Ser-Mien Chia

Multi-layered microcapsules for cell encapsulation.

Mechanical stability, complete encapsulation, selective permeability, and suitable extra-cellular microenvironment, are the major considerations in designing microcapsules for cell encapsulation. We have developed four types of multi-layered microcapsules that allow selective optimization of these parameters. Primary hepatocytes were used as model cells to test these different microcapsule configurations. Type-1 microcapsules with an average diameter of 400 microm were formed by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 microm. Cells in these microcapsules exhibited improved cellular functions over those cultured on collagen monolayers. Type-II microcapsules were formed by encapsulating the Type-I microcapsules in another 2-5 microm ter-polymer shell and a approximately 5 microm collagen layer between the two ter-polymer shells to ensure complete cell encapsulation. Type-II microcapsules comprised of a macro-porous exoskeleton with materials such as alumina sol-gel coated on the Type-I microcapsules. Nano-indendation assay indicated an improved mechanical stability over the Type-I microcapsules. Type-IV microcapsules were created by encapsulating Type-III microcapsules in another 2-5 microm ter-polymer shell, with the aim of imparting a negatively charged smooth surface to minimize plasma protein absorption and ensure complete cell encapsulation. The permeability for nutrient exchange, cellular functions in terms of urea production and mechanical stability of the microcapsules were characterized. The advantages and limitations of these microcapsules for tissue engineering are discussed.

Galactosylated PVDF membrane promotes hepatocyte attachment and functional maintenance

One of the major challenges in BLAD design is to develop functional substrates suitable for hepatocyte attachment and functional maintenance. In the present study, we designed a poly(vinylidene difluoride) (PVDF) surface coated with galactose-tethered Pluronic polymer. The galactose-derived Pluronic F68 (F68-Gal) was adsorbed on PVDF membrane through hydrophobic-hydrophobic interaction between PVDF and the polypropylene oxide segment in Pluronic. The galactose density on the modified PVDF surface increased with the concentration of the F68-Gal solution, reaching 15.4 nmol galactosyl groups per cm2 when a 1 mg/ml of F68-Gal solution was used. The adsorbed F68-Gal remained relatively stable in culture medium. Rat hepatocytes attachment efficiency on F68-Gal modified PVDF membrane was similar to that on collagen-coated surface. The attached hepatocytes on PVDF/F68-Gal membrane self-assembled into multi-cellular spheroids after 1 day of culture. These attached hepatocytes in spheroids exhibited higher cell functions such as albumin synthesis and P450 1A1 detoxification function compared to unmodified PVDF membrane and collagen-coated surface. These results suggest the potential of this galactose-immobilized PVDF membrane as a suitable substrate for hepatocyte culture.

Microcapsules with improved mechanical stability for hepatocyte culture.

Packed-bed or fluidized-bed bioreactor filled with microencapsulated hepatocytes has been proposed as one of the promising designs for bioartificial liver assist device (BLAD) because of potential advantages of high mass transport rate and optimal microenvironment for hepatocyte culture. Recently, we have developed a microcapsule system for the encapsulation of hepatocytes. The microcapsules consist of an inner core of modified collagen and an outer shell of terpolymer of methyl methacrylate, methacrylate and hydroxyethyl methacrylate. Cells encapsulated in these microcapsules exhibit enhanced cellular functions. Improving the mechanical stability of the microcapsules to withstand the shear stress induced by high perfusion rate would be crucial to the success of BLAD applications. In this study, we investigated the effects of terpolymer molecular weight (M(w)) on the mechanical property of these microcapsules and the differentiated functions of encapsulated hepatocytes. Six terpolymers with different M(w) were synthesized using radical polymerization in solution by adjusting the reaction temperature and the initiator concentration. All the terpolymers formed microcapsules with the methylated collagen. While the terpolymer M(w) had little effect on the capsule membrane thickness and permeability of serum albumin, the mechanical property of the microcapsules was significantly improved by the higher M(w) of the terpolymer. Differentiated functions of the hepatocytes cultured in the microcapsules, including urea synthesis, albumin synthesis and cytochrome P450 metabolic activity, were not significantly affected by the terpolymer M(w).

Fibro-C-Index: comprehensive, morphology-based quantification of liver fibrosis using second harmonic generation and two-photon microscopy

We develop a standardized, fully automated, quantification system for liver fibrosis assessment using second harmonic generation microscopy and a morphology-based quantification algorithm. Liver fibrosis is associated with an abnormal increase in collagen as a result of chronic liver diseases. Histopathological scoring is the most commonly used method for liver fibrosis assessment, where a liver biopsy is stained and scored by experienced pathologists. Due to the intrinsic limited sensitivity and operator-dependent variations, there exist high inter- and intraobserver discrepancies. We validate our quantification system, Fibro-C-Index, with a comprehensive animal study and demonstrate its potential application in clinical diagnosis to reduce inter- and intraobserver discrepancies.

The controlled presentation of TGF-β1 to hepatocytes in a 3D-microfluidic cell culture system

3D-microfluidic cell culture systems (3D-microFCCSs) support hepatocyte functions in vitro which can be further enhanced by controlled presentation of 100-200 pg/ml TGF-beta1, thus mimicking the roles of supporting cells in co-cultures. Controlled presentation of TGF-beta1 is achieved by either direct perfusion or in situ controlled release from gelatin microspheres immobilized in the 3D-microFCCS. Primary hepatocytes cultured for 7 days with the in situ controlled released TGF-beta1 exhibited up to four-fold higher albumin secretion and two-fold higher phase I/II enzymatic activities, significantly improving the sensitivity of hepatocytes to acetaminophen-mediated hepatotoxicity, compared to hepatocytes cultured with directly perfused TGF-beta1 or without TGF-beta1. The controlled presentation of TGF-beta1 enhanced hepatocyte functions in microfluidic systems without the complications of co-cultures, allowing for simplifications in drug testing and other hepatocyte-based applications.

HGF regulates the activation of TGF-β1 in rat hepatocytes and hepatic stellate cells.

Hepatocyte growth factor (HGF) ameliorates experimental liver fibrosis through many mechanisms, including degradation of accumulated collagen and decreased expression of fibrotic genes. Investigating an upstream mechanism in which HGF could decrease many fibrotic effectors, we asked whether HGF regulates activation of the fibrotic cytokine transforming growth factor‐beta 1 (TGF‐β1). Specifically, we tested whether HGF decreases the levels of active TGF‐β1, and whether such decrease depends on the predominantly hepatocyte‐secreted protease plasmin, and whether it depends on the TGF‐β1 activator thrombospondin‐1 (TSP‐1). With hepatocyte monocultures, we found HGF‐induced hepatocyte proliferation did increase total levels of plasmin, while decreasing gene expression of fibrotic markers (PAI‐1, TGF‐β1, and TIMP‐2). With in vitro models of fibrotic liver (HSC‐T6 hepatic stellate cells, or co‐cultures of HSC‐T6 and hepatocytes), we found high levels of fibrosis‐associated proteins such as TSP‐1, active TGF‐β1, and Collagen I. HGF treatment on these fibrotic cultures stimulated plasmin levels; increased TSP‐1 protein cleavage; and decreased the levels of active TGF‐β1 and Collagen I. When plasmin was blocked by the inhibitor aprotinin, HGF could no longer decrease TGF‐β1 activation and Collagen I. Meanwhile, the TSP‐1‐specific peptide inhibitor, LSKL, reduced TGF‐β1 to the same level as in the HGF‐treated cultures; combining LSKL and HGF treatments caused no further decrease, suggesting that HGF affects the TSP‐1 dependent pathway of TGF‐β1 activation. Therefore, HGF can decrease TGF‐β1 activation and TGF‐β1‐dependent fibrotic markers, by stimulating hepatocytes to produce plasmin, and by antagonizing TSP‐1‐dependent activation of TGF‐β1. J. Cell. Physiol. 228: 393–401, 2013.

Kinectin-dependent assembly of translation elongation factor-1 complex on endoplasmic reticulum regulates protein synthesis

Kinectin is an integral membrane protein with many isoforms primarily found on the endoplasmic reticulum. It has been found to bind kinesin, Rho GTPase, and translation elongation factor-1δ. None of the existing models for the quaternary organization of the elongation factor-1 complex in higher eukaryotes involves kinectin. We have investigated here the assembly of the elongation factor-1 complex onto endoplasmic reticulum via kinectin using in vitro and in vivo assays. We established that the entire elongation factor-1 complex can be anchored to endoplasmic reticulum via kinectin, and the interacting partners are as follows. Kinectin binds EF-1δ, which in turn binds EF-1γ but not EF-1β; EF-1γ binds EF-1δ and EF-1β but not kinectin. In vivo splice blocking of the kinectin exons 36 and 37 produced kinectin lacking the EF-1δ binding domain, which disrupted the membrane localization of EF-1δ, EF-1γ, and EF-1β on endoplasmic reticulum, similar to the disruptions seen with the overexpression of kinectin fragments containing the EF-1δ binding domain. The disruptions of the EF-1δ/kinectin interaction inhibited expression of membrane proteins but enhanced synthesis of cytosolic proteins in vivo. These findings suggest that anchoring the elongation factor-1 complex onto endoplasmic reticulum via EF-1δ/kinectin interaction is important for regulating protein synthesis in eukaryotic cells.

Automated Algorithm for Standardized Quantification on Liver Fibrosis using Second Harmonic Generation Microscopy

Determining the extent of liver fibrosis has clinically been difficult due to the lack of a simple, objective method that can accurately quantify the amount of collagen in the diseased tissue. Second harmonic generation (SHG) microscopy has been shown to produce bright and robust signals from non-centrosymmetric fibrillar collagen. We designed a SHG system that can objectively measure collagen presence in livers. In addition, we have developed a fully automated algorithm to quantify liver fibrosis in an efficient, standardized and reproducible manner. SHG microscopy was performed on livers harvested from bile duct ligated Wistar rats using a confocal microscope with a mode-locked Ti:Sapphire laser. Images acquired were later analyzed with Otsu method and a custom-developed, fully automated algorithm to measure area of fibers. Both the qualitative and quantitative progression of collagen over time was observed using SHG microscopy. We have compared the modified quantification algorithm with Otsu segmentation algorithm. With the modified algorithm, we have maintained greater amount of collagen after segmentation. We have also shown modified algorithm was able to identify the presence of finer collagen which was completely removed with the normal Otsu method. We have built an imaging system using state-of-the-art SHG microscopy. In addition, a fully-automated algorithm was developed to quantify collagen content in tissue to measure the presence of collagen in high accuracy. By combining SHG microscopy and our quantification algorithm, we can provide sensitive measurement for liver fibrosis which accurately reflects the progression of liver fibrosis, especially in early stages.

Multi-Dimensional Imaging of Cell- and Tissue-Engineered Constructs

The following sections are included: Introduction Natural Spheroids or Engineered Cell Aggregates Micro-capsules Porous Scaffold-based 3-D Tissue Constructs Micro-Fluidic Channels Three-Dimensional Tissue Slices Conclusion and Future Work References

Novel hepatocyte encapsulation enhances cellular functions

We have synthesized a HEMA, MMA and MAA terpolymer to complex with modified collagen for encapsulation of rat hepatocytes. The encapsulated hepatocytes exhibited high level of functions in culture comparable to those of hepatocyte spheroids. The collagen that lined the inner layer of the capsule provided a compatible substrate for hepatocyte culture, and the outer terpolymer shell determined permeability, which was optimized for transport of 60 kDa but not 150 kDa molecules. Besides shorter processing time than spheroid formation, the encapsulation may offer other advantages important in designing a bioartificial liver-assisted device.