Availability of bone marrow stromal cells in three-dimensional coculture with hepatocytes and transplantation into liver-damaged mice

Rat bone marrow stromal cells (BMSCs) were cultured in porous hydroxyapatite (HA) disks for 2 weeks to form a cell layer on the surface. Freshly isolated hepatocytes were then inoculated into both BMSC-cultured and non-treated HA disks. Hepatocytes cocultured with BMSCs secreted significantly more albumin than those in monoculture in vitro. The cell-packed HA disks were implanted into the peritoneal cavity of Nagase analbuminemia rats (NARs), and 4 weeks later, blood samples were collected to measure the albumin concentration. The cotransplantation of BMSCs with hepatocytes significantly increased the serum albumin concentration in NARs. The HA disks coculturing mice hepatocytes and BMSCs were also implanted into mice, in which liver damage had been induced using carbon tetrachloride and phenobarbital. The decreased serum albumin level in liver-damaged mice was completely recovered by the transplantation of hepatocytes and BMSCs. The serum level of IL-6 in liver-damaged mice was also increased by the cotransplantation of BMSCs and hepatocytes. Thus, the transplantation of BMSCs appears to have a systemic effect on recipients through the increase in the serum cytokine level as well as a local effect on cotransplanted hepatocytes.     

The mixed coculture effect of primary rat hepatocytes and bone marrow cells is caused by soluble factors derived from bone marrow cells

Heterospheroids consisting of hepatocytes and bone marrow cells (BMCs) are formed by the mixed coculture of these cells and enhance the expression and maintenance of the liver-specific functions of hepatocytes. Not only the soluble factors derived from these cells, but also functional organoid (heterospheroid) formation, are considered to underlie this coculture effect. Therefore, in the present study, we aimed to clarify the mechanism of this co-culture effect. We performed hepatocyte monoculture with conditioned media prepared from hepatocyte cultures, BMC cultures and a coculture of hepatocytes and BMCs. When using any type of conditioned medium, no hepatocyte spheroids formed, and the hepatocytes formed a monolayer. In addition, an effect for these conditioned media was shown in terms of the albumin production and ammonia metabolism activities of the hepatocytes; conditioned medium from BMCs showed the strongest effect. The monocultured hepatocytes in the conditioned medium derived from BMCs showed equivalent albumin production and ammonia metabolism activities to the cocultured spheroids of hepatocytes and BMCs. Therefore, it was determined that the effect of the coculture of hepatocytes and BMCs was caused by soluble factors derived from BMCs.     

Mag-seeding of rat bone marrow stromal cells into porous hydroxyapatite scaffolds for bone tissue engineering

Bone tissue engineering has been investigated as an alternative strategy for autograft transplantation. In the process of tissue engineering, cell seeding into three-dimensional (3-D) scaffolds is the first step for constructing 3-D tissues. We have proposed a methodology of cell seeding into 3-D porous scaffolds using magnetic force and magnetite nanoparticles, which we term Mag-seeding. In this study, we applied this Mag-seeding technique to bone tissue engineering using bone marrow stromal cells (BMSCs) and 3-D hydroxyapatite (HA) scaffolds. BMSCs were magnetically labeled with our original magnetite cationic liposomes (MCLs) having a positive surface charge to improve adsorption to cell surface. Magnetically labeled BMSCs were seeded onto a scaffold, and a 1-T magnet was placed under the scaffold. By using Mag-seeding, the cells were successfully seeded into the internal space of scaffolds with a high cell density. The cell seeding efficiency into HA scaffolds by Mag-seeding was approximately threefold larger than that by static-seeding (conventional method, without a magnet). After a 14-d cultivation period using the osteogenic induction medium by Mag-seeding, the level of two representative osteogenic markers (alkaline phosphatase and osteocalcin) were significantly higher than those by static-seeding. These results indicated that Mag-seeding of BMSCs into HA scaffolds is an effective approach to bone tissue engineering.     

Effect of temperature on cell population balance in Dexter's culture of murine bone marrow hematopoietic cells with stromal cells

The effect of temperature in the range from 25 to 37 degrees C on the population balance of stromal and multiple-lineage hematopoietic cells from murine bone marrow at various stages of differentiation in Dexter's culture was investigated. The length of time required for stromal cells to reach confluence after inoculation of both cell types harvested from murine bone marrow was shorter at higher temperatures. On the other hand, the hematopoietic cell concentration initially decreased more rapidly at higher temperatures until the confluence of stromal cells, which should then support the proliferation of hematopoietic cells. The concentration of hematopoietic cells began to increase after 2 weeks incubation at 33 and 37 degrees C and after 4 weeks at 29 degrees C. However, the growth of hematopoietic cells was not stable at 37 degrees C, and neither stromal nor hematopoietic cells showed significant growth at 25 degrees C. The specific growth rate of hematopoietic cells at 29 degrees C after 4 weeks was comparable or higher than that at 33 degrees C, while the final concentration of hematopoietic cells was maximal at 33 degrees C. Using a cultivation method in which hematopoietic cells were recharged onto a confluent layer of stromal cells prepared at 33 degrees C, the maintenance and growth of hematopoietic cells were better at 29 degrees C than at higher temperatures. The content of progenitor cells among the hematopoietic cells increased prior to the increase in the hematopoietic cell concentration, and the progenitor cell was greater content at the lower temperature. These results suggest that cultivation at 29 degrees C might be superior to 33 degrees C for the maintenance and growth of hematopoietic cells with a prepared confluent layer of stromal cells.     

Adipogenesis inhibitory effects of Limonium tetragonum in mouse bone marrow stromal D1 cells

Encapsulation of Lactobacillus rhamnosus was performed using spray and freeze-drying. Maltodextrin and gum arabic were used in different combinations for spray-drying. Values of 50% maltodextrin and 40% gum arabic gave best results. Spray-drying was done at temperatures ranging from 110 to 150oC. Survivability, acid tolerance, antibiotic sensitivity testing, and total anthocyanin content and physical properties of moisture content, water activity, color analysis, bulk density, and tap density were analyzed. The moisture content of encapsulated powders ranged from 6.51 to 7.72% (wet basis) and bulk density and tap density values ranged from 0.334 to 0.308 g/cm³ and 0.350 to 0.330 g/cm³, respectively. Total anthocyanin contents were 19.28 and 7.264 mg/100 mL, respectively, for freeze and spray-dried powders. Freeze-dried probiotic pomegranate juice powder yielded best results with high survivability of Lactobacillus rhamnosus, a high total anthocyanin content, and other properties.     

Ex vivo expansion of human cord blood hematopoietic progenitor cells using glutaraldehyde-fixed human bone marrow stromal cells

Human stromal cells were immortalized and fixed with glutaraldehyde to support an ex vivo expansion of human cord blood hematopoietic progenitor cells. In addition, this enabled glutaraldehyde-fixed stromal cells to be stored at 4 degrees C. Although freeze-dried glutaraldehyde-fixed stromal cells did not increase the number of the progenitor cells, the percent decrease in the number of CD34(+) cells in the presence of freeze-dried glutaraldehyde-fixed stromal cells was less than that in the absence of the stromal cells. Thus, glutaraldehyde-fixed stromal cells can serve as a stabilizing device for hematopoietic cell expansion.     

Effect of concentrations of murine stromal and hematopoietic cells on the progenitors expansion in their three-dimensional coculture in nonwoven fabrics

The effects of concentrations of murine stromal and hematopoietic cells on their three-dimensional coculture in nonwoven fabrics were investigated for the construction of an ex vivo expansion system for hematopoietic progenitors without adding expensive cytokines. The increase in initial stromal cell concentration from 0.4 x 10(5) to 2.7 x 10(5) cells/ml increased progenitor concentration, while the increase in initial hematopoietic cell concentration from 0.6 x 10(5) to 6.1 x 10(5) cells/ml decreased the fold increase in hematopoietic cell concentration.     

In vitro proliferation of primary rat hepatocytes expressing ureogenesis activity by coculture with STO cells

A coculture system for in vitro proliferation of rat primary hepatocytes with feeder cells was investigated with the view of constructing a hybrid artificial liver module using human hepatocytes. A mouse cell line, STO, was apparently effective compared with other kinds of feeder cells such as FLS5 and MRC5 in increasing the total cell number in the coculture of rat primary hepatocytes. The parenchymal hepatocyte, which are polygonal cells, spread well as the cultivation progressed. Monitoring of parenchymal hepatocyte colonies under a microscope showed that the area of each colony increased as the single culture and cocultures progressed. The adhesion area per hepatocyte increased little during the coculture with STO cells, while the adhesion area increased markedly in the single culture of hepatocytes. The density of hepatocytes increased more than two times during the coculture for 5 d, while it decreased during the single culture. The production rate of urea by hepatocytes markedly increased during the coculture, while the rate in the single culture was lower than the coculture and increased only slightly. The specific rate of urea production by hepatocytes in the coculture did not decrease even as the hepatocytes grew. Consequently, rat primary hepatocytes could proliferate in vitro by coculture with STO cells without a decrease in urea production activity.     

Porcine bone marrow: extraction procedure and characterization by bone type

Data on porcine and bovine bone marrow composition indicate high calcium content, which may be erroneously elevated owing to the marrow recovery process. A method of bone marrow recovery was developed that involved passing marrow extracted from bone through a filter-press mechanism to remove very fine bone particles and dust, allowing a more accurate analysis of marrow. Calcium values were reduced approximately 90% and ash values reduced more than 50% compared to other reported data. The new recovery method did not require sawing away the hard bone and it removed particulate that may have interfered with analyses. Bone marrow was characterized by bone type. Rib bone marrow had higher protein, iron, non-heme iron and total pigment than scapula, aitch/hip bone or vertebrae marrow. Fat ranged from 17·81 to 26·76% and calcium ranged from 27·25 to 44·33 mg 100 g^-1 among bone types. The pH of bone marrow ranged from 7·14 to 7·53. Bone marrow appears to contribute to some of the properties of meat obtained from advanced meat recovery systems.     

Combination of cytomegalovirus enhancer with human cellular promoters for gene-induced chondrogenesis of human bone marrow mesenchymal stem cells

High-expression plasmid vectors for human mesenchymal stem cells (MSCs) were constructed by combination of cytomegalovirus immediate-early enhancer with cellular promoters. MSCs transfected with the vector showed higher transgene production of a cytokine, which increased the differentiation level to chondrocytes.     

High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells

The effects of the density of human mesenchymal stem cells (MSCs) on their differentiation to chondrocytes in a differentiation medium supplemented with dexamethasone, TGFbeta3, and IGF-1 were investigated for the regenerative therapy of cartilage. The increase in the initial density of MSCs from 0.05 x 10(4) to 0.9 x 10(4) cells/cm(2) accelerated the increase in the expression level of aggrecan mRNA during the differentiation culture for 7 d. The conditioned medium harvested at 7 d from the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2) accelerated the initial increase in the expression level for 3 d in the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2), whereas the conditioned medium harvested at 7 d in the differentiation culture with an initial MSC density of 0.05 x 10(4) cells/cm(2) did not. The differentiation culture after 14 d with an initial MSC concentration of 0.3 x 10(4) cells/cm(2) showed an expression level 1.7-fold that in the case of the culture with an initial MSC concentration of 0.05 x 10(4) cells/cm(2). Thus, a high MSC inoculum density might be appropriate for the rapid differentiation of MSCs to chondrocytes.     

Suppression of thymic stromal lymphopoietin production by rutin in mast cells

Thymic stromal lymphopoietin (TSLP) is a key player in allergic diseases such as asthma and atopic dermatitis. Rutin (RU), a non-nutritive component of many foods, possesses anti-inflammatory, hepatoprotective, and anti-tumour effects. We investigated how RU inhibits the production of TSLP in human mast cell line (HMC-1) cells. RU inhibited the production and mRNA expression of TSLP in HMC-1 cells. The maximal inhibition rate of TSLP production by RU (50 μM) was 56.25 ± 2.81%. Nuclear factor-κB luciferase activity induced by phorbol myristate acetate plus A23187 was inhibited by RU. In the activated HMC-1 cells, the activation of caspase-1 was increased, whereas the activation of caspase-1 was decreased by pre-treatment with RU. Finally, RU inhibited the numbers of TSLP-positive mast cells in lesions of PMA-induced ear oedema model. These results suggest that RU would be helpful for the treatment of inflammatory and atopic diseases through the inhibition of TSLP.     

Shrinkage-free preparation of scaffold-free cartilage-like disk-shaped cell sheet using human bone marrow mesenchymal stem cells

Aiming for the clinical application of cartilage regeneration, a culture method for mesenchymal stem cells (MSCs) derived from human bone marrow to obtain scaffold-free cartilage-like disk-shaped sheet of uniform sizes without the shrinkage was investigated. A disk-shaped cell sheet having the same diameter as that of the membrane without the shrinkage was formed after the cultivation of MSCs (18.6 × 10(5)cells/well) for 3 weeks in a cell culture insert (CCI) containing a flat membrane whose porosity was 12%, while 6.2 and 31.0 × 10(5)MSCs/well, respectively, resulted in the shrinkage of the aggregate and the hole formation in the center part of the sheet. Cell aggregates shrunk also in a 96-well plate and CCIs having lower porosity. The disk-shaped cell sheet showed the comparable thickness (1.2mm) and sulfated glycosaminoglycan (sGAG) density to those of the pellet formed in a pellet culture. The gene expression levels of aggrecan and type II collagen in the disk-shaped cell sheet were not lower than those in the pellet. In conclusion, the usage of CCI having 12% porosity and 18.6 × 10(5)MSCs/well could avoid the shrinkage from the formation of the scaffold-free cartilage-like disk-shaped cell sheet.     

孔石莼多糖对辐射诱发小鼠骨髓微核抑制作用研究

目的:孔石莼中含有4种相对分子量的水溶性多糖(151.7、64.5、58.0ku和28.2ku)。了解不同相对分子量段孔石莼多糖对辐射诱发小鼠骨髓细胞微核形成的抑制作用及效果比较。方法:将通过膜分离纯化得到的3种不同相对分子量段的孔石莼多糖(≥100ku、30～100ku及≤30ku)分别给相应设置的3个实验组小鼠进行灌服,另设辐照对照组和空白对照组;辐照后制备各组动物的骨髓涂片,计数多染性红细胞的微核发生率。结果:低相对分子量段的孔石莼多糖对辐射诱发的小鼠骨髓细胞微核具有一定的抑制效应,其中≤30ku相对分子量的孔石莼多糖对其造成的抑制作用最为显著(P<0.01)。结论:低相对分子量孔石莼多糖对辐射引起小鼠染色体损伤具有一定的抑制作用,由此推断低相对分子量孔石莼多糖具有一定的抗辐射作用。     

Recoverability of Listeria monocytogenes after coculture with Tetrahymena pyriformis

Bactivory by protozoa is a major factor that limits the number of bacteria in nature and may control the presence of Listeria monocytogenes. The effectiveness of Tetrahymena pyriformis destruction of L. monocytogenes was measured. Within 1 hr, 35–40 T. pyriformis cells ingested an average of 1,219 CFU of L. monocytogenes. Gentamicin was then added to kill un-ingested Listeria. In 24 hr, the recoverable bacteria were reduced at an exponential rate to undetectable levels (<1 per culture). A genetically diverse set of L. monocytogenes cultures all reduced Listeria recovery by the same degree. In assays without addition of gentamicin, numbers of attached L. monocytogenes cells were lessened from an average of log 6.5 CFU/2 ml culture to log 4.7 CFU/2 ml culture. T. pyriformis was capable of lowering numbers of both free-swimming and attached L. monocytogenes. This technology may have applications to control L. monocytogenes in food processing environments.     

Enhanced cell aggregation and liver functions using polymers modified with a cell-specific ligand in primary hepatocyte cultures

Hepatocytes cultured as multicellular aggregates called spheroids exhibit enhanced liver functions and maintain them over a long period compared with monolayer culture. We previously reported the induction of hepatocyte spheroids using the synthetic polymer Eudragit (a copolymer of methacrylic acid and methylmethacrylate) as an artificial matrix in a cell suspension system (Yamada et al., J. Biochem., 123, 1017-1023, 1998). In this method, hepatocyte aggregation was promoted by the effects of electrostatic and hydrophobic interactions between cells and the polymer. To enhance the cell aggregation ability and cell-specificity of the polymer, in the present study, we prepared hepatocyte-targeting polymers containing lactone, a ligand of the asialoglycoprotein receptor. Addition of the lactone-modified polymers to the medium promoted cell aggregation and spheroid formation more effectively than unmodified Eudragit. The spheroids induced by the polymers exhibited enhanced liver functions, i.e., albumin secretion, ammonia removal, and urea synthesis, from early in the culture. We also investigated the induction of hetero-spheroids composed of various liver constitutive cells by this method. The hetero-spheroids induced by the polymers showed improved liver functions.     

Identification of genes regulated by interleukin-1beta in human endometrial stromal cells

Interleukin-1beta (IL-1b) is an important immune regulatory factor that in human endometrium plays a role in both menstruation and implantation in the event of pregnancy. It promotes inflammatory-like processes and also stimulates tissue remodelling. We present a cDNA microarray study documenting the major effects of IL-1beta on gene expression in stromal cells from human endometrium. Endometrial stromal cells from five normal healthy women at the mid secretory phase were cultured with or without IL-1beta at 50 and 500 pg/ml for 48 h. cDNA microarrays were used to compare the levels of gene expression in total RNA isolated from cells stimulated with IL-1beta. These cDNA arrays were produced containing 15 164 sequence-verified clones, which included genes known to be important in angiogenesis, immune modulators, apoptosis, cell signalling, extra-cellular matrix (ECM) remodelling and cell cycle regulation. Genes which were regulated by IL-1beta were identified by analysis of the microarray data using the Significance Analysis of Microarrays software package. Upregulated (n = 23) and downregulated (n = 6) different genes were observed, which changed at least 3-fold, at a false discovery rate of less than 2% (P < 0.02). Our results have identified genes regulated by IL-1beta, which are involved in leukocyte recruitment, ECM remodelling and other cellular functions. Changes in three genes, IL-8, colony-stimulating factor 2 and aldoketo reductase family 1 member 1, which were upregulated by IL-1beta, were verified using real-time PCR. Novel functions regulated by IL-1beta in endometrium, including genes involved in free radical protection, and fatty acid metabolism were also identified. These results also provide new insights into the role of IL-1beta in disorders of the endometrium, especially in implantation-related infertility and endometriosis, in which this cytokine plays a major role.     

Control of cyclic AMP concentration in bovine endometrial stromal cells by arachidonic acid

Second messenger signalling through cyclic AMP (cAMP) plays an important role in the response of the endometrium to prostaglandin (PG) E(2) during early pregnancy. Arachidonic acid, which is a by-product of the luteolytic cascade in ruminants, is a potential paracrine signal from the epithelium to the stroma. We investigated the effects of arachidonic acid on the response of the stroma to PGE(2). cAMP was measured in bovine endometrial stromal cells treated with agents known to activate or inhibit adenylyl cyclase, protein kinase C (PKC) or phosphodiesterase (PDE). PGE(2) increased the intracellular cAMP concentration within 10 min, and this effect was attenuated by arachidonic acid and the PKC activator, 4beta-phorbol myristate acetate (PMA). The inhibitory effect of arachidonic acid on PGE(2)-induced cAMP accumulation was prevented by the PKC inhibitor, RO318425, and was absent in cells in which PKC had been downregulated by exposure to PMA for 24 h. The effect of arachidonic acid was also prevented by the PDE inhibitor, 3-isobutyl-1-methylxanthine. Arachidonic acid was shown by immunoblotting to prevent induction of cyclooxygenase-2 by PGE(2), forskolin or dibutyryl cAMP. The results indicate that arachidonic acid activates PDE through a mechanism involving PKC, counteracting a rise in intracellular cAMP in response to PGE(2). The data suggest that arachidonic acid antagonizes PGE(2) signalling through cAMP in the bovine endometrium, possibly acting to ensure a rapid return to oestrus in the case of failure of the maternal recognition of pregnancy.     

Construction of heterotypic cell sheets by magnetic force-based 3-D coculture of HepG2 and NIH3T3 cells

Heterotypic 3-D coculture is essential to mimic tissues and organs, because cell-cell interaction between various types of cells is believed to be important for the activation of cellular functions. In this study, magnetic force was applied to construct a 3-D coculture system of HepG2 and NIH3T3 cells as a model of hepatocytes and mesenchymal cells. Magnetite cationic liposomes (MCLs) were used to label target cells. NIH3T3 cells labeled with MCLs were seeded onto ultralow-attachment plates, whose surface is composed of a covalently bound hydrogel layer that is hydrophilic and neutrally charged. When a magnet was placed under the plate, cells accumulated on the bottom of the well. After a 24-h incubation period, the cells formed a multilayered cell sheet, which contained the major mesenchymal extracellular matrix (ECM) components (fibronectin and type I collagen), suggesting that the use of stromal NIH3T3 cells gave sufficient strength to cell sheets. Both NIH3T3 and HepG2 cells were labeled with MCLs, and cocultured by two methods: NIH3T3 cell sheets were constructed and HepG2 cells were subsequently seeded onto NIH3T3 cell sheets, and then allowed to form layered cell sheets by applying magnetic force; or NIH3T3 and HepG2 cells were mixed and then allowed to form mixed cell sheets by applying magnetic force. These heterotypic multilayered cell sheets were successfully constructed and an enhanced albumin secretion by HepG2 cells was observed. These results suggest that the new tissue engineering technique using magnetite nanoparticles and magnetic force, to which we refer to as magnetic force-based tissue engineering (Mag-TE), is a promising approach to construct multilayered cell sheets consisting of heterotypic cocultured cells.