OBJECTIVETo establish a method for organotypic slice culture of neonatal rat cortex in a modified condition and investigate the effect of spatial signals on neural stem cell (NSC) differentiation.

METHODSThe brain slices (200 µm in thickness) of neonatal SD rats (3 to 5 days old) were prepared and cultured in modified serum-free DMEM/F12 medium at 37 degrees celsius; with 95% O(2) and 5% CO(2). The organotypic slice cultures were observed regularly. NSCs isolated from the cortex of rat embryos (14-15 embryonic days) were cultured in serum-free DMEM/F12 supplemented with B27 and N2, and the passage 3 NSCs were labeled by CM-DiI before transplanted onto the organotypic slices cultured for 2 weeks. The survival of transplanted NSCs was assessed, and the cell differentiation was identified by immunofluorescence staining.

RESULTSThe organotypic slice cultures were well maintained for at least 4 weeks in the modified medium. The thickness of the organotypic slices reduced from 200 µm to 130 µm after 2-week culture in vitro due to the migration of the cells on the edge of the slices. CM-DiI-labeled NSCs survived well and differentiated into GFAP(+) glia and β-tubullin III(+) neurons.

CONCLUSIONNeonatal rat organotypic brain slice can be successfully cultured in a modified condition to serve as a model for studying NSC differentiation induced by spatial signals.

Animals ; Animals, Newborn ; Cell Differentiation ; physiology ; Cerebral Cortex ; cytology ; Coculture Techniques ; methods ; Fetus ; Neural Stem Cells ; cytology ; transplantation ; Organ Culture Techniques ; methods ; Rats

OBJECTIVETo explore the effect of different culture media on viability and expression of tau protein in organotypic hippocampal slice.

METHODSBrain slices (400 microm) from 1, 2, 4, and 8 week-old Wistar rats were prepared and cultured in minimum essential medium (MEM) or Dulbecco's modified eagle medium: nutrient mixture (DMEM/F12) medium respectively for 21 days. Viability of the slices was measured by lactate dehydrogenase (LDH) assay and expression of tau protein was detected by Western blot.

RESULTSThe viability of the slices was not influenced significantly by the two different culture media, while the expression level of tau protein was significantly higher in DMEM/F12 than in MEM (P < 0.05), especially in the slices from 2 and 4 week-old rats.

CONCLUSIONThe slices from 2 or 4 week-old rat hippocampi and DMEM/F12 medium may be the preferred choice for tau associated researches. An ideal Alzheimer's disease model may be established based on the results of these researches.

Animals ; Culture Media ; Hippocampus ; growth & development ; metabolism ; L-Lactate Dehydrogenase ; biosynthesis ; Organ Culture Techniques ; methods ; Rats ; Rats, Wistar ; tau Proteins ; biosynthesis ; genetics

Bone Regeneration/*physiology ; *Bone Substitutes ; Bone and Bones/pathology ; Bone and Bones/surgery ; *Computer-Aided Design ; *Magnetic Resonance Imaging ; Organ Culture Techniques/*methods ; Tissue Engineering/*methods ; *Tomography, X-Ray Computed

Chitosan has similar structure to glycosaminoglycans in the tissue, thus may be a good candidates as tissue engineering scaffold. However, to improve their cell attachment ability, we try to incorporate this natural polymer with collagen by combining it via cross-linking process. In this preliminary study we evaluate the cell attachment ability of chitosan-collagen scaffold versus chitosan scaffold alone. Chitosan and collagen were dissolved in 1% acetic acid and then were frozen for 24 hours before the lyophilizing process. Human skin fibroblasts were seeded into both scaffold and were cultured in F12: DMEM (1:1). Metabolic activity assay were used to evaluate cell attachment ability of scaffold for a period of 1, 3, 7 and 14 days. Scanning electron micrographs shows good cell morphology on chitosan-collagen hybrid scaffold. In conclusion, the incorporation of collagen to chitosan will enhance its cell attachment ability and will be a potential scaffold in tissue engineering.
Cell Adhesion/*physiology ; *Chitosan ; *Collagen ; Energy Metabolism/physiology ; Fibroblasts/cytology ; Microscopy, Electron, Scanning ; Organ Culture Techniques/*methods ; *Polymers ; Tissue Engineering/*methods

In the present study, natural coral of porites species was used as scaffold combined with in vitro expanded bone marrow stem cell derived osteoblasts (BMSC-DO), to develop a tissue-engineered bone graft in a rat model. Coral was molded into the shape of rat mandible seeded with 5x10(6) /ml BMSC-DO subsequently implanted subcutaneously in the back of 5 week Sprague dawely rats for 3 months. Coral alone was implanted as a control. The implants were harvest and processed for gross inspection and histological observations. The results showed that newly bone grafts were successfully formed coral seeded with cells group showed smooth highly vascularized like bone tissue. Histological sections revealed mature bone formation and lots of blood vessel, the bone formation occurred in the manner resemble intramembraneous bone formation. This study demonstrates that coral can be use as a suitable scaffold material for delivering bone marrow mesenchymal stem cells in tissue engineering.
*Anthozoa ; Biodegradation, Environmental ; Bone Marrow Cells/*cytology ; *Bone Transplantation ; *Calcium Carbonate ; Mesenchymal Stem Cells/*cytology ; Microscopy, Electron, Scanning ; Organ Culture Techniques/*methods ; Osteoblasts/*cytology ; Tissue Engineering/*methods

Transformed hairy roots of Polygonium multiflorum Thunb. were obtained by the transformation of Agrobacterium rhizogenes LBA9402. It was clearly demonstrated that T-DNA of A. Rhizogenes Ri plasmid was integrated into the cells of hairy roots by the experiments of PCR and Southern hybridization. Through the screen of basic medium and the research of growth curve of hairy roots, the optimum inoculum time was selected in 30 days or so in the optimum condition-MS medium. On the condition, the content of rhein was 2.85 folds higher in hairy roots than that of natural plants by means of HPLC.
Anthraquinones ; analysis ; metabolism ; Emodin ; analysis ; Organ Culture Techniques ; methods ; Plant Roots ; chemistry ; genetics ; growth & development ; metabolism ; Polygonaceae ; chemistry ; cytology ; Rhizobium ; genetics ; Transformation, Genetic

Fresh rat corneas as well as corneas preserved in several different corneal preservation media were stained with Avidin-Biotin-peroxidase Complex method in order to evaluate major histocompatibility complex (MHC) antigen expression. In fresh corneas, class I antigen was identified in corneal epithelium, stroma and endothelium. Class II antigen was identified only in stroma. In corneas preserved in the media which contained chondroitin and dextran for 7 days, class I antigen was somewhat decreased but class II antigen was increased. In corneas preserved in the medium which contained insulin or epidermal growth factor for 7 days, class II antigens seemed to be increased compaired to the fresh cornea. Expression of MHC antigens of corneas in the medium with fetal bovine serum were similar to those of fresh corneas.
Animals ; Cornea/*metabolism ; Culture Media ; Histocompatibility Antigens Class I/*biosynthesis ; Histocompatibility Antigens Class II/*biosynthesis ; Immunoenzyme Techniques ; Major Histocompatibility Complex ; Organ Preservation/methods ; Rats ; Rats, Inbred Lew

OBJECTIVETo observe the histological characteristics of constructed basement membrane in tissue-engineered skin.

METHODSForskins from circumcision in normal children were obtained with informed consent of the parents, and then the epidermal keratinocytes (KC) and dermal fibroblasts (Fb) were isolated with trypsin and collagenase D digestion in sequence. Tissue engineered skin with composite chitosan was maintained in a submerged state for 3 days, and then at the air-liquid interface. The tissue-engineered skins were fixed in neutral formalin and then embedded in paraffin after culture for 7, 10 and 15 days, respectively for immunohistological examination of the basement membrane component,including the condition of collagen type IV (COL-IV), collagen type VII (COL-VII), and laminin (LN).

RESULTSHE staining showed that the keratinocytes formed a fine stratified squamous epithelium with the presence of basal, spinous, granular and corneous cell layers, and there was various amount of cells in flat and fusiform shape in each layer. It was found that a regular red staining strip situated at the dermal epidermal junction. Positive staining of collagen IV, collagen VII as well as LN was observed by immunohistological examination.

CONCLUSIONThe results suggest that the composite chitosan tissue engineered skin has a good prospect for clinical use because it presents a perfect reconstruction of basement membrane.

Basement Membrane ; cytology ; Cells, Cultured ; Child ; Chitosan ; metabolism ; Collagen Type IV ; metabolism ; Collagen Type VII ; metabolism ; Humans ; Laminin ; metabolism ; Organ Culture Techniques ; Skin, Artificial ; Tissue Engineering ; methods

BACKGROUNDThe development of mechanically active culture systems helps increase the understanding of the role of mechanical stress in intervertebral disc (IVD) degeneration. Motion segment cultures allow for preservation of the native IVD structure, and adjacent vertebral bodies facilitate the application and control of mechanical loads. The purpose of this study was to establish loading and organ culture methods for rabbit IVD motion segments to study the effect of static load on the whole disc organ.

METHODSIVD motion segments were harvested from rabbit lumbar spines and cultured in no-loading 6-well plates (control conditions) or custom-made apparatuses under a constant, compressive load (3 kg, 0.5 MPa) for up to 14 days. Tissue integrity, matrix synthesis, and the matrix gene expression profile were assessed after 3, 7, and 14 days of culturing and compared with those of fresh tissues.

RESULTSThe results showed that ex vivo culturing of motion segments preserved tissue integrity under no-loading conditions for 14 days whereas the static load gradually destroyed the morphology after 3 days. Proteoglycan contents were decreased under both conditions, with a more obvious decrease under static load, and proteoglycan gene expression was also downregulated. However, under static load, immunohistochemical staining intensity and collagen Type II alpha 1 (COL2A1) gene expression were significantly enhanced (61.54 ± 5.91, P = 0.035) and upregulated (1.195 ± 0.040, P = 0.000), respectively, compared with those in the controls (P < 0.05). In contrast, under constant compression, these trends were reversed. Our initial results indicated that short-term static load stimulated the synthesis of collagen Type II alpha 1; however, sustained constant compression led to progressive degeneration and specifically to a decreased proteoglycan content.

CONCLUSIONSA loading and organ culture system for ex vivo rabbit IVD motion segments was developed. Using this system, we were able to study the effects of mechanical stimulation on the biology of IVDs, as well as the pathomechanics of IVD degeneration.

Animals ; Gene Expression Regulation ; Immunohistochemistry ; Intervertebral Disc ; metabolism ; physiology ; Intervertebral Disc Degeneration ; metabolism ; physiopathology ; Male ; Nucleus Pulposus ; metabolism ; physiology ; Organ Culture Techniques ; methods ; Rabbits ; Stress, Mechanical

The strategy used to generate tissue-engineered bone construct, in view of future clinical application is presented here. Osteoprogenitor cells from periosteum of consenting scoliosis patients were isolated. Growth factors viz TGF-B2, bFGF and IGF-1 were used in concert to increase cell proliferation during in vitro cell expansion. Porous tricalcium phosphate (TCP)-hydroxyapatite (HA) scaffold was used as the scaffold to form 3D bone construct. We found that the addition of growth factors, greatly increased cell growth by 2 to 7 fold. TCP/HA proved to be the ideal scaffold for cell attachment and proliferation. Hence, this model will be further carried out on animal trial.
Bone Regeneration/*physiology ; *Bone Transplantation ; Cell Division/physiology ; Collagen/metabolism ; *Mesenchymal Stem Cell Transplantation ; Organ Culture Techniques ; Periosteum/*cytology ; Tissue Engineering/*methods