Bone marrow derived endothelial progenitor cells retain their phenotype and functions after a limited number of culture passages and cryopreservation

Abstract

A critical limitation for tissue engineering and autologous therapeutic applications of bone marrow derived EPCs is their low frequency, which is even lower in number and activity level in patients with cardiovascular risk factors and other diseases. New strategies for obtaining and reserving sufficient ready-to-use EPCs for clinical use have hit major obstacles, because effects of serial passage and cryopreservation on EPC phenotype and functions are still needed to be explored. The present study aims at investigating effects of a limited number of culture passages as well as cryopreservation on EPC phenotype and functions. We isolated EPCs from rat bone marrow and cultured them up to passage 12 (totaling achievements of 40 population doublings). The phenotype and functions of fresh cultured and postcryopreserved EPCs at passages 7 and 12, respectively, were evaluated. EPCs at passage 12 maintained the morphological characteristics, marker phenotype, Dil-ac-LDL uptake and FITC-UEA-1 binding functions, enhanced EPCs proliferation, tube formation and migration, but decreased CD133 expression compared with EPCs at passage 7. Cryopreservation caused limited impairment in EPC phenotype and functions. In brief, our results demonstrated that a limited number of culture passages and cryopreservation did not change EPC phenotype and functions, and can be used for the development of robust strategies and quality control criterion for obtaining sufficient and high-quality ready-to-use EPCs for tissue engineering and therapeutic applications.

Molecular Profiling and Gene Banking of Rabbit EPCs Derived from Two Biological Sources

“…The morphology of rabbit EPCs and BEPCs cultured for less than 2-3 weeks were stable during the culture with spindle-shaped appearance (Figure 1) that agreed with the previous studies [41,42,44]. The same shape of the early EPCs morphology originated from peripheral blood and/or bone marrow was observed for human [54], mouse [30], rat [31][32][33]35], goat [38] or chicken [39]. However, it has been demonstrated that longer culture (more than 3 weeks) of rabbit EPCs caused a change of morphology to cobblestone-like cells, although not in BEPCs culture [42,43].…”

“…In addition, the doubling time of human umbilical cord-derived EPCs was calculated to be about 40 h [ 55 ]. The population doubling times of rat bone marrow-derived EPCs starting at 104 h (P0) decreased with the subsequent passages to 54 h (P4) and up to 40 h at passages 11 and 12 [ 35 ]. Thus, it seems that the proliferation rate of cultured EPCs may increase in the later passages.…”

“…However, this might be caused by the differentiated status of the cultured cells. Another study, which used the same rat cells, but different culture medium, did not observe a significant effect of the passage number on the cell viability [ 35 ].…”

“…Another study showed a high expression levels of CD31, CD34, CD133 and VEGFR-2 in rat EPCs cultured for 10 days, which obviously increased in comparison to the phenotype of freshly isolated bone marrow cells [ 32 ]. A stable expression of CD31, CD34 and VEGFR-2 was, thereafter, noticed in rat EPCs when passages 7 and 12 were compared, whereas a decrease in CD133 expression was observed in the latter mentioned passages [ 35 ]. AcLDL uptake was noticed in the late EPCs too, which were also positive for CD29, CD31, VE-cadherin, VEGFR-2, vWF and eNOS, while negative for CD45 [ 31 , 33 ].…”

“…Beside the human model, EPCs have been already isolated from the peripheral blood and/or bone marrow of mouse [ 30 ], rat [ 31 , 32 , 33 , 34 , 35 ], dog [ 36 , 37 ], sheep [ 22 ] and goat [ 38 ] or even chicken [ 39 ]. Moreover, the EPCs were isolated also from the peripheral blood and bone marrow of rabbits more than ten years ago [ 40 , 41 , 42 , 43 , 44 ].…”