iPSC Growth Medium

This medium is defined, free of serum or Xeno additives and based on the formulation of the E6 medium but modified for increased performance of iPSCs. iPSC growth medium represents a cell culture medium that is suited for the propagation of human induced pluripotent stem cells of various origins. Upon supplementation, it should be used up within 2 weeks due to limited shelf life. It is recommended to prepare the medium for cell culture in aliquots unless the volume of 500 ml will be used within this time range. This medium is tested with iPS cells cultured on Vitronectin and iMatrix-coated 6-well-plates.

iPSC colonies

Figure 1: Examples of induced-pluripotent cells, iPSC-ASC (left) and iPSC-hDPSC (right), the latter derived by reprogramming Mesenchymal Stromal cells isolated from human Dental Pulp cells, cultivated in the CLS iPSC growth medium.

Pluripotency of cells cultured in iPSC growth medium

1. Flow cytometry data

Figure 2: Pluripotency of iPSC-hDPSC (clone 2) by flow cytometry: the cells were propagated in iPSC growth medium in 6-well-plates, detached using Accutase and stained. A) SOX-2, B) Oct3-4, C) Nanog.

2. Immunocytochemistry

Figure 3: Pluripotency of iPSC-hDPSC (clone 1) by immunocytochemical staining: iPSC-hDPSC derived by reprogramming Mesenchymal Stromal cells isolated from human Dental Pulp cells, cultivated in the iPSC growth medium in glass bottom dishes (ibidi). iPSCs expressed the pluripotency markers Oct3/4, SSEA4, TRA-60-1 and SOX2, staining was conducted using the PSC Immunocytochem Kit 4-Marker (Thermofisher).

Media preparation

1. The supplement comes in two parts, the basic medium and the supplement comprising very sensitive ingredients.
2. Due to the reduced shelf life of the media once supplemented, it is recommended to prepare smaller quantities such as 100 mL.
3. To prepare 100 mL of the iPSC growth medium, thaw the supplement at room temperature (18-25°C) or overnight at 4-8°C. Do not thaw at 37°C in a water bath, as this may be harmful to the ingredients. Aliquot the remaining 40 mL adequately and freeze down immediately.
4. Aseptically transfer 90 mL of the iPSC-MG basic medium into a sterile 100 mL bottle and add 10 mL of the supplement. Mix thoroughly. This volume should be used up within 1-2 weeks.
5. If other volumes of media are required, prepare accordingly.
6. For sterile filtration purposes, use 0.2 µm low protein binding polyethersulfone (PES) filter units (e.g. Corning Cat # 431229).

Maintenance

• Keep the basic medium and the supplemented medium refrigerated at +2°C to +8°C in the dark. Freezing as well as warming up to +37°C minimizes the quality of the product.
• Do not heat the medium to more than 37°C or use uncontrollable sources of heat (e.g., microwave appliances).
• If only a part of the medium is to be used, remove this amount from the bottle and warm it up at room temperature.
• Store the supplement at -20°C. Do not leave it for a prolonged time at ambient temperature.
• After supplementation, this medium has a shelf life of about two weeks.