Expansion and Storage of Insect Cells and Virus Stocks.

Healthy insect cell cultures are critical for any method described in this book, including making productive baculovirus banks, protein or AAV expression, and determining viral titers. This chapter describes cell maintenance in shake flasks using serum-free conditions and the expansion of virus stocks from a single plaque purified virus. Insect cells can be passaged over multiple generations, but as the cells may undergo changes over multiple passages, limiting the use of your cells to a defined number of passages such as 50 passages is recommendable. Baculovirus stocks once created using serum-free media are not very stable at 4-8 °C. This chapter also includes a simple method to store cells from an early cell passage and your virus stock in liquid nitrogen.

Targeted metabolomic analysis of serum free fatty acids: Lipidomics disturbance in patients with superior limbic keratoconjunctivitis.

This study aimed to identify and quantify free fatty acids (FFAs), secretory phospholipase A2 group IIa (sPLA2-IIa) and cytosolic phospholipase A2 (cPLA2) in serum of superior limbic keratoconjunctivitis (SLK) patients and explored the association between FFAs, sPLA2-IIa and cPLA2 variations and SLK. Targeted metabolomic analysis of FFAs in serum was performed by gas chromatography tandem mass spectrometry (GC-MS/MS) analysis on 16 SLK patients (43.88 ± 7.88 years; female: 62.50%) and 25 healthy controls (43.12 ± 7.88 years; female: 64.00%). Qualitative and absolute quantitative results of FFAs were obtained and classified according to gender and thyroid tests. Differential lipid metabolites, metabolomic pathways and biomarkers were further evaluated. The serum sPLA2-IIa and cPLA2 were determined by enzyme linked immunosorbent assay (ELISA). Among 40 FFAs identified, 6 FFAs showed significant changes (P < 0.05) in SLK patients, including 4 decreased and 2 increased. They were mainly related to unsaturated fatty acid biosynthesis, α-linolenic acid and linoleic acid metabolism, and fatty acid biosynthesis. When dividing the data by gender or abnormal thyroid tests, some comparable FFAs alterations displayed in SLK patients. The ROC analysis revealed that the AUC values of linoleic acid, γ-linolenic acid, cis-8,11,14-eicosatrienoic acid, stearic acid, and palmitic acid, were all greater than 0.8. The serum concentrations of sPLA2-IIa and cPLA2 in patients with SLK were significantly higher than that in healthy controls. Lipidomics disturbance might be the potential mechanism of SLK. Serum FFA biomarkers associated with SLK have potential for the diagnosis and treatment of the disease.

Advances in serum-free media for CHO cells: From traditional serum substitutes to microbial-derived substances.

The Chinese hamster ovary (CHO) cell is an epithelial-like cell that produces proteins with post-translational modifications similar to human glycosylation. It is widely used in the production of recombinant therapeutic proteins and monoclonal antibodies. Culturing CHO cells typically requires the addition of a certain proportion of fetal bovine serum (FBS) to maintain cell proliferation and passaging. However, serum is characterized by its complex composition, batch-to-batch variability, high cost, and potential risk of exogenous contaminants such as mycoplasma and viruses, which impact the purity and safety of the synthesized proteins. Therefore, search for serum alternatives and development of serum-free media for CHO-based protein biomanufacturing are of great significance. This review systematically summarizes the application advantages of CHO cells and strategies for high-density expression. It highlights the developmental trends of serum substitutes from human platelet lysates to animal-free extracts and microbial-derived substances and elucidates the mechanisms by which these substitutes enhance CHO cell culture performance and recombinant protein production, aiming to provide theoretical guidance for exploring novel serum alternatives and developing serum-free media for CHO cells.

Recombinant Protein Production from Stable CHO Cell Pools.

The continuous improvement of expression platforms is necessary to respond to the increasing demand for recombinant proteins that are required to carry out structural or functional studies as well as for their characterization as biotherapeutics. While transient gene expression (TGE) in mammalian cells constitutes a rapid and well-established approach, non-clonal stably transfected cells, or "pools," represent another option, which is especially attractive when recurring productions of the same protein are required. From a culture volume of just a few liters, stable pools can provide hundreds of milligrams to gram quantities of high-quality secreted recombinant proteins.In this chapter, we describe a highly efficient and cost-effective procedure for the generation of Chinese Hamster Ovary cell stable pools expressing secreted recombinant proteins using commercially available serum-free media and polyethylenimine (PEI) as the transfection reagent. As a specific example of how this protocol can be applied, the production and downstream purification of recombinant His-tagged trimeric SARS-CoV-2 spike protein ectodomain (SmT1) are described.

Plasma-Free Blood as a Potential Alternative to Whole Blood for Transcriptomic Analysis.

RNA sequencing (RNAseq) technology has become increasingly important in precision medicine and clinical diagnostics, and emerged as a powerful tool for identifying protein-coding genes, performing differential gene analysis, and inferring immune cell composition. Human peripheral blood samples are widely used for RNAseq, providing valuable insights into individual biomolecular information. Blood samples can be classified as whole blood (WB), plasma, serum, and remaining sediment samples, including plasma-free blood (PFB) and serum-free blood (SFB) samples that are generally considered less useful byproducts during the processes of plasma and serum separation, respectively. However, the feasibility of using PFB and SFB samples for transcriptome analysis remains unclear. In this study, we aimed to assess the suitability of employing PFB or SFB samples as an alternative RNA source in transcriptomic analysis. We performed a comparative analysis of WB, PFB, and SFB samples for different applications. Our results revealed that PFB samples exhibit greater similarity to WB samples than SFB samples in terms of protein-coding gene expression patterns, detection of differentially expressed genes, and immunological characterizations, suggesting that PFB can serve as a viable alternative to WB for transcriptomic analysis. Our study contributes to the optimization of blood sample utilization and the advancement of precision medicine research. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00121-1.

Development of a Normal Porcine Cell Line Growing in a Heme-Supplemented, Serum-Free Condition for Cultured Meat.

A key element for the cost-effective development of cultured meat is a cell line culturable in serum-free conditions to reduce production costs. Heme supplementation in cultured meat mimics the original meat flavor and color. This study introduced a bacterial extract generated from Corynebacterium that was selected for high-heme expression by directed evolution. A normal porcine cell line, PK15, was used to apply the bacterial heme extract as a supplement. Consistent with prior research, we observed the cytotoxicity of PK15 to the heme extract at 10 mM or higher. However, after long-term exposure, PK15 adapted to tolerate up to 40 mM of heme. An RNA-seq analysis of these heme-adapted PK15 cells (PK15H) revealed a set of altered genes, mainly involved in cell proliferation, metabolism, and inflammation. We found that cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), lactoperoxidase (LPO), and glutathione peroxidase 5 (GPX5) were upregulated in the PK15H heme dose dependently. When we reduced serum serially from 2% to serum free, we derived the PK15H subpopulation that was transiently maintained with 5-10 mM heme extract. Altogether, our study reports a porcine cell culturable in high-heme media that can be maintained in serum-free conditions and proposes a marker gene that plays a critical role in this adaptation process.

An Exploratory Approach of Clinically Useful Biomarkers of Cvid by Logistic Regression.

Despite advancements in genetic and functional studies, the timely diagnosis of common variable immunodeficiency (CVID) remains a significant challenge. This exploratory study was designed to assess the diagnostic performance of a novel panel of biomarkers for CVID, incorporating the sum of κ+λ light chains, soluble B-cell maturation antigen (sBCMA) levels, switched memory B cells (smB) and the VISUAL score. Comparative analyses utilizing logistic regression were performed against established gold-standard tests, specifically antibody responses. Our research encompassed 88 subjects, comprising 27 CVID, 23 selective IgA deficiency (SIgAD), 20 secondary immunodeficiency (SID) patients and 18 healthy controls. We established the diagnostic accuracy of sBCMA and the sum κ+λ, achieving sensitivity (Se) and specificity (Spe) of 89% and 89%, and 90% and 99%, respectively. Importantly, sBCMA showed strong correlations with all evaluated biomarkers (sum κ+λ, smB cell and VISUAL), whereas the sum κ+λ was uniquely independent from smB cells or VISUAL, suggesting its additional diagnostic value. Through a multivariate tree decision model, specific antibody responses and the sum κ+λ emerged as independent, signature biomarkers for CVID, with the model showcasing an area under the curve (AUC) of 0.946, Se 0.85, and Spe 0.95. This tree-decision model promises to enhance diagnostic efficiency for CVID, underscoring the sum κ+λ as a superior CVID classifier and potential diagnostic criterion within the panel.

[The Value of sFLC and Serum Calcium in the Diagnosis and Prognosis of Multiple Myeloma Patients].

OBJECTIVE: To investigate the value of serum free light chain (sFLC) and serum calcium ion in the diagnosis and prognosis of multiple myeloma (MM). METHODS: Forty patients with MM treated in Henan Provincial People's Hospital from January 2018 to January 2022 were selected as the observation group, and 40 healthy volunteers were selected as the control group. The differences of sFLC-κ、sFLC-λ、sFLC-κ/λ, serum calcium ions, etc between the two groups were compared. Meanwhile, the differences of sFLC-κ、sFLC-λ、sFLC-κ/λ, serum calcium ions, etc in different international staging systems (ISS), chemotherapy efficacy and prognosis patients were analyzed. RESULTS: The levels of sFLC-κï¼»(98.39±21.19) vs (12.01±4.45) mg/Lï¼½, sFLC-λï¼»(210.20±45.54) vs (14.10±5.11) mg/Lï¼½ and proportions of hypocalcemia （65% vs 0） in the observation group were significantly higher than those in the control group (P < 0.05), while sFLC-κ/ λ ratio［（0.44±0.10） vs (0.87±0.12)ï¼½ and serum calcium ions ［（1.98±0.46） vs （2.42±0.40）mmol/Lï¼½ were significantly lower than those in the control group (P < 0.05). The sFLC-κ, sFLC-λ, the proportion of hypocalcemia and the course of hypocalcemia in ISS stage III patients in the observation group were significantly higher than those in stage I and II patients (P < 0.05), while sFLC-κ/λ ratio, and serum calcium ions were significantly lower than those in stage I and II patients (P < 0.05). The levels of sFLC-κ ［（107.76±21.22） vs （94.67±20.11）mg/Lï¼½, sFLC- λ［（245.54±41.12） vs （205.54±50.22）mg/Lï¼½ of patients with hypocalcemia in the observation group was significantly higher than those without hypocalcemia (P < 0.05), while the sFLC-κ/λ ratio was significantly lower than those without hypocalcemia ［（0.42±0.04） vs （0.47±0.06）；P < 0.05ï¼½. The levels of sFLC-κ ï¼»(107.29±20.14） vs （ 91.11±18.92）mg/Lï¼½, sFLC-λ［（247.98±42.26） vs （179.29±39.32）mg/Lï¼½ in patients with ineffective chemotherapy were significantly higher than those in patients with effective chemotherapy (P < 0.05), while the sFLC-κ/λ ratio was significantly lower than those in patients with effective chemotherapy ï¼»(0.43±0.10) vs (0.50±0.09)；P < 0.05）］. The area under the ROC curve for sFLC-κ, sFLC-λ, sFLC-κ/λ predicting ineffective chemotherapy was 0.803, 0.793 and 0.699 respectively, P < 0.05. There was no significant difference in sFLC-κ, sFLC-λ, sFLC-κ/λ ratio, serum calcium ion, hypocalcemia ratio and hypocalcemia course between survival and death patients (P >0.05). CONCLUSION: sFLC and serum calcium are related to ISS stage of MM patients. sFLC level has a certain value to predict the curative effect of chemotherapy in MM patients. However, the prognostic values of sFLC and serum calcium are not yet confirmed for MM patients.

The role of minimal residual disease and serum free light chain ratio in the management of multiple myeloma.

Multiple myeloma (MM) denotes a cancerous growth characterized by abnormal proliferation of plasma cells. Growing evidence suggests that the complexity in addressing MM lies in the presence of minimal residual disease (MRD) within the body. MRD assessment is becoming increasingly important for risk assessment in patients with MM. Similarly, the levels of serum free protein light chain and their ratio play a crucial role in assessing the disease burden and changes in MM. In this paper, we review and explore the utilization of MRD and serum free light chain ratio in the treatment of MM, delving into their respective characteristics, advantages, disadvantages, and their interrelation.

Development of a chemically disclosed serum-free medium for mouse pluripotent stem cells.

Mouse embryonic stem cells (mESCs) have been widely used as a model system to study the basic biology of pluripotency and to develop cell-based therapies. Traditionally, mESCs have been cultured in a medium supplemented with fetal bovine serum (FBS). However, serum with its inconsistent chemical composition has been problematic for reproducibility and for studying the role of specific components. While some serum-free media have been reported, these media contain commercial additives whose detailed components have not been disclosed. Recently, we developed a serum-free medium, DA-X medium, which can maintain a wide variety of adherent cancer lines. In this study, we modified the DA-X medium and established a novel serum-free condition for both naïve mESCs in which all components are chemically defined and disclosed (DA-X-modified medium for robust growth of pluripotent stem cells: DARP medium). The DARP medium fully supports the normal transcriptome and differentiation potential in teratoma and the establishment of mESCs from blastocysts that retain the developmental potential in all three germ layers, including germ cells in chimeric embryos. Utility of chemically defined DA-X medium for primed mouse epiblast stem cells (mEpiSCs) revealed that an optimal amount of cholesterol is required for the robust growth of naïve-state mESCs, but is dispensable for the maintenance of primed-state mEpiSCs. Thus, this study provides reliable and reproducible culture methods to investigate the role of specific components regulating self-renewal and pluripotency in a wide range of pluripotent states.

Higher serum free thyroxine levels are associated with increased risk of hip fractures in older men.

Overt and subclinical hyperthyroidism are associated with an increased fracture risk, but whether thyroid hormones are associated with fracture risk in individuals with normal thyroid-stimulating hormone (TSH) has mostly been investigated in women. Therefore, we investigated if serum levels of free thyroxine (FT4) or TSH are associated with fracture risk in Swedish men. We followed (median 12.2 yr) elderly men (n = 1825; mean age 75, range 69-81 yr) participating in the Gothenburg and Malmö subcohorts of the prospective, population-based MrOS-Sweden study. The statistical analyses included Cox proportional hazards regression. Men receiving levothyroxine treatment were excluded. In our total cohort, serum FT4 (per SD increase) was associated with increased risk of major osteoporotic fractures (MOFs; n = 479; fully adjusted hazard ratio [HR] 1.14, 95% CI, 1.05-1.24) and hip fractures (n = 207; HR 1.18, 95% CI, 1.04-1.33). Also, in men with normal TSH (n = 1658), FT4 (per SD increase) was significantly associated with increased risk of MOF and hip fractures. Furthermore, men in the highest FT4 quartile had a 1.5-fold increase in hip fracture risk compared with men in the three lower FT4 quartiles, both in the total population and in men with normal TSH (fully adjusted: HR 1.45, 95% CI, 1.04-2.02 and HR 1.51, 95% CI, 1.07-2.12, respectively). In contrast, the risk of MOF was not statistically different in the highest FT4 quartile compared with the three lower FT4 quartiles. Finally, serum TSH was not associated with fracture risk after full adjustment for covariates. In conclusion, serum FT4, but not serum TSH, is a predictor of hip fracture risk in elderly Swedish men. Additionally, there was an association between FT4 (per SD increase) and the risk of MOF.

Research progress on optimization of in vitro isolation, cultivation and preservation methods of dental pulp stem cells for clinical application.

Due to high proliferative capacity, multipotent differentiation, immunomodulatory abilities, and lack of ethical concerns, dental pulp stem cells (DPSCs) are promising candidates for clinical application. Currently, clinical research on DPSCs is in its early stages. The reason for the failure to obtain clinically effective results may be problems with the production process of DPSCs. Due to the different preparation methods and reagent formulations of DPSCs, cell characteristics may be affected and lead to inconsistent experimental results. Preparation of clinical-grade DPSCs is far from ready. To achieve clinical application, it is essential to transit the manufacturing of stem cells from laboratory grade to clinical grade. This review compares and analyzes experimental data on optimizing the preparation methods of DPSCs from extraction to resuscitation, including research articles, invention patents and clinical trials. The advantages and disadvantages of various methods and potential clinical applications are discussed, and factors that could improve the quality of DPSCs for clinical application are proposed. The aim is to summarize the current manufacture of DPSCs in the establishment of a standardized, reliable, safe, and economic method for future preparation of clinical-grade cell products.

In vitro and in vivo evaluation of periosteum-derived cells and iPSC-derived chondrocytes encapsulated in GelMA for osteochondral tissue engineering.

Osteochondral defects are deep joint surface lesions that affect the articular cartilage and the underlying subchondral bone. In the current study, a tissue engineering approach encompassing individual cells encapsulated in a biocompatible hydrogel is explored in vitro and in vivo. Cell-laden hydrogels containing either human periosteum-derived progenitor cells (PDCs) or human induced pluripotent stem cell (iPSC)-derived chondrocytes encapsulated in gelatin methacryloyl (GelMA) were evaluated for their potential to regenerate the subchondral mineralized bone and the articular cartilage on the joint surface, respectively. PDCs are easily isolated and expanded progenitor cells that are capable of generating mineralized cartilage and bone tissue in vivo via endochondral ossification. iPSC-derived chondrocytes are an unlimited source of stable and highly metabolically active chondrocytes. Cell-laden hydrogel constructs were cultured for up to 28 days in a serum-free chemically defined chondrogenic medium. On day 1 and day 21 of the differentiation period, the cell-laden constructs were implanted subcutaneously in nude mice to evaluate ectopic tissue formation 4 weeks post-implantation. Taken together, the data suggest that iPSC-derived chondrocytes encapsulated in GelMA can generate hyaline cartilage-like tissue constructs with different levels of maturity, while using periosteum-derived cells in the same construct type generates mineralized tissue and cortical bone in vivo. Therefore, the aforementioned cell-laden hydrogels can be an important part of a multi-component strategy for the manufacturing of an osteochondral implant.

Formation of Mycobacterium abscessus colonies in cellular culture in an in vitro infection model.

Mycobacterium abscessus is one of the most important nontuberculous mycobacteria that cause lung diseases. In vitro infection models developed to analyze the immune response are frequently based on the addition of mycobacteria to mononuclear cells or neutrophils from peripheral blood. An important requirement of these assays is that most cells phagocytose mycobacteria, only accomplished by using large multiplicities of infection (1 or more bacteria per cell) which may not adequately reflect the inhalation of a few mycobacteria by the host. We propose modifications that try to mimic some of the conditions in which immune cells deal with mycobacteria. For the preparation of the inoculum mycobacteria are grown in solid media followed by preparation to a single cell suspension. Multiplicities of infection (number of bacteria per cell) are below 0.01. Serum-free cellular media is used to allow the growth of M. abscessus. After several days of incubation Bacterial Colonies in Cellular Culture (BCCC) develop, which are enumerated directly under an inverted microscope. These colonies may represent biofilm formation during chronic infections. •Low multiplicity of infection (below 0.01 bacteria per cell) reflects more realistically conditions encountered by immune cells in the lungs.•The surface of mycobacteria prepared for infection assays that are grown in solid media are less affected than that of mycobacteria grown in liquid media with detergents.•Colony formation in the infected cells may reflect the aggregation and biofilm formation in the lungs during chronic infection.

Establishment of a novel cell line, CHO-MK, derived from Chinese hamster ovary tissues for biologics manufacturing.

Chinese hamster ovary (CHO) cells are widely used as a host for producing recombinant therapeutic proteins due to advantages such as human-like post-translational modification, correct protein folding, higher productivity, and a proven track record in biopharmaceutical development. Much effort has been made to improve the process of recombinant protein production, in terms of its yield and productivity, using conventional CHO cell lines. However, to the best of our knowledge, no attempts have been made to acquire new CHO cell lines from Chinese hamster ovary. In this study, we established and characterized a novel CHO cell line, named CHO-MK, derived from freshly isolated Chinese hamster ovary tissues. Some immortalized cell lines were established via sub-culture derived from primary culture, one of which was selected for further development toward a unique expression system design. After adapting serum-free and suspension culture conditions, the resulting cell line exhibited a considerably shorter doubling time (approximately 10 h) than conventional CHO cell lines (approximately 20 h). Model monoclonal antibody (IgG1)-producing cells were generated, and the IgG1 concentration of fed-batch culture reached approximately 5 g/L on day 8 in a 200-L bioreactor. The cell bank of CHO-MK cells was prepared as a new host and assessed for contamination by adventitious agents, with the results indicating that it was free from any such contaminants, including infectious viruses. Taking these findings together, this study showed the potential of CHO-MK cells with a shorter doubling time/process time and enhanced productivity in biologics manufacturing.

Preliminary study on comparison of egg extraction methods for development of fetal bovine serum substitutes in cultured meat.

Fetal bovine serum (FBS) substitution remains one of the challenges to the realization of cultured meat production in the marketplace. In this study, three methods were developed to extract a substitute for FBS using egg white extract (EWE): using 25 mM CaCl2/2.5 % ammonium sulfate/citric acid (A); ethyl alcohol (B); and 5 % ammonium sulfate/citric acid (C). B EWE can effectively replace up to 50 % of FBS in growth media (10 % of the total). Ovalbumin in the extracts can promote cell proliferation, and components along the 12 kDa protein band have the potential to inhibit cell proliferation. Chick primary muscle cells applied with B EWE, an edible material that improved the cost and time efficiency of cultured meat production, effectively proliferated/differentiated. Therefore, EWE extracted using ethyl alcohol may be used as an FBS substitute to reduce animal sacrifices and should be considered a viable alternative to FBS for cultured meat.

Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture.

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R's (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.

A review: analysis of technical challenges in cultured meat production and its commercialization.

The cultured meat technology has developed rapidly in recent years, but there are still many technical challenges that hinder the large-scale production and commercialization of cultured meat. Firstly, it is necessary to lay the foundation for cultured meat production by obtaining seed cells and maintaining stable cell functions. Next, technologies such as bioreactors are used to expand the scale of cell culture, and three-dimensional culture technologies such as scaffold culture or 3D printing are used to construct the three-dimensional structure of cultured meat. At the same time, it can reduce production costs by developing serum-free medium suitable for cultured meat. Finally, the edible quality of cultured meat is improved by evaluating food safety and sensory flavor, and combining ethical and consumer acceptability issues. Therefore, this review fully demonstrates the current development status and existing technical challenges of the cultured meat production technology with regard to the key points described above, in order to provide research ideas for the industrial production of cultured meat.

Optimization of adaptation parameters from adhesion cell culture in serum-containing media to suspension in chemically defined media by superlative box design.

A new design of experiments-superlative box design (SBD), was adopted to optimize the adaptation of Chinese hamster ovary cells from adhesion culture to serum-free suspension culture. It is a general trend to use a serum-free medium instead of a serum-containing medium. The advantage of serum-free medium (chemically defended) is that it does not contain unknown components and avoids safety issues. SBD requires fewer experiments while ensuring a sufficient number of experiments and uniformity in the distribution of experiments amongst all the factors. Six factors were considered in this experimental design with 43 runs plus three more repeating center runs. The cell line was adapted to serum-free media by gradually reducing serum, and from adherent to suspension by rotating at various speeds in a shake flask. Response surface methodology was applied to find the optimum condition. The optimized cell density reached 7.02 × 105 cells/mL, calculated by the quadratic model. Experiments validated the predicted cell adaptation with the maximum cell density. Three suspension runs were selected randomly to perform in the bioreactor to validate cell stability and production homogeneity. This study provides an efficient method to transfer adherent cells to suspension cells and is the first to successfully use SBD and establish a parameter quadratic optimization model.

Streamlined Adeno-Associated Virus Production Using Suspension HEK293T Cells.

Recombinant adeno-associated viruses (rAAVs) are valuable viral vectors for in vivo gene transfer, also having significant ex vivo therapeutic potential. Continued efforts have focused on various gene therapy applications, capsid engineering, and scalable manufacturing processes. Adherent cells are commonly used for virus production in most basic science laboratories because of their efficiency and cost. Although suspension cells are easier to handle and scale up compared to adherent cells, their use in virus production is hampered by poor transfection efficiency. In this protocol, we developed a simple scalable AAV production protocol using serum-free-media-adapted HEK293T suspension cells and VirusGEN transfection reagent. The established protocol allows AAV production from transfection to quality analysis of purified AAV within two weeks. Typical vector yields for the described suspension system followed by iodixanol purification range from a total of 1 × 1013 to 1.5 × 1013 vg (vector genome) using 90 mL of cell suspension vs. 1 × 1013 to 2 × 1013 vg using a regular adherent cell protocol (10 × 15 cm dishes). Key features • Adeno-associated virus (AAV) production using serum-free-media-adapted HEK293T suspension cells. • Efficient transfection with VirusGEN. • High AAV yield from small-volume cell culture. Graphical overview.