Systematic evaluation of parameters for genome-scale metabolic models of cultured mammalian cells

Genome-scale metabolic models describe cellular metabolism with mechanistic detail. Given their high complexity, such models need to be parameterized correctly to yield accurate predictions and avoid overfitting. Effective parameterization has been well-studied for microbial models, but it remains unclear for higher eukaryotes, including mammalian cells. To address this, we enumerated model parameters that describe key features of cultured mammalian cells – including cellular composition, bioprocess performance metrics, mammalian-specific pathways, and biological assumptions behind model formulation approaches. We tested these parameters by building thousands of metabolic models and evaluating their ability to predict the growth rates of a panel of phenotypically diverse Chinese Hamster Ovary cell clones. We found the following considerations to be most critical for accurate parameterization: (1) cells limit metabolic activity to maintain homeostasis, (2) cell morphology and viability change dynamically during a growth curve, and (3) cellular biomass has a particular macromolecular composition. Depending on parameterization, models predicted different metabolic phenotypes, including contrasting mechanisms of nutrient utilization and energy generation, leading to varying accuracies of growth rate predictions. Notably, accurate parameter values broadly agreed with experimental measurements. These insights will guide future investigations of mammalian metabolism.     

Stem cell culture engineering - process scale up and beyond

Advances in stem cell research and recent work on clinical trials employing stem cells have heightened the prospect of stem cell applications in regenerative medicine. The eventual clinical application of stem cells will require transforming cell production from laboratory practices to robust processes. Most stem cell applications will require extensive ex vivo handling of cells, from isolation, cultivation, and directed differentiation to product cell separation, cell derivation, and final formulation. Some applications require large quantities of cells in each defined batch for clinical use in multiple patients; others may be for autologous use and require only small-scale operations. All share a common requirement: the production must be robust and generate cell products of consistent quality. Unlike the established manufacturing process of recombinant protein biologics, stem cell applications will likely see greater variability in their cell source and more fluctuations in product quality. Nevertheless, in devising stem cell-based bioprocesses, much insight could be gained from the manufacturing of biological materials, including recombinant proteins and anti-viral vaccines. The key to process robustness is thus not only the control of traditional process chemical and physical variables, but also the sustenance of cells in the desired potency or differentiation state through controlling non-traditional variables, such as signaling pathway modulators.     

Stem cell bioprocess engineering towards cGMP production and clinical applications

Stem cells, including mesenchymal stem cells and pluripotent stem cells, are becoming an indispensable tool for various biomedical applications including drug discovery, disease modeling, and tissue engineering. Bioprocess engineering, targeting large scale production, provides a platform to generate a controlled microenvironment that could potentially recreate the stem cell niche to promote stem cell proliferation or lineage-specific differentiation. This survey aims at defining the characteristics of stem cell populations currently in use and the present-day limits in their applications for therapeutic purposes. Furthermore, a bioprocess engineering strategy based on bioreactors and 3-D cultures is discussed in order to achieve the improved stem cell yield, function, and safety required for production under current good manufacturing practices.     

OPP Labeling Enables Total Protein Synthesis Quantification in CHO Production Cell Lines at the Single‐Cell Level

Accurate measurement of global and specific protein synthesis rates is becoming increasingly important, especially in the context of biotechnological applications such as process modeling or selection of production cell clones. While quantification of total protein translation across whole cell populations is easily achieved, methods that are capable of tracking population dynamics at the single‐cell level are still lacking. To address this need, we apply O‐propargyl‐puromycin (OPP) labeling to assess total protein synthesis in single recombinant Chinese hamster ovary (CHO) cells by flow cytometry. Thereby we demonstrate that global protein translation rates slightly increase with progression through the cell cycle during exponential growth. Stable CHO cell lines producing recombinant protein display similar levels of total protein synthesis as their parental CHO host cell line. Global protein translation does not correlate with intracellular product content of three model proteins, but the host cell line with high transient productivity has a higher OPP signal. This indicates that production cell lines with increased overall protein synthesis capacity can be identified by our method at the single‐cell level. In conclusion, OPP‐labeling allows rapid and reproducible assessment of global protein synthesis in single CHO cells, and can be multiplexed with DNA staining or any type of immunolabeling of specific proteins or markers for organelles.     

Theoretical models of wound healing: past successes and future challenges

The complex biology of wound healing is an area in which theoretical modelling has already made a significant impact. In this review article, the authors describe the key features of wound healing biology, divided into four components: epidermal wound healing, remodelling of the dermal extracellular matrix, wound contraction, and angiogenesis. Within each of these categories, previous modelling work is described, and the authors identify what they regard as the main challenges for future theoretical work.     

S-phase synchronized CHO cells show elevated transfection efficiency and expression using CaPi

Various methods exist to transfect mammalian cells in culture. It is generally accepted that individual methods have to be optimized for each of the cell lines or cell types used. Despitethe use of optimized protocols, significant day-to-day variationsin transfection efficiency regularly occur. We postulate that the`status' of cell populations prior to transfection is involved insuch variability. This study evaluates standardized transfectionsdone at different phases of the cell cycle. Cell synchronizationwas achieved using mimosine. Transfection efficiency was monitored by fluorescence quantification of GFP (Green Fluorescent Protein). We show that transfection using the calcium-phosphate-DNA co-precipitation method, at differentphases of the cell cycle, yields variable expression levels of GFP. Highest GFP expression levels were seen when transfecting cell populations with a dominant representation of S-phase-cells.     

Bioprocess engineering: now and beyond 2000

Abstract: Bioprocess engineering may be defined as the translation of life-science discoveries into practical products, processes, or systems capable of serving the needs of society. It is a critical link from discovery to commercialization. Current bioprocess engineering is primarily focused on biopharmaceutical products of high dollar value per gram such as erythropoietin or growth hormones. However, other products of current interest include ethanol, amino acids, organic acids, antibiotics, and specialty chemicals. Current challenges for increased use of bioprocesses for producing bulk and semi-bulk chemicals include both technical and infrastructural barriers. Technical barriers are easy to identify and at times can be overcome by engineering improvements or changes brought about radical developments in science (e.g. recombinant DNA). Infrastructural barriers, such as raw-material substitutions or educational limitations are more difficult to define and change. Recently the National Academy of Sciences examined barriers to bioprocess engineering and issued a report entitled: "Putting Biotechnology to Work: Bioprocess Engineering". A key recommendation was the establishment of a coordinated long-range plan of research, development, training and education in bioprocess engineering involving participation by industry, academe and the federal government. The report was the first national analysis devoted entirely to bioprocess engineering and covered new topics such as space bioprocess engineering. Other topics covered by the author include the current state of the US chemical industry and future directions in three promising areas of bioprocess engineering environmental bioprocess engineering, marine bioprocess engineering and microsystem bioprocess engineering.     

Profiling highly conserved microrna expression in recombinant IgG‐producing and parental Chinese hamster ovary cells

MicroRNAs (miRNAs) play important roles in global gene regulation. Researchers in recombinant protein production have proposed miRNAs as biomarkers and cell engineering targets. However, miRNA expression remains understudied in Chinese Hamster Ovary cells, one of the most commonly used host cell systems for therapeutic protein production. To profile highly conserved miRNA expression, we used the miRCURY? miRNA array for screening miRNAs in CHO cells. The selection criteria for further miRNA profiling included positive hybridization signals and experimentally validated predicted regulatory targets. On the basis of screening, we selected 16 miRNAs for quantitative RT‐PCR profiling. We profiled miR expression in parental CHO DG44 and CHO K1 cell lines as well as four recombinant DG44‐derived CHO lines producing a recombinant human IgG. We observed that miR‐221 and miR‐222 were significantly downregulated in all IgG‐producing cell lines when compared with parental DG44, whereas miR‐125b was significantly downregulated in one IgG‐producing line. In another IgG‐producing line, miR‐19a was significantly upregulated. miRNA expression was also profiled in two of these lines that were amplified by stepwise increase of methotrexate. In both amplified cell lines, let‐7b and miR‐221 were significantly downregulated. In parental CHO K1, let‐7b, miR‐15b, and miR‐17 were significantly downregulated when compared with DG44. The results reported here are the first steps toward profiling highly conserved miRNAs and studying the clonal difference in miRNA expression in CHO cells and may shed light on using miRNAs in cell engineering. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Protein engineering: opportunities and challenges

The extraordinary properties of natural proteins demonstrate that life-like protein engineering is both achievable and valuable. Rapid progress and impressive results have been made towards this goal using rational design and random techniques or a combination of both. However, we still do not have a general theory on how to specify a structure that is suited to a target function nor can we specify a sequence that folds to a target structure. There is also overreliance on the Darwinian blind search to obtain practical results. In the long run, random methods cannot replace insight in constructing life-like proteins. For the near future, however, in enzyme development, we need to rely on a combination of both.     

Sustaining an efficient and effective CHO cell line development platform by incorporation of 24‐deep well plate screening and multivariate analysis

Efficient and effective cell line screening is paramount toward a successful biomanufacturing program. Here we describe the implementation of 24‐deep well plate (24‐DWP) screening of CHO lines as part of the cell line development platform at AbbVie. Incorporation of this approach accelerated the identification of the best candidate lines for process development. In an effort to quantify and predict process performance comparability, we compared cell culture performance in and in shake flasks, for a panel of Chinese Hamster Ovary cell lines expressing a monoclonal antibody. The results in 24‐DWP screening showed reduced growth profiles, but comparable viability profiles. Slow growers in 24‐DWP achieved the highest productivity improvement upon scaling‐up to shake flasks. Product quality of the protein purified from shake flasks and 24‐DWP were also compared. The 24‐DWP culture conditions were found to influence the levels of acidic species, reduce the G0 N‐glycan species, and increase the high‐mannose N‐glycan species. Nevertheless, the identification of undesirable profiles is executed consistently with the scaled‐up culture. We further employed multivariate data analysis to capture differences depending on the two scales and we could demonstrate that cell line profiles were adequately clustered, regardless of the vessel used for the development. In conclusion, the 24‐DWP platform was reasonably predictive of the parameters crucial for upstream process development activities, and has been adapted as part of the AbbVie cell line development platform. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:175–186, 2018     

Stem cell engineering for treatment of heart diseases: Potentials and challenges

Heart disorders are a major health concern worldwide responsible for millions of deaths every year. Among the many disorders of the heart, myocardial infarction, which can lead to the development of congestive heart failure, arrhythmias, or even death, has the most severe social and economic ramifications. Lack of sufficient available donor hearts for heart transplantation, the only currently viable treatment for heart failure other than medical management options (ACE inhibition, beta blockade, use of AICDs, etc.) that improve the survival of patients with heart failure emphasises the need for alternative therapies. One promising alternative replaces cardiac muscle damaged by myocardial infarction with new contractile cardiomyocytes and vessels obtained through stem cell-based regeneration.We report on the state of the art of recovery of cardiac functions by using stem cell engineering. Current research focuses on (a) inducing stem cells into becoming cardiac cells before or after injection into a host, (b) growing replacement heart tissue in vitro, and (c) stimulating the proliferation of the post-mitotic cardiomyocytes in situ. The most promising treatment option for patients is the engineering of new heart tissue that can be implanted into damaged areas. Engineering of cardiac tissue currently employs the use of co-culture of stem cells with scaffold microenvironments engineered to improve tissue survival and enhance differentiation. Growth of heart tissue in vitro using scaffolds, soluble collagen, and cell sheets has unique advantages. To compensate for the loss of ventricular mass and contractility of the injured cardiomyocytes, different stem cell populations have been extensively studied as potential sources of new cells to ameliorate the injured myocardium and eventually restore cardiac function. Unresolved issues including insufficient cell generation survival, growth, and differentiation have led to mixed results in preclinical and clinical studies. Addressing these limitations should ensure the successful production of replacement heart tissue to benefit cardiac patients.     

Corneal stem cells and tissue engineering: Current advances and future perspectives

Major advances are currently being made in regenerative medicine for cornea. Stem cell-based therapies represent a novel strategy that may substitute conventional corneal transplantation, albeit there are many challenges ahead given the singularities of each cellular layer of the cornea. This review recapitulates the current data on corneal epithelial stem cells, corneal stromal stem cells and corneal endothelial cell progenitors. Corneal limbal autografts containing epithelial stem cells have been transplanted in humans for more than 20 years with great successful rates, and researchers now focus on ex vivo cultures and other cell lineages to transplant to the ocular surface. A small population of cells in the corneal endothelium was recently reported to have self-renewal capacity, although they do not proliferate in vivo. Two main obstacles have hindered endothelial cell transplantation to date: culture protocols and cell delivery methods to the posterior cornea in vivo. Human corneal stromal stem cells have been identified shortly after the recognition of precursors of endothelial cells. Stromal stem cells may have the potential to provide a direct cell-based therapeutic approach when injected to corneal scars. Furthermore, they exhibit the ability to deposit organized connective tissue in vitro and may be useful in corneal stroma engineering in the future. Recent advances and future perspectives in the field are discussed.