Stroma‐free mass production of clinical‐grade red blood cells (RBCs) by using poloxamer 188 as an RBC survival enhancer

Baek, Eun Jung; Kim, Han‐Soo; Kim, Ju‐Hye; Kim, Nahn Ju; Kim, Hyun Ok

doi:10.1111/j.1537-2995.2009.02303.x

Cited by 35 publications

(33 citation statements)

References 26 publications

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“…13 The difference in methods was the use of fetal bovine serum instead of cord-blood-derived plasma. Then, the same amount of erythroid cells on day 21 were distributed onto the prepared culture plates at a density of 1Â10 5 /cm 2 to create a monolayer ( Fig.…”

Section: Erythroid Cell Culturementioning

confidence: 99%

“…However, the enucleation was not enhanced that much as compared to the coculture method. As the nucleus extruding motion seems to require a supporting ground for the cytoplasm to push the nucleus out, 13 the role of stromal cells may be to provide an attachment site for the enucleation of the orthochromic erythroblasts to complete the final step in producing RBCs. Therefore, we hypothesized that the optimization of the surface charges of the culture plate would provide an attachment site for erythroblasts due to the highly negative charges of erythroid cell surfaces, and would affect enucleation efficacy via electric interaction between cells and plate surfaces.…”

Section: Introductionmentioning

confidence: 98%

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Enhanced Production of Red Blood Cells in Suspension by Electrostatic Interactions with Culture Plates

Baek

You

Kim

et al. 2010

Tissue Engineering Part C: Methods

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Enucleation of erythroblasts, a critical step in the generation of red blood cells (RBCs), occurs at a low rate without cocultured stromal cells. Previously, the surface properties of the cell culture plate were not considered in the enucleation process, because the cells exist in suspension. Here, we show that a significantly higher rate of enucleation of erythroblasts occurred on the positively charged plates than on the negatively charged surfaces or the both negatively and positively charged plates. Also, the negatively and positively charged plate group showed a significantly higher enucleation than did the hydrophobic plates. Therefore, the plates fully coated with amine groups generated 1.88 times more enucleated RBCs than did the hydrophobic plates. This study suggests an important insight into the effect of surface characteristics of cell culture plates on suspension cell culture. Further, this simple and inexpensive procedure could contribute to a more efficient RBC production system, eliminating the need for robust and expensive coculture procedures.

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Section: Erythroid Cell Culturementioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Enhanced Production of Red Blood Cells in Suspension by Electrostatic Interactions with Culture Plates

Baek

You

Kim

et al. 2010

Tissue Engineering Part C: Methods

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“…5 To generate 3D cell aggregates at days 13-17, cells were seeded at a minimum density of 1 · 10 7 cells/mL and left to settle. The culture conditions were sustained for 1-3 days and supplemented with 2 IU/mL Erythropoietin (EPO; Calbiochem, La Jolla, CA) and 5% CB plasma-derived serum 6 at 37°C and in a 5% CO 2 humidified atmosphere. Half the medium was replaced every 24 h. Cells were cytocentrifuged onto slides and stained with Wright-Giemsa stain to observe their maturation status and integrity.…”

Section: Erythroblast Maturation From Cord Blood Cd34mentioning

confidence: 99%

Red Blood Cell Generation by Three-Dimensional Aggregate Cultivation of Late Erythroblasts

Lee

Han

Choi

et al. 2015

Tissue Engineering Part A

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Stem cell-derived erythroid cells hold great potential for the treatment of blood-loss anemia and for erythropoiesis research; however, cultures using conventional flat plates or bioreactors have failed to show promising results. By mimicking the in vivo bone marrow (BM) environment in which most erythroid cells are physically aggregated, we show that a three-dimensional (3D) aggregate culture system facilitates erythroid cell maturation and red blood cell (RBC) production more effectively than two-dimensional high-density cell cultivation. Late erythroblasts (polychromatic or orthochromatic erythroblasts) were differentiated from cord blood CD34 + cells over 15 days and then allowed to form tight aggregates at a minimum density of 1 · 10 7 cells/mL for 2-3 days. To scale up the cell culture and to make the media supply efficient throughout the cell aggregates, several macroporous microcarriers and porous scaffolds were applied to the 3D culture system. In comparison to control culture conditions, erythroid cells in 3D aggregates were significantly more differentiated toward RBCs with significantly reduced nuclear dysplasia. When 3D culture was performed inside macroporous microcarriers, the cell culture scale was increased and cells exhibited enhanced differentiation and enucleation. Microcarriers with a pore diameter of approximately 400 mm produced more mature cells than those with a smaller pore diameter. In addition, this aggregate culture method minimized the culture space and media volume required. In conclusion, a 3D aggregate culture system can be used to generate transfusable human erythrocytes at the terminal maturation stage, mimicking the in vivo BM microenvironment. Porous structures can efficiently maximize the culture scale, enabling large-scale production of RBCs. These results enhance our understanding of the importance of physical contact among late erythroblasts for their final maturation into RBCs.

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“…16 The medium for erythroid differentiation was added to a series of cytokines in serum-free medium, based on Iscove's modified Dulbecco's medium with several reagents. The basic culture method consisted of four phases.…”

Section: Erythroid Differentiation Of Cd34 + Cellsmentioning

confidence: 99%

“…CD34 + cells were isolated using immuno-magnetic microbead selection (MACS CD34 isolation kit; Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. 16 To prepare the PLL substrates, PLL solution (Sigma, St. Louis, MO) with 0.001%, 0.01%, and 0.1% concentrations was coated onto a 24-well plate for 2 h at room temperature, and then washed twice with distilled water. Selected CD34 + cells were seeded at 1 · 10 4 per cm 2 on a 24-well plate (BD Bioscience, Heidelberg, Germany) that was coated with or without PLL, respectively.…”

Section: Isolation Of Umbilical Cb Cd34 + Cellsmentioning

confidence: 99%

Poly-L-Lysine Increases theEx VivoExpansion and Erythroid Differentiation of Human Hematopoietic Stem Cells, as Well as Erythroid Enucleation Efficacy

Park

Ahn²,

Kim³

et al. 2014

Tissue Engineering Part A

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Hematopoietic stem cells (HSCs) are continuously stimulated by physical interactions with bone marrow or umbilical cord niches as well as by chemical factors found within these niches. The niche can be mimicked by modification of the cytokine composition, elasticity, topography, and/or charge. This work employed cell culture plates coated with several concentrations of poly-L-lysine (PLL), a positively charged synthetic amino-acid chain. Culture substrates that employed relatively high initial coating concentrations of PLL significantly increased the total number of HSCs during ex vivo expansion of CD34(+) cells, as well as erythroid differentiation. Furthermore, the 0.01% PLL substrate stimulated enucleation of erythroid cells, leaving behind a number of extruded nuclei at the bottom of the culture plate, followed by an increase in the number of erythrocytes. Thus, PLL will likely prove useful to enhance the expansion of HSCs and erythroid cells, in addition to the generation of red blood cells.

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Stroma‐free mass production of clinical‐grade red blood cells (RBCs) by using poloxamer 188 as an RBC survival enhancer

Abstract: This RBC production protocol is a simple stroma- and serum-free culture method ensuring enhanced viability of terminally mature erythroid cells and can be easily applicable to mass production of clinical-grade RBCs as well as erythropoiesis research.

Cited by 35 publications

References 26 publications

Enhanced Production of Red Blood Cells in Suspension by Electrostatic Interactions with Culture Plates

Enhanced Production of Red Blood Cells in Suspension by Electrostatic Interactions with Culture Plates

Red Blood Cell Generation by Three-Dimensional Aggregate Cultivation of Late Erythroblasts

Poly-L-Lysine Increases theEx VivoExpansion and Erythroid Differentiation of Human Hematopoietic Stem Cells, as Well as Erythroid Enucleation Efficacy

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