Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology

Use of an ethylene glycol based cryoprotectant solution has been found to be effective for the long-term storage of brain tissue either in block form or as freely floating sections prior to immunocytochemical processing. Storage of tissue in the solution at -20 degrees C or 4 degrees C for up to 3 months produced no adverse effects upon tissue morphology, nor was LHRH immunoreactivity diminished or accompanied by elevated non-specific staining. Furthermore, ultrastructural analysis of cryoprotected tissue revealed excellent preservation of cellular morphology. It is anticipated that this method can find use when it is necessary or desirable for the investigator to retain tissue for later immunocytochemical or electron microscopic processing.     

Optimization of immunohistochemical techniques to detect extracellular matrix proteins in fixed skin specimens

Complete antigen visualization in the context of well-preserved tissue architecture is the goal of all immunohistochemical techniques. Frozen tissue section techniques achieve optimal antigen visualization but preserve tissue architecture poorly. On the other hand, formalin-fixed tissue section techniques preserve tissue architecture very well but result in antigen masking. Enzymatic digestion or salt extraction of formalin-fixed sections has been used to reestablish antigen expression. Recently acid-alcohol-fixed tissue has been used as a successful compromise between tissue architecture preservation and the visualization of cytoskeletal antigens. In an attempt to find an improved immunohistochemical process for non-cytoskeletal antigens, we compared avidin-biotin immunofluorescence staining in frozen, formalin-fixed, and acid-alcohol-fixed tissues. The fixed tissues were either untreated or treated with enzyme digestion or salt extraction. For this study, we examined healing cutaneous wounds in Yorkshire pigs with antibodies to fibronectin, laminin, von Willebrand factor VIII, and keratin. Although tissue architecture was poor, frozen sections provided the best antigen visualization and were therefore used as the standard for complete antigen expression. Formalin-fixed tissues had excellent tissue architecture, but most antigens were completely masked. Pre-treatment technique only partially overcame the antigen masking caused by formalin. In contrast, acid-alcohol fixation preserved tissue architecture almost as well as formalin and sometimes allowed complete antigen visualization; however, laminin and fibronectin were partially masked. Total recovery of the expression of these antigens could be obtained by pre-treating the acid-alcohol-fixed tissue with either hyaluronidase or 1 M NaCl. Therefore, acid-alcohol-fixed tissue appears best for extracellular matrix (ECM) protein immunostaining as well as for cytoskeletal staining. However, certain ECM antigens require hyaluronidase or 1 M NaCl treatment for optimal visualization.     

Cryopreservation of Bovine Ovarian Tissue: Structural Normality of Follicles after Thawing and Culturein Vitro

The recovery of viable follicles from cryopreserved ovarian tissue would be of benefit in many areas of assisted reproduction. Structural integrity needs to be maintained following cryopreservation of ovarian tissue in order to retrieve healthy follicles which can then be cultured in vitro to produce viable oocytes. We have assessed the effect of in vitro culture of bovine tissue for 0, 1, 4, 24, or 48 h after exposure to, or cryopreservation in, dimethylsulphoxide. Immediately after freezing, normality of primary and preantral follicles within the tissue was significantly lower than for tissue exposed to the cryoprotectant without freezing or for control tissue. After 4 h in culture, cryopreserved tissue appeared to have recovered from damage caused by freezing, although the percentage of tissue with normal morphology declined after 24 and 48 h of culture. There was no significant difference between percentage normality in control tissue and tissue exposed to the cryoprotectant without freezing for any of the culture times studied. These data indicate that it is possible to freeze/thaw bovine ovarian tissue while retaining a reasonable yield of morphologically intact follicles and that a short period of post-thaw culture may enhance follicle recovery.     

RAPID CHEMICAL DEHYDRATION OF PLANT MATERIAL FOR LIGHT AND ELECTRON MICROSCOPY WITH 2,2-DIMETHOXYPROPANE AND 2,2-DIETHOXYPROPANE

A rapid and simple procedure was used for chemical dehydration of plant tissue during sample preparation for light and electron microscopy. Chemically fixed tissues were washed with distilled water and then rapidly dehydrated with either 2,2-dimethoxypropane or 2,2-diethoxypropane for 15 minutes. Light microscopic observation of paraffin-embedded tissue or tissue embedded in Spurr's plastic showed excellent preservation. Electron microscopic examination of plastic-embedded tissue showed well maintained ultrastructural morphology. The dehydration procedure was also successfully applied to plant tissue destined for examination in a scanning electron microscope.     

The in vitro maintenance of the apical ectodermal ridge of the chick embryo wing bud: an assay for polarizing activity.

We have devised an in vitro bioassay for limb bud polarizing activity in the chick embryo. This assay has proven to be a relatively quick and effective test for a morphogenetic factor asymmetrically distributed in the limb bud which is capable of maintaining or thickening the apical ectodermal ridge.A small section of the preaxial border of the chick embryo wing bud was cultured alone, with tissue from the posterior border, mid-dorsal or anterior corner of a second donor wing, or from the flank. The tissue from the preaxial border (responding tissue) consisted of mesoderm with overlying ectoderm and apical ectodermal ridge. When the responding tissue was cultured alone, with flank, or with anterior corner limb tissue, the apical ectodermal ridge flattened in 24–36 hr and many macrophages appeared in the underlying mesoderm. When cultured with posterior border limb tissue however, the apical ridge of the responding tissue remained thickened for up to 48 hr., and no macrophages appear in the underlying mesoderm. The behavior of responding tissue was intermediate between these two extremes when cultured with mid-dorsal limb tissue. The morphogenetic activity assayed by this procedure thus seems to be present as a gradient in the wing bud, with activity decreasing from posterior to anterior. Contact with the responding tissue is not required to enable posterior border tissue to elicit ridge thickening and inhibit the cell death.     

Effects of time and temperature during attachment of sections to microscope slides on immunohistochemical detection of antigens.

To study the effects of time and temperature on attachment of tissue sections to microscope slides, we examined the intensity of immunohistochemical staining of selected antigens in nine different neoplastic and normal tissues after attaching sections at different times and temperatures. Typically, both the temperature and time are minimized when tissue sections attached to slides; however, suboptimal times and temperatures during attachment may result in either loss of tissue due to poor attachment or the necessity for inconvenient staining regimens. Using standard immunohistochemical techniques, 5 microm tissue sections were attached at 58 degrees C for 1, 4 and 24 hr. In a separate study, 5 microm tissue sections were attached for 16 hr at 58, 68 and 80 degrees C. The intensity of staining decreased slightly when the tissue sections were heated at 80 degrees C for 16 hr, but there was little or no decrease when tissues were heated at 68 degrees C or lower for 16 hr, or at 58 degrees C for up to 24 hr.     

Freeze-fracture of monolayer cultures

This paper describes a simple method for the freeze-fracturing of cells in monolayers or multi-layer tissue cultures. The method produces high quality replicas and is applicable to the study of virtually any tissue culture or organ culture system. It uses standard materials and equipment for both tissue culture and freeze-fracturing.     

Inductive tissue engineering with protein and DNA-releasing scaffolds

Cellular differentiation, organization, proliferation and apoptosis are determined by a combination of an intrinsic genetic program, matrix/substrate interactions, and extracellular cues received from the local microenvironment. These molecular cues come in the form of soluble (e.g. cytokines) and insoluble (e.g. ECM proteins) factors, as well as signals from surrounding cells that can promote specific cellular processes leading to tissue formation or regeneration. Recent developments in the field of tissue engineering have employed biomaterials to present these cues, providing powerful tools to investigate the cellular processes involved in tissue development, or to devise therapeutic strategies based on cell replacement or tissue regeneration. These inductive scaffolds utilize natural and/or synthetic biomaterials fabricated into three-dimensional structures. This review summarizes the use of scaffolds in the dual role of structural support for cell growth and vehicle for controlled release of tissue inductive factors, or DNA encoding for these factors. The confluence of molecular and cell biology, materials science and engineering provides the tools to create controllable microenvironments that mimic natural developmental processes and direct tissue formation for experimental and therapeutic applications.     

Relationship between luteinizing hormone and decidual luteotropin in the maintenance of luteal steroidogenesis

Between Days 6-11 of pregnancy or pseudopregnancy, the decidual tissue of the rat produces a prolactin-like hormone, decidual luteotropin, which can sustain luteal progesterone production when prolactin is suppressed. However, this effect is dependent upon the presence of the pituitary. The present investigation was undertaken to determine whether decidual luteotropin and luteinizing hormone (LH) act together to sustain luteal steroidogenesis and if so, to find out whether the need for LH is due to the inability of the decidual tissue to produce LH-like material and/or whether LH affects decidual luteotropin production. Pseudopregnant rats with or without decidual tissue were hypophysectomized on Day 8 and treated with either 1.5 IU human chorionic gonadotropin (hCG)/day or with vehicle. Within 24 h, serum progesterone dropped in both vehicle-treated groups and decidual luteotropin levels declined by 80% in the decidual tissue. Human CG administration had no effect on progesterone production in the control group. Yet in rats with decidual tissue, hCG stimulated progesterone production for at least 48 h and maintained the decidual tissue content of decidual luteotropin. Progesterone, but not hCG treatment, maintained decidual luteotropin concentrations in ovariectomized rats. To compare the luteotropic activity of the decidual tissue with that of the placenta, pregnant or pseudopregnant rats with decidual tissue were hypophysectomized on Day 8 and treated with 1.5 IU hCG. Control groups had decidual tissue or placentas removed and were similarly treated. Human CG stimulated progesterone production only in rats with placental or decidual tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue transplantation by stealth—Coherent alginate microcapsules for immunoisolation

Several of today's most prominent diseases arise as a result of aberrant secretory or enzymatic function, or loss of the corresponding tissue. While an obvious approach to treating these diseases is to simply replace the malfunctioning, damaged, or lost tissue, the need to immunosuppress recipients places them at increased risk of pathogenic infection and development of cancer. Limited availability of donor tissue is also problematic, and while xenogenic tissue could be used in some instances, immunosuppression alone is insufficient to prevent rejection. As an alternative, a physical barrier could be employed to mask the implanted tissue from immune surveillance at a local level, without the need for systemic immunosuppression. This approach is embodied through tissue microencapsulation. We review the underlying rationale of alginate based tissue microencapsulation technologies, current challenges, and possible solutions to these challenges in the form of a simple emulsion coating system incorporating functionalized alginate for local immunomodulation.     

Freeze-drying allows double nonradioactive ISH and antigenic labeling.

Because tissue freeze-drying is an excellent way to preserve antigenic conformation, we have tested the feasibility of this technique to reveal nonradioactive in situ hybridization (ISH) of tissue mRNA. We have compared mRNA detection after different methods of tissue preservation, freeze-drying, cryosectioning, and formaldehyde or methanol fixation. Our results show that nonradioactive ISH is more sensitive for tissues preserved by freeze-drying than for other tissue preparations. We have demonstrated that freeze-drying allows combination of ISH and immunohistochemistry for simultaneous detection of mRNA and antigen because with this technique of tissue preservation ISH does not affect the sensitivity or the amount of the detected antigens. This work underscores the fact that tissue freeze-drying is an easy, convenient, and reliable technique for both ISH and immunohistochemistry and achieves excellent structural conditions for nonradioactive detection.     

Effects of high-pressure processing on Toxoplasma gondii tissue cysts in ground pork

Ingestion of Toxoplasma gondii tissue cysts can result in severe disease in immunocompromised individuals and pregnant women. Treatment of meat and meat products to eliminate viable T. gondii tissue cysts would provide a means to protect consumers. In this study, we examined the effects of high-pressure processing (HPP) on ground pork containing viable tissue cysts of the VEG strain of T. gondii. Ground pork containing tissue cysts was exposed to 400, 300, 200, 100, or 0 MPa treatment for 30, 60, or 90 sec in a commercial HPP unit. The HPP-treated ground pork was subjected to acid-pepsin digestion and bioassayed in mice. The results of the mouse bioassay revealed that none of the mice inoculated with tissue cysts exposed to 400 or 300 MPa became infected, whereas all mice inoculated with tissue cysts exposed to 200, 100, or 0 MPa became infected with T. gondii regardless of exposure time. Results indicate that HPP treatment of ground pork with 300 MPa of pressure will render tissue cysts of T. gondii nonviable and make pork safe for human consumption.     

Musculoskeletal tissue engineering by endogenous stem/progenitor cells

From its inception, tissue engineering has had three tenets: cells, biomaterial scaffolds and signaling molecules. Among the triad, cells are the center piece, because cells are the building blocks of tissues. For decades, cell therapies have focused on the procurement, manipulation and delivery of healthy cells for the treatment of diseases or trauma. Given the complexity and potential high cost of cell delivery, there is recent and surging interest to orchestrate endogenous cells for tissue regeneration. Biomaterial scaffolds are vital for many but not all, tissue-engineering applications and serve to accommodate or promote multiple cellular functions. Signaling molecules can be produced by transplanted cells or endogenous cells, or delivered specifically to regulate cell functions. This review highlights recent work in tissue engineering and cell therapies, with a focus on harnessing the capacity of endogenous cells as an alternative or adjunctive approach for tissue regeneration.     

Growth of Pathogenic Virus in a Large-Scale Tissue Culture System

A model system is described for the mass propagation of Rift Valley fever (RVF) virus, utilizing large-volume fermentor units for suspension culture of tissue cells and the subsequent production of virus. Comparisons between laboratory- and fermentor-scale operations of tissue cell growth gave equivalent results. Cell viability dropped 24 to 30 hr postinfection with a subsequent virus yield between 10(8.0) and 10(9.0) mouse intracerebral median lethal doses per milliliter. Infecting volumes of tissue cell culture (20- or 40-liter working volumes) had no apparent effect on virus yields. Tissue cells grown under either oxidation-reduction potential- and pH-controlled or uncontrolled conditions showed little or no difference in their ability to produce RVF virus. We believe this tissue cell virus process to have potential application for large-scale production of vaccines for human or veterinary use or for the mass propagation of certain carcinogenic viruses for cancer research, once use of established lines for this purpose is accepted.     

A novel device for the simple and efficient refinement of liposuctioned tissue

In short, our device allows a surgeon who is harvesting adipose tissue for autologous fat transplantation to immediately, easily, efficiently, and sterilely isolate adipose tissue from the unwanted waste components that are associated with primary liposuction effluent. It does so by "trapping" the fat tissue contained within raw liposuction effluent. Once the tissue fraction has been separated, the device design then allows for direct implantation or subsequent washing/rinsing of the tissue with saline/buffer of choice in preparation for tissue reimplantation.     

Role of plasma as activator and cofactor in phosphorylation catalyzed by protein kinase C

The purpose of this study was to investigate whether plasma can influence the phosphorylation of protein kinase C (PKC). Lysate samples were prepared from normal skin or melanoma tissue and were reacted with a PKC peptide substrate in the presence or absence of plasma. In normal skin tissue lysates, the phosphorylation rates were much lower than those in melanoma tissue lysates. However, the level of phosphorylated peptide was increased in both normal skin and melanoma tissue lysates if plasma was present. Phosphorylation rates in the samples taken from the centre of B16 melanoma tissue were lower than those in samples taken from the edge. Moreover, addition of activator and/or cofactors (diacylglycerol, phosphatidylserine and/or Ca2+) of PKC, or plasma to the lysates contaminated by plasma had no effect on phosphorylation rates for the peptide substrate. These results indicate that plasma can play a role of activator and cofactor for substrate phosphorylation.     

Some biochemical effects of the growth hormone analogue produced by plerocercoids of the tapeworm Spirometra mansonoides on carbohydrate metabolism of adipose tissue from normal, diabetic, and hypophysectomized rats

Plerocercoids of Spirometra mansonoides produce a functional analogue of mammalian growth hormone (GH). Plerocercoid growth factor (PGF) mimics the growth-promoting actions of GH, but has not been shown to duplicate all of the actions reported for GH. The purpose of this study was to determine the effects of plerocercoid infection (chronic PGF treatment) on glucose metabolism of adipose tissue and to compare the effects to those elicited by insulin and GH in intact, diabetic, and hypophysectomized male rats. Groups of rats were constantly exposed to PGF (via plerocercoid infection) or injected twice daily with bovine GH, insulin, or saline for 10 days. Basal oxidation rates of [U-14C]glucose to 14CO2 in adipose tissue segments were measured in vitro immediately after tissue removal. Other aliquots of adipose tissue were preincubated in hormone-free medium for 3 hr prior to testing the ability of the tissue to respond to insulin or human GH (hGH) added in vitro. Adipose tissue from PGF-treated intact and hypophysectomized rats had significantly elevated basal glucose oxidation rates, and the tissue was sensitive to further stimulation by insulin or hGH. The results obtained with intact and hypophysectomized rats were essentially the same, indicating that the effects of PGF were not due to suppression of endogenous GH. The basal glucose oxidation rate in adipose tissue from diabetic rats was stimulated (P less than 0.01) by PGF, but the tissue was not sensitive to insulin added in vitro. Furthermore, PGF had no effect on body growth or blood glucose concentrations of diabetic rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repair and regeneration: opportunities for carcinogenesis from tissue stem cells

This review will discuss the mechanisms of repair and regeneration in various tissue types and how dysregulation of these mechanisms may lead to cancer. Normal tissue homeostasis involves a careful balance between cell loss and cell renewal. Stem and progenitor cells perform these biologic processes as the functional units of regeneration during both tissue homeostasis and repair. The concept of tissue stem cells capable of giving rise to all differentiated cells within a given tissue led to the concept of a cellular hierarchy in tissues and in tumors. Thus, only a few cells may be necessary and sufficient for tissue repair or tumor regeneration. This is known as the hierarchical model of tumorigenesis. This report will compare this model with the stochastic model of tumorigenesis. Under normal circumstances, the processes of tissue regeneration or homeostasis are tightly regulated by several morphogen pathways to prevent excessive or inappropriate cell growth. This review presents the recent evidence that dysregulation of these processes may provide opportunities for carcinogenesis for the long-lived, highly proliferative tissue stem cell population. New findings of cancer initiating tissue stem cells identified in several solid and circulating cancers including breast, brain and hematopoietic tumors will also be reviewed. Finally, this report reviews the cellular biology of cancer and its relevance to the development of more effective cancer treatment protocols.     

组织器官三维构建及原位组织工程概念——组织工程连载之四

组织器官三维构建就是把种子细胞和支架材料结合而获得设计的组织或器官,属于组织工程的核心内容,也最能体现组织工程的技术水平,如血管、气管的构建。由于传统组织工程存在缺陷,Shimizu于1998年首先提出了原位组织工程的概念,它是运用组织工程学基本原理,通过各种方法诱导移植的外源性的种子细胞或内源性的缺损组织局部细胞发生迁移、增殖、分化形成新生组织修复缺损。原位组织工程最大的特点是不依赖体外的细胞培养装置--生物反应器。原位组织工程是传统离体组织工程的有益补充。离体组织工程仍具有广阔的发展前景。     

An abnormally developed embryo of the pill millipede Glomeris marginata that lacks dorsal segmental derivatives

The body of arthropods is subdivided in serially homologous units, the so-called segments. In many arthropods, ventral and dorsal segmental tissue typically is aligned in parallel, but is dependent on different genetic inputs. In the pill millipede Glomeris marginata (Myriapoda: Diplopoda), ventral and dorsal segmental patterning is clearly decoupled providing an excellent model for the investigation of ventral versus dorsal segmentation mechanisms. This paper reports on the finding of a single embryo that lacks dorsal segmental and extraembryonic tissue. Ventral derivatives, however, are widely developed normally. This suggests that ventral and dorsal tissue is not only patterned differently, as shown previously, but also that ventral tissue can develop (or at least persist) independently from dorsal tissue. It also suggests a correlation of dorsal segmentation and function of the extraembryonic tissue. This assumed correlation may involve the guidance of the two dorsal hemispheres of the developing embryo dorsally, or that formation and/or maintenance of extraembryonic tissue depends on the input of dorsal segmental tissue. Whether the observed abnormalities are caused by mutation or are the result of otherwise disturbed early development is unclear.