Isolation and Large Scale Expansion of Human Endothelial Progenitor Cells from Peripheral Blood

emy of Sciences of Ukraine, Kyiv, Ukraine 2Biotechnology Laboratory ilaya.regeneration, Medical Company ilaya®, Kyiv, Ukraine 3State Institute of Genetic and Regenerative Medicine of the National Academy of Medical Sciences of Ukraine, Kyiv, Ukraine 4Laboratory of Immunology and Cell Biotechnology, Immanuel Kant Baltic Federal University, Kaliningrad, Russia 1Інститут молекулярної біології і генетики НАН України, м. Київ, Україна 2Біотехнологічна лабораторія ilaya.regeneration, медична компанія ilaya®, м. Київ, Україна 3Державний інститут генетичної та регенеративної медицини НАМН України, м. Київ, Україна 4Лабораторія імунології і клітинної біотехнології, Балтійський федеральний університет імені Імануїла Канта, м. Калінінград, Росія

The existence of the circulating endothelial progenitor cells (EPCs) was discovered in 1997 [2]. Despite twenty years of intensive investigation, EPCs definition and identifying markers have remained unclear and somewhat controversial [3]. EPCs are often mixed up with tissue resident endothelial cells and pro-angiogenic hematopoietic cells. However, recent findings clarified these aspects and now EPCs could be commonly defined as CD31 + CD34 +/-CD146 + CD309(VEGFR-2) + CD45 -CD133 -cells from peripheral blood, displaying clonal proliferative potential, replating ability, and in vivo vessel forming activity [3,6].
Расчет колониеобразующих единиц (КОЕ). Для оценки потенциала клоногенности EPC 20 мл гепа-approach can explain somewhat non-promising results of some of these studies [7]. Therefore the isolation and in vitro large scale expansion of EPCs in order to obtain a high-quality therapeutic dose have remained an actual goal for innovative cell therapy in ischemic disorders, vascular repair and 3D tissue living equivalent trophic support.
Thus the aim of this study was the development of the low invasive EPC isolation method together with their large-scale expansion in vitro in order to obtain a therapeutic dose.
All the experiments were done in accordance with the bioethics and biological safety norms confirmed by the permission of Medical Company ilaya ® Bioethics Committee. All the donors have signed an informed consent before blood donation.
Colony forming units (CFUs) calculating. To assess the EPC clonogenic potential, 20 ml heparinized whole blood vs. MNC fraction isolated from 20 ml blood over Histopaque ® -1077 density gradient each sample was seeded each per one collagen-coated 175 cm 2 culture flask (SPL, Korea) in growth medium Lonza Clonetics EGM™-2MV BulletKit™ and cultured for 14 days. The cells were both fixed and stained for CFU calculating.
EPCs isolation and in vitro expansion. One of the main difficulties in EPC culture is their isolation as their content among the peripheral blood mononuclear cells is about 1.7 × 10 -8 [10]. We aimed to use the adult venous blood as the source of EPCs as this allowed the less invasive uptake method. Initially we tried seeding the whole venous blood on the collagen-coated cell culture flasks (5 ml of blood per 75 cm 2 flask), but it appeared less effective compared to prior mononuclear cells concentration using Histopaque ® -1077 density gradient, which allowed almost complete erythrocytes elimination. Finally we seeded mononuclear cells from 10 ml of blood per 75 cm 2 collagen-coated flask in M1 or M2 EPC media. The EPC colonies appeared after 5-10 days in culture (Fig. 1A). Interestingly, there was no correlation between the initial number of mononuclear cells seeded and the resulting number of colonies (data were not shown). Obtained cultures successfully expanded until P4 without significant changes in morphology (Fig. 1B). Unfortunately, most cultures showed growth arrest at P5-P6. Nevertheless, this time of in vitro expansion was sufficient to obtain a therapeutic dose of 20 -80 × 10 6 cells. The average PDT value for EPCs cultured in M1 was (35.0 ± ± 2.0) h vs (44.5 ± 2.8) h in M2 (p < 0.05). The number of EPC colonies was 7.1 ± 1.2 (whole blood) vs. (13.3 ± ± 1.7) (MNC fraction: (52.3 ± 4.9) mln MNCs per 20 ml whole blood per donor) per 175 flask (p < 0.02). During all the time of in vitro culture the cells were stable and maintained normal karyotype (Fig. 1C).
EPC tube formation. In order to assess functional properties of EPCs after in vitro expansion, we performed a tube-forming assay. Consistently with the previous reports [6], all of the obtained EPC cultures readily formed capillary-like structures in the Matrigel™ Matrix (Fig. 1D).
Altogether our data demonstrated that EPCs could be successfully isolated from venous blood and expanded in vitro to therapeutic significant doses without losing their properties. These findings open new possibilities for the EPC application in regenerative medicine both for angiogenesis boosting in complex ischemic disorders and 3D tissue equivalents trophic support.