Nanometric self-assembling peptide layers maintain adult hepatocyte phenotype in sandwich cultures
© Wu et al. 2010
Received: 29 September 2010
Accepted: 12 December 2010
Published: 12 December 2010
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© Wu et al. 2010
Received: 29 September 2010
Accepted: 12 December 2010
Published: 12 December 2010
Isolated hepatocytes removed from their microenvironment soon lose their hepatospecific functions when cultured. Normally hepatocytes are commonly maintained under limited culture medium supply as well as scaffold thickness. Thus, the cells are forced into metabolic stress that degenerate liver specific functions. This study aims to improve hepatospecific activity by creating a platform based on classical collagen sandwich cultures.
The modified sandwich cultures replace collagen with self-assembling peptide, RAD16-I, combined with functional peptide motifs such as the integrin-binding sequence RGD and the laminin receptor binding sequence YIG to create a cell-instructive scaffold. In this work, we show that a plasma-deposited coating can be used to obtain a peptide layer thickness in the nanometric range, which in combination with the incorporation of functional peptide motifs have a positive effect on the expression of adult hepatocyte markers including albumin, CYP3A2 and HNF4-alpha.
This study demonstrates the capacity of sandwich cultures with modified instructive self-assembling peptides to promote cell-matrix interaction and the importance of thinner scaffold layers to overcome mass transfer problems. We believe that this bioengineered platform improves the existing hepatocyte culture methods to be used for predictive toxicology and eventually for hepatic assist technologies and future artificial organs.
The liver is an important and complex organ that plays a vital role in metabolism and is responsible for many important functions of the body including glycogen storage, plasma protein production, drug detoxification and xenobiotics metabolization. Due to the importance of this organ in many of the body's daily processes, liver malfunction often leads to death. Most of the activity of the liver can be attributed to hepatocytes, which make up 60-80% of the cytoplasmic mass of the liver [1, 2]. Loss of hepatocyte function can result in acute or chronic liver disease and, as a result, substantially compromise the rest of the organ and the body. Many previous strategies have been implemented to maintain these hepatocyte functions in vitro, including the use of extracellular matrices such as the current standard, collagen [3–6], Matrigel  or liver derived basement membrane matrix . However, the liver carries out and regulates numerous biochemical reactions that require the combined effort of specialized cells and tissues. As a result, isolated hepatocytes removed from their microenvironment soon lose their hepatospecific functions. Therefore, it is important for in vitro cultures to provide a system that closely simulates the local environment of an intact liver. Hepatocyte morphology is known to be closely linked to the functional output of the cells [9, 10]. Standard cell cultures that seed cells on top of a monolayer of extracellular matrix have been used in the past to successfully culture hepatocytes; however, in certain instances hepatocellular functions become compromised because the cell no longer resembles a natural hepatocyte from a live liver. In many cases, specific cellular phenotypes are directly related to the cellular functions including cell survival, proliferation, differentiation, motility and gene expression [11, 12]. Morphogenesis and assembly have been well established to be pertinent in the functional performance of liver-derived cells in vitro [10, 13–15].
The double-gel "sandwich" method has been shown to improve morphology by embedding the cells between two layers to resemble in vivo conditions. Typically, one layer is set on the bottom of a culture dish and an additional layer is placed on top of the hepatocyte monolayer [4, 16, 17]. Under these conditions, hepatocytes have been shown to maintain some function and differentiation for up to several weeks. Verification of hepatocyte function was shown by specific mRNA [5, 18] and protein secretion into culture media [16, 19].
The highly oxygen-demanding hepatocytes are commonly maintained in Petri dishes under oxygen-deficient culture conditions and, thus, the cells are forced into anaerobic metabolic states . Hence, oxygen supply in primary hepatocyte cultures is a crucial issue to be addressed. Generally, in cultures in Petri dishes oxygen consumption is no longer dependent upon hepatocellular uptake rates but it is limited by culture medium thickness as well as ambient oxygen concentrations. However, regardless of these constraints, hepatocytes are able to tolerate the hypoxic conditions by satisfying energy requirements through anaerobic glycolysis . In any case, a previous study has shown that hepatospecific functions are oxygen-dependent, especially demonstrated in the poor production of albumin, urea and drug metabolites over a 14-day study period in common Petri dish models compared to enhanced oxygen delivery cultures on gas-permeable films . Furthermore, it was shown as early as in 1968 that commonly used medium depths of 2-5 mm in Petri dishes rapidly produced hypoxic conditions when hepatocytes respired at their physiological rate . Therefore, because plastic walls and culture medium are efficient barriers of oxygen diffusion, it is important to create a system in which a physiological oxygen supply is maintained [23, 24].
Self-assembling peptide sequences
Since the 70's in microelectronics, non equilibrium, cold, gas plasmas are effective methods utilized in material science and technology, including biomaterials, to tailor surface composition and materials properties. Plasma etching, plasma enhanced chemical vapor deposition (PECVD) and grafting of chemical functionalities by plasma are the three main surface modification processes. Appealing features of plasma techniques are the following: they work at room temperature; modifications are limited within the topmost hundreds nanometers of the materials, with no change of the bulk; use of very low quantities of gas/vapor reagents; no use of solvents; easy integration in industrial process lines . Cold plasmas are used to tailor surface properties of materials intended to be used in biomedical applications. Due to their ability of tuning independently surface chemical composition and topography (e.g., roughness, patterns, etc.), plasma treatments allow processes like: the synthesis of non-fouling coatings, capable of discouraging the adhesion of proteins and cells at the biomaterial surface [39, 40]; the optimization of the adhesion and behaviour of cells onto biomaterials [41–43] and membranes [44, 45]; and the functionalization of surfaces for covalent immobilization of biomolecules like peptides  and saccharides [47, 48] to mimic the extracellular matrix. One example are the plasma-deposited acrylic acid (PdAA) coatings , which are used in the biomedical field to provide the surface of biomaterials with -COOH groups for improving cell adhesion and growth [50–52] or for further immobilization of biomolecules [46–48]. Also, surfaces modified with pentafluorophenyl methacylate (PFM) have been successfully used to anchor biologically active motifs, since this monomer easily reacts with molecules containing primary amines, such as bioactive peptides [53, 54].
Studies have tried cocultures of hepatocytes with other cells such as fibroblasts with the idea that nonparenchymal cell factors may promote and induce specific hepatocyte expression [55, 56]. Others have tried to achieve in vivo level induction by focusing on culture substratum using complex matrices including fibronectin , extracts from liver  and Matrigel . Currently, the best culture conditions for preserving primary hepatocytes are still unresolved. Therefore, in this work we develop a new platform where the hydrogel scaffold dimensions can be several orders of magnitude smaller (from 500 μm down to nanometric scale). Our strategy to control the peptide layer dimensions within a nanometric scale made possible to maintain the CYP3A2 activity for long periods in rat hepatocyte cultures. Briefly, in order to build our new biomaterial platform, we used two biocompatible porous membranes as main structural support for the hydrogel: PEEK-WC-PU, (poly(oxa-1,4-phenylene-oxo-1,4-phenylene-oxa-1,4-phenylene-3,3-(isobenzofurane-1,3-dihydro-1-oxo)-diyl-1,4-phenylene) modified with aliphatic polyurethane)  and PTFE (polytetrafluorethylene). These biocompatible membranes were chemically modified by means of two different plasma modifications in order to immobilize RAD16-I peptides. The anchored RAD16-I molecules directed the self-assembling of additional soluble RAD16-I peptides, which assemble forming a thin scaffold layer. Finally, we were able to obtain expression levels of albumin, CYP3A2 and HNF4-alpha similar to fresh hepatocytes by using the membranes with the controlled self-assembling peptide layer in a sandwich culture system during seven days.
In this work, we attempt to address the concerns of traditional hepatocyte culture methods by combining tissue engineering technologies. Our sandwich culture method is adjusted from the traditional double gel layer "sandwich" technique to address diffusion issues. Instead of culturing the hepatocytes under a thick second layer of peptide, the cells are entrapped under a biocompatible porous membrane (PEEK-WC-PU or PTFE), previously modified through plasma processes to allow dimensional control of a thin hydrogel-coating layer. This self-assembling peptide layer contains signaling peptide sequences to promote specific cell responses, mimicking the cell-matrix interactions that are lost in isolated hepatocytes.
After demonstrating that our substrates were able to promote cell attachment and proper morphology, the following objective was to determine to what extent the self-assembling peptides enhanced hepatocellular function, especially CYP3A2 expression. In a recent publication, we observed that using self-assembling peptide sandwich with layer dimensions between 0.5-1.0 mm, the expression of oxidative enzymes, in particular CYP3A2, in all the conditions tested was highly downregulated .
After 24 hours post-seeding, the cells expressed great levels of albumin and HNF4-alpha (Figure 8A). Albumin expression was close to fresh levels at day 1, then began to slightly decline until day 4 and by day 7, appeared to have improved to -3-fold downregulation. On the other hand, HNF4-alpha expression maintained within a close range to fresh cell levels. CYP3A2 was downregulated at day 1 and slightly evened off around a -7-fold after a week. However, our system at this point is still about 1.5-fold better than the current gold standard method of culturing hepatocytes with collagen or double gel layers of RAD16-I self-assembling peptides (Figure 8B).
Then, in order to see if PTFE membranes were able to increase the expression profile of CYP3A2 due to its bigger pore size and as consequence, possible improvement of mass transfer issues, gene expression relative to freshly isolated hepatocytes over a period of seven days -for modified sandwich cultures using peptide-modified PTFE membranes- was also monitored. In addition we decided to study the effect that functionalized nanofiber network -with biological active motifs- could have on specific cell-receptor signals and therefore improving hepatocyte phenotype. Thus, the nanofiber layer was prepared by using 100% of RAD16-I peptide or blended with small percentages of functionalized ones (RGD and YIG peptides) carrying receptor-binding sequences (Table 1). Different layer compositions were tested: RAD16-I, 5% RGD in RAD16-I, 5% YIG in RAD16-I, and a mix of 2.5% RGD and 2.5% YIG in RAD16-I. RNA samples were collected at day 7 to determine if the peptide layer and the functional motifs enhanced CYP3A2 expression.
We have successfully shown that our novel bioengineering platform can maintain expression levels of albumin, CYP3A2 and HNF4-alpha similar to fresh hepatocytes for as long as a week. This was ultimately done by improving the biophysical features of traditional sandwich cultures by optimizing the top peptide layer dimension to orders of nanometers to facilitate oxygen exchange and nutrient diffusion. Additionally, the biochemical aspects of typical cultures were enhanced by engineering the scaffold with the introduction of functional peptide motifs to strategically target certain cell receptors responsible for the activation of numerous vital cell functions. We believe that our new platform has improved significantly the existing culture methods, opening a new possibility for the pharmacological industry.
Hepatocytes were isolated from male Fisher rats weighing 150-180 g using a modification of the Seglen 2-step collagenase perfusion procedure . Cell yield and viability were determined via trypan blue exclusion and hemocytometry. Typically, 250-300 million hepatocytes were harvested per rat liver with viability ranging from 85-92%. Following isolation, cells were initially suspended in Hepatocyte Culture Medium (HCM, Cambrex, MD, CC-3198), containing 2% fatty acid free BSA (bovine serum albumin), transferrin, insulin, recombinant human EGF (epithelial growth factor), ascorbic acid, hydrocortisone and gentamycin/amphotericin.
PEEK-WC-PU membranes were kindly provided by the De Bartolo Lab (Institute on Membrane Technology, National Research Council of Italy). PTFE membranes were purchased (Biopore, BCGM00010).
PEEK-WC-PU membranes were modified with a PE-CVD surface functionalization process fed with acrylic acid (AA) vapors in order to create a thin stable plasma-deposited acrylic acid (pdAA) coating characterized by a certain density of -COOH groups. Membranes were plasma-coated in a radio frequency (13.56 MHz) driven stainless-steel parallel plate plasma reactor . AA vapors were fed from a liquid reservoir kept a room temperature, at a pressure of 0.2 mbar, and samples were exposed at 100 W for 5 min (PEEK-WC-PU/PdAA). Before use AA was degassed with freeze-thaw cycles. The deposition results in 10 ± 1 nm thick coatings characterized by a surface density of about 4% carboxylic groups over all carbon atoms of the coatings, and a O/C atomic ratio of 0.29, as measured by X-rays photoelectron Spectroscopy . PdAA-coated membranes substrates were then immersed in 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in morpholine ethane sulfonate (MES) buffer to activate -COOH groups. Afterwards, membranes were incubated overnight in a water solution of NH2-GG-RAD16-I (10 mg/ml) at 37°C to obtain the final PEEK-WC-PU membranes with immobilized RAD16-I (PEEK-WC-PU/PdAA/RAD16-I) (see Figure 1A).
Instead, PTFE membranes were modified by graft polymerization of pentafluorophenyl methacrylate (PFM) in a two-step process. The membranes were placed in a cylindrical Pyrex reactor equipped with a copper coil that generates plasma under vacuum conditions (0.02 mbar). Argon was fed into the chamber increasing the pressure to approximately 0.06 mbar and samples were exposed to argon plasma at 50 W for 5 min. Afterwards, argon inflow was closed and PFM vapor was introduced into the reactor and allowed to polymerize for 1 min (PTFE/PgPFM) without plasma. After treatment, membranes were soaked overnight in an aqueous solution of NH2-GG-RAD16-I (10 mg/ml) at 37°C to obtain the final PTFE membranes with immobilized RAD16-I (PTFE/PgPFM/RAD16-I) (see Figure 1B).
All membranes, including the native PEEK-WC-PU, PEEK-WC-PU/PdAA, PEEK-WC-PU/PdAA/RAD16-I, native PTFE, PTFE/PgPFM and PTFE/PgPFM/RAD16-I membranes were sterilized using a 70% ethanol. After the ethanol rinse, the membranes were autoclaved for 20 min at 120°C, followed by a 10 min drying step.
PuraMatrix RAD16-I peptide (BD Biosciences, 354250) was used to coat the surface of the membranes. In cases where modified peptides (RGD or YIG) were included, the modified peptides were blended in a 95:5 proportion with the prototypic peptide RAD16-I (prototypic:modified). A volume of 50 μl of peptide was used to thinly cover the surface of the 0.5 in × 0.5 in square membrane samples. The self-assembling peptide solution becomes a hydrogel through contact with salt-containing buffers or media . However, in order to control the thickness of the peptide layer, gelation was not initialized through the introduction of media, but the soluble peptide was allowed to incubate for an hour to permit any self-assembling to occur with the immobilized peptide strands (Figure 1C). Following the incubation, a rinse step was included that entailed dipping the coated membranes into deionized water ten times in succession to remove any non-assembled peptide.
In order to seed the cells on the peptide-coated membranes, these were incubated with a volume of hepatocyte cell suspension in HCM at a final density of 65,000 cells/cm2 and left to attach in a 37°C incubator for 8 h (Figure 4A). Following the 8 h attachment period (optimized attachment time), the medium was changed to remove dead cells. Meanwhile, the bottom peptide layer was prepared by loading 0.25 ml of peptide into a Millicell tissue culture insert (Millipore, PICM 03050). Then, 1.5 ml of HCM were added underneath the insert membrane to induce gelation, forming a 1 mm-thick gel (Figure 4B). Following the gelation of the peptide, 0.4 ml of HCM were added into the insert and the gel was allowed to equilibrate for 30 min in an incubator at 37°C. To complete the peptide sandwich, the membrane containing the attached cells was inverted on top of the gel layer in the tissue culture insert (Figure 4C). Then, 0.3 ml of HCM was added to the inside of the insert (Figure 4D). Cultures were maintained in a water-jacketed incubator at 37°C and 5% CO2. Media was changed every day so that a fresh reservoir of 1.6 ml surrounded the outside of the insert and 0.3 ml replaced the inside of the insert.
Membrane samples for all conditions (treated/not treated with soluble peptide, incubated/not incubated with hepatocytes) were submerged for 20 min in a fixative mixture containing 2% glutaraldehyde (Sigma, G7526) + 3% paraformaldehyde (Sigma, P6148) in PBS (Invitrogen, 14040). Following the fixing, a series of ethanol dehydration steps were performed, which included incubating the samples for 15 min in 50% ethanol, then 30 min in 75% ethanol, then 60 min in 90% ethanol, followed by two changes of 100% ethanol, where the samples were kept until the next step. Using a Tousimis Super Critical Point Autosamdri815 Dryer, the ethanol-saturated samples underwent a process in which the ethanol was slowly exchanged with CO2 and then dried at the critical pressure and temperature of CO2. The dried samples were subsequently sputter-coated in vacuum (Denton Vacuum, LLC) with gold (30 sec, approximate thickness 4-5 nm). SEM was carried out using a JEOL JSM 6060 Scanning Electron Microscope at an accelerating voltage of 5 kV.
RT-PCR primers of albumin, HNF4-α, CYP3A2, and the housekeeping gene, 18S
We greatly thank Laura Vineyard for isolating and providing the hepatocytes, Loredana de Bartolo for providing the PEEK-WC-PU membranes and Andreas Heilmann for some SEM pictures. NMB acknowledges financial support from DURSI (Generalitat de Catalunya) and the European Social Fund. This work was supported by the grants: NIH I-RO1-EB003805-01A1 to CES and LIVEBIOMAT (6th Frame Program) Project number 013653 to CES and PF.
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