Glycogen storage disease type II (GSDII) or Pompe disease is an inherited disease of glycogen metabolism caused by a lack of functional lysosomal acid α-glucosidase (GAA). Affected individuals store glycogen in lysosomes resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe form. We investigated for the first time the use of lentiviral vectors to correct the GSDII phenotype in human and murine GAA-deficient cells. Fibroblasts from infantile and adult GSDII patients were efficiently transduced by a GAA-expressing lentiviral vector placed under the control of the strong MND promoter, leading to a complete restoration of enzymatic activity. We also developed a muscle-specific lentiviral vector based on the synthetic C5–12 promoter and tested it on deficient myogenic satellite cells derived from a GSDII mouse model. GAA was expressed as a correctly processed protein allowing a complete enzymatic and metabolic correction in myoblasts and differentiated myo-tubes, as well as a significant mannose-6-phosphate (M6P)-dependent secretion reuptake by naive cells. Transduced cells showed lysosomal glycogen clearance, as demonstrated by electron microscopy. These results form the basis for a therapeutic approach of GSDII using lentiviral vector-mediated gene transfer into muscle stem cells.

Manufacturers of vaccines and other biologicals are under increasing pressure from regulatory agencies to develop production methods that are completely animal-component-free. In order to comply with this demand, alternative cell culture substrates to those now on the market, primarily collagen or gelatin, must be found. In this paper, we have tested a number of possible substitutes including recombinant collagen, a 100-kDa recombinant gelatin fragment and a peptide derived from a cell-binding region of type I collagen. The small 15-amino acid peptide did not support attachment of human fibroblasts in monolayer culture. The 100-kDa gelatin fragment supported cell attachment in monolayer culture, but was significantly less active than intact porcine gelatin. Recombinant type I collagen was as successful in promoting cell attachment as native collagen, and both were more effective than porcine gelatin. Based on these data, dextran micro-spheres were treated with the same attachment proteins—porcine gelatin, native collagen, or recombinant collagen. The same trends were observed as in monolayer culture. Concentrations of the recombinant collagen (as well as native collagen) supported cell attachment on dextran microspheres at concentrations as low as 0.01 μg/cm². Treatment of the dextran with a low level of polyethylenimine, a cationic moiety, further enhanced attachment when used in conjunction with the low concentration of recombinant collagen. Where there was increased cell attachment, increased proliferation followed. We are confident, based on these findings, that a fully recombinant substitute could replace gelatin in current microcarrier preparations without losing the cell growth benefits provided by the native protein.

The lack of a suitable flat epithelial preparation isolated directly from the freshwater fish gill has led, in recent years, to the development of cultured gill epithelia on semipermeable supports. To date, their minimal capacity to actively transport ions has limited their utility as ionoregulatory models. The current study describes a new method of culturing gill epithelia consisting either of an enriched population of pavement (PV) cells or a mixed population of PV cells and mitochondria-rich (MR) cells from the gills of adult rainbow trout. Although the cell culture approach is similar to the double-seeded insert (DSI) technique described previously, it makes use of Percoll density centrifugation to first separate populations of PV and MR cells, which are then seeded on cell culture supports in varying proportions on successive days so as to produce preparations enriched in one or the other cell types. Based on rhodamine staining, the MR cell-rich epithelia exhibited a threefold higher enrichment of MR cells compared to traditional DSI preparations. In general, MR cell-rich epithelia developed extremely high transepithelial resistances (TER; >30 kμ cm²) and positive transepithelial potentials (TEP) under symmetrical conditions (i.e., L15 medium on both apical and basolateral sides). Apical exposure of cell cultures to freshwater reduced TER and produced a negative TEP in all the epithelial preparations, although MR cell-rich epithelia maintained relatively high TER and negative TEP for over 2 d under these asymmetrical conditions. Measurement of unidirectional Na fluxes and application of the Ussing flux ratio criterion demonstrated active Na uptake in PV cell-rich and MR cell-rich epithelia under both symmetrical and asymmetrical conditions. In comparison, Ca² uptake and Na /K -ATPase activity were significantly elevated in MR cell-rich preparations relative to the traditional DSI or PV cell-rich cultures under symmetrical conditions. This new methodology enhances our ability to tailor cultured gill epithelia on semipermeable supports with different proportions of PV cells and MR cells, thereby illuminating the ionoregulatory functions of the two cell types.

A central feature of intussusceptive angiogenesis is the development of an intravascular pillar that bridges the opposing sides of the microvessel lumen. In this report, we created polydimethyl siloxane (PDMS) microchannels with geometric proportions based on corrosion casts of the colon microcirculation. The structure of the PDMS microchannels was a bifurcated channel with an intraluminal pillar in the geometric center of the bifurcation. The effect of the intra-luminal pillar on particle flow paths was investigated using an in vitro perfusion system. The microchannels were perfused with fluorescent particles, and the particle movements were recorded using fluorescence videomicroscopy. We found that the presence of an intravascular pillar significantly decreased particle velocity in the bifurcation system (p<0.05). In addition, the pillar altered the trajectory of particles in the center line of the flow stream. The particle trajectory resulted in prolonged pillar contact as well as increased residence time within the bifurcation system (p<0.001). Our results suggest that the intravascular pillar not only provides a mechanism of increasing resistance to blood flow but may also participate in spatial redistribution of cells within the flow stream.

In vitro culture systems are needed to mimic in vivo epithelial cell environments for identifying cell signaling, gene expressions, and molecular mechanisms. One such system is matrix-coated transwell cultures. However, no data exist on culturing Sertoli cells in this manner with respect to testosterone-regulated gene expression. Because the TM4 mouse Sertoli-like cell line expresses androgen receptor, our objective was to determine if testosterone treatment added to the bottom chamber of a matrix-coated transwell system induces some gene expressions found in Sertoli cells in vivo. After serum starvation, transwell-cultured TM4 cells were treated with testosterone or left untreated for 24 h. Microarray analyses initially identified differentially expressed genes either induced or repressed by testosterone treatment. By Northern blot analyses, Pem mRNA, a frequently used marker of Sertoli cell testosterone responsiveness, was induced. Proteins of the transcripts induced by testosterone in the in vitro system were immunolocalized to Sertoli cells in testosterone-dependent stages of spermatogenesis in mouse testes. By immunohistochemistry analyses of sectioned mouse testes, gene expression induced by testosterone in transwell-cultured TM4 cells, profilin as well as secreted protein acidic and rich in cysteines (SPARC) are localized to Sertoli cells in testosterone-dependent stages of spermatogenesis. Findings include localizations of SPARC and profilin, as well as an apparent germ cell communication required for translation of Pem mRNA in Sertoli cells. Taken together, results of these studies suggest that this TM4 transwell-culture system could be used to study these testosterone-regulated Sertoli gene expressions in vitro.

Some functional parameters were identified and assessed in a tissue-engineered self-assembled skin substitute. This skin substitute was produced using fibroblasts and keratinocytes isolated from adult human skin. Keratinocytes were seeded on a dermal layer, composed of two fibroblast sheets cultured for 35 d. The epidermal cells formed a stratified and cornified epidermis and expressed differentiation markers, notably involucrin and transglutaminase. Interestingly and for the first time, the receptor for vitamin D3 was detected in all of the epidermal cell layers of the skin substitute, as it is reported for normal human skin. This observation suggests that keratinocytes retain key receptors during their differentiation in the skin model. A network of collagen fibers was observed by electron microscopy in the dermal layer of the model. In the dermis, collagen fibers remodeling and assembly is dependent on enzymes, notably prolyl-4-hydroxylase. For the first time in a skin construct, the expression of prolyl-4-hydroxylase was detected in dermal fibroblasts by in situ hybridization. The secretion of collage-nases by the cells seeded in our skin substitute was confirmed by zymography. We conclude that the self-assembly approach allows the maintenance of several functional activities of human skin cells in a skin model in vitro.

Primary cell cultures from crustacea have been initiated since the 1960s, yet no permanent cell line is available. Primary cells have a limited proliferative capacity in culture due to cellular senescence, which is regulated by a group of dominant senescence genes. The aim of this research was to manipulate cell cycle regulation by transfecting Cherax quadricarinatus primary cells with oncogenes, in an effort to induce a permanent cell line. Human papillomaviruses (HPV) play a critical role in the formation of anogenital cancer. Research has demonstrated that the HPV-expressed E6 and E7 proteins function concomitantly to disrupt the p53 and retinoblastoma (Rb) tumor suppressor genes, regulators of the cell-cycle checkpoints at the first gap (G₁) phase. HPV E6 and E7 genes were transfected into the C. quadricarinatus cells by lipofection. Successful transfection was demonstrated by the presence of oncogene messenger RNA by reverse transciptase polymerase chain reaction. At day 150, transfected cells still remain viable, although cell proliferation was stagnant. It may be that while transfection of the oncogenes was successful, no proliferation of the C. quadricarinatus cells was evident due to a lack of telomere maintenance.

Serum of animal origin, like foetal calf serum (FCS), is used as a standard supplement for media to cultivate mammalian cells, mostly due to its growth-supporting properties. Unfortunately, animal serum has many disadvantages like the risk of contamination, high costs, fluctuations within the composition of different batches and the high amount of foetuses, which have to be harvested. To avoid all this, it is necessary to provide alternatives, which combine as many positive properties of the animal serum as possible but do not influence the cellular metabolism negatively. Today, several serum-free complete media as well as serum substitutes are commercially available. In the present study, a serum substitute, a serum-reduced medium and a serum-free medium were evaluated concerning their influence on the metabolism on the colon cancer cell line SW-480. The evaluation of morphological changes of the cells was done by microscopic analysis whereas differences in the volatile meta-bolome were analysed by solid phase micro extraction (SPME) followed by gas chromatography/mass spectrometry (GC/MS).

Today there is a concern about the use of animal source proteins and peptides in cell culture applications due to potential contamination by adventitious infectious pathogens. Recombinant production of these proteins using a plant host provides a safe and cost effective alternative. In this paper, we tested the effect of rice-derived recombinant human lactoferrin (rhLF) on mammalian cell growth. The purified rhLF was partially (about 50%) iron-saturated (pis-rhLF). Chemical modification of pis-rhLF generated apo-rhLF (<10% iron saturation) or holo-rhLF (>90% iron saturation). All three forms of rhLF (pis, apo, holo) promoted growth of intestinal cells (HT-29) measured as [³H]-thymidine incorporation or viable cell count, but holo-rhLF was most effective. Holo-rhLF was further tested on hybridoma, osteoblast, and human embryonic kidney cells. Results showed that holo-rhLF promoted cell growth and reduced cell doubling time. The concentration of holo-rhLF in media was critical in promoting cell growth and each cell line had different concentration dependence with the most effective range from 5 to 200 mg/L. The effect of rhLF on antibody production was determined using a hybridoma cell line. Significantly, more antibodies were produced by cells grown with holo-rhLF than cells grown without holo-rhLF. We also compared the effect of holo-rhLF to that of human transferrin, a component commonly used in cell culture media as an iron source. Holo-rhLF was as effective as human transferrin in promoting cell growth and antibody production. Considering all the data obtained, we conclude that rhLF from rice is effective in promoting mammalian cell growth and increasing cell productivity.

Bone marrow stromal cells (BMSCs) have gained considerable attention as a potential source for cell transplantation therapies for a variety of diseases due to their accessibility, proliferative capacity, and multilineage differentiation properties. Canine BMSCs have been shown to contribute to regeneration of osseous tissues, but knowledge about their biology is currently limited. In the present study, we investigated the frequency of adult canine BMSCs in bone marrow, morphological features, growth kinetics, and osteogenic as well as adipogenic differentiation properties in vitro. Our data suggest that adult canine bone marrow contains approximately one BMSC in every 2.38×10⁴ bone marrow mononucleated cells (0.0042± 0.0019%, n=5). Primary BMSC cultures consisted of morphologically heterogeneous adherent cell populations from which spindle-shaped cells grew and became the predominant cell type. Growth kinetics patterns were dependent on the initial cell seeding densities, resulting in the highest fold increase at lower cell density. In the presence of osteogenic and adipogenic inducers, primary BMSCs underwent morphological and phenotypic changes characteristic of osteogenic and adipogenic differentiation, respectively. This study provides insights into basic characterization of adult canine BMSCs.