In vitro chili pepper biotechnology

Summary Chili pepper is an important horticultural crop that can surely benefit from plant biotechnology. However, although it is a Solanaceous member, developments in plant cell, tissue, and organ culture, as well as on plant genetic transformation, have lagged far behind those achieved for other members of the same family, such as tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), and potato (Solanum tuberosum), species frequently used as model systems because of their facility to regenerate organs and eventually whole plants in vitro, and also for their ability to be genetically engineered by the currently available transformation methods. Capsicum members have been shown to be recalcitrant to differentiation and plant regeneration under in vitro conditions, which in turn makes it very difficult or inefficient to apply recombinant DNA technologies via genetic transformation aimed at genetic improvement against pests and diseases. Some approaches, however, have made possible the regeneration of chili pepper plants from in vitro-cultured cells, tissues, and organs through organogenesis or embryogenesis. Anther culture has been successfully applied to obtain haploid and doubledhaploid plants. Organogenic systems have been used for in vitro micropropagation as well as for genetic transformation. Application of both tissue culture and genetic transformation techniques have led to the development of chili pepper plants more resistant to at least one type of virus. Cell and tissue cultures have been applied successfully to the selection of variant cells exhibiting increased resistance to abiotic stresses, but no plants exhibiting the selected traits have been regenerated. Production of capsaicinoids, the hot principle of chili pepper fruits, by cells and callus tissues has been another area of intense research. The advances, limitations, and applications of chili pepper biotechnology are discussed.     

A Novel Feeder-Free Culture System for Expansion of Mouse Spermatogonial Stem Cells

Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost- effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.     

Towards cellulose synthesis in vitro from plant extracts

The synthesis of cellulose in vitro has occupied the attention of numerous biochemists for several decades without success. Since chitin has been synthesized in vitro there does not appear to be any basic reason why the same should not be possible for cellulose. However consideration of the problems involved in the in vitro systems used hitherto from plant cells indicate that the problems involved are not as easily overcome as one might think. Possible alternatives to the previously used systems are discussed which might, after suitable experimentation, provide a better indication of how to solve this major problem of biosynthesis.     

Supporting Partners of the IAPB 14th Quadrennial Congress Dublin as of July 12th 2018

Melatonin is a ubiquitously present indoleamine with a vast capacity for modulating the growth and behavior of plants, animals, and microbes. Though melatonin was discovered in plants decades after its discovery in mammals, its presence has now been confirmed in almost all plant families. Despite this, the in vitro and in vivo mechanisms of action of melatonin are still poorly defined. Although there are an increasingly large number of investigations into the roles of melatonin in plants, few take advantage of in vitro culture systems. Melatonin has been found to possess several important roles in plant growth and development, including functions in rhythmic and cyclic processes, such as chronoregulation, seasonal and senescence processes, as well as modulation of reproductive development, control of root and shoot organogenesis, maintenance of plant tissues, and responses to biotic and abiotic stresses. This review highlights the potential for use of melatonin in several in vitro systems, the roles it plays in plant morphogenesis, and the importance of melatonin in communication within and between plants, and how in vitro systems can be exploited to better understand these understudied functions of melatonin.     

Recent advances in the in vitro cultivation and genetic manipulation of Echinococcus multilocularis metacestodes and germinal cells

In order to elucidate host-parasite interactions and infection strategies of helminths at the molecular level, the availability of suitable in vitro cultivation systems for this group of parasites is of vital importance. One of the few helminth systems for which in vitro cultivation has been relatively successfully carried out in the past is the larval stage of the fox-tapeworm Echinococcus multilocularis, the causative agent of alveolar echinococcosis. Respective ‘first generation’ cultivation systems relied on the co-incubation of larval tissue, isolated from laboratory rodents, with host feeder cells. Although these techniques have been very successful in producing metacestode material for drug screening assays or the establishment of cDNA libraries, the continuous presence of host cells prevented detailed studies on the influence of defined host factors on larval growth. To facilitate such investigations, we have recently introduced the first truly axenic system for long-term in vitro maintenance of metacestode vesicles and used it to establish a technique for parasite cell cultivation. The resulting culture system, which allows the complete in vitro regeneration of metacestode vesicles from germinal cells, is a highly useful tool to study the cellular and molecular basis of a variety of developmental processes that occur during the infection of the mammalian host. Furthermore, it provides a solid basis for establishing transgenic techniques in cestodes for the first time. We consider it an appropriate time point to discuss the characteristics of these ‘second generation’ cultivation systems in comparison with former techniques, to present our first successful attempts to introduce foreign DNA into Echinococcus cells, and to share our ideas on how a fully transgenic Echinococcus strain can be generated in the near future.     

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.     

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.     

Design of experiments (DOE)—history,concepts, and relevance to <Emphasis Type="Italic">in vitro</Emphasis> culture

Design of experiments (DOE) is a large and well-developed field for understanding and improving the performance of complex systems. Because in vitro culture systems are complex and easily manipulated in controlled conditions, they are particularly well-suited for the application of DOE principles and techniques. Successful use of in vitro technologies in horticultural, plant breeding, or genetic applications typically involves improving some aspect of a system’s growth response—organogenesis, somatic embryogenesis, metabolite biosynthesis, or responses required for crop improvement such as ploidy manipulation, embryo rescue, creation and manipulation of chimeras, somaclonal variation, and mutant isolation. How and why DOE is the appropriate research approach for developing and understanding in vitro systems research is explained. The presentation is a narrative of the historical context and the geometric basis of DOE to explain the underlying concepts. Examples illustrate the use of DOE in in vitro plant culture research.     

Detection of chemically induced aneuploidy with plant test systems

The utility of plant test systems for detecting chemically induced aneuploidy was evaluated by using papers published in peer-reviewed journals. A total of 147 papers were provided to the group by the Environmental Mutagen Information Center. Based on the criteria established by the Gene-Tox Committee (Waters and Auletta, 1981), 22 papers were selected for in-depth review. Only those papers listing additional, missing, or lagging chromosomes in the meiotic or mitotic cells were included in this review. Although most plant test systems may be developed to utilize either mitotic or meiotic cells for cytogenetic analysis, only a few have been employed for this purpose. In this review, Allium cepa was found to be the most commonly used test system. Other species used less frequently were Vicia faba, Hordeum vulgare, Sorgham vulgare, and Pennisetum americanum. None of the plant test systems have been sufficiently utilized to warrant judgment for its sensitivity and specificity for detecting induced aneuploidy. A suggested protocol for detecting chromosomal malsegregation in meiotic or mitotic cells is presented. Further development and utilization of plant tissue culture techniques and morphological markers identifiable in the seedling stages is recommended for detecting chemically induced aneuploidy.     

In vitro immunization for production of murine and human monoclonal antibodies: Present status

In vitro immunization is now receiving increasing attention as a novel approach in immunotechnolgy for producing monoclonal antibodies. This concept of immunization in culture will have a major impact on the field of human monoclonal antibodies, because human antibodis against any antigen can be produced without the need for presensitized patients. In this paper I will review the progress of in vitro immunization and why it is important to develop these systems for both murine and human cells.     

Microenvironment Modulates Osteogenic Cell Lineage Commitment in Differentiated Embryonic Stem Cells

Background

Due to their self-renewal, embryonic stem cells (ESCs) are attractive cells for applications in regenerative medicine and tissue engineering. Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best. It is possible that the traditional culture methods, which have been used, are not optimal and that other approaches must be explored.

Methodology/Principal Findings

ESCs were differentiated into osteoblast lineage using a micro-mass approach. In response to osteogenic differentiation medium, many cells underwent apoptosis, while others left the micro-mass, forming small aggregates in suspension. These aggregates were cultured in three different culture conditions (adhesion, static suspension, and stirred suspension), then examined for osteogenic potential in vitro and in vivo. In adhesion culture, ESCs primed to become osteoblasts recommitted to the adipocyte lineage in vitro. In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification. In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells.

Conclusions/Significance

We demonstrate that microenvironment modulates cell fate and subsequent tissue formation during ESC differentiation. For effective tissue engineering using ESCs, it is important to develop optimized cell culture/differentiation conditions based upon the influence of microenvironment.     

Microscopy,Culture, and Quantitative Real-Time PCR Examination Confirm Internalization of Mycobacteria in Plants

The environment is a reservoir of nontuberculous mycobacteria and is considered a source of infection for animals and humans. Mycobacteria can persist in different types of environments for a relatively long time. We have studied their possible internalization into plant tissue through intact, as well as damaged, root systems of different types of plants grown in vitro and under field conditions. The substrate into which plants were seeded was previously contaminated with different strains of Mycobacterium avium (10⁸ to 10¹⁰ cells/g of soil) and feces from animals with paratuberculosis. We detected M. avium subsp. avium, hominissuis, and paratuberculosis in the stems and leaves of the plants by both culture and real-time quantitative PCR. The presence of mycobacteria in the plant tissues was confirmed by microscopy. The concentration of mycobacteria found inside plant tissue was several orders of magnitude lower (up to 10⁴ cells/g of tissue) than the initial concentration of mycobacteria present in the culture medium or substrate. These findings led us to the hypothesis that plants may play a role in the spread and transmission of mycobacteria to other organisms in the environment.     

Molecular Analysis of In Vitro Shoot Organogenesis

Shoot organogenesis is one of the in vitro plant regeneration pathways. It has been widely employed in plant biotechnology for in vitro micropropagation and genetic transformation, as well as in study of plant development. Morphological and physiological aspects of in vitro shoot organogenesis have already been extensively studied in plant tissue culture for more than 50 years. Within the last ten years, given the research progress in plant genetics and molecular biology, our understanding of in vivo plant shoot meristem development, plant cell cycle, and cytokinin signal transduction has advanced significantly. These research advances have provided useful molecular tools and resources for the recent studies on the genetic and molecular aspects of in vitro shoot organogenesis. A few key molecular markers, genes, and probable pathways have been identified from these studies that are shown to be critically involved in in vitro shoot organogenesis. Furthermore, these studies have also indicated that in vitro shoot organogenesis, just as in in vivo shoot development, is a complex, well-coordinated developmental process, and induction of a single molecular event may not be sufficient to induce the occurrence of the entire process. Further study is needed to identify the early molecular event(s) that triggers dedifferentiation of somatic cells and serves as the developmental switch for de novo shoot development.     

A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz’s L15 medium and incubated at 32^°C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10–14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.     

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor’s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.     

Bioreactors to influence stem cell fate: Augmentation of mesenchymal stem cell signaling pathways via dynamic culture systems

Background

Mesenchymal stem cells (MSCs) are a promising cell source for bone and cartilage tissue engineering as they can be easily isolated from the body and differentiated into osteoblasts and chondrocytes. A cell based tissue engineering strategy using MSCs often involves the culture of these cells on three-dimensional scaffolds; however the size of these scaffolds and the cell population they can support can be restricted in traditional static culture. Thus dynamic culture in bioreactor systems provides a promising means to culture and differentiate MSCs in vitro.

Scope of review

This review seeks to characterize key MSC differentiation signaling pathways and provides evidence as to how dynamic culture is augmenting these pathways. Following an overview of dynamic culture systems, discussion will be provided on how these systems can effectively modify and maintain important culture parameters including oxygen content and shear stress. Literature is reviewed for both a highlight of key signaling pathways and evidence for regulation of these signaling pathways via dynamic culture systems.

Major conclusions

The ability to understand how these culture systems are affecting MSC signaling pathways could lead to a shear or oxygen regime to direct stem cell differentiation. In this way the efficacy of in vitro culture and differentiation of MSCs on three-dimensional scaffolds could be greatly increased.

General significance

Bioreactor systems have the ability to control many key differentiation stimuli including mechanical stress and oxygen content. The further integration of cell signaling investigations within dynamic culture systems will lead to a quicker realization of the promise of tissue engineering and regenerative medicine. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

The age-dependent epigenetic and physiological changes in an Arabidopsis T87 cell suspension culture during long-term cultivation

Plant cell suspension cultures represent good model systems applicable for both basic research and biotechnological purposes. Nevertheless, it is widely known that a prolonged in vitro cultivation of plant cells is associated with genetic and epigenetic instabilities, which may limit the usefulness of plant lines. In this study, the age-dependent epigenetic and physiological changes in an asynchronous Arabidopsis T87 cell culture were examined. A prolonged cultivation period was found to be correlated with a decrease in the proliferation rate and a simultaneous increase in the expression of senescence-associated genes, indicating that the aging process started at the late growth phase of the culture. In addition, increases in the heterochromatin-specific epigenetic markers, i.e., global DNA methylation, H3K9 dimethylation, and H3K27 trimethylation, were observed, suggesting the onset of chromatin condensation, a hallmark of the early stages of plant senescence. Although the number of live cells decreased with an increase in the age of the culture, the remaining viable cells retained a high potential to efficiently perform photosynthesis and did not exhibit any symptoms of photosystem II damage.     

Methods for assaying viability of frozen-thawed granulocytes.

Phagocytosis and microbial killing by granulocytes is a complicated process which is not yet completely understood. Innumerable in vitro and in vivo tests have been outlined for the several stages of granulocyte activity leading to microbial killing. No single simple test is sufficient to determine the nature of the lesion observed in abnormal frozen and thawed granulocytes, Several procedures are required to define such lesions before attempts can be made to inhibit or reverse this damage. The tests most commonly in use measure production, mobilization, chemotaxis, opsonization, phagocytosis, degranulation, peroxidation, and microbial killing. A test of microbial killing, either in vitro or in vivo, should always be used as the definitive assay.     

Biotechnological advances in pomegranate (Punica granatum L.)

Pomegranate (Punica granatum L.) is known for its nutritional, medicinal, and ornamental importance. It is conventionally propagated by hardwood and softwood cuttings, but about 1 yr is needed before the rooted cuttings can be transplanted to the field. Propagation by seed is undesirable as populations are heterozygous and seed propagation leads to wide variations in tree and fruit characteristics. Several studies have been conducted on in vitro culture of pomegranate, and protocols have been developed for plant regeneration through organogenesis and embryogenesis from various types of explants. Tissue culture has enabled mass propagation of superior genotypes of both wild and cultivated varieties. However, successful application of tissue culture systems for genetic engineering of pomegranate is still limited. Molecular markers are essential for identification and discrimination of genotypes for genetic conservation, crop improvement, breeding programs, and commercialization of superior genotypes. These techniques may also be applicable to rapid identification and indexing of disease-free planting material. This review focuses on the biotechnological approaches that are being used for pomegranate improvement.     

A Novel Source of Cultured Podocytes

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies.