Biotechnology Journal

During manufacturing batches, Chinese hamster ovary (CHO) cells encounter critical levels of environmental stressors such as ammonia, lactate, and osmolality accumulation that can significantly reduce cell health and productivity. It is therefore crucial that stress adaptation and resistance be factored into cell line development (CLD). In this study, we employee population-based transcriptomic and differential gene expression analysis on stress-induced CHO cells to identify biomarkers displaying both heritable and stress-responsive properties. Using this workflow, 199 genes displayed transcriptional variability characteristic of a bistable system that formed four network communities of co-fluctuating genes. These communities were enriched in genes related to the regulation of apoptotic processes and gene expression/metabolic pathways. Seven genes were identified as promising biomarkers for engineering a stress-resistant phenotype. Genetic engineering methods may be employed in the future to bias clonal populations for higher stress tolerance to manufacturing stress, therefore increasing cell health and productivity in at-scale bioreactors.

Medium optimization is a crucial step of cell culture for biopharmaceutics and regeneration medicine. It remains challenging, as both media and cells are highly complex systems. To address the issue, we tried active learning to fine-tune the culture medium by combining the high-throughput assay and machine learning. As a pilot study, the cell line HeLa-S3 and the gradient-boosting decision tree algorithm were used. The regular and time-saving approaches were developed, and both successfully fine-tuned 29 components to achieve improved cell culture than the original medium. The fine-tuned media showed a significant decrease in fetal bovine serum and the differentiation in vitamins and amino acids. Unexpectedly, the medium optimization raised the cellular NAD(P)H abundance but not the cell concentration owing to the conventional method used for cell culture assay. Our study demonstrated the efficiency of active learning for medium optimization and provided valuable hints for employing machine learning in cell culture.

Nanobody is one special type of single-domain antibody fragment with multiple advantages over traditional antibody. Our previous work established linear-double-stranded DNA (ldsDNA, or PCR amplicon) as novel biological parts for building AND gate genetic circuits in mammalian cells. During this AND-gate circuit formation process, the co-transfected up- and down-stream ldsDNAs could be linked together to form intact gene expression cassette. Here, we employed this ldsDNA-based AND-gate (LBAG) strategy to construct nanobody library in mammalian cells. The sequence complexity of complementary determining regions (CDRs) was introduced into ldsDNA by PCR amplification. After being co-transfected into mammalian cells, the up- and down-stream ldsDNAs undergo AND gate linkage and form full nanobody coding regions, containing CDR1-3. High throughput sequencing identified 22,173 unique oligonucleotide sequences in total generated by this strategy. Thus, we developed a novel method to construct nanobody library, which is a start point for building high content nanobody library in mammalian cells.

Chinese Hamster Ovary (CHO) cell monoclonal antibody (mAb) production in continuous perfusion has witnessed a renewed interest within the biopharmaceutical industry. Widespread implementation of perfusion biomanufacturing, however, remains hindered by long process development timelines and high costs. Use of predictive scale-down platforms to generate large informative metabolic datasets and guide process development decisions is critical to decreasing a molecules time to market. While scale-down platforms based on the pseudo perfusion concept have been previously reported, they have not been rigorously validated. They are often limited by oxygen transport or insufficient metabolic characterization, reducing their role to a preliminary screening tool. Here, we report the design and validation of a pseudo perfusion platform based on a phenotype-driven approach to ascertain that the process emulates continuous perfusion characteristics and is not oxygen limited. Beyond metabolic and cell size steady state, we show that our pseudo perfusion design enables cell cycle subpopulation and intracellular antibody expression steady state. We also demonstrate that pseudo perfusion robustly predicts amino acid demands in continuous perfusion bioreactors with exceptional linear correlation across a broad range of cell-specific perfusion rates (CSPRs). When coupling the pseudo perfusion platform developed here with a workflow for metabolic characterization, we significantly augment the dimensionality and reliability of data which can be generated at this scale to gain actionable insights towards perfusion process design, ultimately reducing process development timelines and the associated costs. HighlightsResidual lactate is a key proxy for oxygen transport in scale down platform design Novel flow cytometry workflow confirms cell cycle and intracellular steady state Pseudo perfusion robustly predicts metabolic phenotypes in continuous perfusion K-means clustering analysis of nutrient rates provides insight into media design

Rapid progress in algal biotechnology has triggered a growing interest in hydrogel-encapsulated microalgal cultivation, especially for the engineering of functional photosynthetic materials and biomass production. An overlooked characteristic of gel-encapsulated cultures is the emergence of cell aggregates, which are the result of the mechanical confinement of the cells. Such aggregates have a dramatic effect on the light management of gel-encapsulated photobioreactors and hence strongly affect the photosynthetic outcome. In order to evaluate such an effect, we experimentally studied the optical response of hydrogels containing algal aggregates and developed optical simulations to study the resultant light intensity profiles. The simulations are validated experimentally via transmittance measurements using an integrating sphere and aggregate volume analysis with confocal microscopy. Specifically, the heterogeneous distribution of cell aggregates in a gel matrix can increase light penetration while alleviating photoinhibition compared to a flat biofilm. Finally, we demonstrate that light harvesting efficiency can be further enhanced with the introduction of scattering particles within the hydrogel matrix, leading to a four-fold increase in biomass growth. Our study, therefore, highlights a new strategy for the design of spatially efficient photosynthetic living materials that have important implications for the engineering of future algal cultivation systems. Significance StatementThe ability to cultivate microalgae at scale efficiently would allow more sustainable production of food and food additives. However, efficient growth of microalgae requires optimised light conditions, which are usually challenging to obtain using biofilm cultivations mode: as the outer layer of cells are necessarily more exposed to incoming light than the inner layer, posing the problem of photoinhibition on the outer cells receiving too much light, and shading the ones below. Here we study both experimentally and numerically, how microalgae aggregates growing in the confinement of a hydrogel can provide an improved light distribution and therefore biomass growth is maximised. This study proposes new strategies on how to engineer future photobioreactors.

Tissue cultures are an important study model for woody plant tissue and can be used to study lignin biosynthesis. The greatest disadvantage of protocols based on extraction of lignin from wood biomass is the almost inevitable alteration of the native structure of lignin. Using a Norway spruce tissue culture with the ability to secrete monolignols into a liquid culture medium, fundamental aspects of lignin have been studied in the past, such as its structure, the enzyme activity related to its polymerization, and its interactions with a secondary cell wall hemicellulose. In this study, parameters that can induce monolignol production and secretion in the tissue culture are investigated via gene expression analysis. The impact of the composition of the solid growth medium, which was in some cases supplemented with xylan, was studied in depth through transcriptomic investigation. We find that the state (i.e. liquid or solid) and the xylan content of the medium can impact gene expression, although microscopic analysis suggests that cellular morphology is consistent. Extracellular lignin was collected from a formulation of liquid medium with the same composition as that used for cellular growth, which was previously presumed to be "non-inducing" of lignin biosynthesis. Chemical analysis of this lignin was performed using nuclear magnetic resonance spectroscopy and size exclusion chromatography, which revealed changes in its structure compared to the polymer produced in the previously developed "inducing" liquid medium. These experiments show that there is still much we do not understand about an oft-used tissue culture system, but show the way to a deeper understanding of the genetic control of lignin biosynthesis.

Microfluidic vortex shedding (VS) can rapidly deliver mRNA to T cells with high yield. The mechanistic underpinning of VS intracellular delivery remains undefined and VS-Cas9 genome editing requires further studies. Herein, we evaluated a series of VS devices containing splitter plates to attenuate vortex shedding and understand the contribution of computed force and frequency on efficiency and viability. We then selected a VS design to knockout the expression of the endogenous T cell receptor in primary human T cells via delivery of CRISPR-Cas9 ribonucleoprotein (RNP) with and without brief exposure to an electric field (eVS). VS alone resulted in an equivalent yield of genome-edited T cells relative to electroporation with improved cell quality. A 1.8-fold increase in editing efficiency was demonstrated with eVS with negligible impact on cell viability. Cumulatively, these results demonstrate the utility of VS and eVS for genome editing human primary T cells with Cas9 RNPs.

The application of CRISPR-based genome editing tools in plants is often challenged by low editing efficiencies, requiring most plant editing workflows to proceed through the delivery of pre-formed ribonucleoprotein (RNP) complexes to protoplasts. Here, we report increases in protoplast-based RNP delivery and genome editing efficiencies through the addition of anionic polymers to standard protoplast transfection protocols. We test addition of various polymers and peptides for their ability to increase genome editing efficiencies in both Nicotiana benthamiana and Arabidopsis thaliana protoplasts, by adding these components to standard PEG-based protoplast transfection protocols: i) non-covalent addition of charged polymers, ii) non-covalent addition of amphiphilic peptide A5K, and iii) tyrosinase-mediated covalent conjugation of various relevant peptide motifs directly to the RNP. Incorporation of the amphiphilic peptide A5K or covalent attachment of peptides to the RNP had no positive effect on editing efficiencies. However, we found that addition of anionic polymer polyglutamic acid to standard PEG transfection protocols significantly improved editing efficiencies in both Nicotiana benthamiana and Arabidopsis thaliana protoplasts relative to RNPs alone without negatively impacting protoplast viability. Our results suggest anionic polymers stabilize the RNP and increase the colloidal stability of the protoplast transfection workflow. This simple and straightforward method of stabilizing Cas9 RNPs can be easily adopted by others working on direct protein delivery to plant protoplasts to increase genome editing efficiencies. Key messageThe addition of anionic polymer polyglutamic acid to standard protoplast PEG transfection workflows enhance CRISPR-Cas9-mediated gene editing in plant protoplasts. Our results suggest the mechanism of increased transfection efficiency is due to colloidal stabilization of RNPs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/635105v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1101f0org.highwire.dtl.DTLVardef@964402org.highwire.dtl.DTLVardef@14afe61org.highwire.dtl.DTLVardef@82a982_HPS_FORMAT_FIGEXP M_FIG C_FIG

Stable and productive CHO cell lines are essential for biopharmaceutical manufacturing, yet early expansion steps are often constrained by prolonged period required for suspension adaptation. Single-cell cloning (SCC) ensures monoclonality and regulatory compliance, but cells transitioning from static to suspension culture frequently exhibit variable recovery, which prolongs timelines and increases process variability. To address this challenge, mixing-based microplate culture systems have been developed to improve early expansion efficiency. The C.NEST platform provides controlled pneumatic mixing and environmental monitoring that facilitates earlier adaptation to suspension conditions. At the 96-well and 24-well stages, this system allows cells to establish stable growth under suspension-like environments, thereby shortening the adaptation period following transfer to shaking culture. In this study, we applied C.NEST to the SCC workflow for developing CHO-K1 stable cell lines. Integrating C.NESTs controlled mixing reduced adaptation time, enhanced the consistency of clone expansion, and improved the ability to identify high-yield clones. These findings highlight the potential of C.NEST to streamline cell line development workflows by accelerating early suspension adaptation and improving clone selection reliability. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/693844v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@ca76bforg.highwire.dtl.DTLVardef@3a3079org.highwire.dtl.DTLVardef@447e01org.highwire.dtl.DTLVardef@ac8d6f_HPS_FORMAT_FIGEXP M_FIG C_FIG C.NEST mixing shortens suspension adaptation, accelerates clone expansion, and enhances early-stage screening. HighlightO_LIThe C.NEST microplate agitation culture system accelerates early CHO-K1 cell line development. C_LIO_LIControlled pneumatic mixing improved oxygen transfer and medium homogeneity, promoting stable growth during early expansion. C_LIO_LIEarly mixing shortens suspension adaptation by approximately one week. C_LIO_LIMixing cultures enabled more accurate clone performance assessment, revealing high-producing outliers. C_LIO_LIC.NEST provides a scalable and reproducible solution for integrating mixing-based culture into single-cell cloning workflows. C_LI

Direct sequencing of total cellular RNA enables a better understanding of a broad spectrum of RNA species controlling cellular processes and organismal function. Current nanopore direct RNA sequencing method, however, only captures polyadenylated RNA for sequencing. To address this issue, we developed a unique 3-end RNA tailing method to capture total RNA for nanopore direct RNA sequencing. Due to the distinct electrical signature of the added tail on nanopore, this method allows simultaneous detection of both non-polyadenylated and polyadenylated RNAs. We demonstrated the effectiveness of this method in capturing the dynamics of transcription and polyadenylation of chloroplast RNAs in plant cell. With its high efficiency in retaining total RNA on nanopore, this method has the potential to be broadly applied to RNA metabolism and functional genomics studies.

Scalable, continuous biomanufacturing processes have grown in importance to meet demand for smaller bioreactor sizes, lowered production costs, and improved quality attributes. The Sf9/recombinant baculovirus (rBV) expression system demonstrates promise for virus-like particle (VLP) vaccine and gene therapy production. Here, we present a continuous rBV platform integrating an infection plug flow reactor (PFR) between stirred tank growth (gCSTR) and production (pCSTR) bioreactors. Cell expansion in the gCSTR included a ramp-up stage followed by continuous growth, reaching a steady state of 5x106 cells/mL and >90% viability. Peclet number-fit tracer studies confirmed near-ideal plug flow in the PFR, yielding a 10 h residence time and progressive infection as measured by gp64 signaling. Finally, a pCSTR with a residence time of 48 h exhibited sustained recombinant protein production. An integrated pilot cascade incorporating all reactors ran continuously for 5 days, maintaining stable CSTR cell densities and a measurable increase in infected cell diameter from 14.5 m to 16.1 m. Western blotting and EM of [~]100 nm VLPs in pCSTR effluent demonstrated platform success. Digital twin mechanistic models across four distinct stages of bioreactor operation and Hill-type relationships for rBV infection kinetics predicted cell growth and death for a 7-day run, demonstrating promise for designing continuous systems in silico and building a quantitative framework for scale-up and optimization. Our multi-stage reactor configuration represents a cell host- and product-agnostic production scheme, particularly for processes prone to product heterogeneity, and paves the way towards a true end-to-end continuous platform for myriad modalities in the future.

The activation of gene circuits can impose a significant burden on cells, leading to heterogeneous expression and reduced productivity. In this work, we focused on the T7 production system in E. coli BL21, a prime example of a burdensome gene circuit, to investigate the main cause for this gene expression heterogeneity and methods to mitigate it. Based on continuous cultivation analyzed and control by automated flow cytometry, we quantified the trade-off between cellular growth and gene expression and tracked the cell-to-cell heterogeneity in gene expression (measured as entropy). We concluded that the growth reduction associated to the activation of the burdensome gene circuit, i.e., the switching cost, is at the origin of the population heterogeneity. The loss of growth rate imposed by the burdensome activation of the gene is compensated at the population level by the overgrowth of less induced cells that safeguard the population by generating entropy. We tried to homogenize the population by pulsing the inducer with increasing frequency but found that the population escapes control through promoter mutation, leading to a genotype exhibiting reduced gene expression, but also, reduced entropy. To engineer a more homogeneous population without sacrificing gene expression, we decreased the switching cost associated to the induction by lowering the quality of the main carbon source. This strategy successfully led to a more homogeneous and productive population. Our approach allows for a precise quantification of the trade-off between growth and gene expression in cell population cultivated under dynamic conditions and highlights the importance of the switching cost for designing efficient approaches of cell population control.

As bioprocess intensification has increased over the last 30 years, yields from mammalian cell processes have increased from 10s of milligrams to over 10s of grams per liter. Most of these gains in productivity have been due to increasing cell numbers in the bioreactors, and with those increases in cell numbers, strategies have been developed to minimize metabolite waste accumulation, such as lactate and ammonia. Unfortunately, cell growth cannot occur without some waste metabolite accumulation, as central metabolism is required to produce the biopharmaceutical. Inevitably, metabolic waste accumulation leads to decline and termination of the culture. While it is understood that the accumulation of these unwanted compounds imparts a less than optimal culture environment, little is known about the genotoxic properties and the influence of these compounds on global genome instability. In this study, we examined the effects on Chinese hamster ovary (CHO) cells genome sequences and physiology due to exposure to elevated ammonia levels. We identified genome-wide de novo mutations, in addition to variants in functional regions of certain genes involved in the mismatch repair (MMR) pathway, such as DNA2, BRCA1 and RAD52, which led to loss-of-function and eventual genome instability. Additionally, we characterized the presence of microsatellites against the most recent Chinese Hamster genome assembly and discovered certain loci are not replicated faithfully in the presence of elevated ammonia, which represents microsatellite instability (MSI). Furthermore, we found 124 candidate loci that may be suitable biomarkers to gauge genome stability in CHO cultures.

Cell-free transcription and translation systems promise to accelerate and simplify the engineering of proteins, biological circuits and metabolic pathways. Their encapsulation on microfluidic platforms can generate millions of cell-free reactions in picoliter volume droplets. However, current methods struggle to create DNA diversity between droplets while also reaching sufficient protein expression levels. In particular, efficient multi-gene expression has remained elusive. We here demonstrate that co-encapsulation of DNA-coated beads with a defined cell-free system allows high protein expression while also supporting genetic diversity between individual droplets. We optimize DNA loading on commercially available microbeads through direct binding as well as through the sequential coupling of up to three genes via a solid-phase Golden Gate assembly or BxB1 integrase-based recombineering. Encapsulation with an off-the-shelf microfluidics device allows for single or multiple protein expression from a single DNA-coated bead per 14 pL droplet. We envision that this approach will help to scale up and parallelize the rapid prototyping of more complex biological systems.

Plasmodesmata are unique channel structures in plants that link the fluid cytoplasm between adjacent cells. Plants have evolved these microchannels to allow trafficking of nutritious substances as well as signaling molecules for intercellular communication. However, tracking the behavior of plasmodesmata in real time is difficult because they are located inside tissues. Hence, we developed a microfluidic device that traps cultured cells and fixes their positions to allow testing of plasmodesmata permeability. The device has 112 tandemly aligned trap zones in the flow channel. Cells of the tobacco line BY-2 were cultured for 7 days and filtered using a sieve and a cell strainer before use to isolate short cell clusters consisting of only a few cells. The isolated cells were introduced into the flow channel, resulting in entrapment of cell clusters at 25 out of 112 trap zones (22.3%). Plasmodesmata permeability was tested from 1 to 4 days after trapping the cells. During this period, the cell numbers increased through cell division. Fluorescence recovery after photobleaching experiments using a transgenic marker line expressing nuclear-localized H2B-GFP demonstrated that cell-to-cell movement of H2B-GFP protein occurred within 200 min of photobleaching. The transport of H2B-GFP protein was not observed when sodium chloride, a compound known to cause plasmodesmata closure, was present in the microfluid channel. Thus, this microfluidic device and one-dimensional plant cell samples allowed us to observe plasmodesmata behavior in real time under controllable conditions.

Effective RNase inhibition is critical for single-cell RNA-sequencing, yet commercial recombinant RNase inhibitors (RRIs) require reducing agents for stability and impose substantial costs. Here, we systematically benchmark SEQURNA, a synthetic thermostable RNase inhibitor, against commercial alternatives using FLASH-seq in human retinal organoids and peripheral blood mononuclear cells (PBMCs). SEQURNA at 0.5-1 U/l achieved 14-50% higher gene detection than Takara RRI, with the greatest improvement in low-RNA PBMCs. Surprisingly, DTT supplementation at standard concentrations (5-10 mM) significantly impaired gene detection across all SEQURNA concentrations without improving RNA quality metrics, challenging established reverse transcription protocols. SEQURNA preserved biological heterogeneity, maintained sample stability during one-month storage at -80{degrees}C, and reduced reagent costs by 70%. We recommend SEQURNA at 1 U/l without DTT as an optimized formulation that simultaneously enhances data quality and cost-effectiveness for full-length single-cell RNA sequencing.

We report here on an attempt to create an engineered structure which can reproduce the physical and mechanical environment of the land plant sporangium. This work is part of a broader effort to understand the stress mechanics of the land plant sporangium and its role in initiating reproductive development. A second purpose is to extend the range of experimental methods available for the study of the physical environment of plant cell growth and the mechanics of trans-cellular signaling in plant development. We describe an experimental protocol, based on the microfluidic encapsulation of living plant protoplasts in multilayered microbeads composed of tunable hydrogel materials whose mechanical properties can be modified to mimic the stress-mechanics of the living sporangium. Our results demonstrate the successful encapsulation of living plant protoplasts in dual-layered microbeads consisting of an inner layer of gelled agarose and an outer layer of cross-linked alginate/methacrylate.

Large plasmids are often avoided in mammalian co-transfection due to the assumption that they transfect poorly, driving the use of multiple smaller plasmids. Here, we pair finite-state-projection modeling with flow cytometry experiments to compare one-, two-, and three-plasmid delivery of GFP/BFP/RFP. Estimated entry rates were size-independent from 4.9 to 16.4 kb, indicating that plasmid length is not the dominant barrier in this range. Our results suggest that using lipofectamine slightly increases co-transfection efficiency due to the ability of lipoplexes to contain multiple plasmids. However, this benefit is limited to only delivering two plasmids. Additionally, we show that contrary to current beliefs, putting all genes onto the same plasmid both increases the probability that a cell will express all genes of interest and results in a tighter correlation of gene expression levels compared to these multi-plasmid systems. Together, these results identify multi-cargo delivery and not plasmid size as the key constraint on co-transfection and show that single-plasmid designs are generally preferable for applications such as viral-vector production. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/675629v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@251928org.highwire.dtl.DTLVardef@196d1cborg.highwire.dtl.DTLVardef@a781e9org.highwire.dtl.DTLVardef@1420928_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microalgal hydrogen production driven by solar energy offers significant promise as a sustainable energy alternative, yet remains economically challenging due to issues of scalability from laboratory to industrial applications. Here, we demonstrate scalable hydrogen production at semi-industrial volumes using the Chlamydomonas reinhardtii pgr5 mutant, employing an optimized cultivation protocol and photobioreactor design. This approach achieves a fivefold increase in hydrogen yield. Notably, the post-production biomass maintains a high-quality protein and nutrients profile, emphasizing microalgae as a "green coin" with energy security on one side and food security on the other. Techno-economic analysis suggests, achievable hydrogen production costs could reach $2.70/kg H2 under projected improvements, and potentially decrease furthur to $1.48/kg with full optimization. By effectively bridging laboratory research and practical industrial implementation, our study establishes a dual-purpose algal hydrogen production platform aligned with circular economy principles, positioning microalgae prominently within sustainable energy and food frameworks.

mRNA lipid nanoparticle (mRNA-LNP) technology has attracted global attention, especially in vaccine development, due to its superior delivery efficiency, molecular stability, and safety profile. However, evaluating mRNA-LNP potency--defined as the quantifiable biological response elicited by the product--remains costly and time-consuming when relying solely on in vitro experiments. Rapid and reliable potency assessment is hindered by limited mechanistic understanding of delivery processes and sparse experimental data. To address these challenges, we present a mechanism-informed, multi-scale kinetic modeling framework that quantitatively captures the coupled dynamics across particle-level, cellular, and macroscopic scales. This model incorporates variability in LNP-cell interactions and accounts for critical factors such as dosage, LNP and cell size distributions, and cell proliferation dynamics--all of which influence delivery efficiency and response variability. By integrating advanced multi-omics assays--such as single-molecule fluorescent in situ hybridization (smFISH), which enables single-cell resolution of mRNA and protein expression--our framework leverages heterogeneous, multi-scale data to support mechanistically grounded modeling of mRNA delivery and enable robust predictions of therapeutic potency. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/679406v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@1f3ec2borg.highwire.dtl.DTLVardef@1163893org.highwire.dtl.DTLVardef@1dc530borg.highwire.dtl.DTLVardef@1d0264a_HPS_FORMAT_FIGEXP M_FIG C_FIG