**2.6. Clinical trial**

**2.5. Bioreactors**

262 Cell Culture

The production of cultured red blood cells for transfusion purposes has been the holy grail for transfusion medicine. However, a main challenge is the well-described limitation in cell density during the expansion phase [171]. A single transfusion unit contains 2 × 10<sup>12</sup> RBCs.

meaning that more than 1000 L of culture would be required for the manufacture of a single transfusion unit [172]. Handling of such large volumes is impractical if static culture conditions are maintained. Thus, multiple bioreactor designs have been proposed to improve the volumetric productivity of the process (produced cells/volume of medium). A static culturing system mimicking BM tissue has been proposed, in which cells are grown in a porous scaffold and nutrients are continuously fed through hollow fibers, while used media containing waste from metabolism are removed [173]. This system allows to have continuous replenishment of spent components in the media, while it is possible to re-use some of the most expensive components such as growth factors and transferrin. Also, it separates cells from large shear stress sources. Although an optimal design of this system could lead to the production of transfusion units at competitive costs compared to the price of rare blood units, it would require significant improvements in transfer of nutrients and matured RBCs between the scaffold and the inflow/outflow streams [174]. Mass transfer limitations in diffusion-governed systems can be tackled with agitation. It is relevant to note that conflicting reports have been made on the effect of shear due to agitation in *in vitro* erythropoiesis [172, 175]. Nevertheless, important advances have been made toward culture of EBLs in stirred reactors. Enucleated RBCs were produced in microbioreactors (<20 mL) in which the effect of shear stress on expansion and enucleation of EBLs was evaluated. Gas sparging caused cell death, whereas stirring enhanced enucleation [172]. Expansion can also be performed in shake flasks (40 mL) and stirred tank bioreactors of larger volumes (500 mL) with similar growth kinetics [176]. Hybrid systems combining mechanical agitation and growth of cells in porous materials have also been proposed. This type of systems could improve mass transfer while protecting cells from shear, but harvesting of mature cells from these carriers is still a challenge that must be addressed. The high sensitivity of MK to shear stress renders culturing and flow cytometry assays challenging. However, it can also be exploited to generate *in vitro* Plts. Multiple techniques can be used to induce the culturing and shedding of Plts like pipetting or the use of flow chambers [157, 165–168]. These approaches mimic more the *in vivo* situation of Plts formation where they are shed in the vasculature, in contrast to static cultures. Plt shedding can be induced by repetitive pipetting of the cultured cells with a hand pipette (p1000) [81]. There is a risk that too much pressure is enforced on the cells, causing them to lyse instead of shed. A more sophisticated method of *in vitro* Plts production is the use of flow chambers, which come in different versions [167, 177]. The most basic one has a linear flow with a surface that is coated to which MKs can attach [165, 167]. These versions were further developed to chambers that include small gaps where MKs are trapped and can extend their cytoplasmic extensions through the gap where they start forming Plts [165, 177]. Another flow chamber setup is the use of pillars in the chamber instead of gaps where the MKs can attach. Coating with a various matrices like fibronectin, von Willebrand factor, or TPO can enhance the efficiency of the Plts generation. In addition, the flow rates that are produced in these chambers can be regulated.

cells/mL,

Conventional cultivation systems using dishes or flasks can reach up to 10 × 10<sup>6</sup>

The ability to produce large numbers of enucleated, hemoglobinized RBCs opens the perspective of producing cultured red blood cells (cRBC) for transfusion purposes. The feasibility to do so has been demonstrated by the team of Luc Douay who cultured 1 mL of packed cRBC from CD34<sup>+</sup> HSCs and transfused it to a healthy volunteer, with a cRBCs half-life of 26 days after injection [133, 179]. Donor-derived RBC transfusion is a cornerstone of modern medicine in the treatment of trauma, chronic anemia, and in surgery. The existence of 30 blood group systems, such as the ABO and Rhesus system, generates at least 300 distinct blood group antigens [180]. Recurrent transfusions carry an inherent risk on alloimmunization to nonidentical blood group antigens, which complicate further transfusions. Besides, this cellular therapy is dependent on donor availability with a potential risk of blood-borne diseases. *In vitro* derivation of cRBCs allows their thorough characterization, therefore providing access to better matched product. Improved cell culture protocols may eventually enable us to generate cRBCs for transfusion purposes from iPSCs that were selected and/or modified to lack most blood group antigens with the advance of donor-independency offered by iPSCs source.

In a recent publication, Ito et al. [178] were able to generate Plts in transfusion quantities, where the functionality of these Plts was shown in *in vivo* mouse experiments. This work gives a feasible prospect of clinical trials, which still requires the system to be converted to GMP grade [178].
