Hybris-8 serum-free medium of 490 ml with an supplement (50-x) of 10 ml
HybriS-8 is a fully defined medium for serum-free and feeder-free culture of induced pluripotent stem cells (iPSC) and embryonic stem cells (ES).
HybriS-8 has defined formulation and contains a minimal set of components which are necessary for survival and proliferation of stem cells. HybriS-8 allow continuous growth as undifferentiated cells during a long series of passages. HybriS-8 contains no ingredients of animal origin such as albumin, fetuin or other undefined components.
The complete HybriS-8 medium is a mixture of mineral salts, amino acids, glucose, vitamins, antioxidants and recombinant human proteins dissolved in purified water.
Without Antibiotics.
This product is intended for research purposes.
Preparation instructions:
- Manipulate the product in aseptic conditions.
- Add a supplement (50x) to the medium HybriS-8, diluting it in a ratio of 1:50.
- Add L-GLutamine to the medium at final concentration of 365 mg/l.
- Add antibiotics (if necessary).
SHELF LIFE: 1 year.
STORAGE CONDITIONS:
- Store the complete medium at temperature of +2 to +8 °C for no more than 2 weeks.
- Store the medium at temperature of +2 to +8 °C.
- Store the supplement at temperature from -15 to -20 °C.
General recommendations for iPSC culture in Hybris-8
GirbiS-8 allows the use of various commercially available proteins to coat culture plastics: solubilized basement membrane matrix from Engelbreth-Holm-Swarm sarcoma (Matrigel by Corning) or recombinant human vitronectin (VTN-N).
Stem cells culture in HybriS-8 can be manipulated without contact with serum. Use Trypsin and a Trypsin Inhibitor from soybean or enzyme-free protocol with Versene solution to cell detachment.
For long-term routine cultivation in HybriS-8 medium we recommend to take stem cells in small aggregates (50-200 microns in diameter), when their colonies cover 60-70% of the available area or when their colonies are very large with multilayer structure.
The optimal time between passages varies from 3 to 8 days. It is recommended to dilute the suspension of cells in the ratio from 1:3 to 1:12 (according to the desired crop maintenance schedule).
A small number of differentiated cells are allowed in culture that do not require removal.
It is necessary to change the medium every day.
HybriS-8 supports the growth of stem cell cultures for 2-3 passages when seeding with single cells with the addition of a Rho-associated protein kinase (ROCK) (Y-27632) on the 1st- 2nd day. However, in this case, the probability of differentiation of all or most cells, disorders in the karyotype and a decrease in the intensity of cell proliferation and survival significantly increases.
Results of testing the Hybrid-8 medium in a model experiment
To test the HybriS-8 medium, we used the iPSC culture. The cells were cultured in T25 vials coated with Corning Matrigel at 5% CO2, 37°C and 95% humidity. The cells were replanted with aggregates every 7-8 days. When replanting, the suspension of cells was diluted in a ratio of 1:10. We have shown that HybriS-8 supports iPSC proliferation for at least 8 passages. At the same time, for 7-8 days after sowing, the initial iPSC colonies areas(observed 24 hours after sowing) increased by 10-50 times. During testing, we did not observe any cell differentiation signs.
Micrographs (magnification – 10x) of the same section of the culture vial on days 3, 4 and 7 are below.
