Optimization of Novel Human Acellular Dermal Dressing Sterilization for Routine Use in Clinical Practice.

Gamma rays and electrons with kinetic energy up to 10 MeV are routinely used to sterilize biomaterials. To date, the effects of irradiation upon human acellular dermal matrices (hADMs) remain to be fully elucidated. The optimal irradiation dosage remains a critical parameter affecting the final product structure and, by extension, its therapeutic potential. ADM slides were prepared by various digestion methods. The influence of various doses of radiation sterilization using a high-energy electron beam on the structure of collagen, the formation of free radicals and immune responses to non-irradiated (native) and irradiated hADM was investigated. The study of the structure changes was carried out using the following methods: immunohistology, immunoblotting, and electron paramagnetic resonance (EPR) spectroscopy. It was shown that radiation sterilization did not change the architecture and three-dimensional structure of hADM; however, it significantly influenced the degradation of collagen fibers and induced the production of free radicals in a dose-dependent manner. More importantly, the observed effects did not disrupt the therapeutic potential of the new transplants. Therefore, radiation sterilization at a dose of 35kGy can ensure high sterility of the dressing while maintaining its therapeutic potential.

Maternal predictors and quality of umbilical cord blood units.

The aim of the study was to determine the relationship between the maternal age at delivery and selected properties of the cord blood stem cells. The study included 50 pregnant women aged between 18 and 38 years in which spontaneous labors or elective cesarean sections were performed. Umbilical cord blood was collected immediately after the women were delivered of newborns. The samples were analyzed in the Polish Stem Cells Bank in Warsaw. The highest mean WBC level (p < 0.05) was observed in the umbilical blood collected from patients aged 35 years and more. Similarly, the highest mean cell viability was observed in the umbilical cord blood collected from patients aged 35 and more. There were no statistically significant correlations between the CD34+ cells count and mean cell viability in the umbilical cord blood and the maternal age. With the significance level at p < 0.001, the females after spontaneous labor revealed a visibly higher WBC level than patients after a cesarean section. The higher mean WBC concentration (24.95 thousand/µl) was observed in the umbilical cord blood of patients aged 35 and more after spontaneous labors. In the same group, the umbilical cord blood was also characterized by the highest mean cell viability (98.72%). The number of nucleated cells in the umbilical cord blood collected in the perinatal period increases together with the maternal age. In the course of physiological spontaneous labors, the collected umbilical cord blood has more nucleated cells as compared with elective caesarian sections.

Comprehensive study of hydrostatic pressure treated human umbilical cord blood cells via response surface method.

Amelioration of the survival parameters of cryopreserved samples after thawing has already been addressed through several techniques including vitrification to avoid the formation of ice cores. However, this approach cannot be followed in the case of samples with higher volumes. Hydrostatic pressure (HP) treatment has been proven to increase some qualifying parameters (e.g., motility, insemination efficiency) of certain biological samples. Accordingly, the preparation of umbilical cord blood (UCB) samples through an active (mechanical) pre-stressing process to increase the survival rate of cryopreserved samples can be regarded as a novel strategy that calls for basic experimental studies. The goal of our study was to assess the effects of HP treatment on the qualifying parameters (DNA fragmentation by agarose gel electrophoresis and capillary electrophoresis, Total Nucleotide Cell (TNC) count, CD34+/CD45+ count, and superoxide dismutase activity (SOD) of human umbilical cord blood (UCB) derived cells). The experimental arrangement was set to provide data for response-surface analysis to take into account the common effects of the individual variables of pressure and time exposure. 3D visualization of experimental data revealed that 50-min long HP treatment at 12.5 MPa can significantly (α = 0.05) enhance white blood cell (WBC) and CD34+/CD45+ cell counts. However no DNA fragmentation was observed even at higher pressures, SOD activity was triggered over 15.0 MPa. As a conclusion, HP treatment may contribute to the optimal cryopreservation of UCB cells by significantly increasing WBC and CD34+/CD45+ cell counts without adverse effects neither on DNA stability nor on triggering SOD activity.

[The influence of pH and pCO2 levels of umbilical cord blood obtained perinatally on selected parameters of stem cells]. / Wplyw pH i pCO2 krwi pepowinowej uzyskiwanej okoloporodowo na wybrane parametry komórek macierzystych.

OBJECTIVE: The aim of the study was to demonstrate a correlation between pH and pCO2 levels in umbilical cord blood and the quality of the harvested material. Additionally the effect of pH and pCO2 on the number of cord blood CD34+ cells and their vitality was analyzed. MATERIAL AND METHODS: The study included 50 pregnant women after vaginal delivery at term or elective cesarean section. Umbilical cord blood was collected immediately after birth. The probes were analyzed at the Polish Stem Cell Bank in Warsaw. RESULTS: The number of CD34+ cells ranged from 0.1-0.2 in white blood cells count over 12 thousand/ml and pH of > 7.3. If pH ranged between 7.35-7.40, the number of CD34+ was 0.3-0.4. The highest number of CD34+ cells was noted for pH of 7.30-7.35 and amounted to 0.4-0.5. Analysis of stem cell vitality showed that the highest level, over 98%, was obtained when pH was < 7.3 and > or = 7.4. The study revealed the viability of stem cells to drop to 97-98% at pH level of 7.3-7.4. Low values of CD34+ (0.01-0.09) were related to pCO2 of > 40.0 mmHg. For pCO2 < 38 mmHg, the value of CD34+ cells was 0.2-0.3%, which is relatively high. However when pCO2 was > or = 38 mmHg, the number of CD34+ ranged between 0.1-0.2. CONCLUSIONS: Viability of the umbilical cord stem cells increases along with the decrease of pH and pCO2 levels. The mode of delivery does not influence the viability of the stem cells.

Microbiological Aspects of Pharmaceutical Manufacturing of Adipose-Derived Stem Cell-Based Medicinal Products.

Subcutaneous adipose tissue is an excellent source of mesenchymal stem cells (ADSCs), which can be used in cell therapies as an active substance in advanced therapy medicinal products (ATMPs). Because of the short shelf-life of ATMPs and the time needed to obtain the results of microbiological analysis, the final product is often administered to the patient before sterility is confirmed. Because the tissue used for cell isolation is not sterilized to maintain cell viability, controlling and ensuring microbiological purity at all stages of production is crucial. This study presents the results of monitoring the contamination incidence during ADSC-based ATMP manufacturing over two years. It was found that more than 40% of lipoaspirates were contaminated with thirteen different microorganisms, which were identified as being physiological flora from human skin. Such contamination was successfully eliminated from the final ATMPs through the implementation of additional microbiological monitoring and decontamination steps at various stages of production. Environmental monitoring revealed incidental bacterial or fungal growth, which did not result in any product contamination and was reduced thanks to an effective quality assurance system. To conclude, the tissue used for ADSC-based ATMP manufacturing should be considered contaminated; therefore, good manufacturing practices specific to this type of product must be elaborated and implemented by the manufacturer and the clinic in order to obtain a sterile product.

Direct use of the comet assay to study cell cycle distribution and its application to study cell cycle-dependent DNA damage formation.

The comet assay or single cell gel electrophoresis has proven to be a versatile and sensitive method of measuring the induction and repair of DNA damage in individual cells. However, one of the drawbacks of the assay is the bias caused by changes in the ability of cells to repair DNA damage in different cell cycle phases. Whereas the bias seems less important when G0 peripheral blood lymphocytes are studied, it might cause problems when proliferating cells are investigated. In this paper, we validate the assumption that the total comet fluorescence intensity corresponds to the position of the cell in the cell cycle and can be used to assign single cells to specific cell cycle phases. To validate the approach, we used a very homogenous blood mononuclear CD34(+) cell population in G0 phase (unstimulated) or stimulated to enter the cell cycle. An analysis of the cell cycle distribution revealed that the 15 comet intensity classes and the 100 comets usually analyzed in a typical comet experiment are sufficient to obtain a reliable cell cycle distribution comparable with the results obtained by the flow cytometry for the same cell population. The effect of the cell cycle position on the results obtained by the comet assay for proliferating and non-proliferating cell populations irradiated with 3 Gy of X-radiation is also discussed.

Chronic Diabetic Wounds and Their Treatment with Skin Substitutes.

With the global prevalence of type 2 diabetes mellitus steeply rising, instances of chronic, hard-healing, or non-healing diabetic wounds and ulcers are predicted to increase. The growing understanding of healing and regenerative mechanisms has elucidated critical regulators of this process, including key cellular and humoral components. Despite this, the management and successful treatment of diabetic wounds represents a significant therapeutic challenge. To this end, the development of novel therapies and biological dressings has gained increased interest. Here we review key differences between normal and chronic non-healing diabetic wounds, and elaborate on recent advances in wound healing treatments with a particular focus on biological dressings and their effect on key wound healing pathways.

Assessment of the Neuroprotective and Stemness Properties of Human Wharton's Jelly-Derived Mesenchymal Stem Cells under Variable (5% vs. 21%) Aerobic Conditions.

To optimise the culture conditions for human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) intended for clinical use, we investigated ten different properties of these cells cultured under 21% (atmospheric) and 5% (physiological normoxia) oxygen concentrations. The obtained results indicate that 5% O2 has beneficial effects on the proliferation rate, clonogenicity, and slowdown of senescence of hWJ-MSCs; however, the oxygen level did not have an influence on the cell morphology, immunophenotype, or neuroprotective effect of the hWJ-MSCs. Nonetheless, the potential to differentiate into adipocytes, osteocytes, and chondrocytes was comparable under both oxygen conditions. However, spontaneous differentiation of hWJ-MSCs into neuronal lineages was observed and enhanced under atmospheric oxygen conditions. The cells relied more on mitochondrial respiration than glycolysis, regardless of the oxygen conditions. Based on these results, we can conclude that hWJ-MSCs could be effectively cultured and prepared under both oxygen conditions for cell-based therapy. However, the 5% oxygen level seemed to create a more balanced and appropriate environment for hWJ-MSCs.

Mesenchymal Stromal Cells from Different Parts of Umbilical Cord: Approach to Comparison & Characteristics.

Mesenchymal stromal/stem cells (MSCs) are a unique population of cells that play an important role in the regeneration potential of the body. MSCs exhibit a characteristic phenotype and are capable of modulating the immune response. MSCs can be isolated from various tissues such as: bone marrow, adipose tissue, placenta, umbilical cord and others. The umbilical cord as a source of MSCs, has strong advantages, such as no-risk procedure of tissue retrieval after birth and easiness of the MSCs isolation. As the umbilical cord (UC) is a complex organ and we decided to evaluate, whether the cells derived from different regions of umbilical cord show similar or distinct properties. In this study we characterized and compared MSCs from three regions of the umbilical cord: Wharton's Jelly (WJ), the perivascular space (PRV) and the umbilical membrane (UCM). The analysis was carried out in terms of morphology, phenotype, immunomodulation potential and secretome. Based on the obtained results, we were able to conclude, that MSCs derived from distinct UC regions differ in their properties. According to our result WJ-MSCs have high and stabile proliferation potential and phenotype, when compare with other MSCs and can be treated as a preferable source of cells for medical application.

Banking of AT-MSC and its Influence on Their Application to Clinical Procedures.

Processing of MSCs to obtain a therapeutic product consists of two main steps: 1) the in vitro expansion of the cells until an appropriate number of them is obtained, and 2) freezing and storage of the expanded cells. The last step is critical and must be optimized so that after thawing the cells retain all their physiological properties including the secretory function. In this paper, we evaluated physiological parameters of AT-MSC's after a full cycle of their processing, particularly freezing and storing at the liquid nitrogen vapor temperature. Based on the recovered proliferative and secretory capacities of the thawed cells, we have designed the optimal technique for processing of MSCs for clinical applications. In our work, we tried to select the best DMSO-based cryoprotectant mixture on the base of post thawing fully retain their properties. We have demonstrated the effectiveness of the use of DMSO in various configurations of the constituent cryoprotective fluids. We have also shown that AT-MSCs that show control levels in most standard tests (viability, shape, culture behaviour, and proliferative properties) after thawing, may show transient variations in some important physiological properties, such as the level of secreted growth factors. Obtained results let us to indicate how to optimize the AT-MSC preparation process for clinical applications. We suggest that before their clinical application the cells should be cultured for at least one passage to recover their physiological stability and thus assure their optimal therapeutic potential.

Abdominoplasty Skin-Based Dressing for Deep Wound Treatment-Evaluation of Different Methods of Preparation on Therapeutic Potential.

The management of hard-to-heal wounds is a significant clinical challenge. Acellular dermal matrices (ADMs) have been successfully introduced to enhance the healing process. Here, we aimed to develop protocol for the preparation of novel ADMs from abdominoplasty skin. We used three different decellularization protocols for skin processing, namely, 1M NaCl and sodium dodecyl sulfate (SDS, in ADM1); 2M NaCl and sodium dodecyl sulfate (SDS, in ADM1); and a combination of recombinant trypsin and Triton X-100 (in hADM 3). We assessed the effectiveness of decellularization and ADM's structure by using histochemical and immunochemical staining. In addition, we evaluated the therapeutic potential of novel ADMs in a murine model of wound healing. Furthermore, targeted transcriptomic profiling of genes associated with wound healing was performed. First, we found that all three proposed methods of decellularization effectively removed cellular components from abdominoplasty skin. We showed, however, significant differences in the presence of class I human leukocyte antigen (HLA class I ABC), Talin 1/2, and chondroitin sulfate proteoglycan (NG2). In addition, we found that protocols, when utilized differentially, influenced the preservation of types I, III, IV, and VII collagens. Finally, we showed that abdominoplasty skin-derived ADMs might serve as an effective and safe option for deep wound treatment. More importantly, our novel dressing (ADM1) improves the kinetics of wound closure and scar maturation in the proliferative and remodeling phases of wound healing. In conclusion, we developed a protocol for abdominoplasty skin decellularization suitable for the preparation of biological dressings. We showed that different decellularization methods affect the purity, structure, and therapeutic properties of ADMs.

Safety of intrathecal injection of Wharton's jelly-derived mesenchymal stem cells in amyotrophic lateral sclerosis therapy.

Animal experiments have confirmed that mesenchymal stem cells can inhibit motor neuron apoptosis and inflammatory factor expression and increase neurotrophic factor expression. Therefore, mesenchymal stem cells have been shown to exhibit prospects in the treatment of amyotrophic lateral sclerosis. However, the safety of their clinical application needs to be validated. To investigate the safety of intrathecal injection of Wharton's jelly-derived mesenchymal stem cells in amyotrophic lateral sclerosis therapy, 43 patients (16 females and 27 males, mean age of 57.3 years) received an average dose of 0.42 × 106 cells/kg through intrathecal administration at the cervical, thoracic or lumbar region depending on the clinical symptoms. There was a 2 month interval between two injections. The adverse events occurring during a 6-month treatment period were evaluated. No adverse events occurred. Headache occurred in one case only after first injection of stem cells. This suggests that intrathecal injection of Wharton's Jelly-derived mesenchymal stem cells is well tolerated in patients with amyotrophic lateral sclerosis. This study was approved by the Bioethical Committee of School of Medicine, University of Warmia and Mazury in Olsztyn, Poland (approval No. 36/2014 and approval No. 8/2016). This study was registered with the ClinicalTrials.gov (identifier: NCT02881476) on August 29, 2016.

Cartilage Repair in the Knee Using Umbilical Cord Wharton's Jelly-Derived Mesenchymal Stem Cells Embedded Onto Collagen Scaffolding and Implanted Under Dry Arthroscopy.

Cell-based cartilage repair procedures are becoming more widely available and have shown promising potential to treat a wide range of cartilage lesion types and sizes, particularly in the knee joint. More recently, techniques have evolved from 2-step techniques that use autologous chondrocyte expansion to 1-step techniques that make use of mesenchymal stem cells (MSCs) embedded onto biocompatible scaffolding. Our 1-step technique has been further developed to provide cell-based cartilage repair using MSCs that have the potential to be used in an off-the-shelf manner, without the need for autologous tissue harvest. Precursor MSCs can be isolated in abundance from the Wharton's jelly of umbilical cord tissue. These cells have been shown to have the desired capacity for proliferation, differentiation, and release of trophic factors that make them an excellent candidate for use in the clinical setting to provide cell-based restoration of hyaline-like cartilage. Although allogeneic in nature, these cells stimulate little or no host immune response and can be stored for long periods while maintaining viability. We present a technique of cartilage repair in the knee using Wharton's jelly-derived MSCs embedded onto scaffolding and implanted in a minimally invasive fashion using dry arthroscopy.

Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy.

Dendritic cells (DC) generated from human umbilical cord blood might replace patients' DC in attempts to elicit tumor-specific immune response in cancer patients. We studied the efficiency of transfection of human cord blood DC with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene, to test if nonviral gene transfer would be a method to load DC with protein antigens for immunotherapy purposes. Cord blood mononuclear cells were cultured in serum-free medium in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) and Flt-3 ligand (FL), to generate DC from their precursors, and thereafter transfected by electroporation. Maturation of DC was induced by stimulation with GM-CSF, SCF, FL and phorbol myristate acetate (PMA). Transfected DC strongly expressed EGFP, but transfection efficiency of DC, defined as HLA-DR(+) cells lacking lineage-specific markers, did not exceed 2.5%. Expression of the reporter gene was also demonstrated in the DC generated from transfected, purified CD34(+) cord blood cells, by stimulation with GM-CSF, SCF, FL, and tumor necrosis factor alpha (TNF-alpha). Transfection of CD34(+) cells was very efficient, but proliferation of the transfected cells was much reduced as compared to the untransfected cells. Therefore, the yield of transgene-expressing DC was relatively low. In conclusion, nonviral transfection of cord blood DC proved feasible, but considering the requirements for immunotherapy in cancer patients, transfection of differentiated DC or generation of DC from transfected hematopoietic stem cells provide only a limited number of DC expressing the transgene.

Interleukin 12-based immunotherapy improves the antitumor effectiveness of a low-dose 5-Aza-2'-deoxycitidine treatment in L1210 leukemia and B16F10 melanoma models in mice.

PURPOSE: Recent findings indicating that many genes related to cancer development are silenced by an aberrant DNA methylation suggest that inhibitors of this process may be effective cancer therapeutics. In this study we investigated the efficacy of low-dose 5-aza-2'-deoxycitydine (DAC), a methylation inhibitor, with interleukin (IL) 12, one of the most potent cytokines with antitumor activity. EXPERIMENTAL DESIGN: Mice inoculated with L1210 leukemia cells or with B16F10 melanoma cells were treated with 7 daily injections of low-dose DAC (0.2 mg/kg) and/or 7 daily doses of IL-12 (100 ng/dose). Scid/scid mice as well as monoclonal antibodies against CD4, CD8, and NK1.1 were used to investigate the mechanisms of the antitumor effects of the combination treatment. The activity of murine lymphocytes was measured with enzyme-linked immunospot and (51)Cr release assays. RESULTS: Treatment with DAC or IL-12 given alone produced moderate antitumor effects. In both tumor models combined treatment resulted in potentiated antitumor effects and produced 70% long-term survivors among mice inoculated with L1210 cells. The antitumor efficacy of combined treatment was abrogated in scid/scid mice, and after depletion of CD4(+) and CD8(+) T cells. Mice inoculated with B16F10 melanoma cells had significantly delayed tumor growth after combined treatment with DAC and IL-12. Strong antitumor effect correlated with a significant activation of lymph node-derived CD8(+) and CD4(+) cells. Transient neutropenia was observed in mice under treatment of DAC alone, but remarkably this effect was not potentiated by IL-12. CONCLUSIONS: This study provides the first evidence that antitumor effects of DAC can be strongly potentiated by IL-12 and could be beneficial in an effective low-dose-based antitumor therapy.

Nonhematopoietic stem cells of fetal origin--how much of today's enthusiasm will pass the time test?

Stem cells originating at fetal age are for many reasons superior as a material for the regenerative medicine purposes, when compared to their adult counterparts. While hematopoietic cells, isolated from fetal liver or cord blood, have been well known for a long time and have passed practical tests as clinical transplantation material, the non-hematopoietic cells are newly recognized, and the knowledge of their phenotype and differentiation potential is rather insufficient. We, and the others, have identified a subpopulation of cord blood cells phenotypically different from hematopoietic cells (CD34-, CD45-, CD29+, CD44+, CD51+, CD105+, SH-2, SH-3), in vitro plastic adherent, and capable of multilineage differentiation. The other candidates for multipotential stem cells are cells extracted from umbilical cord or placental tissue. The preliminary observations suggest, that these cells, phenotypically similar to the nonhematopoietic cord blood cells, are capable of extensive replication in vitro and of multilineage differentiation into a variety of tissues including cardiac muscle, bone and cartilage, adipocytes, and nerve cells. The other possible medical applications include "rejuvenation" of selected tissues and systems in senile patients, and therapeutical cloning - for both purposes, cells at the fetal stage of genetic regulation may be more useful than cells collected from adult donors. There is still, however, a high level of uncertainty concerning future medical applications of fetal stem cells. Their numbers and characteristics may differ from the preliminary observations, and their behavior in vivo may not fulfill the expectations originating from the in vitro studies. Finally, the autologous applications of stem cells collected at the stage of birth may need the involvement of technical and financial resources for the storage of frozen cell samples throughout the period of life of their potential user. Such procedure seems possible from technical point of view, but may be inadequately substantiated by the eventual advantages.

Differentiation potential of the fetal rat liver-derived cells.

Mesenchymal stem cells derived from bone marrow or several fetal tissues can be expanded and differentiated into other cell lines. The fetal liver is the source of early hematopoietic cells and also, as a fetal tissue, may be considered as a source of pluripotent stem cells. The differentiation potential of fetal rat liver cells have been examined. Freshly isolated liver cells from 14-d fetuses were cultured in Dulbecco medium supplemented with 10% FCS. The plastic-adherent cells were then passaged up to 10 times. Freshly isolated cells and cells from every passage were cultured in hematopoiesis-promoting environment that consists of methylcelulose supplemented with FCS, rat IL-3, human IL-6 and Epo. Parallely these cells were incubated in co-culture with rat muscle satellite cells (Dulbecco medium with 10% FCS and 10% HS) to examine their myogenic potential. Culture in methylcelulose resulted in a high number of GM and Mix colonies in case of freshly isolated liver cells and the number of colonies decreased according to the number of passages. In case of cells from 4th passage, there ware no hematopoietic colonies in culture. In contrast--freshly isolated cells were not able to fuse with rat satellite cells and form the myotubes. This ability appeared in plastic-adherent cells just from the second passage and increases to 5th passage. The cells from every next passage up to 10th when co-cultured with satellite cells participated in myotube formation at the same high level. This result may suggest that in the 14-d rat liver there exist at least two subpopulations of cells: the non-adherent hematopoietic cell population, and the population of plastic-adherent cells capable of differentiating into myotubes. Since the attempts to redifferentiate hematopoietic subpopulation into myopoiesis, or myopoietic subpopulation into hematopoiesis failed, it may be concluded that at least under our experimental conditions the fetal liver cells do not reveal the "plasticity" features.

Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood.

Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34(+) hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34(+) hematopoietic cells were separated on immunomagnetic MiniMACS columns. Pure population of CD34(+) cells was incubated in a serum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of various pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 microF, 550 V/cm, and 10 microg of DNA per 500 microl. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capacitance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34(+) hematopoietic cells derived from human umbilical cord blood.

[Analysis of factors influencing the cord blood sample collection for clinical purposes]. / Analiza czynników wplywajacych na objetosc pozyskiwanych porcji krwi pepowinowej dla potrzeb klinicznych.

The survival rate of patients after cord blood transplantation depends on the number of nucleated cell transplants. The number of nucleated cells available for transplantation closely correlates with collected volume of cord blood. The influence of several obstetric factors on the volume of cord blood donation was investigated. Cord blood was obtained from 32 normal full-term deliveries. Length of gestation, age of mother, weight of placenta and length of umbilical cord were analysed for their impact on the volume of cord blood. Mean volume of collected cord blood was 103 ml. We did not establish the correlation between the volume of collection and the weight of placenta (measured after blood collection), the length of gestation and the age of mothers. A close and significant correlation (p < 0.05, r = 0.78) concerned the length of umbilical cord and the volume of cord blood donation. Length of umbilical cord is the parameter which may be useful for selection of the most promising cases in the collection of cord blood.

Intracerebroventricular Transplantation of Cord Blood-Derived Neural Progenitors in a Child With Severe Global Brain Ischemic Injury.

Transplantation of neural stem/precursor cells has recently been proposed as a promising, albeit still controversial, approach to brain repair. Human umbilical cord blood could be a source of such therapeutic cells, proven beneficial in several preclinical models of stroke. Intracerebroventricular infusion of neutrally committed cord blood-derived cells allows their broad distribution in the CNS, whereas additional labeling with iron oxide nanoparticles (SPIO) enables to follow the fate of engrafted cells by MRI. A 16-month-old child at 7 months after the onset of cardiac arrest-induced global hypoxic/ischemic brain injury, resulting in a permanent vegetative state, was subjected to intracerebroventricular transplantation of the autologous neutrally committed cord blood cells. These cells obtained by 10-day culture in vitro in neurogenic conditions were tagged with SPIO nanoparticles and grafted monthly by three serial injections (12 × 10(6) cells/0.5 ml) into lateral ventricle of the brain. Neural conversion of cord blood cells and superparamagnetic labeling efficiency was confirmed by gene expression, immunocytochemistry, and phantom study. MRI examination revealed the discrete hypointense areas appearing immediately after transplantation in the vicinity of lateral ventricles wall with subsequent lowering of the signal during entire period of observation. The child was followed up for 6 months after the last transplantation and his neurological status slightly but significantly improved. No clinically significant adverse events were noted. This report indicates that intracerebroventricular transplantation of autologous, neutrally committed cord blood cells is a feasible, well tolerated, and safe procedure, at least during 6 months of our observation period. Moreover, a cell-related MRI signal persisted at a wall of lateral ventricle for more than 4 months and could be monitored in transplanted brain hemisphere.