The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel''s cartilage, nasal glands, tongue, and ear.     

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.     

灯盏细辛的组织培养与快速繁殖

本研究以野生高含量灯盏花的叶片为外植体,在7个不同浓度NAA和BA组合的MS培养基上进行愈伤组织诱导、不定芽分化和增殖及生根的培养,确定了灯盏花快繁体系的最适培养条件：(1)初代培养基：(MS+6-BA) 0.5 mg.L-1+NAA0.1 mg.L-1;(2)丛生芽增殖培养基：(MS+6-BA) 2 mg.L-1+NAA 0.7 mg.L-1;(3) 生根培养基：(2/3MS+NAA) 0.5 mg.L-1+IBA0.8。     

两步外植体法高频再生麝香百合

采用两步外植体法,即以百合鳞片叶为初始外植体,以从初始外植体上长出的芽为次级外植体,成功建立了麝香百合的高频离体再生系统。对不同的BA浓度及次级外植体的不同部位对再生效果的影响,以及组培苗移栽前低温处理的影响进行了研究。结果表明:不同部位的次级外植体中,以短缩茎切片出芽快、整齐、芽数多且粗壮;以MS附加1.0 mg L-1 BA和0.1 mg L-1 NAA的培养基最适于麝香百合的分化。一个中等大小已脱春化的鳞茎通过两步外植体法能扩繁出54 000株左右的新植株,从鳞片叶开始至开花仅需8个月,而且4!低温处理对开花期的影响不大。     

A Novel Technique for the Reattachment of Large Coral Reef Sponges

Sponges are dominant components of coral reef ecosystems, often exceeding reef-building corals in abundance. Large sponges, often more than 1 m in diameter, may be hundreds to thousands of years old. When damaged or dislodged, large sponges usually die because they are unable to reattach to the reef substratum. Because suitable methods for reattaching dislodged sponges are lacking, they are typically excluded from coral reef restoration efforts. Here we present a novel technique for the reattachment of large sponges that was tested using the Caribbean Giant barrel sponge, Xestospongia muta . Transplants of X. muta were conducted at 15- and 30-m depth off Key Largo, Florida. Despite the active hurricane season of 2005, 90% of deep and 35% of shallow transplants survived, with nearly 80% reattaching to the substratum and growing after 2.3–3 years. This technique may be generally adapted for securing large sponges in coral reef restoration efforts.     

灯盏细辛的组织培养与快速繁殖

本研究以野生高含量灯盏花的叶片为外植体,在7个不同浓度NAA和BA组合的MS培养基上进行愈伤组织诱导、不定芽分化和增殖及生根的培养,确定了灯盏花快繁体系的最适培养条件:(1)初代培养基:(MS 6-BA)0.5 mg·L-1 NAA0.1 mg·L-1;(2)丛生芽增殖培养基:(MS 6-BA)2 mg·L-1 NAA0.7 mg·L-1;(3)生根培养基:(2/3MS NAA)0.5 mg·L-1 IBA0.8.     

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine''s micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells'' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells'' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.     

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease¹. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms² and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson''s disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.     

Micropropagation of Indian Rosewood by Tissue Culture

Multiple shoots were induced on excised hypocotyl segments andshoot tips of in vitro germinated seedlings of Indian rosewood(Dalbergia latifolia) on Murashige and Skoog's medium supplementedwith cytokinins and auxins. Roots were induced when individualshoots were treated first with half strength MS medium supplementedwith NAA, IAA and IBA (1 mg 1^–1 each) and subsequentlytransferred to hormone-free half-strength MS medium. The plantletswere then transferred to pots and grown in the greenhouse. Dalbergia latifolia, micropropagation, tissue culture, clonal propagation     

A Novel Culture Method for High Level Production of Heterologous Protein in Saccharomyces cerevisiae

A high level production system for heterologous protein by cold culture of yeast transformants at 15°C was developed. The yeast transformants, carrying a plasmid containing cDNA for Aspergillus oryzae α-amylase (Taka-amylase A) or human lysozyme synthetic DNA, were cultivated in a selective medium for 1 or 2 days until full growth at 30°C. The yeast cells were harvested by centrifugation from the culture fluid and then were transferred to YPD medium. These inoculated broths were incubated for 2 days at 15°C and then for another 2 days at 30°C. By the cold culture method described above, higher amounts of Taka-amylase A (28.6 mg/liter) and human lysozyme (6.1 mg/liter) were produced by the yeast transformants compared to those by conventional methods.

Heterologous protein productions using YEp, YCp, and YIp types of yeast expression vectors with ADH1 or GAPDH promoter by the cold culture method showed effective productivity of about 2-fold compared to those by the conventional method of culture at 30°C. The high level production of heterologous protein by this method was not specific to the S. cerevisiae strains examined.     

象草腋芽外植体消毒方法的筛选

为了解决象草组织培养中外植体污染问题,提高象草组织培养的成功率。本研究运用正交设计实验设置自来水冲洗、0.1%升汞浸泡和2%次氯酸钠浸泡3因子的不同消毒组合,通过直观分析和方差分析对象草腋芽外植体消毒灭菌结果进行分析。结果表明,象草腋芽外植体消毒的最佳组合为:自来水冲洗60min,0.1%升汞浸泡25min,2%次氯酸钠浸泡20min。该组合不仅能使污染率降低到13.33%,同时又在外植体的承受范围之内,没有对外植体造成伤害,本文结果将为象草的离体再生培养提供技术参考。     

一种研究和鉴别悬浮培养红豆杉细胞凋亡与坏死的新方法

将在动物细胞凋亡研究中应用的Hoechst-PI双重荧光染色法与琥珀酸脱氢酶(SDH)染色法相结合,建立了一种更加完善的、能同时鉴别和研究悬浮培养的植物细胞凋亡及坏死的新方法——Hoechst-PI-SDH三重染色法(H-P-S法)。该方法可直接用于红豆杉悬浮培养细胞,无需对细胞进行去壁、固定及切片等其它方法所必需的步骤,在荧光显微镜下可鉴别活细胞、死细胞及凋亡细胞,并可同时观测细胞凋亡的全部过程。该方法简单、快速、准确,而且克服了因细胞膜通透性差异引起的对死、活细胞判断的困难,可在植物细胞凋亡的研究中广泛应用。     

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 µm in diameter, in PA gels, within 1.6 μm of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.     

植物组织培养新技术:光自养微繁

系统地综述了常规植物组织培养存在的不足,如易染菌、生长周期长、生产成本高等,从而引出了光自养微繁的概念、研究现状、控制方向以及它的优势。如植株长势较好、生长周期短、生产成本低等,并对该技术做了展望。光自养微繁技术作为一种新型的组织培养方法,克服了传统组培无法克服的缺陷,必将成为今后组培生产的一种重要手段。     

New Method for Counting Bacteria Associated with Coral Mucus

The ability to count bacteria associated with reef-building corals in a rapid, reliable, and cost-effective manner has been hindered by the viscous and highly autofluorescent nature of the coral mucus layer (CML) in which they live. We present a new method that disperses bacterial cells by trypsinization prior to 4′,6-diamidino-2-phenylindole (DAPI) staining and quantification by epifluorescence microscopy. We sampled seawater and coral mucus from Porites lobata from 6 reef sites influenced by wastewater intrusion and 2 reef sites unaffected by wastewater in Hawaii. Bacterial and zooxanthella abundances and cell sizes were quantified for each sample. Bacteria were more abundant in coral mucus (ranging from 5.3 × 10⁵ ± 1.0 × 10⁵ cells ml⁻¹ to 1.8 × 10⁶ ± 0.2 × 10⁶ cells ml⁻¹) than in the surrounding seawater (1.9 × 10⁵ ± 0.1 × 10⁵ cells ml⁻¹ to 4.2 × 10⁵ ± 0.2 × 10⁵ cells ml⁻¹), and the mucus-associated cells were significantly smaller than their seawater counterparts at all sites (P < 0.0001). The difference in cell size between mucus- and seawater-associated bacteria decreased at wastewater-influenced sites, where simultaneously mucus bacteria were larger and seawater bacteria were smaller than those at uninfluenced sites. The abundance of zooxanthellae in mucus ranged from 1.1 × 10⁵ ± 0.1 × 10⁵ cells ml⁻¹ to 3.4 × 10⁵ ± 0.3 × 10⁵ cells ml⁻¹. The frequency of dividing cells (FDC) was higher in the surrounding seawater than in mucus, despite finding that a 1,000-fold-higher zooxanthella biovolume than bacterial biovolume existed in the CML. Establishment of a standardized protocol for enumeration will provide the field of coral microbial ecology with the urgently needed ability to compare observations across studies and regions.The extremely viscous and highly autofluorescent nature of coral mucus has been a major challenge in developing enumeration techniques and has limited our ability to study the ecological interactions among coral mucus layer (CML)-associated microbial communities. Only a few studies have used direct counts to quantify bacteria in the CML, and the methods and subsequent results vary widely. The techniques have included scanning electron microscopy (SEM) (34), phase-contrast microscopy (27), and epifluorescent microscopy using a variety of stains (acridine orange staining [8], SYBR gold [20], and 4′,6-diamidino-2-phenylindole [DAPI] [3]). Bacterial abundances reported from these studies spanned more than 5 orders of magnitude (from 1.6 × 10² cells [cm²]⁻¹ using acridine orange [8] to 6.2 × 10⁷ cells [cm²]⁻¹ using SYBR gold [20]), and some of the studies are difficult to compare to each other because different units were used, such as cells ml⁻¹ of mucus and cells (cm²)⁻¹ of coral. Some variation in abundance is likely due to differences in mucus sampling methods and differences among coral species. However, the enormous quantity of autofluorescence emitted in green and red wavelengths found in most coral species creates a substantial challenge for reliably counting fluorescently stained cells in that portion of the spectrum, because many of the particles are bacterium sized. Many of these same particles could be visible with phase-contrast microscopy as well. Thus far, researchers quantifying CML-associated bacteria using epifluorescence microscopy have prepared their samples by following well-established protocols that were developed for seawater. We suggest that the viscous and autofluorescent nature of coral mucus may require some modifications from standard seawater protocols for epifluorescence microscopy to be most effective.SEM is an alternative to fluorescence-dependent techniques. It has the advantage of acquiring images with sufficient detail to distinguish among particles and cells, but this method is time-consuming, visualizes only the surface of the sample, and is not widely available or affordable enough for it to be a standard field protocol. An additional limitation is that most studies that have employed SEM for CML observation have found bacteria to be too dispersed to count in a reasonable number of micrographs (8, 19).Here we present a new method that disperses bacterial cells by enzymatically digesting the mucus with trypsin (an adaptation of routine cellular biology cell line culture procedures) and subsequently staining the cells with DAPI for rapid quantification using epifluorescence microscopy. DAPI fluoresces in the blue end of the spectrum, and its emission does not overlap with the autofluorescence of the mucus samples. This method is rapid, uses reagents and equipment readily available in microbial ecology laboratories, and can provide necessary information for studies of the ecology of microbial cells associated with mucus. It may also be helpful for studies of other aquatic gel-associated microbial communities.This visualization capability revealed that bacteria living with the reef-building coral Porites lobata were significantly smaller than their water-associated counterparts and that this difference is reduced in reefs heavily influenced by anthropogenic impacts. There is only one other report that we are aware of that observed small bacterial cell size in mucus from corals (of the genus Fungia), but that study did not quantify cell size (34). Given that mucus is a carbon-rich environment (6, 11, 12, 18, 24, 25, 31), this discovery is counterintuitive. It highlights questions regarding the ecological interactions that must occur in situ to select for small cell size in such a rich environment (3, 4, 7, 8, 11, 25, 34).     

The “Flexi-Chamber”: A Novel Cost-Effective In Situ Respirometry Chamber for Coral Physiological Measurements

Coral reefs are threatened worldwide, with environmental stressors increasingly affecting the ability of reef-building corals to sustain growth from calcification (G), photosynthesis (P) and respiration (R). These processes support the foundation of coral reefs by directly influencing biogeochemical nutrient cycles and complex ecological interactions and therefore represent key knowledge required for effective reef management. However, metabolic rates are not trivial to quantify and typically rely on the use of cumbersome in situ respirometry chambers and/or the need to remove material and examine ex situ, thereby fundamentally limiting the scale, resolution and possibly the accuracy of the rate data. Here we describe a novel low-cost in situ respirometry bag that mitigates many constraints of traditional glass and plexi-glass incubation chambers. We subsequently demonstrate the effectiveness of our novel “Flexi-Chamber” approach via two case studies: 1) the Flexi-Chamber provides values of P, R and G for the reef-building coral Siderastrea cf. stellata collected from reefs close to Salvador, Brazil, which were statistically similar to values collected from a traditional glass respirometry vessel; and 2) wide-scale application of obtaining P, R and G rates for different species across different habitats to obtain inter- and intra-species differences. Our novel cost-effective design allows us to increase sampling scale of metabolic rate measurements in situ without the need for destructive sampling and thus significantly expands on existing research potential, not only for corals as we have demonstrated here, but also other important benthic groups.     

Organ Explant Culture of Neonatal Rat Ventricles: A New Model to Study Gene and Cell Therapy

Testing cardiac gene and cell therapies in vitro requires a tissue substrate that survives for several days in culture while maintaining its physiological properties. The purpose of this study was to test whether culture of intact cardiac tissue of neonatal rat ventricles (organ explant culture) may be used as a model to study gene and cell therapy. We compared (immuno) histology and electrophysiology of organ explant cultures to both freshly isolated neonatal rat ventricular tissue and monolayers. (Immuno) histologic studies showed that organ explant cultures retained their fiber orientation, and that expression patterns of α-actinin, connexin-43, and α-smooth muscle actin did not change during culture. Intracellular voltage recordings showed that spontaneous beating was rare in organ explant cultures (20%) and freshly isolated tissue (17%), but common (82%) in monolayers. Accordingly, resting membrane potential was -83.9±4.4 mV in organ explant cultures, −80.5±3.5 mV in freshly isolated tissue, and −60.9±4.3 mV in monolayers. Conduction velocity, measured by optical mapping, was 18.2±1.0 cm/s in organ explant cultures, 18.0±1.2 cm/s in freshly isolated tissue, and 24.3±0.7 cm/s in monolayers. We found no differences in action potential duration (APD) between organ explant cultures and freshly isolated tissue, while APD of monolayers was prolonged (APD at 70% repolarization 88.8±7.8, 79.1±2.9, and 134.0±4.5 ms, respectively). Organ explant cultures and freshly isolated tissue could be paced up to frequencies within the normal range for neonatal rat (CL 150 ms), while monolayers could not. Successful lentiviral (LV) transduction was shown via Egfp gene transfer. Co-culture of organ explant cultures with spontaneously beating cardiomyocytes increased the occurrence of spontaneous beating activity of organ explant cultures to 86%. We conclude that organ explant cultures of neonatal rat ventricle are structurally and electrophysiologically similar to freshly isolated tissue and a suitable new model to study the effects of gene and cell therapy.     

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.