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Erythroid Culture Based on Differentiation of Progenitor Cells from Leftover Buffy Coats Produced in the Reveos Automated Blood Processing System

Vidovic, Karina LU ; Nylander, Anja LU ; Alattar, Abdul Ghani LU ; Akkök, Çiǧdem Akalin and Olsson, Martin L LU (2017) 27th Regional Congress of the International Society of Blood Transfusion, ISBT 2017 In Vox Sanguinis 112(supp 1). p.288-288
Abstract
Background: Efforts to culture red blood cells (RBCs) for transfusion purposes are ongoing in numerous research laboratories worldwide. In addition, smaller-scale erythroid culture is one of many approaches toward better understanding of erythropoiesis, enucleation and RBC biology. Our center recently changed from traditional blood component separation to the Reveos(R) automated blood- processing system. Most buffy coats (BCs) after whole- blood donations were previously used for platelet production but leftover BCs are now discarded. BCs are still popular among scientists, mainly as a source of peripheral leukocytes but we asked the question if these leftover bags could be useful for erythroid culture. Aims: We aimed to set up and... (More)
Background: Efforts to culture red blood cells (RBCs) for transfusion purposes are ongoing in numerous research laboratories worldwide. In addition, smaller-scale erythroid culture is one of many approaches toward better understanding of erythropoiesis, enucleation and RBC biology. Our center recently changed from traditional blood component separation to the Reveos(R) automated blood- processing system. Most buffy coats (BCs) after whole- blood donations were previously used for platelet production but leftover BCs are now discarded. BCs are still popular among scientists, mainly as a source of peripheral leukocytes but we asked the question if these leftover bags could be useful for erythroid culture. Aims: We aimed to set up and evaluate a protocol for in vitro erythroid culture of CD34+ progenitor cells extracted from Reveos(R) BCs. Methods: Total CD34+ cells in Reveos(R) BCs were measured using flow cytometry and the BD Cell Enumeration Kit. Following pre-enrichment and fractionation on a density gradient, the mononuclear cells were labeled with anti-CD34 and separated by magnetic particles using an EasySep™ magnet. Culture conditions for the extracted CD34+ cells were divided into phases I-III, each lasting for 7 days. Phase I is an expansion phase, phase II an erythroid differentiation phase and phase III a terminal differentiation phase. StemSpan™ SFEM II culture medium containing bovine serum albumin, human insulin and iron-saturated human transferrin was used in all phases. In phase I, SFEM II was supplemented with SCF, IL-3, FLT3L, TPO and dexamethasone, in phase II with SCF, IL-3, EPO and dexamethasone and in phase III with 30% serum, EPO and holo-transferrin. Fetal bovine serum (FBS) or human serum (HS) were used initially in parallel cultures, to compare their influence on proliferation rate and terminal differentiation. In all cultures, the total number of cells and cell viability were determined by cell counting in a Bürker chamber after trypan blue dye staining. The cultures were carefully monitored and diluted to 0.4 × 106/ml when entering phase II. Erythroid differentiation was analyzed on day 19, 21 and 23 by flow cytometry with anti-GPA-APC, anti-Band3-PE and anti- CD49d-PE-Cy7. The frequency of erythroblasts and enucleated cells present in the cultures were counted on May-Grünwald-Giemsa stained cytospin samples. Results: We found the CD34+ cell content by flow cytometry in BCs (n = 11, volume of 9.5-13.5 ml) to be 0.2-1.5 × 106 with a mean of 93% viability. Thereafter progenitor cells were isolated and subjected to a 3-phase erythroid culture procedure to evaluate their potential to expand and differentiate. Cells cultured in 30% HS in phase III were less viable but showed slightly higher co-expression of CD49d and Band 3 compared to cultures grown in 30% FBS (86% vs 75% on day 19, and 95% vs 86% on day 21), as well as a higher proportion Band 3-positive, CD49d-negative cells, indicating an increased proportion of late erythroblasts and reticulocytes. In order to promote end-stage maturation, HS cultures were favored. After 23 days in culture with HS in phase III, 8-21% of the cells were enucleated and the cultures contained ∼60% erythroblasts. Summary/Conclusions: Extraction of CD34+ hematopoietic progenitors at acceptable viability from leftover Reveos(R) BCs is feasible and convenient. Despite their small volume and the processing cycle, these BCs provide suitable starting material for small-scale, experimental erythroid cultures directly from single donors. Conditions in this culture model can be fine-tuned further to focus on different aspects of erythropoiesis. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Vox Sanguinis
volume
112
issue
supp 1
pages
288 - 288
publisher
Federation of European Neuroscience Societies and Blackwell Publishing Ltd
conference name
27th Regional Congress of the International Society of Blood Transfusion, ISBT 2017
ISSN
1423-0410
DOI
10.1111/vox.12530
language
English
LU publication?
yes
id
daae56bb-acb2-426a-9c4a-4a004daef737
date added to LUP
2017-12-21 15:02:46
date last changed
2018-10-03 11:42:22
@misc{daae56bb-acb2-426a-9c4a-4a004daef737,
  abstract     = {Background: Efforts to culture red blood cells (RBCs) for transfusion purposes are ongoing in numerous research laboratories worldwide. In addition, smaller-scale erythroid culture is one of many approaches toward better understanding of erythropoiesis, enucleation and RBC biology. Our center recently changed from traditional blood component separation to the Reveos(R) automated blood- processing system. Most buffy coats (BCs) after whole- blood donations were previously used for platelet production but leftover BCs are now discarded. BCs are still popular among scientists, mainly as a source of peripheral leukocytes but we asked the question if these leftover bags could be useful for erythroid culture. Aims: We aimed to set up and evaluate a protocol for in vitro erythroid culture of CD34+ progenitor cells extracted from Reveos(R) BCs. Methods: Total CD34+ cells in Reveos(R) BCs were measured using flow cytometry and the BD Cell Enumeration Kit. Following pre-enrichment and fractionation on a density gradient, the mononuclear cells were labeled with anti-CD34 and separated by magnetic particles using an EasySep™ magnet. Culture conditions for the extracted CD34+ cells were divided into phases I-III, each lasting for 7 days. Phase I is an expansion phase, phase II an erythroid differentiation phase and phase III a terminal differentiation phase. StemSpan™ SFEM II culture medium containing bovine serum albumin, human insulin and iron-saturated human transferrin was used in all phases. In phase I, SFEM II was supplemented with SCF, IL-3, FLT3L, TPO and dexamethasone, in phase II with SCF, IL-3, EPO and dexamethasone and in phase III with 30% serum, EPO and holo-transferrin. Fetal bovine serum (FBS) or human serum (HS) were used initially in parallel cultures, to compare their influence on proliferation rate and terminal differentiation. In all cultures, the total number of cells and cell viability were determined by cell counting in a Bürker chamber after trypan blue dye staining. The cultures were carefully monitored and diluted to 0.4 × 106/ml when entering phase II. Erythroid differentiation was analyzed on day 19, 21 and 23 by flow cytometry with anti-GPA-APC, anti-Band3-PE and anti- CD49d-PE-Cy7. The frequency of erythroblasts and enucleated cells present in the cultures were counted on May-Grünwald-Giemsa stained cytospin samples. Results: We found the CD34+ cell content by flow cytometry in BCs (n = 11, volume of 9.5-13.5 ml) to be 0.2-1.5 × 106 with a mean of 93% viability. Thereafter progenitor cells were isolated and subjected to a 3-phase erythroid culture procedure to evaluate their potential to expand and differentiate. Cells cultured in 30% HS in phase III were less viable but showed slightly higher co-expression of CD49d and Band 3 compared to cultures grown in 30% FBS (86% vs 75% on day 19, and 95% vs 86% on day 21), as well as a higher proportion Band 3-positive, CD49d-negative cells, indicating an increased proportion of late erythroblasts and reticulocytes. In order to promote end-stage maturation, HS cultures were favored. After 23 days in culture with HS in phase III, 8-21% of the cells were enucleated and the cultures contained ∼60% erythroblasts. Summary/Conclusions: Extraction of CD34+ hematopoietic progenitors at acceptable viability from leftover Reveos(R) BCs is feasible and convenient. Despite their small volume and the processing cycle, these BCs provide suitable starting material for small-scale, experimental erythroid cultures directly from single donors. Conditions in this culture model can be fine-tuned further to focus on different aspects of erythropoiesis.},
  articleno    = {P-731},
  author       = {Vidovic, Karina and Nylander, Anja and Alattar, Abdul Ghani and Akkök, Çiǧdem Akalin and Olsson, Martin L},
  issn         = {1423-0410},
  language     = {eng},
  note         = {Conference Abstract},
  number       = {supp 1},
  pages        = {288--288},
  publisher    = {Federation of European Neuroscience Societies and Blackwell Publishing Ltd},
  series       = {Vox Sanguinis},
  title        = {Erythroid Culture Based on Differentiation of Progenitor Cells from Leftover Buffy Coats Produced in the Reveos Automated Blood Processing System},
  url          = {http://dx.doi.org/10.1111/vox.12530},
  volume       = {112},
  year         = {2017},
}