Can we grow a supply of red blood cells by differentiating stem cells to replace donor blood?

Obtain CD34+ cells and thaw following cell line specific thawing procedure and documentation.

Subculture the cells into at a 1:1 ratio. Incubate the original culture for 48 hours until it has reached the original cell density.

Follow the procedure to culture and expand subcultures.

1. Isolated CD34+ cells are seeded in 5-7.5 ml of medium at a density of 1105cells/mL in T25 flasks at 37℃ with 5% CO2 with 100ng/mlSCF.

2. Iscove’s Modified Dulbecco’s Medium( IMDM) is used in combination with nutritional supplements transferrin (5 ng/mL) and ascorbic acid (10 μg/mL).

3. Exchange medium every 2 days.

Let the cells proliferate for 5 days to maximum cell density, and then transfect the cells with the supplied plasmids.

Wait 48 hours for maximum protein expression and isolate successfully transfected cells. Culture in iron positive and negative conditions.

1. Iron positive condition can be created by the addition of 500 μg/ml transferrin with 300-600 ppm iron content.

Use RFP assay to measure RFP levels. For each new stable cell line, determine if RFP level is above or below optimal levels compared to exogenously introduced EPO and SCF.