Adherent Cell Cultures Research Articles

Integrated Colony Imaging, Analysis, and Selection Device for Regenerative Medicine

Stem and progenitor cells derived from human tissues are being developed as cell sources for cell-based assays and therapies. However, tissue-derived stem and progenitor cells are heterogeneous. Differences in observed clones of stem cells likely reflect important aspects of the underlying state of the source cells, as well as future potency for cell therapies. This paper describes a colony analysis and picking device that provides quantitative analysis of heterogeneous cell populations and precise tools for cell picking for research or biomanufacturing applications. We describe an integrated robotic system that enables image acquisition and automated image analysis to be coupled with rapid automated selection of individual colonies in adherent cell cultures. Other automated systems have demonstrated feasibility with picking from semisolid media or off feeder layers. We demonstrate the capability to pick adherent bone-derived stem cells from tissue culture plastic. Cells are efficiently picked from a target site and transferred to a recipient well plate. Cells demonstrate viability and adherence and maintain biologic potential for surface markers CD73 and CD90 based on phase contrast and fluorescence imaging 6 days after transfer. Methods developed here can be applied to the study of other stem cell types and automated culture of cells.

Positive Feedback Amplifies the Response of Mitochondrial Membrane Potential to Glucose Concentration in Clonal Pancreatic Beta Cells

Analysis of the cellular mechanisms of metabolic disorders, including type 2 diabetes mellitus, is complicated by the large number of reactions and interactions in metabolic networks. Metabolic control analysis with appropriate modularization is a powerful method for simplifying and analyzing these networks. To analyze control of cellular energy metabolism in adherent cell cultures of the INS-1 832/13 pancreatic β-cell model we adapted our microscopy assay of absolute mitochondrial membrane potential (ΔψM) to a fluorescence microplate reader format, and applied it in conjunction with cell respirometry. In these cells the sensitive response of ΔψM to extracellular glucose concentration drives glucose-stimulated insulin secretion. Using metabolic control analysis we identified the control properties that generate this sensitive response. Force-flux relationships between ΔψM and respiration were used to calculate kinetic responses to ΔψM of processes both upstream (glucose oxidation) and downstream (proton leak and ATP turnover) of ΔψM. The analysis revealed that glucose-evoked ΔψM hyperpolarization is amplified by increased glucose oxidation activity caused by factors downstream of ΔψM. At high glucose, the hyperpolarized ΔψM is stabilized almost completely by the action of glucose oxidation, whereas proton leak also contributes to the homeostatic control of ΔψM at low glucose. These findings suggest a strong positive feedback loop in the regulation of β-cell energetics, and a possible regulatory role of proton leak in the fasting state. Analysis of islet bioenergetics from published cases of type 2 diabetes suggests that disruption of this feedback can explain the damaged bioenergetic response of β-cells to glucose. This article is part of a Special Issue entitled: Oxidative Stress and Mitochondrial Quality in Diabetes/Obesity and Critical Illness Spectrum of Diseases - edited by P. Hemachandra Reddy.

Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture

Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87% of the basic fibroblast growth factor and 80% of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1±0.1)×106cellsmL-1 was obtained, with 99.5±0.2% OCT 3/4 and 78.3±1.1% TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.

Metastatic Ovarian Cancer Can Be Efficiently Treated by Genetically Modified Mesenchymal Stromal Cells.

Due to late diagnosis, often recurrence, formation of metastases and resistance to commonly used chemotherapeutics human ovarian carcinoma represents a serious disease with high mortality. Adipose tissue-derived mesenchymal stromal cells (AT-MSC) can serve as vehicles for therapeutic genes and we engineered AT-MSC to express either Herpes simplex virus thymidine kinase (HSVtk-MSC), which phosphorylates ganciclovir (GCV) to its toxic metabolites or yeast fused cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT-MSC), which converts 5-fluorocytosine (5-FC) to highly toxic 5-fluorouracil (5-FU). Here, we reported different responses of cytotoxicity mediated by CD::UPRT-MSC/5-FC treatment on human ovarian carcinoma cell lines-SKOV-3 and A2780 used in adherent or three-dimensional (3D) cell culture and we proved high potential of 3D model to predict results in our in vivo experiments. Both tumor cell lines showed similarly high chemosensitivity to the used treatment in adherent culture, but 3D model revealed severe discrepancy-only 36% of SKOV-3 cells but even 90% of A2780 cells were eliminated. This result served as a prognostic marker-we were able to achieve significantly decreased tumor volumes of subcutaneous xenografts of A2780 cells in nude mice and we prolonged tumor-free survival in 33% of animals bearing highly metastatic ovarian carcinoma after CD::UPRT-MSC/5-FC treatment.

Abstract 378: Cholecystokinin Upregulates Cholesterol Absorption in Human Intestinal Epithelial Cells

Excessive absorption of intestinal cholesterol is a risk factor for atherosclerosis. We previously reported that cholecystokinin (CCK) increases intestinal cholesterol absorption and plasma cholesterol level in mouse models. The goal of this study was to investigate the effect of CCK on cholesterol absorption and Niemann-Pick C1 Like 1 (NPC1L1) expression in human primary intestinal epithelial cells (HPIECs). Normal HPIECs were isolated from small bowl resection specimens, and purchased from the Lonza Group (Walkersville, MD) . Cholesterol absorption was determined by measuring transcellular cholesterol transport in adherent cell culture and cholesterol association and release in suspension cell culture. Surface NPC1L1 was isolated using a biotinylation kit and detected by western blotting. Our data demonstrate that HPIECs express both CCK receptor-1 and -2 (CCK1R; CCK2R). Treatment of HPIECs with 3 nM [Thr28, Nle31]-CCK for 60 min increased transcellular cholesterol transport, cholesterol association and release by ~38, 32 and 44%. Selective inhibition of CCK1R and CCK2R with antagonists (1 μM lorglumide or L365260) or selective knockdown of CCK1R and CCK2R with siRNAs attenuated CCK-induced cholesterol absorption. In the cells cultured on transwell membranes, CCK increased the level of NPC1L1 in the apical membrane by ~35% but did not alter the total NPC1L1 protein expression. Inhibition or knockdown of NPC1L1 attenuated CCK-induced cholesterol absorption. These data imply that activation of CCK1R/2R enhances cholesterol absorption by induction of NPC1L membrane translocation. [This study was supported by NIH grants U54MD0007593, UL1TR000445, and SC1HL101431]

Real‐time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device

Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real‐time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time‐course data for bulk and peri‐cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non‐invasive and label‐free approach. Additionally, we confirmed non‐invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell−1 s−1, and 5 and 35 amol cell−1 s−1 were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non‐invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell‐based therapies.

Técnica de Tinta China en células adherentes en cultivo

Implementar la utilización de Tinta China como alternativa para visualizar, cambios a nivel de matriz y pared celular, en células vivas adheridas en cultivo, antes y después de la exposición a una sustancia toxica. Métodos. Se implementó la Tinta China, como técnica de contraste en microscopia óptica, comparando la nitidez observada (adecuada apreciación del borde de las estructuras) entre frascos de cultivo de células troncales de médula ósea de rata (CTMO) expuestos al glicoalcaloide tóxico α-solanina. Las diferencias de nitidez se compararon entre los diversos tratamientos, con test exacto de Fisher. Resultados. La tinción con Tinta China permitió identificar con nitidez los cambios fenotípicos celulares anormales, secundarios a la exposición del citotóxico en células adherentes en cultivo (p <0.0001). Conclusiones. La Tinta China es útil en la visualización nítida de las células CTMO y de los efectos producidos por el glicoalcaloide α-solanina en células adheridas en cultivo. Es un método sencillo que aporta al entendimiento del efecto que diversas sustancias producen en las CTMO en cultivo.

309. Optimization of Production of AAVrh.10 Viral Vectors

With the increasing interest in the use of adeno-associated virus (AAV) vectors to treat human disorders, there is increasing focus on optimizing AAV production to improve yield, purity and potency. The production of AAV vectors can be conceptualized in two stages: upstream cell culture and downstream purification and formulation. As an example of how AAV production can be optimized, we assessed upstream (cell density at transfection, plasmid and transfection reagent concentration, cell culture nutrients and harvest timing) and downstream (virus recovery, purification methods, potency) steps in the production of AAVrh. 10, a nonhuman primate vector, using adherent 293T cells and a 2 plasmid transfection system using polyethylenimine (PEI). Upstream, vector yield, as measured by quantitative PCR in the cell harvest from transfections at each of several different cell densities peaked at 8 × 104 cell/cm2. Total vector yield as a function of time after transfection peaked at day 3 post-transfection with diminishing returns and greater partitioning into the cell supernatant at longer times. Higher vector yield correlated with a greater total quantity of plasmid and PEI, but changing the PEI:plasmid ratio from 1:1 diminished productivity. Fetal bovine serum concentration had minimal effect. Downstream, freeze-thaw methods were not reproducible. Physically lysing the cells was not as effective for AAV recovery as the detergents, triton or tween, which were equally effective, with the lowest effective detergent concentration important to minimize the residual detergent in the final product. While iodixanol gradients are the traditional AAV purification systems, depth filtration followed by tangential flow filtration worked well for small scale production (>90%), but for large scale was less efficient (>70%). Affinity chromatography using AVB (GE Healthcare Life Sciences), provided an effective purification with maximum vector loads of up to 3 × 1012 vector genomes per ml of packed column. Finally, concentration of the final bulk AAV product was critically dependent on a formulation that stabilizes the solubility of the AAV monomer, with 0.01% pluronic acid providing AAV recovery of nearly 80%. Together, the optimized methods for the best yield, purity and potency relied on transfection with equal amounts of plasmid and PEI and a cell density of 8 × 104 cells/cm2 combined with the use of detergent to disrupt and recover AAV and sequential purification by depth filtration, tangential flow filtration and affinity chromatography. This process improved the yield from harvest to final product from 9% to 40% and achieved final purified AAV at 7 × 109 vector genomes/cm2 of adherent cell culture. These systematic refinements to the individual steps of AAV production demonstrate improved yield and facilitates transfer of a method for GMP production for clinical use.

The RNA Binding Protein IMP2 Preserves Glioblastoma Stem Cells by Preventing let-7 Target Gene Silencing.

Cancer stem cells (CSCs) can drive tumor growth, and their maintenance may rely on post-transcriptional regulation of gene expression, including that mediated by microRNAs (miRNAs). The let-7 miRNA family has been shown to induce differentiation by silencing stem cell programs. Let-7-mediated target gene suppression is prevented by LIN28A/B, which reduce let-7 biogenesis in normal embryonic and some cancer stem cells and ensure maintenance ofstemness. Here, we find that glioblastoma stem cells (GSCs) lack LIN28 and express both let-7 and their target genes, suggesting LIN28-independent protection from let-7 silencing. Using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), we show that insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) binds to let-7 miRNA recognition elements (MREs) and prevents let-7 target gene silencing. Our observations define the RNA-binding repertoire of IMP2 and identify a mechanism whereby it supports GSC and neural stem cell specification.

Evaluation of cell culture in microfluidic chips for application in monoclonal antibody production

Microfluidic chips are useful devices for cell culture that allow cell growth under highly controlled conditions, as is required for production of therapeutic recombinant proteins. To understand the optimal conditions for growth of cells amenable of recombinant protein expression in these devices, we cultured HEK-293T cells under different microfluidic experimental conditions. The cells were cultured in polymethyl methacrylate (PMMA) and polydimethylsiloxane (PDMS) microdevices, in the absence or presence of the cell adhesion agent poly-d-lysine. Different microchannel geometries and thicknesses, as well as the influence of the flow rate have also been tested, showing their great influence in cell adhesion and growth. Results show that the presence of poly-d-lysine improves the adhesion and viability of the cells in continuous or discontinuous flow. Moreover, the optimal adhesion of cells was observed in the corners of the microchannels, as well as in wide channels possibly due to the decrease in the flow rate in these areas. These studies provide insight into the optimal architecture of microchannels for long-term culture of adherent cells in order to use microfluidics devices as bioreactors for monoclonal antibodies production.

Modulatory Effect of Mouse Compact Bone-derived Suspending MSC on T Cells and It's Related Mechanisms

To investigate the modulatory effect of the MSC derived from low attaching culture systems (suspending MSC) on T lymphocytes and the related mechanisms. The suspending MSC were generated from mouse compact bones by using low attaching plates and adherent cell culture flasks, respectively. The morphology of suspending MSC was observed under the inverted microscope and the cells were induced to differentiate into osteoblasts and adipocytes. Further, the surface antigen profile of MSC was analyzed with flow cytometry. In addition, the culture medium (CM) of suspending MSC and adherent MSC was collected and added into the activated T cell cultures before detection of the proliferation by CFSE assay. Moreover, the modulaory effects of the CM on the T cell-derived cytokines were detected by quantitative PCR. Also, the mRNA expression of cytokines of MSC was detected. The suspending MSC grew in floating cell spheres and differentiated into osteoblasts and adipocytes in the induction medium. Furthermore, the suspending MSC shared the typical immuno-phenotype with their adherent counterparts. In addition, the results of CFSE assay demonstrated that suspending MSC derived CM suppressed ConA induced T cell proliferation. The results of quantitative PCR revealed that suspending MSC expressed transforming factor β1 and interleukin-6 at a higher level and suppressed the T cell expressing interferon γ and interleukine-17A. The suspending MSC exerted an unique modulatoy effect on T cells, which is quite different to adherent MSC.

Determining Maximum Glycolytic Capacity Using Extracellular Flux Measurements.

Measurements of glycolytic rate and maximum glycolytic capacity using extracellular flux analysis can give crucial information about cell status and phenotype during normal operation, development of pathology, differentiation, and malignant transformation. They are also of great use when assessing the effects of chemical or drug treatments. Here, we experimentally define maximum glycolytic capacity, demonstrate how it differs from glycolytic rate, and provide a protocol for determining the basal glycolytic rate and maximum glycolytic capacity in cells using extracellular flux measurements. The results illustrate the power of extracellular flux analysis to describe the energetics of adherent cells in culture in a fully quantitative way.

The role of the equatorial ligands for the redox behavior, mode of cellular accumulation and cytotoxicity of platinum(IV) prodrugs

The current study aims to elucidate the possible reasons for the significantly different pharmacological behavior of platinum(IV) complexes with cisplatin-, carboplatin- or nedaplatin-like cores and how this difference can be related to their main physicochemical properties. Chlorido-containing complexes are reduced fast (within hours) by ascorbate and are able to unwind plasmid DNA in the presence of ascorbate, while their tri- and tetracarboxylato analogs are generally inert under the same conditions. Comparison of the lipophilicity, cellular accumulation and cytotoxicity of the investigated platinum compounds revealed the necessity to define new structure-property/activity relationships (SPRs and SARs). The higher activity and improved accumulation of platinum(IV) complexes bearing Cl− in equatorial position cannot only be attributed to passive diffusion facilitated by their lipophilicity. Therefore, further platinum accumulation experiments under conditions where active/facilitated transport mechanisms are suppressed were performed. Under hypothermic conditions (4°C), accumulation of dichloridoplatinum(IV) complexes is reduced down to 10% of the amount determined at 37°C. These findings suggest the involvement of active and/or facilitated transport in cellular uptake of platinum(IV) complexes with a cisplatin-like core. Studies with ATP depletion mediated by oligomycin and low glucose partially confirmed these observations, but their feasibility was severely limited in the adherent cell culture setting.

CD271(+) stromal cells expand in arthritic synovium and exhibit a proinflammatory phenotype.

BackgroundCD271+ stromal cells (SCs) with multipotent stem cell capacity have been identified in synovial tissues, but their functional significance is unknown. We analyzed the distribution of CD271+ cells in inflammatory synovial tissues as well as their ex vivo immunomodulatory and inflammatory phenotypes.MethodsCD271 expression was analyzed by immunohistochemistry in synovial tissues and by flow cytometry in primary adherent synovial cell cultures from rheumatoid arthritis (RA), osteoarthritis (OA), and non-inflammatory control tissues. Isolation of CD271+ synovial SCs was carried out by magnetic cell sorting. Allogeneic T-cell/SC cocultures were performed to analyze the regulatory capacity of these cells on T-cell proliferation and cytokine production. The production of inflammatory mediators was analyzed in cultures of sorted CD271+/− SCs. The capacity of CD271+/− SCs to induce inflammatory cell recruitment in vivo was evaluated in subcutaneous implants in immunodeficient mice.ResultsCD271+ SC were detected in non-inflammatory as well as in arthritic synovial tissues with a specific perivascular distribution. CD271+ SC density was increased in RA and OA compared with normal synovial tissues. T-cell proliferation and cytokine synthesis were similarly modified by CD271+ and CD271− SCs. Sorted CD271+ SCs from OA synovial tissues released significantly more interleukin (IL)-6, matrix metalloproteinase (MMP)-1, and MMP-3 than CD271− SCs. In immunodeficient mice, implants of CD271+ SCs induced significantly higher myeloid cell infiltration than CD271− SCs.ConclusionsOur results demonstrate that CD271+ perivascular SCs expand in RA and OA synovial tissues. CD271+ cells showed enhanced proinflammatory properties ex vivo and in vivo, whereas immunoregulatory properties were equivalent in CD271+ and CD271− SC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-0966-5) contains supplementary material, which is available to authorized users.

FOXA1 expression affects the proliferation activity of luminal breast cancer stem cell populations.

The expression of estrogen receptor is the key in most breast cancers (BC) and binding of estrogen receptor to the genome correlates to Forkhead protein (FOXA1) expression. We herein assessed the correlation between the cancer stem cell (CSC) population and FOXA1 expression in luminal BC. We established luminal BC cells derived from metastatic pleural effusion and analyzed the potency of CSC and related factors with established luminal BC cell lines. We also confirmed that mammosphere cultures have an increased aldehyde dehydrogenase‐positive population, which is one of the CSC markers, compared with adherent culture cells. Using a quantitative PCR analysis, we found that mammosphere forming cells showed a higher expression of FOXA1 and stemness‐related genes compared with adherent culture cells. Furthermore, the growth activity and colony‐forming activity of 4‐hydroxytamoxifen‐treated BC cells were inhibited in a mammosphere assay. Interestingly, 4‐hydroxytamoxifen‐resistant cells had significantly increased FOXA1 gene expression levels. Finally, we established short hairpin RNA of FOXA1 (shFOXA1) MCF‐7 cells and investigated the relationship between self‐renewal potential and FOXA1 expression. As a result, we found no significant difference in the number of mammospheres but decreased colony formation in shFOXA1 MCF‐7 cells compared with control. These results suggest that the expression of FOXA1 appears to be involved in the proliferation of immature BC cells rather than the induction of stemness‐related genes and self‐renewal potency of CSCs.

THE THIOREDOXIN SYSTEM IN REGULATING MCF-7 CELL PROLIFERATION UNDER REDOX STATUS MODULATION

Introduction. Despite the available data on tumor cell functioning under the conditions of free radical-mediated oxidation, the mechanisms of redox regulation, cell proliferation management and apoptosis avoidance remain understudied. The objective of the study was to identify the role of the thioredoxin system in regulating MCF-7 breast cancer cell proliferation under redox status modulation with 1.4-dithioerythritol. Material and methods. The studies were conducted on the MCF-7 breast cancer cell line, grown in adherent cell culture. Cell redox status was modulated with5 mM N-ethylmaleimide – an SH group and peptide inhibitor and5 mM 1.4-dithioerythritol – a thiol group protector. The cell cycle was evaluated by flow cytometry, the same technique was used to measure the reactive oxygen species concentration. The levels of reduced and oxidized glutathione and the activity of thioredoxin reductase were identified by spectrophotometry. The intracellular concentrations of thioredoxin, cyclin E and cyclin-dependent kinase 2 were determined by Western blot analysis. Results and discussion. The essential role of the thioredoxin system in regulating MCF-7 breast cancer cell proliferation was exhibited. S-phase arrest under the effect of N-ethylmaleimide and G0/G1-phase arrest under the effect of 1.4-dithioerythritol are associated with the changes in the activity of redox-sensitive protein complexes (cyclins and cyclin-dependent kinases) that regulate cell proliferation. Conclusion. Redoxdependent modulation of proliferation regulating intracellular protein activity occurs due to the thioredoxin system. This is a promising research area for seeking molecular targets of breast cell malignization.

Fast Sampling of Adherent Cell Cultures for Optimal Metabolomics Results

Metabolomics is a valuable tool to gain mechanistic insight into biological processes. It is frequently used to obtain complementary details to other ‘omics technologies such as transcriptomics or proteomics. For knowledge generation, reproducible measurements of physiological, intact, and artifact-free metabolite levels are imperative necessitating further standardization of best practices to improve reliability of research outcomes. Here we report a novel cell sample preparation method (MxP® CellCollect) for metabolomics applications using adherent mammalian cells, which reduces the time consumption and physiological stress of conventional methods such as trypsinization or cell scraping. The most common sampling procedures to detach cells from their growth surface, trypsinization and scraping were compared to the MxP® CellCollect method investigating metabolite profiles of two breast cancer cell lines (MDA-MB-231 and MCF7). Metabolite levels as well as direction of metabolite changes differed tremendously revealing issues with trypsinization and scraping risking non-physiological or misleading results in contrast to MxP® CellCollect. Differences in metabolic profiles of cells harvested by trypsinization as compared to MxP® CellCollect or scraping can be directly attributed to prolonged, medium-free incubation time during cell detachment leading to a severely energy-depleted intracellular state. Labile metabolites or metabolites with fast intracellular turnover rates such as glycolysis and TCA cycle intermediates were strongly and significantly decreased by trypsinization. The same was true for amino acids and nucleoside triphosphates. Results obtained with scraping using methanol as solvent were multifaceted. Even mild evaporation of methanol prior metabolite extraction led to temperature- and/or light-dependent degradation of labile metabolites such as nucleoside triphosphates into di- and monophosphates liberating pyrophosphate. Furthermore, lipid metabolites, in particular cell membrane lipids, were found to have significantly lower levels than measured by trypsinization or MxP® CellCollect, indicating that lipid metabolites are insufficiently detached and/or unspecifically adsorb to the hydrophobic dish and scraping tool.

Bioreactor Expansion of Skin-Derived Precursor Schwann Cells.

Scaling up the production of cells in a culture process is a critical step when trying to develop cell-based regenerative therapies. Static cultures often cannot be easily scaled up to clinically relevant cell numbers. Alternatively, bioreactors offer a highly valuable means to develop a clinical-ready process. To culture adherent cells in suspension, such as skin-derived precursor Schwann cells (SKP-SCs), microcarriers need to be used. Microcarriers are small spherical beads suspended within the vessel that allow for higher growth surface area to volume ratio. Here we describe the procedure of combining microcarriers with the controllability of bioreactors to generate higher cell densities in smaller reactor volumes leading to a more efficient and cost-effective cell production for applications in regenerative medicine.

Comparative characteristics cultural properties of adherent cells derived from bone marrow of multiorgan donors and tissues donors

Purpose of work - compare cultural properties of adherent cells derived from bone marrow of multiorgan donors and tissues donors, to assess the possibility of using these cells in clinical practice. Materials and methods. Adherent culture cells from cadavers bone marrow were obtained using standart methods of isolation, scaling, and passaging cells. In culture, counted the number of CFU-F, proliferating activity of cell , their complies with the minimal criteria for defining multipotent mesenchymal stromal cells (hMSC) and compatibility with allogeneic tissue grafts. The Results. In cultures of cells derived from multiorgan donors and tissues donors the number of CFU-F was 0,0004% and 0,00024%, while the duration of the phase "rest proliferative" was 3-4 and 10-12 days respectively. All examined cell culture after the second passage have gained a high degree of homogeneity, and the cells were complies the minimum criteria hMSC. Conclusion: bone marrow of multiorgan donors and tissues donors can be used for obtain hMSC, suitable for creating a combined cellular-tissue grafts for clinical use.

Spheroid Culture of Mesenchymal Stem Cells

Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs) cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown.