Developing direction cellular medicine is the use of theunique properties of progenitor cells with high biologicalactivity and potential for differentiation. Multipotentmesenchymal stromal cells (MMSC) are pluripotent stemcells, has several important properties for clinical use. Theuse of MMSC cultured «ex vivo» opens the question aboutthe quality and safety culture for clinical use. The purposeof this review was to develop a program of cultivation andstudy of important properties of samples of human MMSCfor clinical application. The review shows the characteristicphases of assessing the quality and safety of MMSC, includingthe cultivation of cells «ex vivo», immunological assessment,growth, immunomodulatory, and regenerative properties ofthe progenitor, an assessment of genetic and microbiologicalsafety. Analyzed the assessment the quality and safety ofMMSC «in vitro» tests. It is shown that the severity of theproperties of each samples different and depends on thesource and culture conditions MMSC.
Implantation of artifitial heart valve prostheses is assotiatedwith several serious complications. Therefore, there is a needin new approaches of preparing heart valve prostheses.One of such approaches is heart valve tissue engineering.Tissue engineered heart valves are to be biocompatible,durable, long-life, no necessity in anti-trombotic therapy, andthey have potential to grow and regenerate with host. Themost developed method of heart valve tissue engineering isdecellularization, i.e. preparing an extracellular matrix, whichhas potential in recellularize with stem host-cells and thenimplant it. This review focuses on methods of decellularizationof heart valves and their ability to change structural andbiomechanical properties of allo- and xenografts.
In vitro proliferation and myeloid differentiation of humanumbilical cord blood (UCB) CD34+ cells with/without bonemarrow multipotential mesenchymal stromal cells (BMMMSCs)feeding layer in serum-free medium supplementedwith IL-3, IL-6, SCF and Flt3/l were investigated.Mononuclear cells (MCs) were isolated from the healthydonor UCB specimens (n = 4), CD34-enriched MCs wereobtained by positive immunomagnetic selection (CD34+-rich).Both cell fractions (MCs and CD34+-rich) were expanded fortwo weeks in vitro. Cells were seeded to the new vesselswhen necessary. The percentage of CD34/CD45-positive cellswas accounted by flow cytometry, subpopulations of myeloidprogenitors were assessed in colony-forming tests.Upon two weeks of culture the number of CD34+ cells inMC fraction augmented by 70±29 and 980±414-fold withand without BM-MMSC feeding layer respectively and wasnot significantly differed from that in CD34+-rich fraction.Progenitor cell subpopulations were not altered during thefirst week of culture but granulocytopoiesis was superiorover erythropoiesis during the second week. CD34 andCD45 expression patterns revealed two hematopoietic cellpopulations, one of them differentiating on the first week ofexpansion, and the other - on the second one. BM-MMSCfeeder promoted significantly higher (p < 0,05) expansionrates of CD34+ cells and myeloid progenitors in comparisonwith non-feeding culture. BM-MMSC feeder also causedprogenitor cells redistribution between suspension andadhesive fractions by predominantly binding erythroid andmultipotential progenitors. Optimum expansion protocol formultipotential progenitors was culturing CD34+-rich cellpopulation with BM-MMSC feeding layer for a week whichresulted in the increase the amount of the nucleated cells,CD34+ cells, multi-potential progenitors, and colony-formingunits by 56, 36, 45, and 60-fold respectively.
The content of the main pro- and anti- inflammatory cytokinesand growth factors in autologous cell products, designedfor personalized cell-based therapy in corneal endotheliumlesions was studied. It was established that cell productcontains substances that mediate the effectiveness of celltherapy. The influence of processing conditions on the concentrationof cytokines and growth factors in cell products isclarified. It is established that cytokine profile of the cell productis composed of pre-existing substances in freshly bloodserum and plasma (TGF-β1, TGF-β2, b-FGF, PDGF-AB, VEGF,IL-4, IF-γ) and those concentration of which increased as aresult of processing the cell product - incubation for 4 hoursat 37°C (IL-8) and stimulation with polyA:U (IL-1, 6, IF-α andTNF-α). The evaluation of cytokine profile of cell preparationreveals implementation mechanisms of therapeutic effect ofpersonalized cell-based therapy.
In this paper we have show the possibility of cryopreservationof placental tissue for the reason to obtain viable hematopoieticprogenitor 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 cryopreservedplacental tissue. Also we first described the differencesexpression of CD90 and CD31on the CD34+/CD45low/SSClowcells of umbilical cord blood and placenta. Clonal analysis ofcells revealed the presence in cryopreserved placental tissuesof precursors of granulocytes, monocytes and erythrocytesthat forming colonies of granulocyte-monocyte, monocytes,granulocytes and erythrocytes. Also it was obtained culturesof stromal cells from cryopreserved placental tissues withimmunophenotype CD90+/CD73+/CD105+/HLA-ABClow/CD45-/CD34-/CD133-/CD14- and adipogenic and osteogenic potentialin vitro.
In parallel to the hypertrophy of major cardiac myocytepopulation, we detected immunohistochemically the formationof colonies consisting of small (dm = 6,20,5 m) residentc-kit+ and Sca+ stem cells (SC) and Isl1+-positive cardiacmyocyte progenitors (CMP) in the primary culture of neonatalrat cardiac myocytes. First contracting colonies (~1-2clones per 100000 cells) were registered starting from 8thday of culture. The cells of the colonies were capable of spontaneousdifferentiation, demonstrating the maturation of contractilemachinery and Ca2+ responses caffeine (5 мМ) andK+ (120 мМ). The full-scale development of electromechanicalcoupling with typical for cardiac muscle Ca2+-induced Ca2+release was obvious at 3 weeks of culture. At first, the local,weak, spontaneous, asynchronous, and arrhythmic contractionsat a rate of 2-3 beats/min were registered. However,with time the contractions became synchronous and involvedall cells of the colony with the rate of contractions being58-60 beats/min at the end of the month. First contractingclones comprised Isl1+ CMP, while c-kit+-colonies started tocontract 9-10 days later possibly owing to a more prolongedperiod of proliferation.Thus, we first demonstrated and characterized thecontracting colonies originating from SC and CMP whenthose were co-cultivated with mature cardiac myocytes.The process described in this study is akin to regenerativecardiomyogenesis encompassing the pathway from residentprogenitor cell to the colony of mature contracting cardiacmyocytes. It follows, therefore, that contracting myocytecolony is a suitable model for basic research, testing of drugs,and the investigation of regenerative capacity of SC and CMPaimed at future applications of resident progenitor cells incell-based treatment of cardiac injury.
The study addresses consequences of subcutaneousimplantation of film matrices prepared from different PHAsto laboratory animals. No negative effects of subcutaneousimplantation of PHA matrices on physiological and biochemicalcharacteristics of the animals were determined. Independentlyof the matrices composition and duration of the contact withthe internal environment of the organism we did not observeany deviations in the behavior of animals, their growth anddevelopment, as well as blood functions. Response of thetissues to PHA matrices was comparable with the response topolylactide, but substantially less expressed at the earlier timeperiods after implantation. Tissues response to implantation ofPHA of all types is characterized by short-term (up to 2 weeks)post-traumatic inflammation with formation of fibrous capsulesby 30th-60th days with the thickness less than 100 microns,which get thinner down to 40-60 microns by 180th day as theresult of involution. No differences in response of tissues andthe whole organism were observed for the matrices producedfrom the homopolymer of 3-hydroxybutyric acid (P3HB),copolymers of 3-hydroxybutyric and 4-hydroxybutyric acids(P3HB/4HB), 3-hydroxybutyric acid and 3-hydroxyvalerianicacids (P3HB/3HV), 3-hydroxybutyric and 3-hydroxyhexanoateacids (P3HB/3HH). Macrophages and foreign-body giant cellsactively participate in the response of the tissues to PHAs. Inthe studied conditions matrices from the copolymers containing3-hydroxyhexanoate and 4 hydroxybutyrate were determined asmore actively degraded PHA. The next less degraded matriceswere matrices from the copolymer of P3HB/3HV and the mostresistant were P3HB matrices. The slower degradation of PHAmatrices was accompanied by delayed development of giantcellsresponse. The studied PHA matrices can be placed in thefollowing range by their degradation: P3HB/3HH - P3HB/4HB -P3HB/HV - P3HB.
The article presents a comparative experimental data ofmorphological studies with biological plastic materials, activelyused in various fields of reconstructive surgery in Russia. Areaof implantation was the lower jaw of rabbits, made by originalequipment of the model and experiment. Observation periodswere 10, 20, 30, 60 and 90 days. Based on morphologicalanalysis of the regenerative abilities assessed followed usedmaterials «Osteomatrix», «CollapAn», «Osteoplast-T» and«Perfoost».
Rapid development of cell technologies stipulates the needfor informative and express methods for the analysis of viabilityand physiological activity of human cells. We studied analyticalpossibilities of novel tools for comprehensive characterizationof bioelectrochemical properties of living cells, e.g. surfacecharge of cellular membrane and redox activity of metabolites.Using Malvern Zetasizer dynamic light scattering analyzer weproposed an approach to assessment of zeta potential of humancells and detection of phosphatidylserine on their surface asan early apoptotic marker. On the basis of modified electrodeswe designed sensors exhibiting high sensitivity towardselectroactive cellular metabolites including antioxidantsand macroergic molecules. The sensors were applied forassessment of metabolic activity/energetic status of humancells (blood cells and cell cultures). Electrochemical signal ofadenine nucleotides of cells on sensor surface correlated withintracellular level of ATP according to luciferase assay and wasfound to be more sensitive to alteration in cell viability thanconventional MTS test. On the basis of disposable screenprintedelectrodes we fabricated a prototype of portableanalyzer for rapid analysis of cell health (e.g. for donor cells)in less than 5 l volume of cell sample. Proposed tools andmethods are of interest in cell transplantology, basic researchand cell-based medical diagnostics.
In this paper the bio-photonic mechanism of the activationof cellular programmes is analyzed. Our basic hypothesis isthat cells and individual molecules radiate certain waves thatare close to monochromatic (i.e., have a narrow range ofradiation frequencies). The cell receptors, for their part, takein certain monochromatic signals. In order to estimate thevalue of the intensity of radiation it is necessary to refer tothe fundamental laws of electro-magnetic waves propagationand photon emission.The research was conducted using a model of thehaemopoietic precursors proliferation induction and theapoptosis of the mononuclear cells of peripheral blood ex vivo.The granulocytic colony-stimulating factor (G-CSF) in differentconcentrations, including ultra-low (homeopathic) ones, wasused as the inductor of the proliferation and the inhibitor ofapoptosis. Some homeopathic preparations in high dilutionswere also included in the study. The study showed the highefficacy of low and ultra-low (homeopathic) concentrations ofthe preparations. This phenomenon may be explained with thehelp of the destructive and constructive interference theory.A calculation of the interaction of the monochromic wavesradiated by active molecules was done based on their sizeand molecular mass as well as the size of the target cells.The authors developed an original method of calculating theeffective concentration of the preparation.
The clinical and experimental study on the restoration ofbone tissue with tissue engineering scaffold based on stemcells of adipose tissue and resorbable osteoplastic matrix. In apilot study proved the effectiveness of the transplant, foundedthe dates of dental implantation. Use of tissue engineeringscaffold led to organotypic reconstruction of bone tissue inthe field of transplantation in patients with severe atrophy ofthe alveolar process of the maxilla and mandible. As part of aclinical trial were treated 20 patients.
4 experimental alternatives has been developed fororgan transplantation: 1. Cell therapy as a suspension ofdisorganized somatic and stem cells. 2. The Grafts of welldifferentiated cells on 3D-matrix to compensate (restore)the damaged function. 3. Translational medicine - singleselected pilot project of personalized medicine of auto- stemcell sphere transplantation with a new emergent potential fortissue morphogenesis and regeneration. 4. High throughputcellular serial fabrics for multiple generation of standardmicro-tissues and 3D-mini-tissues. They are especiallydevoted to this essay.