Hirotoshi Miyoshi

Novel cell immobilization method utilizing centrifugal force to achieve high-density hepatocyte culture in porous scaffold

Cell seeding is one of the key procedures in the construction of tissue-engineered organs. In our previous efforts to create a bioartificial liver, high-density cultures of hepatocytes (>1 x 10(7) cells/1 cm(3)-substrate) and long-term maintenance of metabolic function were achieved with a packed-bed reactor utilizing porous poly(vinyl formal) (PVF) resin as a scaffold. However, a low seeding efficiency of about 30% remains a major obstacle to the scaleup of the reactor. In the present study, a new cell seeding method, centrifugal cell immobilization (CCI), which is based on alternating centrifugation and resuspension, was used to achieve high-density seeding and improve the seeding efficiency. Using the CCI method, the maximum density of the immobilized hepatocytes reached 3.8 x 10(7) cells/1 cm(3)-PVF, and the seeding efficiency was improved to about 43% after a relatively short immobilization process (about 15 min). Moreover, further improvement of the seeding efficiency was obtained by serial immobilization procedures. Thus, we concluded that this method is useful and effective for seeding cells into 3-dimensional scaffolds.

EFFECTS OF SHEAR STRESS ON METABOLIC FUNCTION OF THE CO-CULTURE SYSTEM OF HEPATOCYTE/NONPARENCHYMAL CELLS FOR A BIOARTIFICIAL LIVER

To improve the culture conditions of hepatocytes for use as a bioartificial liver, the effects of shear flow on the co-culture system of hepatocytes/nonparenchymal cells (NPC) were investigated. A flow chamber with a collagen coated rectangular glass plate, where hepatocytes (5 x 10(4) cell/cm2) and NPC (2 x 10(5) cell/cm2) were seeded, was used to attain a shear stress of 4.7 dyne/cm2. Concentrations of ammonia and urea in the medium were measured daily during the 2 week experiment. The metabolic activity of hepatocytes in the homotypic culture were lower than those of the co-culture, especially when the cultivation time exceeded 1 week. In addition, the applied shear flow promoted activity of the co-culture system. An enhancement in the rates of ammonium removal and urea synthesis was obtained in the perfusion systems. Morphologic observation revealed that aggregates of hepatocytes formed abundantly in the perfusion system and hepatocytes developed a cuboid shape. This suggested that perfusion affected the function and morphology of hepatocytes in the co-culture system. Shear flow could induce cell-cell interactions and secretion of extracellular matrix through the activation of NPC.

Long-term culture of fetal liver cells using a three-dimensional porous polymer substrate

To develop a bioartificial liver, long-term culture of fetal liver cells over a month’s time was performed under three different culture conditions, i.e., stationary cultures and shaken-flask cultures, both by using a substratum made of porous polyvinyl formal (PVF) resin and conventional monolayer dish cultures as controls. Time course changes in cell numbers and albumin secretion were evaluated in cultures using Williams’ E medium (WE) or minimum essential medium alpha (aMEM) supplemented with serum and hormones. In the WE medium, the numbers of fetal liver cells in all culture conditions gradually decreased with time, and albumin secretion rates rapidly decreased. In the stationary cultures using PVF, however, a significant increase in albumin secretion was observed after two weeks of culture. When cells were cultured in aMEM, the fetal liver cells exhibited sufficient proliferation in stationary and monolayer cultures, although albumin secretion rates per single cell were lower than those in WE. On the basis of these results, another series of culture experiments were performed, in which aMEM was used for the first 10 days to encourage cell proliferation, and the medium was changed to WE afterward. In these cultures, albumin secretion rates in the stationary cultures dramatically increased after the medium exchanges and were maintained at these high levels throughout the remaining culture period.

Improvement of Metabolic Performance of Hepatocytes Cultured In Vitro in a Packed-Bed Reactor for Use as a Bioartificial Liver

A packed-bed reactor using reticulated polyvinyl formal (PVF) resin as a support material is a useful configuration to achieve high density culture of hepatocytes for use as a bioartificial liver. The authors investigated the effects of oxygen concentrations of the culture medium on the metabolic performance of hepatocytes cultured in the reactor. A packed-bed reactor loaded with 250 PVF resin cubes (2 x 2 x 2 mm) was used. Hepatocytes obtained from male Wistar rats were inoculated into the reactor. Culture medium was perfused from the reservoir into the reactor through an oxygenator using a roller pump. Concentration of the dissolved oxygen in the medium was controlled by changing the gas mixture ratio supplied to the oxygenator. Hepatocytes cultured in the packed-bed reactor (cell density: 8.6 x 10(6) cells/cm3 PVF) under conditions of high dissolved oxygen concentrations ranging from 260 to 460 micromol/L showed 30% higher ammonium metabolic activity and 85% higher albumin secretion activity compared with those from the monolayer culture in the earlier culture stage (up to 2 days). However, low oxygen concentrations in the medium (<100 micromol/L) impaired activities of cultured hepatocytes.

Hepatocyte culture utilizing porous polyvinyl formal resin maintains long-term stable albumin secretion activity

To investigate the effects of culture conditions on the maintenance of metabolic functions of cultured hepatocytes, long-term hepatocyte culture lasting 20 days was performed under two different culture conditions, i.e. stationary cultures utilizing porous polymer (polyvinyl formal (PVF) resin) as a substratum and conventional monolayer dish cultures without PVF. Metabolic activities specific to hepatocytes were evaluated in terms of ammonia metabolism, urea synthesis, and albumin secretion. Concerning ammonia metabolic and urea synthetic activities, no significant differences in maintenance of these activities were found between the two culture conditions, and these activities rapidly decreased with the elapse of the culture period, especially during the early stage of the experiments. However, after day 10, these activities in the stationary cultures were maintained at a slightly more favorable level than in the monolayer cultures. On the other hand, compared with ammonia metabolism and urea synthesis, stable and well-maintained albumin secretion of hepatocytes (60% of the activity in day 1) was exhibited in the stationary culture experiments, despite that this particular activity under the monolayer culture condition gradually reduced to a very low level (5.7% of that on day 1) at the end of the culture. From the morphological observations, hepatocytes immobilized in the PVF resin revealed individual spherical shapes without forming multicellular aggregation, and it was suggested that this characteristic structure contributed to good albumin secretion of hepatocytes. In conclusion, the advantages of the hepatocyte culture technique utilizing PVF resin over the conventional dish culture in maintaining some representative metabolic function specific to hepatocytes were clarified.

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.

New type of matrix support for bone marrow cell cultures: in vitro culture and in vivo transplantation experiments.

A new type of bone marrow cell culture system was developed by using a highly porous substrate matrix, i.e., porous polyvinyl formal (PVF) resin. Murine bone marrow (BM) cells were cultured without the use of exogenous growth factors in a three-dimensional matrix support made of collagen coated porous PVF resin. To examine the optimal conditions for highest stromal cell density, short-term and long-term in vitro culture experiments using PVF were performed. In the short-term culture experiments, it was found that cubes of PVF (10 × 10 × 2 mm and 130 &mgr;m in pore size) coated with type I collagen with a seeding density of 2 × 107 BM cells offered the most appropriate culture conditions. In the long-term cultures, BM cells in PVF maintained their viability for up to 6 weeks. In another series of re-inoculation experiments, freshly isolated BM cells were inoculated onto the already developed stromal layer. In this study, a higher cell density of the stromal layer was obtained in the PVF culture compared with those in the control dish culture. Based upon the results of in vitro experiments, in vivo transplantation studies were also performed. Histologic examinations of the subcutaneously transplanted PVF with stroma revealed host derived hematopoiesis inside the PVF matrix. Moreover, survival of approximately 15% of the transplanted BM cells that were cultured in PVF were confirmed in X-ray irradiated recipients. From these results, it is suggested that PVF resin is a promising three-dimensional substrate for BM cell culture and that it can maintain hematopoietic stem cells or progenitor cells after transplantation.

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.

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 studys 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. Copyright