Megakaryocytic Potential of Unseparated Thawed Cord Blood Cells.
Authors: Möttönen Sari , Eskola Mikko , Juutistenaho Sari , Kekomäki Riitta 
Published: April 2020

Abstract
BACKGROUND: Growth of megakaryocytic lineage in vitro from isolated CD34+ cells from fresh cord blood (CB) has been in the focus of recent research, whereas megakaryocytic potential of thawed unseparated CB bank products has remained less well characterized. The aim of this study was to evaluate further the megakaryocytic potential of unseparated thawed CB cells in liquid and collagen matrices using thrombopoietin (TPO), interleukin 3 (IL-3) and IL-6.
MATERIALS & METHODS: Viability (trypan blue), nucleated cell (NC) and CD34+ cell concentration and colony-forming cells (CFC) were assessed pre-freezing and post-thawing from CB units processed and thawed by current CB bank procedures. NCs from unseparated thawed CB units (red blood cell and plasma reduced before freezing; n=5; range 0.06–0.5x106/ml; CD34+ cell range 0.2–3.5x103/ml) and fresh CB (Ficoll separated mononuclear cells, n=3; 0.1x106/ml; CD34+ cell range 0.3–0.8x103/ml) were cultured in protein free medium (PF01, MacoPharma) supplemented with albumin, insulin and transferrin at 37°C for 9–15 days and in semisolid medium (MegaCult-C, StemCell Technologies Inc.) for 14 days. Megakaryocytic colony formation was evaluated as total score (0–3, 0=no growth, 3=maximum growth; Eskola et al. Transfusion2008;48:884-892). Flow cytometry, cytocentrifugation and immunohistochemical staining were used to detect hematopoietic and megakaryocytic cells.
RESULTS: Viability of NCs varied from 83 to 99% and the recovery of CD34+ cells from 76 to 147% post-thawing. Clonogenic progenitors were preserved after thawing. Total fold increase of cells was up to 27 and 10 in cultures of thawed and fresh cells, respectively. Megakaryocytic cells were observed clearly on cytospin slides at days 12–14. Notably, CD41+ cell aggregates of similar size as megakaryocytic cells were observed already at day 5. In flow cytometry, the amount of large cells with moderate and high CD41 expression increased up to 7-fold from day 5 on in liquid cultures of both thawed and fresh cells. Proportions of CD34+ cells among large CD41+ cells were 76% (mean; range 61–87%, n=3) and 62% (mean; range 35–75%, n=3) for cultures of thawed and fresh CB at days 12–15. Throughout the culture particles of the same size as adult platelets were present. Megakaryocytic growth potential of different thawed CB units was variable in liquid culture, whereas good growth from each CB unit was detectable in semisolid collagen matrix. The scores varied from 2.0–2.5 for thawed and 1.8–3.0 for fresh cells. Both thawed and fresh CB units with the largest numbers of NCs, CD34+ cells and total CFC proliferated well and produced the largest amounts of CD41+ cells. The liquid as well as semisolid culture conditions also allowed proliferation of other hematopoietic cell lines.
CONCLUSIONS: Unseparated cells from thawed CB units can produce megakaryocytic cells in vitro. Megakaryocytic differentiation potential of thawed cells in liquid culture varies between the CB units. At its best it was comparable to megakaryocytic growth potential in collagen matrix when identical exogenous cytokines were applied. Current CB bank procedures seem not to cause freezing lesions detectable on the phenotype of megakaryocytic progenitors in short term cultures. Distinguishing cell aggregates from large megakaryocytes requires careful morphological analysis. Best growth of megakaryocytic cells was detectable in cultures of CB units with largest numbers of NCs and CD34+ cells.