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Manufactured red blood cells
cyte stability and filtration efficiency. Another way is to use culture manipulation to try and maintain stemness and proliferation potential. For example, there are small molecule inhibitors that are reported to enhance cord blood HSPC self-renewal, such as UM17140 and the aryl hydrocarbon receptor agonist SR-1.41 There are also fac- tors such as angiopoietin-like 5 and IGBFP242 and notch ligand.43 Although there is evidence that these small mole- cules or factors enhance HSPC expansion, there are no data yet to suggest that they can enhance the yield of reticulocytes when cultured on a large scale.
Perhaps the most exciting advancement in erythroid pro- genitor manipulation is the recently reported overexpres- sion of BMI1 in human HSPC, which gave a 1012 fold increase of erythroblasts.44 Not only does the extensive expansion give the potential for higher yields (assuming the cells do not differentiate spontaneously when grown in large volumes), it also confers the opportunity for further genetic manipulation due to the extended time frame. Genetic manipulation in the form of YTHDF2 knockdown also generated a reported 14.3-fold increase in CD34+ fre- quency in the culture conditions used by a separate group of researchers.45 Alternatively, better biomimicry of the stem cell niche to recapitulate conditions ex vivo has the potential to maintain HSPC stemness for longer periods and therefore increase yield; however, these technologies still require further development.46-52 It will be very interest- ing to see if applications of these innovations can translate into higher yields for large-scale mRBC production.
Media composition and optimization
As well as exploiting cell-intrinsic properties, the base medium composition could be further developed and sup- plemented. Erythroid progenitors are generally cultured in Iscove modified Dulbecco medium (IMDM) and laborato-
ries maintain consistency with suppliers whenever possi- ble. There are many different IMDM commercially avail- able, some better than others in terms of supporting the proliferation and enucleation of erythroid progenitors. Studies are needed to determine exactly what nutrients are required to support the highest proliferation rates of HSPC and erythroid progenitors in culture, particularly important when culturing at high cell densities. Interestingly Heshusius et al. supplemented their IMDM with nucleo- sides and a range of trace elements to make a more defined GMP-compliant medium.7 Zhang and colleagues13 added folic acid and selenium to their large-scale cultures of human cord blood CD34+ cells. An experimental approach, using parallel stirred tank micro-bioreactors, is needed to identify the definitive media and supplements to use for erythroid culture.
The lipid sources added to base media by different labo- ratories also vary significantly, with some groups favoring different amounts of plasma, serum (human or bovine) or serum-free conditions supplemented with animal, human or plant-derived lipid-rich reagents. For compliance with GMP, animal sources must eventually be substituted, which can have an impact on yields. In their recent report Heshuvius et al. also highlighted the importance of albumin purity for proliferation.7 Interestingly, Wilkinson et al.53 showed that 0.1% human serum albumin can be replaced by 0.1% polyvinyl alcohol for cultures of human umbilical cord blood-derived CD34+ cells but as yet this observation has not been tested on a large scale.
Glucocorticoids
The importance of glucocorticoids in promoting stress erythropoiesis was originally discovered in avian and mice studies54,55 and glucocorticoids have been used to increase the yield of human mRBC.26,29,56,57 Three of the larger-scale
Table 1. Summary of recently published, large-scale erythroid culture systems, with expansion and enucleation rates where provided as well as bioreactor and GMP/non-GMP media constituents where applicable. Exhaustive reviews of small-scale erythroid cultures can be found elsewhere.20,22,25
Source and culture period
Cord blood, 33 days
Peripheral blood CD34+ cells
Peripheral blood CD34+ cells,
20 days
Peripheral blood and cord blood CD34+ cells 20 days
Cord blood CD34+ cells
Peripheral blood MNC (no CD34+ cell isolation) 21 to 37 days
(due to expansion stage)
General protocol
Two-stage Three-stage
Three-stage
Three-stage
Four-stage Three-stage
Expansion
2.3x108
by extrapolation
6.15x104 fold Large cultures (actual yield)
>104 fold Large cultures (actual yield)
>105 fold Large cultures (actual yield)
2.9x105 fold Large cultures (actual yield)
107 fold
by extrapolation
Enucleation rate
>90% 68%
55-95%
63%
> 90%
Key points
First demonstration of bioreactor use; 1 L cultures in wave-type bioreactor; non-GMP (use of BSA)
1 culture of 2.5 mL packed filtered mRBC under GMP conditions autologous human transfusion
5 mL packed filtered mRBC, constant batch feeding in spinner flasks (no medium changes); non GMP
Large scale cultures (~25 L)
10 mL packed filtered mRBC using constant batch feeding in spinner flasks under GMP conditions
Reference
Timmins et al., 2011 Giarratana et al., 2011
Griffiths et al., 2012
Kupzig et al., 2017
Large scale culture in rotating wall vessels; non-GMP (use of 15% FBS
in steps 2 & 3)
G-Rex bioreactor
GMP compliant: serum-free and plant derived lipids for expansion;
5% human plasma for differentiation
Zhang et al., 2017 Heshusius et al., 2019
GMP: Good Manufacturing Practice; BSA: bovine serum albumin, mRBC: manufactured red blood cells, FBS: fetal bovine serum; MNC: mononuclear cells.
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