Abstract
Background -Rabbits provide an excellent model for many animal and human diseases, such as cardiovascular diseases, for the development of new vaccines in wound healing management and in the field of tissue engineering of tendon, cartilage, bone and skin.The study presented herein aims to investigate the biological properties of bone marrow rabbit MSCs cultured in different conditions, in order to provide a basis for their clinical applications in veterinary medicine.Findings -MSCs were isolated from 5 New Zealand rabbits. Fold increase, CFU number, doubling time, differentiation ability and immunophenotype were analyzed.With the plating density of 10 cells/cm2 the fold increase was significantly lower with DMEM-20%FCS and MSCs growth was significantly higher with αMEM-hEGF. The highest clonogenic ability was found at 100 cell/cm2 with MSCBM and at 10 cell/cm2 with M199. Both at 10 and 100 cells/cm2, in αMEM medium, the highest CFU increase was obtained by adding bFGF. Supplementing culture media with 10%FCS-10%HS determined a significant increase of CFU.Conclusion -Our data suggest that different progenitor cells with differential sensitivity to media, sera and growth factors exist and the choice of culture conditions has to be carefully considered for MSC management.
Highlights
Bone marrow contains at least two major stem cell lineages, hematopoietic [1,2] and mesenchymal cells [3,4,5]
Determination of influence of culture passage and plating density on cell proliferation and clonogenic potential (CFU-F assay) (Fig. 2) Doubling time at P3 was always significantly lower than at P1 and P5 (p < 0.001), and within each passage it was significantly affected by cell density
We found that at both 10 and 100 cells/cm2 plating density was significantly lower with DMEM than α-MEM
Summary
Bone marrow contains at least two major stem cell lineages, hematopoietic [1,2] and mesenchymal (stromal) cells [3,4,5]. A standard in vitro assay for bone marrow stromal cell activity is the Fibroblastic Colony-Forming Unit (CFU-F) assay in which adherent fibroblastic cells are cultured by plating bone marrow cells either directly or following gradient separation [6]. The phenotype of these cells appears to vary depending on the culture conditions and the specific cell preparation [7,8]. The aim of the present study was to compare the basic biological properties of the cells obtained with different culture media, supplements, and protocols of rabbit Mesenchymal Stem Cell (rMSC) culture, in order to establish the optimal culturing conditions providing a basis for further studies on the biological heterogeneity of rMSC A broad range of colony sizes has been obtained, with varying growth rates and different cell morphologies [9], probably reflecting the presence of a mixed population of multi-, bi-, and unipotential progenitors [10,11], whose features and biological properties have been only partially investigated.
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