Microfluidic cell culture models for tissue engineering

Microfluidic systems have emerged as revolutionary new platform technologies for a range of applications, from consumer products such as inkjet printer cartridges to lab-on-a-chip diagnostic systems. Recent developments have opened the door to a new set of opportunities for microfluidic systems, in the field of tissue and organ engineering. Advances in the design of physiologically relevant structures and networks, fabrication processes for biomaterials suitable for in vivo use, and techniques for scaling towards large, three-dimensional constructs, are converging towards therapeutic applications of microfluidic technologies in engineering complex tissues and organs. These advances herald a new generation of microfluidics-based approaches designed for specific tissue and organ applications, incorporating microvascular networks, structures for transport and filtration, and a three-dimensional microenvironment suitable for supporting phenotypic cell behavior, tissue function, and implantation and host integration.     

微重力条件下细胞培养和组织工程研究进展

随着空间生命科学研究的发展,人们将细胞、组织培养技术与微重力环境相结合产生了组织工程研究的一个新领域——微重力组织工程。模拟微重力条件下细胞培养和组织构建研究表明,微重力环境有利于细胞的三维生长,形成具有功能的组织样结构,培养后的三维组织无论从形态上还是基因表达上都更接近于正常的机体组织。这种微重力对细胞的作用效应,将可能为未来组织工程和再生医学研究提供一条新途径。该文概述了近十年来国内外微重力组织工程相关研究的最新进展。     

Hair follicular cell/organ culture in tissue engineering and regenerative medicine

Hair follicles are complex organs composed of the dermal papilla (DP), dermal sheath (DS), outer root sheath (ORS), inner root sheath (IRS) and hair shaft. Development of hair follicles begins towards the end of the first trimester of pregnancy and is controlled by epidermal–mesenchymal interaction (EMI), which is a signaling cascade between epidermal and mesenchymal cell populations. Hair grows in cycles of various phases. Specifically, anagen is the growth phase, catagen is the involuting or regressing phase and telogen is the resting or quiescent phase. Alopecia is not life threatening, but alopecia often causes severe mental stress. In addition, the number of individuals afflicted by alopecia patients has been increasing steadily. Currently there are two methods employed to treat alopecia, drug or natural substance therapy and human hair transplantation. Although drug or natural substance therapy may retard the progress of alopecia or prevent future hair loss, it may also accelerate hair loss when the medication is stopped after prolonged use. Conversely, the transplantation of human hair involves taking plugs of natural hair from areas in which occipital hair is growing and transplanting them to bald areas. However, the number of hairs that can be transplanted is limited in that only three such operations can generally be performed. To overcome such problems, many researchers have attempted to revive hair follicles by culturing hair follicle cells or mesenchymal cells in vitro and then implanting them in the treatment area.     

Recombinant human gelatin substitute with photoreactive properties for cell culture and tissue engineering

The human recombinant collagen I α1 chain monomer (rh‐gelatin) was modified by the incorporation of an azidophenyl group to prepare photoreactive human gelatin (Az‐rh‐gelatin), with approximately 90% of the lysine residues conjugated with azidobenzoic acid. Slight changes in conformation (circular dichroism spectra) and thermal properties (gelation and melting points) were noticed after modification. Ultraviolet (UV) irradiation could immobilize the Az‐rh‐gelatin on polymer surfaces, such as polystyrene and polytetrafluoroethylene. Az‐rh‐gelatin was stably retained on the polymer surfaces, while unmodified gelatin was mostly lost by brief washing. Human mesenchymal cells grew more efficiently on the immobilized surface than on the coated surface. The immobilized Az‐rh‐gelatin on the polymer surfaces was able to capture engineered growth factors with collagen affinity, and the bound growth factors stimulated the growth of cells dose‐dependently. It was also possible to immobilize Az‐rh‐gelatin in micropatterns (stripe, grid, and so on) using photomasks, and the cells grew according to the patterns. These results suggest that the photoreactive human gelatin, in combination with collagen‐binding growth factors, will be clinically useful for surface modification of synthetic materials for cell culture systems and tissue engineering. Biotechnol. Bioeng. 2011;108: 2468–2476. © 2011 Wiley Periodicals, Inc.     

Mulberry improvements via plastid transformation and tissue culture engineering

The in vitro tissue culture and micropropagation studies for Morus spp., a pivotal sericulture plant, are well established. The rapid and reproducible in vitro response to plant growth regulator treatments has emerged as an essential complement of transformation studies for this plant species. A major area of study is the use of protoplast culture and fusion techniques where advantages to mulberry improvement can be applied. The advancements in genetic transformation of mulberry are reviewed, and a section on strategy for transforming plastids (chloroplasts) of mulberry is included. A role for mulberry in “molecular farming” is envisioned. The conclusions and future prospects for improvement of this economically important tree species are proposed.Key words: molecular farming, Morus spp., plastid transformation, protoplast electrofusion, sericultureThe importance of silk production is well recognized in sericulture industry that involves cultivation of host plants for silkworm rearing. India is one of the countries where sericulture is an important agro-based cottage industry involved in production of five different silk types—mori, muga, eri, tasar and oak types. This classification comes from type of host plant that act as feed for silkworm, and thus sericulture industry largely depends on the availability of host plant species. Bombyx mori (mulberry silkworm) feeds on mulberry leaves, Philosomia ricini (eri silkworm) on castor leaves, Anthraea assama (muga silkworm) on som and soalu leaves, Anthraea proylei (temperate/oak tasar silkworm) on oak leaves, and Anthraea mylitta (tropical tasar silkworm) on Terminalia leaves. A systematic and proper cultivation of novel primary and/or secondary host plants showing high yield, suitability to silkworm rearing, and resistance to different abiotic stress conditions i.e., tolerance to water stress, alkalinity and salinity are recommended for sericulture improvement.The genus Morus (commonly known as mulberry) belongs to the family Moraceae, is a group of dioecious woody trees/shrubs. Many varieties of these species are cultivated on a commercial scale in India, China, Japan and Korea for the sericulture industry.¹ In India, six species are found, namely, M. alba L., M. indica L., M. nigra L., M. atropurpurea Roxb., M. serrata Roxb. and M. laevigata Wall.² Due to higher economic return and greater employment potential, attempts are been made to increase productivity by developing high yielding mulberry varieties. At present, Mysore local, Bomaypiasbari, Kanva-2 (K2), Bilidevalaya, Kajli, Sujanpur-1 (S1), BC (2) 59, C776, RFS-175, S36 and Victory-1 are being cultivated extensively in different parts of India.     

Historical reflections on cell culture engineering

Cell culture engineering has enabled the commercial marketing of about a dozen human therapeutic products derived from rDNA technology and numerous monoclonal antibody products as well. A variety of technologies have proven useful in bringing products to the marketplace. Comparisons of the technologies available 15 years ago are contrasted with those available today. A number of improvements in unit operations have greatly improved the robustness of the processes during the past 15 years. Further evolution of the technology is expected in several directions driven by commercial and regulatory pressures. Some problems remain for the next generation of cell culture engineers to solve. This revised version was published online in August 2006 with corrections to the Cover Date.     

Scaffold-free cartilage by rotational culture for tissue engineering

Our objective was to investigate the hypothesis that tissue-engineered cartilage with promising biochemical, mechanical properties can be formed by loading mechanical stress under existing cell-cell interactions analogous to those that occur in condensation during embryonic development. By loading dedifferentiated chondrocytes with mechanical stress under existing cell-cell interactions, we could first form a scaffold-free cartilage tissue with arbitrary shapes and a large size with promising biological, mechanical properties. The cartilage tissue which constituted of chondrocytes and ECM produced by inoculated dedifferentiated chondrocytes to a high porous simple mold has arbitrary shapes, and did not need any biodegradable scaffold to control the shape. In contrast, scaffold-free cartilage tissue cultured under static conditions could not keep their shapes; it was fragile tissue. The possibility of scaffold-free organ design was suggested because the cartilage tissue increases steadily in size with culture time; indeed, the growth of cartilage tissue starting from an arbitrary shape might be predictable by mathematical expression. For tissue-engineered cartilage formation with arbitrary shapes, biochemical and mechanical properties, loading dedifferentiated chondrocytes with mechanical stress under existing cell-cell interactions has prominent effects. Therefore, our scaffold-free cartilage model loaded mechanical stress based on a simple mold system may be applicable for tissue-engineered cartilage.     

观赏植物组织培养与基因工程研究进展(综述)

本文综述近年来观赏植物组织培养和基因工程的研究进展。     

Simplifying the extracellular matrix for 3-D cell culture and tissue engineering: a pragmatic approach

The common technique of growing cells on tissue culture plastic (TCP) is gradually being supplanted by methods for culturing cells in two-dimensions (2-D) on matrices with more appropriate physical and biological properties or by encapsulation of cells in three-dimensions (3-D). The universal acceptance of the new 3-D paradigm is currently constrained by the lack of a biocompatible material in the marketplace that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. In this Prospect, I argue that the standard for 3-D cell culture should be bio-inspired, biomimetic materials that can be used "as is" in drug discovery, toxicology, cell banking, and ultimately in medicine. Such biomaterials must therefore be highly reproducible, manufacturable, approvable, and affordable. To obtain integrated, functional, multicellular systems that recapitulate tissues and organs, the needs of the true end-users-physicians and patients-must dictate the key design criteria. Herein I describe the development of one such material that meets these requirements: a covalently crosslinked, biodegradable, simplified mimic of the extracellular matrix (ECM) that permits 3-D culture of cells in vitro and enables tissue formation in vivo. In contrast to materials that were designed for in vitro cell culture and then found unsuitable for clinical use, these semi-synthetic hyaluronan-derived materials were developed for in vivo tissue repair, and are now being re-engineered for in vitro applications in research.     

Mammalian cell culture: engineering principles and scale-up

If biological products such as monoclonal antibodies, interferons, vaccines, plasminogen activators and many others are to be obtained at an economic cost from mammalian cells, a number of engineering problems must be solved (Tables 1 and 2). The two most imposing barriers to the scale-up of this technology from those listed are the inability to provide sufficient oxygen to high density cultures of mammalian cells grown in large volumes, and the high cost of serum usage. This review focuses on: (i) techniques used to cultivate mammalian cells; (ii) technical barriers to scale-up; and (iii) comparing methods of producing cells with regards to their ability to overcome these barriers.     

Surface functionalization of nanobiomaterials for application in stem cell culture,tissue engineering,and regenerative medicine

Stem cell‐based approaches offer great application potential in tissue engineering and regenerative medicine owing to their ability of sensing the microenvironment and respond accordingly (dynamic behavior). Recently, the combination of nanobiomaterials with stem cells has paved a great way for further exploration. Nanobiomaterials with engineered surfaces could mimic the native microenvironment to which the seeded stem cells could adhere and migrate. Surface functionalized nanobiomaterial‐based scaffolds could then be used to regulate or control the cellular functions to culture stem cells and regenerate damaged tissues or organs. Therefore, controlling the interactions between nanobiomaterials and stem cells is a critical factor. However, surface functionalization or modification techniques has provided an alternative approach for tailoring the nanobiomaterials surface in accordance to the physiological surrounding of a living cells; thereby, enhancing the structural and functional properties of the engineered tissues and organs. Currently, there are a variety of methods and technologies available to modify the surface of biomaterials according to the specific cell or tissue properties to be regenerated. This review highlights the trends in surface modification techniques for nanobiomaterials and the biological relevance in stem cell‐based tissue engineering and regenerative medicine. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:554–567, 2016     

Transmissible spongiform encephalopathies and tissue cell culture

The discovery of prion proteins and the diseases which are associated with them still present scientists and clinicians with a number of problems. There are clearly risks with the use of living cells and materials of animal origin to produce therapeutic compounds with respect to the transmission of prion protein. However the medical benefit many of these compounds has to be weighed against this. It is clear a number of groups are continuing to unravel the highly complex relationships of prion biology and pathology and it is only when this is clearly established that the community can decide on these issues. Until this time the scientific community must rely on the best research available and provide guidance from this. This revised version was published online in August 2006 with corrections to the Cover Date.     

Production and engineering of terpenoids in plant cell culture

Terpenoids are a diverse class of natural products that have many functions in the plant kingdom and in human health and nutrition. Their chemical diversity has led to the discovery of over 40,000 different structures, with several classes serving as important pharmaceutical agents, including the anticancer agents paclitaxel (Taxol) and terpenoid-derived indole alkaloids. Many terpenoid compounds are found in low yield from natural sources, so plant cell cultures have been investigated as an alternate production strategy. Metabolic engineering of whole plants and plant cell cultures is an effective tool to both increase terpenoid yield and alter terpenoid distribution for desired properties such as enhanced flavor, fragrance or color. Recent advances in defining terpenoid metabolic pathways, particularly in secondary metabolism, enhanced knowledge concerning regulation of terpenoid accumulation, and application of emerging plant systems biology approaches, have enabled metabolic engineering of terpenoid production. This paper reviews the current state of knowledge of terpenoid metabolism, with a special focus on production of important pharmaceutically active secondary metabolic terpenoids in plant cell cultures. Strategies for defining pathways and uncovering rate-influencing steps in global metabolism, and applying this information for successful terpenoid metabolic engineering, are emphasized.     

The complete automation of cell culture: improvements for high-throughput and high-content screening

Genomic approaches provide enormous amounts of raw data with regard to genetic variation, the diversity of RNA species, and protein complement. High-throughput (HT) and high-content (HC) cellular screens are ideally suited to contextualize the information gathered from other "omic" approaches into networks and can be used for the identification of therapeutic targets. Current methods used for HT-HC screens are laborious, time-consuming, and prone to human error. The authors thus developed an automated high-throughput system with an integrated fluorescent imager for HC screens called the AI.CELLHOST. The implementation of user-defined culturing and assay plate setup parameters allows parallel operation of multiple screens in diverse mammalian cell types. The authors demonstrate that such a system is able to successfully maintain different cell lines in culture for extended periods of time as well as significantly increasing throughput, accuracy, and reproducibility of HT and HC screens.     

Genomic and proteomic perspectives in cell culture engineering.

In the last few years, the number of biologics produced by mammalian cells have been steadily increasing. The advances in cell culture engineering science have contributed significantly to this increase. A common path of product and process development has emerged in the last decade and the host cell lines frequently used have converged to only a few. Selection of cell clones, their adaptation to a desired growth environment, and improving their productivity has been key to developing a new process. However, the fundamental understanding of changes during the selection and adaptation process is still lacking. Some cells may undergo irreversible alteration at the genome level, some may exhibit changes in their gene expression pattern, while others may incur neither genetic reconstruction nor gene expression changes, but only modulation of various fluxes by changing nutrient/metabolite concentrations and enzyme activities. It is likely that the selection of cell clones and their adaptation to various culture conditions may involve alterations not only in cellular machinery directly related to the selected marker or adapted behavior, but also those which may or may not be essential for selection or adaptation. The genomic and proteomic research tools enable one to globally survey the alterations at mRNA and protein levels and to unveil their regulation. Undoubtedly, a better understanding of these cellular processes at the molecular level will lead to a better strategy for 'designing' producing cells. Herein the genomic and proteomic tools are briefly reviewed and their impact on cell culture engineering is discussed.     

Microscale tissue engineering using gravity-enforced cell assembly

Designing artificial microtissues by reaggregation of monodispersed primary cells, neoplastic or engineered cell lines is providing insight into cell-cell interactions and underlying regulatory networks. Recent advances in microtissue production have highlighted the potential of scaffold-free cell aggregates in maintaining tissue-specific functionality, supporting seamless integration of implants into host tissues, and providing complex feeder structures for difficult-to-differentiate cell types. Furthermore, these tissues are amenable to therapeutic and phenotype-modulating interventions using latest-generation transduction technologies. Microtissues produce therapeutic transgenes at increased levels and offer tissue-like assay environments to improve drug-function correlations in current discovery programs. Here, we outline scaffold-free microtissue design in liver, heart and cartilage, and discuss how this technology could significantly impact regenerative medicine.     

Computational study of culture conditions and nutrient supply in cartilage tissue engineering

Different culture conditions for cartilage tissue engineering were evaluated with respect to the supply of oxygen and glucose and the accumulation of lactate. A computational approach was adopted in which the culture configurations were modeled as a batch process and transport was considered within constructs seeded at high cell concentrations and of clinically relevant dimensions. To assess the extent to which mass transfer can be influenced theoretically, extreme cases were distinguished in which the culture medium surrounding the construct was assumed either completely static or well mixed and fully oxygenated. It can be concluded that severe oxygen depletion and lactate accumulation can occur within constructs for cartilage tissue engineering. However, the results also indicate that transport restrictions are not insurmountable, providing that the medium is well homogenized and oxygenated and the construct's surfaces are sufficiently exposed to the medium. The large variation in uptake rates of chondrocytes indicates that for any specific application the quantification of cellular utilization rates, depending on the cell source and culture conditions, is an essential starting point for optimizing culture protocols.