1. Large-scale production of red blood cells from stem cells: What are the technical challenges ahead?

2. cytokine

3. Main advances in RBC culture in view of the large-scale production of clinical grade cells A biological odyssey

4. On the way to clinical applications (i) Are there any fundamental barriers to large-scale manufacture of RBC? (ii) Is it possible to generate clinically relevant numbers of cells? Three economic questions arise

5. The price ① 5 × 10 7 cells/mL, it would take 40 liters of culture media with a total cost of $8330 to generate enough cells (without considering the investment in facilities) ② Zeuner et al. proposed a production price $8000 – 15 000 per unit of blood. Timmins, N. E., Nielsen, L. K., Blood cell manufacture: current methods and future challenges. Trends Biotechnol. 2009, 27, 415–422. Zeuner, A., Martelli, F., Vaglio, S., Federici, G. et al., Concise review: stem cell-derived erythrocytes as upcoming players in blood transfusion. Stem Cells 2012, 30, 1587– 1596. $700–1200 hospital cost

6. biotechnological breakthrough seven 175-cm 2 flasks expansion and dilution CD34 + RBC 2 tennis courts

7. medium price

9. control of the pH pH lactate cell metabolism slows down with higher cell density ammonia metabolic byproducts pH decrease modification of osmolarity direct growth inhibitor glucose and glutamine metabolisms

10. DO level Dissolved oxygen

11. How to improve the culture conditions It will be necessary to monitor at the same time the differentiation of the cells, which will imply a compromise between expansion and maturation. For RBC production, the complete medium is too expensive, with growth factors and other proteins representing more than 90% of the price. Thus, a new method to eliminate waste products while conserving the expensive molecules is needed. All the downstream process will have to be revised, with critical improvements to implement in the separation and purification procedures.

12. RBC production in bioreactors

13. Day 21 expanded cell populations comprised a pure population of erythroid cells of mixed maturity EBs at different stages of maturation 21 Day

14. Ultra-high-yield manufacture of RBC CD71 CD235 10 7 10 8 500 units of RBC per UCB donation per UCB donation (5 × 10 6 CD34+ cells/ donation)

15. hollow-fiber bioreactor

16. Bioreactor perfusion schematic

17. Bioreactor parameters