Novel method to obtain highly enriched cultures of adult rat Schwann cells

Schwann cells (SCs) can be used to repair both the peripheral and central nervous systems. Therefore, establishment of a procedure to obtain activated, highly proliferative SCs, in an appropriate time for clinical applications, is a prerequisite. Purification is complicated by contamination with fibroblasts which often become the predominant cell type in an in vitro SC culture. This study describes a novel and efficient method to enrich SCs by utilizing the differential detachment properties of the two cell types. In culture, cells were treated with two different media and the chelator, EGTA, which detached SCs faster than fibroblasts and allowed for easy isolation of SCs. Within seven days, high yields of SCs with a purity of greater than 99% were achieved. This was confirmed by immunostaining characterization and flow-cytometric analyses using an antibody against the p75 low affinity nerve growth factor receptor (p75LNGFR). The entire procedure was completed in approximately 21 days. This method has the advantage of being technically easier, faster, and more efficient than other previously described methods. An SC culture that was about 99% homogenous was achieved.     

Comparison of non-viral transfection methods in melanoma cell primary cultures

Melanoma primary cultures were transiently transfected via electroporation and lipofection for comparison. Transfection efficiency was superior with electroporation (58+/-9%) as compared to lipofection (23+/-9%) as determined by enhanced green fluorescent plasmid (EGFP) transfection. Secretion of IL-2 persisted for up to 3 weeks after electroporation. The increase in sensitivity against immunologic effector cells by transfection with IL-2 was not significant. Our results show the feasibility of a gene transfer into primary human melanoma cells, different from retroviral transduction.     

A new protocol for cultivation of predegenerated adult rat Schwann cells

The purpose of this study was to optimize the methodology of cultivation of predegenerated Schwann cells (SCs). SCs were isolated from 7-day-predegenerated sciatic nerves of adult rats. We applied commercially available culture medium for cultivation of endothelial cells endothelial cell culture medium (EBM-2) instead of Dulbecco’s Modified Eagle’s Medium commonly used to culture adult Schwann cells. Additionally, cell culture medium was supplemented with factors specifically supporting SCs growth as: bovine pituitary extract (5 μg/ml), heregulin (40 ng/ml) and insulin (2.5 ng/ml). Similarly to the reports of others authors, we did not observe any beneficial effects of Forskolin application, so we didn’t supplement our medium with it. Cell culture purity was determined by counting the ratio of GFAP, N-Cadherin and NGFR p75-positive cells to total number of cells. About 94–97 % of cells were confirmed as Schwann cells. As a result, we obtained sufficient number and purity of Schwann cells to be applied in different experimental models in rats. EBM-2 medium coated with fibronectin was the best for cultivation of adult rat Schwann cells.     

Optimized protocol for efficient transfection of dendritic cells without cell maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection¹. Detection of pathogenic antigen by naïve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response^2,3. Thus, DCs link the innate and adaptive immune systems. The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs⁴.Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)^5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs^9,10,11,12. Electroporation has been used with mixed results^13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation¹⁶. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFNβ. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor^16,17, at both the mRNA and protein levels. Download video file.^{(52M, mov)}     

A modified enrichment protocol for adult caprine skeletal muscle stem cell

To establish an adequate model to study the proliferation and differentiation of adult caprine skeletal muscle in response to bioactive compounds, a pool of satellite cells (SC) was derived from the rectus abdominis muscle of adult goat. Skeletal muscle contains a population of adult stem cells, named as satellite cells that reside beneath the basal lamina of skeletal muscle fiber and other populations of cells. These SC are multipotent stem cells, since cells cultured in the presence of specific cell lineage inducing cocktails can differentiate into several types of mesenchymal lineage, such as osteocytes and adipocytes. In the present study, we have developed a modified protocol for isolating satellite cells (>90%) and examined their myogenic and contractile properties in vitro.     

An efficient system for selection and culture of Schwann cells from adult rat peripheral nerves

Schwann cells (SCs), the supporting cells of the peripheral nerves, are indispensable for regenerating the peripheral and central nervous system. Copious preparation of these cells in a well-defined manner is to be a privileged position. SCs cultivation is overwhelmed by contaminating fibroblasts which are often outgrowing as the predominant cell type in an in vitro culture. This study introduces a technically simple and efficient procedure for SCs isolation and enrichment based on implementing recombinant and defined supplements. Collected adult rat sciatic nerves were cultured for 10 days as in vitro predegeneration. After dissociation and plating, the medium changed to knockout serum replacement supplemented DMDM/F12 medium containing various growth factors. The whole procedure took 3 weeks and SCs purity was then evaluated through implementing specific cytoplasmic and membranous markers. The viability of enriched SCs were evaluated by MTT assay. Within 10 days, over 99 % homogenous SCs were achieved and confirmed through immunofluorescence staining and flow-cytometry for P75^NTR and S100 markers, respectively. MTT data revealed that the viability and metabolic activities of purified SCs were increased in expansion medium. This study provides a technically easy and efficient method with the benefits of not utilizing bovine serum or other animal products for SCs isolation and enrichment.     

Differential expression of adult type MHC in satellite cell cultures from regenerating fast and slow rat muscles.

The local anaesthetic (Bupivacaine (1-n-butyl-DL-piperidine-2-carboxylic acid-2, 6-dimethyl anilide hydrochloride) has been used to induce myofiber damage (and thus satellite cells proliferation) and thereby represents a tool for increasing the yield of myoblasts from adult muscles. Replicating satellite cells were isolated by enzymatic dissociation from soleus (slow type) and tibialis anterior (fast type) muscles of adult rats, and categorized by the isoform (embryonic, fast and slow) of myosin heavy chain (MHC) expressed following myotube formation in a similar in vitro environment. According to light microscopic criteria, no morphological differences exist between the satellite cell cultures obtained from adult fast and slow muscles after Bupivacaine injection. On the other hand the derived myotubes express, beside the embryonic type, the peculiar myosin heavy chains which characterize the myosin pattern of the donor muscles.     

Novel non-viral method for transfection of primary leukemia cells and cell lines

BACKGROUND: Tumor cells such as leukemia and lymphoma cells are possible targets for gene therapy. However, previously leukemia and lymphoma cells have been demonstrated to be resistant to most of non-viral gene transfer methods. METHODS: The aim of this study was to analyze various methods for transfection of primary leukemia cells and leukemia cell lines and to improve the efficiency of gene delivery. Here, we evaluated a novel electroporation based technique called nucleofection. This novel technique uses a combination of special electrical parameters and specific solutions to deliver the DNA directly to the cell nucleus under mild conditions. RESULTS: Using this technique for gene transfer up to 75% of primary cells derived from three acute myeloid leukemia (AML) patients and K562 cells were transfected with the green flourescent protein (GFP) reporter gene with low cytotoxicity. In addition, 49(+/- 9.7%) of HL60 leukemia cells showed expression of GFP. CONCLUSION: The non-viral transfection method described here may have an impact on the use of primary leukemia cells and leukemia cell lines in cancer gene therapy.     

Conditions affecting primary cell cultures of functional adult rat hepatocytes

Summary Primary monolayer cell cultures of adult rat hepatocytes underwent change in morphology and substantial cell loss between 1 and 3 days postinoculation. Dexamethasone-supplementation (1μM) of the culture medium maintained the polygonal epithelial morphology of the hepatocytes and increased longevity such that over 80% of the cells survived for 3 days and at least 30% for 8 or 9 days. This enhancement of survival was obtained up to 48 hr postinoculation, but the earlier the time of dexamethasone supplementation the greater the effect. Removal of dexamethasone resulted in a decrease in longevity. The positive effect of dexamethasone on longevity was observed following dexamethasone replacement of insulin in supplemented cultures, but the combination of insulin and dexamethasone resulted in poorer survival than with dexamethasone alone. The results are interpreted to indicate that dexamethasone provided a requirement of the in vitro environment for survival and suggest that elaboration of a complex medium is required to maintain hepatocytes in culture. This study was supported by an Alexander Ralston Peacock Memorial Grant for Cancer Research (No. BC-133A) from the American Cancer Society.     

Conditions affecting primary cell cultures of functional adult rat hepatocytes

Summary The conditions for obtaining representative, primary adult rat hepatocyte cultures were explored. The methods applied included enzymatic liver perfusion which was nondestructive to hepatocytes, the prevention of aggregation of dissociated cells and the selective attachment of viable cells. These procedures yielded a recovery of 50% of the liver cells which gave rise to cultures representing 14% of the total liver cells. The cultures were composed of homogeneous epithelial-like cells cytologically similar to hepatocytes and possessed a number of liver-specific enzymes. There was virtually no cell division initially and most cells died between 24 and 48 hr. Insulin enhanced the attachment of the liver cells, altered their morphology, but did not prolong cell survival. This study was supported by grant no. BC 133 from the American Cancer Society.     

A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles

Delivery of DNA and siRNA into mammalian cells is a powerful technique in treating various diseases caused by single gene defects. Herein, we report a highly efficient delivery system using 1,4-butanediol diglycidyl ether (bisepoxide) crosslinked polyethylenimine (PEI) nanoparticles (PN). The nanoparticle/DNA complexes (nanoplexes) exibited approximately 2.5- to 5.0-fold gene transfer efficacy and decreased cytotoxicity in cultured cell lines, compared to the native PEI (25 kDa) (gold standard) and commercially available transfection agents such as Lipofectamine 2000 and Fugene. The bisepoxide crosslinking results in change in amine ratio in PEI; however, it retains the net charge on PN unaltered. A series of nanoparticles obtained by varying the degree of crosslinking was found to be in the size range of 69-77 nm and the zeta potential varying from +35 to 40 mV. The proposed system was also found to deliver siRNA efficiently into HEK cells, resulting in approximately 70% suppression of the targetted gene (GFP).     

Improved non-viral transfection of glial and adult neural stem cell lines and of primary astrocytes by combining agents with complementary modes of action

BACKGROUND: Rational design of gene vectors for therapeutic applications requires understanding of transfection mechanisms. In this study, multiple transfection assays revealed complementary mechanisms between two commonly used transfection agents. This finding was then exploited to produce improved transfection outcomes. METHODS AND RESULTS: Rat C6 glial cells, adult rat hippocampal progenitor cells and primary astrocytes were transfected using Lipofectamine (LA) or polyethylenimine (PEI), in vitro. Although LA- and PEI-transfected populations expressed the same total level of transgene product, LA transfected considerably more cells than PEI (approximately 20 vs. 14%). A fluorescently labelled plasmid and time-course analysis, involving both flow cytometry and confocal microscopy, were used to explain this apparent discrepancy. Results showed that LA delivered more plasmid DNA to the cytoplasm and achieved transgene expression in more cells than PEI. In contrast, PEI transfected fewer cells but, on average, produced more transgene product per transfected cell. CONCLUSIONS: A comparative transfection model was developed to explain these different characteristics. According to this model, transfection is a multistage process with different transfection agents exerting their primary effect at different stages in this process. This model forecast that it should be possible to prepare a chimeric complex with a transfection efficiency that exceeded that achievable with Lipofectamine or polyethylenimine alone. This prediction was tested and shown to hold for glioma cells, primary astrocytes, and adult neural stems cells.     

Glycogen metabolism in adult rat liver parenchymal cell primary cultures.

Parenchymal cells were isolated from adult rat liver with an enzyme perfusion technique. The single-cell suspension, representing 40-50% of the liver's hepatocytes was suspended in medium and maintained in primary culture for up to four days. The cells were found to carry out glycogen synthesis for the first eight hours in culture after which time the accumulated glycogen was gradually degraded. The ability of the liver cell cultures to accumulate glycogen was found to be dependent upon the metabolic state of the animal prior to cell isolation. Cells prepared during the feeding period from animals on the 8+16 feeding schedule had markedly different capacities for glycogen accumulation. Changes in glycogen metabolism were found to be due, in part, to changes in the fraction of cells involved in metabolism at any given time. High concentrations of glucose stimulated the cells to deposit glycogen but the response was reduced the longer the cells were in culture over a 3-day period. This loss of glycogen synthesizing capacity appears to be due to a decrease in glycogen synthetase activity. The activities of pyruvate kinase, hexokinase and aldolase also decrease during the culture period.     

Microbubble‐mediated sonoporation for highly efficient transfection of recalcitrant human B‐ cell lines

Sonoporation has not been widely explored as a strategy for the transfection of heterologous genes into notoriously difficult‐to‐transfect mammalian cell lines such as B cells. This technology utilizes ultrasound to create transient pores in the cell membrane, thus allowing the uptake of extraneous DNA into eukaryotic and prokaryotic cells, which is further enhanced by cationic microbubbles. This study investigates the use of sonoporation to deliver a plasmid encoding green fluorescent protein (GFP) into three human B‐cell lines (Ramos, Raji, Daudi). A higher transfection efficiency (TE) of >42% was achieved using sonoporation compared with <3% TE using the conventional lipofectamine method for Ramos cells. Upon further antibiotic selection of the transfected population for two weeks, we successfully enriched a stable population of GFP‐positive Ramos cells (>70%). Using the same strategy, Raji and Daudi B cells were also successfully transfected and enriched to 67 and 99% GFP‐positive cells, respectively. Here, we present sonoporation as a feasible non‐viral strategy for stable and highly efficient heterologous transfection of recalcitrant B‐cell lines. This is the first demonstration of a non‐viral method yielding transfection efficiencies significantly higher (42%) than the best reported values of electroporation (30%) for Ramos B‐cell lines.     

Fibronectin promotes rat Schwann cell growth and motility

Techniques are now available for culturing well characterized and purified Schwann cells. Therefore, we investigated the role of fibronectin in the adhesion, growth, and migration of cultured rat Schwann cells. Double-immunolabeling shows that, in primary cultures of rat sciatic nerve, Schwann cells (90%) rarely express fibronectin, whereas fibroblasts (10%) exhibit a granular cytoplasmic and fibrillar surface-associated fibronectin. Secondary cultures of purified Schwann cells do not express fibronectin. Exogenous fibronectin has a small effect on promoting the adhesion of Schwann cells to the substrate and does not significantly affect cell morphology, but it produced a surface fibrillar network on fibronectin on the secondary Schwann cells. Tritiated thymidine autoradiography revealed that addition of fibronectin to the medium, even at low concentrations, markedly stimulates Schwann cell proliferation, in both primary and secondary cultures. In addition, when cell migration was measured in a Boyden chamber assay, fibronectin was found to moderately, but clearly, stimulate directed migration or chemotaxis.     

Simple and efficient control of CHO cell cultures

Cell cultures must tightly be kept under control in order to guarantee a sufficiently small variability in the protein product quality. A simple and efficient technique for CHO-cell cultures is presented that allows keeping the viable cell count X(v) and the specific growth rate μ of the cells on predefined trajectories. As X(v) and μ cannot directly be measured online, they are controlled indirectly via the total mass of oxygen consumed. Online values of the latter can precisely be estimated from off gas analysis, i.e. from the O? volume ratio measured in the vent line and air flow rate measurements. In glutamine-limited fed-batch cultivations, the glutamine feed rate can be manipulated in such a way that the viable cell density and the specific growth rate are kept on predefined profiles for nearly the entire cultivation time. The viability of the cells is not affected by the closed loop control actions. The technique was validated with CHO-cells cultured in a 2.5-L fully instrumented stirred tank bioreactor. It is shown that the controller is able to run the process exactly on predefined tracks with a high batch-to-batch reproducibility. By means of six fed-batch cultivations of CHO cells it was shown that a remarkable reproducibility of viable cell concentration could be achieved throughout 140 h cultivation time.     

Characteristics of small cell colonies developing in primary cultures of adult rat hepatocytes

Phenotypes of the cells developing into small colonies after days of primary culture of adult rat hepatocytes in serum-free modified Dulbecco Modified Eagles’ medium containing 10 mM nicotinamide and 10 ng/ml epidermal growth factor were analyzed immunocytochemically, cytochemically and ultrastructurally. Albumin, cytokeratin 8 and 18 were seen by immunocytochemical techniques in the cells of the small colonies at Day 6. Transferrin, α-antitrypsin, ceruloplasmin, and haptoglobin, proteins secreted by mature hepatocytes, were faintly stained in these cells as was α-fetoprotein. These proteins were secreted into the culture medium as evidenced by immunoblot analysis. γ-Glutamyltransferase, alkaline phosphatase and glucose 6-phosphatase were not present in the cells of the small colonies as well as the surrounding hepatocytes at Day 6 of culture. In addition, ultrastructural examinations of the cells in the small colonies indicated that these cells not only had many characteristic mitochondria and desmosomes, but also a few small peroxisomes. Such cells, even after 20 days in culture were proliferating, as evidenced by the intranuclear presence of the proliferating cell nuclear antigen. The potential relation of these cells to hepatocytes which may serve as the principal reserve for replicating hepatocytes is discussed.