Large-scale production of red blood cells from stem cells: What are the technical challeng

es ahead?

cytokine

Main advances in RBC culture in view of the large-scale production of clinical grade cells

A biological odyssey

On the way to clinical applications

(i) Are there any fundamental barriers to large-scalemanufacture of RBC?

(ii) Is it possible to generate clinically relevant numbers of cells?

Three economic questions arise

The price

① 5 × 107 cells/mL, it would take 40 liters of culture media with a total cost of $8330 to generate enough cells (without consideringthe investment in facilities)

②Zeuner 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

biotechnological breakthrough

seven 175-cm2 flasks

expansion and dilution

CD34+ RBC

2 tennis courts

medium price

control of the pH

pH

lactate

cell metabolism slows down with higher cell density

ammoniametabolic byproducts

pH decreasemodification of osmolarity

direct growth inhibitor

glucose and glutamine metabolisms

DO level

Dissolved oxygen

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.

RBC production in bioreactors

Day 21 expanded cell populations comprised a pure population of erythroid cells of mixed maturity

EBs at different stages of maturation

21 Day

Ultra-high-yield manufacture of RBC

CD71

CD235

107 108

500 units of RBC per UCB donation

per UCB donation (5 × 106 CD34+ cells/ donation)

hollow-fiber bioreactor

Bioreactor perfusion schematic

Bioreactor parameters