Perfusion culture of fetal human hepatocytes in microfluidic environments

Various types of bioreactors composed of microstructured PDMS (Polydimethylsiloxane) layers have recently been fabricated for perfusion culture of mammalian cells such as adult rat hepatocytes. As a new feature of those bioreactors, in this study, cultivation of fetal human hepatocytes (FHHs) was attempted, because they have high possibility to mature in vitro with preserving their normality, which is suitable for inplantation of liver tissue equivalents reconstituted in vitro. During the perfusion culture in the PDMS bioreactors for 1 week, cells showed good attachment, spreading and reached their confluence over the channels. In addition, their albumin production was significantly enhanced in the perfusion culture using the PDMS bioreactors up to about four times during the FHH perfusion culture when compared in dish-level static culture. Hep G2 cell cultures were also performed and have also shown under perfusion conditions an enhanced cell activity multiplied by 2 compared to static conditions. Although, the cellular activities of FHH cells are still low even compared to those of the Hep G2 cells, the conclusions of this work is encouraging toward future liver tissue engineering based on in vitro propagation and maturation of hepatocyte progenitors combined with microfabrication technologies.     

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research, the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems. Recently, researchers have been actively developing and evaluating three-dimensional (3D) cell culture-based platforms using microfluidic technologies, such as organ-on-a-chip and organoid-on-a-chip platforms, and they have achieved promising breakthroughs in stem cell engineering. In this review, we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery. In a subsequent section, we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research. In addition, some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.     

Application of process quality engineering techniques to improve the understanding of the in vitro processing of stem cells for therapeutic use

The translation of experimental cell-based therapies to volume produced commercially successful clinical products requires the development of capable, economic, scaleable (and therefore frequently necessarily automated) manufacturing processes. Application of proven quality engineering techniques will be required to interrogate, optimise, and control in vitro cell culture processes to regulatory and clinically acceptable specifications. We have used a Six Sigma inspired quality engineering approach to design and conduct a factorial screening experiment to investigate the expansion process of a population of primary bone marrow-derived human mesenchymal stem cells on a scaleable automated cell culture platform. Key cell culture process inputs (seeding density, serum concentration, media quantity and incubation time) and important cell culture process responses (cell number and the expression of alkaline phosphatase, STRO-1, CD105 and CD71) were identified as experimental variables. The results rank the culture factors and significant culture factor interactions by the magnitude of their effect on each of the process responses. This level of information is not available from conventional single factor cell culture studies but is essential to efficiently identify sources of variation and foci for further process optimisation. Systematic quality engineering approaches such as those described here will be essential for the design of regulated cell therapy manufacturing processes because of their focus on identifying the sources of and the control of variation, an issue that is at the core of current Good Manufacturing Practice.     

Regulatory aspects of the in vitro development of retinal Müller glial cells

Utilizing immunochemical and biochemical methods we have examined the maturation of retinal Müller cells in vitro both in monolayer cultures of dissociated tissue as well as rotation-mediated suspension culture of reaggregated embryonic retina cells. We have manipulated heterotypic cell-cell interactions through the use of such cell surface probes as plant lectins and monoclonal antibodies. In this report we show that the succinylated derivative of Con-A is capable of blocking neuronal-glial interactions in reaggregation cultures resulting in neuronal-glial segregation and failure of glial maturation. Furthermore, we describe a new monoclonal antibody which also inhibits glial maturation in vitro. This antibody recognizes an antigen which is present on retinoblast cells in general early in development, but becomes gradually restricted to Müller cells and to a much lesser extent photoreceptor cells during tissue maturation. The results further substantiate the regulatory influence of heterotypic cell-cell interactions in the development of retinal Müller cells and establishes probes for the analysis of the molecular basis of this phenomenon.     

Terminal differentiation of murine erythroleukemia cells: physical stabilization of end- stage cells

An important limitation in the use of the murine erythroleukenia (MEL) cell system as an in vitro system for the study of terminal erythroid differentiation has been the inability to produce significant numbers of cells which represent the end-point of the pathway in vitro. We show here that a major reason for the failure to observe end-stage cells in vitro is that such cells are physically unstable under the standard culture conditions used for MEL cell differentiation. Modification of these culture conditions by the addition of either bovine serum albumin or Ficoll leads to physical stabilization of end-stage cells. Under such culture conditions, uniform cultures of terminally differentiated MEL cells with morphological characteristics similar to those of normal mouse orthochromatophilic erythroblasts and reticulocytes are observed. Examination of physical and biochemical parameters of these cell populations give values which are similar to values characteristic of mouse reticulocytes. A physically stabilized MEL cell shows a narrow cell volume distribution with an average value of approximately 100 mum(3), similar to the cell volume distribution observed for mouse reticulocytes, while a typical MEL cell culture treated with DMSO but without a stabilizing agent exhibits a broader, more heterogeneous cell volume distribution with an average value of approximately 500 mum(3). Globin mRNA levels and levels of globin synthesis reach values almost equal to those in mouse reticulocytes in cultures of physically stabilized MEL cells while differentiating cultures not treated with a stabilizing agent reach substantially lower values for these parameters. We suggest that the ability to produce populations of MEL cells which undergo complete terminal erythroid differentiation in vitro will allow the analysis of the molecular mechanisms which control the terminal stages of the erythroid differentiation process.     

Bioreactor systems for in vitro production of foreign proteins using plant cell cultures

Plant cells have been demonstrated to be an attractive heterologous expression host (using whole plants and in vitro plant cell cultures) for foreign protein production in the past 20years. In recent years in vitro liquid cultures of plant cells in a fully contained bioreactor have become promising alternatives to traditional microbial fermentation and mammalian cell cultures as a foreign protein expression platform, due to the unique features of plant cells as a production host including product safety, cost-effective biomanufacturing, and the capacity for complex protein post-translational modifications. Heterologous proteins such as therapeutics, antibodies, vaccines and enzymes for pharmaceutical and industrial applications have been successfully expressed in plant cell culture-based bioreactor systems including suspended dedifferentiated plant cells, moss, and hairy roots, etc. In this article, the current status and emerging trends of plant cell culture for in vitro production of foreign proteins will be discussed with emphasis on the technological progress that has been made in plant cell culture bioreactor systems.     

Micro- and nanoengineering for stem cell biology: the promise with a caution

Current techniques used in stem cell research only crudely mimic the physiological complexity of the stem cell niches. Recent advances in the field of micro- and nanoengineering have brought an array of in vitro cell culture models that have enabled development of novel, highly precise and standardized tools that capture physiological details in a single platform, with greater control, consistency, and throughput. In this review, we describe the micro- and nanotechnology-driven modern toolkit for stem cell biologists to design novel experiments in more physiological microenvironments with increased precision and standardization, and caution them against potential challenges that the modern technologies might present.     

Understanding cellular networks to improve hematopoietic stem cell expansion cultures

Efforts to develop culture technologies capable of eliciting robust human blood stem cell growth have met with limited success. Considering that adult stem cell cultures are complex systems, comprising multiple cell types with dynamically changing intracellular signalling environments and cellular compositions, this is not surprising. Typically treated as single-input single-output systems, adult stem cell cultures are better described as complex, non-linear, multiple-input multiple-output systems wherein the proliferation of subpopulations of cells leads to the formation of intercellular endogenously secreted protein interaction networks. Genomic and proteomic tools need to be applied to generate high-throughput (and ideally high-content) biological measurements of stem cell culture evolution. Datasets describing cellular interaction networks need to be integrated into predictive models of in vitro stem cell development. Ultimately, such models will serve as a starting point for the rational design of blood stem cell expansion bioprocesses utilizing dynamic system perturbations to achieve the preferential expansion of target cell populations.     

Stimulation of osteoblast growth by an electromagnetic field in a model of bone-like construct

The histogenesis of bone tissue is strongly influenced by physical forces, including magnetic fields. Recent advances in tissue engineering has permitted the generation of three dimensional bone-like constructs. We have investigated the effects of electromagnetic stimulation on human osteoblast cells grown in a hydrophobic polyurethane scaffold. Bone-like constructs were stimulated by pulsed electromagnetic fields in a bioreactor. Proliferation, bone protein expression and calcified matrix production by osteoblasts were measured using histochemical methods. In stimulated cultures, the number of cells was significantly higher compared to static (control) cultures. In both stimulated and control cultures, cells were immunoreactive to osteoblast markers, including type-I collagen, osteocalcin and osteopontin, thus suggesting that the expression of bone-related markers was maintained throughout the in vitro experiments. Morphometric analysis of von Kossa-stained sections revealed that stimulation with electromagnetic field significantly increased matrix calcification. The data lend support to the view that the application of a magnetic field can be used to stimulate cell growth in bone-like constructs in vitro. This finding may be of interest for the production of biomaterials designed for clinical applications.     

Muscle cell culture as a tool in animal growth research

Muscle cell culture techniques have been used for several years in research on muscle growth and development. Several types of culture systems have been devised, including primary cultures from embryonic or postnatal muscle and myogenic cell lines. In addition, serum-free and serum-containing media have been developed to address specific muscle development questions. Many of these questions center around muscle cell differentiation and muscle cell physiology; and, more recently, muscle cell cultures have been used as bioassay tools for examining growth physiology in domestic animals. In our laboratory, skeletal muscle satellite cells have been studied in vitro to evaluate the effect of several protein hormones and growth factors on satellite cell proliferation and differentiation. Of the hormones examined, only the insulin-like growth factors/somatomedins and fibroblast growth factor have been shown to have a stimulatory effect on proliferation that could be physiologically significant. None of the major anterior pituitary hormones interacted directly with satellite cells to stimulate proliferation. With advances in serum-free medium formulations and cell separation techniques, more information can be obtained from experiments with muscle cell cultures. With appropriate design and interpretation, our knowledge of muscle growth in domestic animals will be expanded.     

Monitoring and control of the physiological state of cell cultures

Advances in bioprocess engineering depends ultimately on the level of understanding and control of the physiological state of the cell population. Process efficiency is strongly influenced by changes in the cellular state which should be monitored, interpreted, and, if possible, properly manipulated. In most control systems this function is not explicitly considered, which hampers process development and optimization. Conventional control logic is based on direct mapping of the growth environment into process efficiency, thereby bypassing the cell state as an intermediate control objective. Today, this limitation is well realized, and explicit monitoring and control of cellular physiology are considered to be among the most challenging tasks of modern bioprocess engineering. We present here a generic methodology for the design of systems capable of performing these advanced monitoring and control functions.The term "physiological state" is quantified by a vector composed of several process variables that convey significant information about cellular state. These variables can be selected among different classes, including specific metabolic rates, metabolic rate ratios, degees of limitation, and others. The real-time monitoring of many of these is possible using commercial sensors. The definition and calculation of representative sets of physiological state variables is demonstrated with examples from several fermentor cultures: recombinant Escherichia coli for phenylalanine production, bioluminescent E. coli (harboring lux genes driven by a heat shock protein promoter) for detection of environmental pollutants, plant cell culture of Perilla frutescensfor anthocyanin production, and perfusion cultures of recombinant mammalian cells (NS0 and CHO) for therapeutic protein production.If the physiological state vector is on-line calculated, the fermentation process can be described by its trajectory in a space defined by the vector components. Then, the goal of the control system is to maintain the physiological state of the cell as close as possible to the trajectory, providing maximum efficiency. A control structure meant to perform this function is proposed, along with the mechanism for its design. In contrast to conventional systems which work in a closed loop in respect to the cell environment, this scheme operates in a closed loop in respect to the cell state. The discussed control concept has been successfully applied to the recombinant phenylalanine production, resulting in physiologically consistent operation, total computer control, and high process efficiency. Initial results from the application of the method to perfusion mammalian cell cultures are also presented. (c) 1996 John Wiley & Sons, Inc.     

Application of plant tissue cultures in phytoremediation research: Incentives and limitations

The aim of this review is to critically assess the benefits and limitations associated with the use of in vitro plant cell and organ cultures as research tools in phytoremediation studies. Plant tissue cultures such as callus, cell suspensions, and hairy roots are applied frequently in phytoremediation research as model plant systems. In vitro cultures offer a range of experimental advantages in studies aimed at examining the intrinsic metabolic capabilities of plant cells and their capacity for toxicity tolerance. The ability to identify the contributions of plant cells to pollutant uptake and detoxification without interference from microorganisms is of particular significance in the search for fundamental knowledge about plants. However, if the ultimate goal of plant tissue culture experiments is the development of practical phytoremediation technology, the limitations inherent in the use of in vitro cultures as a representative of whole plants in the field must be recognized. The bioavailability of contaminants and the processes of pollutant uptake and metabolite distribution are likely to be substantially different in the two systems; this can lead to qualitative as well as quantitative differences in metabolic profiles and tolerance characteristics. Yet, many studies have demonstrated that plant tissue cultures are an extremely valuable tool in phytoremediation research. The results derived from tissue cultures can be used to predict the responses of plants to environmental contaminants, and to improve the design and thus reduce the cost of subsequent conventional whole plant experiments. Biotechnol. Bioeng. 2009;103: 60–76.     

Intelligent bioprocessing for haemotopoietic cell cultures using monitoring and design of experiments

The need for successful ex-vivo expansion and directed differentiation of haematopoietic stem cells (HSCs) for therapeutic applications has increased over the past decade. Haematopoietic cell cultures are complex and full characterisation of the process environment has yet to be achieved. The complexity and transient nature of HSC cultures make the identification, maintenance and control of optimal operating conditions challenging. Application of real-time, on-line monitoring techniques and process control strategies enhances the ability to operate bioprocesses of desired reproducibility and high product quality. In this review, we discussed the methods by which in vitro culture information necessary for bioprocess control may be obtained, including process considerations, monitoring and analytical tools, and design of experiments (DOE). The successful application of these tools may result in time- and cost-effective cultures for directed differentiation and expansion of haematopoietic components intended for clinical use.     

Establishment of life-span extended bovine fibroblast cells carrying the characterization of primary cells

Although primary bovine embryonic fibroblast (BEF) cells have previously been used as nucleus-donors for nuclear transfer (NT), it has now been proposed to use BEF cells to generate cloned cows that were genetically modified by transgenic or a knock-out system. A major limitation to gene targeting somatic cells, however, is the overall life-span of the cell. In this study, we first examined in vitro life-span of primary BEF cells. Primary BEF cells were found to be replicative senescent at passage 10th-12th, similar to primary murine embryonic fibroblast cells. To overcome this short in vitro life-span, we have optimized culture conditions to extend the life-span and determined growth characteristics of BEF cell lines. Two life-span extended BEF cell lines (designated CGFR -BO-1 and CGFR-BO-2) were shown to grow much faster than their parental primary counterparts. Both cell lines did not display any potential for abnormal growth such as foci formations in either soft-agar or confluent culture condition. In cloning experiments using these cell lines as a nuclear donor, the reconstructed karyoblasts underwent apoptosis, reprogramming and development in the blastocyst stage, at a similar frequency to those observed with parental as well as adult primary fibroblasts. Furthermore, these cell lines targeted with green fluorescence protein (GFP) were successfully transduced, selected and reprogrammed by NT to develop into a blastocyst stage with GFP expression. Our results suggested methods to extend life-span of donor cells with tremendous implications for the genetic engineering of bovine fibroblast cells.     

Overview of Cell Models: From Organs Cultured in a Petri Dish to Organs-on-Chips

In this review, we tried to elucidate the origin and development of different animal and human cell culture methodologies used to evaluate the effects of various factors and substances in vitro. Organ cultures and conventional two-dimensional cultures of dissociated cells of various types, such as primary, tumor, induced pluripotent, stem, etc., have their advantages and drawbacks but usually do not represent accurate models for studying biological processes that take place in living organisms. Nowadays, high-throughput cell assays on the basis of various methods of signal detection (optical utilizing colorimetric, luminescent and fluorescent methods of detection, and electrochemical) are widely used at early stages of drug development for selection of the most active compounds and evaluation of their cytotoxic effects. The use of animals as models for drug testing is being criticized because of the lack of correlation between the results obtained in studies on them and on humans, and also because of the high cost and ethical issues. Therefore, much effort is put to create models based on human cells. This is how cultures emerged that utilize a three-dimensional network to simulate the architecture of tissues in vivo, and then so-called organs-on-chips—microfluidic microfabricated devices combining several types of cells—that replicate physical and chemical parameters of the microenvironment of cells in living organisms. In summary, experimental cell models have come a long way from the whole organs cultivated in a growth medium to almost complete reconstruction of organs in vitro based on the cutting-edge engineering approach with the use of different cell types. This currently enables one to replicate complex biological processes and study the influence of different substances and factors on them more successfully.     

Real‐time monitoring and control of soluble signaling factors enables enhanced progenitor cell outputs from human cord blood stem cell cultures

Monitoring and control of primary cell cultures is challenging as they are heterogenous and dynamically complex systems. Feedback signaling proteins produced from off‐target cell populations can accumulate, inhibiting the production of the desired cell populations. Although culture strategies have been developed to reduce feedback inhibition, they are typically optimized for a narrow range of process parameters and do not allow for a dynamically regulated response. Here we describe the development of a microbead‐based process control system for the monitoring and control of endogenously produced signaling factors. This system uses quantum dot barcoded microbeads to assay endogenously produced signaling proteins in the culture media, allowing for the dynamic manipulation of protein concentrations. This monitoring system was incorporated into a fed‐batch bioreactor to regulate the accumulation of TGF‐β1 in an umbilical cord blood cell expansion system. By maintaining the concentration of TGF‐β1 below an upper threshold throughout the culture, we demonstrate enhanced ex vivo expansion of hematopoietic progenitor cells at higher input cell densities and over longer culture periods. This study demonstrates the potential of a fully automated and integrated real‐time control strategy in stem cell culture systems, and provides a powerful strategy to achieve highly regulated and intensified in vitro cell manufacturing systems. Biotechnol. Bioeng. 2014;111: 1258–1264. © 2013 The Authors Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Induction of enamel matrix protein expression in an ameloblast cell line co-cultured with a mesenchymal cell line in vitro

Interactions between epithelium and mesenchyme are important for organ and tissue development. In this study, in order to mimic interactions between epithelium and mesenchyme during native tooth development, we constructed three-dimensional culture systems in vitro using a collagen membrane. Two types of collagen membrane-based in vitro culture systems were constructed in which dental epithelial and dental follicle cell lines were cultured. One co-culture method involved inoculation of one cell line into one side of the collagen membrane, and the other cell line into the opposite side of the membrane (sandwich co-culture). As a control, the second method involved culture of one of the cell lines on a culture dish and the second cell line on a collagen membrane, facing away from the first cell line (separate co-culture). The HAT-7 cells were also grown as a monolayer culture on collagen. Ameloblast differentiation in these cultures was investigated by analysis of the mRNA and/or protein expression of ameloblastin and amelogenin. Our results suggest that interaction of epithelial and mesenchymal cells via the extracellular matrix is important for tooth differentiation in vitro. Our culture system should be a useful method for investigation of epithelial-mesenchymal interactions.     

Gas‐permeable membranes and co‐culture with fibroblasts enable high‐density hepatocyte culture as multilayered liver tissues

To engineer reliable in vitro liver tissue equivalents expressing differentiated hepatic functions at a high level and over a long period of time, it appears necessary to have liver cells organized into a three‐dimensional (3D) multicellular structure closely resembling in vivo liver cytoarchitecture and promoting both homotypic and heterotypic cell–cell contacts. In addition, such high density 3D hepatocyte cultures should be adequately supplied with nutrients and particularly with oxygen since it is one of the most limiting nutrients in hepatocyte cultures. Here we propose a novel but simple hepatocyte culture system in a microplate‐based format, enabling high density hepatocyte culture as a stable 3D‐multilayer. Multilayered co‐cultures of hepatocytes and 3T3 fibroblasts were engineered on collagen‐conjugated thin polydimethylsiloxane (PDMS) membranes which were assembled on bottomless frames to enable oxygen diffusion through the membrane. To achieve high density multilayered co‐cultures, primary rat hepatocytes were seeded in large excess what was rendered possible due to the removal of oxygen shortage generally encountered in microplate‐based hepatocyte cultures. Hepatocyte/3T3 fibroblasts multilayered co‐cultures were maintained for at least 1 week; the so‐cultured cells were normoxic and sustained differentiated metabolic functions like albumin and urea synthesis at higher levels than hepatocytes monocultures. Such a microplate‐based cell culture system appears suitable for engineering in vitro miniature liver tissues for implantation, bioartificial liver (BAL) development, or chemical/drug screening. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.     

Cover Image,Volume 116, Number 9, September 2019

The new and rapid advancement in the complexity of biologics drug discovery has been driven by a deeper understanding of biological systems combined with innovative new therapeutic modalities, paving the way to breakthrough therapies for previously intractable diseases. These exciting times in biomedical innovation require the development of novel technologies to facilitate the sophisticated, multifaceted, high-paced workflows necessary to support modern large molecule drug discovery. A high-level aspiration is a true integration of “lab-on-a-chip” methods that vastly miniaturize cellulmical experiments could transform the speed, cost, and success of multiple workstreams in biologics development. Several microscale bioprocess technologies have been established that incrementally address these needs, yet each is inflexibly designed for a very specific process thus limiting an integrated holistic application. A more fully integrated nanoscale approach that incorporates manipulation, culture, analytics, and traceable digital record keeping of thousands of single cells in a relevant nanoenvironment would be a transformative technology capable of keeping pace with today's rapid and complex drug discovery demands. The recent advent of optical manipulation of cells using light-induced electrokinetics with micro- and nanoscale cell culture is poised to revolutionize both fundamental and applied biological research. In this review, we summarize the current state of the art for optical manipulation techniques and discuss emerging biological applications of this technology. In particular, we focus on promising prospects for drug discovery workflows, including antibody discovery, bioassay development, antibody engineering, and cell line development, which are enabled by the automation and industrialization of an integrated optoelectronic single-cell manipulation and culture platform. Continued development of such platforms will be well positioned to overcome many of the challenges currently associated with fragmented, low-throughput bioprocess workflows in biopharma and life science research.