Continuous Cell Culture Research Articles

Attempt to Isolate Elephant Endotheliotropic Herpesvirus (EEHV) Using a Continuous Cell Culture System.

Simple SummaryElephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is one of the most important viral infectious diseases in young Asian elephants (Elephas maximus). To date, in vitro isolation or propagation of EEHV has so far unsuccessful. Findings in the present study suggest that the U937 cells, a cell line derived from the human myeloid leukemia patient, can be used to isolate and propagate EEHV in vitro. Replication of EEHV in the U937 cells is determined by the presence of EEHV DNA polymerase antigens in the infected cells. However, the replication in these cells was shown to be restricted and observed only in the early passages of virus infection. Although EEHV replication in U937 cells has only occurred in the early passages, our findings have shed some light on the feasibility of using this cell line for further in vitro EEHV isolation.Elephant endotheliotropic herpesvirus (EEHV) infection is known to cause acute fatal hemorrhagic disease, which has killed many young Asian elephants (Elephas maximus). Until recently, in vitro isolation and propagation of the virus have not been successful. This study aimed to isolate and propagate EEHV using continuous cell lines derived from human and/or animal origins. Human cell lines, including EA. hy926, A549, U937, RKO, SW620, HCT-116 and HT-29, and animal cell lines, including CT26.CL25 and sp2/0-Ag14, were investigated in this study. Mixed frozen tissue samples of the heart, lung, liver, spleen and kidney obtained from fatal EEHV1A- or EEHV4-infected cases were homogenized and used for cell inoculation. At 6, 24, 48 and 72 h post infection (hpi), EEHV-inoculated cells were observed for cytopathic effects (CPEs) or were assessed for EEHV infection by immunoperoxidase monolayer assay (IPMA) or quantitative PCR. The results were then compared to those of the mock-infected controls. Replication of EEHV in the tested cells was further determined by immunohistochemistry of cell pellets using anti-EEHV DNA polymerase antibodies or re-inoculated cells with supernatants obtained from passages 2 or 3 of the culture medium. The results reveal that no CPEs were observed in the tested cells, while immunolabeling for EEHV gB was observed in only U937 human myeloid leukemia cells. However, quantitation values of the EEHV terminase gene, as well as those of the EEHV gB or EEHV DNA polymerase proteins in U937 cells, gradually declined from passage 1 to passage 3. The findings of this study indicate that despite poor adaptation in U937 cells, this cell line displays promise and potential to be used for the isolation of EEHV1 and EEHV4 in vitro.

TB-02 Comprehensive analysis of expandable benign pituitary adenomas without genetic manipulations

Background: Since most pituitary adenomas are benign, there is no model of pituitary adenomas that can be continuously cultured without genetic manipulation. In this study, we analyzed the genetic characteristics of the established cell lines by continuous culture of benign pituitary adenomas without genetic manipulation. Method: The pituitary adenoma was continuously cultured by modifying the conditional reprogramming (CR) method, and the morphology was observed and fluorescent immunostaining (IF) was performed. In addition, genetic characteristics were comprehensively analyzed, differences from the surgical samples were analyzed by enrichment analysis using GSEA, and network analysis was performed using cytoscape based on the protein-protein interaction database. (Results) 1) Of the 14 cases of pituitary adenoma that had undergone primary culture (median MIB-1 labeling index 1.5%), 12 cases were capable of continuous culture for more than 2 months. (6 months or more: 5 cases) 2) After 2 weeks of culturing, the cell morphology changed dramatically. These cultured cells were positive for follicular cell marker s100, ectodermal nervous system markers Nestin, Notch1, and mesodermal marker Vimentin. In these cells, synaptophysin, which was strongly positive in the surgical specimen collected at the same time as the culture, disappeared. 3) Enrichment analysis revealed that gene expression such as epithelial mesenchymal transition(EMT), angiogenesis, IL6 signaling via NFkB, extracellular degradation, integrin cell surface interaction increased in cultured cells, and gene expression such as neurotransmitter including synaptophysin decreased significantly.(Conclusion) 1) The modified CR method allowed continuous culture of pituitary adenoma cells. 2) The cultured cells showed characteristic change of EMT, which was seen in invasive pituitary adenoma.

Towards Reduction or Substitution of Cytotoxic DMSO in Biobanking of Functional Bioengineered Megakaryocytes.

Donor platelet transfusion is currently the only efficient treatment of life-threatening thrombocytopenia, but it is highly challenged by immunological, quality, and contamination issues, as well as short shelf life of the donor material. Ex vivo produced megakaryocytes and platelets represent a promising alternative strategy to the conventional platelet transfusion. However, practical implementation of such strategy demands availability of reliable biobanking techniques, which would permit eliminating continuous cell culture maintenance, ensure time for quality testing, enable stock management and logistics, as well as availability in a ready-to-use manner. At the same time, protocols applying DMSO-based cryopreservation media were associated with increased risks of adverse long-term side effects after patient use. Here, we show the possibility to develop cryopreservation techniques for iPSC-derived megakaryocytes under defined xeno-free conditions with significant reduction or complete elimination of DMSO. Comprehensive phenotypic and functional in vitro characterization of megakaryocytes has been performed before and after cryopreservation. Megakaryocytes cryopreserved DMSO-free, or using low DMSO concentrations, showed the capability to produce platelets in vivo after transfusion in a mouse model. These findings propose biobanking approaches essential for development of megakaryocyte-based replacement and regenerative therapies.

Polymer fraction including exosomes derived from Chinese hamster ovary cells promoted their growth during serum-free repeated batch culture

While continuous (perfusion) culture of mammalian cells might reduce the reactor size owing to the high cell density, there is the problem of higher medium cost; however, this problem is expected to be solved by the reuse of growth-promoting components in the culture supernatant. The polymer fraction (PF, 10kDa-220nm) collected from the supernatant of serum-free repeated-batch culture of Chinese hamster ovary (CHO) cells in not only adhesion but also suspension promoted the cell growth in respective serum-free cultures. PF contained CD81-positive exosomes and proteins, both of which were necessary for its growth-promoting activity. Consequently, the medium cost for the continuous (perfusion) serum-free suspension culture of CHO cells may be decreased by the repeated collection and addition of PF that contains exosomes and growth factor proteins.

Optimization of cultivation parameters for bovine respiratory syncytial virus strain Vologda/2019

There are currently many controversial issues in the study of bovine respiratory syncytial infection. In this regard, it is relevant to study the biological properties of the virus, optimize the methods of its cultivation and select the most technologically advanced methods of designing diagnostic and prevention tools for this disease. The aim of this work was to select sensitive cell systems and to optimize the cultivation parameters in selected cell cultures. The Vologda/2019 strain of the bovine respiratory syncytial infection virus isolated from biological material obtained from a calf with respiratory symptoms was used in the experiment. The strain was adapted to the continuous cell culture derived from bovine turbinate tissue (BT) and deposited in the State collection of microorganism strains at FGBI “ARRIAH”. It was established that the continuous cell lines of fetal bovine trachea (FBT) and calf kidney (RBT) are the most sensitive cell systems for the reproduction of the bovine respiratory syncytial virus strain Vologda/2019, the maximum accumulation of the virus was observed in these cell cultures. The cytopathic activity of the virus in the FBT cell culture ranged from 4.78 ± 0.18 to 5.50 ± 0.16 lg TCID 50 /cm 3 , and in the RBT cell culture – from 4.00 ± 0.23 to 4.75 ± 0.20 lg TCID 50 /cm 3 at days 4–5 of cultivation. It was determined that in case of multiplicity of inoculation of FBT and RBT cell cultures with the virus at 0.1 lg TCD 50 /cell and the use of 2% glutamine in the maintenance nutrient medium, as well as 2% horse or cattle blood serum, it is possible to obtain virus material with high cytopathic activity.

Tracking leukemic T-cell transcriptional dynamics in vivo with a blood-based reporter assay.

Transcriptional dynamics of cancer cells govern cell fate decisions and are therapeutically actionable drug targets. In this study, we engineered a circulating cancer cell line that secretes a luciferase reporter to capture constitutive and circadian clock-driven transcription dynamics over the course of a day. Engineered human leukemic T cells (Jurkat) were observed to rhythmically secrete luciferase in a continuous flow cell culture system. When transplanted invivo, engineered leukemic cells caused circadian plasma luciferase activity and had expected pathological signs of leukemic disease. This technique is rapid and noninvasive, requiring only a few microliters of media or blood, and can aid in investigating relationships between invivo cancer cell signaling and behavior, such as diet or sleep.

Investigation of essential cell cycle regulator genes as candidates for immortalized shrimp cell line establishment based on the effect of in vitro culturing on gene expression of shrimp primary cells

Continuous shrimp cell cultures offer opportunities for studying shrimp pathogens at the cellular and molecular level and developing diagnostic tools. However, no continuous shrimp cell lines have yet been successfully established. This might be due to the lack of information on the molecular mechanisms that control shrimp cell proliferation and cell arrest under culture conditions. In this study, differentially expressed genes (DEGs) in shrimp primary culture cells were determined by comparison with normal tissue (in vivo). This study aimed to comprehensively identify key regulator genes that control cell proliferation and cell cycle arrest in culture conditions. The primary shrimp cells were derived from testicular tissue and cultured in vitro. RNA sequencing (RNA-Seq) was performed to investigate change in gene expression level across the entire transcriptome between shrimp primary cells and normal tissue (in vivo). RNA-seq results revealed over 100 genes with distinct gene expression patterns between primary cells and normal tissue. The DEG results and functional gene analysis showed a clear difference in gene expression patterns of cell cycle-related genes between primary cells and normal tissue. LRWD1, TMEM127, CDCA3, PPP2R1A, and GOLGA2, which are cell cycle-related genes, were downregulated in shrimp primary cells. These genes are required for G1/S phase transition, entry into mitosis, and cell proliferation control. However, a gene that induces cell cycle arrest, ARAF, was upregulated in culture conditions. The results demonstrated that these genes might play essential roles in cell proliferation and cell arrest under culture conditions. These candidate genes may be alternative potential targets for genetic manipulation to maintain cell proliferation and establishment of a shrimp continuous cell line.

Evervac: phase I/II study of immunogenicity and safety of a new adjuvant-free TBE vaccine cultivated in Vero cell culture

ABSTRACT Approximately 10,000 cases of tick-borne encephalitis (TBE), a serious disease of the central nervous system caused by tick-borne encephalitis virus (TBEV), are registered worldwide every year. Vaccination against TBE remains the most essential measure of preventing the disease. Unlike available TBE vaccines, a new inactivated lyophilized candidate vaccine Evervac is produced in Vero continuous cell culture and its final formulation does not include aluminum-based adjuvants. To study the safety and immunogenicity of Evervac, healthy adults 18–60 y of age were immunized twice at 30-d intervals. The study was single-blind, randomized, comparative, controlled, and was conducted in TBE-endemic areas. The commercial lyophilized vaccine TBE-Moscow was used as a comparison treatment. The subjects were observed for incidence, severity, and duration of adverse reactions. It was shown that the severity of local and systemic reactions in the Evervac vaccine group was mild to moderate. There were no significant differences in the incidence of adverse reactions between the Evervac and TBE-Moscow vaccine groups. Immunization with Evervac produced a significant increase in geometric mean titer (GMT) of anti-TBEV antibodies in both initially seronegative and seropositive recipients. The seroconversion rate for the initially seronegative recipients was 69% (GMT = 1:214) after the first dose and reached 100% after the second dose. In these parameters, there were no significant differences between the study and control vaccine groups. Thus, the adjuvant-free Vero-based vaccine Evervac was well tolerated, had low reactogenicity, induced a pronounced immune response, and was overall non-inferior to the commercial adjuvanted TBE vaccine used as a control.

Study of biological properties of field isolates of cattle minor infections agents on homological cell cultures

Biological properties of field isolates of bovine immunodeficiency virus and bovine foamy virus on homological cell cultures (fetal bovine lung and bovine coronary artery endothelial cells) were investigated. Pathogens of bovine slow infections, namely bovine immunodeficiency virus and bovine foamy virus, are able to integrate into cell cultures of homologous to cattle type, which is confirmed by the results of PCR. There has been determined the presence of genetic material of pathogens of bovine immunodeficiency (BIV) and spumavirus infection (BFV) in the cultivation of lymphocytes of field isolates in the culture of bovine coronary artery endothelial cells (BCAEC) at the level of 5th passage, and in the cell culture of fetal bovine lung (FBL) — at the level of 10th passage. In the process of integration of pathogens of immunodeficiency and spumavirus infection of cattle in continuous cell cultures FBL and BCAEC, morphological changes in the state of the monolayer by the principle of syncytiation and vacuolation are observed

Complex of technical tools for fish cells cultivation

The solution of problems of viral diseases of fishes which are quite often arising in an aquaculture requires holding diagnostic actions with use of cell cultures, at the same time creation of new cell lines will allow to expand researches as much as possible. Technological means for simplification and mechanization of preparation of cellular suspension and also for large-scale cultivation of the continuous cell cultures in a monolayer are presented in this article. The offered devices are intended for laboratory or for industrial use as a part of technology park for the solution of tasks in the field of bionanotechnology.

Physiologic expansion of human heart-derived cells enhances therapeutic repair of injured myocardium

BackgroundSerum-free xenogen-free defined media and continuous controlled physiological cell culture conditions have been developed for stem cell therapeutics, but the effect of these conditions on the relative potency of the cell product is unknown. As such, we conducted a head-to-head comparison of cell culture conditions on human heart explant-derived cells using established in vitro measures of cell potency and in vivo functional repair.MethodsHeart explant-derived cells cultured from human atrial or ventricular biopsies within a serum-free xenogen-free media and a continuous physiological culture environment were compared to cells cultured under traditional (high serum) cell culture conditions in a standard clean room facility.ResultsTransitioning from traditional high serum cell culture conditions to serum-free xenogen-free conditions had no effect on cell culture yields but provided a smaller, more homogenous, cell product with only minor antigenic changes. Culture within continuous physiologic conditions markedly boosted cell proliferation while increasing the expression of stem cell-related antigens and ability of cells to stimulate angiogenesis. Intramyocardial injection of physiologic cultured cells into immunodeficient mice 1 week after coronary ligation translated into improved cardiac function and reduced scar burden which was attributable to increased production of pro-healing cytokines, extracellular vesicles, and microRNAs.ConclusionsContinuous physiological cell culture increased cell growth, paracrine output, and treatment outcomes to provide the greatest functional benefit after experimental myocardial infarction.

Polyampholytes as Emerging Macromolecular Cryoprotectants.

Cellular cryopreservation is a platform technology which underpins cell biology, biochemistry, biomaterials, diagnostics, and the cold chain for emerging cell-based therapies. This technique relies on effective methods for banking and shipping to avoid the need for continuous cell culture. The most common method to achieve cryopreservation is to use large volumes of organic solvent cryoprotective agents which can promote either a vitreous (ice free) phase or dehydrate and protect the cells. These methods are very successful but are not perfect: not all cell types can be cryopreserved and recovered, and the cells do not always retain their phenotype and function post-thaw. This Perspective will introduce polyampholytes as emerging macromolecular cryoprotective agents and demonstrate they have the potential to impact a range of fields from cell-based therapies to basic cell biology and may be able to improve, or replace, current solvent-based cryoprotective agents. Polyampholytes have been shown to be remarkable (mammalian cell) cryopreservation enhancers, but their mechanism of action is unclear, which may include membrane protection, solvent replacement, or a yet unknown protective mechanism, but it seems the modulation of ice growth (recrystallization) may only play a minor role in their function, unlike other macromolecular cryoprotectants. This Perspective will discuss their synthesis and summarize the state-of-the-art, including hypotheses of how they function, to introduce this exciting area of biomacromolecular science.

The design of a thermoresponsive surface for the continuous culture of human pluripotent stem cells

Commonly, stem cell culture is based on batch-type culture, which is laborious and expensive. We continuously cultured human pluripotent stem cells (hPSCs) on thermoresponsive dish surfaces, where hPSCs were partially detached on the same thermoresponsive dish by decreasing the temperature of the thermoresponsive dish to be below the lower critical solution temperature for only 30 min. Then, the remaining cells were continuously cultured in fresh culture medium, and the detached stem cells were harvested in the exchanged culture medium. hPSCs were continuously cultured for ten cycles on the thermoresponsive dish surface, which was prepared by coating the surface with poly(N-isopropylacrylamide-co-styrene) and oligovitronectin-grafted poly(acrylic acid-co-styrene) or recombinant vitronectin for hPSC binding sites to maintain hPSC pluripotency. After ten cycles of continuous culture on the thermoresponsive dish surface, the detached cells expressed pluripotency proteins and had the ability to differentiate into cells derived from the three germ layers in vitro and in vivo. Furthermore, the detached cells differentiated into specific cell lineages, such as cardiomyocytes, with high efficiency.

Establishing a Cultivable Cell Line of the Tick Dermacentor marginatus

Owing to the changes in the general ecological situation in Russia, livestock losses from ovine anaplasmosis have rapidly increased in recent years. The development of a vaccine against this disease is therefore all the more relevant. A continuous culture of tick cells could be used as an appropriate substrate for producing biomass of the pathogen of ovine anaplasmosis, Anaplasma ovis, for the purpose of manufacturing a cell-cultural inactivated vaccine. Such a continuous cell line of the tick Dermacentor marginatus, the definitive reservoir host of A. ovis, has been obtained by cultivation of homogenized tick eggs on a modified Leibovitz medium L15. The established cell line DM-77 has an undifferentiated phenotype: the cells are rounded, with large nuclei and well-visible nucleoli. By the end of the study, the cell line was passaged eight times and did not express signs of growth rate decrease. The duplication time was 2.8 ± 0.3 days for cultivation in a medium with 10% fetal serum at 32°C.

VIABILITY OF VERO E6 CELLS EXPOSED TO THE Bothrops leucurus SNAKE CRUDE VENOM

Primary and continuous cell culture systems are a good in vitro alternative for the quantitative assessment of snake venom toxicity. There are still few studies evaluating the cellular response against Bothrops leucurus venom (BlV). This study aimed to evaluate the cytotoxic effect of crude BlV on culture of VERO E6 cells. Neutral Red incorporation (NR) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) tests were performed for viable cells, and Trypan Blue (TB) dye exclusion test was performed for non-viable cells. The differences between the groups were evaluated by ANOVA-One-Way and Dunnet through the program Graph Pad 5.0 with significance of 5%. The 50% cytotoxic concentration (CC50) of BlV was 7.86 μg/mL. There was a trend of dose-dependent reduction on cell viability in all assays evaluated. However, MTT and NR tests were more sensitive for the evaluation of BlV cytotoxicity on the VERO E6 cell line.

Abstract 4842: Overcoming EGFR and ERK-mediated resistance to enzalutamide in castration-resistant prostate cancer

Abstract The present project was undertaken to determine whether the activation of the EGFR family (EGFR/ErbB2/ErbB3/ErbB4) and its downstream signaling effector ERK1/2 plays a role in enzalutamide resistance and whether treatment with ErbB or ERK inhibitors overcome this resistance. C4-2B (enzalutamide sensitive) and 22Rv1 (partially enzalutamide resistant) cell lines cultured for prolonged periods in enzalutamide, developed resistance to this drug (C4-2B/MDVR and 22Rv1/MDVR, respectively). Whole genome comparison of enza-resistant to parental lines demonstrated that the ErbB signaling network was significantly upregulated in the enza-resistant lines. Comparison of various enza-sensitive and resistant lines demonstrated a strong correlation between phosphorylation at EGFR(Y1068) and enza resistance. Inhibition of EGFR, but not that of ErbB2 or ErbB3 prevented cell viability. The EGFR inhibitors erlotinib and dacomitinib, but not the ErbB2 inhibitor lapatinib, suppressed viability in combination with enzalutamide. We hypothesized that enza-resistant tumors expressing higher levels of EGFR would be more responsive to erlotinib. To test this hypothesis, we compared the effects of erlotinib and enzalutamide, individually or in combination, in organelles from PDX tumors expressing high or low EGFR levels. Significantly, tumors expressing high EGFR, but not those expressing low EGFR, were responsive to the combination. Finally, we investigated the cause for greater efficacy with erlotinib compared to lapatinib in PCa. Comparison of the effects of different ligands revealed that while both lapatinib and erlotinib inhibited EGFR phosphorylation, only erlotinib but not lapatinib was able to inhibit EGF-induced ERK phosphorylation. This indicated an important role for ERK in the mediation of EGFR ligand induced enzalutamide resistance. Continuous culture of enza-sensitive C4 cells in enzalutamide resulted in the development of ERK phosphorylation in these PTEN-null cells that normally do not express phosphorylated ERK, and the newly ERK phosphorylated cells also were more susceptible to erlotinib as well. In support of a role for ERK in mediating the effects of EGFR activation in enza-resistant cells, inhibition of EGFR but not ErbB2 or ErbB3 inhibited ERK phosphorylation, and the ERK inhibitor ulitertinib inhibited viability of enza-resistant lines. We conclude that enzalutamide treatment causes an increase in EGFR ligands that result in the phosphorylation of EGFR at Y1068, which further resulted in phosphorylation of ERK. EGFR inhibitors that prevent ERK phosphorylation were effective in suppressing viability of enza-resistant CRPC cells, whereas ErbB2 inhibitors that did not affect ERK phosphorylation were unable to affect cell viability. These results demonstrate why certain ErbB inhibitors failed to affect enza-resistant CRPC tumors but provide encouragement for others. Citation Format: Thomas M. Steele, Maitreyee K. Jathal, Salma Siddiqui, Sisi Qin, Clifford G. Tepper, Ralph W. deVere White, Manish Kohli, Liewei Wang, Allen C. Gao, Paramita M. Ghosh. Overcoming EGFR and ERK-mediated resistance to enzalutamide in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4842.

The means of combating persistent infection of viral diarrhea

The paper presents results of research into the means of inhibiting persistent infection caused by noncytopathic biotype of bovine viral diarrhea virus. It was shown that bovine viral diarrhea virus, widespread in the cattle population in the whole world, can be a primary cause of pathology development of the respiratory and reproductive organs, and can cause a persistent form of infection in animals. The virus can contaminate fetal serum, cell cultures, trypsin, and other biological products, which leads to a decrease in the quality of biotech products from these materials and the spread of bovine viral diarrhea virus in a population of susceptible animals. The study was done on the model of a continuous calf coronary cell culture persistently infected with the noncytopathic biotype of the bovine viral diarrhea virus. The antiviral effect of two commercial preparations Ribavirin-Lipint and Reaferon-EC-Lipint was studied. The effectiveness of antiviral treatment of the infected cell culture was determined by the reverse transcription polymerase chain reaction, carried out in two versions: with electrophoresis and in real-time mode. It was found that these drugs in doses of 0.05 mg / ml and 30000 IU / ml, respectively, when added to growth medium for 24 consecutive passages, led to a decrease in the infectious activity of the bovine viral diarrhea virus in the cell culture, but did not cure it completely from the persistent infection caused by the noncytopathic biotype of the virus. Application of antiviral drugs is one of the ways to decrease economic losses caused by biological product contamination, the use of persistently infected animals for pedigree purposes and restrictions in international cattle trade.

Metabolic flux analysis during galactose and lactate co-consumption reveals enhanced energy metabolism in continuous CHO cell cultures

Chinese Hamster Ovary (CHO) cells are the main expression system for production of therapeutic recombinant (r-) proteins. The increased demand for these therapeutics has boosted the development of more robust production processes. Use of optimised feed/media has dramatically improved the performance of CHO cells in culture. However, such progress in biomass synthesis and r-protein production often come with an increased accumulation of lactate. In this study, we present a combined feeding strategy that uses galactose and lactate to replace glucose in CHO cell cultures. Replacement of glucose by galactose and lactate sustained cell growth and r-protein production in CHO cells. This strategy supported a better-balanced and more efficient metabolism, observed by an overall decreased consumption of carbon sources and amino acids, associated with an increased ATP production per C-mol consumed. Our results provide new insights of CHO cell metabolism in glucose-free media based on galactose and lactate.

Semi-continuous scale-down models for clone and operating parameter screening in perfusion bioreactors.

Perfusion cell culture, confined traditionally to the production of fragile molecules, is currently gaining broader attention in the biomanufacturing of therapeutic proteins. The development of these processes is made difficult by the limited availability of appropriate scale-down models. This is due to the continuous operation that requires complex control and cell retention capacity. For example, the determination of an optimal perfusion and bleed rate for continuous cell culture is often performed in scale-down bioreactors and requires a substantial amount of time and effort. To increase the experimental throughput and decrease the required workload, a semi-continuous procedure, referred to as the VCDmax (viable cell density) approach, has been developed on the basis of shake tubes (ST) and deepwell plates (96-DWP). Its effectiveness has been demonstrated for 12 different CHO-K1-SV cell lines expressing an IgG1. Further, its reliability has been investigated through proper comparisons with perfusion runs in lab-scale bioreactors. It was found that the volumetric productivity and the CSPRmin (cell specific perfusion rate) determined using the ST and 96-DWP models were successfully (mostly within the experimental error) confirmed in lab-scale bioreactors, which then covered a significant scale-up from the half milliliter to the liter scale. These scale-down models are very useful to design and scale-up optimal bioreactor operating conditions as well as screening for different media and cell lines.

Continuous Live-Cell Culture Imaging and Single-Cell Tracking by Computational Lensfree LED Microscopy.

Continuous cell culture monitoring as a way of investigating growth, proliferation, and kinetics of biological experiments is in high demand. However, commercially available solutions are typically expensive and large in size. Digital inline-holographic microscopes (DIHM) can provide a cost-effective alternative to conventional microscopes, bridging the gap towards live-cell culture imaging. In this work, a DIHM is built from inexpensive components and applied to different cell cultures. The images are reconstructed by computational methods and the data are analyzed with particle detection and tracking methods. Counting of cells as well as movement tracking of living cells is demonstrated, showing the feasibility of using a field-portable DIHM for basic cell culture investigation and bringing about the potential to deeply understand cell motility.