Elsevier

Journal of Biotechnology

Volume 161, Issue 3, 31 October 2012, Pages 308-319
Journal of Biotechnology

Mathematical approach for the optimal expansion of erythroid progenitors in monolayer culture

https://doi.org/10.1016/j.jbiotec.2012.06.003Get rights and content

Abstract

The continuous production of large numbers of red blood cells (RBCs) ex vivo is a challenging task due to process economics and complex culture conditions. In any serial passaging process, the culture conditions and operation mode are important criteria for achieving high proliferation with optimal passage lengths.

The optimal inoculation concentration for serial passaging is a factor that affects both the kinetics and the total expansion performance. As part of our attempt to develop a scalable, economical and reproducible system for production of RBCs we used mathematical expressions to define the growth curves of peripheral blood derived erythroid progenitors over the course of their expansion process. We used a Gompertz function to evaluate the specific growth rate for the optimisation of inoculation concentration and passage lengths to achieve optimal expansion. This led to values of 3 × 105 cells/ml as the optimum inoculation concentration and 36 h as the optimum passage length. Also the variations in growth curves confirmed the altered growth kinetics of erythroid progenitors during sequential passaging in expansion process.

Cost analysis suggested a 60-h passage length at every passage, resulting in a 42.9% process-cost reduction. However, this has increased the process duration in achieving the similar expansion factor. This methodology for optimising the expansion process of peripheral derived erythroid progenitors based on optimum culture conditions could provide us with a direction and an improved performance for scale-up applications.

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)

  • E.J. Baek et al.

    In vitro clinical-grade generation of red blood cells from human umbilical cord blood CD34+ cells

    Transfusion

    (2008)
  • Cited by (3)

    • Auxenochlorella pyrenoidosa extract supplementation replacing fetal bovine serum for Carassius auratus muscle cell culture under low-serum conditions

      2023, Food Research International
      Citation Excerpt :

      Choi et al. (2020) found that skeletal muscle growth medium-2 containing epidermal growth factor (EGF), dexamethasone, and p38 inhibitor were suitable for maintaining pig muscle stem cell expansion culture within two passages. Lei et al. (2022) reported that the mixture of four cytokines, including long chain human insulin growth factor-1, platelet derived growth factor BB, basic fibroblast growth factor, and epidermal growth factor, could be used to reduce the required concentration of FBS by at least 5% for the maintenance of long-term porcine muscle stem cell proliferation for 28 days. Fang et al. (2022) found that vitamin C was an excellent supplement for the culture of porcine MuSCs ex vivo for 29 days.

    View full text