Development of membranes for the cultivation of kidney epithelial cells

The development of biohybrid organs (BHO) will benefit from improved membranes regarding transport and cell contacting properties. Here we describe in a first study the development and testing of membranes made of polyacrylonitrile (PAN) and polysulfone (PSU) for the immobilisation of kidney epithelial cells. Comparative investigations on overall polymer toxicity tested with 3T3 fibroblasts, and morphology and proliferation of Madin-Darby canine kidney (MDCK) cells cultured on the membranes could show that these materials have comparable cell contacting properties like Millicell membranes. Since PAN and PSU have superior membrane forming properties with regard to membrane geometry, i.e. for the preparation of hollow fibres, and porosity, i.e. for immuno isolation, both materials or modifications thereof seem to be suitable for the application in BHO such as biohybrid kidney.     

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.     

Transcellular water transport and stability of expression in aquaporin 1-transfected LLC-PK1 cells in the development of a portable bioartificial renal tubule device

We investigated a portable bioartificial renal tubule device (BRTD) consisting of renal tubule cells and hollow fibers, to improve the quality of life of patients. It is necessary for a BRTD system to be compact. A compact portable BRTB requires transfection of an appropriate water channel or electrical pump genes in tubular epithelial cells, which should be based on physiological similarities to human kidney function. LLC-PK(1) cells, into which rat kidney aquaporin 1 (AQP1) cDNA was stably transfected, were evaluated for water transport ability. The expression and localization of water AQP1 were examined by Western blotting, RT-PCR, and immunofluorescence. To measure transcellular water permeation, a simple method was applied, using phenol red as a cell-impermeant marker of concentration. In contrast to wild-type LLC-PK(1) cells, rat AQP1-transfected cells had high transcellular osmotic water permeability. The expression of rat AQP1 mRNA (ratio of AQP1 to beta-actin mRNA) and protein bands (a 28-kDa band and a broad, 35- to 45-kDa band) was confirmed to be stably maintained until a population doubling level of 24. In AQP1-transfected LLCPK(1) cells, the protein was localized mainly to the basolateral side, but also the apical side, of the plasma membrane. Wild-type LLC-PK(1) cells were not stained at the plasma membrane. It is possible that enough AQP1-transfected tubule epithelial cells were supplied for a bioartificial renal tubule device.     

Transmission electron microscopic study of hepatocytes in bioartificial liver

A bioartificial liver (BAL) was prepared by simple inoculation of hepatocytes into the inner space of hollow fibers of a hemodialyzer and it was maintained in a closed circuit for in vitro culture. Morphology of hepatocytes in the hollow fibers was studied in detail using transmission electron microscopy (TEM). The hepatocytes formed three-dimensional, rod-shaped aggregates of 200 microm in diameter throughout the whole dimension of the hollow fibers after 1 day of culture. Approximately five hepatocyte layers existed from the surface to the center of the aggregate. The hepatocytes in the aggregate displayed mostly polygonal shapes and were surrounded by five to six cells. Abundant bile canaliculi were formed between the hepatocytes and were sealed by tight junctions. The distance between the adjacent hepatocytes except the bile canaliculus domain was approximately 20 nm, and interdigitation was observed between some hepatocytes. These observations indicate that the hepatocytes formed functionally associated aggregates, that is, organoids. Although the cells facing the inner surface of the hollow fiber lost their polygonal shape and became flattened during the following several-day culture, no drastic change was observed in the morphology of the hepatocytes located inside the aggregate. After 14 days of culture, the number of living cells decreased and most of these had a deformed nucleus, few numbers of organelles, and intermittent lipid droplets.     

生物降解聚乳酸中空纤维的制备及其药物体外释放的研究

采用干／湿相转化法制备聚乳酸中空纤维得到四个不同纺制体系的中空纤维，扫描电镜测定了中空纤维的膜结构和尺寸，灌药（ＮＥＴ）及熔封后的中空纤维的体外释药研究表明，ＮＥＴ的释放与中空纤维的膜壁结构有关，文章对释放机理进行初步探讨。     

Role of tight junctions of polarized epithelial MDCK cells in the replication of herpes simplex virus type 1

Before completion of polarization, Madin-Darby canine kidney (MDCK) cells showed high infectivity and progeny production of herpes simplex virus type 1 infection. After polarization or formation of tight junctions, the infectivity and virus replication in MDCK cells was restricted significantly. The disruption of tight junctions by depletion of Ca²⁺ resulted in increasing virus infectivity and productivity. Mechanical disruption of tight junctions by scratching the cell monolayers with injection needle allowed markedly the replication of HSV-I in the cells aligned along the injured area. In polarized MDCK cells the progeny were released preferentially from the apical surface of the cells. These data suggest that because polarized MDCK cells mimic the epithelial cell layers, this cell line is helpful for determining the factors which regulate viral transmission in the human body. © Wiley-Liss, Inc.     

Function of a new internal bioartificial liver: an in vitro study

The goal of this study was to determine whether a new internal bioartificial liver utilizing porcine hepatocytes can perform detoxification and other metabolic functions. Such a system might aid in treating patients with moderate to severe liver failure and prolong patient survival until a matching organ is found for transplantation. Porcine hepatocytes were attached to a microcarrier and an internal artificial liver was constructed by perfusing the hepatocytes into a polysulfon hollow fiber. The 4 experimental groups were: (a) control group, (b) microcarrier group, (c) hollow fiber group, and (d) internal bioartificial liver group. Viability of hepatocytes, alanine aminotransferase (ALT) and lactate dehydrogenase (LD) activities in the medium, urea production, diazepam transformation, protein synthesis, and glucose-6-phosphatase activity of cells were monitored during a 7-day culture period. Viability of porcine hepatocytes in the internal bioartificial liver group was maintained at >80% during the culture period, and alanine aminotransferase and lactate dehydrogenase activities did not fluctuate significantly. These enzyme activities were significantly lower in the internal bioartificial liver group than in the control or microcarrier groups. Urea production, diazepam transformation, [3H]-leucine incorporation, and glucose-6-phosphatase activity were significantly higher in the internal bioartificial liver group than in the control and hollow fiber groups. These results show that the new internal bioartificial liver produces small amounts of ALT and LD and exhibits detoxification and protein synthetic functions.     

Growth and differentiated properties of a kidney epithelial cell line (MDCK)

The MDCK dog kidney epithelial cell line has been shown to retain the capacity for vectorial salt and fluid transport, sensitivity to growth regulation, and the ability to regenerate kidney tubular-like structures when injected into athymic nude mice. MDCK cells grown in tissue culture or in baby nude mice have the morphological properties of distal tubular cells, form tight and gap junctions, lack proximal tubular enzyme markers, and possess appreciable activities of Na+-K4-ATPase, ectoleucine aminopeptidase, and ectoalkaline phosphatase. Adenylate cyclase in intact cells is responsive to vasopressin, prostaglandins E1 and E2, and glucagon. Two Na+ transport systems have been characterized: a Na+-H+ antiport system, sensitive to amiloride inhibition, and a NaCl-KCl cotransport system, dependent on metabolic energy and sensitive to furosemide inhibition. Genetic techniques have been used to modify the properties of the cells. The results suggest that the MDCK cell line has retained the differentiated properties of the kidney epithelial cells of origin and that a clonally isolated cell possesses the receptor, transmission, and target enzyme systems necessary for the regulation of transcellular salt and fluid transport.     

Two-dimensional manipulation of differentiated Madin-Darby canine kidney (MDCK) cell sheets: the noninvasive harvest from temperature-responsive culture dishes and transfer to other surfaces

A renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, adheres, spreads, and proliferates to confluency on our developed temperature-responsive culture dishes grafted with a poly(N-isopropylacrylamide) (PIPAAm) at 37 degrees C. In addition to other cell types, including hepatocytes and endothelial cells, MDCK cell sheets noninvasively were harvested from PIPAAm-grafted dishes merely by reducing the temperature. However, during the early stage of culture (up to 3 weeks), confluent MDCK cell detachment is greatly repressed. In the present study, we succeeded in the rapid harvest of confluent MDCK cell sheets and intact transfer to other culture dishes by utilizing hydrophilically modified poly(vinylidene difluoride) (PVDF) membranes as supporting materials. Immunocytochemistry with anti-beta-catenin antibody revealed that the functional cell-cell junctions were well organized in the transferred MDCK cell sheets. The viability assay showed that the transferred cells were not damaged during the two-dimensional cell-sheet manipulation. By transmission electron microscopy it was confirmed that the harvested MDCK cells retained differentiated phenotypes and had many microvilli and tight junctions at the apical and lateral plasma membranes, respectively. This two-dimensional cell-sheet manipulation technique promises to be useful in tissue engineering as well as in the investigation of epithelial cell sheets.     

Fiber density of electrospun gelatin scaffolds regulates morphogenesis of dermal-epidermal skin substitutes

Porous, nowoven fibrous gelatin scaffolds were prepared using electrospinning. Electrospun scaffolds with varying fiber diameter, interfiber distance, and porosity were fabricated by altering the concentration of the electrospinning solution. Solution concentration was a significant predictor of fiber diameter, interfiber distance, and porosity with higher solution concentration correlated with larger fiber diameters and interfiber distances. The potential of electrospun gelatin as a scaffolding material for dermal and epidermal tissue regeneration was also evaluated. Interfiber distances >5.5 microm allowed deeper penetration of human dermal fibroblasts into the scaffold, whereas cells in scaffolds with more densely packed fibers were able to infiltrate only into the upper regions. Scaffolds with interfiber distances 相似文献   

Excimer laser channel creation in polyethersulfone hollow fibers for compartmentalized in vitro neuronal cell culture scaffolds

Hollow fiber scaffolds that compartmentalize axonal processes from their cell bodies can enable neuronal cultures with directed neurite outgrowth within a three-dimensional (3-D) space for controlling neuronal cell networking in vitro. Controllable 3-D neuronal networks in vitro could provide tools for studying neurobiological events. In order to create such a scaffold, polyethersulfone (PES) microporous hollow fibers were ablated with a KrF excimer laser to generate specifically designed channels for directing neurite outgrowth into the luminal compartments of the fibers. Excimer laser modification is demonstrated as a reproducible method to generate 5microm diameter channels within PES hollow fiber walls that allow compartmentalization of neuronal cell bodies from their axons. Laser modification of counterpart flat sheet PES membranes with peak surface fluences of 1.2Jcm(-2) results in increased hydrophobicity and laminin adsorption on the surface compared with the unmodified PES surface. This is correlated to enhanced PC12 cell adhesion with increasing fluence onto laser-modified PES membrane surfaces coated with laminin when compared with unmodified surfaces. Adult rat neural progenitor cells differentiated on PES fibers with laser-created channels resulted in spontaneous cell process growth into the channels of the scaffold wall while preventing entrance of cell bodies. Therefore, laser-modified PES fibers serve as scaffolds with channels conducive to directing neuronal cell process growth. These hollow fiber scaffolds can potentially be used in combination with perfusion and oxygenation hollow fiber membrane sets to construct a hollow fiber-based 3-D bioreactor for controlling and studying in vitro neuronal networking in three dimensions between compartmentalized cultures.     

A novel configuration of bioartificial liver support system based on circulating microcarrier culture

The purpose of this investigation is to initiate a new bioartificial liver support system that utilizes circulating microcarrier cultures in the extracapillary space of a hollow fiber cartridge. The material exchange occurs on the membranes of the hollow fiber. Toxins are metabolized by the circulating cells on the microcarriers driven by a centrifugal pump. We inoculated 2-3 x 10E8 Hep G2 cells on 2.5 grams of Cytodex 3 microcarriers, and allowed them flowing in the extracapillary space of a modified plasma filter. 10% FCS Medium was pumped through the capillaries at different rates. Cells keep morphological integrity and functionality during the circulation. These preliminary results suggest that this configuration of a bioartificial liver support system offers a future investigation.     

Self-standing aligned fiber scaffold fabrication by two photon photopolymerization

Development of materials and fabrication techniques lead the growth of three-dimensional cell culture matrices in biomedical engineering. In this work, we present a method for fabricating self-standing fiber scaffolds by two-photon polymerization induced by a femtosecond laser. The aligned fibers are 330 μm long with a diameter of 6–9 μm. Depending on the pitch of the aligned fibers, various cell morphologies are distinguished via three-dimensional images. Furthermore, the morphologies of fibroblast cells (NIH-3T3) and epithelial cells (MDCK) on the fiber scaffolds are studied to show the effect of high curvature (3–4.5 μm radii) on cell morphology. NIH-3T3 cells that contain straight pattern of actin microfilament bundles are extended and partly wrap single fibers or tend to reside between fibers. On the other hand, MDCK cells that contain circular pattern of actin microfilament bundles cover the fiber peripheral surface exhibiting high aspect ratio elongation. These results indicate that cell morphology on fiber scaffolds is influenced by the pattern of actin microfilament bundles.     

Bioartificial liver with whole blood perfusion

In our series of studies, we have made an effort to develop a bioartificial liver (BAL) system through which whole blood can be perfused as in hemodialysis therapy. In this study, BAL cartridges containing porcine hepatocytes were prepared and perfused in an extracapillary space with human whole blood in vitro. Lidocaine loading tests were performed to evaluate the detoxification ability of the BAL. The clearance value of lidocaine decreased during the initial 6 hours to about 50% of the initial value. After that, it was almost stable until 48 hours. After 48 hours perfusion, thin sections of the hollow fibers containing hepatocytes were prepared and stained with hematoxylin-eosin and immunohistochemically stained for membrane attack complex (MAC). The porcine hepatocytes formed aggregates in the hollow fibers, nuclei in the cells were observed clearly, and MAC was not seen on the porcine hepatocyte aggregation. A hollow fiber that can reject 90% of molecules with molecular weight of 50 kDa effectively protects porcine hepatocytes from humoral immunity. The in vivo assessment of the BAL cartridge was performed using a canine model for 24 hours. No significant hemolysis or thrombus that affected the BAL system and the canine were observed. These results suggest that our BAL system is a promising liver assist device through which patients' whole blood can be perfused.     

Differentiated growth of human renal tubule cells on thin-film and nanostructured materials

Over 300,000 Americans are dependent on hemodialysis as treatment for renal failure, and kidney transplantation is limited by scarcity of donor organs. This shortage has prompted research into tissue engineering of renal replacement therapy. Existing bioartificial kidneys are large and their use labor intensive, but they have shown improved survival compared to conventional therapy in preclinical studies and an US Food and Drug Administration-approved phase 2 clinical trial. This hybrid technology will require miniaturization of hemofilters, cell culture substrates, sensors, and integration of control electronics. Using the same harvesting and isolation techniques used in preparing bioartificial kidneys for clinical use, we characterized human renal tubule cell growth on a variety of silicon and related thin-film material substrates commonly used in the construction of microelectromechanical systems (MEMS), as well as novel silicon nanopore membranes (SNMs). Human cortical tubular epithelial cells (HCTC) were seeded onto samples of single-crystal silicon, polycrystalline silicon, silicon dioxide, silicon nitride, SU-8 photoresist, SNMs, and polyester tissue culture inserts, and grown to confluence. The cells formed confluent monolayers with tight junctions and central cilia. Transepithelial resistances were similar between SNMs and polyester membranes. The differentiated growth of human tubular epithelial cells on MEMS materials strongly suggests that miniaturization of the existing bioartificial kidney will be feasible, paving the way for widespread application of this novel technology.     

Evaluation of Na+ active transport and morphological changes for bioartificial renal tubule cell device using Madin-Darby canine kidney cells

The function of current hemodialysis as an artificial kidney is insufficient because of the lack of reabsorptive function. In this study, we intend to develop a bioartificial renal tubule cell device (RTD) using tubular epithelial cells and artificial membranes and to evaluate the reabsorptive function of the confluent layers. Madin-Darby canine kidney (MDCK) cells were cultured on a nucleopore polycarbonate membrane for up to 4 weeks after confluence to examine the influence of the culture period on their ability to transport Na+ actively using Na+/K+ATPase (NKA). The results were (1) active Na+ transport of the cells averaged 24.8 mM/m(2) x 24 h during the initial 2 weeks after confluence and then decreased to about 4.2 mM/m(2) x 24 h during the next 2 weeks; (2) NKA localized on the basal-lateral sides of the cells during the initial 2 weeks, whereas it also localized on the apical side of the cells during the next 2 weeks; (3) long-term culture resulted in an increased number of upheaving cell mass, increased fatty droplets in the cells, and necrosis; and (4) scanning electron microscopy showed fewer microvilli 3-four weeks after confluence. It is concluded that the culture period is critical for developing RTD using cultured renal tubular epithelial cells.     

体外生物人工肝支持系统生物功能的体外研究

用２×１０８ 个混合聚集球形培养的人肝细胞、肝非实质细胞与中空纤维型生物反应器组成体外生物人工肝支持系统（ＥＢＬＳＳ）,通过循环液（ＰＲＩＭ １６４０）中肝细胞合成分泌产物的分析,评价其生物功能。结果表明,经过６ ｈ 的体外循环,循环液中总蛋白、白蛋白、甲胎蛋白和尿素含量均显著增加,该循环液使体外培养大鼠肝细胞ＤＮＡ 合成率显著增加（与对照组比较Ｐ＜ ００１）,实验后球形体肝细胞仍保持良好的活力。提示由培养人肝细胞和中空纤维型生物反应器组成的体外生物人工肝具备肝脏生物合成功能,并有刺激肝细胞增殖的能力。     

Morphologic studies of hepatocytes entrapped in hollow fibers of a bioartificial liver

A bioartificial liver cartridge was prepared by inoculating porcine hepatocytes into the inner space of hollow fibers of a hemodialyzer. The hepatocytes formed rod shaped cell aggregates during in vitro perfusion culture within 1 day. Morphologic examination was carried out on the aggregates by optical and electron microscopy. Each hepatocyte was in direct contact with adjacent cells and a bile canaliculus-like structure was occasionally seen between hepatocytes. High magnification observation showed that the canaliculus was separated from the remainder of the intercellular space by a tight junction. These facts suggest that the hepatocytes formed functionally associated cell aggregates with a compact morphology not unlike hepatocyte spheroids. These structures were well maintained for 7 days in culture, and then the amorphous area in the aggregates and the nonviable cell number increased with lengthening culture period. The bioartificial liver maintained the ability to metabolize lidocaine, ammonia, and galactose for 7 days and then deteriorated with time.     

Evaluation of proliferation and functional differentiation of LLC-PK1 cells on porous polymer membranes for the development of a bioartificial renal tubule device

To develop a bioartificial renal tubule system using renal tubular cells and porous polymer membrane hollow fibers, long-term maintenance of a confluent monolayer and the functionally differentiated condition of cells is essential. We examined the proliferation and functional differentiation of LLC-PK1 (Lewis-lung cancer porcine kidney 1) cells on two types of membranes: polysulfone and cellulose acetate. Cell proliferation was significantly higher on the polysulfone membrane than on the cellulose acetate membrane, and was enhanced by coating the membranes with various extracellular matrices. Confluent monolayer formation of cells was observed on matrix-coated polysulfone membrane but not on matrix-coated cellulose acetate membrane within 1 week. Cell proliferation continued for 3 weeks after confluent monolayer formation. Messenger RNA (mRNA) expression of glucose transporters, indicators of the functional differentiation of the LLC-PK1 cells, was observed in the polysulfone and cellulose acetate membrane groups, but was not observed in the nonporous polystyrene plate group under subconfluent conditions. Expression of glucose transporters mRNA was maintained for 3 weeks after confluent monolayer formation. Polysulfone membrane is more suitable than cellulose acetate membrane for a bioartificial renal tubule system with regard to LLC-PK1 cell proliferation. Extracellular matrix coating of the membrane further improves cell proliferation.     

The effect of poly (L-lactic acid) nanofiber orientation on osteogenic responses of human osteoblast-like MG63 cells

In this study, poly (L-lactic acid) (PLLA)/trifluoroethanol (TFE) solution was electrospun to fabricate fibrous scaffolds with different fiber orientations. Random and parallel PLLA nanofiber alignments were achieved by using a metal plate and a rolling rod as the receiver, respectively. The parallel PLLA fibrous scaffolds were further hot-stretched to obtain hyperparallel PLLA fibrous scaffolds. The PLLA fibrous scaffolds were characterized by fiber diameter, interfiber distance, fiber array angle, water contact angle, morphology and mechanical strength. The tensile strength of hyperparallel nano-fibers was approximately 5- and 14-times the parallel and random fibers, respectively. Osteoblast-like MG63 cells were cultured on the PLLA scaffolds to study the effects of fiber orientation on cell morphology, proliferation and differentiation. The cells on the randomly-oriented scaffolds showed irregular forms, while the cells exhibited shuttle-like shapes on the parallel scaffolds and had larger aspect ratios along the fiber direction of the hyperparallel scaffolds. Alkaline phosphatase (ALP) activity and collagen I (placeStateCol I) and osteocalcin (OC) deposition exhibited fiber orientation dependence. With an increase in parallelism of the fibers, there was a decrease in ALP activity and placeStateCol I and OC production. These results suggest that exploitation of PLLA fiber orientation may be used to control osteoblast-like cell responses.