Patent Application
20120052525
The present invention relates to the provision of certain polymers to which hepatocytes are able to attach and display hepatocyte function for a period of time. There is also provided use of certain polymers for attachment and maintenance of function of hepatocytes. There is further provided apparatus formed of, or comprising a coating of the polymers of the present invention for use in the attachment and maintenance of functional hepatocytes.
The cost of drug development is heavily influenced by compound attrition rate. For every new drug that reaches the market, ˜5000 to 10,000 compounds have been tested in preclinical trials with ˜250 reaching animal trials. Following animal trials ˜5 possible drugs make it to full-scale human clinical trials with only 1 obtaining final approval [1]. These figures demonstrate there is a clear requirement for developing more accurate predictive toxicity models. The generation of human hepatocyte like cells (HLCs) from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) is one such approach. We have recently developed in vitro models of human liver function [2-6], although efficient maintenance of their long-term hepatocyte function has proven elusive.
The present invention is based on the identification of a class of polymers which have been shown to possess the features of allowing attachment of hepatocyte cells thereto, which attached cells display good hepatocyte functional properties.
In a first aspect there is provided a polymer substrate for use in the attachment and functioning of hepatocyte and hepatocyte like cells.
The present inventors have observed that the polyurethane surface, formed by polymerising PHNGAD, MDI and an extender, provides a supportive effect to re-plated hepatic endoderm (hepatocytes are not usually replatable). Additionally the polyurethane surface plays an instructive effect/role in maintaining hepatocyte identity and stable function for at least 15 days post-replating. The bio-active nature of the polyurethane surface, formed by polymerising PHNGAD, MDI and an extender, utilised in these studies may also be applicable to other eukaryotic, especially mammalian cell types and therefore provide generic and defined extra-cellular support.
In one embodiment of the invention the polymer is a polyurethane polymer formed from polymerising PHNGAD, MDI and an extender molecule.
PHNGAD is Poly[1,6-hexanediol/neopentyl glycol/di(ethylene glycol)-alt-adipic acid]diol.
MDI is 4,4′-Methylenebis(phenyl isocyanate) and the extender serves to increase physical parameters, such as elasticity, wettability and/or surface topography and/or biochemical properties such as the ability to absorb extracellular matrix proteins.
Suitable extender molecules include 1,4-butanediol (BD); 3-dimethylamino-1,2-propanediol (DMAPD); 3-diethylamino-1,2-propanediol (DEAPD); (BD), 2,2,3,3,4,4,5,5-octafluoro-1,6-hexanediol (OFHD), 1,3-propylene glycol (PG), 1,2-ethylene glycol (EG), 2-nitro-2-methyl-1,3-propanediol (NMPD), diethyl-bis-(hydroxymethyl)-malonate (DHM), 1,12 dedecanediol, cyclododecanediol, hydroquinone bis(2-hydroxylethyl)ether, 2,2,3,3-tetrafluoro-1,4-butanediol, 2,2,3,3-tetrafluoro-1,4-butanediol, 2-ethyl-1,3-hexanediol (EHD), N,N-diisopropanolanaline (DIPA), ethylenediamine, m-phenylene 4-diaminosulfonic acid (PDSA).
The present inventors have observed that polymers formed from all three components (PHNGAD, MDI and extender) are able to allow attachment of hepatocytes which display appropriate functional activity, whereas polymers which do not comprise an extender and are only formed from PHNGAD and MDI do not bind hepatocyte cells.
A preferred polymer for use in the present invention is identified herein as polymer 134 (see
Hepatocytes as used herein may include hepatocyte cells which have been obtained directly from the liver, by way of for example a biopsy, of a subject. However, preferred hepatocytes are derived from embryonic stem cells or embryonic stem cell lines which have been differentiated into hepatocytes or hepatocyte like cells. Examples of such cells are described in [3]. Also hepatocyte like cells which have been obtained from reprogrammed adult cells [6] known in the art, may be employed (Takahashi & Yamanaka, (2006), Cell, 126, p663-676 and Takahashi et al, (2007), Cell, 131, p861-872).
In a further aspect there is provided use of a polymer as described herein for attachment of functional hepatocytes.
As used herein the terms “function” or “functional” refers to metabolic activity commonly associated with hepatocytes. Thus, the hepatocytes of the present invention desirably display more active endocrine and exocrine functions: the elevated production of human serum proteins—Fibronectin, Fibrinogen and Transthyretin and the expression of one or more cytochrome p450 enzymes, such as CYP3A4 and CYP1A2 are key examples. Moreover, such metabolic activity may be increased and/or be of longer duration than when the cells are attached to other substrates. Both MG and 134 show differing levels of hepatocyte maintenance 15 days post-replating, with 134 exhibiting ˜2 fold increase in CYP3A4, Fibronectin, Fibrinogen, Transthyretin and ˜6 fold increase in CYP1A2 activity.
The hepatocyte cells may be attached directly to the polymer of the present invention, with the polymer being formed into a suitable form. Alternatively the polymer may be physically or chemically coated using appropriate techniques such as spin coating, grafting or dip coating onto a suitable substrate. One of skill in the art will appreciate that spin coating is used to coat 2D- and 3D-substrates by spinning the substrate at certain rpm while a solution of the material used to coat the substrate is deposited on top of the surface. One of skill in the art will appreciate that dip coating comprises immersing a substrate into a solution of material used to coat the substrate at a certain velocity. One of skill in the art will appreciate that grafting consists of a chemical process between the substrate and the material to be used. The substrate provides a surface for polymeric coating. Examples of a suitable substrate include, but are not limited to, polymeric and ceramic materials, glasses, ceramics, natural fibres, synthetic fibres, silicones, metals, and composites thereof. In accordance with one embodiment of the present invention, the substrate may be fabricated of a polymeric material, such as polypropylene, polystyrene, polycarbonate, polyethylene, polysulfone, PVDF, Teflon, their composites, blends, or derivatives and the polyfibre core of a bio-artificial liver—a non-woven hydrophilic polyester matrix which is conducive to the immobilisation and high-density cultivation of hepatocytes.
The polymer or polymer coated substrate may take any suitable form and may be made into a porous or non-porous form. In accordance with another embodiment of the present invention, polymer or polymer coated substrate of the present invention may be in a form of threads, sheets, films, gels, membranes, beads, plates and like structures. In accordance with a further embodiment of the present invention, the polymer or polymer coated substrate may be fabricated in the form of a planar device having discrete isolated areas in the form of wells, troughs, pedestals, hydrophobic or hydrophilic patches, diecut adhesive reservoirs or laminated gasket diecuts that form wells, or other physical barriers to fluid flow. Examples of such a solid support include, but are not limited to, a microplate or the like.
In essence any suitable structure may be envisaged providing the hepatocyte cells are able to attach thereto. For example, in one embodiment the polymer may be coated onto wells formed in a microplate or printed in discrete locations on a substrate such that ordered assays of hepatocytes can be formed so as to allow testing of drugs and the like. It may also be appropriate to coat the substrate initially or in the areas to which the polymer is not bound, with a material which inhibits cell adhesion.
In another embodiment, the polymer or polymer coated substrate may take the form of a device designed to act as a bio-artificial liver or detoxifier which is designed to be used to metabolise agents which are added to it. Such a device may find application as a temporary device in subjects who have damaged livers. Alternatively, it may be used to identify metabolites of chemical agents, which may be of therapeutic use.
The present invention will now be further described by way of example and with reference to the Figures which show:
hESCs were differentiated to hepatocyte like cells (HLCs) using an established method. At day 23 hESC-derived HLCs were incubated in 1 ml of hepatocyte culture medium for 24 hours. The following morning culture supernatants were harvested and serum protein production measured by ELISA and quoted as ng/mg of cellular protein. HLCs cultured on polymer 134 exhibited the greatest effect on hepatic function with a greater than 2 fold induction of fibrinogen (A), transthyretin (B) and fibronectin (C) when compared to the other polymers or matrigel extracellular matrices, (n=3);
(A) Hepatic endoderm (HE) morphology was granular on all polymers screened, except for polymer 134 which exhibited a healthy morphology (see also
(A) hESC-derived HLCs morphology plated on matrigel (MG) or polyurethane 134 were compared. In general hepatocytes maintained on 134 looked healthier with a less grainy appearance. (B) Protein lysates were prepared from HE maintained on MG or 134. Extracts were Western blotted, blocked and probed for p-Akt, p-FAK, p-ERK, p15, p21, E-Cadherin, N-Cadherin, Albumin, hPXR and Cyp3A4. hESC-HE maintained on polymer 134, but not MG, displayed increased, Akt, FAK and ERK signalling; cell cycle inhibitors, p15 and p21, expression; adhesion molecule, E-Cadherin and N-Cadherin, expression and hPXR and Cyp3A4 expression. 2 house keeping genes, B-Actin and GAPDH, were employed as loading controls. Similar expression of B-Actin was observed in both MG and 134 samples, whereas greater expression of GAPDH was detected in MG protein samples. In addition HE maintained on polymer 134 exhibited the presence of a phosphorylated upper band consistent with drug inducible hPXR function. The levels of albumin remained similar on hepatocyte like cells maintained on polyurethane 134 and matrigel. (C) hESC-derived HLCs and primary human hepatocytes were incubated with hepatocyte culture media supplemented with 50 μM of CYP3A4 pGlo™ substrate as per manufacturers instructions. 5 hours post-treatment a 50 μl sample of the supernatant was removed and read on a luminometer (POLARstar optima). CYP3A4 activity was greater on cells maintained on 134 than MG and activity is expressed as relative light units (R.L.U.)/mg protein (* p<0.05 by the Student's t-test, (n=3)).
Diethylene glycol, 1,6-hexanediol, neopentyl glycol and adipic acid were purchased from Aldrich. Stannous octoate and titanium(IV) butoxide were of commercial grade (Aldrich), and used without further purification. 4,4′-methylenebis(phenylisocyanate) (MDI) was used as a diisocyanate and the chain extenders (3-dimethylamino-1,2-propanediol (DMAPD), 3-diethylamino-1,2-propanediol (DEAPD), 1,4-butanediol (BD) and 2,2,3,3,4,4,5,5-octafluoro-1,6-hexanediol (OFHD)) were used for polyurethane synthesis (Aldrich).
The synthesis of the PHNGAD polyol was performed using a melting technique of the monomers without any organic solvents. Initially, all monomers were subjected to heat treatment at 60° C. for 48 hours under vacuum to ensure the removal of water. The required amount of monomers, 1,6-hexanediol (0.22 mol), di(ethylene glycol) (0.22 mol), neopentyl glycol (0.22 mol) and adipic acid (0.55 mol) were charged into the reaction flask. The whole assembly was kept in an oven at 40° C. for 6 hours, to avoid any moisture absorption during charging the chemical into the flask. Following drying the required amount of catalyst either stannous octoate or titanium (IV) butoxide was injected through a needle, drop by drop, and the reaction mixture was heated to 180° C., stirred under N2 atmosphere and water was collected through a condenser. The reaction was performed up to the desired time. The molecular weight distribution of the polyol can be controlled by varying the compositions of the monomers, catalyst, reaction time and temperature.
An alternative method for synthesis of the polyol is the solution technique, in which the monomers and other additives are dissolved in an organic solvent. In this case the reaction can be performed at much lower temperature than the melting one, and the solvents removed by evaporation.
The synthesis of polyurethanes was performed by a two-step polymerisation method. The polyol of one equivalent was first reacted with two equivalents of diisocyante, and subsequently one equivalent of a chain extender added to the reaction solution to give copolymer product.
One or more catalysts may be used in the polyurethane synthesis. Particularly preferred catalysts are dibutyltin dilaurate, dimethyltin dicarboxylate, stannous octoate, iron(III) acetylacetonate. The preferred amount of catalysts are in the range of between 0 to 5% by weight.
Additionally, further additives may also be added such as antifoams or adhesive promoters during the reaction, which reduces the surface tension of a solution, thus inhibiting or modifying the formation of a foam.
Various solvents such as N,N′-dimethyl formamide (DMF), toluene, tetrahydrofuran (THF), chloroform, N-methyl-2-pyrrolidone (NMP), 1,2-dichloroethane, dioxane, dimethyl sulfoxide (DMSO), etc may be used. One or more solvents may be used to dissolve the starting materials in the reaction system, and the solvents are dry. Binary solvents may also be used in the synthesis of polyurethane.
The synthesis of polyurethane's may be performed at various temperatures, and the particular preferable temperature range is from 50° C. to 140° C. The reaction may be prolonged up to 96 hours, in an inert atmosphere, and preferably with N2 or Argon purging.
Following the reaction, the polyurethane was collected by precipitation in which a poor solvent can be added drop wise into the reaction solution until the precipitation occurs. Finally, the polyurethanes were separated from the solution and analysed.
Various analytical techniques and methods were used to characterise these materials (such as gel permeation chromatography (GPC), NMR, FTIR spectroscope, differential scanning calorimeter (DSC), etc), to assure the molecular weight distribution and the functional groups of the polymers, and the melting and glass transition temperature, etc).
Various molecular weight distributions (Mw, Mn and D) (table 1) of polyurethanes may be achieved by varying the reaction conditions (such as reaction time, concentration of initiator, etc), the example of molecular weight range 5 kDa to 400 kDa preferable for this invention.
hESC culture was carried out as previously described [2,3]. hESCs were differentiated to hepatocyte like cells using activin and wnt3a as published [3]. At day 9 in the differentiation process the cells were removed from their substrate using a 5 minute 37° C. incubation with Trypsin/EDTA (Invitrogen). Following this hepatocyte like cells were seeded onto the polymer array, polymer coated coverslips or matrigel coated plasticware. The iPS cell line 33D-6 was cultured, propagated and differentiated to hepatic endoderm as previously described [6]. At day 9 in the differentiation protocol the cells were removed from their substrate by a 5 minute incubation with Trypsin/EDTA (Invitrogen). Following this hepatocyte like cells (HLCs) were plated onto polymer 134 or MG, and cultured in L-15 maturation medium as [6].
Immunostaining was carried out as previously described [3].
ELISAs were carried out as previously described [3].
p450 Assay
CYP3A4 and CYP1A2 activity were assessed using the pGlo kit from Promega and carried out as per manufacturers instructions for non-lytic CYP450 activity estimation, (http://www.promega.com/tbs/tb325/tb325.pdf). CYP Activities are expressed as relative light units (RLU) per milligram of protein.
Western blotting was carried out as previously described [4]. Primary antibodies to the proteins are shown in the table below:
Coverslips were coated with the six polymers as previously reported [7].
Polymer microarrays were fabricated by contact printing 380 generic polyurethane and polyacrylate polymers onto an agarose coated glass microscope slide [7, 8]. Once printed, the slides were dried overnight and sterilised by UV irradiation prior to cell plating. We screened this polymer library for stem cell derived hepatic endoderm (HE) attachment, stabilisation and promotion of function. Direct differentiation of human embryonic stem cells (hESCs) to HE was initiated using a recently developed highly efficient tissue culture model (
Human-ESCs were differentiated and replated as detailed. Cell function was assessed at 15 days post re-plating and defined by expression of a panel of hepatocyte specific genes and export of essential serum proteins. Using this strategy, polymer 134 [7] was identified as the most effective cellular support associated with enhanced expression of Fibrinogen [9] (
Following the observations of polymer 134 and its potential utility, further related polymers (prepared in accordance with earlier papers 7, 8) were studied and others identified (see Table 1) which also allow attachment and maintained function of hepatocytes.
Our further studies focussed on HE morphology, signalling, gene expression and drug metabolism on two extracellular matrices, Matrigel and polymer 134. Matrigel was used as our control as it has previously been shown to improve hepatocyte performance in vitro and is currently considered the “gold standard”. We observed a significant change in HE morphology (day 24), thus HE passaged and maintained on matrigel or polymers 2BG9, 212, 9G7, 3AA7 and 223 (
We employed the polyfibre core (PFC), the cell matrix, used in a bio-artificial liver (BAL) device. The PFC was used in its native form or coated with polymer 134. Upon adopting a hepatic fate (Day 9), HE was detached from the biological extracellular matrix and replated onto native or polymer coated PFC and cultured for a further 15 days (day 24) in conditions that support hepatic identity. At day 24 we fixed HE attached to the uncoated (FIG. 6Aa) and polymer coated BAL matrix (FIG. 6Ac) and examined cell structure by electron microscopy. hESC-derived HE maintained on uncoated PFC demonstrated cell attachment and cell processes resembling stress fibres (FIG. 6Ab) whereas HE maintained on polymer 134 coated PFC exhibited a smooth tissue like appearance (FIG. 6Ad) which may limit the effects of fluid shear stress on HE in the BAL.
These data exemplify the value of polymer 134 and hESC-derived HE in a BAL setting. In addition to p450 drug inducibility polymer 134 also promoted human albumin production measured prior to drug induction (days 17 to 21) (
These data exemplify the value of polymer 134 and hESC-derived HE in a BAL setting. In addition to p450 drug inducibility polymer 134 also promoted human urease activity (day 24) (
A polyurethane matrix (polymer 134) plays an important role in hepatocyte functionality by facilitating the culture of highly functional iPSC-derived hepatic endoderm (HE).
In conclusion, screening allowed the identification of a new class of polymer matrix that promotes long-term hepatocellular differentiated function before and after passaging. These attributes bypass current limitations associated with adult human hepatocytes, and will play important roles in developing in vitro models of drug toxicology and may help to reduce drug attrition rates. Additionally our in vitro derived cells provide a resource for the construction of extra-corporeal devices and facilitate novel studies of human liver development and disease.
| Number | Date | Country | Kind |
|---|---|---|---|
| 09048374.9 | Mar 2009 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2010/000523 | 3/19/2010 | WO | 00 | 11/18/2011 |
