3D Transfection System3D Transfection System

Why 3D Technology?Why 3D Technology?Introduction to Three Dimensional (3D) Cell CultureThe 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, differentia-tion 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.

3D Cell Culture Enhances the Biological Relevance of Your ResearchThere 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 sur-faces. 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.

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.

Effects of Cell Culture on Cell Growth and FunctionTraditional 2D Cell Culture

Alvetex® 3D Cell Culture

Normal In Vivo Environment

General Dimensions and Physical DifferencesMaximum distance of cell from the source of nutrients 0 0-100 µm 0-200 µmResemblance to in vivo cellular environment Low High n/aAppearance of cell morphology Flattened 3D Shape 3D ShapePotential for 3 dimensional cell to cell interactions Very Low High HighAbility to form complex 3 dimensional cellular structures Very Low High HighUpon Initial Seeding of Cells onto PlasticwareDegree of cellular stress placed upon cell structure High Low n/aChanges to protein and gene expression High Low n/aCell surface area in contact with plastic Approaches 50% 0-50% n/aPost Seeding PhaseOngoing changes to cellular shape High Low n/aNeed for remodelling of cytoskeletal architecture High Low n/aDeviation from normal in vivo morphology High Low n/aCell surface area in contact with plastic Approaches 50% 0-50% n/aOpportunity for enhanced in vitro cell functionality Low High n/a

2D cell growth 3D alvetex® cell growth2D Cell Growth 3D alvetex® Cell Growth

TransIT®-3D Transfection ReagentMirus 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

3D Transfection of Multiple Cell Types. In collaboration with Reinnervate, methods have been developed that enable the transfection of cells grown in 3D culture. Com-mon 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 post-transfection 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.

1,500,000,000

1,000,000,000

750,000,000

300,000,000

250,000,000

200,000,000

150,000,000

100,000,000

50,000,000

0

Luci

fera

se A

ctiv

ity (R

LUs)

Reagent-to-DNA RatioCHO-K1 HeLa HepG2 MCF-7 NIH-3T3

2:1 3:1 2:1 3:1 2:1

1,250,000,000

1. Add cells to hydrated alvetex® 3D scaffold

15-30 minutes2 days 24+ hours

2. Add plasmid DNA 3. Add TransIT®-3D Reagent

4. Add transfection complexes

5. Perform assay

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 PlatesReinnervate is a market leading company in the fast growing area of 3D cell cul-ture. 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 func-tion.

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 in-teracting with adjacent cells and laying down extra-cellular matrix which often 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. Scien-tists 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 200 µm thick; so no cell is ever more than 100 µm from a source of nutrients and gasses. This compares favourably to the typical in vivo environment where cells are generally no more than 150-200 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 200 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.

Presentation of alvetex® in a 12-well plate format.

3D Transfection System3D Transfection SystemRelevant 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 (3:1 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 LSM510). The data shows a 40 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 two-dimensional (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.

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 indi-cated. 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.

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CHO-K1

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HepG2

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MCF7

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2:1 3:1 4:1

NIH3T3

고 객 (학술/제품문의) 02-2081-2510

지원센터 support@takara.co.kr

Tel. 02-2081-2510

Tel. 042-828-6525

본 전 지 사대 전 지 사

3D Transfection System3D Transfection SystemProduct InformationProduct Information3D Transfection SystemProduct Name Product No. Quantity 0.4 ml TransIT®-3D Transfection Reagent & 2 alvetex® 12-well plates MIR 5820 1

TransIT®-3D Transfection ReagentProduct Name Product No. Quantity TransIT-3D Transfection Reagent MIR 5804 1 x 0.4 ml MIR 5800 1 x 1.0 ml MIR 5805 5 x 1.0 ml MIR 5806 10 x 1.0 ml

alvetex® Plates and Inserts for 3D CultureProduct Name Product No. Description 12-Well Plate AVP002 Single plastic plate with alvetex inserted 6-Well Insert AVP004-3 3 individually sealed 6-well inserts 12-Well Insert AVP005-3 3 individually sealed 12-well inserts Well Insert Holder in a Petri Dish AVP015 1 holder and Petri dish Note: All alvetex® products are gamma irradiated and supplied sterile.

To order separate components of the 3D Transfection System,please contact the respective manufacturers

Order TransIT®-3D Transfection Reagent

from Mirus Bio:www.mirusbio.com

608.441.2852888.530.0801 (U.S. only)

Order alvetex® 3D Culture Plates

from Reinnervate:www.reinnervate.com

+44 (0) 1740 625266