Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
Induced pluripotent stem cells (iPSCs) hold immense promise for the future of regenerative medicine and personalized therapeutic treatments for a myriad of diseases and conditions. Invitrogen Lipofectamine 3000 Transfection Reagent was developed to unleash the power of stem cells by providing a highly efficient, cost-effective nucleic acid delivery alternative to electroporation. This advanced lipid nanoparticle technology minimizes the stress on cells caused by electroporation, simplifies the reprogramming workflow, and enables advanced gene editing technologies.
Lipofectamine 3000 reagent is designed to provide:
Patient-derived iPSCs offer exciting potential by enabling access to cell populations that are otherwise unavailable from living donors. Efficient reprogramming of donor somatic cells to iPSCs plays a key role in the realization of this potential. In this study, fibroblasts from skin biopsies of three donors were reprogrammed to iPSCs using the Invitrogen Epi5 Episomal iPSC Reprogramming Kit* and Lipofectamine 3000 reagent (Figures 1 and 2). Reprograming efficiencies equivalent to those seen with electroporation were observed (Figure 3). This advancement in delivery helps to minimize stress on the cells caused by electroporation, simplifies the reprogramming workflow, and results in transgene-free, virus-free human iPSCs at efficiencies in the range of 0.04% to 0.3%.
* Designed by Dr. Okita in the laboratory of Professor Yamanaka at the Center for iPS Cell Research and Application (CiRA), Kyoto University.
Figure 1. Lipofectamine 3000 reagent for pluripotent stem cell applications. High transfection efficiency enables Lipofectamine 3000 to be used for a variety of applications, including reprogramming of fibroblasts, gene editing, and transfection of difficult cells.
Figure 2. Protocol outline for generating iPSCs using the Epi5 reprogramming kit and Lipofectamine 3000 reagent. Colonies can be picked and expanded less than 21 days after fibroblast transfection. (Transfection medium: Fibroblast Medium; Culture media: (1) N2B27 Medium (N-2 supplement and B-27 supplement, with bFGF) and (2) E8 Medium).
Figure 3. Reprogramming efficiency of Lipofectamine 3000 reagent compared to electroporation. BJ fibroblasts as well as neonatal (HDFn) and adult (HDFa) human dermal fibroblasts were reprogrammed to iPSCs by transfection of Epi5 vectors using either Lipofectamine 3000 reagent or the Invitrogen Neon Transfection System. Colonies were (A) visualized by brightfield microscopy and (B) stained for alkaline phosphatase.
Genome editing technology derived from clustered regularly interspaced short palindromic repeats (CRISPRs) allows precise cleavage of DNA at specific loci. However, the effectiveness of genome editing is contingent upon the intrinsic properties of the locus of interest, efficiency of delivery, and the painstaking downstream processes of generating stable cell lines and knockout models to study the phenotypic effects of the genetic modifications. Here we demonstrate that Lipofectamine 3000 reagent can deliver plasmid DNA into various iPSC clones for genome engineering purposes using targeted CRISPR vectors. More than a 2-fold increase in gene modification efficiency compared to Invitrogen Lipofectamine 2000 reagent was observed using Lipofectamine 3000 reagent, and with a lower amount of lipid (Figure 4).
Figure 4. Lipofectamine 3000 reagent improves cleavage efficiency of CRISPR vectors in iPSCs. An Invitrogen GeneArt CRISPR vector targeting a specific locus was transiently transfected using Lipofectamine 2000 or Lipofectamine 3000 reagent. Cleavage efficiency was assessed using the Invitrogen GeneArt Genomic Cleavage Detection Kit.
Lipofectamine 3000 Transfection Reagent was used to deliver DNA into difficult-to-transfect stem cells (H9 ESCs and iPSCs) growing as colonies in Gibco Geltrex matrix–coated plates (Figure 5). Transfection efficiencies of 40–70% were observed, with high mean fluorescence intensity (Figure 6).
Figure 5. Protocol outline for transfection of pluripotent stem cells with Lipofectamine 3000 reagent. Cells were transferred into multiwell plates from Geltrex matrix–coated plates and transfected with Lipofectamine 3000 reagent. Transfection efficiency was assessed using flow cytometry and fluorescence imaging.
Figure 6. Transfection of stem cells. (A) H9 ESCs or (B) iPSCs were transfected using Lipofectamine 3000 reagent. Cells were visualized by fluorescence microscopy and processed using flow cytometry to determine transfection efficiency.
Lipofectamine 3000 Transfection Reagent was developed to improve transfection efficiency in difficult-to-transfect cells. In this study, we demonstrated that using a superior transfection reagent such as Lipofectamine 3000 reagent in conjunction with the Epi5 Episomal iPSC Reprogramming Kit enables highly efficient reprogramming of somatic cells without the need for electroporation.
Lipofectamine 3000 Transfection Reagent, used with the Epi5 Episomal iPSC Reprogramming Kit, enables highly efficient reprogramming of somatic cells without electroporation.
Reprogramming timeline | ||||
---|---|---|---|---|
Day –1 | Day 0 | Days 1–14 | Days 15–20 | Days 21+ |
Seed cells on Geltrex matrix–coated 6-well dishes | Transfect in fibroblast growth medium for 24 hr | N2B27 Medium with 100 ng/mL FGF (change medium daily) | Essential 8 Medium (change medium daily) | Colonies ready for picking for further culture and expansion |
Cells and reagents | Equipment |
---|---|
Epi5 Episomal iPSC Reprogramming Kit (Cat. No. A15960) | Sterile cell culture hood (i.e., biosafety cabinet) equipped with a stereomicroscope |
Lipofectamine 3000 Transfection Reagent (Cat. No. L3000015) | Inverted microscope |
Opti-MEM I Reduced Serum Medium (Cat. No. 31985062) | Incubator set at 37°C, 5% CO2 |
Basic Fibroblast Growth Factor (bFGF), Recombinant Human Protein (Cat. No. PHG0261) | Water bath set at 37°C |
KnockOut Serum Replacement (Cat. No. 10828010) | Sterile serological pipettes (5 mL, 10 mL) |
Essential 8 Medium (Cat. No. A1517001) | Centrifuge |
DMEM/F-12, GlutaMAX Supplement (Cat. No. 10565018) | 15 mL centrifuge tubes |
N-2 Supplement (100X) (Cat. No. 17502048) | 6-well tissue culture treated plates |
B-27 Supplement (50X), Serum Free (Cat. No. 17504044) | 10 μL, 200 μL, and 1,000 μL micropipettors with tips |
100X MEM Non-Essential Amino Acids (NEAA) (Cat. No. 11140050) | |
β-Mercaptoethanol (Cat. No. 21985023) | |
Fetal Bovine Serum, embryonic stem cell–qualified (Cat. No. 16141061) | |
DMEM, High Glucose, GlutaMAX Supplement (Cat. No. 10566016) | |
0.05% Trypsin-EDTA (1X), phenol red (Cat. No. 25300054) | |
Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix (Cat. No. A1413301) | |
Dulbecco’s PBS (DPBS) without Calcium and Magnesium (Cat. No. 14190) |
I. Media preparation
Fibroblast Medium
To prepare 500 mL of Fibroblast Medium, aseptically mix the following components. Fibroblast Medium can be stored at 4˚C for up to 1 month.
N2B27 Medium
To prepare 500 mL of complete N2B27 Medium, aseptically mix the following components. N2B27 Medium (without bFGF) can be stored at 2–8°C for up to 1 week.
bFGF (100 µg/mL)
bFGF: 100 µg
DPBS without Ca2+ and Mg2+: 999 µL
KnockOut Serum Replacement: 1 µL
Essential 8 Medium
II. Preparation of Geltrex matrix–coated dishes
III. Fibroblast culturing
IV. Epi5 episomal reprogramming
Seeding human fibroblasts on Geltrex matrix–coated dishes
Plate 50,000 to 100,000 cells per well into a 6-well plate at ~30–60% confluence (see Figure 7) in 2 mL Fibroblast Medium and culture overnight at 37˚C and 5% CO2.
Transfection of human fibroblasts in a 6-well dish using Lipofectamine 3000 reagent
Figure 7. Seeding densities of neonatal (HDFn) and adult (HDFa) human dermal fibroblasts.
Preparation of contents of Tube A and Tube B on a per-well basis of a 6-well dish is outlined below:
Tube A
Tube B
Removal of transfection reagent and recovery of cells
Transition to Essential 8 Medium
Figure 8. Alkaline phosphatase–positive colonies obtained after reprogramming of neonatal (HDFn) and adult (HDFa) human dermal fibroblasts.
Cell Culture & Transfection Learning Center
Access cell culture and transfection educational resources for better experiment planning and execution.
Media Formulation Tool
Find the right Gibco media formulation for DMEM, DMEM/F-12, MEM, and RPMI-1640 media.
Cell Culture & Transfection Support Center
Find technical support recommendations for your cell culture and transfection workflows, including tips for experimental setup and in-depth troubleshooting help.
Need technical support? Contact our expert team for technical and application support of Laboratory Products
For Research Use Only. Not for use in diagnostic procedures.