Decellularized liver matrix modified cryogel scaffolds as potential

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Decellularized liver matrix modified cryogel scaffolds as potential hepatocyte carriers in bioartificial liver support systems and implantable liver constructs Apeksha Damania, Anupam Kumar, Arun Kumar Teotia, Kimura Haruna, Masamichi Kamihira, Hiroyuki Ijima, Shiv Kumar Sarin, and Ashok Kumar ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13727 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 9, 2017

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ACS Applied Materials & Interfaces

Decellularized liver matrix modified cryogel scaffolds as potential hepatocyte carriers in bioartificial liver support systems and implantable liver constructs Apeksha Damania1, Anupam Kumar2, Arun K. Teotia1, Kimura Haruna3, Masamichi Kamihira3, Hiroyuki Ijima3, Shiv Kumar Sarin2 and Ashok Kumar1, * 1

Department of Biological Sciences and Bioengineering, Indian Institute of Technology

Kanpur, Kanpur-208016, UP, INDIA 2

Institute of Liver and Biliary Sciences, Vasant Kunj, New Delhi, INDIA

3

Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Fukuoka-

8190395, JAPAN

*

For correspondence:

Department of Biological Sciences and Bioengineering Indian Institute of Technology Kanpur, Kanpur-208016, UP, India Tel. +91-512-2594051 Fax. +91-512-2594010 E-mail: [email protected]

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Abstract Recent progress in the use of decellularized organ scaffolds as regenerative matrices for tissue engineering holds great promise in addressing the issue of donor organ shortage. Decellularization preserves the mechanical integrity, composition, and microvasculature critical for zonation of hepatocytes in the liver. Earlier studies have reported the possibility of repopulating decellularized matrices with hepatic cell lines or stem cells to improve liver regeneration. In this work, we study the versatility of decellularized liver matrix as a substrate coating of 3D cryogel scaffolds. The coated cryogels were analysed for their ability to maintain hepatic cell growth and functionality in vitro, which was found to be significantly better than the uncoated cryogel scaffolds. The decellularized liver matrix coated cryogel scaffolds were evaluated for their potential application as a cell loaded bioreactor for bioartificial liver support and as an implantable liver construct. Extracorporeal connection of the coated cryogel bioreactor to a liver failure model showed improvement in liver function parameters. Additionally, offline clinical evaluation of the bioreactor using patient derived liver failure plasma showed its efficacy in improving liver failure conditions by approximately 30-60%. Furthermore, implantation of the decellularized matrix coated cryogel showed complete integration with the native tissue as confirmed by H&E staining of tissue sections. HepG2 cells and primary human hepatocytes seeded in the coated cryogel scaffolds implanted in the liver failure model maintained functionality in terms of albumin synthesis and cytochrome P450 activity post 2 weeks of implantation. In addition, a 20-60% improvement in liver function parameters was observed post implantation. These results put together, suggest a possibility of using the decellularized matrix coated cryogel scaffolds for liver tissue engineering application.

Keywords: decellularized liver, cryogel, liver failure, regeneration, bioartificial liver, implantable liver construct


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Introduction Scaffolds play a critical role in providing the right form of microenvironment for the cells to attach to and subsequently develop into a functional tissue 1. Numerous groups have shown the importance of providing components of the extracellular matrix (ECM) as a substratum for culturing and maintaining cells in vitro

2-3

. Specific components from the ECM are now

commonly used as additives or an integral component of the porous scaffolds used in tissue engineering due to their profound effects on cells with respect to attachment, survival and maintenance of functions 3. While these components have shown a positive effect on the cellular behaviour, they do not recapitulate the in vivo ECM in terms of the tissue-specific combinations and/or ratios of proteins or polysaccharides 2, 4. Over the last decade, a significant advancement has been made in the field of bio scaffold design for liver tissue engineering, with the use of decellularized matrix derived from discarded donor organs. The first report of a transplantable liver biomatrix showed the use of several million primary rat hepatocytes 5. More recently, repopulation of a decellularized human liver matrix with human liver cells

6

has also been explored. Hence, the use of

decellularized matrices for liver tissue engineering has come a long way. Although most of the studies have focused on the preservation of native microvascular network as the primary advantage for use of the decellularized whole-liver constructs, there are few reports on the versatility of the decellularized liver tissue to be used as a coating substance 2-3. Owing to a growing increase in shortage of donors, alternatives to liver transplantation, such as liver support systems, stem cell therapy and implantable liver constructs are being extensively explored. One of the major challenges that has limited the use of implantable liver grafts in liver tissue engineering is the formation of a necrotic core due to limited 3 ACS Paragon Plus Environment


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oxygen and nutrient transfer because of the distance between the cells in the scaffold and the native capillary network

7-8

. On the other hand, immobilisation of a large cell number of an

optimum cell type seems to be a major challenge with liver support systems 9. Cryogels exhibit a characteristic feature of the interconnected porosity, which unlike their hydrogel counterparts, provides a large surface-area-to-volume ratio for cellular attachment. The inherent interconnected macroporous network of the cryogels favours convection transport mediated nutrient transfer as well as promotes oxygen diffusion, enabling the proper growth and proliferation of cells present deep within the porous scaffold 7, 10. In this work, we explore the use of decellularized liver matrix obtained from rat and porcine liver as a component of cryogel scaffolds synthesised for liver tissue engineering. The decellularized liver matrix was used as a substrate coating for cryogel scaffolds and growth and functionality of liver cells monitored in vitro. Further, potential application of the coated cryogel scaffolds as a cell-seeded bioreactor in bioartificial liver (BAL) devices and an implantable liver construct was explored using rodent models of liver failure.

Materials and Methods Animals Male Wistar rats (250-350 g) were used for the decellularization of liver tissue and generation of liver failure model as per the approval of Institute Animal Ethics Committee (IITK/IAEC/2014/1022) in accordance with the relevant guidelines and regulations of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The animals were housed in climate-controlled environment with alternate 12 h light and dark cycles and standard food and water provided. Liver decellularization protocol


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The hepatic portal vein was cannulated using an 18G catheter and blood flushed out using calcium and magnesium free phosphate buffered saline (CMF-PBS) for 30 min. Triton X-100 (4 %) was perfused through the liver for 6-8 h. The Triton X-100 was then replaced with CMF-PBS with 0.2 mg/mL DNase and 0.2 mg/mL RNase for another 1 h. Finally, the liver was perfused via the portal vein with CMF-PBS with 2% antibiotic for 2 h (at a flow rate of 1-2 mL/min) before being harvested 11. The porcine liver was obtained from the Fukuoka City Central Wholesale Market- Meat Market Corporation (Fukuoka, Japan). Liver from the pig type LWD was used. The LWD is the most common pig in Japan and is a crossbreed of Landrace, Large White and Duroc pigs. The porcine liver was sliced (1cm x 1cm x 2mm) and 30-35 mg of liver soaked in 300 ml of Triton X-100 (1%). The liver was stirred at 4 °C for 4 days with change in solution every day. The surfactant was then washed with an equal volume of CMF-PBS for 24-48 h. The liver was dialyzed through a Spectra/Por® dialysis membrane (molecular weight cutoff:1000, Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) with dialysate of water (2 L) at 4 °C for 48 h. Decellularization assessment (i)

Scanning electron microscopy (SEM) and Energy dispersive X-ray spectroscopy (EDX) analysis

The decellularized liver tissue was fixed in 10% neutral buffered formalin for 48 h. After slicing the tissue (1 cm x 1 cm), a gradient ethanol wash was carried out and the samples allowed to vacuum dry in a dessicator. Dried tissue samples were coated with gold using a sputter coater and analyzed using SEM/EDX. (ii)

Histological analysis



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For histological examination, the decellularized and native liver tissues were fixed in 10% neutral buffered formalin for 48 h. The fixed samples were then paraffin embedded and the sections (5 µm thickness) stained by standard haematoxylin and eosin (H&E) protocol to validate the complete removal of cells from the liver. Stained samples were examined using a light microscope. (iii)

Fluorescence microscopy

The 5 µm thick sections of native and decellularized liver tissues were stained using the nuclear stain, 4',6-diamidino-2-phenylindole (DAPI) for 5-10 min in the dark at room temperature. Stained samples were examined using a fluorescent microscope. (iv)

DNA content quantification

DNA content was used as an indicator of residual cells in the decellularized liver matrix. First, approximately 10 mg of native and decellularized liver matrix were frozen in liquid nitrogen and lyophilised overnight. The samples were re-weighed to obtain the dry weight and incubated in 1 mL papain solution (1.056 mg/mL papain, in 0.1 M dibasic sodium phosphate, 5 mM cysteine HCl and 5 mM EDTA, pH 6.5) overnight in a water bath at 60 ºC. DNA content was estimated using the Hoechst 33258 assay method, wherein 100 µL of 0.5 µg/mL Hoechst 33258 working solution was added to 50 µL of the papain digested tissue sample. Fluorescence was measured at an excitation/emission wavelength of 360/460 nm. (v)

Glycosaminoglycan (GAG) content quantification

GAG content was used to indicate any alteration in the structural component of the ECM on decellularization. Again, 10 mg of native and decellularized liver matrix was frozen in liquid nitrogen and lyophilised. After re-weighing, the samples were incubated in 1 mL papain solution overnight in a water bath at 60 ºC for digestion. GAG content was quantified using the 1,9-dimethylmethylene blue assay

12-13

. Absorbance was measured at 540 nm and

concentration calculated using a standard curve of varying concentrations of chondroitin-46 ACS Paragon Plus Environment

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sulphate. The concentration values obtained were normalised to the dry weight of the matrices 14. Preparation of decellularized liver ECM coated cryogel scaffolds Decellularized rat liver ECM was prepared by freezing the decellularized liver in liquid nitrogen and grinding the tissue to a powder-like consistency. Approximately 100 mg of this powdered ECM was solubilised in 0.1N HCl supplemented with 10 mg/mL pepsin for 72 h at 4 ºC. The solubilised ECM was dialysed (molecular weight cut-off:1000 Da, Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) against distilled water for 48 h at 4 ºC. After freeze-drying the dialysed ECM, the powder obtained could be used further for coating by dissolving in 0.001N HCl (pH 3). Similarly, porcine derived decellularized liver matrix was dissolved in 0.001N HCl (pH 3) for further coating of the cryogel scaffolds. Poly(Nisopropylacrylamide)-chitosan (poly(NiPAAm)-chitosan) cryogels were synthesized using the method described earlier

15

. One mg/mL solution of decellularized liver matrix was

poured over the cryogel discs (3 mm thickness) just enough to cover the disc and allowed to air-dry overnight at room temperature on a clean bench. The liver matrix was later crosslinked using 1% glutaraldehyde for 3 h at room temperature. Residual glutaraldehyde was removed by washing thrice in basal media. Culture and seeding of HepG2 and primary human hepatocytes HepG2 cells, obtained from National Centre for Cell Science (NCCS), Pune, India, were cultured in Dulbecco’s Modified Eagles’ Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, USA) and 1% antibiotic (HiMedia, India) at 37 ºC in a humidified incubator with 5% CO2. Cells were passaged at 70% confluency and media was replaced every alternate day. Primary human hepatocytes were obtained from cadaveric donor livers (n=2) found to be unsuitable for transplantation at the Institute of Liver and 7 ACS Paragon Plus Environment


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Biliary Sciences. The experimental protocol involving use of human primary hepatocytes was approved by the human ethics and safety committee of the Institute of Liver and Biliary Sciences, New Delhi, India (IEC/2017/49/MA14) and the methods followed were as per the guidelines and regulations of this committee. The cells were cultured in William’s medium E supplemented with 10% fetal bovine serum, 1% antibiotic, epidermal growth factor (EGF) (10ng/mL), insulin (5µg/mL), hydrocortisone (0.5µg/mL) and dexamethasone (400ng/mL) at 37 ºC in a humidified incubator with 5% CO2. The dry normal cryogel scaffolds were sterilised with 70% alcohol overnight followed by washing thrice with PBS for 15-20 min each. The cryogels were then equilibrated in complete media for 4-5 h. The coated cryogel scaffolds after removal of residual glutaraldehyde, were washed thrice in PBS (15-20 min) followed by equilibration in complete media for 4-5 h. Both HepG2 and primary human hepatocytes were seeded into the coated and normal cryogel discs (diameter 8 mm, thickness 3 mm) at a density of 1.5x105 cells/scaffold. Cell proliferation assay and analysis of hepatic functions in vitro Cell proliferation was assayed at regular time intervals using the MTT assay as well as by quantitating the DNA content and amount of aspartate transaminase (AST) leakage. Complete media was replaced with a 0.5 mg/mL MTT solution made in basal media and incubated with the cells for 4-5 h. Post incubation, the MTT containing media was removed and 1.5 mL dimethyl sulfoxide (DMSO) added to each well to dissolve the formazan crystals formed. Absorbance was read at 570 nm using a spectrophotometer. To quantify DNA, the cryogel scaffolds loaded with cells were harvested at regular time intervals and crushed. Total genomic DNA was isolated using an isolation kit (Qiagen, India) as per the manufacturer’s protocol and quantified using a nano-drop (ThermoFisher, India).

Further, media was 8

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collected at regular time points and AST leakage measured using an AST assay kit (Sigma Aldrich, USA) as per the manufacturer’s protocol. Hepatic functions, primarily albumin and urea synthesis were monitored on both coated and normal cryogels. Albumin was estimated using the human albumin enzyme-linked immunosorbent assay (ELISA) quantitation set (Bethyl Laboratories, Montgomery, TX, USA), as per the manufacturer's protocol using flat bottomed microtiter plates (Nunc MaxiSorp®, ThermoFisher Scientific, India)

16-17

. Urea was measured as mentioned earlier

using the diacetyl monoxime method 18-19 . Cell seeded cryogels were fixed using 10% neutral buffered formalin for histological analysis using standard H&E and Ki67 staining protocols as reported earlier 20. For scanning electron microscopy (SEM) the gels were fixed using 2.5% glutaraldehyde and gold coated using a sputter coater after a gradient ethanol wash followed by vacuum drying. For confocal and fluorescence microscopy, the cell seeded gels were fixed in 4% paraformaldehyde followed by staining with Sytox green (ThermoFisher Scientific, 1: 30,000) and phalloidin TRITC (Sigma Aldrich, 50µg/mL) and DAPI (Sigma Aldrich). The cells were imaged using a Zeiss confocal microscope with LSM710 confocal scan head (20X objective lens) and a Leica fluorescence microscope (20X objective lens). Application of the decellularized liver ECM coated cryogel scaffold as a bioartificial liver support Preparation of the decellularized liver matrix coated cryogel scaffold and normal cryogel scaffold for use as a bioartificial liver support was carried out using a method previously described 21. Briefly, the coated and normal cryogel scaffolds (height 40 mm; diameter 8 mm) were seeded with HepG2 cells at a density of 1.5x106 cells/scaffold. Five-day cell-seeded cryogels were used for further animal and offline studies. Partial hepatectomy (70%) 9 ACS Paragon Plus Environment


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followed by mild ischemia (PHx) was used to induce acute liver injury in male Wistar rats. After making a mid-abdominal incision the upper abdomen and lateral lower portions of the hemi thoraces were compressed to partly exteriorize the liver. The right and left medial lobes together with the left lateral lobe were tied down near the hilum. The hepatic portal vein and hepatic artery were clamped for 40 min to create ischemia. The knotted lobes were then resected en bloc and the abdomen sutured using prolene sutures. A modified version of the extracorporeal BAL system used by Flendrig et al.

22

was used to connect the liver failure

model to the cryogel based bioreactor as previously described

21

. Briefly, the carotid artery

and jugular vein of the model were cannulated. Blood from the carotid artery was pumped into a plasma separator and the separated-out plasma pumped into the cryogel based bioreactor. Treated plasma was pumped back into circulation via the jugular vein. Experimental groups constituted of healthy rats and liver failure models connected to an uncoated cryogel based bioreactor and to a decellularized matrix coated cryogel based bioreactor (n=5 per group). Control groups (n=5) consisted of healthy and liver failure rats not connected to any bioreactor. The animals are connected to the bioreactors 1-2 h post injury induction. Plasma samples (0.2-0.5 mL) collected at regular time intervals were assayed for various liver function parameters using a biochemical analyser (ERBA Mannheim, Germany). Further, human albumin was assayed in the plasma samples using sandwich ELISA as per standard protocol. Offline clinical evaluation of decellularized liver matrix coated cryogel based bioreactor using alcoholic acute liver failure (ALF) patient plasma The decellularized liver matrix coated cryogel based bioreactor and its uncoated counterpart were subjected to offline evaluation using plasma from alcoholic ALF patients taken for routine clinical examination using a method previously described

21

. Briefly, a 5-day old 10

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HepG2 loaded cryogel bioreactor was washed with PBS and perfused with plasma for 3 h (n=5). The plasma was monitored for changes in liver function parameters using a biochemical analyser. Informed consent was obtained from each of the patients prior to the experiments. The experimental protocol involving humans was approved by the human ethics and safety committee of the Institute of Liver and Biliary Sciences, New Delhi, India (IEC/IRB no: 21/6 dated 26/06/2013) and the methods followed were as per the guidelines and regulations of this committee. Application of decellularized liver matrix coated cryogel scaffold as an implantable liver construct All the animals were orally administered cyclosporine daily (10 mg/kg/day) starting 7 days prior to surgical implantation till end of experiment, to minimize immune mediated implant rejection. After PHx, before removing the clamp on the hepatic portal vein and suturing the abdomen, porcine decellularized matrix coated poly(NiPAAm)-chitosan cryogel scaffolds (8 mm length, 4 mm width, 2mm thick) alone or seeded either with primary human hepatocytes or HepG2 cells (1.5 x 106 cells/scaffold) were implanted onto the remnant lobe of animals (n=5 each). For implantation, a capsule-like opening was created on the posterior side of the remnant lobe. The cryogel scaffolds were placed very carefully in this opening and sutured in place using 6-0 Vicryl sutures (Figure 1). The clamp from the hepatic portal vein was removed to resume blood flow and the abdomen sutured closed. The control group (n=3) had no matrix implanted onto the remnant lobe. The animals were monitored over a period of two weeks for their response to injury.



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Figure 1: Implantation of coated cryogel scaffold in the remnant lobe of the liver after partial hepatectomy. Capsule-like opening made to place the scaffold (A); after the matrix (yellow arrow) is placed in the opening (B) it is sutured in place using 6-0 Vicryl sutures (C). Histological evaluation of integration of decellularized liver matrix coated cryogel scaffolds with the native liver tissue To evaluate integration of the cryogel matrix with the native tissue, the remnant liver lobe onto which the matrix had been implanted was excised at the end of one-week post implantation. The tissue was fixed in 10% neutral buffered formalin for 48 h followed by paraffin embedding. Sections (5 µm) of the tissue were stained by standard H&E protocol and examined using a light microscope. In vivo functional evaluation of hepatic cells seeded on decellularized liver matrix coated cryogel scaffolds post implantation in liver failure model Functionality of the HepG2 cells and primary human hepatocytes seeded on the decellularized liver matrix coated poly(NiPAAm)-chitosan cryogel post implantation was evaluated as an indicator of the cells’ ability to maintain functionality post transplantation. Blood collected from the rats at the end of one-week post implantation was analysed for presence of human albumin secreted by the HepG2 cells using the human albumin ELISA quantitation kit. The same was done at the end of two weeks post implantation in the group implanted

with

primary

human

hepatocytes

seeded

cryogels.

In

addition,

immunohistochemistry was used to stain for human albumin in tissue sections obtained from models implanted with coated cryogel scaffolds seeded with HepG2 cells and primary human 12 ACS Paragon Plus Environment

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hepatocytes. For this, 5 µm tissue sections were incubated with anti-human albumin (1:200; Sigma Aldrich, USA) at 4 ºC overnight, followed by staining with secondary antibody (1:100, Sigma Aldrich, USA) for 2 h. Counterstaining of the nuclei was done using DAPI for 5-10 min in the dark. The stained samples were examined using a fluorescence microscope. Further, the 7-hydroxylation of coumarin by the catalytic activity of cytochrome P450 (CYP2A6 enzyme) was analysed after injecting the rats with coumarin (80 mg/kg) and collecting the urine 24 h after injection. Effect of implanted decellularized liver matrix coated cryogel scaffold on recovery of liver functions in liver failure model Blood samples were collected every 24 h and analysed for liver function biochemical parameters such as AST, alanine transaminase (ALT), bilirubin and albumin using a blood biochemical analyser.

Results and Discussion Decellularization assessment Upon decellularization, the lobes of the liver become increasingly translucent due to the dissolution of the cells (Figure 2A). There are well established methods for qualitatively determining decellularization of the native tissue, such as fluorescence staining for the nuclei, histological analysis, and SEM

5, 23-24

. We confirmed the removal of the cellular components

using SEM, nuclei staining, and histological analysis. SEM image of the matrix did not show any cellular presence and indicated the porous network of the liver matrix (Figure 2B). Further, EDX analysis of the decellularized liver showed the presence of distinct peaks of carbon (C), oxygen (O), and nitrogen (N) typical of the components of the extracellular matrix. (Figure S1A). Sections of the decellularized liver matrix stained with DAPI showed 13 ACS Paragon Plus Environment


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very little presence of nuclei as compared to the native liver tissue (Figure 2C & 2D) suggesting the removal of most of the cellular components. This was further corroborated by H&E staining (Figure 2E & 2F). The DNA content was measured as one of the quantitative indicators of complete decellularization. DNA in the decellularized liver matrix was found to be significantly lower than that in the native liver (Figure 2G). DNA content in decellularized matrices is generally reported to be less than 3-4% of that in the native tissues

5, 24

, hence the results obtained

suggest successful decellularization. Some of the decellularization techniques that involve the use of harsh detergents result in structural damage to the native extracellular matrix

25

. The GAG content of the native and

decellularized matrix shows a higher GAG content per gram of matrix as compared to the native tissue (Figure 2H), indicating the structural preservation of the ECM. GAGs and proteoglycans, which make up most of the native ECM have been reported to play a critical role in hepatocyte functionality

10, 24

. Hence, preservation of the GAG content in the

decellularized liver matrix may enhance the role it may play in improving cellular functionality when combined with the cryogel scaffolds.


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Figure 2: Determination of liver decellularization. Digital image of decellularized liver (A); SEM image of decellularized liver (Scale bar: 20 µm) (B); nuclei staining by DAPI in the native liver (C) and decellularized liver (D); H&E staining of the native liver (E) and decellularized liver (F); DNA quantification in the decellularized and native liver (G); GAG content quantification in the decellularized liver and the native liver (Scale bar: 100 µm) (H). The liver was decellularized using Triton X-100, which is a non-ionic surfactant and a mild detergent. In this study, the liver was decellularized using a chemical method. It is reported that the lungs and liver, which are relatively low in strength, are typically decellularized using a chemical method

26

. Studies have reported Triton X-100 as a preferred detergent for the

decellularization process over other detergents such as sodium dodecyl sulphate (SDS) and enzymatic treatment using trypsin due to its effectiveness in reducing the DNA content without causing any GAG depletion 14, 27. This effect of Triton X-100 is clearly visible in the normalised DNA and GAG content of the decellularized matrix. It is important to note that normalisation to dry weight was used in calculating the DNA and GAG content. It has been 15 ACS Paragon Plus Environment


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reported that different decellularization protocols may extract various ECM constituents with different efficacies. This affects water‐binding capacity and hence may compromise normalization to final wet weight 14. DNase and RNase were used in combination with the Triton X-100 treatment to reduce the nucleic acid content to as low as possible, while retaining majority of the ECM proteins. Decellularization by perfusing through the hepatic portal vein ensures the equal distribution of the decellularizing agents across the mass of the organ

25

. The porcine liver in this study was decellularized by chopping the liver to acquire

only the necessary amount of decellularized liver. Several researchers have decellularized the liver using the same method

3, 28

. Since it is difficult to decellularize the whole liver

uniformly and requires large volumes of surfactant solutions liver slices are made for uniform and easy decellularization.

Decellularized liver matrix coating improves in vitro growth and functionality of hepatic cells on cryogel scaffolds There was a significant difference (p< 0.01) in the cell proliferation rate of HepG2 cells on cryogel scaffolds coated with decellularized liver matrix as compared to the uncoated scaffolds (Figure 3A). Significantly high amount of DNA in the coated scaffolds as compared to the uncoated scaffolds (Figure 3B) further corroborated the differences observed in the MTT assay. In addition, AST leakage was found to be relatively higher in the uncoated scaffolds in comparison to the coated scaffolds (Figure 3C). Since AST leakage is a common marker for cell death and cytotoxicity, the results suggest reduced cell death on the coated cryogel scaffolds as compared to the uncoated scaffolds. In terms of functionality, both albumin and urea synthesis are seen to be significantly higher (p< 0.05 and p< 0.01, respectively) in the coated scaffolds as compared to the uncoated scaffolds (Figure 3D & 3E). 16 ACS Paragon Plus Environment

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Primary human hepatocytes also showed a significantly higher proliferation rate (p< 0.01) on the coated cryogel scaffolds as compared to the uncoated gels (Figure 3F). However, it was observed that the primary hepatocytes begin to lose their viability and functionality post the 3rd day of in vitro culture on the both the coated as well as uncoated cryogel scaffolds. Furthermore, both albumin and urea synthesis of the primary human hepatocytes was found to be better in the coated cryogel scaffolds over their uncoated counterparts (Figure 3G & 3H). Primary hepatocytes have been observed to lack the ability to maintain growth and functionality in vitro for a prolonged time period 7. Despite the cryogel scaffold providing the cells with a three-dimensional architecture, the cells are unable to maintain a steady growth rate over a prolonged period and by the end of 10 days completely lose their viability and functionality. The representative confocal, fluorescence and scanning electron microscopic images of the human hepatocytes seeded coated cryogel scaffolds showed the cells to be distributed across the cryogel matrix. The cells could form spheroids, an inherent feature of hepatocytes, which were distributed well across the pores of the cryogel matrix (Figure 3I(i-iii)). The scanning electron micrographs of the uncoated cryogel scaffolds shows the presence of smooth pore walls. However, upon coating with the decellularized liver matrix, the roughness in the pore walls increased (Figure S1B). Cell-material interactions are known to be governed by several factors including the topography of the scaffold. Surface roughness has been shown to significantly influence cell adhesion and growth on biological scaffolds 29. Hence, it may be concluded that coating the cryogel scaffold with a solution of decellularized liver matrix increases the surface roughness of the scaffold allowing for better cell adhesion, growth, and functionality.



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Figure 3: Cell proliferation and hepatic functions on the decellularized liver matrix coated cryogel scaffolds. MTT assay of HepG2 cells seeded on coated and uncoated cryogel scaffolds (A); DNA content measured over a period of 6 days, post seeding of HepG2 cells on coated and uncoated cryogel scaffolds (B); AST leakage from coated and uncoated cryogel scaffolds seeded with HepG2 cells (C); albumin (D) and urea (E) synthesis on coated cryogel scaffolds versus uncoated cryogel scaffolds; MTT assay of primary human hepatocytes seeded on coated and uncoated cryogel scaffolds (F); albumin (G) and urea (H) synthesis by primary human hepatocytes on the coated vs uncoated scaffolds; representative confocal (green stain: Sytox Green; red stain: Phalloidin), fluorescence (blue stain: DAPI) and SEM image of the primary human hepatocytes seeded on the coated poly(NiPAAm)chitosan cryogel scaffold (I) (Scale bar: 200 µm (i-ii); 20 µm (iii)).

Histological analysis of the HepG2 cells on both coated and uncoated cryogel scaffolds showed a dense cellular aggregation on the decellularized liver matrix coated cryogel scaffolds as compared to their uncoated counterpart (Figure 4A-D). Further, SEM images of the coated cryogel scaffolds also show dense cellular population in the macropores of the 18 ACS Paragon Plus Environment

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scaffold as compared to the uncoated scaffold (Figure 4E & 4F), forming spheroid-like aggregates- a feature characteristic to the hepatocytes and beneficial for their overall functionality 15, 30. Several proteins and peptides present in the decellularized matrix solution including elastin, fibrinogen and collagen along with their precursors have previously been identified 24. The improved proliferation as well as functionality on the coated scaffolds may be attributed to the better attachment of the cells on these scaffolds due to the presence of these liver-specific ECM components.

Figure 4: Microscopic analysis of cells on decellularized liver matrix coated cryogel scaffolds versus uncoated cryogel scaffolds. H&E staining of cells on sections of uncoated and coated cryogel scaffolds seeded with HepG2 cells (A and B, respectively) Scale bar: 100 µm; Ki67 staining of HepG2 cells on uncoated and coated cryogel scaffolds (C and D, respectively) Scale bar: 100 µm; SEM image of HepG2 cells seeded on uncoated and coated cryogel scaffolds showing dense cellular aggregation on the coated scaffolds (some 19 ACS Paragon Plus Environment


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representative spheroids are shown in the yellow circles) (E and F, respectively). Scale bar: 20 µm.

Decellularized liver matrix was also integrated within the cryogel during the cryogelation process. The solution of decellularized liver matrix was mixed with the mixture of chitosan and N-isopropylacrylamide/methyl-bisacrylamide along with crosslinkers glutaraldehyde and TEMED. This mixture was frozen at -12 ºC for 16 h in appropriate moulds (detailed method in Supporting Information). Although the integrated cryogel scaffolds supported the growth and functionality of the HepG2 cells, it was still lower than that on coated cryogel scaffolds (Figure S2). It was observed that there was no significant difference in the proliferation of the HepG2 cells on cryogel scaffolds integrated with the decellularized liver matrix during the synthesis process over the unintegrated cryogel scaffolds (Figure S2A). Although functionality in terms of albumin and urea synthesis was slightly better in the integrated cryogel scaffolds as compared to the unintegrated cryogel scaffolds (Figure S2 B & C). On comparison with the coated cryogel scaffolds it was found that the coated cryogel scaffolds performed better than the integrated cryogel scaffolds. Similar in vitro studies using porcine-derived decellularized liver matrix showed lower proliferation and functionality of the cells (Figure S3 A-C) on the decellularized liver matrix integrated cryogel scaffolds than on the coated cryogel scaffolds. To evaluate this difference, the protein concentration of the coated cryogel scaffolds as well as the integrated matrix cryogel scaffolds was measured and found to be significantly higher (p 70%)

37

. On evaluating the CYP2A6 activity, it was found that the

activity was significantly higher in the rat models in which HepG2 cells were seeded in the ECM coated cryogels implanted in the remnant lobe as compared to the other groups where only the cryogel matrix was implanted or no scaffold was implanted (Figure 8E). These results together suggest the ability of the ECM coated cryogel scaffolds to act as potential hepatocyte carriers enabling them to maintain their functionality in vivo even one-week post implantation.


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Figure 8: Functional evaluation of HepG2 cells seeded on decellularized liver matrix coated cryogel scaffolds post implantation in rodent liver failure model. ELISA quantitation of human albumin present in rat plasma in the group implanted with HepG2 cells seeded coated cryogel scaffolds (A); Immuno-histochemical staining for human albumin and counterstaining for nuclei using DAPI (Scale bar: 100 µm) (B (i)); Immunohistochemical staining for human albumin and counterstaining for nuclei using DAPI in control group implanted with only matrix and no cells (B (ii)); ELISA quantitation of human albumin present in rat plasma in the group implanted with primary human hepatocytes seeded coated cryogel

scaffolds

(C);

Immuno-histochemical

staining

for

human

albumin

and

counterstaining for nuclei using DAPI (Scale bar: 100 µm) (D (i-ii)); Cytochrome p450 activity, specifically CYP2A6 enzyme activity in hydroxylation of coumarin in rats (E). Implanted decellularized liver matrix coated cryogel scaffold improves recovery of liver functions in rodent liver failure model Finally, the effect of the hepatocyte seeded ECM coated cryogel scaffolds on recovery of liver functions in a liver failure model was evaluated to assay its potential application in the treatment of liver failure conditions. Typical biochemical parameters associated with liver function such as AST, ALT, bilirubin, and albumin were analysed. As mentioned earlier, the values of AST and ALT generally increase within the first few hours of liver injury indicating cell death and cytotoxicity to the liver cells. As the liver recuperates and regeneration sets in, with time AST and ALT levels also reduce. 29 ACS Paragon Plus Environment


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Interestingly, the levels of both AST and ALT in the group implanted with cell seeded cryogel scaffold are significantly lower than those in the group implanted with only cryogel scaffold and still lower than the control with no scaffold implanted, within the first 24-48 h post injury (Figure 9A and 9B). Similarly, the level of bilirubin is significantly lower in the group with cell seeded cryogel scaffold implanted whereas albumin levels are significantly higher (Figure 9C and 9D). These results indicate the ability of the cells in the implanted scaffold to alleviate the conditions of liver injury to some extent.

Figure 9: Effect of implanted decellularized liver matrix coated cryogel scaffold seeded with HepG2 cells on recovery of liver functions in a liver failure model. Levels of AST (A), ALT (B), bilirubin (C), and albumin (D) at regular time intervals post implantation of coated cryogel scaffolds. Statistical analysis using Student’s t-test; * p

Decellularized liver matrix modified cryogel scaffolds as potential

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