Pei transfection protocol hek293 suspension. PEI-based transfection is widely used for transient tra.

Pei transfection protocol hek293 suspension 1 PREPARATION OF THE CELLS Here, we describe an optimized PEI-based virus production process for high-yielding viral vector production, compatible with different cell culture adherent and suspension systems. Greater than 90% of XDC293 cells and 98% of HeLa cells transfected using our method were positive for EGFP expression We have developed three nonviral DNA transfer protocols for suspension-adapted HEK-293 and CHO cells: (1) a calcium phosphate based method (Ca-Pi), (2) a calcium-mediated method called Calfection, and (3) a polyethylenimine-based method (PEI). The final transfection concentration is 1 μg pDNA for each mL of culture to be transfected. 6-well plate: 9 µl of PEI (1 µg/µl) = 9 µg PEI to 3 µg DNA b. Sep 19, 2017 · Here we describe the methodology to produce VLPs in a 3L bioreactor, using suspension HEK 293 cells and PEIPro as a transfection reagent. This ratio can be optimized to maximize transfection efficiency. Jun 27, 2024 · Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital shaken bioreactors and the subsequent purification of vector particles. Once a batch of PEI is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. With the available quality grades of PEI, PEIpro® and PEIpro-HQ®, Polyplus-transfection offers a reliable PEI transfection method suitable for process development up to viral vector production for clinical and commercial manufacturing. Sep 13, 2022 · Second, we demonstrated that cell-penetrating peptide-based transfection achieves significantly higher protein yields compared to PEI and lipoplex methods in both CHO and HEK293 producer cell lines. Ramón Román, Martí Lecina and Jordi Joan Cairó. Mar 10, 2022 · In this study, we describe our optimized protocol to produce rAAV by polyethyleneimine (PEI)-mediated transfection in suspension HEK293 cells, along with a side-by-side comparison to our high-performing system using the herpes simplex virus (HSV). In addition, the cell uptake and the endosome escape functions of PEI modified was enhanced significantly. May 14, 2017 · Polyplus-transfection now provides PEIpro® and PEIpro®-HQ, the unique PEI-based transfection reagents suitable for use in process development and in cGMP biomanufacturing, respectively. Mar 26, 2018 · PEI-mediated transient transfection is an efficient and cost-effective approach to produce viral vectors. Cells were seeded at 0. In this study, we describe our optimized protocol to produce rAAV by polyethyleneimine (PEI)-mediated transfection in suspension HEK293 cells, along with a side-by-side comparison to our high-performing system using the herpes simplex virus (HSV). The suspension HEK293 were grown in adherent conditions, with 20% FBS supplementation. Transfection is inhibited by serum. First prepare 20 μg/mL of pDNA using 5% final culture volume in a clean vial. Invitrogen Corporation (“Invitrogen”) grants to you, its customer, a non-exclusive, non-transferable license to access the Invitrogen transfection protocol applicable to Invitrogen products, as set forth below (the “Protocol”). This protocol describes how to transfect suspension HEK293 cells with recombinant antibody plasmids using Polyethylenimine Max as a transfection reagent. Abstract The production of lentiviral vectors (LVs) in human embryonic kidney 293 (HEK293) cells using serum-free medium in a suspension culture for the transduction of chimeric antigen receptor T-cells (CAR-T) can be achieved by different methods. A total of 15 combinations were designed according to Box-Behnken design to identify the effects of DNA concentration, polyethylenimine concentration and incubation time on transient transfection efficiency. 25 μg/mL) following the standard protocols. This new platform has produced ~160 ± 20 mg/L of therapeutic antibody 10 days after transfection. For both lentivirus and AAV transient transfection production processes, the transfection complex is formed by a charge-charge interaction between the plasmid DNA and the transfection reagent, typically PEI. This procedure can be modified for alternative packaging cell lines or transfection reagents. Only use media that is reduced-serum, serum-free or chemically defined. Our protocol is to transfect when cells are ~50% confluent Jun 10, 2025 · This property contributes significantly to its effectiveness as a transfection reagent, particularly for transient expression systems. I am trying to transfect a GPCR into HEK-293 cells. There are multiple transfection reagents that can be used; this protocol is based on the use of polyethylenimine (PEI) with the HEK 293T cell line. Many cell lines can be transfected successfully with PEI but in our experience these two cell lines express the highest level of protein compared to other cells. On the second day, when the cell density reached 70% - 80%, transfection can be carried out. As a result, this method is generally not considered suitable for suspension HEK293 transient transfection and AAV production scale-up. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension PEI transfection (lentivirus → HEK293T) 1. Apr 8, 2025 · In this study, we developed an upstream platform for batch rAAV production using an easy to procure suspension HEK293 cell line that was adapted into a chemically defined medium with a formulation developed in-house. transfection. Nov 11, 2021 · Different AAV production platforms can be used, and this webcast will feature process development of AAV2 and AAV5 production using triple plasmid transient transfection with polyethylenimine (PEI In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293 (EBNA) cells grown in serum-free suspension culture. . The first two methods have already been scaled up to 14 L and 100 L for HEK-293 cells in bioreactors. This protocol is appropriate for two suspension cell lines, CHO-S and HEK 293 GnTi -. Optimized LVV production protocol using HEK293T suspension cells The DoE study was designed as a full factorial design, including five parameters (VCD, incubation time, enhancer concentration, enhancer time, and DNA concentration), to evaluate the optimal conditions for transfection. springer. The approach uses transient co-transfection of a Human Embryonic Kidney (HEK) cell line (e. There are many different ways to transfect cells; this is just one set of guidelines. Apr 27, 2020 · Ansorge S, Lanthier S, Transfiguracion J, Durocher Y, Henry O, Kamen A. It can be used to generate functional multi-specific antibodies in high amounts. Nov 7, 2023 · Pro-Tip The ratio of µg DNA:µg PEI needs to be empirically determined. When using suspension-adapted HEK293 Comparison of different DNA transfection protocols with HEK293 cells in suspension cultures. INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. In addition, stable viral vector producer cell lines have been established. PEIs acylation was determined by 1 Thank you Marzieh Rostaminejad for sharing the protocol. , Jordan et al. Transient Transfection of HEK-293F Suspension Cultures using PEI Cells are grown in suspension on a platform shaker in a humidified 37°C CO2 incubator with rotation at ~150 rpm. The cell line was cultured in HyQSFM4TransFx293 (HyQ) (Hyclone, Logan, UT, USA), a commercial medium specially developed for the transfection of HEK293 cells, supplemented with 5% Cell Boost 5 (CB5, Hyclone). rpm. However, this method presents some limitations in large scale bioreactors: inadequate transfection protocol, reduced transfection efficiency and lower productivity. Conclusions This protocol is the first describing transfection of the human Expi293 cells with PEI. SEAP expression levels were quantified 5 days post-transfection using phosphatase reporter dye and UV/Vis absorbance. 8 μg DNA/mL), PEI (1 μg DNA/mL), and FreeStyleTM MAX (1. Apr 19, 2012 · This protocol outlines steps for optimizing the transfection of adherent primary mammalian cells using the readily available off-the-shelf cationic polymer, 25-kDa branched polyethylenimine These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. PEI is the basis of most commercially available transfection agents and acts as a very cost effective transfection vector. The evaluation of DNA/PEI complexes over time was followed by DLS and NTA. After transfection and expression, the recombinant antibody can be purified for use in a variety of applications. The system includes FreeStyleTM 293-F cells that have been adapted to serum-free, suspension culture in FreeStyleTM 293 Expression Medium. 2. DMEM was the best transfection medium for adherent HEK 293T This protocol is appropriate for two suspension cell lines, CHO-S and HEK 293 GnTi-. rAAV was produced using an optimized transient transfection process based on PEI‐mediated plasmid DNA transfer, and vectors Jul 20, 2013 · The toxicity of Gag-GFP plasmid DNA/PEI polyplexes upon transfection of HEK 293 cells growing in supplemented Freestyle medium was evaluated (A and B). PEI is more scalable and cost-effective than lipid-mediated transfection, and significantly more reliable than calcium-mediated transfection. For a given cell line, the efciency of PEI-mediated transfection depends on many parameters such as the culture medium in which the transfection occurs, the mode of cell culture (i. Our linear PEI protocol is as effective as commercial reagents in the transfection of HeLa cells and XDC293 cells, a derivative of HEK293 cells, but at a fraction of the cost. May 25, 2022 · The findings provide a better understanding of the role of DNA/PEI aggregates in transient gene expression approaches, in particular considering that similar complexation protocols and saline solutions are widely used for the transfection of mammalian cell cultures. We determined the optimal DNA:PEI ratios for maximal expression of the reporter gene SEAP while monitoring cytotoxicity following transfection. Lysates were RELATED INFORMATION A number of protocols are available for large-scale transfection of mammalian cells for production of milligram to gram quantities of r-proteins (e. Oct 1, 2023 · To generate stable suspension cells expressing the RBD-SrtA, a single two plasmids transfection was performed, with hygromycin selection. Stable complexation with DNA, efficient entry into the cell, and ability to escape the endosome makes PEI a highly efficient transfection reagent which is compatible for a wide range of cell lines/types including the most commonly used HEK293 and CHO cells grown in adherent and suspension cultures. Sep 22, 2018 · This chapter describes all the steps needed for a successful protein expression following PEI-mediated transfection of HEK293 cells grown in serum-free media in suspension cultures. MCE PEI Transfection Reagent is based on 25 kDa PEI, it is modified by introducing functional genes to enhance the binding ability of DNA, and reduced the cytotoxicity. 1998; Schlaeger and Christensen 1999; Durocher et al. In In HEK293 HEK293 and and CHO CHO cell cell expression expression systems, systems, PEI PEI provides provides excellent excellent transfection transfection results results at at different different sizes sizes (from (from 96-well 96-well plates plates to to 1 Introduction The FreeStyleTM 293 Expression System is designed to allow large-scale transfection of suspension 293 human embryonic kidney cells in a defined, serum-free medium. This protocol describes the steps needed for successful transfection of HEK293 cells adapted to serum-supplemented or serum-free medium in adherent culture using branched PEI. 5 × 106 cells). This is often the best place to start, especially in a new cell line. , 2015 ). adherent vs sus-pension), the cell density at time of transfection, the DNA concen-tration in the culture medium and the DNA:PEI ratio. Jun 10, 2016 · Three commercially available linear polyethylenimines (25 kDa LPEI, 40 kDa PEI“Max” and PEIpro™) were compared regarding their potency to transfect serum-free growing and suspension-adapted HEK293 and CHO cells. Sep 15, 2017 · This protocol is the first describing transfection of the human Expi293 cells with PEI. 5 × 10 6 cells/mL in 125-mL flasks the day before transfection. For a number of various nonantibody proteins, we found that this process expressed product at levels either similar to or higher than what was expressed Sep 22, 2018 · This chapter describes all the steps needed for a successful protein expression following PEI-mediated transfection of HEK293 cells grown in serum-free media in suspension cultures. The transfection (PEI-DNA) complexes that are formed can change in size over time. Numerous cell lines can be effectively transfected with PEI, however in our experience, these two cell lines express the most protein compared to other cells. rAAV was produced using an optimized transient transfection process based on PEI-mediated plasmid DNA transfer, and vectors were Abstract Transient transfection of mammalian cells is used in the biotechnology industry to quickly supply recombinant protein for research and large molecule drug development. (Inoculation density: 6 orifice plate: 0. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly Application of PEI STAR™ transfection reagent. Unfiltered LV vectors produced using the LV-MAX Lentiviral Production System were compared to PEI-mediated LV vector production in adherent HEK293T/FT cells and suspension HEK293 cells. Feb 20, 2022 · The transient transfection of mammalian cells is a rapid and versatile platform for the manufacture of recombinant proteins, but industrial processes depend on reliable scalability and efficient conversion from adherent to suspension cell cultures. Nov 10, 2019 · The production of lentiviral vectors (LVs) in human embryonic kidney 293 (HEK293) cells using serum-free medium in a suspension culture for the transduction of chimeric antigen receptor T-cells (CAR-T) can be achieved by different methods. PEI-based transfection is widely used for transient tra This is one obvious advantage of the PEI-based transfection of HEK293 cells in suspension, because highly pure plasma DNA remains one of the main cost drivers of transient production processes. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. At Cytion, we've observed that fresh preparation of transfection reagents substantially improves efficiency—particularly for calcium phosphate methods. HEK293 cells, ~50,000 cells/cm 2, seeded the day prior to 1 HEK293F suspension culture Transfection protocol: Moremen lab, 12/15/11. Transfection protocol for virus production in suspension cells PEIpro® is perfectly suited for DNA transfection of cells grown in suspension in shaker flasks, cell culture bags or bioreactors in serum-free media. Abstract Response surface methodology was undertaken to optimize the polyethylenimine-mediated transient transfection of suspension cultured HEK 293-F cells. Scheme for the Side-by-Side Comparison of Production Methods and Purification Procedures HEK293 cells were transfected using calcium phosphate transfection for adherent AAV293 cells grown in cell factories and PEI for HEKExpress suspension cells grown in Tubespin 600 bioreactors. Polyethylenimine (PEI) transfection is one of the most widely used methods to co-transfect rAAV plasmids into HEK293 cells. Protocol for transient gene expression n HEK293 suspensions using PEI STAR™ transfection reagent. PEIpro® is compatible with the use of antibiotics in the cell culture medium. The current study presents a detailed side-by-side comparison of a PEI transfection and an HSV infection platform in suspension. Polyethylenimine (PEI) transfection is one of the most widely used methods to co-transfect plasmids into HEK293 cells. The working solution of PEI is 1mg/1ml (1:1000). NOTES There are many different ways to transfect cells; this is just one set of guidelines. In this study, we developed an upstream platform for batch rAAV production using an easy to procure suspension HEK293 cell line that was adapted into a chemically defined medium with a formulation developed in‐house. PEI is a very cost effective transfection factor which allows for external DNA to be endocytosed, and subsequently gain access to mammalian host DNA. A) Suspension HEK-293T cells were seeded at 1 x 106cells/ml in serum-free medium and transfected with PEIpro® and another PEI-based reagent following the recommended protocols. However, the low yield of such biomanufacturing challenges bioprocess engineers to develop more efficient strategies capable of increasing volumetric productivity. The volume of PEI used is based on a 3:1 ratio of PEI (µg):total DNA (µg). A total of 5 × 10 8 cells (500 mL) per batch were transfected and harvested 72 hr post-transfection. Transient Transfection of HEK-293F Suspension Cultures using PEI. 4, 0. In this protocol, we developed a simple scalable AAV production protocol using serum-free-media-adapted HEK293T suspension cells and VirusGEN transfection reagent. The aim of the present study was to optimize the polyethylenimine (PEI)‑mediated transfection method in order to simplify the efficient production of lentiviral vectors (LvVs), and to compare the CaPO4‑ and PEI‑mediated transfection methods for producing LvVs. Three nonviral DNA transfer methods were developed: Ca-Pi, Calfection, and PEI for protein expression. Description: Protocol for recombinant Adeno-Associated Virus (rAAV) production with PEI STAR™ transfection in suspension HEK293 cells. Feb 15, 2008 · Published protocols for transfection of suspension-adapted HEK-293 cells with polyethyleneimine have shown great promise in overcoming some of these bottlenecks, but still require a priori complex formation for optimal yields and limit the choice of transfection and production media. Here, we present the optimized protocols, currently in use in our lab, for the maintenance and transfection of HEK293-F cells, a commercially available HEK293 cell line suitable for high-density culture and transient transfection in suspension ( Nettleship et al. PEI is the most popular reagent for transient transfection of HEK293 and CHO suspension cultures. Oct 25, 2022 · The highest transfection efficiency was over 90% and obtained by using polyethyleneimine (PEI) 25 K and by media adaptation in IVY without using any transfection enhancer. I used to do HEK293T transfection with 25 uM of Chloroquine with CaCl2 protocol. a. Based on the protocol, I understand that 2ug of DNA and 8/10 ul of PEI gave the best transfection efficiency, but what about the Designed specifically for transfection of high density suspension cell culture, with matching transfection enhancers that boost transfection performance and protein expression Achieves protein yields 2- to 10-fold higher than other transfection reagents used on high density 293 cell cultures Employs the same transient expression protocols typically used in current low-density 293 suspension Then, a critical assessment of the challenges of large-scale transient transfection follows, focusing on suspension cell cultures transfected with polyethylenimine (PEI), which is the most widely Transient gene expression in HEK-293 cells enables rapid production of 1-500 mg of r-proteins. Here we describe the optimized transfection of HEK 293T cells in both culture formats. Recent advancements in transient transfection technologies, cell line and media development have demonstrated that viral vector production can be performed using serum-free suspension-based transfection process in chemically defined cell culture medium. Our laboratory uses PEI over other cell transfection reagents because of its low cost. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct Furthermore, transfection with calcium phosphate can be unreliable and require extensive process optimization and strict process adherence to achieve adequate transfection and productivity [2,3]. Feb 18, 2022 · This protocol describes how to transfect suspension HEK293 cells with recombinant antibody plasmids using Polyethylenimine Max as a transfection reagent. It is highly efficient for transfection of suspension CHO and HEK-293 cells in various serum-free media, using low DNA amount (< 1 µg/ml of cell culture). Cells are grown in suspension on a platform shaker in a humidified 37 C CO2 incubator with rotation at ~150. Sharing speeds science. Additional protocols can be found in this issue, including Transfection of HEK293-EBNA1 Cells in Suspension with Linear PEI for Polyethylenimine (PEI) transfection is one of the most widely used methods to co-transfect the rAAV plasmids into HEK293 cells. This protocol describes the steps needed for successful transfection of HEK293 cells adapted to serum-supplemented or serum-free medium in suspension culture using linear PEI. PEI STAR™ performance data comparison (HEK293): HEK 293 - 20 mL cultures containing HEK293 suspensions were transfected with a CMV-SEAP plasmid at optimized PEI/DNA ratios using either PEI STAR™ (3:1) or leading competitor PEI. It is a core component of the Expi293TM Expression System and supports high density culture of Expi293FTM cell lines for scalable transient protein expression. Within 24 hours before transfection, HEK293T cells were inoculated into a six well plate for culture (2mL DMEM medium containing 10% FBS per well). Oct 16, 2014 · The approach uses transient co-transfection of a Human Embryonic Kidney (HEK) cell line (e. First, we describe two optimized PEI-based production protocols in suspension HEK293 cells, as well as a novel downstream protocol to support the purification of both PEI- and HSV-produced rAAV9. We would like to show you a description here but the site won’t allow us. The protocol for expression The suspension HEK293 cells were initially grown in SFM4Transfx-293 media and were seeded at 1 × 10 6 viable cells/ml in a 30 ml volume in 125 ml shaker flasks on the day of transfection. See full list on link. The protocol for a 24-well transfection reaction with HEK293 cells is here: Plate 10,000-15,000 HEK293 cells per well in 0. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as th … The current study presents a detailed side-by-side comparison of a PEI transfection and an HSV infection platform in suspension. Mar 1, 2008 · This protocol describes the steps needed for successful transfection of HEK293 cells adapted to serum-supplemented or serum-free medium in suspension culture using linear PEI. Suspension-adapted CHO-K1 cells were seeded following the recommended protocol, and transfected with FectoPRO® (0. Here, we describe a method for high throughput transient transfection of Human Embryonic Kidney 293 (HEK293) cells in 30 mL tubespins using polyethylenimine (PEI) as a transfection reagent. During our process development, the goal was to achieve the following criteria: 1010 virus particles (VP/mL Apr 1, 2024 · By identifying 24-hour transfection with high level cell survival and GFP expression was achieved with PEI as low as 0. PEI STARTM has been shown to be an effective option for creating highly infectious rAAVs in independent, peer-reviewed We describe a scalable process using HEK293T suspension cell line for transient transfection of lentiviral vector (LVV) production in single-use bioreactors. HEK293 expression medium or DMEM maintained at 37°C. To simplify the transfection optimization process, we have created a transfection selection tool that provides recommended transfection reagent with optimized protocol for 120 cell lines. Greater than 90% of XD … Introduction This protocol can be used to produce lentivirus from a lentiviral vector transfected into 293T cells using a polyethylenimine (PEI) transfection protocol. Known for its stable complexation with DNA, effective cellular entry, and superior endosomal escape, PEI MAX® achieves high transfection efficiency across a broad range of cell types, including HEK293 and CHO cells, in both adherent and suspension cultures. 6 and 0. In particular, the linear isoform of PEI is more effective for transfecting cells in suspension. Multiple transfection agents can be used, however this protocol is based on the use of polyethylenimine (PEI) with the HEK293T cell line. The highest integral optic Mar 25, 2019 · DNA/PEI complexes used in a standard transient transfection protocol of HEK 293 cells were examined with particle tracking techniques. TRANSIENT TRANSFECTION PROTOCOL FectoPRO® kit is perfectly suited for DNA transfection of suspension CHO and HEK-293 cells grown in suspension under agitation in serum-free media in deep-well plates, filter top tubes, shaker flasks, spinners, cell culture bags or bioreactors. Transfection Protocols Our Lipofectamine Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. e. Only use media that is reduced-serum, serum-free or chemically defined. You will need 200 µL serum-free DMEM + 3 µgDNA+9 µg PEI for each well of a 6-well plate you plan to transfect. PEI Prime™️ is a high-performance transfection reagent designed for robust, low-cost and scalable transient gene expression. This chapter describes LV production by transient transfection, induction of stable packaging cell lines, and induction of stable producer cell lines. Successful scaling of transfection methods reached up to 100 L in bioreactors for HEK-293 cells. Standard protocol for the transfection of HEK-293 in a flask 75 cm2: On the day of transfection, measure the cell density and determine transfection parameters (DNA amount and PEIpro® volume per million cells) according to Table 4. g. For laboratories new to lentiviral production, we recommend starting with our optimized PEI protocol using HEK293T Cells at passages 5-15. Jul 24, 2017 · Hello, I have been having some issues with getting efficient transfection using PEI. We have developed three nonviral DNA transfer protocols for suspension-adapted HEK-293 and CHO cells: (1) a calcium phosphate based method (Ca-Pi), (2) a calcium-mediated method called Calfection, and (3) a polyethylenimine-based method (PEI). 02 µl/ml this allowed us to determine whether this level of PEI would provide efficient transfection of HEK293T in suspension. HEK293T - Human embryonic kidney cell line (293T CRL-3216, from the ATCC cell bank, USA or 293T ACC 635 from the DSMZ cell bank, Germany). For the first time the feasibility of HEK293 cells, which were adapted to grow in suspension culture by Florabio and IVY media, were tested for virus production. 2005). Follow this protocol to produce adeno-associated virus (AAV) in HEK293 cells. We added the chloroquine 5 minutes before transfection. PEI STARTM has been shown to be an effective option for creating highly infectious rAAVs in independent, peer-reviewed The FreeStyle™ 293 Expression System is designed to allow large-scale transfection of suspension 293 human embryonic kidney cells in a defined, serum-free medium. Jun 14, 2019 · Additionally, we conducted a side-by-side comparison of AAV2/9 production in adherent and suspension-adapted HEK293 cells to validate and demonstrate bioequivalence. Oct 27, 2024 · Significant volumetric TGE productivities of secreted recombinant proteins have been achieved using polyethyleneimine (PEI) for DNA delivery into either Human Embryonic Kidney 293 (HEK293) or Chinese hamster ovary (CHO) cells in suspension culture [1, 2, 3, 4]. , Freestyle HEK 293F cells). Maintain cultures for 5-7 passages prior to performing transfections to ensure stable growth patterns. PEI FectoPRO® shows a remarkable transfection efficiency in CHO-K1 cells in comparison to PEI and FreeStyleTM MAX. Afterward, several of the clones were adapted to suspension cell culture in a chemically defined medium, EX-CELL ® CD HEK293 Viral Vector Media. Apr 6, 2024 · This procedure is suitable for CHO-S and HEK 293 GnTi- suspension cell lines. Check the cells 1–2 days after transfection to determine what ratio gives the highest percentage of GFP positive cells. Product Qualification: HEK293 Transfection Reagent is tested functionally by transfection of HEK293 cells with a small interfering RNAs targeting 3 different genes (Lamin A/C, GAPDH, Cyclophilin B). CRL-1573) using Lipofectamine LTX Reagent. Apr 26, 2025 · A majority of suspension HEK293 cell-based rAAV production protocols reported rely on a triple transfection at cell density below 2 × 10 6 cells/mL. Nov 24, 2011 · Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0. Introduction Invitrogen™ Lipofectamine™ LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. Transient Gene Expression in Adherent HEK293 Cells using PEI STARTM Transfection Reagent Materials PEI STARTM, 1 mg/mL, pH neutralized, sterile-filtered HEK293 expression medium or DMEM maintained at 37°C transfection is inhibited by serum. An automated liquid handler We have developed a simple protocol to transfect mammalian cells using linear polyethylenimine (PEI). Nov 4, 2020 · In order to look into gene expression variations correlating with adherent and suspension HEK 293 cell lines, differential expression analysis between adherent and suspension HEK293 progeny cell Transient transfection of suspension cells is the most commonly used production platform, as it offers significant flexibility for cell and gene therapy development. First, we describe two optimized PEI-based production protocols in suspension HEK293 cells, as well as a novel downstream protocol to support the puri cation of both PEI- and fi HSV-produced rAAV9. com In our experience, HEK293T cells are easily transfected and the percentage of transfection can be around 80% by using the present protocol. The cell line presents great transfection potential and it grows in suspension under serum-free conditions. 2002; Baldi et al. Due to its cost-effectiveness, ease of use, and scalability, PEI is especially popular in large-scale transient transfection protocols using mammalian suspension cell lines like HEK293 and CHO. For a summary of We would like to show you a description here but the site won’t allow us. Sep 5, 2008 · We have developed three nonviral DNA transfer protocols for suspension-adapted HEK-293 and CHO cells: (1) a calcium phosphate based method (Ca-Pi), (2) a calcium-mediated method called Calfection, and (3) a polyethylenimine-based method (PEI). Cells are grown in the bioreactor to 1 × 10 6 cells/mL and transfected with a plasmid DNA–PEI complex at a ratio of 1:2. After transfection and Transfection Protocols Our Lipofectamine Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. 1. Different titration methods of LvV stocks, as well as different culture media, culture durations, cell densities and DNA Mar 10, 2022 · The current study presents a detailed side-by-side comparison of a PEI transfection and an HSV infection platform in suspension. Application of PEI STAR™ transfection reagent. Sep 17, 2019 · FectoPRO® transfection reagent was designed after extensive screening of numerous chemical structures based on their transfection efficiency, protein production yield and cell viability. 8 μg/106 cells) and repeated transfections done at 34° and 37°C. These cells have been derived from the HEK 293 cell line, are adapted to grow in suspension cultures, reaching high densities using serum-free media (such as FreeStyle 293 Expression Medium). Jan 1, 2013 · We have generated a high-yielding transient transfection platform based on suspension-adapted CHO K1 cells for generating recombinant proteins. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. PEI Prime™️ is a choice reagent for production for recombinant proteins, antibodies and viruses in mammalian expression systems. The adherent cells were transfected following an established protocol for calcium phosphate transfection. Learn the fundamentals of protein expression using the Gibco Expi293 Expression Systems with this protocol guide that includes protocol videos, a protein expression protocol calculator, required material checklists, visual step-by-step guides, and helpful tips. Protocol for transient gene expression in adherent HEK293 cells using PEI STAR™ transfection reagent Materials PEI STAR™, 1 mg/mL, pH neutralized, sterile-filtered. Feb 5, 2024 · 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. Also, when used in conjunction with six-well CellBIND plates, branched PEI can be used to adhere transfected cells when establishing stable cell lines. 5 ml of complete growth medium 12-24 hours prior to transfection Expi293TM Expression Medium is a chemically defined, serum-free, protein-free, animal origin-free medium for growth and transfection of suspension-adapted HEK 293 cells. PEI is the basis of most commercially available transfection agents and alone acts as a very cost effective transfection vector. Stable complexation with DNA, efficient entry into the cell, and ability to escape the endosome makes PEI MAX a highly efficient transfection reagent which is compatible for a wide range of cell lines/types including the most commonly used HEK293 and CHO cells grown in adherent and suspension cultures. PEIpro® produces more virus with less reagent and lower DNA amount compared to another PEI-based reagent and Calcium Phosphate transfection. Sep 14, 2017 · Polyethyleneimine (PEI), a cationic polymer vehicle, forms a complex with DNA which then can carry anionic nucleic acids into eukaryotic cells. Beginning with an adherent HEK293 cell line, we selected clones for high growth and high transfection capability. Transfection Reagent is tested for absence of nuclease contamination and microbial contamination. This chapter describes LV cell cell transfection. Jul 1, 2024 · A rational study upon DNA/PEI complex formation, stability and delivery during transfection of HEK293 cell cultures has been conducted, providing insights on the mechanisms for polyplexes uptake at low cell density and disruption at high cell density. PEI STARTM is more scalable and cost-effective than lipid-mediated transfection, and significantly more reliable than calcium-mediated transfection. Transient Transfection Dec 1, 2006 · We have developed a simple protocol to transfect mammalian cells using linear polyethylenimine (PEI). PEI STAR™ is more scalable and cost-effective than lipid-mediated transfection, and significantly more reliable than calcium-mediated transfection. Jun 14, 2019 · This is one obvious advantage of the PEI-based transfection of HEK293 cells in suspension, because highly pure plasma DNA remains one of the main cost drivers of transient production processes. This reference provides a recommended procedure to transfect plasmid DNA into HEK 293, human embryonic kidney cells (ATCC No. zgmpm foy khcq uver nwqp mezzd jbgl ifcpeo paltyr bwru zesxwl tyime eng jqfu thfukvkt