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S. Pellegrin et al.
erythroid culture protocols reported to date incorporate glu- cocorticoids, using either dexamethasone in serum-free conditions7 or hydrocortisone in the presence of 5% plas- ma5 or in serum-free conditions.27
Recent studies have highlighted both the importance of lipid metabolism during terminal erythroid differentiation58 and the fact that exposure to glucocorticoids can affect the lipid metabolism in cultured erythroid cells.59,60 For erythroid cells cultured in the presence of hydrocortisone, the defect
in lipid metabolism and resulting fragility of mRBC can be counteracted by supplementing the medium with choles- terol-rich lipids.60 Interestingly though, addition of choles- terol-rich lipids slightly accelerated differentiation60 and Heshuvius et al. reported that the addition of plasma caused premature differentiation in the presence of dexametha- sone.7 The interplay between glucocorticoid exposure time and lipid metabolism needs further investigation to maxi- mize yield without compromising mRBC quality.
Table 2. Highlighted strategies to improve current culture systems for manufactured red blood cells. Improving yield & quality of mRBC
Starting material
Media composition and supplements
Filtration
Storage
GMP-compliance and reproducibility
Starting material
Media composition and supplements
Bioreactors and filtration
Other considerations
Cost reduction
Starting material
Media composition and supplements
Bioreactors and filtration
• selection of donors with genetic markers linked to specific RBC traits (high hemoglobin, packed cell volume or RBC count)
• selection of donors with consistently high HSPC counts
• use of whole mononuclear cell population (PBMNC) not just CD34+ cells
• genetic manipulation of starting material to enhance proliferation and compatibility
• slow down attrition of HSPC self-renewal capacity
• maximize expansion of erythroid progenitors (CFU-E)
• maximize enucleation and maintain nascent reticulocyte viability
• novel filtration technology to minimize loss of mRBC
• maturation of mRBC to erythrocytes will enhance filtration efficiency
• optimal reticulocyte storage conditions to minimize loss of filtered mRBC until transfusion • maturation of mRBC to erythrocytes to enhance storage times
• minimize stem cell donor variability and cell loss during isolation
• use defined reagents of known reliability and controlled provenance • multiple suppliers identified for key reagents
• supplier surveys and site visits
• closed systems
• scalable GMP-compliant bioreactors with ease of use • close monitoring of the culture growth
• GMP-compliant filtration process
• define release criteria of product and storage times • identification of optimum therapeutic dose of mRBC
• use of PBMNC to circumvent expensive CD34+ cell isolation
• defined in-house media constituents
• in-house growth factors
• replace or reduce the most expensive constituents (e.g. holotransferrin)
• develop protocols/bioreactors that use less medium overall without affecting the yield and quality of mRBC obtained
• enhance media to enable increases in cell density
• fully automated culture processes
• automated filtration
• minimal footprint and labor requirement
RBC: red blood cells; mRBC: manufactured RBC; HSPC: hematopoietic stem and progenitor cells; PBMNC: peripheral blood mononuclear cells; CFU-E: colony-forming unit – erythroid; GMP: Good Manufacturing Practice.
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haematologica | 2021; 106(9)