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.

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 × 10 cells/cm) into porous polymer scaffolds with or without stromal cell layers and HCs were expanded in the cultures for 2 weeks without exogenous cytokines.

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).

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).

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.

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.

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.

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.

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.

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.

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%).

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.

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.

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.

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.

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.

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.

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.

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.