Three-dimensional culture of rat exocrine pancreatic cells using collagen gels.

Rat pancreatic cells were dissociated using a combined enzyme and EDTA method, grown on a plastic surface and then overlayed with collagen gel. Our studies have shown that exocrine pancreatic cells grown in this way have the ability to rearrange themselves into a three-dimensional organoid structure in which well defined epithelial lumina have been identified by ultrastructural and light microscopic examination. This in vitro system has advantages in examining the cytodifferentiation of pancreatic cells and may be exploited in studying pancreatic carcinogenesis.     

Three-dimensional two-layer collagen matrix gel culture model for evaluating complex biological functions of monocyte-derived dendritic cells

Dendritic cell-like cells (Mo-DCs) generated from peripheral blood monocytes with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been used as tools to treat cancer patients (DC-vaccines). Because Mo-DCs have multiple antigen presentation-related functions, including phagocytosis, migration, cytokine production, and T cell stimulation, establishment of a method for simultaneously evaluating the various functions of Mo-DCs is important. We developed a new in vitro three-dimensional two-layer collagen matrix culture model that consists of a collagen gel containing Mo-DCs as the lower layer and a collagen gel containing necrotic GCTM-1 tumor cells and/or T cells as the upper layer. We used this system to observe simultaneously multiple functions of Mo-DCs by phase-contrast or fluorescence microscopy and to assess IL-12 secretion during more than 2 weeks of culture. We also observed interactions between Mo-DCs and necrotic GCTM-1 or T cells on an individual cell basis by time-lapse videomicroscopy. In addition, we collected Mo-DCs from the collagen gels by collagenase treatment and analyzed the expression of antigen presentation-related molecules such as HLA-DR, CD80, CD83, and CD86 on Mo-DCs. This model may be a useful tool for evaluation of the various functions of Mo-DCs used as DC vaccines and for studies of the complex behaviors of Mo-DCs in vivo.     

Three-dimensional culture and differentiation of human osteogenic cells in an injectable hydroxypropylmethylcellulose hydrogel

The present work evaluates a newly developed silated hydroxypropylmethylcellulose (Si-HPMC)-based hydrogel as a scaffold for 3D culture of osteogenic cells. The pH variation at room temperature catalyzes the reticulation and self-hardening of the viscous polymer solution into a gelatine state. We designed reticulation time, final consistency and pH in order to obtain an easy handling matrice, suitable for in vitro culture and in vivo injection. Three human osteogenic cell lines and normal human osteogenic (HOST) cells were cultured in 3D inside this Si-HPMC hydrogel. We show here that osteosarcoma cells proliferate as clonogenic spheroids and that HOST colonies survive for at least 3 weeks. Mineralization assay and gene expression analysis of osteoblastic markers and cytokines, indicate that all the cells cultured in 3D into this hydrogel, exhibited a more mature differentiation status than cells cultured in monolayer on plastic. This study demonstrates that this Si-HPMC hydrogel is well suited to support osteoblastic survival, proliferation and differentiation when used as a new scaffold for 3D culture and represents also a potential basis for an innovative bone repair material.     

Three-dimensional culture of rat exocrine pancreatic cells using collagen gels

Rat pancreatic cells were dissociated using a combined enzyme and EDTA method, grown on a plastic surface and then overlayed with collagen gel. Our studies have shown that exocrine pancreatic cells grown in this way have the ability to rearrange themselves into a three-dimensional organoid structure in which well defined epithelial lumina have been identified by ultrastructural and light microscopic examination. This in vitro system has advantages in examining the cytodifferentiation of pancreatic cells and may be exploited in studying pancreatic carcinogenesis.     

Primary culture of rabbit gallbladder epithelial cells in collagen gel matrix

Primary culture of gallbladder epithelial cells obtained from normal rabbits was attempted in collagen gel matrix for up to 6 weeks. Fluid medium containing 0.02% ethylenediaminetetraacetic acid and 0.25% trypsin was poured into the gallbladder lumen. The pellets obtained by centrifugation of recovered fluid contained many isolated epithelial cells and a few small cell clumps. These pellets were dispersed and embedded inside collagen gel matrix and cultured in William's medium E supplemented with fetal calf serum and epidermal growth factor. The three-dimensional outgrowth from individual cells and small cell clumps consisted predominantly of spherical cystic masses 2-4 days later. These cysts contained mucin and were covered by a single layer of cuboidal or low columnar epithelial cells. Electron microscopy revealed the epithelial arrangement of cells lining the cyst walls, and these cells were similar to gallbladder epithelial cells in vivo. These epithelial cells showed active mucin secretion into the cystic cavities. Cytokeratin was diffusely present in the cytoplasm. This isolation and culture system provides a reproducible and consistent method for sustained growth of normal gallbladder epithelial cells from normal tissue in primary culture and seems valuable for investigating pathologic conditions of the gallbladder.     

All-trans-retinoic acid suppresses matrix metalloproteinase activity and increases collagen synthesis in diabetic human skin in organ culture

Diabetes increases susceptibility to chronic skin ulceration. The etiology of chronic wound formation in diabetic individuals is multifactoral but may be accelerated by changes in the structure and function of the skin secondary to impaired fibroblast proliferation, decreased collagen synthesis, and increased matrix metalloproteinase (MMP) expression. This study explored the effects of all-trans-retinoic acid (RA) on cellular and biochemical features of diabetic human skin in organ culture. Two-mm skin biopsies from hip or ankle were obtained from diabetic subjects and incubated for 9 days in the absence or presence of 2 micro mol/L RA. Hip skin from non-diabetic individuals served as control. Following organ culture incubation, untreated and RA-treated tissue was examined histologically after staining with hematoxylin and eosin. In parallel, organ culture-conditioned medium collected on days 5 and 7 was assayed for levels of active and total MMP-1 (interstitial collagenase) and MMP-9 (gelatinase B). The same organ culture fluids were assayed for the presence of soluble collagen. In comparison with skin from non-diabetic individuals, diabetic skin demonstrated no major differences in overall epidermal thickness or collagen production (both were increased in RA-treated tissue as compared to non-RA-treated tissue). In contrast, levels of MMP-9 (active forms) were elevated in organ culture fluid from diabetic skin as compared to non-diabetic control skin. In the presence of RA, active forms of both MMP-1 and MMP-9 were reduced. Together, these data suggest that RA has the capacity to improve structure and function of diabetic skin, and that a major effect is on reduction of collagen-degrading MMPs.     

Three-dimensional in vitro cocultivation of lung carcinoma cells with human bronchial organ culture as a model for bronchial carcinoma.

We describe the development of a three-dimensional in vitro organ culture model for bronchial carcinoma using bronchial mucosa organ cultures and three different human non-small cell lung cancer cell lines. During precultivation, bronchial fragments obtained as biopsies during routine bronchoscopy had regenerated a complete epithelial covering with a well-preserved organotypic architecture around a nucleus consisting of connective tissue. To create cocultures, different types of confrontation between tumor cells and organ cultures were applied. Histologic light microscopy and scanning electron microscopy were used in analysis. When tumor cells were confronted with completely epithelialized organ cultures, they showed a low incidence of attachment. When organ cultures were wounded before confrontation, tumor cells always attached to the wounded side and showed a progressive invasion into the stromal tissue. Measurements of the penetration depth of tumor cells into the organ cultures after different incubation times permitted the quantitative evaluation of invasion. Histologic studies revealed well-differentiated normal epithelium in spite of long culture periods. Histologic features of the tumors were those of an invasive undifferentiated carcinoma and showed marked similarities to the situation in vivo. The coculture model permits internal controls because it contains both normal human epithelium and human tumor cells in the same organotypic culture. Therefore it offers opportunities for various in vitro investigations on therapeutic and diagnostic modalities of lung cancer, as indicated in this paper by an example of photodynamic procedures with 5-aminolevulinic acid.     

星形胶质细胞在凝胶环境下的三维培养

目的:制备不同浓度的三维琼脂糖凝胶以进行星形胶质细胞的体外培养,从而寻找适合细胞生长的理想环境。方法:分别制备1%、2%、3%的琼脂糖凝胶,利用纳米压痕仪测量其弹性模量。星形胶质细胞在凝胶中培养1、3、5、7 d,观察细胞活性以及细胞骨架的变化。结果:随着凝胶浓度的增加,琼脂糖凝胶弹性模量逐渐增加。2%的琼脂糖凝胶的弹性模量最接近筛板组织的弹性模量;在2%、3%琼脂糖凝胶环境下,细胞活性具有较高水平。随着细胞在凝胶中培养的时间增加,星形胶质细胞的突起逐渐伸出,细胞从球形向梭形或星形转变,更接近细胞真实的生长状态。结论:2%琼脂糖凝胶最接近星形胶质细胞在体内的生长环境,细胞成活率较高,是细胞体外三维培养的理想环境。 【关键词】星形胶质细胞;琼脂糖凝胶;三维培养;力学特性     

Reconstruction of alveolus-like structure from alveolar type II epithelial cells in three-dimensional collagen gel matrix culture.

The purpose of this study is to reconstruct an alveolus-like structure from alveolar type II epithelial cells in a culture condition. Isolated alveolar type II epithelial cells of the rat were cultured in a three-dimensional collagen gel matrix. Single type II cells formed cellular aggregates that had a lumen after cell division in this culture condition. Through proliferation of the component cells, these aggregates grew to assume a globular or branching structure, part of which in turn developed into a large, cystic alveolus-like structure. This structure consisted of flattened epithelial cells intermingled with cuboidal epithelial cells. In these structures, the surfactant production was confirmed by immunohistochemistry and electron microscopy. To our knowledge, this is the first report of a reconstruction of an alveolus-like structure in a three-dimensional collagen gel matrix culture. This culture system seems to provide an appropriate physiological environment in which to study the differentiation and disorders of pulmonary alveoli.     

In vitro spermatogenesis by three-dimensional culture of rat testicular cells in collagen gel matrix

In an effort to improve in vitro spermatogenesis by potentiating the interactions between developing germ cells, somatic cells, and the extracellular matrix (ECM), the efficiency of the germ cell-somatic cell coculture in a three-dimensional (3D) collagen gel matrix was examined. Cells isolated from rat seminiferous tubules 18 days after birth were cultured for 22 days in a monolayer without ECM, collagen gel (CG), or collagen+Matrigel (CGM). After culture, the viabilities of the cultured cells in the monolayer, CG, and CGM culture were 42.8%, 70.7% and 76.1%, respectively. Occludin-positive cells in a cyst-like structure were found in the ECM gel matrix together with 3beta hydroxysteroid dehydrogenase-positive cells, suggesting the presence of functional Sertoli cells and Leydig cells, respectively. Flow cytometric analysis of DNA content revealed a significant increase in the haploid cell population in the CG and CGM compared to the monolayer culture. Transition protein 2 (TP2) and protamine 2-positive cells were found together with a significant increase in TP2 mRNA levels in cells cultured in CG and CGM over those in monolayer culture, suggesting the occurrence of the post-meiotic differentiation of spermatogenetic cells. Taken together, a 3D in vitro culture system for testicular cells using a collagen gel matrix could enhance viability, meiosis, and post-meiotic differentiation of germ cells to presumptive differentiating spermatids.     

Perfusion culture enhances osteogenic differentiation of rat mesenchymal stem cells in collagen sponge reinforced with poly(glycolic Acid) fiber

The objective of this study was to obtain fundamental knowledge about in vitro culture systems to enhance the proliferation and differentiation of mesenchymal stem cells (MSCs) in collagen sponge reinforced by the incorporation of poly(glycolic acid) (PGA) fiber. A collagen solution with PGA fiber homogeneously localized at PGA:collagen weight ratios of 0.67, 1.25, 2.5, and 5 was freezedried, followed by cross-linking of combined dehydrothermal, glutaraldehyde, and ultraviolet treatment. Scanning electron microscopy revealed that collagen sponges exhibited homogeneous and interconnected pore structures with an average size of 180 microm, irrespective of PGA fiber incorporation. When rat MSCs were seeded into collagen sponge with or without PGA fiber incorporation, more attached cells were observed in collagen sponge incorporating PGA fiber than in collagen sponge without PGA fiber incorporation, irrespective of the PGA:collagen ratio. The proliferation and osteogenic differentiation of MSCs in PGA-reinforced sponge at a weight ratio of 5 were greatly influenced by the culture method and growth conditions. Alkaline phosphatase (ALP) activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method became maximum at a flow rate of 0.2 mL/min, although they increased with culture time period. It may be concluded that appropriate perfusion conditions enable MSCs to positively improve the extent of proliferation and differentiation.     

Calvaria derived osteogenic cells: phenotypic expression in culture

The osteoblast phenotype is characterized by its ability to (a) synthesize a well defined mineralized collagenous matrix, (b) regulate the remodeling process by synthesizing local hormones (PGE2) and specific molecules (osteocalcin) and enzymes (alkaline phosphatase and collagenase), (c) respond to a variety of hormones (PTH, PGs, vitamin-D metabolites, steroids and growth factors), (d) respond to mechanical stimulation. Most of osteoblast culture systems meet many of the above qualifications though most fail to show the PTH effect on DNA synthesis, (c), and mechanical stimulation (d). Here we show that by using trypsin digestion and serum-containing low calcium medium (0.25 mM), all the above listed osteoblast phenotypic characteristics are demonstrated including their responsiveness to mechanical stimulation and the PTH effect on DNA synthesis.     

Three-dimensional porous hydroxyapatite/collagen composite with rubber-like elasticity

A three-dimensional porous hydroxyapatite/collagen (HAp/Col) composite with a random pore structure was fabricated using freeze-drying processes; the self-organized HAp/Col nanocomposite with a weight ratio of 80.5:19.5, freeze-dried, was kneaded in 100 mM sodium phosphate buffer, frozen at -20 degrees C and freeze-dried. The cross-linkage of Col molecules was introduced dehydrothermally at 140 degrees C in vacuo. The porous composite had a porosity of 94.7% with pore sizes between 200 and 500 microm. The compressive stress for the wet porous composite in phosphate buffer saline (PBS) was gradually decreased during 20 days incubation with a small amount of weight loss. The cyclic and time-course compression tests showed good repeatability of stress and well-recovery of its height, and caused no collapse of the porous composite. The implantation of the porous composite in rat bone holes showed the biodegradable property and new bone formation occurred in the pores without inflammatory response. The porous composite fabricated has good flexibility and rubber-like elasticity, and is a promising bone regenerative material.     

Three-dimensional porous hydroxyapatite/collagen composite with rubber-like elasticity

A three-dimensional porous hydroxyapatite/collagen (HAp/Col) composite with a random pore structure was fabricated using freeze-drying processes; the self-organized HAp/Col nanocomposite with a weight ratio of 80.5:19.5, freeze-dried, was kneaded in 100 mM sodium phosphate buffer, frozen at ?20°C and freeze-dried. The cross-linkage of Col molecules was introduced dehydrothermally at 140°C in vacuo. The porous composite had a porosity of 94.7% with pore sizes between 200 and 500 μm. The compressive stress for the wet porous composite in phosphate buffer saline (PBS) was gradually decreased during 20 days incubation with a small amount of weight loss. The cyclic and time-course compression tests showed good repeatability of stress and well-recovery of its height, and caused no collapse of the porous composite. The implantation of the porous composite in rat bone holes showed the biodegradable property and new bone formation occurred in the pores without inflammatory response. The porous composite fabricated has good flexibility and rubber-like elasticity, and is a promising bone regenerative material.     

Medium hydrated collagen gel as an explant support in organ culture

Summary This report describes the preparation of medium hydrated collagen gels for use as the physical support material for explant culture. Collagen gels are simple to prepare from rat tail tendon-derived stock collagen. These gels provide a firm, fluid-permeable substrate for submersion or interface culture of organ explants. Collagen gels can be conveniently applied to serum-free medium conditions.     

Differentiation of olfactory receptor cells in organ culture

Presumptive olfactory mucosa was excised from the heads of rat fetuses in the eleventh and twelfth days of gestation and explanted in organ cultures. At the time of explantation, the presumptive olfactory cells were recognizable by their long narrow apical processes and basally located nuclei. However at this stage they were in an early phase of differentiation as indicated by the large numbers of free ribosomes and virtual absence of microtubules and cilia in the apical cytoplasm. After three to eight days in culture, there was a progressive increase in the total number of cells in the epithelium. Differentiation in olfactory receptor cells was detectable by the appearance in the apical processes of axially oriented microtubules and centrioles or basal bodies. Some of which generated cilia. At their basal ends, the cytoplasm narrowed into axons and bundles of these axons, arranged in the unique manner of olfactory nerve axons, entered the connective tissue. Olfactory receptor cells, as defined by morphological criteria, differentiated under suitable organ culture conditions in the absence of any tissue from the central nervous system.     

Three-dimensional culture of human disc cells within agarose or a collagen sponge: assessment of proteoglycan production

The objective of the present study was to assess proteoglycan production by human intervertebral disc cells cultured in vitro in selected cell carriers. Based on previous studies which evaluated disc cells seeded into collagen sponge, collagen gel, agarose, alginate or fibrin gel three-dimensional (3D) cell carriers, collagen sponge and agarose were found to provide superior microenvironments for formation of extracellular matrix (ECM). A standardized test design was used to evaluate ECM formed after 14 days of culture using the 1,9-dimethylmethylene blue (DMB) assay to assess sulfated glycosaminoglycan (S-GAG) production. Although agarose culture showed higher S-GAG levels compared to collagen sponge (2.94+/-2.20 (19) microg/ml S-GAG (mean+/-S.D. (n)) vs. 0.94+/-0.77 (22), respectively, p=0.0003), this is off-set by the significantly lower proliferation rate associated with culture of disc cells in agarose.     

Three-dimensional culture of hepatocytes on porcine liver tissue-derived extracellular matrix

There is currently no optimal system to expand and maintain the function of human adult hepatocytes in culture. Recent studies have demonstrated that specific tissue-derived extracellular matrix (ECM) can serve as a culture substrate and that cells tend to proliferate and differentiate best on ECM derived from their tissue of origin. The goal of this study was to investigate whether three-dimensional (3D) ECM derived from porcine liver can facilitate the growth and maintenance of physiological functions of liver cells. Optimized decellularization/oxidation procedures removed up to 93% of the cellular components from porcine liver tissue and preserved key molecular components in the ECM, including collagen-I, -III, and -IV, proteoglycans, glycosaminoglycans, fibronectin, elastin, and laminin. When HepG2 cells or human hepatocytes were seeded onto ECM discs, uniform multi-layer constructs of both cell types were formed. Dynamic culture conditions yielded better cellular infiltration into the ECM discs. Human hepatocytes cultured on ECM discs expressed significantly higher levels of albumin over a 21-day culture period compared to cells cultured in traditional polystyrene cultureware or in a collagen gel "sandwich". The culture of hepatocytes on 3D liver-specific ECM resulted in considerably improved cell growth and maintained cell function; therefore, this system could potentially be used in liver tissue regeneration, drug discovery or toxicology studies.     

Type VIII collagen. Synthesis by normal and malignant cells in culture

A novel protein belonging to the collagen family was originally purified from the culture medium of bovine aortic endothelial cells. This endothelial collagen, termed EC, was also found to comprise the major collagen type synthesized by a malignant astrocytoma-derived cell line. Examination of several cell strains derived from normal tissues revealed that EC was not synthesized by all endothelial cells; it was absent from human endothelial cells cultured from both large and small vessels but was present in bovine cells, including those from capillaries. Human foreskin fibroblasts also secreted this protein in small amounts relative to interstitial procollagens, but it was not detected in two human epithelial cell strains. EC was consistently observed in human cell lines derived from several carcinomas and comprised the major collagenous protein secreted by cells cultured from a Ewing's sarcoma. In contrast, malignant or transformed murine cells did not produce EC in vitro. In addition, the protein was not apparent after metabolic labeling of human cells from an epidermoid carcinoma, a fibrosarcoma, and two Wilms' tumors. EC-like proteins were isolated from cell culture medium by ion-exchange chromatography and were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after cleavage with vertebrate collagenase, mast cell protease, and CNBr. In addition to the homologies displayed by comparative peptide mapping, these collagens exhibited other unusual properties that collectively were characteristic of EC from endothelial and astrocytoma-derived cells. These studies support the existence of a novel class of collagenous proteins that are secreted by a wide variety of cells derived from both normal and neoplastic tissues. This class of proteins, which manifests several unusual structural characteristics, has been designated type VIII collagen.