Noninvasive quality estimation of adherent mammalian cells for transplantation

The noninvasive quality estimation of adherent mammalian cells for transplantation is reviewed. The quality and heterogeneity of cells should be estimated before transplantation because cultured cells are not homogeneous but heterogeneous. The estimation of cell quality should be performed noninvasively because most protocols of regenerative medicine are autologous cell system. The differentiation level and contamination of other cell lineage could be estimated by two-dimensional cell morphology analysis and tracking using a conventional phase contrast microscope. The noninvasive determination of the laser phase shift of a cell using a phase-shifting laser microscope, which might be more noninvasive, and more useful than the atomic force microscope and digital holographic microscope, was carried out to determine the three-dimensional cell morphology, and the estimation of the cell cycle phase of each adhesive cell and the mean proliferation activity of a cell population. Chemical analysis of the culture supernatant by conventional analytical methods such as ELISA was also useful to estimate the differentiation level of a cell population. Chemical analysis of cell membrane and intracellular components using a probe beam, an infrared beam, and Raman spectroscopy was useful for diagnosing the viability, apoptosis, and differentiation of each adhesive cell.     

Cell culture quality control by rapid isoenzymatic characterization

Procedures that involve cell cultures require careful quality control to avoid inter- and intraspecies contamination. We have developed an electrophoresis technique that can be used routinely in cell culture laboratories to monitor cell line integrity. The method involves the isoenzymatic separation of nine polymorphic enzymes, three of which can be used for cell line species determinations and seven of which can be used for human cell line characterizations. Examples of how the system has been applied to both inter- and intraspecies identifications are described. The routine application of this protocol would be a valuable asset for laboratories concerned with establishing effective cell culture quality control.     

Tailoring recombinant protein quality by rational media design

Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C‐ & N‐terminal modifications), aggregates, low‐molecular‐weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high‐throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015     

Quality prediction of cell substrate using gene expression profiling

Changes in cell culture conditions influence the metabolism of cells, which consequently affects the quality of the products that they produce, such as viral vectors, recombinant proteins, or vaccines. Currently there is no effective technique available to monitor global quality of cells in cell culture. Here we describe a new method using gene expression profiling by microarray to predict the quality of cell substrates. Human embryonic kidney 293 cells are a commonly used cell substrate in the production of biological products. We demonstrate that the yield of adenoviral vectors was lower in over-confluent 293 cells, compared to 40 or 90% confluent cells. Total RNA derived from these cells of different confluence states was reverse transcribed, labeled, and used to hybridize 10K cDNA arrays to determine biomarkers for confluence states. Phenotype scatter-plot analysis and cluster analysis were used for class discovery. Based on this approach, we identified genes that were either up-regulated or down-modulated in response to different cell confluence states. By multivariate predictive models we identified a set of 37 genes that were either down-regulated or up-regulated compared to 90% confluent cells as a predictor of cell confluence and quality of 293 cell cultures. The predictive accuracy of these models was assessed by the leave-one-out cross-validation method. The expression of selected gene predictors was validated by quantitative PCR analysis. Our results demonstrate that gene expression profiling can assess the quality of cell substrates prior to large-scale production of a biological product.     

The use of multidimensional image-based analysis to accurately monitor cell growth in 3D bioreactor culture

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells.     

Quality control in haematology: Report of interlaboratory trials in britain

An interlaboratory quality control scheme has been established in Britain by the British Committee for Standards in Haematology. In the first instance this has been confined to haemoglobin, red blood cell count, and packed cell volume. The materials which have been circulated include whole blood, stabilized red cell preparations, lysates, cyanmethaemoglobin solutions, and cyanmethaemoglobin reference preparations. The first two trials have been completed, and there are implications for instrument calibration, dilution techniques, and the use of standards. Periodic interlaboratory trials, at a national level, in conjunction with regular individual intralaboratory quality control procedures, are necessary in order to achieve acceptable levels of accuracy and precision.     

Cell culture quality control by rapid isoenzymatic characterization

Summary Procedures that involve cell cultures require careful quality control to avoid inter- and intraspecies contamination. We have developed and electrophoresis technique that can be used routinely in cell culture laboratories to monitor cell line integrity. The method involves the isoenzymatic separation of nine polymorphic enzymes, three of which can be used for cell line species determinations and seven of which can be used for human cell line characterizations. Examples of how the system has been applied to both inter- and intraspecies identifications are described. The routine application of this protocol would be a valuable asset for laboratories concerned with establishing effective cell culture quality control. This work was supported by Contract N01-CP-9-1003 from the National Cancer Institute, Bethesda, MD.     

Integration of high-throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi-subunit vaccine antigen

Mammalian cell line generation typically includes stable pool generation, single cell cloning and several rounds of clone selection based on cell growth, productivity and product quality criteria. Individual clone expansion and phenotype-based ranking is performed initially for hundreds or thousands of mini-scale cultures, representing the major operational challenge during cell line development. Automated cell culture and analytics systems have been developed to enable high complexity clone selection workflows; while ensuring traceability, safety, and quality of cell lines intended for biopharmaceutical applications. Here we show that comprehensive and quantitative assessment of cell growth, productivity, and product quality attributes are feasible at the 200–1,200 cell colony stage, within 14 days of the single cell cloning in static 96-well plate culture. The early cell line characterization performed prior to the clone expansion in suspension culture can be used for a single-step, direct selection of high quality clones. Such clones were comparable, both in terms of productivity and critical quality attributes (CQAs), to the top-ranked clones identified using an established iterative clone screening approach. Using a complex, multi-subunit antigen as a model protein, we observed stable CQA profiles independently of the cell culture format during the clonal expansion as well as in the batch and fed-batch processes. In conclusion, we propose an accelerated clone selection approach that can be readily incorporated into various cell line development workstreams, leading to significant reduction of the project timelines and resource requirements.     

Standardization in animal cell technology

Continuous cell lines offer a level of reproducibility, and thus standardization, which cannot normally be achieved using primary cells. However, even with continuous cell lines adoption of correct cell banking and appropriate quality control procedures are critical to the provision of reliable, reproducible and safe cell stocks. These procedures enable establishment of cryopreserved stocks of pure cultures of correct identity and phenotype which are free from adventitious agents. In addition to quality control techniques, culture conditions and growth medium used often require standardization. In particular different sources of serum, growth factors and cell attachment substrates may lead to significant variation in the ‘performance’ of cell lines. To ensure a high degree of reliability it is essential to obtain cells from authenticated and quality controlled sources. In culture collections, the principles of correct cell banking should be applied with appropriate quality control for which the minimum standard should be confirmation of viability and mycoplasma testing. Such approaches will afford increased confidence in research data and avoid the waste of time and resources which result from the use of cross-contaminated or infected cells. Presented at the FEMS Symposium “Novel Methods and Standardization in Microbiology”, Košice (Slovakia) 1996.     

Integrating Protein Homeostasis Strategies in Prokaryotes

Bacterial cells are frequently exposed to dramatic fluctuations in their environment, which cause perturbation in protein homeostasis and lead to protein misfolding. Bacteria have therefore evolved powerful quality control networks consisting of chaperones and proteases that cooperate to monitor the folding states of proteins and to remove misfolded conformers through either refolding or degradation. The levels of the quality control components are adjusted to the folding state of the cellular proteome through the induction of compartment specific stress responses. In addition, the activities of several quality control components are directly controlled by these stresses, allowing for fast activation. Severe stress can, however, overcome the protective function of the proteostasis network leading to the formation of protein aggregates, which are sequestered at the cell poles. Protein aggregates are either solubilized by AAA+ chaperones or eliminated through cell division, allowing for the generation of damage-free daughter cells.     

细胞药物的标准及质量控制——细胞药物连载之四

细胞药物最终用于人体,必须建立相应的质量标准,进行质量控制。系统地贯彻到供者筛查、组织采集、细胞分离、培养、冻存、复苏、放行、运输、使用等全过程,确保产品的安全性、有效性和稳定性。近年来,我国逐渐改变了把细胞治疗作为第三类医疗技术管理的思路。一方面,已有第三类医疗技术取消行政审批;另一方面,又把除自体外的干细胞移植纳入药物管理,并建立了相应的质量标准和质量管理办法。     

Evaluating Cell Processes,Quality, and Biomarkers in Pluripotent Stem Cells Using Video Bioinformatics

There is a foundational need for quality control tools in stem cell laboratories engaged in basic research, regenerative therapies, and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging, expansion, maintenance, and differentiation. In this paper, an unbiased, automated high-content profiling toolkit, StemCellQC, is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy, unhealthy, and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups, and these features were linked to growth, motility, and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis, which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features, cell types, treatments, and differentiating cells.     

Production of tPA in recombinant CHO cells under oxygen-limited conditions

Animal cell bioreactors are often limited by the oxygen supply. The reduction in oxygen consumption per cell that occurs under hypoxic conditions may be exploited as a method for increasing reactor capacity if additional glucose is provided to offset increased glycolytic activity. The effects of oxygen deprivation on recombinant tPA (tissue-type plasminogen activator) production were investigated using midexponential and slowly growing CHO cells. The specific oxygen consumption rate can be reduced by at least 50% (mild hypoxic conditions) without affecting the cell growth rate, maximum cell concentration, tPA production rate, or tPA quality (as characterized by the tPA-specific activity and SDS-PAGE analysis). This suggests that mild-hypoxic conditions (with sufficient glucose) can be used to double the cell concentration and volumetric tPA production rate (at a constant volumetric oxygen supply rate) without sacrificing product quality. However, anoxic conditions should be avoided. When slowly growing cultures were exposed to anoxia, the tPA production rate decreased by 80% without affecting tPA quality. However, when midexponential cultures were exposed to anoxia, the drop in tPA production was accompanied by a decrease in tPA quality that ranged from a 40% decrease in tPA specific activity to extensive tPA degradation. (c) 1993 John Wiley & Sons, Inc.     

讲座细胞药物的制备工艺——细胞药物连载之二

细胞药物制备的质量直接关系到细胞治疗的效果。由于细胞治疗所用细胞是具有生物学效应的,细胞药物的制备技术和应用方案具有多样性、复杂性和特殊性,不像一般生物药物那样有统一的制作标准。细胞药物的制备过程主要包括供者筛查、供者检测、采集、加工、分离纯化、储存等,以造血干细胞、间充质干细胞、肝细胞及树突状细胞为例对其进行简要介绍。     

Cell surface targeting of VPAC1 receptors: evidence for implication of a quality control system and the proteasome

Like for most transmembrane proteins, translation of G protein-coupled receptors (GPCRs) mRNA takes place at the endoplasmic reticulum (ER) where they are synthesized, folded and assembled. The molecular mechanisms involved in the transport process of GPCRs from ER to the plasma membrane are poorly investigated. Here we studied the mechanisms involved in glycosylation-dependent cell surface expression and quality control of the receptor for Vasoactive Intestinal Polypeptide (VIP) VPAC1, a member of the B family of GPCRs. Using biochemical and pharmacological techniques and fluorescence microscopy, we have shown that only a fraction of newly synthesized VPAC1 attains properly conformation that allows their cell surface targeting. Misfolded or immature VPAC1 are taken in charge by co- and post-translational quality control that involves: 1) calnexin-dependent folding strictly through a glycan-dependent mechanism, 2) BiP-dependant folding, 3) translocation to the cytoplasm and proteasome-dependent degradation of improper proteins, and 4) post-ER quality control check points. Our data suggest that VPAC1 expression/trafficking pathways are under the control of complex and precise molecular mechanisms to ensure that only proper VPAC1 reaches the cell surface.     

Stem cell applications for pathologies of the urinary bladder

New stem cell based therapies are undergoing intense research and are widely investigated in clinical fields including the urinary system. The urinary bladder performs critical complex functions that rely on its highly coordinated anatomical composition and multiplex of regulatory mechanisms. Bladder pathologies resulting in severe dysfunction are common clinical encounter and often cause significant impairment of patient’s quality of life. Current surgical and medical interventions to correct urinary dysfunction or to replace an absent or defective bladder are sub-optimal and are associated with notable complications. As a result, stem cell based therapies for the urinary bladder are hoped to offer new venues that could make up for limitations of existing therapies. In this article, we review research efforts that describe the use of different types of stem cells in bladder reconstruction, urinary incontinence and retention disorders. In particular, stress urinary incontinence has been a popular target for stem cell based therapies in reported clinical trials. Furthermore, we discuss the relevance of the cancer stem cell hypothesis to the development of bladder cancer. A key subject that should not be overlooked is the safety and quality of stem cell based therapies introduced to human subjects either in a research or a clinical context.     

饮用水微生物的安全快速检测

【目的】为了更好地分析饮用水中的微生物含量。【方法】利用流式细胞术(Flowcytometry,FCM)、ATP测定方法检测瓶装无气饮用水中的微生物数量、可同化有机碳(Assimilable organic carbon,AOC)含量以及微生物活性,并将检测结果与传统的饮用水微生物检测技术相对照。【结果】FCM方法可快速区分水样中的活性细菌和非活性细菌,AOC含量反映了水样中微生物再生能力;而ATP检测方法也能比异养细菌平板计数法(Heterotrophic plate count,HPC)更好地反映瓶装无气饮用水中的实际微生物含量。【结论】FCM、ATP测定方法要明显优于依赖于培养的传统方法。     

细胞培养工艺条件对抗体异质性影响的研究进展

抗体药物具有靶向明确、副作用小等优势,近年来受到国内外药企的广泛关注。然而,动物细胞大规模培养和抗体质量分析已然成为我国的抗体药物产业化的主要限制因素,尤其是细胞培养工艺条件对抗体异质性的影响非常显著,如何有效通过优化工艺条件来满足抗体药物开发要求成为迫在眉睫的问题。文中简要综述了近年来细胞培养工艺条件对抗体异质性影响的技术进展,并对未来国内抗体药物开发作了展望。     

A 24-microwell plate with improved mixing and scalable performance for high throughput cell cultures

Multi-well plates are widely used in high throughput drug screening, cell clone development, media design and cell culture optimization in the biotechnology industry. The reproducibility and data quality of cell cultures in multi-well plates are greatly affected by mixing, aeration, and evaporation. A novel 24-microwell plate (MWP) with static mixers for improved mixing and aeration, and gas permeable lids for reduced evaporation was developed for cell cultures. Mixing, oxygen transfer, evaporation, and cell proliferation as affected by the static mixer, shape of the well and agitation rate were studied. The static mixer improved mixing pattern and reduced cell aggregation under orbital shaking conditions. Consequently, the static mixer also improved cell proliferation with a significantly higher specific growth rate in round wells. In general, consistent growth kinetics was observed for cells cultured on the plate. Overall, the MWP improved the data quality with smaller standard deviations and better reproducibility. Furthermore, CHO cells cultured in the MWP gave similar kinetics in glucose consumption, lactate production, cell growth and viability, and antibody production in a serum-free medium to those cultured in spinner flasks, demonstrating its scalable performance and potential application in high throughput screening for cell culture process development.