Mathematical approach for the optimal expansion of erythroid progenitors in monolayer culture
Highlights
► We examine culture conditions for inoculation and passaging of erythroid progenitors. ► We use mathematical expressions to define the growth curves of peripheral blood derived erythroid progenitors over the course of their expansion process. ► Significantly different growth curves are obtained at each passage confirming the altered growth kinetics of erythroid progenitors during sequential passaging in expansion process. ► Cost analysis suggest a 60 h passage length at every passage, resulting in a 43% process-cost reduction.
Introduction
Blood transfusion saves lives and improves health but millions of patients worldwide do not have timely access to safe blood or sufficient blood supply. Although cell free blood substitutes like recombinant haemoglobin and oxygen transporters (perfluorocarbons) have been explored but results have been disappointing (Cohn and Cushing, 2009, Greenburg, 2009). An alternative is the ex vivo generation of RBCs from adult haematopoietic stem cells (HSCs). In the past decade researchers have made extensive progress in the ex vivo expansion of erythroid progenitors from CD34+ derived from various sources such as umbilical cord blood (CB), mobilised peripheral blood (mPB), bone marrow (BM), and peripheral blood (PB), for potential clinical applications (Baek et al., 2009, Baek et al., 2008, Boehm et al., 2009, Douay and Andreu, 2007, Fujimi et al., 2008, Giarratana et al., 2005, Ivanovic et al., 2006, Neildez-Nguyen et al., 2002, Timmins and Nielsen, 2009). High expansion factor and high enucleation efficiency have been recently achieved making this approach at least methodologically feasible (Boehm et al., 2009, Kim and Baek, 2011), however the expansion and differentiation of CD34+ cells to produce RBC's industrially, within the norms of good manufacturing practice (GMP), still remains a challenge and far from realisation.
Process control and optimisation of culture conditions during the ex vivo expansion of HSCs is complicated by variability of cell types, transient cell kinetics and intracellular interactions posing significant challenges to process characterisation and scale-up. However, a better understanding can be achieved by modelling adult human stem cell growth kinetics.
Various attempts have been made to optimise the expansion of different cell lines for ex vivo culture conditions (Melero-Martin et al., 2006, Melero-Martin et al., 2007). Here, we provide a rational methodology for assessing the culture conditions and operational modes required to optimise cell expansion in a serial passaging process of erythroids from PB derived CD34+.
The key issues raised in the large-scale growth of any primary cell lines are inoculation concentration and culture conditions based on feeding strategy. These factors influence the growth rate and expansion of erythroid progenitors that can be achieved in a scale-up system. Here, we have identified the optimum cell concentration for inoculation in serial passaging and developed a feeding strategy based on passage length using a mathematical approach.
The majority of researchers have modelled the growth of primary cells using Gompertz, logistics, inverse cube root and power functions (Buchanan et al., 1997, Deakin, 1970, Kasper et al., 2009) to understand the growth kinetics, with Gompertz growth models being the most broadly applied and successful of these models for normal primary cells (Melero-Martin et al., 2006, Melero-Martin et al., 2007) and tumours (Heegaard et al., 2003, Kasper et al., 2009, Lloyd, 1975, Spratt et al., 1993).
By using a Gompertz growth model, we have determined the optimum inoculation concentration and passaging conditions to achieve optimal expansion factor and growth kinetics. Additionally, the costing was considered equally vital in determining the suitability of the evaluated process parameters.
Section snippets
Isolation of PB derived CD34+ cells
Mononuclear cells were isolated from buffy coats through density gradient centrifugation using Histopaque-1077 (Sigma–Aldrich) and CD34+ cells were purified through MiniMACS using CD34 microbead kit (Miltenyi, Germany). Cells were cultivated in IMDM based medium (Biochrom, Germany) enriched with 1% bovine serum albumin, 10 μg/ml insulin, 120 μg/ml iron saturated human transferring, 90 ng/ml ferric nitrate, 900 ng/ml ferrous sulphate, 40 μg/ml inositol, 10 μg/ml folic acid, 1.6 × 10−4 M monothioglycerol
Effect of cell seeding concentration on cell proliferation
Erythroid development in the bone marrow takes place in a very crowded and packed environment with other haematopoietic cells. Hence, the concentration of cells at the inoculation of every passage can critically affect the cell proliferation kinetics and the total cell expansion process ex vivo.
Fig. 2 shows the growth curves at four different time points of cell expansion with six different cell-seeding concentrations after fitting to the four coefficients Gompertz equation, and the values of
Conclusions
This work provides an in-depth study of the culture conditions for inoculation and passaging of erythroid progenitors in the optimal operation mode for the ex vivo expansion of PB-derived erythroid progenitors. Our results and analysis showed that the optimal cell concentration for inoculation in serial passaging involved a compromise between the growth kinetics and the expansion factor. Results from this study can be summarised as follows: operating in no feeding mode, the cell concentration
Acknowledgement
This work was funded by the Irish Blood Transfusion Service (IBTS).
References (23)
- et al.
The potential of human peripheral blood derived CD34+ cells for ex vivo red blood cell production
Journal of Biotechnology
(2009) - et al.
When is simple good enough: a comparison of the Gompertz Baranyi, and three-phase linear models for fitting bacterial growth curves
Food Microbiology
(1997) - et al.
Oxygen therapeutics: perfluorocarbons and blood substitute safety
Critical Care Clinics
(2009) - et al.
Ex vivo production of human red blood cells from hematopoietic stem cells: what is the future in transfusion?
Transfusion Medicine Reviews
(2007) The ideal blood substitute
Critical Care Clinics
(2009)- et al.
Generation of induced pluripotent stem cells from human blood
Blood
(2009) - et al.
In vivo vasculogenic potential of human blood-derived endothelial progenitor cells
Blood
(2007) - et al.
Reprogramming of human peripheral blood cells to induced pluripotent stem cells
Cell Stem Cell
(2010) - et al.
Blood cell manufacture: current methods and future challenges
Trends in Biotechnology
(2009) - et al.
Stroma-free mass production of clinical-grade red blood cells (RBCs) by using poloxamer 188 as an RBC survival enhancer
Transfusion
(2009)
In vitro clinical-grade generation of red blood cells from human umbilical cord blood CD34+ cells
Transfusion
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