Non-adherent cells are difficult to transfect with chemical-mediated delivery methods. Electroporation is an attractive strategy to transfer the molecules of interest into suspension cells. Care must be taken with the viability of the transfected cells since parameters, which increase cell membrane permeability, subsequently increase transfection efficiency, leading to higher cell death indices. We intended to evaluate the distribution of hard-to-transfect UT-7 cells among different subpopulations: transfected/viable, untransfected/viable, transfected/dead, and untransfected/dead populations, for a better understanding of the relation between gene electrotransfer efficacy and cell death. The following electroporation parameters were tested: pulse strength, duration, plasmid DNA concentration, and ZnSO4 as DNase inhibitor. BTX T820 square-wave generator was used, and 48 h after electroporation, cells were observed for viability and fluorescence analysis. Increasing pulse strength correlated directly with an increased ratio of pEGFP-positive cells and inversely with cell viability. The best results, representing 21% pEGFP positive/viable cells, were obtained after EP with 1 HV 1400 V/cm pulse of 250 µs duration using 200 µg/mL plasmid concentration. Results demonstrated that plasmid concentration played the most significant role in pEGFP electrotransfer into UT-7 cells. These results can represent a relevant improvement of gene electrotransfer to obtain genetically modified suspension cells for further downstream experiments.

Over 50 years have passed since discovering mesenchymal stromal cells (MSCs). Initially, despite gaps in the knowledge of the identity of these cells, their therapeutic aspects were recognized. Consequently, MSCs became candidates for treating a wide range of diseases. However, the therapeutic effects of MSCs are not stable in the long term, and there are inconsistent data on their clinical efficacy. Even though more than 1000 MSC-based clinical trials have been registered, and the safety of MSCbased cell therapies has been proven, data on the clinical efficacy of MSCs have not been enough to warrant FDA approval for clinical treatment and marketing purposes. The available information on MSCs still contains some controversies, perhaps owing to little progress in understanding their in vivo identity. MSCs have been used for therapeutic purposes despite poor knowledge of their in vivo origin or functions. Hence, perhaps we need to go back to the basics of MSCs and spend more time understanding the biology of these cells. An improved understanding of MSCs\' location and function within tissues may improve their therapeutic efficacy and, consequently, their establishment as a cell therapy product.

The interest around the graphene family of materials is constantly growing due to their potential application in biomedical fields. The effect of graphene and its derivatives on cells varies amongst studies depending on the cell and tissue type. Since the toxicity against non-adherent cell lines has barely been studied, we investigated the effect of graphene and two different graphene oxides against four multiple myeloma cell lines, namely KMS-12-BM, H929, U226, and MM.1S, as well as two non-Hodgkin lymphoma cells lines, namely KARPAS299 and DOHH-2. We performed two types of viability assays, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide conversion) and ATP (adenosine triphosphate detection), flow cytometry analysis of apoptosis induction and cell cycle, cell morphology, and direct interaction analysis using two approaches-visualization of living cells by two different systems, and visualization of fixed and dyed cells. Our results revealed that graphene and graphene oxides exhibit low to moderate cytotoxicity against cells, despite visible interaction between the cells and graphene oxide. This creates possibilities for the application of the selected graphene materials for drug delivery systems or theragnostics in hematological malignancies; however, further detailed studies are necessary to explain the nature of interactions between the cells and the materials.

BACKGROUND: Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy.
METHODS: We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm.
RESULTS: We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells.
CONCLUSIONS: The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients\' cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field.

OBJECTIVE: The aim of present study was to identify characteristic and response of mouse bone marrow (BM) derived low-adherent bone marrow mesenchymal stem cells (BMMSCs) obtained by quantification of extracellular matrix (ECM).
METHODS: Non-adherent cells acquired by ECM coated dishes were termed low-adherent BMMSCs and these cells were analyzed by in vitro and in vivo methods, including colony forming unit fibroblast (CFU-f), bromodeoxyuridine (BrdU), multi-potential differentiation, flow cytometry and transplantation into nude mouse to measure the bone formation ability of these low-adherent BMMSCs. Titanium (Ti) discs with machined and anodized surfaces were prepared. Adherent and low-adherent BMMSCs were cultured on the Ti discs for testing their proliferation.
RESULTS: The amount of CFU-f cells was significantly higher when non-adherent cells were cultured on ECM coated dishes, which was made by 7 days culturing of adherent BMMSCs. Low-adherent BMMSCs had proliferation and differentiation potential as adherent BMMSCs in vitro. The mean amount bone formation of adherent and low-adherent BMMSCs was also investigated in vivo. There was higher cell proliferation appearance in adherent and low-adherent BMMSCs seeded on anodized Ti discs than machined Ti discs by time.
CONCLUSIONS: Low-adherent BMMSCs acquired by ECM from non-adherent cell populations maintained potential characteristic similar to those of the adherent BMMSCs and therefore could be used effectively as adherent BMMSCs in clinic.

We tried here to optimize the proliferation of both Hematopoietic and Mesenchymal stem cells of Umbilical Cord blood in minimal cytokine growth condition. Failing to get good results of expansion of non-adherent Hematopoietic Total Nucleated Cells and adherent Fibroblastic Mesenchymal Stem Cells derived from 10-12 ml of collected Cord blood, we designed the further experimental study by increasing the volume of Cord blood sample up to 65-70 ml. We harvested the non-adherent as well as adherent fraction separately derived from the primary culture of Umbilical Cord blood stem cells under the influence of growth promoting Cytokines or Growth Factors. The proliferation study was conducted by taking different combinations of two hematopoietic growth stimulatory Cytokines like stem cell factor (SCF) and Fms like tyrosine kinase-3Ligand (Flt3L) at concentrations (10 ng/ml, 100 ng/ml) while we preferred Mesenchymal specific growth factor i.e. basic Fibroblast growth factor (FGF-β) at its 10 ng/ml concentration for adherent cells to get optimal results. The Hematopoietic and Fibroblast Colony forming abilities of the expanded stem cells were performed through Colony Forming Unit assay. Culture Medium containing cytokine combination like SCF 100 ng/ml with Flt3L 10 ng/ml was found to be optimal for the proliferation of hematopoietic stem cells. But the number of hematopoietic colonies like Erythroid colonies generated were less in case of media supplemented with SCF & Flt3L while more number of Myeloid colonies were observed in Growth factor supplemented media in comparison to the control one. The FGF-β supplemented media successfully enhanced the proliferation of Mesenchymal Stem Cells and exhibited its efficient Fibroblast colony forming ability. Our experimental study supports the minimal utilization of cytokines for haematopoietic and mesenchymal stem cell proliferation which may help in future safe Cord blood stem cell infusion.