Human Brain Slice Culture: A Useful Tool to Study Brain Disorders and Potential Therapeutic Compounds / 神经科学通报·英文版

Investigating the pathophysiological mechanisms underlying brain disorders is a priority if novel therapeutic strategies are to be developed. In vivo studies of animal models and in vitro studies of cell lines/primary cell cultures may provide useful tools to study certain aspects of brain disorders. However, discrepancies among these studies or unsuccessful translation from animal/cell studies to human/clinical studies often occur, because these models generally represent only some symptoms of a neuropsychiatric disorder rather than the complete disorder. Human brain slice cultures from postmortem tissue or resected tissue from operations have shown that, in vitro, neurons and glia can stay alive for long periods of time, while their morphological and physiological characteristics, and their ability to respond to experimental manipulations are maintained. Human brain slices can thus provide a close representation of neuronal networks in vivo, be a valuable tool for investigation of the basis of neuropsychiatric disorders, and provide a platform for the evaluation of novel pharmacological treatments of human brain diseases. A brain bank needs to provide the necessary infrastructure to bring together donors, hospitals, and researchers who want to investigate human brain slices in cultures of clinically and neuropathologically well-documented material.

Primary human airway epithelial cell cultures: an update on technique and application in virology / 中华微生物学和免疫学杂志

Well-differentiated primary human airway epithelial cell culture (HAE), an organotypic human airway culture system, has been increasingly used as a linkage between researches conducted on animal models and humans.In this review, we focus on progress in technological development and its application in virology.The following aspects are covered in this review: (1)Structure and function of the normal airway, (2)Role of respiratory epithelial cells in pulmonary innate immunity, (3)Development of primary human airway epithelial cell cultures, (4)Application of primary human airway epithelial cell cultures in virology.

Vascular Endothelial Growth Factor Enhances Axonal Outgrowth in Organotypic Spinal Cord Slices via Vascular Endothelial Growth Factor Receptor 1 and 2

Enhancing adult nerve regeneration is a potential therapeutic strategy for treating spinal cord injury. Vascular endothelial growth factor (VEGF) is a major contributor to angiogenesis, which can reduce the spinal cord injury by inhibiting the inflammation and improve recovery after spinal cord injury. We have previously demonstrated that exogenous VEGF has neurotrophic effects on injured spinal nerves in organotypic spinal cord slice cultures. However, the mechanisms underlying the neurite growth by exogenous VEGF remain to be explored in spinal cord. In this study, we found out that exogenous VEGF mediated axonal outgrowth through VEGF receptor 1 (VEGFR1) and VEGFR2, both of which were expressed on organotypic spinal cord slices. Although VEGFR1 and VEGFR2 were constitutively expressed in some cells of control spinal cord slices, VEGF treatment upregulated expression of VEGFR1 and VEGFR2. Both VEGFR1 and VEGFR2 were expressed in neuronal cells as well as glial cells of organotypic spinal cord slices. We also observed that VEGF-induced axonal outgrowth was attenuated by a specific mitogen-activated protein kinase (MAPK) inhibitor PD98059 and a specific phosphoinositide 3-kinase (PI3K) inhibitor wortmannin. Thus, these findings suggest that these MAPK and PI3K pathways have important roles in regulating VEGF-induced axonal outgrowth in the postnatal spinal cord.

A Promotive Effect of Low Level Laser on Hair Cell Viability in Postnatal Organotypic Culture of Rat Utricles

BACKGROUND AND OBJECTIVES: To culture and maintain mammalian hair cells is still a big challenge. In this study, long-term organotypic culture of rat utricular maculae was established to study vestibular hair cell. The effects of low level laser on hair cell viability in postnatal organotypic culture of rat utricles were investigated. MATERIALS AND METHOD: Uticular explants were prepared from postnatal 2 to 7 rats and cultured. To improve hair cell survival, the utricles were irradiated daily with low level laser. Whole-mount utricles were stained with FM1-43 which is known to be an efficient marker to identify live hair cells in cultured tissues. Such cells visualized directly through tissue culture dish with cover glass bottom by Confocal laser scanning microscope at specific time points. RESULTS: The explanted utricular hair cells were cultured for up to 31 days in in vitro culture system. In low level laser irradiation group, utricular hair cells were more survived at 24 DIV and 31 DIV. CONCLUSION: These results suggest that low level laser promotes hair cell viability in utricular explants.

A Promotive Effect of Low Level Laser on Hair Cell Viability in Postnatal Organotypic Culture of Rat Utricles

BACKGROUND AND OBJECTIVES: To culture and maintain mammalian hair cells is still a big challenge. In this study, long-term organotypic culture of rat utricular maculae was established to study vestibular hair cell. The effects of low level laser on hair cell viability in postnatal organotypic culture of rat utricles were investigated. MATERIALS AND METHOD: Uticular explants were prepared from postnatal 2 to 7 rats and cultured. To improve hair cell survival, the utricles were irradiated daily with low level laser. Whole-mount utricles were stained with FM1-43 which is known to be an efficient marker to identify live hair cells in cultured tissues. Such cells visualized directly through tissue culture dish with cover glass bottom by Confocal laser scanning microscope at specific time points. RESULTS: The explanted utricular hair cells were cultured for up to 31 days in in vitro culture system. In low level laser irradiation group, utricular hair cells were more survived at 24 DIV and 31 DIV. CONCLUSION: These results suggest that low level laser promotes hair cell viability in utricular explants.

The Bcl-2 and NeuN Expressions and Morphological Changes in Organotypic Explant Culture of Rat Hippocampus

BACKGROUND: Bcl-2 protein is essential for neurodevelopment and it is implicated in various neurodegenerative diseases. However, little is known about the normal developmental expression of Bcl-2 and NeuN protein in hippocampus slice cultures. We performed this study to assess their morphological changes and developmental expressions of Bcl-2 and NeuN protein. METHODS: We cultured the hippocampus of an eight days postnatal Sprague-Dawley (SD) rat in slices of 450 m. We observed the morphological development and differentiation of the hippocampus slices. Additionally, we measured the developmental expression of Bcl-2 and NeuN, -actin protein by western blotting. RESULTS: The hippocampal slice cultures revealed matured structures as early as 6 days in vitro (DIV) and a relatively high degree of tissue organization until 18 DIV. After 21 DIV, there were migration of the cells away from the margins of the slices. NeuN expressions at 7, 13, and 19 DIV were 1.42 +/- 0.38 (meanSD, NeuN/ beta-actin OD ratio, insert number=4, each insert had 5 culture slices), 2.95 +/- 0.53, 1.58 +/- 0.42 respectively. Bcl-2 expression at 6, 12, and 18 DIV were 0.56 +/- 0.18 (mean +/- SD, Bcl-2/beta-actin OD ratio, insert number=4, each insert had 5 culture slices), 1.22 +/- 0.28, 1.03 +/- 0.25. CONCLUSIONS: Organotypic slice cultures of a rat's hippocampus shows morphological maturation within 14 DIV and the highest NeuN and Bcl-2 level at about 13 DIV. It is expected that these findings would be useful as baseline data in the field of research on neuronal development and aging processes.

Change of Stratification of Three Dimensional Culture by Gingival Keratinocytes & Fibroblasts / 대한치주과학회지

Epithelial-mesenchymal interaction plays a important role in cell growth and differentiation. This interaction is already well known to have an importance during the organ development as well as cell growth and differentiation. However, in vitro experimental model is not well developed to reproduce in vivo cellular microenvironment which provide a epithelial-mesenchymal interaction. Because conventional monolayer culture lacks epithelial-mensenchymal interaction, cultivated cells have an morphologic, biochemical, and functional characteristics differ from in vivo tissue. Moreover, it's condition is not able to induce cellular differention due to submerged culture condition. Therefore, the aims of this study were to develop and evaualte the in vitro experimental model that maintains epithelial-mesenchymal interaction by organotypic raft culture, and to characterize biologic properties of three-dimensionally reconstituted oral keratinocytes by histological and immunohistochemical analysis. The results were as follow; 1. Gingival keratinocytes reconstituted by three-dimensional organotypic culture revealed similar morphologic characteristics to biopsied patient specimen showing stratification, hyperkeratinosis, matutation of epithelial architecture. 2. Connective tissue structure was matured, and there is no difference during stratification period of epithelial 3-dimensional culture. 3. The longer of air-exposure culture on three-dimensionally reconstituted cells, the more epithelial maturation, increased epithelial thickness and surface keratinization 4. In reconstitued mucosa, the whole epidermis was positively stained by anti-involucrin antibody, and there is no difference according to air-exposured culture period. 5. The Hsp was expressed in the epithelial layer of three-dimensionally cultured cells, especially basal layer of epidermis. The change of Hsp expression was not significant by culture stratification. 6. Connexin 43, marker of cell-cell communication was revealed mild immunodeposition in reconstitued epithelium, and there is no significant expression change during stratification. These results suggest that three-dimensional oragnotypic co-culture of normal gingival keratinocytes with dermal equivalent consisting type I collagen and gingival fibroblasts results in similar morphologic and immunohistochemical characteristics to in vivo patient specimens. And this culture system seems to provide adequate micro-environment for in vitro tissue reconstitution. Therefore, further study will be focused to study of in vitro gingivitis model, development of novel perioodntal disease therapeutics and epithelial-mensenchymal interaction.

Ultrastructural Study on Microtumor Spheroids from Human Malignant Glioma Specimens Maintained in Organotypic Culture

The Present study investigated not only the feasibility of organotypic spheroid culture system taken from human malignant gliomas, but also the similarities and differences between surgical specimens and cultured spheroids using light microscopy, electron microscopy, and flow cytometric examination. Surgically resected tumor specimens from eighteen human malignant gliomas were minced and explanted into agarose-coated culture wells. After three to five days, these microtumor fragments emerged as spheroids and could be maintained as organotypic spheroids for more than eight weeks. Measurements of the spheroids showed that they decreased during the initial two to three weeks and afterwards remained unaltered over a specific period of time. This growth pattern of the spheroids was consistent with the condition of tumors in vivo suggesting the linkage of cell proliferation and loss. Light microscopic and electron microscopic studies of the spheroids demonstrated that morphological structures were similar to those of the original tumor tissue in vivo and histopathologic characteristics of the original tumor were maintained over a long culture period. The spheroids contained connective tissues, blood vessels, macrophages, and neutrophils maintaining a three-dimensional architectural resemblance to the original tumors. Of three pairs of the surgical and spheroid specimen examined by the flow cytometry, one showed a change of ploidy pattern and two contained increased fractions of proliferating cells. It is concluded that this microtumor spheroid system can maintain the characteristics of the original tumors, and may serve as an alternative to the in vivo xenograft model for the research of brain tumor biology, invasion and immunology while providing a valuable technique for the evaluation of new therapies, such as biological response modifiers.