Cell Culture Process Research Articles

Characterisation of mesenchymal stem cells conditioned media obtained at different conditioning times: their effect on glial cells in in vitro scratch model

In this study, the bone marrow mesenchymal stem cells conditioned media (BMMSC-CM) obtained by conditioning for 24(CM24), 48(CM48) and 72(CM72) hours was characterised. In vitro, the impact of BMMSC-CM on the astrocyte migratory response and oligodendrocyte density was evaluated using the scratch model. The proteomic profiles of individual secretomes were analysed by mass spectrometry and the concentrations of four selected neurotrophins (BDNF, NGF, GDNF and VEGF) were determined by ELISA. Our results revealed an increased number of proteins at CM72, many of which are involved in neuroregenerative processes. ELISA documented a gradual increase in the concentration of two neurotrophins (NGF, VEGF), peaking at CM72. In vitro, the different effect of individual BMMSC-CM on astrocyte migration response and oligodendrocyte density was observed, most pronounced with CM72. The outcomes demonstrate that the prolonged conditioning results in increased release of detectable proteins, neurotrophic factors concentration and stronger effect on reparative processes in neural cell cultures.

Maximizing yield of perfusion cell culture processes: Evaluation and scale-up of continuous bleed recycling

Bleed recycling is a novel method to increase the yield of steady-state perfusion processes by concentrating process bleed to selectively remove biomass and recycle the liquid fraction. This results in significant product saving which otherwise would go to waste. As long as cells can be concentrated and separated, existing cell separation devices can be used for such an application. However, limited information comparing operation modes and efficiency for bleed recycling applications is available. For the first time, inclined gravity settling has been used as bleed recycling technology and was compared to acoustic separation. Except for lower debris removal, inclined gravity settling showed similar bleed recycling efficiency and no negative impact on cell viabilities, nutrient and metabolite levels and product quality. Additionally considering reduced system complexity and facilitated scale-up, inclined gravity settling was the preferred technology for further evaluation during a 42-day lab-scale perfusion process. Up to a 3.5-fold bleed reduction and an average harvest rate increase of 19% was achieved. Scalability was subsequently tested with a large-scale inclined gravity settler suitable for a 2000 L perfusion process confirming performance of lab-scale experiments. Bleed recycling characterization data from screening experiments combined with scalability demonstration facilitates decision making when considering bleed recycling for novel perfusion process settings to reduce perfusion waste, increase process sustainability and boost overall process yield.

Development of a novel capillary electrophoresis method for quantitative measurements of intracellular recombinant protein titer

The measurement of volumetric titer is an integral step in the assessment and selection of a production cell line and cell culture process. The production of monoclonal antibodies (mAbs), a major class of therapeutic proteins, in Chinese Hamster Ovary (CHO) cell lines is challenging due to the clone-to-clone variations in the intrinsic capability to secrete a biologically complex protein. The measurement of intracellular mAb concentration could be a valuable tool to determine the ratio of intracellular to secreted product and be part of the evaluation of potential mAb productive cell lines. High throughput automation is a valuable tool that is used in bioprocess development to reduce work intensive steps. When coupled with the Simple Western (Wes) platform, automated capillary electrophoresis is an efficient method to measure recombinant protein concentration. In this study, we demonstrate the utility of using the automated Wes to rapidly measure intracellular titer and then compare the intracellular titer, volumetric titer and specific productivity between high and low production CHO clones expressing a model human IgG1 mAb.

Accelerated cell culture process development and characterization for cilgavimab/tixagevimab (AZD7442) for the prevention and treatment of COVID-19.

The global COVID-19 pandemic ignited an unprecedented race to develop vaccines and antibody therapeutics. AstraZeneca's pursuit to provide AZD7442 (EVUSHELD), two long-acting, SARS-CoV-2 spike receptor binding domain-specific neutralizing monoclonal antibodies, to individuals at risk on highly accelerated timelines challenged our traditional ways of process development and spurred the rapid adoption of novel approaches. Conventional upstream development processes were replaced by agile strategies that combined technological advances and highly accelerated workflows. With calculated business risks and close cross-functional collaborations, this process paved the way for hyper accelerated antibody development from discovery through manufacturing, process validation, emergency use authorization filing, and global regulatory approvals. The result was initiation of commercial manufacturing at a contract manufacturing organization less than 6 months from the selection of cilgavimab and tixagevimab-a process that historically has taken close to 10 years.

Identification of Cell Culture Factors Influencing Afucosylation Levels in Monoclonal Antibodies by Partial Least-Squares Regression and Variable Importance Metrics

Retrospective analysis of historic data for cell culture processes is a powerful tool to develop further process understanding. In particular, deploying retrospective analyses can identify important cell culture process parameters for controlling critical quality attributes, e.g., afucosylation, for the production of monoclonal antibodies (mAbs). However, a challenge of analyzing large cell culture data is the high correlation between regressors (particularly media composition), which makes traditional analyses, such as analysis of variance and multivariate linear regression, inappropriate. Instead, partial least-squares regression (PLSR) models, in combination with machine learning techniques such as variable importance metrics, are an orthogonal or alternative approach to identifying important regressors and overcoming the challenge of a highly covariant data structure. A specific workflow for the retrospective analysis of cell culture data is proposed that covers data curation, PLS regression, model analysis, and further steps. In this study, the proposed workflow was applied to data from four mAb products in an industrial cell culture process to identify significant process parameters that influence the afucosylation levels. The PLSR workflow successfully identified several significant parameters, such as temperature and media composition, to enhance process understanding of the relationship between cell culture processes and afucosylation levels.

CDK8/19 inhibition plays an important role in pancreatic β-cell induction from human iPSCs

BackgroundTransplantation of differentiated cells from human-induced pluripotent stem cells (hiPSCs) holds great promise for clinical treatments. Eliminating the risk factor of malignant cell transformation is essential for ensuring the safety of such cells. This study was aimed at assessing and mitigating mutagenicity that may arise during the cell culture process in the protocol of pancreatic islet cell (iPIC) differentiation from hiPSCs.MethodsWe evaluated the mutagenicity of differentiation factors used for hiPSC-derived pancreatic islet-like cells (iPICs). We employed Ames mutagenicity assay, flow cytometry analysis, immunostaining, time-resolved fluorescence resonance energy transfer-based (TR-FRET) cell-free dose–response assays, single-cell RNA-sequencing and in vivo efficacy study.ResultsWe observed a mutagenic effect of activin receptor-like kinase 5 inhibitor II (ALK5iII). ALK5iII is a widely used β-cell inducer but no other tested ALK5 inhibitors induced β-cells. We obtained kinase inhibition profiles and found that only ALK5iII inhibited cyclin-dependent kinases 8 and 19 (CDK8/19) among all ALK5 inhibitors tested. Consistently, CDK8/19 inhibitors efficiently induced β-cells in the absence of ALK5iII. A combination treatment with non-mutagenic ALK5 inhibitor SB431542 and CDK8/19 inhibitor senexin B afforded generation of iPICs with in vitro cellular composition and in vivo efficacy comparable to those observed with ALK5iII.ConclusionOur findings suggest a new risk mitigation approach for cell therapy and advance our understanding of the β-cell differentiation mechanism.

Effect of Tryptophan Metabolites on Cell Damage Revealed by Bacteria-Cell Interactions in Hydrogel Microspheres.

This work presented an alternative approach for studying bacteria-cell interactions in three-dimensional (3D) hydrogel microspheres formed by the cross-linking reaction of alginate and calcium-ethylenediaminetetraacetic acid (EDTA-Ca) produced in a microfluidic chip. During the co-culture process of hepatocytes (HepG2) and Escherichia coli (E. coli) 25922, we concluded that the content change of tryptophan metabolites detected via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was related to the cell damage level and the change of interleukin (IL-22) detected by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was related to the ways of co-cultivation. Compared to the two-dimensional (2D) adherent cell culture process in a Petri dish (2D), the co-culture process of HepG2 and E. coli 25922 in hydrogel microspheres indicated more information about metabolism such as the appearance of indole-3-propionic acid (IPA) and possibly IL-22. The method provides a new perspective to investigate the bacteria-cell interaction and it could be a promising tool in the study of gut microbiota and human health.

Industrial batch monitoring for cell culture processes during process scale-ups: data challenges and solutions

Cell culture process scale-ups are common in biologics manufacturing as the process moves from the early stages of process development to commercial manufacturing. Statistical batch process monitoring (BPM) is frequently deployed for efficient real-time monitoring and control of cell culture processes. A BPM platform relies on historical process data to train in-control model for real-time monitoring of future batches. Deploying a BPM platform during or immediately after process scale-up is nontrivial due to limited batch campaigns at the commercial scale. In the absence of available at-scale commercial data during scale-ups, we propose an approach to reuse data sets generated at bench or pilot-scale for training BPM models at the commercial scale. The proposed method uses a scaling model to learn complex data scaling relations between bench/pilot-commercial scales. The model is then used to synthesise data sets at a commercial scale using existing in-control bench/pilot data. Using synthesised at-scale commercial scale data, a BPM model can be trained and deployed soon after the scale-up, which would not have been possible otherwise. The efficacy of the proposed approach is illustrated in an industrial cell culture process.

Role of geneticin in isolation and culturing of skin melanocytes and melanoma cells

Abstract Introduction Development of an effective, suitable, and reliable method for both the isolation and culturing of melanocytes is crucial for studies on pathomechanisms of skin diseases originating from melanocyte disorders. In this study, we have investigated the influence of geneticin (G418), a substance used for melanocyte selection, in the view of the frequency of presence of cells such as keratinocytes and fibroblasts, widely known as contaminators of melanocyte-originating cell cultures. Materials and Methods Study was conducted on primary, freshly isolated melanocytes, keratinocytes, fibroblasts, and melanoma cells as well as on commercially available melanoma cell lines MeWo, G-361, and A375. Cells were cultured in different culture media supplemented with various concentrations of geneticin ranging from 0.05 to 1 mg/mL. Cell viability, proliferation rate and detection of apoptotic/necrotic cells was assessed. Results Choice of culture media supplemented with various concentrations of geneticin (0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL and 1 mg/mL) strongly affect viability of melanocytes, fibroblasts, and keratinocytes. Selective culture media without FBS facilitate the process of melanocytes and melanoma pure cell culture, yet without geneticin supplementation are insufficient for complete eradication of fibroblast contamination from cell culture. Conclusions In this study we provide, for the first time, the dose-response action of keratinocytes and fibroblasts upon geneticin stimulation in different culture media and show that a low concentration (0.05 mg/mL) of geneticin added to the selective culture media may be safely implemented to facilitate the production of melanocyte and melanoma cell cultures that are free from frequent cell contaminants.

Effect of Fetal Bovine Serum Concentration towards Vero Cells Growth on Culture in DMEM Medium

Cell culture requires a suitable medium and environment for in vivo conditions. Conditions are created by regulating temperature, pH, oxygen, CO2, osmotic pressure, surfaces to adhere to cells, nutrients, and vitamins, protection against toxic substances, hormones, and growth factors that regulate cell growth and differentiation. In general, good cell growth occurs at a pH of 7.0 - 7.6. Fetal Bovine Serum (FBS) is one of the supplement media to support cell growth. The purpose of this study was to determine the effect of using FBS supplementation with various concentrations on vero cell culture media on DMEM (Dulbecco's modified eagle medium) media. Vero cell culture on DMEM media has been successfully carried out using FBS by observing its growth for 14 days. Vero cell culture process begins with the manufacture of media consisting of DMEM and FBS. The cell density of each well at 5% and 10% concentration media was 1017 cells/well. By varying the concentration of FBS in the culture medium, the difference in the number of cells produced can be observed under the microscope. The results showed that the number of Vero cells produced from DMEM media with 10% FBS concentration was higher than 5% FBS concentration. Thus, this study proves that the higher the concentration of FBS in the medium, the more it supports the cell growth process in the Vero cell culture process.

Development of Auto-PIVOT: Automated Platform In Vitro for Cell Tissue Culture

Bioreactors are growing in popularity among network engineers. Bioreactors are generally chambers used for cell culture processes with predetermined parameters. Pivot is a bioreactor system for cell culture that is controlled automatically and remotely using multi-chambers that allow inflow of fresh liquid or output for sample collection. This system facilitates gas exchange between the culture medium and ambient gas. The system provides a storage bag for fresh media and a peristaltic pump is used to move the media in a closed loop during perfusion and stirring in the cell culture chamber. The flow rates used is 2 mL/min. The system has a container for direct sample measurement to obtain pH, oxygen, and carbon dioxide parameters. The bioreactor system is also equipped with a mixing vessel which allows the addition of desired nutrients or additives to the system. The results of a computational fluid dynamics (CFD) and shear stress-based flow analysis have been conducted. The simulation results demonstrate that the applied Pivot parameters correspond to an ideal environment to hepatocyte cell viability and growth. This research is expected to increase the number of cells produced without reducing the quality of each chamber and be carried out simultaneously, automatically, and remotely controlled.

Improved corrosion resistance and biocompatibility of AZ31 alloy by acid pickling pretreatment and (H+) hydroxyapatite/chitosan composite coating

As a new generation of biodegradable hard tissue repair implants, the excessive corrosion rate in a liquid environment and the potential cytotoxicity of magnesium and its alloys have limited their further clinical applications to some extent. Because of this, in this study, a network-like porous structural surface with relatively uniform pore size was designed and prepared on the surface of AZ31 magnesium alloy by acid etching treatment, and the HAp prepared on this surface exhibited the advantage of high interfacial bonding strength, and the (H+)HAp /CS composite coating was prepared by using biocompatible CS as the sealing layer of the HAp coating pores, and the acid etching was investigated by electrochemical tests. The corrosion resistance of HAp prepared under acid-etching conditions and HAp prepared on AZ31 surface without acid-pickling treatment were investigated by electrochemical tests, while the composite coatings were characterized by X-ray diffraction (XRD), Fourier transforms infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and EDS, and further tests such as electrochemical experiments, hydrogen precipitation experiments and cytocompatibility assessment concluded that the composite coating has good corrosion resistance, can effectively reduce the degradation rate of magnesium alloy, low magnesium ion precipitation concentration in the cell culture process has less effect on cell growth, and the coating can promote the attachment and proliferation of BMSCs osteoblasts, showing excellent cytocompatibility. Therefore, this study provides a new idea for the design of AZ31 surface morphology and the improvement of corrosion resistance and biocompatibility of AZ31 magnesium alloy, which is beneficial to promote the further development of AZ31 magnesium alloy as a bone repair material.

Application of semi-supervised convolutional neural network regression model based on data augmentation and process spectral labeling in Raman predictive modeling of cell culture processes

Raman spectroscopy coupled to chemometric model-based technology have a great potential for real-time monitoring of critical process parameters in animal cell culture. The measurement of off-line reference values required for calibration of monitoring procedures is complex, time- and resource-intensive, whereas the acquisition of spectra is a seconds- or minutes-based process. Usually, less than 5% spectra is labeled with off-line data during calibration steps, thus a large amount of spectra is wasted. In this study, we proposed a Semi-Supervised Convolutional Neural Network Regression (SSCNNR) model framework for Raman model establishment based on data augmentation and process spectra labeling. Compared with the original CNNR model, the size of the calibration dataset was expanded from 132 samples to nearly 8500, and the constructed SSCNNR models obtained a significantly improved accuracy in predicting glucose, glutamine, asparagine, and ammonium, with RMSEP values decreased by 29.1%, 37.3%, 38.3% and 7%, respectively. Compared with traditional modeling approach of PLSR and SVR, the SSCNNR models were proved to accurately predict the trend of substance concentration during frequent feeding process. Based on this framework, potential efforts included improving model prediction accuracy by hyperparameters optimization and accommodating scenarios of culture change by transfer learning.

Sustainable Future Protein Foods: The Challenges and the Future of Cultivated Meat.

Global pressure from consumers to improve animal welfare, and reduce microbiological risks or the use of antibiotics pose new challenges for the meat industry. Today's livestock production, despite many undertaken measures, is still far from being sustainable. This forced the need to work on alternative protein types that come from plants, insects, fungi, or cell culture processes. Due to some technical and legal barriers, cultivated meat is not present on the European market, however, in 2020 it was approved in Singapore and in 2022 in the USA. While the technology of obtaining cell cultures from animal muscles has been known and successfully practiced for years, the production of a stable piece of meat with appropriate texture, taste, and smell, is still a problem for several scientific groups related to subsequent companies trying to obtain the highest quality product, in line with the expectations of customers. Although the work on optimal cell meat production has been going on for years, it is still in an early stage, mainly due to several limitations that represent milestones for industrial production. The most important are: the culture media (without animal serum), which will provide an environment for optimal muscle development, natural or close to natural (but still safe for the consumer) stable scaffolds for growing cells. Here, we review the actual knowledge about the above-mentioned challenges which make the production of cellular meat not yet developed on an industrial scale.

Quantitation of Coxsackievirus A21 Viral Proteins in Mixtures of Empty and Full Capsids Using Capillary Western.

A prototype strain of Coxsackievirus A21 (CVA21) is being evaluated as an oncolytic virus immunotherapy. CVA21 preferentially lyses cells that upregulate the expression of intercellular adhesion molecule 1, which includes some types of tumor cells. CVA21 has an icosahedral capsid structure made up of 60 protein subunits encapsidating a viral RNA genome with a particle diameter size of 30 nm. Rapid and robust analytical methods to quantify CVA21 total, empty, and full virus particles are important to support the process development, meet regulatory requirements, and validate manufacturing processes. In this study, we demonstrate the detection of all four CVA21 capsid proteins, VP1, VP2, VP3, and VP4, as well as VP0, a surrogate for empty particles, using in-house-generated antibodies. An automated and quantitative capillary Western blot assay, Simple Western, was developed using these antibodies to quantify CVA21 total particles through VP1, empty particles through VP0, relative ratio of empty to full particles through VP0 and VP4, and the absolute ratio of empty to total particles through VP0 and VP1. Finally, this Simple Western method was used to support CVA21 cell culture and purification process optimization as a high-throughput analytical tool to make rapid process decisions.

Near-infrared spectroscopy based monitoring of all 20 amino acids in mammalian cell culture broth

The biopharmaceutical industry extensively employs Chinese hamster ovary (CHO) cell culture for monoclonal antibody production. Amino acids represent an essential source of nutrients in all CHO cell culture media, and their concentration is known to significantly impact cell viability, titre, and monoclonal antibody critical quality attributes. In this study, a robust Fourier transform near-infrared spectroscopy (FT-NIR) based quantification method has been developed for of all 20 amino acids (0–24 mM), as well as concentrations of glucose (0–6.7 mg mL−1), lactate (0–2.7 mg mL−1), and trastuzumab (0–2.5 mg mL−1) in the CHO cell culture. Near infra-red absorbance spectrum in the range of 4000–11,000 cm−1 were acquired, and spectra pre-processing through smoothening and derivatives were employed to enhance key characteristic signals. High-performance liquid chromatography with pre-column derivatization was used as the orthogonal analytical tool for quantification. Principal component analysis and partial least squares regression were employed for region selection and calibration model development, respectively. The results demonstrate that a good calibration statistic with the acceptable coefficient of determinations for both calibration (Rc2 = 0.94–0.99) and prediction (Rp2 = 0.83–0.98) could be achieved, along with high RPD values (>3) for all components except alanine (2.4). The external validation study also exhibited a satisfactory outcome (REV2 = 0.89–0.99, RMSE = 0.04–1.04), validating the model's ability to predict the concentrations of the respective species. The calibration models were successfully applied for at-line monitoring of two perfusion runs on a 10 L scale. To our knowledge, this is the first application where NIR spectroscopy-based measurement of all 20 amino acids in mammalian cell culture samples has been demonstrated. The proposed tool can play a critical role as biopharma manufacturers implement continuous processing as well as for facilitating process analytical technology-based control of mammalian cell culture processes.

Proteomics reveals that cell density could affect the efficacy of drug treatment

In vitro cell biology study plays a fundamental role in biological and drug development research, but the repeatability and accuracy of cell studies remain to be low. Various uncertainties during the cell culture process could introduce bias into drug research. In this study, we evaluate the potential effects and underlying mechanisms induced by cell number differences in the cell seeding process. Normally, drug experiments are initiated 24 h after cell seeding, and the difference in the cell number at the time of inoculation leads to the difference in cell confluence (cell density) when drug research is conducted. While cell confluence is closely related to intercellular communication, surface protein interaction, cell autocrine as well as paracrine protein expression of cells, it might have a potential impact on the effect of biological studies such as drug treatment. This study used proteomics technology to comprehensively explore the different protein expression patterns between cells with different confluences. Due to the high sensitivity and high throughput of liquid chromatography-mass spectrometry (LC-MS/MS) detection, it was hired to evaluate the protein expression differences of Hep3B cells with 3 different confluences (30%, 50%, and 70%). The differential expressed proteins were analyzed by the Reactome pathway and the Gene Ontology (GO) pathway. Significant differences were identified across three confluences in terms of the number of proteins identified, the protein expression pattern, and the expression level of certain KEGG pathways. We found that those proteins involved in the cell cycle pathway were differently expressed: the higher the cell confluence, the higher these proteins expressed. A cell cycle inhibitor palbociclib was selected to further verify this observation. Palbociclib in the same dose was applied to cells with different confluence, the results indicated that the growth inhibition effect of palbociclib increases along with the increasing trend of cell cycle protein expression. The result indicated that cell density did influence the effect of drug treatment. Furthermore, three other drugs, cisplatin, paclitaxel, and imatinib, were used to treat the three liver cancer cell lines Hep3B, SUN387, and MHCC97, and a similar observation was obtained that drug effect would be different when the cell confluences were different. Therefore, selecting an appropriate number of cells for plating is vitally important at the beginning of a drug study.

Pharmaceutical process optimisation: Decision support under high uncertainty

Optimisation of cell culture processes is an extremely challenging problem for the biomanufacturing industry. Limited data availability, coupled with biological complexity in modelling highly variable living cells, necessitates a decision support methodology that is performant under high levels of uncertainty. Raw materials, experimental & manufacturing facilities, and human expertise are all steeply expensive and availability is tightly constrained — planning their allocation is subject to the core uncertainties underlying the behaviour of living cells.This paper presents a novel decision support methodology for optimisation under high uncertainty. Optimisation techniques require an “objective function” that maps decision variables to the quantity being optimised, so that the decision space can be explored to find an optimum. By learning multiple types of objective function candidates with different levels of fidelity to real-world processes, our method mitigates the risk of picking a poor approximation of the objective function due to sampling effects and algorithmic randomness.Wet lab experimentation on a biomanufacturing feed optimisation problem verified that inferred candidates can successfully support domain experts in designing a new optimised feed strategy with significantly higher product yield than the current industrial control strategy. Our results indicate potential for extending our methodology to the optimisation of other complex industrial processes.

Automated assembly of hybrid dynamic models for CHO cell culture processes

The emergent realisation of Industry 4.0 principles across biomanufacturing, through recent endeavours, will markedly enhance the development and manufacture of modern therapeutics. Through implementation of digital process models, a greater understanding of the intricate relationship between product quality attributes and manufacturing process performance may be established. While contributing towards accelerated process development, representative process models enable advanced optimisation of process parameters, thus having a tangible impact on the assurance of product quality and manufacturing robustness. Hybrid approaches, which couple mechanistic interpretability with statistical data-fitting, are posed to broaden the value and utility of digital models. To augment the advancement in modelling techniques and high-throughput technology, there is a growing requirement for automated approaches towards data processing and model assembly. In this study, a novel strategy is proposed, which leverages saturation and sigmoidal relationships, along with an underlying material balance framework, for the automated assembly of hybrid dynamic models of cell growth. The proposed hybrid model is compared against an equivalent mechanistic model based on Monod expressions. While both models achieve a reasonable fit against experimental data, the hybrid model demonstrates superior predictive performance. Development of automated hybrid models, as demonstrated in this study, may greatly accelerate process digitalisation across biopharmaceutical manufacture.

Abstract B015: A non-genetic engineering platform for rapidly generating and expanding cancer-specific armed T cells

Abstract Genetically engineered T cell technology, especially the chimeric antigen receptor (CAR) T cell, has successfully treated hematological tumors in clinical practice. However, the gene transfer technology used in current CAR-T cells is still mainly based on lentivirus systems, which poses a potential carcinogenic risk. Additionally, the laborious protocols for manufacturing and quality control of CAR-T cell products take over 20 days and delay treating patients. Our team presents a non-genetic engineering technology for rapidly manufacturing cancer-specific T cells. Using an anti-cancer/anti-CD3 bispecific antibody (BsAb) with a high-affinity CD3-binding arm to culture patients’ peripheral blood mononuclear cells (PBMCs), a large amount of cancer-specific BsAb-armed T cells can be rapidly generated with a purity of over 90% in 7 days. The BsAb-armed T cells efficiently accumulated at tumor sites in vitro and in vivo. The release of cytotoxins (perforin and granzyme) and cytokines (TNF-α and IFN-γ) from the BsAb-armed T cells dramatically increased after they contacted cancer cells, resulting in a remarkable anti-cancer efficacy. Preclinical animal studies have shown that the BsAb-armed T cells can effectively treat blood cancers, various solid tumors, and malignant metastatic tumors without severe cellular cytokine release syndrome (CRS) and tissue toxicity. Our first candidate (anti-EGFR/anti-CD3 BsAb armed T cell product) is ready to initiate an investigator-initiated clinical trial in EGFR expressed Kras, Braf, and Nras mutations in end-stage untreatable metastatic colorectal cancer. The technical schematic and representative results of the BsAb-armed T cell technology present in this linked video: https://youtu.be/TlDos-vPg3o According to the results mentioned above, we conclude that the BsAb-armed T cell technology has the following essential advantages: 1. The BsAb armed T cell technology is a non-viral method for rapidly producing cancer-specific T cells with more than 90% purity. 2. Easily controlling the surface amounts of BsAb on T cells allows us to generate customized BsAb-armed T cells with higher therapeutic efficacy and lower CRS risk for individual patients 3. The T cells armed with 10% to 100% of the maximum BsAb loading amount exhibited similar tumor-killing ability. 4. During the T cell culturing process, the BsAbs could bind directly to the surfaces of ex vivo amplified effector T cells but not pro-inflammatory Th17 or immunosuppressive Treg cells, which were able to provide the cancer-targeting ability for these effector T cells. 5. This strategy provides flexibility to choose the appropriate cancer target by simply changing the anti-cancer portion of BsAbs. The BsAb-armed T cell technology, as compared to current genetically engineered T cell technology, represents a simple, time-saving, highly safe, and cost-effective method to generate highly pure cancer-specific effector T cells, thereby providing a better T cell immunotherapy to patients. Citation Format: Kuo-Hsiang Chuang. A non-genetic engineering platform for rapidly generating and expanding cancer-specific armed T cells [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B015.