3D Transfection System

3D Transfection System

Why 3D Technology? Introduction to Three Dimensional (3D) Cell Culture The goal of three-dimensional (3D) cell culture is to eliminate the stress and artificial responses cells experience as a result of adaptation to flat, 2D growth surfaces and to create suitable surroundings for optimal cell growth, differentiation and function. Genuine 3D cell culture allows individual cells to maintain their normal 3D shape and structure with minimal exogenous support and interference. Cells are freely able to form complex interactions with adjacent cells and receive and transmit signals, enabling a more natural environment to foster the creation of native architecture found in tissues. Scanning electron microscopy images of HepG2 cells grown in 2D culture versus alvetex scaffold. Cells in alvetex grow more homogeneously and develop a 3D form characteristic of liver tissues in the body. 2D Cell Growth 3D alvetex Cell Growth 3D Cell Culture Enhances the Biological Relevance of Your Research There is an increasing demand for in vitro systems that more accurately mimic natural in vivo tissue environments. Cells maintained in 3D using alvetex exhibit enhanced functionality compared with those cultured on traditional 2D surfaces. As 3D culture technologies become more integral for research, a wide range of applications including cell-based assays and molecular biology techniques such as transfection need to be developed specifically for 3D matrices. Effects of Cell Culture on Cell Growth and Function Traditional 2D Cell Culture Alvetex 3D Cell Culture Normal In Vivo Environment General Dimensions and Physical Differences Maximum distance of cell from the source of nutrients -1 µm -2 µm Resemblance to in vivo cellular environment Low High n/a Appearance of cell morphology Flattened 3D Shape 3D Shape Potential for 3 dimensional cell to cell interactions Very Low High High Ability to form complex 3 dimensional cellular structures Very Low High High Upon Initial Seeding of Cells onto Plasticware Degree of cellular stress placed upon cell structure High Low n/a Changes to protein and gene expression High Low n/a Cell surface area in contact with plastic Approaches 5% -5% n/a Post Seeding Phase Ongoing changes to cellular shape High Low n/a Need for remodelling of cytoskeletal architecture High Low n/a Deviation from normal in vivo morphology High Low n/a Cell surface area in contact with plastic Approaches 5% -5% n/a Opportunity for enhanced in vitro cell functionality Low High n/a

TransIT -3D Transfection Reagent Mirus Bio serves the global research market through an expansive and innovative range of nucleic acid delivery product offerings. TransIT broad spectrum transfection reagents effectively deliver nucleic acid into the vast majority of cell types while specialized reagents are optimized for individual cell types. Applying this expertise to novel applications has led to development of the TransIT -3D Transfection Reagent, a delivery technology enabling high efficiency, low toxicity transfection of cells in 3D culture. Features and benefits of the new TransIT -3D Transfection Reagent include: x High Efficiency DNA Delivery Achieve high expression in a wide range of cell types x Easy-to-use User-friendly protocol that requires minimal optimization x Biologically Relevant Data Perform experiments in 3D, mimicking an in vivo environment 1,5,, Luciferase Activity (RLUs) 1,25,, 1,,, 75,, 3,, 25,, 2,, 15,, 1,, 3D Transfection of Multiple Cell Types. In collaboration with Reinnervate, methods have been developed that enable the transfection of cells grown in 3D culture. Common cell types (HeLa, HepG2, MCF-7, NIH-3T3 and CHO-K1) were seeded at optimized cell densities in 12-well alvetex 3D plates and adapted to 3D culture conditions for 48 hours. After adaptation, cells were transfected with TransIT -3D Transfection Reagent combined with a plasmid encoding firefly luciferase at the reagent-to-dna ratios indicated beneath the bars. Luciferase activity was measured 24 hours posttransfection using a conventional assay. High expression was detected in all cell types demonstrating the efficiency of TransIT -3D Transfection Reagent when used with alvetex 3D culture plates. 5,, Reagent-to-DNA Ratio CHO-K1 HeLa HepG2 MCF-7 NIH-3T3 1. Add cells to hydrated alvetex 3D scaffold 2. Add plasmid DNA 3. Add TransIT -3D Reagent 4. Add transfection complexes 5. Perform assay 15-3 minutes 2 days 24+ hours Transfection in 3D Culture Using TransIT -3D. Transfection in alvetex with TransIT -3D is as easy to perform as 2D tissue culture formats. TransIT -3D exhibits low toxicity and is serum compatible.

Alvetex 3D Culture Plates Reinnervate is a market leading company in the fast growing area of 3D cell culture. The alvetex family of 3D cell culture products provides a flexible platform that enable scientists in any life science laboratory to quickly and easily establish better in vitro assays and models that better mimic in vivo cell growth and function. By accurately recreating the complex organization and environment experienced by cells within their native tissues, alvetex enables more accurate investigation into the study of cell behavior and function than is possible by culturing cells as mono-layers within conventional 2D model systems. Cells maintain their natural 3D shape and structure within alvetex, freely interacting with adjacent cells and laying down extra-cellular matrix which often Presentation of alvetex in a 12-well plate format. leads to the formation of mini slabs of tissue-like structures. Using alvetex, the cell biologist can create in vitro models which more accurately mimic the tissue environment, gaining a much deeper insight into the complexities of cell function and behavior. The following attributes make alvetex the perfect product for reliable and reproducible 3D cell culture: x Genuine 3D Cell Growth a highly porous and consistent scaffold structure provides the ideal environment for cells to grow and proliferate in three dimensions generating highly consistent and reproducible 3D cell growth. x Easily Switch Between 2D and 3D Cell Growth made from the same inert polystyrene material as standard 2D cell culture plasticware, alvetex gives researchers the confidence to easily switch between 2D and 3D cell culture. Scientists can continue to use the same cell culture media and reagents in both types of studies! x Ideal Geometry for 3D Cell Growth specifically designed to mimic the typical in vivo cellular environment, alvetex is 2 µm thick; so no cell is ever more than 1 µm from a source of nutrients and gasses. This compares favourably to the typical in vivo environment where cells are generally no more than 15-2 µm away from a capillary. x Ideal for Long-term Studies inert and highly stable, even during long-term cell culture, alvetex eliminates the potential of any experimental variability even during long-term studies. x No New Equipment is Required compatible with existing standard cell culture plates from most manufacturers and does not require the use of additional equipment. Alvetex Scaffold for 3D Cell Culture. Left photo: Alvetex is a highly porous cross-linked polystyrene scaffold, which as been sectioned into 2 µm thick membranes. Right photo: cross section of HaCaT cells grown for 3 days in submerged media conditions and 14 days at the air interface.

3D Transfection System Relevant Results TransIT -3D successfully transfects fibroblasts grown using 3D alvetex scaffold technology. Fibroblasts were transfected using TransIT -3D Transfection Reagent and a GFP expressing plasmid ( reagent-to-dna ratio). Cells were seeded at 48 hours prior to transfection, and the cultures were fixed 24 hours post-transfection. Cells were imaged using a confocal microscope (Zeiss LSM51). The data shows a 4 micron integrated stack of multiple images as viewed from above the intact 3D culture. The position of all the cell nuclei are visualized with Hoechst 33342 (blue), and the positively transfected cells express GFP (green). Three-dimensional (3D) cell culture is an emerging trend in cell biology as it permits complex multicellular organization that more closely mimics the mechanical and biochemical cues present in native tissues as compared to routinely used twodimensional (2D) cell culture. Transfection is a technique frequently used by cell biologists in 2D cell culture and requires further development to be effectively adapted to 3D cell culture. Unlike current 3D transfection protocols, which require transfection of cells in suspension prior to plating in 3D matrices, Mirus has established a system where cells are effectively transfected with TransIT -3D Transfection Reagent within the alvetex scaffold. The 3D Transfection System, comprised of the Mirus TransIT -3D Transfection Reagent and the Reinnervate alvetex scaffold, is a novel 3D transfection technology that provides users biologically relevant results. CHO-K1 6 3 5 25 NIH3T3 MCF7 HepG2 4 25 35 2 2 1 15 1 5 25 2 15 1 15 1 5 5 3 2 4 3 Transfection efficiency measured by flow cytometry. Cells were seeded at optimized cell densities in 12-well alvetex 3D plates and adapted to 3D culture conditions for 48 hours. After adaptation, cells were transfected with TransIT -3D combined with a plasmid encoding Green Fluorescent Protein at the reagent-to-dna ratios indicated. Efficiency of delivery was measured 24 hours later (CHO-K1 and NIH3T3) or 48 hours later (MCF7 and HepG2) using a BD LSRII flow cytometer.

Product Information 3D Transfection System Product Name Product No. Quantity.4 ml TransIT -3D Transfection Reagent & 2 alvetex 12-well plates MIR 582 1 To order separate components of the 3D Transfection System, please contact the respective manufacturers TransIT -3D Transfection Reagent Product Name Product No. Quantity TransIT-3D Transfection Reagent MIR 584 1 x.4 ml MIR 58 1 x 1. ml MIR 585 5 x 1. ml MIR 586 1 x 1. ml alvetex Plates and Inserts for 3D Culture Product Name Product No. Description 12-Well Plate AVP2 Single plastic plate with alvetex inserted 6-Well Insert AVP4-3 3 individually sealed 6-well inserts 12-Well Insert AVP5-3 3 individually sealed 12-well inserts Well Insert Holder in a Petri Dish AVP15 1 holder and Petri dish Note: All alvetex products are gamma irradiated and supplied sterile. Order TransIT -3D Transfection Reagent from Mirus Bio: www.mirusbio.com 68.441.2852 888.53.81 (U.S. only) Order alvetex 3D Culture Plates from Reinnervate: www.reinnervate.com +44 () 174 625266 고객 ( 학술 / 제품문의 ) 2-281-251 지원센터 support@takara.co.kr 본전지사대전지사 Tel. 2-281-251 Tel. 42-828-6525