Engineering Protective Polymer Coatings for Liver Microtissues

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Engineering Protective Polymer Coatings for Liver Microtissues Xi CHEN, Wen Jiang, Ayman Ahmed, Clare S. Mahon, Markus Müllner, Bocheng Cao, and Tian Xia Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00120 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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Chemical Research in Toxicology

Engineering Protective Polymer Coatings for Liver Microtissues Xi Chen,a,b⊥ Wen Jiang,a,c⊥ Ayman Ahmed,a Clare S. Mahon,d Markus Müllner,d,e Bocheng Cao,f Tian Xiaa,g* aCenter

for Environmental Implications of Nanotechnology, California NanoSystems

Institute, University of California, Los Angeles, California 90095, USA. bWyss

Institute for Biologically Inspired Engineering, Harvard University, Boston, MA

02115, USA. cDepartment

dKey

of Chemistry, University of Georgia, Athens, Georgia 30602, USA.

Centre for Polymers and Colloids, School of Chemistry, The University of Sydney,

Sydney NSW 2006, Australia. eThe

University of Sydney Nano Institute (Sydney Nano), Sydney NSW 2006, Australia.

fDepartment

of Chemistry and Biochemistry, University of California, Los Angeles,

California 90095, USA.

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Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

gDivision

of NanoMedicine, Department of Medicine, University of California, Los

Angeles, California 90095, USA.


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ABSTRACT: Three-dimensional (3D) hepatocyte microtissues (MT), also known as spheroids, have proven to be advantageous in providing more accurate information and physiologically relevant and predictive data for liver-related in vivo tests; therefore, spheroids have increasingly been used to study hepatotoxicity, drug delivery to the liver, and tissue engineering. However, variabilities in the generation of 3D MT remain a major challenge. Methods which encapsulate and protect hepatocytes offer a promising pathway in prolonging cell survival, as well as maintaining its pristine cell functions. Herein, we studied the encapsulation and resultant protective effects of hydrogen bonded, biocompatible polymer coatings for hepatocytes MT in 3D cell culture. We exposed the MT to hepatotoxic nanomaterials (NMs), such as graphene oxide (GO) and cobalt oxide (Co3O4), to assess the protective effects of poly(vinylpyrrolidone) (PVPON) and tannic acid (TA) coatings. The polymer coating allowed the MT to maintain its morphology. More significantly, it increased the viability of hepatocytes-composed MT by hampering the cellular interaction between hostile NMs and hepatocytes. Based on alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, the liver cell 4 ACS Paragon Plus Environment

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function was maintained throughout the coating process, including after NMs treatment. The study provides a straightforward and safe methodology for maintaining the morphology as well as cellular function of hepatocyte MT in vitro.



INTRODUCTION The liver is the largest internal organ and gland in the human body and maintains many vital functions, including bile production to break down fats, detoxification of drugs and chemicals, the synthesis of blood plasma proteins, and the regulation of the mononuclear phagocyte system.1 Hepatocytes are the predominant cell type in the liver. These are epithelial cells that perform important roles in metabolic, endocrine and secretory functions of the body.2 Many studies have used hepatocytes as an in vitro model to investigate liver functions; however, isolated hepatocytes generally dedifferentiate when cultured in traditional two-dimensional (2D) monolayers in vitro, resulting in the loss of phenotype and specific functions.3 For example, the detection of metabolism-mediated hepatotoxicity of drugs in vitro often fails, as hepatocytes in 2D monolayers rapidly lose their morphologies and functions.4 In addition, various bioartificial liver (BAL) systems have been used for clinical trials as a bridge for liver transplantations in patients with liver failure.5 Long term and stable liver-specific functions of hepatocytes should be considered in the development of BAL support 6 ACS Paragon Plus Environment

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systems. Therefore, in the context of mimicking and predicting biological responses of liver hepatocytes, various approaches have emerged to advance in vitro hepatocytes cell culture; examples include co-cultures with non-parenchymal cells and threedimensional (3D) cell culture methods.6,7 Culturing hepatocytes in 3D typically yielding spherical microtissue (MT) also known as hepatocyte spheroids is an effective method in the upregulation of liver-specific functions of hepatocytes due to the high similarity to real tissues.8,9 Moreover, spheroidal MT present high biological relevance due to the tighter intercellular interactions, establishing a more realistic ‘cell architecture’ which also features active uptake and secretion pathways.10,11 A persistent problem of multicellular spheroids is their variability of morphologies or lack of uniformity, often linked to medium composition, cell density and duration in cell culture. For example, liver spheroids from commercial sources may show a loss in uniformity, morphology and integrity after transportation (see Figure S1). These hurdles affecting reproducibility and proliferation rates in 3D cell culture need to be overcome to warrant comparable data evaluation.12 In this context, the protective encapsulation of 7 ACS Paragon Plus Environment


the hepatocytes provides a feasible avenue to prolong cell survival in vitro, while maintaining the protein secretion and avoiding hepatotoxicity.13 Encapsulation using biocompatible polymers provides a semi-permeable protective barrier for the environmental pathogens (e.g. engineered nanomaterials), while permitting the diffusion of nutrients, oxygen and metabolic products which maintain cell survival and function, which could be useful for toxicity screening of small molecule drugs but not nanomaterials.14 Mitry et al. have recently shown that human hepatocytes can be embedded into alginate to protect the cells during transplantation into a patient with acute liver failure – who then benefited from improved cell viability and liver function compared to non-encapsulated hepatocytes.15 Specifically, the encapsulation and cell surface modification to preserve cell function can be achieved using layer-by-layer (LbL) assembly methods.16,17 The LbL technique is a robust methodology allowing for alternating deposition of polymer layers on surfaces. This in turn permits the wrapping of substrates (e.g. cells) with ultra-thin coatings of controllable thickness and composition.18 Hydrogen-bonding systems based on tannic acid (TA), a natural organic 8 ACS Paragon Plus Environment

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polyphenol, have received increasing interest as effective protective coating due to their intrinsic antioxidant and antibacterial properties.19 Kozlovskaya et al. have used TA and its hydrogen-bonding interaction with poly(N-vinylpyrrolidone) (PVPON) to perform multi-layer coating of pancreatic islet cells.6 The TA/PVPON layers deposited on the islets surface could maintain the cell viability and β-cell functionality for at least 96 h in

vitro, which has a great potential use in the pancreatic islets transplantation. In this context, to enhance cell viability, and maintain the cell morphologies and functionalities of MT, we hypothesized that a protective encapsulation of the MT may provide a feasible avenue to prolong cell survival in vitro, while maintaining the protein secretion and avoiding hepatotoxicity induced by environmental pathogens exposure including nanomaterials.13 Herein, we created hepatocyte MT using hanging-drop 3D cell culture technique and fabricated MT encapsulation with multi-layer PVPON/TA coating through hydrogen bonding via LbL assembly. To test the protective effects of PVPON/TA coating, we used two type of nanomaterials (NMs), graphene oxide (GO) nanosheet and cobalt oxide (Co3O4) nanoparticle, as model environmental toxicants to 9 ACS Paragon Plus Environment


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study the cytotoxicity, cellular uptake, hepatocytes function, as well as the interaction of NMs with coated and un-coated spheroids. These two nanomaterials have been demonstrated by us to be able to induce cytotoxicity in vitro (Haiyuan and Ruibin references). We found that the polymer coating significantly decreased the toxicity induced by NMs, but maintained the hepatocytes functions upon exposure to NMs. Our study demonstrates an avenue to preserve hepatocytes MT in vitro functions in the presence of toxic nanomaterials.

EXPERIMENTAL SECTION Chemicals. Tannic Acid (TA), Polyvinylpyrrolidone (PVPON, MW 10K), Fluorescein

isothiocyanate-modified

Hydroxysuccinimide

(NHS),

and

bovine

serum

albumin

(BSA-FITC),

N-

N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide

hydrochloride (EDC) were purchased from Sigma-Aldrich. Reagents were purchased from Aldrich and used as received unless otherwise indicated. Vinylpyrrolidone (VPon, 97%) and hydroxyethyl acrylate (HEA, 96%) were destabilised using a silica gel column. 10 ACS Paragon Plus Environment

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High purity methanol was received from Sigma-Aldrich and diethyl ether was received from Merck. Deuterium oxide (D2O) and deuterated chloroform (CDCl3) were purchased from Cambridge Isotopes Laboratories. CH2Cl2 was dried over 4 Å molecular sieves prior to use. Azobisisobutyronitrile (AIBN) and 2-[(ethoxythioxomethyl)thio] propanoic acid (Xanthate) were recrystallised and provided by the Key Centre for Polymers & Colloids, The University of Sydney, Australia. The Co3O4 nanoparticles were from Lutz Madler’s lab at the University of Bremen and GO were from Mark Hersam’s lab at Northwestern University. Preparation of Fluorescently Labeled PVPON. (1) 7-(diethylamino)-2-oxo-2Hchromene-3-carboxylic acid (compound 1), 4-diethylsalicylaldehyde (3.86 g, 20.0 mmol), diethyl malonate (6.38 g, 40.0 mmol) and piperidine (2 mL) were combined in EtOH (60 mL) and the mixture was heated under reflux for 16 h. 10 % v/v NaOH(aq) (60 mL) was added, yielding immediately a yellow precipitate which re-dissolved to leave a brown solution, which was heated under reflux for a further 15 min. The solution was then cooled to room temperature before acidification to pH 2 using 37 % HCl(aq). The 11 ACS Paragon Plus Environment


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orange solid produced was isolated by filtration and recrystallised from EtOH, to afford the title product as orange needle-like crystals (0.389 g, 8 %).20 1H NMR (200 MHz, CDCl3) δ 1.26 (6H, t J = 7.1 Hz, CH2CH3), 3.50 (4H, q, J = 6.4 Hz, CH2CH3), 6.53 (1H, d, J = 2.9 Hz) 6.70 (1H, dd, J = 3.0, 9.0 Hz), 7.45 (1H, d, J = 8.8 Hz) 8.65 (1H, s), 12.32 (1H,s, OH)

13C

NMR (75 MHz, CDCl3) δ 12.4, 45.3, 96.8, 105.4, 108.6, 110.9, 132.0,

150.2, 153.8, 158.0, 164.4, 165.5. Melting point: 223.5 – 225 ° C. (2) Synthesis of poly(vinylpyrrolidone-co-hydroxyethyl

acrylate)

copolymer

–

poly(VPon-co-HEA).

Xanthate (9.60 mg, 0.05 mmol), VPon (1.00 g, 9.01 mmol), HEA (0.06 g, 0.51 mmol) and 2.0 mL MeOH were mixed in a Schlenk flask and degassed via three freeze-pumpthaw cycles. The polymerisation was carried out at 70 °C and stopped after 6 h by cooling and exposing the mixture to air. The polymer was purified via precipitation into cold diethyl ether, a subsequent dialysis into MeOH and finally freeze-dried from water. (3) Diethylaminocoumarin-labelled poly(VPon-co-HEA). Poly(VPon-co-HEA) (20 mg, 2.1 x 10-6 mol, 1.0 eq.) was dissolved in CH2Cl2. Compound 1 (0.5 mg, 2.0 x 10-6 mol, 1.0 eq.) and dimethylaminopyridine (0.02 mg, 2.0 x 10-7 mol, 0.1 eq.) were added before 12 ACS Paragon Plus Environment

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addition of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (0.4 mg, 2.2 x 10-6 mol, 1.1 eq.). The solution was left to stir at room temperature for 8 h, then dialysed against CH2Cl2 (MWCO 2 kDa). The solution was evaporated to dryness, yielding a yellow film which was taken up in H2O and dialysed again against H2O. The solution was lyophilised to afford the title product (15 mg, 71 %). Fluorescent Labeling GO with BSA-FITC. BSA-FITC labeled GO samples were prepared via a diimide-activated amidation as described before.21 5 mg of N-(3Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and 10 mg of NHydroxysuccinimide (NHS) were dissolved in 2 mL GO sample suspensions (100 µg mL-1) in DI water, and the mixture solutions were then stirred at room temperature for 2 h. Subsequently, the GO pellets were washed twice with DI water and collected by centrifugation at 15,000 rpm for 10 min. The obtained GO samples were then reacted with 1 mL BSA-FITC solution (0.1 mg mL-1 in DI water). After a 12 h reaction with stirring, the resultant BSA-FITC labeled GO samples were washed three times with DI water and then re-suspended in cell culture water for future use. 13 ACS Paragon Plus Environment


Characterizations of Nanomaterials and Polymers. The sizes and morphologies of GO nanosheets and Co3O4 nanoparticles samples were determined using transmission electron microscopy (TEM JEOL 1200 EX microscope) operating at 120 kV. The average size of all samples was obtained by analyzing at least 30 individual nanomaterials. Hydrodynamic size and ζ-Potential measurements of all GO and Co3O4 suspensions with a concentration of 50 µg mL-1 in DI water or cell culture medium were evaluated at 25 °C using a ZetaPALS instrument (Brookhaven Instrument, Holtsville, US). NMR spectra were recorded on a Bruker Avance 300 spectrometer, with 1H at 300 MHz and

13C

at 75 MHz, or on a Bruker Avance 200 spectrometer, with 1H at 200 MHz,

with the residual solvent as an internal standard. Gel permeation chromatography (GPC) was performed on an UFLC Shimadzu Prominence system at 50 °C using DMAc/LiBr as eluent and a flow rate of 1.0 mL·min-1. The GPC sample was taken from the reaction mixture, diluted with DMAc and pressed through a 220 nm Nylon filter prior to injection. The GPC system was calibrated using PMMA standards.


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Maintenance the Hepatocytes and Preparation of Hepatocytes Microtissues. Mouse liver hepatocytes (Hepa 1-6, obtained from ATCC, US) were maintained in completed Dulbecco's Modified Eagle's Medium (DMEM containing 10% fetal bovine serum, 100 units mL-1 penicillin and 100 g mL-1 streptomycin) at 37 °C in a 5% CO2 humidified

atmosphere

and

sub-cultured

prior

to

confluence

using

trypsin-

ethylenediaminetetraacetic acid (EDTA) solution with a concentration of 0.25% (w/v) trypsin - 0.53 mM EDTA. To prepare the 3D hepatocytes tissues, a number of 500 Hepa 1-6 cells in 40 µL cell culture medium were seeded into each well in a 96-well hanging drop plate (HDP1096 Perfecta3D®) on the 1st day. To facilitate the tissue growth, 5 µL of fresh medium was added to the each well every day from 2nd day to 6th day. The tissues were then harvested by transferring into a 96-well U-shape plate once the tissue spheroids formed on 7th day. Specifically, the hanging drop plate was lined up on top of the U-shape plate, and then 40 µL of fresh medium was added to each well to flush the tissue spheroids from the hanging drop plate down to the U-shape plate (Ultra low attachment, Corning Costar, US). 15 ACS Paragon Plus Environment


Fabrication of LbL Polymer (PVPON/TA) Coated Hepatocytes Microtissues. To fabricate the LbL polymer coated hepatocyte tissues, a group of 8 tissues was plated into each well of a 12-well plate (FalconTM polystyrene microplates. Corning, US). The tissues were then re-suspended in 0.5 µL PVPON solution (1 mg mL-1 in cell culture medium), followed by mixing and gently shaking (50 rpm) at 37 °C (performed on Thermo Scientific MaxQ 4450 shaker) for 3 min to allow for polymer adsorption. After incubation, the polymer-coated tissues were washed by two cycles of removal and redispersion in fresh cell medium. 0.5 µL TA solution (0.3 mg mL-1 in cell culture medium) was then added to the PVPON-coated tissues and incubated for 3 min. The tissues were washed as described above. The sequential adsorption of PVPON and TA was repeated until 2/4/6 bilayers were deposited on the tissues. Characterization of 3D Hepatocytes Microtissues. The growth of 3D tissues from 1st day to 7th day was monitored by a differential interference contrast (DIC) microscopy (Observer D1, Zeiss, US) with a 2.5× objective. Fluorescently labeled tissues were imaged on a fluorescence microscopy (Observer D1, Zeiss, US) equipped with a 16 ACS Paragon Plus Environment

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standard FITC filter cube. Transmission electron microscope (TEM) was also used to observe the morphology of the 3D hepatocytes tissue. Harvested tissues were fixed with 2% glutaraldehyde in 0.1 M phosphate-buffered saline (1× PBS) for 2 h at room temperature. After post-fixation in 1% osmium tetroxide (OsO4) in PBS for 1 h, the tissues were dehydrated in a graded ethanol series (30%, 50%, 70%, 95% and 100%), and then treated with propylene oxide and embedded in Epon for 48 h. Approximately 50-70 nm thick sections were cut on a Reichert-Jung Ultracut E ultramicrotome and picked up on Formvar-coated copper grids. The sections were stained with uranyl acetate (UA) and Reynolds lead citrate and imaged on a TEM (JEOL 100 CX, US) at 80 kV in the UCLA BRI Electron Microscopy Core. Further, to analyze the histology of 3D tissues, tissues were fixed in 4% paraformaldehyde (PFA) and embedded in histogel (Thermo Scientific Richard-Allan Scientific Specimen Processing Gel). The samples were further processed for resin embedding, sagittal sectioning, and hematoxylin/eosin (H&E) staining in the Pathology and Laboratory Medicine Department at UCLA. Histological images of tissues were obtained using a light microscopy (Digital 17 ACS Paragon Plus Environment


micromaster premier microscopy, Fisher Scientific, US). In addition, to quantify the number of cells in each 3D hepatocytes tissue, a calibration curve of cell ATP value versus cell number was first set up. Specifically, a serial number of cells, 0, 250, 500, 750, 1000, 2000, 3000 and 4000, were seeded into a 96-well flat-bottom white plate, respectively. After incubation overnight to allow the cell attachment, an ATP assay was performed according to manufacturer’s protocol (ATPlite Luminescence Assay, Perkin Elmer, US) and the ATP values were determined by a microplate reader (SpectraMax M5, Molecular Devices, US). A calibration curve of cell ATP values versus cell numbers was then obtained. Meanwhile, the ATP value of 3D tissues was evaluated by using a 3D cell viability ATP assay (CellTiter-Glo® 3D Cell Viability Assay, Promega, US). The obtained ATP value was then interpreted into the above calibration curve, the number of cells in each tissue was then determined. Assessment of Toxicity of Microtissues Using 3D Cell ATP Viability Assay. A single tissue in 80 µL cell medium was seeded in each well of a 96-well clear bottom white plate. Tissues were then treated with to 20 µL GO nanosheets or Co3O4 18 ACS Paragon Plus Environment

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nanoparticle suspensions (final concentration from 12.5, 25, 50, 100 µg mL-1). After 24 h treatment, the tissues were washed with PBS three times to remove free materials. Cell viability determined by cellular ATP content was accessed by the CellTiter-Glo® 3D Cell Viability Assay (Promega, USA) according to the manufacturer’s instructions. The luminescence intensity was read on a SpectraMax M5 microplate spectrophotometer (Molecular Devices, US). Assessment of Cellular Uptake of Nanomaterials in the Microtissue. Freshly prepared Co3O4 and GO nanomaterials at the concentration of 12.5 to 100 μg mL-1 were incubated with liver microtissues for 24 h in Corning® Costar® Ultra-Low attachment multiwell plates. Microtissues were then transferred to a new U bottom 96-well plate with 100 µL PBS in each well and then centrifuged at 1500 rpm for 5 min to settle down into the bottom of the plate. The absorbance of the nanomaterials in the microtissues were read at 460 nm, and particle standards for both Co3O4 and GO at the dose ranged from 0 to 50 ppm were tested at the same time. The cellular uptake amount of the



nanomaterials in each microtissues was calculated based on the standards. At least 5 microtissues were used at each dose. Assessment of ALT and AST Content in the Microtissues after Treatment with Nanomaterials. Five tissues in 80 µL cell medium were seeded in each well of a 96-well clear bottom white plate (for CYP study) or 96-well clear bottom plate (for ALT and AST study). Tissues were then treated with to 20 µL GO nanosheet or Co3O4 nanoparticle suspensions (final concentration was 50 µg mL-1). After 24 h treatment, the tissues were washed with PBS three times to remove free materials. ALT (Alanine Transaminase Activity Assay Kit, Abcam, Canada) and AST (Aspartate Aminotransferase Activity Assay Kit, Abcam, Canada) contents were determined according to the manufacturer’s instructions. The absorption intensity was read on a SpectraMax M5 microplate spectrophotometer (Molecular Devices, US).

RESULTS AND DISCUSSION


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Preparation and Characterization of Polymer-Coated 3D Hepatocyte Spheroids. Individual 3D hepatocyte spheroids were prepared using the hanging-drop approach as illustrated in Figure 1A. 500 hepatocytes were seeded into each well of a hanging-drop plate at day 1, and cells continued to aggregate and assemble from day 2 to 5. The spheroids started to form at day 5 and were harvested into a U-shaped plate at day 7. The hepatocytes and obtained spheroids were imaged by a differential interference contrast (DIC) microscopy (Figure 1B). The average diameter of the spheroids harvested at day 7 was 230 ± 70 μm (analyzing at least 25 spheroids). The innerstructure of the spheroid was observed using a transmission election microscopy (TEM), revealing tight cell-cell contact at the MT edges and a hollow center (Figure S2). The cell number of each spheroid was quantified based on an ATP-cell number calibration curve (Figure S3), and each spheroid was composed of 1500 ± 180 hepatocytes after 7 days incubation. The spheroid was subsequently coated with a protective polymer shell using the LbL approach as illustrated in Figure 1C. Briefly, PVPON and TA were sequentially deposited on the surface of spheroid to yield a multi21 ACS Paragon Plus Environment


layered protective coating. FITC-labelled PVPON (the synthesis procedure of fluorescent-labeled PVPON is drew in Figure S4) was used to visualize the polymer coating (Figure 1D).


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Figure 1. Preparation and Characterization of polymer coated 3D Hepatocyte Spheroids. (A) Scheme of 3D hepatocyte spheroid fabrication (B) The spheroid was imaged using a DIC microscopy at day 2, 5 and 7, respectively. Scale bars are 100 μm. (C) Schematic figure of polymer coating (PVPON/TA) on the spheroid via LbL assembly. (D) FITC-labeled PVPON was used to visualize the polymer coating (4 bilayers, termed as 4L) by fluorescence microscopy. Scale bars are 100 μm.

Assessment of Cellular Toxicity of NMs in the Hepatocyte Spheroid. To investigate whether the polymer coating affects cell viability, and to assess the protective effect for spheroids upon exposure to NMs, we treated spheroids with NMs (GO and Co3O4) known to induce hepatotoxicity.21,22 The physicochemical properties of the NMs were characterized before exposure to spheroids (Figure S5). To determine the toxicity of NMs on the un-coated and coated spheroids, cell viability was quantified by a CellTiter-Glo® 3D ATP assay. We first examined the effect of polymer layers on the protection of spheroids by tailoring the number of polymer layers (Figure 2). With the 23 ACS Paragon Plus Environment


outer polymer layer number increased from 0L (un-coated) to 6L (6 bi-layers), a negligible cell viability decrease was observed in the spheroids, confirming the polymer itself did not induce cytotoxicity in the spheroids. However, upon the exposure to GO (50 μg mL-1), spheroids showed a significant increase in cell viability with the increase of polymer layer number. In contrast, this layer effect (between 2L to 6L) was not observed in spheroids after treatment with CO3O4 (50 μg mL-1), even though multi-layer coated spheroids showed a significant increase in cell viability compared to un-coated spheroids. It is notable that there were negligible differences in the cell viability (for both GO and Co3O4 treatment) between the spheroids coating with 4L and 6L polymer, indicating 4L polymer coating exhibiting nearly ultimate protective effect on the spheroids. Hence, we continued to use 4L-coated spheroids to examine the protective effect of polymer coating under exposure to NMs with a dose range from 12.5 to 100 μg mL-1.


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Figure 2. Cytotoxicity of polymer coated (0-6 L) hepatocyte spheroids after 24 h incubation with GO and Co3O4 with the concentration of 50 µg mL-1, respectively. Cell viability was quantified by a CellTiter-Glo® 3D ATP assay (Promega, US). Data represent the mean ± standard error from three independent experiments, and at least five spheroids were analyzed in each experiment. * P

Engineering Protective Polymer Coatings for Liver Microtissues

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