Contributions
Abstract: S294
Type: Oral Presentation
Session title: Transfusion medicine
Background
Transfusion of donor-derived red blood cells to alleviate anaemia is the oldest and most common form of cellular therapy. In addition, red blood cells hold great promise as delivery agents of e.g. specific drugs of enzymes. However, the source depends on donor availability, and carries a potential risk of alloimmunization and blood borne diseases. In vitro cultured, customizable red blood cells (cRBC) negate these concerns and introduce precision medicine both in transfusion medicine as well as in drug delivery applications.
Aims
Here we investigated the requirements to produce red blood cells from erythroid cultures in stirred bioreactors in order to upscale the process.
Methods
Using inhouse generated serum-free GMP-grade media we optimize a protocol to expand erythroblast and differentiate cultures using 0.5 and 3.0 L stirred bioreactors. Culture parameters, e.g. oxygen availability, shear stress, impellor speed, culture density and perfusion speeds were measured. Sheer stress-induced effects on cells was evaluated by assessing specific signal transduction pathways and globally by RNA-sequencing. Cultured red blood cells were subjected to quality assessment including deformability by Automated Rheoscope and Cell Analyser (ARCA) and oxygen equilibrium curves as measured with a Hemox analyser. Flow cytometry was used to asses enucleation rates and differentiation progression.
Results
In stationary cultures, more than 90% of the cells enucleated and expressed adult haemoglobin as well as the correct blood group antigens. Passaging cRBC through a leukodepletion filter yielded 100% enucleated, stable cRBC. Deformability and oxygen association/dissociation were similar in cRBC and peripheral blood erythrocytes. RNA sequencing and proteomics was performed daily during differentiation and revealed expression dynamics of important erythroid processes, e.g. increased expression of genes involved in blood group expression, globin regulation, erythroid specific metabolic enzymes, and autophage/mitophage degradation of intracellular organels as well as / downregulation of processes involved in cell proliferation. Next, we optimized a protocol to expand erythroblast cultures using 0.5 and 3.0 L stirred bioreactors. Erythroblasts produced in this system showed similar proliferation potential compared to static conditions. Oxygen availability is a critical process parameter and erythroblasts could be cultured at oxygen concentrations as low as 0.7 mg/L, controlled by intermittent sparging. Maximum shear stress at the impeller tip, predicted to be ~1.8 Pa, was mimicked in an orbital shaker setup to evaluate the effect of mixing on the culture. This level of shear stress induced Ca2+-dependent signaling pathways, including the calcineurin/NFAT pathway, and PKC-enhanced STAT5 signaling. As a result, erythroid maturation under differentiation conditions is accelerated by this level of shear stress. Cell erythroblast densities of up to 10 x 106 cells/mL were reached upon continuous media perfusion, 5X higher compared to the current expansion culture systems. Erythroblasts cultured at high density with perfusion maintained high viability and low spontaneous differentiation. This perfusion strategy leads to smaller culture volumes.
Conclusion
Our culture protocols enable us to culture RBC from adult peripheral blood mononuclear cells both in static and bioreactor settings. The use of bioreactor is projected to be significantly more cost-effective compared to static cultures. Currently we prepare for a clinical trial with cRBC.
Keyword(s): ABO blood group, Erythroid differentiation, Signaling, Transfusion
Abstract: S294
Type: Oral Presentation
Session title: Transfusion medicine
Background
Transfusion of donor-derived red blood cells to alleviate anaemia is the oldest and most common form of cellular therapy. In addition, red blood cells hold great promise as delivery agents of e.g. specific drugs of enzymes. However, the source depends on donor availability, and carries a potential risk of alloimmunization and blood borne diseases. In vitro cultured, customizable red blood cells (cRBC) negate these concerns and introduce precision medicine both in transfusion medicine as well as in drug delivery applications.
Aims
Here we investigated the requirements to produce red blood cells from erythroid cultures in stirred bioreactors in order to upscale the process.
Methods
Using inhouse generated serum-free GMP-grade media we optimize a protocol to expand erythroblast and differentiate cultures using 0.5 and 3.0 L stirred bioreactors. Culture parameters, e.g. oxygen availability, shear stress, impellor speed, culture density and perfusion speeds were measured. Sheer stress-induced effects on cells was evaluated by assessing specific signal transduction pathways and globally by RNA-sequencing. Cultured red blood cells were subjected to quality assessment including deformability by Automated Rheoscope and Cell Analyser (ARCA) and oxygen equilibrium curves as measured with a Hemox analyser. Flow cytometry was used to asses enucleation rates and differentiation progression.
Results
In stationary cultures, more than 90% of the cells enucleated and expressed adult haemoglobin as well as the correct blood group antigens. Passaging cRBC through a leukodepletion filter yielded 100% enucleated, stable cRBC. Deformability and oxygen association/dissociation were similar in cRBC and peripheral blood erythrocytes. RNA sequencing and proteomics was performed daily during differentiation and revealed expression dynamics of important erythroid processes, e.g. increased expression of genes involved in blood group expression, globin regulation, erythroid specific metabolic enzymes, and autophage/mitophage degradation of intracellular organels as well as / downregulation of processes involved in cell proliferation. Next, we optimized a protocol to expand erythroblast cultures using 0.5 and 3.0 L stirred bioreactors. Erythroblasts produced in this system showed similar proliferation potential compared to static conditions. Oxygen availability is a critical process parameter and erythroblasts could be cultured at oxygen concentrations as low as 0.7 mg/L, controlled by intermittent sparging. Maximum shear stress at the impeller tip, predicted to be ~1.8 Pa, was mimicked in an orbital shaker setup to evaluate the effect of mixing on the culture. This level of shear stress induced Ca2+-dependent signaling pathways, including the calcineurin/NFAT pathway, and PKC-enhanced STAT5 signaling. As a result, erythroid maturation under differentiation conditions is accelerated by this level of shear stress. Cell erythroblast densities of up to 10 x 106 cells/mL were reached upon continuous media perfusion, 5X higher compared to the current expansion culture systems. Erythroblasts cultured at high density with perfusion maintained high viability and low spontaneous differentiation. This perfusion strategy leads to smaller culture volumes.
Conclusion
Our culture protocols enable us to culture RBC from adult peripheral blood mononuclear cells both in static and bioreactor settings. The use of bioreactor is projected to be significantly more cost-effective compared to static cultures. Currently we prepare for a clinical trial with cRBC.
Keyword(s): ABO blood group, Erythroid differentiation, Signaling, Transfusion