Effects of mouse fetal liver cell culture density on hematopoietic cell expansion in three-dimensional cocultures with stromal cells.

OBJECTIVE: An effective ex vivo expansion system of primitive hematopoietic cells (HCs) is required for wider application of hematopoietic stem cell transplantation. In this study, we examined effects of culture density on mouse fetal liver cells (FLCs) used as an HC source for the expansion of primitive HCs in three-dimensional (3D) cocultures with two kinds of mouse stromal cell lines (OP9 or C3H10T1/2). MATERIALS AND METHODS: FLCs were seeded at different densities (1, 2, and 10 × 107 cells/cm3) into porous polymer scaffolds with or without stromal cell layers and HCs were expanded in the cultures for 2 weeks without exogenous cytokines. RESULTS: Differential effects of culture density on HC expansion were observed between cocultures and solitary FLC controls. In stromal cell cocultures, high expansion of HCs was achieved when FLCs were seeded at low densities. In contrast, the expansion in the controls was enhanced with increasing culture densities. With respect to expansion of primitive HCs existing in the FLCs, cocultures with C3H10T1/2 cells were superior to those with OP9 cells with a 29.3-fold expansion for c-kit+ hematopoietic progenitor cells and 8.3-fold expansion for CD34+ hematopoietic stem cells. In the controls, HC expansion was lower than in any cocultures, demonstrating the advantages of coculturing for HC expansion. CONCLUSION: Stromal cell lines are useful in expanding primitive HCs derived from FLCs in 3D cocultures. Culture density is a pivotal factor for the effective expansion of primitive HCs and this effect differs by culture condition.

Polyvinyl alcohol scaffolds and supplementation support 3D and sphere culturing of human cancer cell lines by reducing apoptosis and promoting cellular proliferation.

Three-dimensional (3D) culturing mimics the heterogeneous cellular conditions of the in vivo tumor microenvironment compared to 2D monolayer-cultured cells and 3D cultures of established cancer cell lines (sphere culture) or patient-derived cancer cells (organoid culture) are frequently used for cancer research or drug screening and evaluation. To establish more cost and time-efficient 3D culture methods for cancer cell lines, we supplemented sphere culture medium with polyvinyl alcohol (PVA) and found that 3D sphere cultures of breast and pancreatic cancer cell lines were significantly increased. Mechanistically, we found that PVA prevented cell death and promoted cellular proliferation while maintaining levels of stemness-related gene expression. Furthermore, we showed that polyvinyl formal resin (PVF) 3D scaffolds made by cross-linked PVA can function in serum-free, long-term 3D cultures to support maintenance of sphere- or tumor-like cell masses for diverse cancer cell types. Taken together, we demonstrate the effectiveness of PVA and PVF in human cancer cell line culture protocols.

Expansion of mouse primitive hematopoietic cells in three-dimensional cultures on chemically fixed stromal cell layers.

To establish a practical and convenient method to expand hematopoietic cells (HCs), we applied chemically-fixed stromal cell layers formed within three-dimensional (3D) scaffolds to feeder of HC cultures. The HCs were expanded using two successive cultures. First, stromal cells were cultured within porous polymer scaffolds and formed tissue-engineered constructs (TECs); the scaffolds containing stromal cells, were fixed using aldehyde (formaldehyde or glutaraldehyde) or organic solvents (acetone, methanol or ethanol). Second, mouse fetal liver cells (FLCs), as a source of HCs, were cultured on the TECs for 2 weeks, and the effects of fixative solutions on expansion of primitive HCs (c-kit+ and CD34+ cells) were examined. In the cultures on aldehyde-fixed TECs, primitive HCs were expanded 2.5- to 5.1-fold in the cultures on TECs fixed with glutaraldehyde, whereas no expansions were detected in those fixed with formaldehyde. However, we achieved expansion of primitive HCs > fivefold in the cultures using TECs fixed with organic solvents. Among these solvents, the highest expansions-of roughly tenfold-were obtained using acetone fixation. Ethanol-fixed TECs also supported the expansion of the primitive HCs well (6.6- to 8.0-fold). In addition to these sufficient expansions, the procedure and storage of fixed TECs is fairly easy. Thus, HC expansion on chemically-fixed TECs may be a practical method for expanding primitive HCs.

Growth and albumin secretion of mouse fetal liver cells cryopreserved within porous polymer scaffolds as a viable cell source for bioartificial livers.

To clinically apply bioartificial livers (BALs), an effective liver cell cryopreservation method is required for a stable cell supply. In this study, we performed tissue-engineered construct (TEC) cryopreservation of fetal liver cells (FLCs) in which FLCs cultured within a porous polymer scaffold were cryopreserved. Growth and albumin secretion in TEC-cryopreserved FLCs after thawing were compared to freshly isolated FLCs (control experiments). The effect of preculture duration prior to cryopreservation (0-3 weeks) on these functions was also examined. In the three-dimensional cultures, the TEC-cryopreserved FLCs with preculturing showed constant growth, and this growth was comparable to controls. On the contrary, the TEC-cryopreserved FLCs without preculturing did not proliferate after thawing. Albumin secretion of TEC-cryopreserved FLCs with preculturing rapidly increased up to day 12 and high secretory activity comparable to controls was maintained thereafter in FLCs with 1- or 2-week preculturing, suggesting this as an appropriate preculture duration. Compared to conventionally cryopreserved FLCs, growth and albumin secretion in the TEC-cryopreserved FLCs were significantly higher, indicating their usefulness as a potent cell source for BALs.

Expansion of mouse hematopoietic stem/progenitor cells in three-dimensional cocultures on growth-suppressed stromal cell layer.

With the aim of establishing an effective method to expand hematopoietic stem/progenitor cells for application in hematopoietic stem cell transplantation, we performed ex vivo expansion of hematopoietic stem/progenitor cells derived from mouse fetal liver cells in three-dimensional cocultures with stromal cells. In these cocultures, stromal cells were first cultured within three-dimensional scaffolds to form stromal layers and then fetal liver cells containing hematopoietic cells were seeded on these scaffolds to expand the hematopoietic cells over the 2 weeks of coculture in a serum-containing medium without the addition of cytokines. Prior to coculture, stromal cell growth was suppressed by treatment with the DNA synthesis inhibitor mitomycin C, and its effect on hematopoietic stem/progenitor cell expansion was compared with that in control cocultures in which fetal liver cells were cocultured with three-dimensional freeze-thawed stromal cells. After coculture with mitomycin C-treated stromal cells, we achieved a several-fold expansion of the primitive hematopoietic cells (c-kit+ hematopoietic progenitor cells >7.8-fold, and CD34+ hematopoietic stem/progenitor cells >3.5-fold). Compared with control cocultures, expansion of hematopoietic stem/progenitor cells tended to be lower, although that of hematopoietic progenitor cells was comparable. Thus, our results suggest that three-dimensional freeze-thawed stromal cells have higher potential to expand hematopoietic stem/progenitor cells compared with mitomycin C-treated stromal cells.

Design of antioxidative biointerface for separation of hematopoietic stem cells with high maintenance of undifferentiated phenotype.

During cell cultivation, excessively generated reactive oxygen species (ROS) affect cellular properties and functions. Although cell cultivation media contain several types of low-molecular-weight antioxidants, these small antioxidants are internalized into the mitochondria and they disrupt regulated redox balance. Here, we developed a novel biointerface that effectively eliminates ROS on a cell culture surface. Poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)aminomethylstyrene] (PEG-b-PMNT) was synthesized and covalently coated on a carboxyl group-activated culture dish using sec-amino groups on a PMNT segment followed by immobilization of anti-CD34 antibodies. CD34-positive hematopoietic stem progenitor cells (HSPCs) were separated from mice fetal liver cells using our polymer-coated cell culture dish. The separated HSPCs possessed intact mitochondrial membrane potential compared with those in the conventional cell cultivation system. In addition, the expression level of CD34 was maintained for an extended period on our culture dish with the antioxidative biointerface. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2080-2085, 2016.

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.

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.

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.

Spheroid array of fetal mouse liver cells constructed on a PEG-gel micropatterned surface: upregulation of hepatic functions by co-culture with nonparenchymal liver cells.

A spheroid array of fetal mouse liver cells, which comprise various immature cells, was constructed on a PEG-gel micropatterned surface and its hepatic activity and degree of differentiation induction were significantly upregulated by co-culture with nonparenchymal liver cells as feeder-cells.

Stimulating effects of fibroblast growth factors on hepatic function of fetal liver cells synergistically with oncostatin M in three-dimensional culture.

Fetal liver cells (FLCs) are regarded as a feasible cell source of bioartificial liver (BAL), because the FLCs have proliferating ability even in vitro. However, the cellular functions of FLCs are considerably lower compared with mature hepatocytes. Thus, maturation of cultured FLCs is essential for enhancing the performance of the BAL using the FLCs. In the present study, the effects of fibroblast growth factors (FGF-1, FGF-2, and FGF-4) on cell growth and the liver-specific functions of mouse FLCs were investigated in the presence or absence of oncostatin M (OSM), under both three-dimensional (3-D) and monolayer culture conditions. When FGF-2 was used, no stimulating effects on the albumin secretion activities of the FLCs were observed either in the 3-D or monolayer cultures, although cell growth was improved in these cultures. In the cases of FGF-1 and FGF-4, these factors also had no effect on the albumin secretion activities in the absence of OSM. However, in the presence of OSM, FGF-1 and FGF-4 significantly enhanced the activities of the FLCs but only in the 3-D cultures. From scanning electron microscopic observation, the 3-D culture FLCs formed big cell aggregates on the surface of a porous scaffold. In conclusion, it was clarified that FGF-1 and FGF-4 facilitate the maturation of 3-D culture FLCs synergistically with OSM.

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.

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.

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.

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.

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.

Effect of growth factors on ex vivo bone marrow cell expansion using three-dimensional matrix support.

To develop a culture system for bone marrow (BM) cell expansion, we examined the effect of growth factors (GFs) on the proliferation and differentiation of BM cells cultured in three-dimensional (3D) scaffolds of porous polyvinyl formal (PVF) resin. Murine BM cells were cultured for 2 weeks in the PVF resin or in culture dishes as a control, in the presence or absence of 4 GFs (erythropoietin, stem cell factor, interleukin [IL]-3, and IL-6). These GFs remarkably stimulated cell proliferation both in PVF and dish cultures. In addition, the PVF cultures showed enhanced cell proliferation in comparison with the corresponding dish cultures. Moreover, PVF cultures with GFs revealed the highest number of colony-forming units and the highest percentage of hematopoietic progenitor cells (HPCs) among all the cultures examined. Therefore, this 3D PVF culture system with GFs is considered as a potential alternative method for the ex vivo expansion of HPCs.