Functionally Enhanced Human Stem Cell Derived Hepatocytes in Galactosylated Cellulosic Sponges for Hepatotoxicity Testing.

Pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) are an attractive alternative to primary human hepatocytes (PHHs) used in applications ranging from therapeutics to drug safety testing studies. It would be critical to improve and maintain mature hepatocyte functions of the hPSC-HLCs, especially for long-term studies. If 3D culture systems were to be used for such purposes, it would be important that the system can support formation and maintenance of optimal-sized spheroids for long periods of time, and can also be directly deployed in liver drug testing assays. We report the use of 3-dimensional (3D) cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold has a macroporous network which helps to control the formation and maintenance of the spheroids for weeks. Our results show that culturing hPSC-HLCs in 3D cellulosic scaffolds increases functionality, as demonstrated by improved urea production and hepatic marker expression. In addition, hPSC-HLCs in the scaffolds exhibit a more mature phenotype, as shown by enhanced cytochrome P450 activity and induction. This enables the system to show a higher sensitivity to hepatotoxicants and a higher degree of similarity to PHHs when compared to conventional 2D systems. These results suggest that 3D cellulosic scaffolds are ideal for the long-term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs with improved cellular function can be continually maintained in the scaffolds and directly used for hepatotoxicity assays, making this system highly attractive for drug testing applications.

Scalable spheroid model of human hepatocytes for hepatitis C infection and replication.

Developing effective new drugs against hepatitis C (HCV) virus has been challenging due to the lack of appropriate small animal and in vitro models recapitulating the entire life cycle of the virus. Current in vitro models fail to recapitulate the complexity of human liver physiology. Here we present a method to study HCV infection and replication on spheroid cultures of Huh 7.5 cells and primary human hepatocytes. Spheroid cultures are constructed using a galactosylated cellulosic sponge with homogeneous macroporosity, enabling the formation and maintenance of uniformly sized spheroids. This facilitates easy handling of the tissue-engineered constructs and overcomes limitations inherent of traditional spheroid cultures. Spheroids formed in the galactosylated cellulosic sponge show enhanced hepatic functions in Huh 7.5 cells and maintain liver-specific functions of primary human hepatocytes for 2 weeks in culture. Establishment of apical and basolateral polarity along with the expression and localization of all HCV specific entry proteins allow for a 9-fold increase in viral entry in spheroid cultures over conventional monolayer cultures. Huh 7.5 cells cultured in the galactosylated cellulosic sponge also support replication of the HCV clone, JFH (Japanese fulminant hepatitis)-1 at higher levels than in monolayer cultures. The advantages of our system in maintaining liver-specific functions and allowing HCV infection together with its ease of handling make it suitable for the study of HCV biology in basic research and pharmaceutical R&D.

Nucleic Acid Plate Culture: Label-Free and Naked-Eye-Based Digital Loop-Mediated Isothermal Amplification in Hydrogel with Machine Learning.

Digital nucleic acid amplification enables the absolute quantification of single molecules. However, due to the ultrasmall reaction volume in the digital system (i.e., short light path), most digital systems are limited to fluorescence signals, while label-free and naked-eye readout remain challenging. In this work, we report a digital nucleic acid plate culture method for label-free, ultrasimple, and naked-eye nucleic acid analysis. As simple as the bacteria culture, the nanoconfined digital loop-mediated isothermal amplification was performed by using polyacrylamide (PAM) hydrogel as the amplification matrix. The nanoconfinement of PAM hydrogel with an ionic polymer chain can remarkably accelerate the amplification of target nucleic acids and the growth of inorganic byproducts, namely, magnesium pyrophosphate particles (MPPs). Compared to that in aqueous solutions, MPPs trapped in the hydrogel with enhanced light scattering characteristics are clearly visible to the naked eye, forming white "colony" spots that can be simply counted in a label-free and instrument-free manner. The MPPs can also be photographed by a smartphone and automatically counted by a machine-learning algorithm to realize the absolute quantification of antibiotic-resistant pathogens in diverse real samples.

Shape-Anisotropic Microembolics Generated by Microfluidic Synthesis for Transarterial Embolization Treatment.

Particulate embolic agents with calibrated sizes, which employ interventional procedures to achieve endovascular embolization, have recently attracted tremendous interest in therapeutic embolotherapies for a wide plethora of diseases. However, the particulate shape effect, which may play a critical role in embolization performances, has been rarely investigated. Here, polyvinyl alcohol (PVA)-based shape-anisotropic microembolics are developed using a facile droplet-based microfluidic fabrication method via heat-accelerated PVA-glutaraldehyde crosslinking reaction at a mild temperature of 38 ° C. Precise geometrical controls of the microembolics are achieved with a nearly capsule shape through regulating surfactant concentration and flow rate ratio between dispersed phase and continuous phase in the microfluidics. Two specific models are employed, i.e., in vitro decellularized rabbit liver embolization model and in vivo rabbit ear embolization model, to systematically evaluate the embolization behaviors of the nonspherical microembolics. Compared to microspheres of the same volume, the elongated microembolics demonstrated advantageous endovascular navigation capability, penetration depth and embolization stability due to their comparatively smaller radial diameter and their central cylindrical part providing larger contact area with distal vessels. Such nonspherical microembolics present a promising platform to apply shape anisotropy to achieve distinctive therapeutic effects for endovascular treatments.

Tethered primary hepatocyte spheroids on polystyrene multi-well plates for high-throughput drug safety testing.

Hepatocyte spheroids are useful models for mimicking liver phenotypes in vitro because of their three-dimensionality. However, the lack of a biomaterial platform which allows the facile manipulation of spheroid cultures on a large scale severely limits their application in automated high-throughput drug safety testing. In addition, there is not yet a robust way of controlling spheroid size, homogeneity and integrity during extended culture. This work addresses these bottlenecks to the automation of hepatocyte spheroid culture by tethering 3D hepatocyte spheroids directly onto surface-modified polystyrene (PS) multi-well plates. However, polystyrene surfaces are inert toward functionalization, and this makes the uniform conjugation of bioactive ligands very challenging. Surface modification of polystyrene well plates is achieved herein using a three-step sequence, resulting in a homogeneous distribution of bioactive RGD and galactose ligands required for spheroid tethering and formation. Importantly, treatment of polystyrene tethered spheroids with vehicle and paradigm hepatotoxicant (chlorpromazine) treatment using an automated liquid handling platform shows low signal deviation, intact 3D spheroidal morphology and Z' values above 0.5, and hence confirming their amenability to high-throughput automation. Functional analyses performance (i.e. urea and albumin production, cytochrome P450 activity and induction studies) of the polystyrene tethered spheroids reveal significant improvements over hepatocytes cultured as collagen monolayers. This is the first demonstration of automated hepatotoxicant treatment on functional 3D hepatocyte spheroids tethered directly on polystyrene multi-well plates, and will serve as an important advancement in the application of 3D tethered spheroid models to high throughput drug screening.

Cell culture on MEMS platforms: a review.

Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bio-incompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bio-incompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented.

Synthetic sandwich culture of 3D hepatocyte monolayer.

The sandwich culture of hepatocytes, between double layers of extra-cellular matrix (ECM), is a well-established in vitro model for re-establishing hepatic polarity and maintaining differentiated functions. Applications of the ECM-based sandwich culture are limited by the mass transfer barriers induced by the top gelled ECM layer, complex molecular composition of ECM with batch-to-batch variation and uncontrollable coating of the ECM double layers. We have addressed these limitations of the ECM-based sandwich culture by developing an 'ECM-free' synthetic sandwich culture, which is constructed by sandwiching a 3D hepatocyte monolayer between a glycine-arginine-glycine-aspatic acid-serine (GRGDS)-modified polyethylene terephthalate (PET) track-etched membrane (top support) and a galactosylated PET film (bottom substratum). The bioactive top support and bottom substratum in the synthetic sandwich culture substituted for the functionalities of the ECM in the ECM-based sandwich culture with further improvement in mass transfer and optimal material properties. The 3D hepatocyte monolayer in the synthetic sandwich culture exhibited a similar process of hepatic polarity formation, better cell-cell interaction and improved differentiated functions over 14-day culture compared to the hepatocytes in collagen sandwich culture. The novel 3D hepatocyte monolayer sandwich culture using bioactive synthetic materials may readily replace the ECM-based sandwich culture for liver tissue engineering applications, such as drug metabolism/toxicity testing and hepatocyte-based bioreactors.

A gel-free 3D microfluidic cell culture system.

3D microfluidic cell culture systems offer a biologically relevant model to conduct micro-scale mammalian cell-based research and applications. Various natural and synthetic hydrogels have been successfully incorporated into microfluidic systems to support mammalian cells in 3D. However, embedment of cells in hydrogels introduces operational complexity, potentially hinders mass transfer, and is not suitable for establishing cell-dense, ECM-poor constructs. We present here a gel-free method for seeding and culturing mammalian cells three-dimensionally in a microfluidic channel. A combination of transient inter-cellular polymeric linker and micro-fabricated pillar arrays was used for the in situ formation and immobilization of 3D multi-cellular aggregates in a microfluidic channel. 3D cellular constructs formed this way are relieved of hydrogel embedment for cellular support. Two mammalian cell lines (A549 and C3A) and a primary mammalian cell (bone marrow mesenchymal stem cells) were cultured in the gel-free 3D microfluidic cell culture system. The cells displayed 3D cellular morphology, cellular functions and differentiation capability, affirming the versatility of the system as a 3D cell perfusion culture platform for anchorage-dependent mammalian cells.

Microfabricated silicon nitride membranes for hepatocyte sandwich culture.

We have developed a hepatocyte sandwich culture with improved mass transport properties based on ultra-thin microfabricated porous silicon nitride (Si(3)N(4)) membranes. The dimensions and uniformity of the membrane pores can be configurable, which confers more control over the mass transport. Instead of collagen gels used in conventional sandwich culture, we utilized galactose ligands immobilized on the Si(3)N(4) membranes to support hepatocyte attachment and function in the sandwich culture. Diffusion studies using FITC-dextrans confirmed that mass transport of the microfabricated Si(3)N(4) membrane based sandwich was significantly better than conventional collagen gel sandwich and can be configured by varying the porosity of the Si(3)N(4) membrane. Hepatocytes cultured in the microfabricated Si(3)N(4) membrane based sandwich culture exhibited earlier apical repolarization and biliary excretion, improved differentiated functions and enhanced drug sensitivity compared to hepatocytes cultured in a collagen gel sandwich. The Si(3)N(4) membrane based sandwich culture allows for a systematic optimization of the mass transport properties of hepatocyte culture by changing the pore size and inter-pore distance. This will enable more effective drug testing applications where optimal mass transport is required for hepatocyte function maintenance and drug accessibility.

Transient inter-cellular polymeric linker.

Three-dimensional (3D) tissue-engineered constructs with bio-mimicry cell-cell and cell-matrix interactions are useful in regenerative medicine. In cell-dense and matrix-poor tissues of the internal organs, cells support one another via cell-cell interactions, supplemented by small amount of the extra-cellular matrices (ECM) secreted by the cells. Here we connect HepG2 cells directly but transiently with inter-cellular polymeric linker to facilitate cell-cell interaction and aggregation. The linker consists of a non-toxic low molecular-weight polyethyleneimine (PEI) backbone conjugated with multiple hydrazide groups that can aggregate cells within 30 min by reacting with the aldehyde handles on the chemically modified cell-surface glycoproteins. The cells in the cellular aggregates proliferated; and maintained the cortical actin distribution of the 3D cell morphology while non-aggregated cells died over 7 days of suspension culture. The aggregates lost distinguishable cell-cell boundaries within 3 days; and the ECM fibers became visible around cells from day 3 onwards while the inter-cellular polymeric linker disappeared from the cell surfaces over time. The transient inter-cellular polymeric linker can be useful for forming 3D cellular and tissue constructs without bulk biomaterials or extensive network of engineered ECM for various applications.

Vitreous cryopreservation of cell-biomaterial constructs involving encapsulated hepatocytes.

We put forward a new strategy for cryopreservation, namely vitrification or ice-free preservation, of cell-biomaterial constructs for tissue-engineering applications. In this study, for a period of 6 days, we tested vitrified and control hepatocytes entrapped at 2 different cell densities (1.5 x 10(6) and 5 x 10(6) cells/mL) in 2 types of engineered collagen matrices (M- and G-collagen) as models to evaluate efficacy and universality of the developed vitrification method. The nature of collagens caused differences in capsule sizes (100-200 microm versus 350-450 microm). The developed method included rapid step-wise introduction of microencapsulated hepatocytes to vitrification solution (40v/v% ethylene glycol 0.6 M sucrose in medium) and their direct immersion in liquid nitrogen. Vitrification did not affect viability and functions of the microencapsulated hepatocytes, which exhibited trends similar to those of untreated controls in the decline of their functions and the rate of cell death during continuous culture, irrespective of physical and chemical properties of the biomaterial and cell density. For control and vitrification, the percentage of live cells varied from 80.3% +/- 0.9% to 82.3% +/- 1.4% in capsules formed by M-collagen, from 82.8% +/- 1.1% to 85.0% +/- 3.3% in capsules formed by G-collagen with cells entrapped at low density, and from 84.4% +/- 1.3% to 86.8% +/- 0.6% in capsules formed by G-collagen with cells entrapped at high density (p > 0.05). Within the same day, the maximum relative change in cell viability and functions between control and vitrification was 4% and 16%, respectively. The developed vitrification approach, which is an alternative to freezing, can be applied to other tissue-engineered constructs with comparable sizes, various cell numbers, and various properties of the biomaterials involved.

Identification and characterization of a novel prespheroid 3-dimensional hepatocyte monolayer on galactosylated substratum.

Three-dimensional (3D) hepatocyte spheroids mimicking the structural and functional characteristics of hepatocytes in vivo were self-assembled onto a galactosylated polyethylene terephthalate (PET) substratum, and the dynamic process of spheroid formation was investigated using time-lapse confocal microscopy. Hepatocytes cultured on this galactosylated substratum formed small cell-aggregates within 12 h, which gradually merged into "island-like" clusters at approximately 1 day and spread to form prespheroid monolayer within 2 days; the prespheroid monolayer was stretched to fold into compact and larger 3D spheroids after 3 days. We compared the expressions of F-actin (cytoskeleton), phosphorylated focal adhesion kinase (p-FAK, cell-substratum interactions) and E-cadherin (cell-cell interactions) during the dynamic process of 3D hepatocyte spheroid formation with the dynamic process of 2D hepatocyte monolayer formation on collagen substratum. Hepatocytes in the prespheroid monolayer stage exhibited the strongest cell-substratum interactions of all 4 stages during spheroid formation with cell-cell interactions and F-actin distribution comparable with those of the 3D hepatocyte spheroids. The prespheroid monolayer also exhibited better hepatocyte polarity (multidrug resistance protein 2) and tight junction (zonula occludens-1) formation, more-differentiated hepatocyte functions (albumin production and cytochrome P450 1 A activity), and higher sensitivity to hepatotoxicity than the conventional 2D hepatocyte monolayer. The transient prespheroid 3D monolayer could be stabilized on a hybrid glycine-arginine-glycine-aspartic acid-serine (GRGDS)/galactose-PET substratum for up to 1 week and destabilized to form 3D spheroids in excess soluble GRGDS peptide.

3D hepatocyte monolayer on hybrid RGD/galactose substratum.

Hepatocyte-based applications such as xenobiotics metabolism and toxicity studies usually require hepatocytes anchoring onto flat substrata that support their functional maintenance. Conventional cell culture plates coated with natural matrices or synthetic ligands allow hepatocytes to adhere tightly as two-dimensional (2D) monolayer but these tightly anchored hepatocytes rapidly lose their differentiated functions. On galactosylated substrata, hepatocytes adhere loosely; and readily form three-dimensional (3D) spheroids that can maintain high levels of cellular functions. These spheroids detach easily from the substrata and exhibit poor mass transport properties unsuitable for many applications. Here, we have developed a hybrid RGD/galactose substratum based on polyethylene terephthalate film conjugated with both RGD peptide and galactose ligand to enhance cell adhesion and functions synergistically. Primary hepatocytes adhere effectively onto the transparent hybrid substratum in 96-well plates as monolayer while exhibiting high levels of liver-specific functions, morphology and cell-cell interactions typically seen in the 3D hepatocyte spheroids. The hepatocytes cultured onto the hybrid substratum also exhibit high levels of sensitivity to a model drug acetaminophen similar to the 3D hepatocyte spheroids. The monolayer of hepatocytes exhibiting the 3D cell behaviors on this flat hybrid substratum can be useful for various applications requiring both effective mass transfer and cellular support.

[Encapsulating hepatocytes with chitosan in physiological conditions].

Prepared from 15.3% N-acetylated chitosan (FNC), half N-acetylated chitosan (HNC) possesses a good solubility in a weak basic solution, guaranteeing the formation of microcapsules by the coacervating reaction between HNC and methacrylic acid (MAA)-hydroxyethyl methacrylate (HEMA)-methyl methacrylate (MMA) (MAA-HEMA-MMA) terpolymer under physiological conditions. When hepatocytes were encapsulated in such 3-dimensional microenvironment, as compared to monolayer culture, cell functions, including P450 activity, urea production and albumin release, were well supported. The prepared microcapsules have good mechanical stability and permeability.

Constrained spheroids for prolonged hepatocyte culture.

Liver-specific functions in primary hepatocytes can be maintained over extended duration in vitro using spheroid culture. However, the undesired loss of cells over time is still a major unaddressed problem, which consequently generates large variations in downstream assays such as drug screening. In static culture, the turbulence generated by medium change can cause spheroids to detach from the culture substrate. Under perfusion, the momentum generated by Stokes force similarly results in spheroid detachment. To overcome this problem, we developed a Constrained Spheroids (CS) culture system that immobilizes spheroids between a glass coverslip and an ultra-thin porous Parylene C membrane, both surface-modified with poly(ethylene glycol) and galactose ligands for optimum spheroid formation and maintenance. In this configuration, cell loss was minimized even when perfusion was introduced. When compared to the standard collagen sandwich model, hepatocytes cultured as CS under perfusion exhibited significantly enhanced hepatocyte functions such as urea secretion, and CYP1A1 and CYP3A2 metabolic activity. We propose the use of the CS culture as an improved culture platform to current hepatocyte spheroid-based culture systems.

Application of a polyelectrolyte complex coacervation method to improve seeding efficiency of bone marrow stromal cells in a 3D culture system.

High seeding efficiency with homogenous distribution of limited cell sources such as bone marrow stromal cells (BMSCs) are of clinical relevance in scaffold-based tissue engineering. Therefore, considerable research efforts have been invested to ameliorate the seeding efficiency in 3D scaffolds. Preliminary data demonstrated that indeed BMSCs were viable and were able to proliferate in a model 3D scaffold, i.e. Cytomatrix scaffold. However, the eventual practical application of BMSCs in such 3D scaffolds is limited by the low seeding efficiency of the cells within the scaffold. Here, we demonstrated that the cell seeding efficiency of BMSCs in the Cytomatrix scaffold can be improved significantly (t-test, p<0.05) by means of macroencapsulating the scaffold via the complex coacervation of a methylated collagen and terpolymer. The thickness and density of the polyeletrolyte complex can be modulated by the contact time between the methylated collagen and terpolymer to balance between cell entrapment efficacy and mass transfer impedance imparted by the complex. Porcine BMSCs were macroencapsulated in Cytomatrix scaffolds using various polyelectrolyte contact time and cultured under both static and dynamic conditions. Throughout the range of contact time investigated, macroencapsulation did not affect the viability of the porcine BMSCs in dynamic culture. However, the viability of the cells under static cultures was compromised with longer polyelectrolyte contact time. Therefore, this proposed method of macroencapsulation enables customization to achieve enhanced seeding efficiency without mass transfer impedance for different culture configurations.

Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices.

Hepatocytes are anchorage-dependent cells sensitive to microenvironment; the control of the physicochemical properties of the extra-cellular matrices may be useful to the maintenance of hepatocyte functions in vitro for various applications. In a microcapsule-based 3-D hepatocyte culture microenvironment, we could control the physical properties of the collagen nano-fibres by fine-tuning the complex-coacervation reaction between methylated collagen and terpolymer of hydroxylethyl methacrylate-methyl methacrylate-methylacrylic acid. The physical properties of the nano-fibres were quantitatively characterized using back-scattering confocal microscopy to help optimize the physical support for hepatocyte functions. We further enhanced the chemical properties of the collagen nano-fibres by incorporating galactose onto collagen, which can specifically interact with the asialoglycoprotein receptor on hepatocytes. By correlating a range of collagen nano-fibres of different physicochemical properties with hepatocyte functions, we have identified a specific combination of methylated and galactosylated collagen nano-fibres optimal for maintaining hepatocyte functions in vitro. A model of how the physical and chemical supports interplay to maintain hepatocyte functions is discussed.

Galactosylated poly(vinylidene difluoride) hollow fiber bioreactor for hepatocyte culture.

To overcome the limitations of long-term expression of highly differentiated hepatocyte functions, we have developed a novel bioreactor in which hepatocytes are seeded in a ligand-immobilized hollow fiber cartridge. Galactosylated Pluronic polymer is immobilized on poly(vinylidene difluoride) (PVDF) hollow fiber surface through an adsorption scheme yielding a substrate with hepatocyte-specific ligand and a hydrophilic surface layer, which can resist nonspecific protein adsorption and facilitate cell binding to the galactose ligand. Interestingly, the galactosylated PVDF hollow fiber shows enhanced serum albumin diffusion across the membrane. Freshly isolated rat hepatocytes were seeded and cultured in the extralumenal space of the hollow fiber cartridge for 18 days in a continuously circulated system. Albumin secretion function of the seeded hepatocytes was monitored by analyzing circulating medium by enzyme-linked immunosorbent assay. Urea synthesis and P-450 function (7-ethoxycoumarin dealkylase activity) were measured periodically by doping the circulating medium with NH4Cl and 7-ethoxycoumarin, respectively. Hepatocytes cultured on galactosylated PVDF hollow fibers maintained better albumin secretion and P-450 functions than on unmodified and serum-coated PVDF hollow fibers when cultured in serum-containing medium. Morphological examination by scanning electron microscopy showed that hepatocytes cultured on galactosylated PVDF hollow fibers developed significant aggregation, in contrast to those cultured on unmodified PVDF fibers or on serum-coated PVDF fibers. Transmission electron microscopy images revealed that tight junctions and canaliculus-like structures formed in these aggregates. These results suggest the potential application of this galactosylated PVDF hollow fiber cartridge for the design of a bioartificial liver assist device.

Encapsulating live cells with water-soluble chitosan in physiological conditions.

A new class of microcapsules was prepared under physiological conditions by polyelectrolyte complexation between two oppositely-charged, water-soluble polymers. The microcapsules consisted of an inner core of half N-acetylated chitosan and an outer shell of methacrylic acid (MAA) (20.4%)-hydroxyethyl methacrylate (HEMA) (27.4%)-methyl methacrylate (MMA) (52.2%) (MAA-HEMA-MMA) terpolymer. Both 400 and 150 kDa half N-acetylated chitosans maintained good water solubility and supplied enough protonated amino groups to coacervate with terpolymer at pH 7.0-7.4, in contrast to other chitosan-based microcapsules which must be prepared at pH <6.5. The viscosity of half N-acetylated chitosan solutions between 80 and 3000 cPas allowed the formation of microcapsules with spherical shape. Molar mass, pH and concentration of half N-acetylated chitosan, and reaction time, influenced the morphology, thickness and porosity of the microcapsules. Microcapsules formed with high concentration of half N-acetylated chitosan exhibited improved mechanical stability, whereas microcapsules formed with low concentration of half N-acetylated chitosan exhibited good permeability. This 3D microenvironment has been configured to cultivate sensitive anchorage-dependent cells such as hepatocytes to maintain high level of functions.

Characterization of amphoteric multilayered thin films by means of zeta potential measurements.

Multilayer films of amphoteric methylated collagen were assembled on SOURCE 15S or SOURCE 15Q beads by sequential electrostatic deposition with negatively charged methylacrylic acid-hydroxyethyl methacrylate-methyl methacrylate (MAA-HEMA-MMA) terpolymer. Methylated collagen and terpolymer were deposited under conditions where they were oppositely charged to one another, thereby facilitating growth of the films through electrostatic interactions. Measurements revealed alternating positive and negative zeta-potential with the deposition of each methylated collagen and terpolymer layer, respectively. Assembly pH had a remarkable influence on zeta-potential of the assembled multilayers and the deposition of methylated collagen will be frustrated when the assembly pH is up to 9.0. In addition, ionic strength (NaCl concentration) showed an intricate effect on zeta-potential of the films of amphoteric methylated collagen.