Biological validation of bio-engineered red blood cell productions
Introduction
For many years, blood transfusion has been confronted with constant difficulties in obtaining supplies, linked to the increasing need for blood products and a lack of donors. It has become an urgent necessity to find alternatives to the classical blood donations. Hence the attempts to generate erythroid cells from cells of diverse origins make good sense. If the use of fetal cells or human embryonic or induced pluripotent cells still requires adjustments notably in terms of the level of enucleation [1], [2], [3], [4], [5], [6], the mass production in vitro of functional enucleated cells from hematopoietic stem cells isolated from cord blood or by leukapheresis has marked an important step [7], [8], [9], [10], [11], [12], [13]. In this domain, our team has established the concept of the transfusion of cultured RBC (cRBC) by showing that these cells generated in vitro and presenting the characteristics of reticulocytes survive in the blood circulation after injection into humans just as transfused cells do [14].
However, the in vitro production of functional cRBC is the culmination of a multiparametric technological procedure, which makes it difficult to control the products and is susceptible to generate damaged cells. The quality of cultured cells depends on many parameters and the control of one or more of them can easily escape the manipulator (to name a few factors: the quality of the samples, culture media and materials; control of the input of nutrients and the accumulation of catabolites; control of the gas diffusion and physicochemical parameters; control of the mechanical stress in stirred or cell rocking systems ....). Defective monitoring of the culture conditions can lead to osmotic shock, oxidative, metabolic or shear stresses which may potentially be responsible for cell damages [15]. The transfusion efficacy should depend directly on the quality of the injected cells in so far as damaged cRBC would have a limited survival. Consequently, the qualitative evaluation of cultured RBC should contribute to their validation before their injection into humans. The present study aims at determining a standardized method to globally validate cRBC which, in the future, will be destined for transfusion to patients.
A cascade of events occurs in vivo during the senescence of RBC leading to their rigidity [16], [17] and to their clearance by macrophages [18]. The phagocytosis is preceded by significant membrane changes of the aging RBC such as (i) the relocation of phosphatidylserine (PS) on the outer membrane, acting as an opsonization signal [19], [20], (ii) the decrease of CD47 expression which has been reported to trigger the phagocytosis by macrophages [21], (iii) the emergence of autoantibodies that binds to the senescent band 3 protein and targeted by the Fc receptors of macrophages [22]. The opsonization of antibodies and PS at the surface of the aging RBC highly contributes to the decrease in cell flexibility. More recently, novel mechanisms of erythrocyte recognition have been proposed; thus, RBC may be recognized by macrophages via the externalization of Arg-Gly-Asp (RGD) motifs of ankyrin on the surface of RBC which serve as receptors for cell adhesion [23], while nucleolin (a lactoferrin binding protein) has been identified as a macrophage receptor for oxidized RBC [24]. In addition, other events occur at the intracellular level during apoptosis as the decrease in metabolic activity [25] that promotes the oxidation of cell components [26]. On the other hand, RBC may display in vitro signs of alteration that are alike the in vivo process of aging. Thus, during poorly controlled storage conditions for RBC one can observe membrane vesiculations, the decrease in erythrocyte mechanical properties, the decrease in CD47 level and the increase of PS expression [27], [28], [29], [30]. Ultimately, both in vivo aging or in vitro conditions inducing cell damages may result in erythrophagocytosis [31], [32].
In mammals, phagocytosis is performed not only by professional phagocytes (polynuclear granulocytes, monocytes, dendritic cells and macrophages) but also by non professional phagocytes (fibroblasts and epithelial and endothelial cells) [33]. The latter dispose of neither Fc receptors nor bactericidal mechanisms. Nevertheless, they can incorporate into their cytoplasm diverse particles, beads coated with fibronectin or collagen, microorganisms, apoptotic cells or erythrocytes [34], [35], [36]. Although the efficient immunological mechanisms of recognition are lacking for the non-professional cells, the latter dispose of a panel of receptors that is certainly more restrictive than that of their professional counterparts but enabling the selective adhesion of the target cells before their engulfment. Thus, by using vitronectin receptors or specific mannose/fucose lectins, fibroblasts are fully capable of recognition and of subsequent clearance of the only apoptotic cells [37]. Fibroblasts can discriminate apoptotic cells presenting PS on the outer leaflet from the non-apoptotic cells, via a putative PS-receptor [38]. Moreover, Fens and co-workers have established that endothelial cells have phagocytic properties for lactadherin-opsonized cells in a model of Arg-Gly-Asp (RGD)-modified erythrocytes [39]. In addition, Taylor et al. established that purified lactadherin promoted RGD-dependent cell adhesion of murine fibroblast via αv-integrins [40]. Furthermore, lactadherin has been identified in fibroblast exosomes [41]. Besides, nucleolin has been reported as a receptor for early apoptotic cells [42]. Recently, this lectin-like protein was detected not only in macrophages but also on the surface of various cell types as endothelial cells which bound and endocytozed lactoferrin in target cells [43]. All these data strongly suggest that non-professional cells, including fibroblast cells, can specifically adhere to target cells prior to phagocytosing them.
Starting from these observations, we developed an in vitro erythrophagocytosis test with the murine fibroblast cell line MS-5 and we showed that this cell line was efficient for revealing the presence among cRBC of damaged erythrocytes. The EP assay using MS-5 cells was compared to the approach using human macrophages (reference method). Then, EP results were confronted to other parameters known to precede or to be involved in the cell clearance, such as (i) cell viability, (ii) CD47 expression, (iii) phosphatidylserine (PS) externalization, and (iv) cell deformability.
We show that the EP test developed represents an efficient tool to evaluate the batch to batch variations of cultured cells.
Section snippets
Sample preparation
All samples were collected after obtaining informed consent from the donors of cells.
Calibration of the erythrophagocytosis test
The test was calibrated to determine (i) the optimal duration of erythrophagocytosis, (ii) the optimal erythrocyte/phagocyte ratio and (iii) the sensitivity of the test after the optimization of the conditions. This procedure was necessary because each type of phagocyte has its own characteristics. The EP tests were performed comparatively using the murine fibroblast cell line MS-5 and as the reference technique human macrophages (MΦ).
Duration of incubation and optimal quantity of target cells
Preliminary experiments showed that aged RBC (a-RBC) were
Discussion
Many teams have developed EP tests using cell lines [48], [49], [50], [51]. However, some of these cells require prior induction for several days with vitamin D3 or phorbol esters [48]. Others, like the THP-1 line, must be maintained at low concentrations to avoid modifying their phagocytic properties [52]. The MS-5 cell line does not present these disadvantages as its growth is stopped by contact inhibition and its response is constant irrespective of the number of passages (evaluated up to
Authors' disclosures of potential conflicts of interest
The authors indicated no potential conflicts of interest.
Author contributions
Conception, design, analysis and manuscript writing: Marie-Catherine Giarratana.
Experiments conduction: Tiffany Marie and Dhouha Darghouth.
Head of laboratory and designed and supervised the overall research: Luc Douay.
Final approval of the manuscript: all authors.
Acknowledgments
This work was supported by grants from the Etablissement Français du Sang (EFS) and Association “Combattre la leucémie”.
The authors are indebted to Pierre Buffet (UMR 945, Université Pierre et Marie Curie, Paris, France) for kindly putting his laboratory premises and the LORCA at our disposal. We thank Séverine Jolly (UPMC, Etablissement Français du Sang) for expert technical assistance.
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