Cell Density Cultivation Research Articles

Dynamics of long-term continuous culture of Limnospira indica in an air-lift photobioreactor.

SummaryMELiSSA (Microecological Life Support System Alternative) is a developing technology for regenerative life support to enable long‐term human missions in Space and has developed a demonstration Pilot Plant. One of the components of the MELiSSA Pilot Plant system is an 83L external loop air‐lift photobioreactor (PBR) where Limnospira indica (previously named Arthrospira sp. PC8005) is axenically cultivated in a continuous operation mode for long‐periods. Its mission is to provide O2 and consume CO2 while producing edible material. Biological and process characterization of this PBR is performed by analysing the effect of two main variables, dilution rate (D) and PFD (Photon Flux Density) illumination. A maximum oxygen productivity (rO2) of 1.35 mmol l−1 h−1 is obtained at a D of 0.025 h−1 and PFD of 930 µmol m−2 s−1. Photoinhibition can occur when a 1 g l−1 cell density culture is exposed to PFD higher than 1700 µmol m−2 s−1. This process is reversible if the illumination is returned to dim light (150 µmol m−2 s−1), proving the cell adaptability and capacity to respond at different illumination conditions. Influence of light intensity in cell composition is also described. Specific photon flux density (qPFD) has a direct effect on phycobiliproteins and chlorophyll content causing a decrease of 62.5% and 47.8%, respectively, when qPFD increases from 6.1 to 19.2 µmol g−1 s−1. The same trend is observed for proteins and the opposite for carbohydrate content. Morphological and spiral structural features of L. indica are studied by confocal microscopy, and size distribution parameters are quantified. A direct effect between trichome width and CDW/OD ratio is observed. Changes in size distribution are not correlated with environmental factors, further confirms the adaptation capacity of the cells. The systematic analysis performed provides valuable insights to understand the key performance criteria of continuous culture in air‐lift PBRs.

A cell engineering approach to enzyme-based fed-batch fermentation

BackgroundA fundamental problem associated with E. coli fermentations is the difficulty in achieving high cell densities in batch cultures, attributed in large part to the production and accumulation of acetate through a phenomenon known as overflow metabolism when supplying enough glucose for the cell density desired. Although a fed-batch configuration is the standard method for reducing such issues, traditional fed-batch systems require components which become problematic when applying them at smaller scale. One alternative has been the development of a system whereby the enzymatic degradation of starch is used to release glucose at a controlled rate. However, to date, amylolytic enzymes have only been applied to the culture exogenously, whereas our goal is to design and construct a self-secreting amylolytic chassis capable of self-regulated enzyme-based fed-batch fermentation.ResultsA putative glucoamylase from C. violaceum has been cloned and expressed in E. coli BL21(DE3) and W3110, which exhibits significant glucose releasing amylolytic activity. Extracellular amylolytic activity was enhanced following a replacement of the enzymes native signal peptide with the DsbA signal sequence, contributing to a glucoamylase secreting strain capable of utilising starch as a sole carbon source in defined media. Introduction of PcstA, a glucose sensitive K12 compatible promoter, and the incorporation of this alongside C. violaceum glucoamylase in E. coli W3110, gave rise to increased cell densities in cultures grown on starch (OD600 ∼ 30) compared to those grown on an equivalent amount of glucose (OD600 ∼ 15). Lastly, a novel self-secreting enzyme-based fed-batch fermentation system was demonstrated via the simultaneous expression of the C. violaceum glucoamylase and a recombinant protein of interest (eGFP), resulting in a fourfold increase in yield when grown in media containing starch compared with the glucose equivalent.ConclusionsThis study has developed, through the secretion of a previously uncharacterised bacterial glucoamylase, a novel amylolytic E. coli strain capable of direct starch to glucose conversion. The ability of this strain to achieve increased cell densities as well as an associated increase in recombinant protein yield when grown on starch compared with an equivalent amount of glucose, demonstrates for the first time a cell engineering approach to enzyme-based fed-batch fermentation.

Multilayered Cell Sheets of Cardiac Reprogrammed Cells for the Evaluation of Drug Cytotoxicity.

Various cell-culture systems have been used to evaluate drug toxicity in vitro. However, factors that affect cytotoxicity outcomes in drug toxicity evaluation systems remain elusive. In this study, we used multilayered sheets of cardiac-mimetic cells, which were reprogrammed from human fibroblasts, to investigate the effects of the layer number on drug cytotoxicity outcomes. Cell sheets of cardiac-mimetic cells were fabricated by reprogramming of human fibroblasts into cardiac-mimetic cells via coculture with cardiac cells and electric stimulation, as previously described. Double-layered cell sheets were prepared by stacking the cell sheets. The mono- and double-layered cell sheets were treated with 5-fluorouracil (5-FU), an anticancer drug, in vitro. Subsequently, apoptosis and lipid peroxidation were analyzed. Furthermore, effects of cardiac-mimetic cell density on cytotoxicity outcomes were evaluated by culturing cells in monolayer at various cell densities. The double-layered cell sheets exhibited lower cytotoxicity in terms of apoptosis and lipid peroxidation than the mono-layered sheets at the same 5-FU dose. In addition, the double-layered cell sheets showed better preservation of mitochondrial function and plasma membrane integrity than the monolayer sheets. The lower cytotoxicity outcomes in the double-layered cell sheets may be due to the higher intercellular interactions, as the cytotoxicity of 5-FU decreased with cell density in monolayer cultures of cardiac-mimetic cells. The layer number of cardiac-mimetic cell sheets affects drug cytotoxicity outcomes in drug toxicity tests. The in vitro cellular configuration that more closely mimics the in vivo configuration in the evaluation systems seems to exhibit lower cytotoxicity in response to drug.

Cell-cell and cell-substratum contacts in the regulation of MAPK and Akt signalling: Importance in therapy, biopharmacy and bioproduction

The use of cultured cells as a tool for research, precision medicine, biopharmacy, and biomanufacturing is constantly increasing. In parallel, the role of cell-cell and cell-substratum contacts in cell functioning is increasingly validated. Adhesion signalling plays a key role here. The activity of cell fate-regulating signalling molecules is an important factor in determining cell behaviour, as well as their response to treatment, depending on cell phenotypic status and location in the body. Three cellular state models (adherent, single cells in suspension, and aggregated cells) were compared for cell signalling, including focal adhesion (FAK), mitogen-activated (MAPK), as well as Akt protein kinases, and transcription factor cJun, by using lung adenocarcinoma A549, muscle-derived stem Myo, as well as primary lung cancer cell lines. Survival of both A549 and Myo cells was dependent on kinases Akt and ERK in detached conditions. Intercellular contacts in aggregates promoted activation of Akt and ERK, and cell survival. Loss of contacts with the substrate increased phosphorylation of MAP kinases JNK and p38, while decreased Akt phosphorylation by processes involving FAK. Unexpectedly, detachment increased phosphorylation of antiapoptotic kinase ERK in A549, while in Myo stem cells ERK phosphorylation was downregulated. JNK target transcription factor cJun protein level was markedly diminished by contacts between cells possibly involving mechanism of proteasomal degradation. Furthermore, studies revealed the opposite dependence of molecules of the same signalling pathway – phospho-cJun and phospho-JNK – on cell culture density. Differences in ERK activation under detachment conditions indicate that targeting of prosurvival kinases during anoikis should be different in different cells. Moreover, the outcome of JNK activation in cells may depend on the amount of cJun, which is determined by cell-cell contacts.

Effects of mouse fetal liver cell culture density on hematopoietic cell expansion in three-dimensional cocultures with stromal cells.

An effective ex vivo expansion system of primitive hematopoietic cells (HCs) is required for wider application of hematopoietic stem cell transplantation. In this study, we examined effects of culture density on mouse fetal liver cells (FLCs) used as an HC source for the expansion of primitive HCs in three-dimensional (3D) cocultures with two kinds of mouse stromal cell lines (OP9 or C3H10T1/2). FLCs were seeded at different densities (1, 2, and 10 × 107 cells/cm3) into porous polymer scaffolds with or without stromal cell layers and HCs were expanded in the cultures for 2 weeks without exogenous cytokines. Differential effects of culture density on HC expansion were observed between cocultures and solitary FLC controls. In stromal cell cocultures, high expansion of HCs was achieved when FLCs were seeded at low densities. In contrast, the expansion in the controls was enhanced with increasing culture densities. With respect to expansion of primitive HCs existing in the FLCs, cocultures with C3H10T1/2 cells were superior to those with OP9 cells with a 29.3-fold expansion for c-kit+ hematopoietic progenitor cells and 8.3-fold expansion for CD34+ hematopoietic stem cells. In the controls, HC expansion was lower than in any cocultures, demonstrating the advantages of coculturing for HC expansion. Stromal cell lines are useful in expanding primitive HCs derived from FLCs in 3D cocultures. Culture density is a pivotal factor for the effective expansion of primitive HCs and this effect differs by culture condition.

The damage and tolerance mechanisms of Phaffia rhodozyma mutant strain MK19 grown at 28\xa0\xb0C

BackgroundPhaffia rhodozyma has many desirable properties for astaxanthin production, including rapid heterotrophic metabolism and high cell densities in fermenter culture. The low optimal temperature range (17–21 °C) for cell growth and astaxanthin synthesis in this species presents an obstacle to efficient industrial-scale astaxanthin production. The inhibition mechanism of cell growth at > 21 °C in P. rhodozyma have not been investigated.ResultsMK19, a mutant P. rhodozyma strain grows well at moderate temperatures, its cell growth was also inhibited at 28 °C, but such inhibition was mitigated, and low biomass 6 g/L was obtained after 100 h culture. Transcriptome analysis indicated that low biomass at 28 °C resulted from strong suppression of DNA and RNA synthesis in MK19. Growth inhibition at 28 °C was due to cell membrane damage with a characteristic of low mRNA content of fatty acid (f.a.) pathway transcripts (acc, fas1, fas2), and consequent low f.a. content. Thinning of cell wall and low mannose content (leading to loss of cell wall integrity) also contributed to reduced cell growth at 28 °C in MK19. Levels of astaxanthin and ergosterol, two end-products of isoprenoid biosynthesis (a shunt pathway of f.a. biosynthesis), reached 2000 µg/g and 7500 µg/g respectively; ~2-fold higher than levels at 21 or 25 °C. Abundance of ergosterol, an important cell membrane component, compensated for lack of f.a., making possible the biomass production of 6 g/L for MK19 at 28 °C.ConclusionsInhibition of growth of P. rhodozyma at 28 °C results from blocking of DNA, RNA, f.a., and cell wall biosynthesis. In MK19, abundant ergosterol made possible biomass production 6 g/L at 28 °C. Significant accumulation of astaxanthin and ergosterol indicated an active MVA pathway in MK19 at 28 °C. Strengthening of the MVA pathway can be a feasible metabolic engineering approach for enhancement of astaxanthin synthesis in P. rhodozyma. The present findings provide useful mechanistic insights regarding adaptation of P. rhodozyma to 28 °C, and improved understanding of feasible metabolic engineering techniques for industrial scale astaxanthin production by this economically important yeast species.

Microalgal Biomass Generation via Electroflotation: A Cost-Effective Dewatering Technology

Microalgae are an excellent source of bioactive compounds for the production of a wide range of vital consumer products in the biofuel, pharmaceutical, food, cosmetics, and agricultural industries, in addition to huge upstream benefits relating to carbon dioxide biosequestration and wastewater treatment. However, energy-efficient, cost-effective, and scalable microalgal technologies for commercial-scale applications are limited, and this has significantly impacted the full-scale implementation of microalgal biosystems for bioproduct development, phycoremediation, and biorefinery applications. Microalgae culture dewatering continues to be a major challenge to large-scale biomass generation, and this is primarily due to the low cell densities of microalgal cultures and the small hydrodynamic size of microalgal cells. With such biophysical characteristics, energy-intensive solid–liquid separation processes such as centrifugation and filtration are generally used for continuous generation of biomass in large-scale settings, making dewatering a major contributor to the microalgae bioprocess economics. This article analyzes the potential of electroflotation as a cost-effective dewatering process that can be integrated into microalgae bioprocesses for continuous biomass production. Electroflotation hinges on the generation of fine bubbles at the surface of an electrode system to entrain microalgal particulates to the surface. A modification of electroflotation, which combines electrocoagulation to catalyze the coalescence of microalgae cells before gaseous entrainment, is also discussed. A technoeconomic appraisal of the prospects of electroflotation compared with other dewatering technologies is presented.

Lactate adsorption by layered double hydroxides in aqueous solution and cell culture medium

When cells proliferate in a culture medium, metabolites such as lactic acid gradually accumulate and eventually hinder cell growth. Herein, we considered various layered double hydroxides (LDH) and layered double oxides (LDO) for the selective removal of lactate in aqueous solution and a culture medium by anion exchange. NO3•Mg-Al LDH showed high lactate adsorption, low adsorption of glucose (a nutrient in culture medium), appropriate pH stability, and low cytotoxicity within a limited contact time. In contrast, NO3•Ni-Al LDH, NO3•Ca-Al LDH, Mg-Al LDO, and Cl•Mg-Al LDH had a lower performance than NO3•Mg-Al LDH. To further improve the performance, the anion of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, a common pH buffer in culture media) was tested as the intercalated anion in Mg-Al LDH. HEPES•Mg-Al LDH (Mg/Al = 2) was found to be the most promising adsorbent for culture medium regeneration because its adsorption behavior was similar to that of NO3•Mg-Al LDH, while it had a much lower cytotoxicity. The traditional methods for regenerating a culture medium by removing accumulated lactate to increase the cell density in large-scale cell culture and lower the costs, including electrolysis, cell modification, and modification of the culture medium’s composition, are complicated to operate and expensive. The adsorption method using HEPES•Mg-Al LDH presented in this study, instead, can be easily operated and has low costs. Thus, it is expected to be applied in actual cases for regenerating culture media.

Calibration Curves of Culture Density Assessed by Spectrophotometer for Three Microalgae (Nephroselmis sp., Amphidinium carterae and Phormidium sp.)

This study concerns the finding of an effective operational equation relating the measured absorpbance (or O.D. - optical density) in a spectrophotometer of the suspended cells of microalgae to their cell density (c.d.) in culture in order to construct calibration curves for use in culture operations. The microalgae examined were the chlorophyte Nephroselmis sp., the dinophyte Amphidinium carterae and the filamentous cyanobacterium Phormidium sp. Wavelengths of 430 and 680 nm were selected that correspond to chlorophyll-a peaks of their absorption spectra were used and additionally 750 and 570 nm where absorbance was not peaked. From all equations extracted best fitness with strong predictive values were those of a logarithmic type: O.D.=0,9328*ln(c.d.)-14,108 (R2=0,9943) at 680 nm for Nephroselmis, a power equation: O.D.=0,0000009*(c.d.)0,9195 (R2=0,9936) at 680 nm for Amphidinium and a polynomial second order: O.D.=0,9869(c.d.)2+2,4393(c.d.)+0,2666 (R2=0,9737) at 570 nm for Phormidium.

Effects of Fibroblast Growth Factor-2 and Other Microsupplements on the Productivity of IgG- and IgA-Producing Cell Lines

Several serum-free media have been developed and are successfully used at present for the cultivation of various recombinant eukaryotic cell lines. Therapeutic recombinant IgA antibodies have recently been increasingly used along with numerous IgG1 isotype antibodies. Therefore, it became necessary to improve the growth characteristics, metabolism, and productivity of cell cultures producing these antibodies. The stimulating effect of a complex supplement containing Zn salts and FGF-2 on cell cultivation on basal media, DMEM and IMDM, was revealed in this work. The addition of dextran sulfate sodium salt and iron citrate to the basal medium was accompanied by improved productivity of stable IgG- and IgA-producing cell lines, as well as the homogeneity and density of cell cultures, which is most important in IgA-antibody production.

Sustainable Production of Monoraphidium Microalgae Biomass as a Source of Bioenergy

Microalgae are a renewable source of unconventional biomass with potential application in the production of various biofuels. The production of carbon-neutral fuels is necessary for protecting the environment. This work determined the possibility of producing biomass of microalgae belonging to Monoraphidium genus using saline wastewater resulting from proecological salmon farming in the recirculating aquaculture system. The tests were carried out in tubular photobioreactors using LED light. As a part of the analyses, the growth and productivity of microalgal biomass, cell density in culture, and lipid concentration and ash content in biomass were determined. In addition, the concentration of selected phosphorus and nitrogen forms present in wastewater corresponding to the degree of their use by microalgae as a nutrient substrate was determined. The biomass concentration estimated in the tests was 3.79 g·L−1, while the maximum biomass productivity was 0.46 g·L−1·d−1. The cells’ optical density in culture measured at 680 nm was 0.648. The lipid content in biomass was 18.53% (dry basis), and the ash content was 32.34%. It was found that microalgae of the genus Monoraphidium effectively used the nitrogen as well as phosphorus forms present in the wastewater for their growth. The total nitrogen content in the sewage decreased by 82.62%, and total phosphorus content by over 99%. The analysis of the individual forms of nitrogen showed that N-NO3 was reduced by 85.37% and N-NO2 by 78.43%, while orthophosphate (V) dissolved in water was reduced by 99%. However, the content of N-NH4 in wastewater from the beginning till the end of the experiment remained <0.05 mg·L−1.

Syncytium cell growth increases Kir2.1 contribution in human iPSC-cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enable cardiotoxicity testing and personalized medicine. However, their maturity is of concern, including relatively depolarized resting membrane potential and more spontaneous activity compared with adult cardiomyocytes, implicating low or lacking inward rectifier potassium current (Ik1). Here, protein quantification confirms Kir2.1 expression in hiPSC-CM syncytia, albeit several times lower than in adult heart tissue. We find that hiPSC-CM culture density influences Kir2.1 expression at the mRNA level (potassium inwardly rectifying channel subfamily J member 2) and at the protein level and its associated electrophysiology phenotype. Namely, all-optical cardiac electrophysiology and pharmacological treatments reveal reduction of spontaneous and irregular activity and increase in action potential upstroke in denser cultures. Blocking Ik1-like currents with BaCl2 increased spontaneous frequency and blunted action potential upstrokes during pacing in a dose-dependent manner only in the highest-density cultures, in line with Ik1’s role in regulating the resting membrane potential. Our results emphasize the importance of syncytial growth of hiPSC-CMs for more physiologically relevant phenotype and the power of all-optical electrophysiology to study cardiomyocytes in their multicellular setting.NEW & NOTEWORTHY We identify cell culture density and cell-cell contact as an important factor in determining the expression of a key ion channel at the transcriptional and the protein levels, KCNJ2/Kir2.1, and its contribution to the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes. Our results indicate that studies on isolated cells, out of tissue context, may underestimate the cellular ion channel properties being characterized.

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.

Bioindication of heavy metals in aquatic environment using photosynthetic pigments in cyanobacteria

Freshwater resources is seriously threatened by heavy metals pollution. Thus developing an effective screening method for the presence of heavy metals is required. In this paper, the potential of using cyanobacteria for heavy metals detection in water bodies is determined. The cyanobacteria Anabaena cylindrica had been cultured, immobilized, and exposed to Cu, Pb, and Cd with the concentrations of 0.01 – 10.00 mg/L. The responses of the cells to the heavy metals were measured for 60 min. The effect of the cell culture age and cell density were determined as well. The cells with 7 days of culture age, and the amount of cell with density of OD= 0.5 A (measured at 700 nm wavelength) were found to produce highest photosynthetic fluorescence for the detection for heavy metals. The cells were capable to provide detectable fluorescence emission within 10 min of exposure. Thus the cyanobacteria was identified as a good candidate to serve as bioindicator for the heavy metals in water.

Cell Density-Dependent Fibroblast Growth Factor-2 Signaling Regulates Syndecan-4 Expression in Cultured Vascular Endothelial Cells.

Syndecan-4 is a member of the syndecan family of transmembrane heparan sulfate proteoglycans, and is involved in cell protection, proliferation, and the blood coagulation-fibrinolytic system in vascular endothelial cells. Heparan sulfate chains enable fibroblast growth factor-2 (FGF-2) to form a complex with its receptor and to transduce the cell growth signal. In the present study, bovine aortic endothelial cells were cultured, and the intracellular signal pathways that mediate the regulation of syndecan-4 expression in dense and sparse cultures by FGF-2 were analyzed. We demonstrated the cell density-dependent differential regulation of syndecan-4 expression. Specifically, we found that FGF-2 upregulated the synthesis of syndecan-4 in vascular endothelial cells via the MEK1/2-ERK1/2 pathway in dense cell cultures, with only a transcriptional induction of syndecan-4 at a low cell density via the Akt pathway. This study highlights a critical mechanism underlying the regulation of endothelial cell functions by proteoglycans.

Improving HIV Outgrowth by Optimizing Cell-Culture Conditions and Supplementing With all-trans Retinoic Acid.

The persistence of replication-competent HIV reservoirs in people living with HIV (PLWH) receiving antiretroviral therapy (ART) is a barrier to cure. Therefore, their accurate quantification is essential for evaluating the efficacy of new therapeutic interventions and orienting the decision to interrupt ART. Quantitative viral outgrowth assays (QVOAs) represent the “gold standard” for measuring the size of replication-competent HIV reservoirs. However, they require large numbers of cells and are technically challenging. This justifies the need for the development of novel simplified methods adapted for small biological samples. Herein, we sought to simplify the viral outgrowth procedure (VOP) by (i) using memory CD4+ T-cells, documented to be enriched in HIV reservoirs (ii) optimizing cell-culture conditions, and (iii) supplementing with all-trans retinoic acid (ATRA), a positive regulator of HIV replication. Memory CD4+ T-cells were sorted from the peripheral blood of ART-treated (HIV+ART; n = 14) and untreated (HIV+; n = 5) PLWH. The VOP was first performed with one original replicate of 1 × 106 cells/well in 48-well plates. Cells were stimulated via CD3/CD28 for 3 days, washed to remove residual CD3/CD28 Abs, split every 3 days for optimal cell density, and cultured in the presence or the absence of ATRA for 12 days. Soluble and intracellular HIV-p24 levels were quantified by ELISA and flow cytometry, respectively. Optimal cell-culture density achieved by splitting improved HIV outgrowth detection. ATRA promoted superior/accelerated detection of replication-competent HIV in all HIV+ART individuals tested, including those with low/undetectable viral outgrowth in the absence of ATRA. Finally, this VOP was used to design a simplified ATRA-based QVOA by including 4 and 6 original replicates of 1 × 106 cells/well in 48-well plates and 2 × 105 cells/well in 96-well plates, respectively. Consistently, the number of infectious units per million cells (IUPM) was significantly increased in the presence of ATRA. In conclusion, we demonstrate that memory CD4+ T-cell splitting for optimal density in culture and ATRA supplementation significantly improved the efficacy of HIV outgrowth in a simplified ATRA-based QVOA performed in the absence of feeder/target cells or indicator cell lines.

Important considerations for cell therapy manufacturing of mesenchymal stem cell

Background & Aim Recent advances in cell therapy manufacturing, e.g. microcarrier-based 3D culture and perfusion, have paved the way for cost-effective large-scale expansion of mesenchymal stem cells (MSCs). In particular, perfusion and enhanced feed boosts allow the culture to reach increasingly high cell densities. Although the high cell density is favorable to customers due to the low cost and high cell dose requirements, we should not overlook its impact on cell quality. Here, we studied the correlation between 3D culture cell density with a variety of commonly used cell quality parameters for both fresh and post-thaw cells. Methods, Results & Conclusion MSC 3D cultures were carried out using different microcarriers (Cultispher-G, dissolvable Corning Synthemax, and Pall SoloHill collagen) and media in spinner flasks and stirred-tank bioreactors. MSCs were able to expand on all microcarriers (Figure1), reaching a final density of 0.6 to 1 million cells/mL. Attachment and proliferation capacities of 3D-expanded MSCs, harvested at different cell densities, were assessed by culturing them post-harvest on 2D flatware. Figure2 shows that MSCs harvested at low cell densities (day4) proliferated significantly higher than MSCs harvested at high cell densities (day5-8). Similar pattern was observed for 3D-cultured MSCs post-thaw. Cell viability was also evaluated for both fresh and post-thaw MSCs harvested at low and high cell densities. For most cases, MSCs harvested at both low and high cell densities, showed above 90% viability over 0-6 hours (Figure3). MSCs expanded using SoloHill microcarriers displayed >90% post-thaw viability for both low and high cell densities. However, dissolvable Corning-Synthemax exhibited up to 12% and 30% drops, and Cultispher-G up to 24% and 51% drops for low and high cell density groups, respectively. Figure3 also shows that the extent of decline in post-thaw vibility was higher for MSCs harvested at high cell densities versus low cell densities. In conclusions, our findings indicated that the extended 3D culture (i.e. harvesting at higher cell densities) led to compromising attachment and proliferation capacities of MSCs, regardless of the choice of microcarrier. For Cultispher-G and dissolvable Coning-Synthemax, we observed 20-30% lower cell viabilities for MSCs harvested at high densities compared to low densities.

Pre-stage perfusion and ultra-high seeding cell density in CHO fed-batch culture: a case study for process intensification guided by systems biotechnology

Process intensification strategies are needed in the field of therapeutic protein production for higher productivities, lower cost of goods and improved facility utilization. This work describes an intensification approach, which connects a tangential-flow-filtration (TFF) based pre-stage perfusion process with a concentrated fed-batch production culture inoculated with an ultra-high seeding density (uHSD). This strategy shifted biomass production towards the pre-stage, reaching up to 45 × 106 cells/mL in perfusion mode. Subsequently, production in the intensified fed-batch started immediately and the product titer was almost doubled (1.9-fold) in an equivalent runtime and with comparable product quality compared to low-seeded cultures. Driven by mechanistic modelling and next-generation sequencing (NGS) the process had been optimized by selecting the media composition in a way that minimized cellular adaptation between perfusion and production culture. As a main feature, lactate feeding was applied in the intensified approach to promote cell culture performance and process scalability was proven via transfer to pilot-scale i.e., 20 L pre-stage perfusion and 80 L production reactor. Moreover, an earlier shift from a growth associated to a production stage associated gene expression pattern was identified for uHSD cultures compared to the reference. Overall, we showed that the described intensification strategy yielded in a higher volumetric productivity and is applicable for existing or already approved molecules in common, commercial fed-batch facilities. This work provides an in-depth molecular understanding of cellular processes that are detrimental during process intensification.

Using SERS Tags to Image the Three‐Dimensional Structure of Complex Cell Models

AbstractMethods to image complex 3D cell cultures are limited by issues such as fluorophore photobleaching and decomposition, poor excitation light penetration, and lack of complementary techniques to verify the 3D structure. Although it remains insufficiently demonstrated, surface‐enhanced Raman scattering (SERS) imaging is a promising tool for the characterization of biological complex systems. To this aim, a controllable 3D cell culture model which spans nearly 1 cm2 in surface footprint is designed. This structure is composed of fibroblasts containing SERS‐encoded nanoparticles (i.e., SERS tags), arranged in an alternating layered structure. This “sandwich” type structure allows monitoring of the SERS signals in the z‐axis and with mm dimensions in the xy‐axis. Taking advantage of correlative microscopy techniques such as electron microscopy, it is possible to corroborate nanoparticle positioning and distances in z‐depths of up to 150 µm. This study reveals a proof‐of‐concept method for detailed 3D SERS imaging of a complex, dense 3D cell culture model.

The Influence of Cell Culture Density on the Cytotoxicity of Adipose-Derived Stem Cells Induced by L-Ascorbic Acid-2-Phosphate

Ascorbic acid-2-phosphate (A2-P) is an oxidation-resistant derivative of ascorbic acid that has been widely employed in culturing adipose-derived stem cells (ASCs) for faster expansion and cell sheet formation. While high dose ascorbic acid is known to induce cellular apoptosis via metabolic stress and genotoxic effects, potential cytotoxic effects of A2-P at high concentrations has not been explored. In this study, the relationship between ASC seeding density and A2-P-induced cytotoxicity was investigated. Spheroid-derived ASCs with smaller cellular dimensions were generated to investigate the effect of cell-cell contact on the resistance to A2-P-induced cytotoxicity. Decreased viability of ASC, fibroblast, and spheroid-derived ASC was noted at higher A2-P concentration, and it could be reverted with high seeding density. Compared to control ASCs, spheroid-derived ASCs seeded at the same density exhibited decreased viability in the A2-P-supplemented medium. The expression of antioxidant enzymes (catalase, SOD1, and SOD2) was enhanced in ASCs at higher seeding densities. However, their enhanced expression in spheroid-derived ASCs was less evident. Furthermore, we found that co-administration of catalase or N-acetylcysteine nullified the observed cytotoxicity. Collectively, A2-P can induce ASC cytotoxicity at higher concentrations, which can be prevented by seeding ASCs at high density or co-administration of another antioxidant.