ReproFF2

Product Classification : hES/iPSC Culture Media / ReagentsResearch Theme : ES/iPS cells

ReproFF2

Feeder-Free Culture Medium for human ES/iPS cells

  • Require culture maintenance only three days per week. (i.e. Monday, Wednesday and Friday)
  • Reduces medium usage by 50% compared to medium that require change everyday.
  • Equivalent performance to feeder dependent medium in maintaining undifferentiated state.
  • Maintain differentiation capability. (differentiated into cadiomyocytes, neurons and others)
  • Each batch Serum-free is functionally tested by using human iPS cells. Osmolarity, pH, sterility, and mycoplasma-contamination is tested to ensure batch-to-batch consistency.
  • Serum-free.

Note: bFGF is not included in ReproFF2. Please purchase bFGF separately and add it before you culture human ES cells and iPS cells.

Culture maintenance only on Monday, Wednesday and Friday

With ReproFF2, medium change is not required everyday.
Culture doesn’t have to be cared during the weekend if passaging is done on Friday.

Suggested weekly schedule

Overview

ReproFF2 is an epoch-making feeder-free culture medium for human ES cells and iPS cells. ReproFF2 was developed in order to simplify culture maintenance. While conventional stem cell medium require medium change every day, ReproFF2 requires culture maintenance only three days in a week. For example, you can change medium on Monday and Wednesday, and then passage cells on Friday. Consequently, cost and usage amount of medium will be halved .

*: not required for all cell lines

Human iPS cells (201B7) grown with ReproFF2 on a Laminin-5 coated plate.

Human iPS cells (201B7) grown with ReproFF2 on a Laminin-5 coated plate.

Data

Maintenance of an undifferentiated state by using ReproFF2

Cells cultured with ReproFF2 retain the key characteristics of pluripotent cells

Maintanance of undifferentiated human iPS cells with ReproFF2

human iPS cell line 201B7
Feeder-free culture with ReproFF2: Passage 10, day 7
Feeder-dependent culture on MEF: Passage 48, day 3

1. Morphology and ALP activity

Human iPS cell line 201B7 (established in Kyoto University) was kept undifferentiated in feeder-free culture with ReproFF2, as confirmed by morphology (A) and alkaline phosphatase staining (B). These results looked comparable to those of feeder-dependently cultured iPS cells (C and D).

2. Expression of pluripotency markers

Human iPS cells cultured with ReproFF2 retained expression of pluripotency marker genes Oct 3/4, SSEA-4, TRA-1-60 and Nanog, similarly to cells cultured on feeder cells.

3. FACS analysis of pluripotency marker expression in iPS cells

Human iPS cells cultured with ReproFF2 showed profiles comparable to those of feeder-dependently cultured cells when analysed with FACS for pluripotency marker genes SSEA-4, TRA-1-60 and TRA-1-81.

4. Karyotype analysis of human iPS cells after long term culture with ReproFF2

Human iPS cells cultured for 30 passages using ReproFF2 showed no abnormality in karyotype analysis. This result shows ReproFF2 is suitable for long term culture of human iPS cells.

Maintanance of undifferentiated human ES cells with ReproFF2

Human ES cell line KhES-1 (established in Kyoto University) remained undifferentiated in feeder-free culture with ReproFF2, as confirmed by morphology (A), alkaline phosphatase staining (B) and Oct 3/4 expression (C). In collaboration with Kyoto University.

Protocol

Protocol using Gentle Cell Dissociation Reagent(STEM CELL TECHNOLOGIES, 07174)and Vitronectin-N(LIFE TECHNOLOGIES, REFA14700)

ReproFF2 Protocol (Switch fron feeder-dependent/feeder-free using GCDR&VTN)

Protocol using Dissociation Solution (RCHETP002) and Laminin-5 (RCHEOT004) / Matrigel (Corning)

ReproFF2 Protocol (Switch from feeder-dependent using DS/Laminin/Matrigel)

ReproFF2 Protocol (Switch from feeder-free cultivation)

Data Sheet

Safety Data Sheet

Publications

  • Morizane R, et al. “Nephron organoids derived from human pluripotent stem cells model kidney development and injury.” Nat Biotechnol. 2015 Oct 12. doi: 10.1038/nbt.3392.
  • Naoki Nishishita, et al. “Generation and Maintenance of iPSCs From CD34+Cord Blood Cells on Artificial Cell Attachment Substrate.” Biochemistry, Genetics and Molecular Biology, Chapter 5. http://dx.doi.org/10.5772/58591
  • Kanemura, Hoshimi, et al. “Tumorigenicity Studies of Induced Pluripotent Stem Cell (iPSC)-Derived Retinal Pigment Epithelium (RPE) for the Treatment of Age-Related Macular Degeneration.” PloS one 9.1 (2014): e85336.
  • Hasegawa, Tsuyoshi, et al. “Cancer‐associated fibroblasts might sustain the stemness of scirrhous gastric cancer cells via transforming growth factor‐β signaling.” International Journal of Cancer (2013).
  • Kanemura, Hoshimi, et al. “Pigment epithelium-derived factor secreted from retinal pigment epithelium facilitates apoptotic cell death of iPSC.” Scientific reports 3 (2013).
  • Lam, Albert Q., et al. “Rapid and Efficient Differentiation of Human Pluripotent Stem Cells into Intermediate Mesoderm That Forms Tubules Expressing Kidney Proximal Tubular Markers.” Journal of the American Society of Nephrology: JASN (2013).
  • Murakami, Masashi, et al. “The use of granulocyte-colony stimulating factor induced mobilization for isolation of dental pulp stem cells with high regenerative potential.” Biomaterials 34.36 (2013): 9036-9047.
  • Nishishita, Naoki, et al. “Generation of virus-free induced pluripotent stem cell clones on a synthetic matrix via a single cell subcloning in the naive state.” PloS one 7.6 (2012): e38389.
  • Sugiyama, Norikazu, et al. “Label-free characterization of living human induced pluripotent stem cells by subcellular topographic imaging technique using full-field quantitative phase microscopy coupled with interference reflection microscopy.” Biomedical optics express 3.9 (2012): 2175-2183.

Product list

Feeder-Free Culture Reagents

Catalog No. Product Price
RCHEMD006 ReproFF2 (500mL) Ask
RCHEMD006A ReproFF2 (500mL) x3 Ask
RCHEMD006B ReproFF2 (500mL) x3 & bFGF(25μL) pack Ask

Related Products

Feeder-Dependent Culture Reagents

Catalog No. Product Price
RCHEMD001 Primate ES cell medium (500mL) Ask
RCHEMD001A Primate ES cell medium (500mL) x5 Ask
RCHEMD001B Primate ES cell medium (500mL) x5 & bFGF(25μL) pack Ask
RCHEMD005 ReproStem (500mL) Ask
RCHEMD005A ReproStem (500mL) x5 Ask
RCHEMD005B ReproStem (500mL) x5 & bFGF(25μL) pack Ask
RCHEOT001 ReproCoat (500mL) Ask
RCHECK002 Primate ES cell culture kit Ask

Feeder-Free Culture Reagents

Catalog No. Product Price
RCHEMD007 ReproXF (500mL) Ask
RCHEMD004 ReproFF (500mL) Ask
RCHEMD004A ReproFF (500mL) x5 Ask
RCHEMD004B ReproFF (500mL) x5 & bFGF(25μL) pack Ask

Growth Factor

Catalog No. Product Price
RCHEOT002 bFGF (25μL) Ask
RCHEOT003 bFGF (250μL) Ask

Othe Culture Reagents

Catalog No. Product Price
RCHETP002 Dissociation solution for human ES/iPS cells (30mL) Ask
RCHEFM001 Freezing medium for human ES/iPS cells (25mL) Ask

Antibodies for Pluripotency Markers

Catalog No. Product Price
RCAB001P Anti mouse Nanog antibody (200μL) Ask
RCAB002P-F Anti mouse Nanog antibody (100μL) Ask
RCAB003P Anti human Nanog antibody (200μL) Ask
RCAB004P-F Anti human Nanog antibody (100μL) Ask