Isolating and Expanding Keratinocytes
Supplements available for EpiLife® Basal Medium range from the defined, animal origin–free Supplement S7, to the standard supplement (Human Keratinocyte Growth Supplement) containing bovine pituitary extract. The classic keratinocyte growth systems include the tried-and-true Keratinocyte-SFM, which contains bovine pituitary extract, and the defined, xeno-free medium Defined Keratinocyte-SFM.
Figure 1. Extended lifespan of keratinocytes grown in EpiLife® medium compared to growth in a competitor’s medium. Normal Neonatal Human Keratinocytes were grown in EpiLife® medium and in a keratinocyte medium from a leading competitor. Cultures grown in EpiLife® medium demonstrated population doublings over an extended period compared to cells grown in the competitor’s medium.
|KERATINOCYTE ISOLATION AND EXPANSION|
|Human Epidermal Keratinocytes, adult (HEKa)||≥500,000 viable cells|
|Human Epidermal Keratinocytes, neonatal (HEKn)||≥500,000 viable cells|
|EpiLife® Medium||500 mL|
|Supplement S7||5 mL|
|Human Keratinocyte Growth Supplement||5 mL, 100X|
|EpiLife® Defined Growth Supplement||5 mL, 100X|
|Defined Keratinocyte-SFM||500 mL|
|FIBROBLAST ISOLATION AND EXPANSION|
|Human Dermal Fibroblasts, adult (HDFa)||≥500,000 viable cells|
|Human Dermal Fibroblasts, neonatal (HDFn)||≥500,000 viable cells|
|Medium 106||500 mL|
|Low Serum Growth Supplement||10 mL, 50X|
|Synth-a-Freeze® Cryopreservation Medium||50 mL|
|Fetal Bovine Serum, ES Cell-Qualified||500 mL|
|SOMATIC CELL REPROGRAMMING|
|iPSC Lentivirus—Nanog||100 μL, 6.92 x 107 particles|
|iPSC Lentivirus—Klf4||100 μL, 9.90 x 107 particles|
|iPSC Lentivirus—Lin28||100 μL, 3.64 x 107 particles|
|iPSC Lentivirus—Sox2||100 μL, 1.57 x 105 particles|
|iPSC Lentivirus—Oct4||100 μL, 6.50 x 106 particles|
|iPSC Lentivirus—cMyc||100 μL, 4.90 x 107 particles|
Isolating and Expanding Fibroblasts
Save Time with Ready-to-Use Somatic Cells
Methods for Reprogramming
Retroviruses and lentiviruses are randomly integrating viruses that are extremely efficient at transfecting multiple cell types and provide high levels of short-term expression of the required transgenes. While retroviruses are only able to infect dividing cells, lentiviruses can infect both dividing and nondividing cells , making them a preferred choice for reprogramming experiments.
Deliver Reprogramming Factors with Maximum Efficiency
Drug-inducible lentiviral particles allow temporal control of reprogramming factor expression, providing efficient tools for reprogramming factor screens and optimization of reprogramming conditions. ViraPower™ HiPerform™ iPSC lentiviral particles were constructed with the pLenti6.3 tetO DEST vector, allowing inducible expression of the reprogramming factor(s) of interest and precise control of expression level. Cells cotransduced with Lenti6.3/TetR virus particles and ViraPower™ HiPerform™ iPSC lentivirus particles do not express the reprogramming factors until the addition of tetracycline.
Figure 2. Human dermal fibroblasts reprogrammed with ViraPower™ HiPerform™ iPSC lentiviral particles. (Left) Human dermal fibroblasts (HDFs) from a 34-year old female were transduced at day 0 with ViraPower™ HiPerform™ iPSC lentiviral particles containing Oct4, Sox2, Klf4, and c-Myc. (Center) iPSC colony surrounded by HDFs at day 14. (Right) Large iPSC colony at day 75. Data provided by Yasuyuki Asai, ReproCELL Inc., Tokyo, Japan.
- Aasen T, Raya A, Barrero MJ et al. (2008) Nat Biotechnol 26:1276–1284.
- Takahashi K, Yamanaka S (2006) Cell 126:663–676.
- Takahashi K, Tanabe K, Ohnuki M et al. (2007) Cell 131:861–872.
- Yu J, Vodyanik MA, Smuga-Otto K et al. (2007) Science 318:1917–1920.
- Maherali N, Hochedlinger K (2008) Cell Stem Cell 3:595–605.
- Naldini L, Blömer U, Gallay P et al. (1996) Science 272:263–267.
Find more information on our stem cell research reagents.
- Learn More about Tools for Induced Pluripotent Stem Cell Research