the HepG2 cells. This new method to create the decellularized matrix would be useful to engineer a broad array of physiologically relevant organoids. Moreover, the decellularized matrix of MSC spheroids will be useful to repair and recreate damaged liver tissues. ASSOCIATED CONTENT Supporting Information. The following file is available free of charge. Graph of the analysis of total protein concentration of HepG2 cell lysates in HepG2 cell


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clusters, clusters in 2D decellularized matrix of MSCs, and clusters in spheroidal decellularized matrix of MSCs. (PDF) AUTHOR INFORMATION Corresponding Author. Correspondence to: [email protected]

Author Contributions. JK, EK, MJK, YJK, and HJK had major contributions to the concept and design. JP, JK, KMS, and SB acquired the data. JP, JK, KMS, SB, MJK, and HJK analyzed and interpreted the data. JP, EK, YJK, and HJK participated in drafting the article. KMS and HJK revised the article. Funding Sources. This work was supported by Korean Institute of Science and TechnologyEurope (Joint research 11791 to H.K.), National Science Foundation (STC-EBICS Grant CBET-0939511

to

H.K.),

and

National

Research

Foundation

of

Korea

(2015R1A6A3A03015834 to J.Y.P.), and Overseas Training Program of Seoul National University Dental Hospital (to M.J.K).



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For Table of Contents Use Only A Decellularized Matrix Produced by Mesenchymal Stem Cells Modulates Growth and Metabolic Activity of Hepatic Cell Cluster Jooyeon Park, Joyeon Kim, Kathryn Sullivan, Seungyun Baik, Eunkyung Ko, Myung-Joo Kim, Young Jun Kim, Hyunjoon Kong*


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A Decellularized Matrix Produced by Mesenchymal Stem Cells Modulates Growth and Metabolic Activity of Hepatic Cell Cluster Jooyeon Park,1 Joyeon Kim,2 Kathryn Michele Sullivan,3 Seungyun Baik,4 Eunkyung Ko,3 MyungJoo Kim,5 Young Jun Kim,4 Hyunjoon Kong*,1,3, 6 1

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-


Department of Materials Engineering, University of Illinois at Urbana-Champaign, Urbana, IL

61801, USA 3

Department of Bioengineering, Institute for Genomic Biology, University of Illinois at Urbana-


Environmental Safety Group, Korea Institute of Science and Technology (KIST Europe), Campus

E 7.1, Universitaet des Saarlandes, Saarbrucken 66123, Germany 5

Department of Prosthodontics and Dental Research Institute, School of Dentistry, Seoul National

University, Seoul 110-749, Korea 6

Carl L. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign,

Urbana, IL 61801, USA

Graphics for Manuscript: full-sized figures



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Figure 1. Schematic description of the processes to prepare decellularized matrices of mesenchymal stem cells (MSCs). (a) After culture on a cell culture flask at confluency over 3 days, MSCs were detached and incubated in the water-surfactant mixture for decellularization. (b) MSCs spheroids assembled via the Hanging drop method were incubated in the water-surfactant mixture for decellularization.


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Figure 2. Scanning electron microscopy images of decellularized matrices. (a) Decellularized matrix formed by MSCs cultured on the 2D substrate (b) Decellularized matrix of MSC spheroids. In (b), red dotted line represents the boundary of a pore and white arrows represent the fibrous matrix.



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Figure 3. Chromatograms for samples run with LC-MS full scan mode; (a) Base peak chromatograms (BPC) in 40 min of running. (b) Extracted ion chromatograms (EIC) from the major peak (1). (c) Extracted ion chromatograms (EIC) from the major peak (2). For all chromatograms, black, blue and red lines indicated medium blank, the decellularized matrix of single MSCs and the decellularized matrix of MSC spheroids, respectively. Ranges beyond retention time selected (3), retention time between 20 and 40 minutes, was neglected for EIC due to existence of same peaks from medium blank.


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Figure 4. Immunohistochemical analysis of HepG2 cell clusters formed by three different processes. (a) Schematic description of the processes to prepare HepG2 cell clusters by culturing cells on the non-adherent agarose gel. In (b)-(d), Condition I represents the HepG2 cell clusters formed on the non-adherent agarose gel. Condition II represents the HepG2 cell clusters formed in the decellularized matrix of MSCs cultured on the 2D substrate. Condition III represents the HepG2 cell clusters formed in the decellularized matrix of MSC spheroids. (b) Immunostained images of F-actin (green color) and DAPI (blue color) of HepG2 cell clusters. (c) The relative number of metabolically active cells in each condition. The number of cells was normalized to the number of metabolically active cells in Condition I. Three samples were used for analyses. The difference of values for each condition was statistically significant (*p < 0.05). (d) Staining of Ecadherin (red color) and Integrin β1 (green color) to examine the cellular adhesion to neighboring cells and the decellularized matrix. Scale bars in (b) and (d) represent 20 µm.



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Figure 5. Evaluation of the metabolic activity of HepG2 cell clusters. (a) Quantification of the VEGF secretion demonstrated that the HepG2 cell clusters formed on the agarose gel (Condition I) present the largest secretion activity. The measured VEGF concentration was normalized to the total protein concentration from HepG2 cell lysates measured on Day 3. Conditions II and III represent HepG2 cell clusters formed in the decellularized matrix of MSCs and those formed in the decellularized matrix of MSC spheroids. Four samples were used for analyses. The difference of the values between each condition was statistically significant (*p < 0.05). (b) Cytochrome P450 detoxification assay to examine the hepatocyte-specific metabolic activity. HepG2 cell clusters formed in the decellularized matrix of MSC spheroids (Condition III) compared with HepG2 cell clusters formed on the agarose gel (Condition I) and HepG2 cell clusters formed in the decellularized matrix of MSCs cultured on the 2D substrate (Condition II). The scale bar represents 100 µm.


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