Vol 7, No 1 (2012)

Developing direction cellular medicine is the use of the unique properties of progenitor cells with high biological activity and potential for differentiation. Multipotent mesenchymal stromal cells (MMSC) are pluripotent stem cells, has several important properties for clinical use. The use of MMSC cultured «ex vivo» opens the question about the quality and safety culture for clinical use. The purpose of this review was to develop a program of cultivation and study of important properties of samples of human MMSC for clinical application. The review shows the characteristic phases of assessing the quality and safety of MMSC, including the cultivation of cells «ex vivo», immunological assessment, growth, immunomodulatory, and regenerative properties of the progenitor, an assessment of genetic and microbiological safety. Analyzed the assessment the quality and safety of MMSC «in vitro» tests. It is shown that the severity of the properties of each samples different and depends on the source and culture conditions MMSC.

In vitro proliferation and myeloid differentiation of human umbilical cord blood (UCB) CD34+ cells with/without bone marrow multipotential mesenchymal stromal cells (BMMMSCs) feeding layer in serum-free medium supplemented with IL-3, IL-6, SCF and Flt3/l were investigated. Mononuclear cells (MCs) were isolated from the healthy donor UCB specimens (n = 4), CD34-enriched MCs were obtained by positive immunomagnetic selection (CD34+-rich). Both cell fractions (MCs and CD34+-rich) were expanded for two weeks in vitro. Cells were seeded to the new vessels when necessary. The percentage of CD34/CD45-positive cells was accounted by flow cytometry, subpopulations of myeloid progenitors were assessed in colony-forming tests. Upon two weeks of culture the number of CD34+ cells in MC fraction augmented by 70±29 and 980±414-fold with and without BM-MMSC feeding layer respectively and was not significantly differed from that in CD34+-rich fraction. Progenitor cell subpopulations were not altered during the first week of culture but granulocytopoiesis was superior over erythropoiesis during the second week. CD34 and CD45 expression patterns revealed two hematopoietic cell populations, one of them differentiating on the first week of expansion, and the other - on the second one. BM-MMSC feeder promoted significantly higher (p < 0,05) expansion rates of CD34+ cells and myeloid progenitors in comparison with non-feeding culture. BM-MMSC feeder also caused progenitor cells redistribution between suspension and adhesive fractions by predominantly binding erythroid and multipotential progenitors. Optimum expansion protocol for multipotential progenitors was culturing CD34+-rich cell population with BM-MMSC feeding layer for a week which resulted in the increase the amount of the nucleated cells, CD34+ cells, multi-potential progenitors, and colony-forming units by 56, 36, 45, and 60-fold respectively.

In this paper we have show the possibility of cryopreservation of placental tissue for the reason to obtain viable hematopoietic progenitor cells and multipotent mesenchymal stromal cells. It is established, that the relative content of the cells of CD34+/ SSClow/CD45low cells significantly higher in the cryopreserved placental tissue. Also we first described the differences expression of CD90 and CD31on the CD34+/CD45low/SSClow cells of umbilical cord blood and placenta. Clonal analysis of cells revealed the presence in cryopreserved placental tissues of precursors of granulocytes, monocytes and erythrocytes that forming colonies of granulocyte-monocyte, monocytes, granulocytes and erythrocytes. Also it was obtained cultures of stromal cells from cryopreserved placental tissues with immunophenotype CD90+/CD73+/CD105+/HLA-ABClow/CD45-/ CD34-/CD133-/CD14- and adipogenic and osteogenic potential in vitro.

The study addresses consequences of subcutaneous implantation of film matrices prepared from different PHAs to laboratory animals. No negative effects of subcutaneous implantation of PHA matrices on physiological and biochemical characteristics of the animals were determined. Independently of the matrices composition and duration of the contact with the internal environment of the organism we did not observe any deviations in the behavior of animals, their growth and development, as well as blood functions. Response of the tissues to PHA matrices was comparable with the response to polylactide, but substantially less expressed at the earlier time periods after implantation. Tissues response to implantation of PHA of all types is characterized by short-term (up to 2 weeks) post-traumatic inflammation with formation of fibrous capsules by 30th-60th days with the thickness less than 100 microns, which get thinner down to 40-60 microns by 180th day as the result of involution. No differences in response of tissues and the whole organism were observed for the matrices produced from the homopolymer of 3-hydroxybutyric acid (P3HB), copolymers of 3-hydroxybutyric and 4-hydroxybutyric acids (P3HB/4HB), 3-hydroxybutyric acid and 3-hydroxyvalerianic acids (P3HB/3HV), 3-hydroxybutyric and 3-hydroxyhexanoate acids (P3HB/3HH). Macrophages and foreign-body giant cells actively participate in the response of the tissues to PHAs. In the studied conditions matrices from the copolymers containing 3-hydroxyhexanoate and 4 hydroxybutyrate were determined as more actively degraded PHA. The next less degraded matrices were matrices from the copolymer of P3HB/3HV and the most resistant were P3HB matrices. The slower degradation of PHA matrices was accompanied by delayed development of giantcells response. The studied PHA matrices can be placed in the following range by their degradation: P3HB/3HH - P3HB/4HB - P3HB/HV - P3HB.

Rapid development of cell technologies stipulates the need for informative and express methods for the analysis of viability and physiological activity of human cells. We studied analytical possibilities of novel tools for comprehensive characterization of bioelectrochemical properties of living cells, e.g. surface charge of cellular membrane and redox activity of metabolites. Using Malvern Zetasizer dynamic light scattering analyzer we proposed an approach to assessment of zeta potential of human cells and detection of phosphatidylserine on their surface as an early apoptotic marker. On the basis of modified electrodes we designed sensors exhibiting high sensitivity towards electroactive cellular metabolites including antioxidants and macroergic molecules. The sensors were applied for assessment of metabolic activity/energetic status of human cells (blood cells and cell cultures). Electrochemical signal of adenine nucleotides of cells on sensor surface correlated with intracellular level of ATP according to luciferase assay and was found to be more sensitive to alteration in cell viability than conventional MTS test. On the basis of disposable screenprinted electrodes we fabricated a prototype of portable analyzer for rapid analysis of cell health (e.g. for donor cells) in less than 5 ƒl volume of cell sample. Proposed tools and methods are of interest in cell transplantology, basic research and cell-based medical diagnostics.

The clinical and experimental study on the restoration of bone tissue with tissue engineering scaffold based on stem cells of adipose tissue and resorbable osteoplastic matrix. In a pilot study proved the effectiveness of the transplant, founded the dates of dental implantation. Use of tissue engineering scaffold led to organotypic reconstruction of bone tissue in the field of transplantation in patients with severe atrophy of the alveolar process of the maxilla and mandible. As part of a clinical trial were treated 20 patients.