Cryopreservation of fibroblasts immobilized within a porous scaffold: effects of preculture and collagen coating of scaffold on performance of three-dimensional cryopreservation.

As a preliminary investigation to establish a cryopreservation method suited for bioartificial livers (BALs), three-dimensional (3-D) cryopreservation experiments with fibroblasts were performed, in which the cells were firstly seeded into a porous scaffold, and the scaffold containing the cells was then cryopreserved. After thawing, 65% of the initially applied cells were still attached to the scaffold, and this efficiency was significantly higher than that in the control experiments (39%), in which fibroblasts cryopreserved in a suspension were seeded into the scaffold. This higher efficiency was mainly caused by higher immobilization efficiency at the time of cell seeding (83%) than in the controls (54%). Collagen coating of the scaffold in the 3-D cryopreservation enhanced immobilization efficiency at the time of cell seeding, and 1-day precultures before the 3-D cryopreservation considerably improved cell growth after thawing. From these favorable results, this 3-D cryopreservation method may become useful for developing BALs.

Effects of oncostatin M on secretion of vascular endothelial growth factor and reconstruction of liver-like structure by fetal liver cells in monolayer and three-dimensional cultures.

Vascular endothelial growth factor (VEGF) is crucial for the development and regeneration of the liver. However, there have been no reports about VEGF secretion by cultured fetal liver cells (FLCs). In the present study, the effects of oncostatin M (OSM), which strongly stimulates the growth and albumin secretion of FLCs, on VEGF secretion and morphological changes of long-term cultured FLCs were investigated under three-dimensional (3-D) and monolayer conditions. The cultured FLCs proliferated well and showed stable secretion of VEGF for up to 1 month under both monolayer and 3-D culture conditions. The addition of OSM to cultured cells strongly enhanced VEGF secretion. Compared with 3-D cultures, VEGF secretion per cell was higher in monolayer cultures. After 1 month in culture, the FLCs in 3-D cultures formed large aggregates like liver tissue, and FLCs also formed colonies and duct-like structures after several months of culture even under monolayer conditions. In conclusion, OSM stimulated the secretion of VEGF by cultured FLCs, which seemed to contribute to the development of a liver-like structure.

Three-dimensional culture of porcine fetal liver cells for a bioartificial liver.

A three-dimensional (3-D) culture experiment of porcine fetal liver cells (FLCs) was performed using a porous resin substrate, for the purpose of developing a bioartificial liver. A long-term 3-D culture and monolayer culture as the control were performed for more than 1 month. To promote cell growth and maturation, human oncostatin M (OSM), the human leukemia inhibitory factor (LIF), or cortisol was added to the cultures, and the effect of each agent on cell proliferation and liver-specific cellular functions was investigated. The cell numbers in both the monolayer and 3-D cultures increased gradually with time, irrespective of the supplementation of the stimulating agents. In the monolayer culture, the albumin secretion of FLCs decreased rapidly, and scarce activity was detected from 2 weeks onward under all culture conditions tested. In the 3-D cultures, neither human OSM nor human LIF had any definite effect on the albumin secretion of FLCs. However, in the cultures with cortisol, albumin secretion was maintained for a considerably long period. These findings suggest that a bioartificial liver can be developed by culturing porcine FLCs with cortisol as the stimulant.

Engineering approaches to the microcirculation studies.

This review focused on a few methodologies which the author, with a background of chemical engineering, has developed in the physiological studies of microcirculation. (1) Fluorescent tracers to visualize mass transfer and hemodynamics: By means of a high sensitive SIT camera equipped in an intravital microscope system, dynamic processes of the permeation of a fluorescent dye from the microvessels through the extravascular space to lymphatics was made to be visualized. Dynamic behaviors of the formed elements were also quantitatively analyzed by the selective fluorescent labeling technique. (2) The dye/light method to induce platelet thrombus in vivo: Intravascular platelet aggregation and subsequent thrombus formation leading to the complete occlusion of the vessels were produced in the microvasculature by the irradiation of filtered light in combination with the intravascular administration of sodium fluorescein. This method enables quantitative evaluation of thrombus formation process in terms of thrombus formation times. Effects of hemodynamic parameters on thrombogenesis in vivo were quantitatively analyzed. (3) Establishment of peritoneal disseminated tumor model: Colon tumor cells (RCN-9) were inoculated into the peritoneal cavity of male Fischer rats, and the intravital microscopic observation of angiogenic vascular growth accompanying tumor growth was made possible. Dynamic behavior of leukocytes in the microcirculation of solid tumor tissue was visualized using a fluorescent labeling technique combined with the use of a real-time confocal laser-scanning microscope.

Effects of cryotherapy after contusion using real-time intravital microscopy.

PURPOSE: To examine effects of local tissue cooling on contusion-induced microvascular hemodynamics and leukocytes behavior using real-time intravital microscopy. METHODS: Male Wistar rats (N = 21, 130-150 g) were randomly assigned to intensive cooling group (3 degrees C, N = 7), a moderate cooling group (27 degrees C, N = 7), or control group (37 degrees C, N = 7). Contusion was induced by dropping a plastic ball on exposed cremaster muscle. After 5 min, the cremaster muscle was superfused with a saline solution for 10 min at controlled temperature of either 3 degrees C (cooling), 27 degrees C (moderate cooling), or 37 degrees C (control). Microvascular hemodynamics (vessel internal diameter, blood flow rate and erythrocyte velocity) and leukocyte behavior (rolling and adhesion) were measured from recorded videotapes in the same venules before and after contusion, and after cooling. RESULTS: Cooling-induced vasoconstriction was marked at 3 degrees C and moderate at 27 degrees C compared with that at 37 degrees C. Blood flow rate and erythrocyte velocity were markedly lower at 3 degrees C compared to 37 degrees C. At 27 degrees C, erythrocyte velocity was higher than that at 37 degrees C, but blood flow rate was maintained at a level similar to that at 37 degrees C. The number of rolling and adhering leukocytes at 3 degrees C and 27 degrees C were significantly less than at 37 degrees C. CONCLUSION: Our results suggest that local tissue cooling, similar to cryotherapy, improves edema and inflammatory reaction, and may be useful for reducing inflammatory response without inhibiting blood flow after contusion.

Effects of electroacupuncture stimulation applied to limb and back on mesenteric microvascular hemodynamics.

The effects of electroacupuncture stimulation (EAS) of the hind paw and the back on the mesenteric microhemodynamics in anesthetized rats were investigated using an intravital microscope system. Red blood cell (RBC) velocity in the mesenteric arterioles was measured by the dual-sensor method developed by the authors. Electrical stimulation was applied using two acupuncture needles inserted into the skin and the underlying muscles of the hind paw and the dorsal Th13-L1 level area. The hind-paw EAS evoked intensity-dependent pressor responses and increase responses in RBC velocity in mesenteric precapillary arterioles, while the back EAS evoked depressor responses and decrease responses in RBC velocity. Heart rate showed increase responses accompanying EAS either on the hind paw or the back. The pressor responses and increase responses in RBC velocity in mesenteric precapillary arterioles accompanying the hind paw EAS were abolished by an intravenous administration of alpha-adrenergic receptor antagonist (phenoxybenzamine; POB), and the tachycardiac responses were abolished by administration of beta-adrenergic receptor antagonist (propranolol). Occasional but notable reflex vasoconstrictions in the mesenteric terminal arteriole were induced by EAS either on the hind paw or the back. These vasoconstrictive responses were not affected by the administration of POB. The present study directly demonstrated that hemodynamic changes at the level of precapillary arterioles accompanying EAS either on the hind paw or the back mainly depend on the changes of systemic arterial pressure regardless of stimulus current intensities. Moreover, the results in the present study suggest some receptors other than alpha-adrenergic receptor might be involved in the mechanism of EAS-induced vasoconstriction in the mesenteric arteriole.

Expansion of mouse hematopoietic progenitor cells in three-dimensional cocultures on frozen-thawed stromal cell layers formed within porous scaffolds.

To establish a highly efficient method of ex vivo expansion of hematopoietic cells (HCs), three-dimensional (3D) cocultures of HCs and stromal cell lines were performed using porous polymer scaffolds. Hematopoietic cells derived from mouse fetal livers were expanded by two successive cultures without the use of exogenous cytokines, namely, 3D cultures of stromal cells (DAS 104-8 cell line) to form stromal layers within the scaffolds, and, subsequently, by cocultures of the HCs on the stromal cell layers for 2 weeks. To expand the HCs more conveniently, in some experiments the stromal layers formed within the scaffolds were frozen (3D freezing) before the cocultures, then stored and applied to the cocultures after thawing. When the HCs were cocultured on the stromal layers of the DAS 104-8 cells, primitive HCs (c-kit(+) and CD34(+) cells) were expanded several fold during the cocultures. In contrast, the expansion of these primitive HCs was remarkably enhanced in the cocultures using the 3D frozen-thawed DAS 104-8 stromal layers (c-kit(+) cells > fifteenfold and CD34(+) cells > thirtyfold), and these expansions were significantly higher than those without the 3D freezing. The expansions enhanced by cocultures on the 3D frozen-thawed stromal layers were also observed in the cocultures with another stromal cell line (DAS 104-4). Because 3D frozen-thawed stromal cell lines are easy to handle, 3D coculture of HCs on frozen-thawed stromal cell lines may be an effective and convenient method for expanding primitive HCs.

Especially polymorphonuclear leukocytes, but also monomorphonuclear leukocytes, roll spontaneously in venules of intact rat skin: involvement of E-selectin.

White blood cells roll spontaneously in venules of intact, noninflamed rat skin. We investigated noninvasively in two experimental series which leukocyte subtypes participate in this phenomenon and the possible involvement of E-selectin. Male Lewis rats were anesthetized with sodium pentobarbital, and intravital video microscopy was performed on postcapillary venules in the nail-fold of a hind leg. In series 1 acridine yellow was infused for 15 min (50 mg per kg intravenously) to stain the leukocyte nuclei in situ. With the use of fluorescence microscopy rolling leukocytes could be classified unequivocally as polymorphonuclear (granulocytes) or monomorphonuclear (lymphocytes/monocytes) by the shape of their nucleus. Irrespective of vessel depth beneath the skin surface (25-45 microm), most identified rolling leukocytes were classified as granulocytes (72%-100%; median 89%). This percentage was independent of total rolling leukocyte flux, systemic leukocyte count, or their in vitro differentiation pattern. In series 2, rats were treated with either a synthetic, highly selective E-selectin blocking peptide or a control peptide (intravenously, 12 mg peptide per kg bolus, followed by 50 mg per kg per h). E-selectin blockade significantly reduced the leukocyte rolling level to about 50% of baseline (p <0.01), whereas the rolling velocity increased (p <0.01); the control peptide had no effect. In summary, most of the leukocytes rolling spontaneously in postcapillary venules of intact rat skin are granulocytes, despite the absence of an acute inflammatory reaction. One of the adhesion molecules involved in this phenomenon is E-selectin.

Perfusion of medium with supplemented growth factors changes metabolic activities and cell morphology of hepatocyte-nonparenchymal cell coculture.

To develop a feasible perfusion-type bioartificial liver device, perfusion of hepatocyte-nonparenchymal cell (NPC) cocultures with medium supplemented with hepatocyte growth factor (HGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) was carried out. On day 1 of culture, perfusion at a constant shear stress of 1.3 dyn/cm2 enhanced ammonia metabolic and urea synthetic activities of hepatocytes. These enhanced activities were sustained up to day 7 only when growth factors were present. In contrast, no beneficial effects of growth factors on these activities were observed in static cultures. In perfusion cultures, three-dimensional cell aggregates were formed. On the surface of these aggregates, flattened cell layers composed mainly of NPCs were found, and the central cluster of cell aggregates was composed of round-shaped hepatocytes and reticulin fibrils. These observations strongly suggested that the reconstruction of different types of liver cells and connective tissues formed tissue-mimicking cell aggregates in the perfusion culture that was able to modulate the liver-specific functions of hepatocytes. Thus, perfusion culture conditions of the hepatocyte--NPC coculture system should be appropriately designed to induce suitable reconstruction of the cultured cells for use as a bioartificial liver device.

Vasodilatation of muscle microvessels induced by somatic afferent stimulation is mediated by calcitonin gene-related peptide release in the rat.

In anesthesized rats, the effects of electrical stimulation (ES) to the saphenous nerve on the microcirculation of the gracilis muscle were assessed through the measurement of two different hemodynamic parameters: (a). the muscle blood flow (MBF) using a laser Doppler flowmeter; and (b). the changes in diameter of the muscle arterioles observed directly using an intravital microscope system. Ipsilateral ES (5 V, 20 Hz, for 30 s) produced increases in MBF and mean arterial pressure (47+/-10% and 18+/-5%) over the baseline, while no significant changes in MBF were observed in the contralateral muscle. Neither selective nor simultaneous alpha- and beta-adrenergic blockade altered the increases in MBF induced by ipsilateral ES. The arteriolar diameter was found to increase by 38.9+/-5% following ipsilateral ES. This response in diameter was abolished after the topical application of a calcitonin gene-related peptide receptor antagonist (CGRP(8-37)). Contralateral ES produced a decrease in arteriolar diameter by 26+/-14%. Thus, ipsilateral nerve ES produced vasodilative responses in the muscle accompanied by increases in MBF independently of the sympathetic activity. Furthermore, CGRP was found directly involved in the reflex neural regulation of the muscle microcirculation, which suggests the participation of an axon reflex mechanism.

Electro-acupuncture stimulation to muscle afferents in anesthetized rats modulates the blood flow to the knee joint through autonomic reflexes and nitric oxide.

Recent reports have focused on the mechanisms of the action of electro-acupuncture stimulation (EAS) in the regulation of blood flow to different tissues. In the knee joint, blood flow is known to be modulated mainly by sympathetic postganglionic fibers, but recently the release or induction of nitric oxide (NO) synthesis in response to electrical stimulation has also been suggested. Therefore, a direct observation of the microcirculation is needed to further understand the mechanism by which blood flow is regulated by somatic afferent stimulation. In the present study, the effects of EAS to the vastus medialis muscle on systemic hemodynamics and the knee joint microcirculation were observed in vivo using a real-time confocal laser-scanning microscope system (CLMS). Electrical stimulation (5 mA, 0.5 ms, 5 Hz) was applied for 30 min using a pair of acupuncture needles introduced into the vastus medialis muscle. To clarify a plausible involvement of NO in the responses to EAS, the stimulus was applied either in the presence or absence of N(omega)-nitro-L-arginine methyl ester (L-NAME). Stimulation to either the muscle or the skin of the thigh after blockade of neuromuscular transmission was performed to determine the involvement of muscle contraction during EAS treatment. The changes in mean arterial pressure (MAP) and diameter of the arterioles supplying the knee joint were monitored continuously until 60 min poststimulus. Significant and persistent increases in arteriolar diameter by 26 +/- 6% and MAP by 17 +/- 2%, respectively, were observed after EAS to the muscle. Electro-acupuncture to the vastus medialis in the presence of L-NAME produced a strong decrease in diameter of the knee joint arterioles by -38 +/- 14% under the baseline with a simultaneous increase of 35 +/- 5% in MAP. EAS to the skin did not produce changes in arteriolar diameter while a slight increase in MAP by 12 +/- 6% over the baseline occurred after the stimulus. EAS to the muscle after neuromuscular blockade did not produce significant changes in diameter, while an increase in MAP by 24 +/- 8% was still observed, which facts suggest that the muscle contraction is required to produce vasodilatation. These responses suggest that a dynamic balance between the autonomic nervous system and the release of NO is the primary mechanism mediating the EAS effects on knee joint microcirculation.

Effects of electrical stimulation of the dorsal skin on systemic and mesenteric microvascular hemodynamics in anesthetized rats.

The effects of electrical stimulation of the dorsal skin area on the mesenteric arterioles were investigated in anesthetized rats by the use of an intravital microscope-television system. Changes in the diameter of the mesenteric precapillary arterioles (10-40 microm in diameter) were measured with an image processor. Blood flow velocity in the mesenteric precapillary arterioles was monitored by the dual sensor method developed by the authors. Electrical stimulation was performed through two platinum electrodes placed at the right dorsal Th5-12 level skin area by the use of an electrical stimulator (0.2 ms, 20 Hz). Continuous stimulation lasting for 30 s (1-10 mA) and intermittent stimulation lasting for 10 min (3 mA) were applied. The pressor response following the depressor response was induced by a stimulus current above 8 mA. The decrease in mesenteric blood flow velocity was induced by stimulus current above 10 mA. These responses were abolished by lidocaine injection into the subcutaneous area where the electrodes were attached. No significant change in arteriolar diameter or heart rate were induced by the stimulation for 30 s. Electrical stimulation of the skin for 10 min evoked a decrease in the diameter of arterioles (-3.4 +/- 2%, p < 0.01, n = 12). In the adrenalectomized group, electrical stimulation of the skin for 10 min elicited a slight increase in the diameter (1.1 +/- 0.5%, n = 6). It is therefore suggested that electrical stimulation of the skin for 30 s reflexly evoked decreases in MAP and in blood flow velocity, and that the constriction of the mesenteric precapillary arterioles induced by the stimulation for 10 min was mediated by humoral adrenaline and noradrenaline released by somato-adrenal medullary reflex.

Leukocyte behavior in angiogenic vessels is affected by tumor-derived nitric oxide.

Recent studies indicate a possible role of nitric oxide (NO) in regulating leukocyte-endothelial cell interactions, which plays a key role in the tumor immunity. The purpose of the present study is aimed to observe the tumor hemodynamics intravitally and to clarify the effect of NO on tumor microcirculation by means of a real-time confocal laser-scanning microscope using NO-reactive indicators. Visualization of localization of NO and the leukocyte behavior was made in the mesenteric microvessels of an experimental tumor model rat. Production of NO was clearly visualized along the endothelium of the tumor-free rats, but scarcely found in the newly formed tumor microvessels. A higher level of NO production was observed in a solid tumor region, where a more marked decrease in the leukocyte-endothelial cell interactions was observed. Local administration of a NO synthase (NOS) inhibitor increased leukocyte adhesion. This indicates that tumor-derived NOS creates the tumor specific microenvironment of the immature angiogenic tumor vessels, thereby modulating leukocyte behavior.

Endovascular stent configuration affects intraluminal flow dynamics and in vitro endothelialization.

Neointimal hyperplasia influenced by intravascular hemodynamics is considered partly responsible for restenosis after endovascular stenting. To evaluate the effect of stent configuration on fluid flow behavior, we visualized flow near stents, and measured the proliferation of cultured endothelial cells (ECs). A single-coil stent (coil pitch; CP = 2.5, 5, or 10 mm) was inserted into a glass tube and perfused at 30-90 ml/min, while the flow pattern was determined by particle imaging velocimetry. The reduction of the flow velocity near the wall was correlated with the decrease in the coil interval of the stent. In perfusion cultures with stents, the proliferation of ECs was influenced by the local flow velocity distribution. When a stent with a CP value of 10 mm was used, the doubling time of ECs was 30.7 h, while the doubling time was 38.5 h when the CP was 5 mm. The doubling time of ECs was shorter at sites upstream of the stent wire where the velocity was higher than downstream of the wire. In conclusion, a single-coil stent can be used to modify hemodynamic factors, suggesting that improved stent design may facilitate rapid endothelialization after stent implantation.

Three-dimensional culture of mouse bone marrow cells on stroma formed within a porous scaffold: influence of scaffold shape and cryopreservation of the stromal layer on expansion of haematopoietic progenitor cells.

This study's primary goal was to develop an effective ex vivo expansion method for haematopoietic cells. 3D culture of mouse bone marrow cells was performed in porous scaffolds using a sheet or cube shape. Bone marrow cells were cultured on bone marrow-derived stromal layers formed within the scaffolds and the effect of scaffold shape on the expansion of haematopoietic cells was examined. In some experiments, stromal layers within cubic scaffolds were frozen and then used to culture bone marrow cells after thawing. Results show that after comparison, total cell density and expansion of haematopoietic cells were greater in cultures using the cubic scaffold, suggesting that it was superior to the sheet-like scaffold for expanding haematopoietic cells. When cryopreserved stroma was used, it effectively supported the expansion of haematopoietic cells, and a greater expansion of haematopoietic cells [(erythroid and haematopoietic progenitor cells (HPCs)] was achieved than in cultures with stromal cells that had not been cryopreserved. Expansion of cells using cryopreserved stroma had several other advantages such as a shorter culture period than the conventional method, a stable supply of stromal cells, and ease of handling and scaling up. As a result, this is an attractive method for ex vivo expansion of haematopoietic stem cells (HSCs) and HPCs for clinical use.

Three-dimensional culture of mouse bone marrow cells within a porous polymer scaffold: effects of oxygen concentration and stromal layer on expansion of haematopoietic progenitor cells.

To establish an ex vivo expansion method of haematopoietic progenitor cells (HPCs) and erythroid cells, three-dimensional (3D) cultures of mouse bone marrow cells were performed, employing a porous polyvinyl formal (PVF) resin as a scaffold. In these cultures, the effects of oxygen concentration and co-cultures with stromal cells on the expansion of HPCs and erythroid cells were investigated. When bone marrow cells were cultured under 3D conditions, HPCs and erythroid cells expanded without supplementation of exogenous cytokines, irrespective of the presence of stromal cells. On the contrary, slight expansion of HPCs or erythroid cells was observed in monolayer cultures as controls, indicating that the 3D cultures using the PVF scaffold were far better in expanding HPCs and erythroid cells than the monolayer cultures. Under hypoxic conditions, bone marrow stromal cells allowed for a 3D culture of erythroid cells and HPCs at higher cell densities compared to cultures without stromal cells, and the duration of the expansion of HPCs and erythroid cells after initiating the 3D co-cultures was prolonged. The number of these cells increased throughout the culture period up to 3 weeks under hypoxic conditions, although the number decreased after 2 weeks under normoxic conditions. In conclusion, the 3D co-culture method of haematopoietic cells with stromal cells under hypoxic conditions was confirmed to be effective in expanding HPCs and erythroid cells, and this method seemed to be useful for developing an ex vivo expansion method for haematopoietic cells.

Three-dimensional perfusion cultures of mouse and pig fetal liver cells in a packed-bed reactor: effect of medium flow rate on cell numbers and hepatic functions.

To develop a tissue-engineered bioartificial liver (BAL), perfusion cultures of mouse and pig fetal liver cells (FLCs) immobilized within a three-dimensional (3D) porous scaffold were performed utilizing a packed-bed reactor system. These FLCs were cultured under different medium flow rate conditions, and the effects of the flow rates on cell growth and the hepatic functions of the FLCs were investigated. In the cultures of mouse FLCs, the medium flow suppressed cell growth and the albumin secretion activity of the FLCs, and considerably lower albumin secretion than that in the 3D stationary control cultures was obtained in the perfusion cultures. In the case of pig FLCs, cell growth was also inhibited by the medium flow, however, the cells exhibited higher tolerance to the medium flow compared with mouse FLCs. The albumin secretion activity of pig FLCs was well maintained under an extremely low flow rate condition (4.8 mm/min in the reactor), and activity higher than the 3D stationary cultures was detected at a later stage (after 20 days in the perfusion cultures). These results revealed that FLCs are quite sensitive to medium flow and an extremely low shear stress is required for the perfusion cultures of FLCs.

Efficient proliferation and maturation of fetal liver cells in three-dimensional culture by stimulation of oncostatin M, epidermal growth factor, and dimethyl sulfoxide.

For the purpose of applying fetal liver cells (FLCs) as a cell source to tissue-engineered bioartificial livers, three-dimensional (3-D) cultures of FLCs using a porous polymer scaffold, as well as monolayer cultures as a control, were simultaneously performed. To achieve efficient growth and differentiation, the FLCs were cultured in the growth medium for the first 3 weeks and then cultured in the differentiation medium for 3 more weeks. In these cultures, stimulating factors (oncostatin M (OSM), epidermal growth factor (EGF), hepatocyte growth factor (HGF), or dimethyl sulfoxide (DMSO)) were added to the media, and their effects were examined. When the growth medium containing OSM and EGF was used, EGF stimulated the growth of FLCs synergistically with OSM. For the differentiation of FLCs into mature hepatocytes, DMSO added to the differentiation medium remarkably enhanced albumin secretion in the 3-D and monolayer cultures, although HGF was effective only in the monolayer culture. Microscopic observation proved that FLCs exhibited hepatocyte-like morphology only in the media containing DMSO. In conclusion, successive supply of the growth medium containing EGF and OSM and the differentiation medium containing DMSO efficiently induced the growth of the 3-D cultured FLCs and their differentiation into mature hepatocytes.

Oncostatin M stimulates proliferation and functions of mouse fetal liver cells in three-dimensional cultures.

In order to develop a tissue engineered bioartificial liver (BAL), long-term three-dimensional (3-D) culture of fetal liver cells (FLCs) utilizing porous polymer as a scaffold was performed for up to 1 month. The effects of the basal medium and supplementation with oncostatin M (OSM) on the proliferation and differentiation of mouse FLCs were examined in both 3-D culture and conventional monolayer dish culture. Compared with monolayer culture, cell numbers and hepatic function of FLCs were better maintained by 3-D culture. When two kinds of basal media were tested in this study, Williams' medium E (WE) was superior to minimum essential medium alpha (alphaMEM) in expressing hepatic function of FLCs in both 3-D and monolayer cultures, although higher cell densities were obtained with alphaMEM. OSM potently stimulated both cell growth and metabolic activity, especially in 3-D culture. When WE supplemented with OSM was used for 3-D culture, albumin secretion by FLCs increased dramatically after day 5, and a high level of secretion was maintained until the end of culture. During a period of over 1 month, no decrease of albumin secretion was observed. In conclusion, this 3-D culture method was expected to be one of the realistic attempts to develop a tissue engineered BAL.

Chondroinduction of mouse mesenchymal stem cells in three-dimensional highly porous matrix scaffolds.

Porous polyvinyl formal (PVF) resin and poly(lactide-caprolactone) [P(LA/CL)] sponges were examined as three-dimensional matrices for chondroinduction of cultured bone marrow mesenchymal stem cells (MSCs). Approximately 5 x 10(5) mouse MSCs were seeded in porous PVF resin or P(LA/CL) sponges and were cultured for up to 1 month in serum-free high-glucose Dulbecco's modified Eagle's medium containing 10 ng/mL transforming growth factor-beta3 and 100 nM dexamethasone for chondroinduction. After the 1-month culture period, the PVF resin and P(LA/CL) sponges contained approximately twice the amount of glycosaminoglycans compared with the control pellet. Safranin-O staining of PVF and P(LA/CL) after 1 month of culture revealed a cartilage-like extracellular matrix containing glycosaminoglycans and collagen. When implanted into nude mice, PVF and P(LA/CL) seeded with MSCs were found to be both biocompatible and chondroinductive. These highly porous scaffolds can maintain a large number of cells in a three-dimensional structure. Both are potentially promising for the chondroinduction of bone marrow MSCs for research and clinical applications.