Ceiling Culture Research Articles

Ceiling Culture-Derived Proliferative Adipocytes are A Possible Delivery Vehicle for Enzyme Replacement Therapy in Lecithin: Cholesterol Acyltransferase Deficiency

Human proliferative adipocytes propagated via ceiling culture technique from subcutaneous fat tissue (desig- nated as ccdPA) were herein evaluated for their potential as a recipient for retroviral vector-mediated gene transduction of a therapeutic protein delivery. Exposure to the ZsGreen-expressing vector supernatant using a cell preparation generated by a 7-day ceiling culture induced a 40-50% transduction efficiency, with less than two integrated copies of viral genome per cell on average. The lcat gene-transduced human ccdPA secreted functional LCAT protein, correlating with the inte- grated copy number of vector genome. The gene-transduced cells could be expanded up to nearly 10 12 cells from 1 g of fat tissue within one month after fat tissue preparation. The cells also maintained the potential to differentiate into adipocytes in vitro. The presence of human LCAT protein in serum was immunologically identified upon transplantation of lcat- expressing ccdPA into the adipose tissue of immune-deficient mice. These results indicated that human ccdPA has a novel therapeutic potential for LCAT-deficient patients. The clinical application in combination with cell transplantation shed a light on a development of a life-long protein replacement therapy for LCAT-deficient patients.

Effects of MicroRNA-143 in the differentiation and proliferation of bovine intramuscular preadipocytes

MicroRNAs (miRNAs) are short non-coding RNA that post-transcriptionally regulates gene expression. miRNA-143 has been proposed to play a role in the differentiation of preadipocytes. However, effects and mechanism of miRNA-143 in the differentiation of mammals intramuscular adipocytes is unknown. In this study, the fibroblast-like preadipocytes were cultured from the marbling muscle tissue of holstein steers by the ceiling culture method. The in vitro studies showed that the fibroblast-like preadipocytes could differentiated into mature adipocytes with up-regulated expression of miRNA-143. Furthermore, the transfection of the fibroblast-like preadipocytes with miRNA-143 antisense inhibitor induced a significant suppression of differentiation, and indicated by decreased storage of lipid droplets and down-regulated expression of key adipocytes regulatory genes such as CCAAT/enhancer-binding protein-α (C/EBPα) and fatty acid binding proteins-4 (FABP-4). On the contrary, cells proliferation were increased with miRNA-143 inhibitor transfection. Taken together, our study provide the first evidence for stimulation of endogenous miRNA-143 in the differentiation of bovine intramuscular fat, which in part contribute to the regulated expression of adipocyte genes.

Human mesenchymal adipose stromal cells from mature adipocyte fraction

AbstractMore effective techniques should be employed for isolation of human mesenchymal stromal cells derived from adipose tissue (ADSC), seeking to make adipose tissue biopsies smaller in volume and thus less invasive. In this study, we compared properties of ADSC isolated by several different methods from the same samples of adipose tissue in order to enhance yields of potential ADSC. The mature adipocyte fraction was investigated using the ceiling culture method, including both ceiling and bottom cell fractions, and the control culture method with standard amount of medium. The results were also compared using the stromal vascular fraction from the same samples. The most efficient was the bottom cell population isolated from the mature adipocyte fraction by ceiling culture method. These cells readily differentiated into osteogenic, adipogenic and chondrogenic lineages and, similar to stromal vascular fraction cells, displayed high proliferation potential. Cultures of mature adipocyte fractions with standard amount of medium were considerably less effective. Mature adipocyte fractions yields large quantities of adipose-derived stem cells that have properties comparable with stromal vascular fraction cells suitable for tissue regeneration, especially when only small biopsies can be taken.

Dedifferentiated fat cells convert to cardiomyocyte phenotype and repair infarcted cardiac tissue in rats

Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes, indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model, transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (p<0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition, transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease.

Links between De Novo Fatty Acid Synthesis and Leptin Secretion in Bovine Adipocytes

Leptin secretion by adipose tissue is involved in many physiological control systems, including those that determine growth, development, body composition, milk production, and reproductive function. In the adipocyte of monogastric animals, malonyl CoA (coenzyme A) seems to link the flux of energy substrates to the control of leptin production. In this study, we tested this for ruminants by examining the effect of cerulenin, an inhibitor of de novo fatty acid synthesis at the step from malonyl CoA to palmitate, on leptin production by cultured bovine adipocytes derived from intermuscular fat. Purified preadipocytes were obtained by the ceiling culture method, and adipogenic media were used to induce their differentiation into adipocytes. We found that leptin concentrations increased significantly with time in culture, and with increases in glucose concentration. Addition of 2-deoxy-D-glucose to the medium, a competitive inhibitor of glucose transport and metabolism, suppressed leptin secretion. In media with high glucose concentrations, cerulenin enhanced leptin secretion. We conclude that, as in monogastrics, malonyl CoA may play a key role in the control of leptin secretion in ruminants.

Ceiling culture of mature human adipocytes: use in studies of adipocyte functions.

Adipocytes contain large lipid droplets in their cytoplasm. When cultured, they float on top of the medium, clump together, and do not gain equal and sufficient access to the medium. Morphological changes cannot be observed and the majority of adipocytes undergo cell lysis within 72 h of isolation. We have used a ceiling culture method for human mature adipocytes which uses their buoyant property to allow them to adhere to a floating glass surface, where they remain viable for several weeks. Using confocal immunofluorescence microscopy we showed the cellular expression and subcellular localization of leptin in ceiling-cultured adipocytes. The secretion of leptin was increased from ceiling cultures following tumour necrosis factor-alpha treatment. Proliferation of mature human adipocytes in serum-containing medium was demonstrated by incorporation of bromodeoxyuridine, 2% of adipocytes showing positive incorporation after 4 h labelling. Proliferation was also evident from the budding of daughter cells. Apoptosis in the ceiling cultures was increased by 48 h serum deprivation (30-35 vs 10-15% in the control) and was assayed by propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling. Lipolysis, analysed by liquid scintillation counting, was increased by forskolin (10 microM for 90 min) and lipogenesis, shown by autoradiography, was stimulated by insulin (10 and 100 nM for 4 h). These findings indicate that ceiling-cultured adipocytes maintain adipocyte-specific functions and that ceiling culture, which overcomes the shortcomings of adipocyte suspension culture, can be used to study adipocyte cell biology.

Thoughts on the source of tissue on subsequent cell culture success.

This paper describes attempts to initiate equine adipocyte cultures from necropsy cases with varying intervals from time of death to isolation and culture. Equine adipocytes were isolated from 21 necropsy cases, regardless of the interval from time after death to establishment in primary ceiling cultures. However, while all cultures produced adipocytes, only 2 attempts to produce long-term equine adipocyte cultures from the subcutaneous rump fat depots were successful and not contaminated. Findings from these experiments indicate that it is possible to collect and culture equine adipocytes from necropsy cases with varying intervals of time of death to culturing provided that the issue of contamination is addressed. Viable cells were produced from tissue with an interval of 38.5 hours as well as 45 minutes. This result encourages the continuation of research using equine necropsy cases as a source of adipose tissue.

Intrinsic site-specific differences in the expression of leptin in human adipocytes and its autocrine effects on glucose uptake.

Leptin, the ob gene product of adipocytes, regulates body weight by actions on the satiety center in the hypothalamus, but it may also have peripheral effects on the metabolic actions of insulin. In human mature adipocytes isolated from omental (OM) and s.c. tissue, we found that leptin (10 and 100 ng/mL) significantly reduced insulin-mediated glucose uptake by 40% (P<0.05). The effects were rapid and sustained. A U-shaped dose-response curve was obtained, and high leptin concentrations (>100 ng/mL) were without effect. Leptin did not affect basal glucose uptake in adipocytes and had no effect on insulin-stimulated glucose uptake in human preadipocytes. Because leptin may thus have autocrine effects, we examined leptin production from OM and s.c. adipocytes. Western blotting of leptin from 96-h conditioned medium showed greater leptin secretion from s.c. than OM adipocytes, with a ratio of 3.2 (SE +/-0.3, P<0.01). Long-term ceiling cultures were used to examine intrinsic differences in leptin expression under closely controlled conditions. Confocal immunofluorescence microscopy of 12- to 16-day-old ceiling-cultured adipocytes showed that sc adipocytes contained 3.4-fold more leptin (SE +/-0.5, P<0.01) than OM adipocytes, indicating an intrinsic site-specific difference in leptin production. The autocrine effects of leptin to inhibit insulin-stimulated glucose uptake and subsequent lipogenesis in adipose tissue may, therefore, be less in OM adipocytes and may play a role in determining visceral obesity.

Proliferation and differentiation of unilocular fat cells in the bone marrow.

Fat cells contribute not only to systemic lipid metabolism, but also to osteogenesis and hemopoiesis in the bone marrow. The present study represents the first attempt to culture mature unilocular fat cells of the bone marrow. Two methods devised in our laboratory were employed: one is the "ceiling culture method" that utilizes the floating property of the cells; the other, a three-dimensional collagen gel matrix culture that captures unilocular fat cells in the gel matrix. Using these methods, the proliferation of unilocular fat cells from the bovine metacarpal bone marrow was demonstrated. First, we confirmed the proliferative ability of unilocular fat cells derived from the bone marrow, using autoradiography to study 3H-thymidine incorporation into the nuclei. The unilocular fat cells de-differentiated into fibroblast-like fat cells and then proceeded to proliferate. When they underwent a contact inhibition of growth, re-differentiation from fibroblast-like fat cells into unilocular fat cells occurred at a high rate. A specific enzymatic marker of the fat cell, alpha-glycerophosphate dehydrogenase activity related to lipogenesis, was then demonstrated in the cultured fat cells. We examined the functional reactivity of the fat cells by treatment with insulin and cyclic-AMP, and both lipogenesis and lipolysis were also confirmed in them. We concluded that unilocular fat cells from the bone marrow de-differentiated, proliferated and re-differentiated in culture. The present results may help to clarify the various functions of fat cells in the bone marrow.

A Versatile, Cheap, and Easy Self-Made Cell Culture Dish

Abstract Anybody who has ever worked with cells or tissues in culture knows that the dish in which to grow and look at the specimen can be of crucial importance to the results obtained, With the growing importance of live cell microscopy, be it simple phase contrast or sophisticated multilabel confocal laser scanning microscopy (CLSM), people have been looking for dishes that combine optimal growth conditions with optimal optical properties. As follows, I will describe a solution that has worked for us with a multitude of different cells, tissues and applications. Apart from that, it is cheap and easily self-made. The bottom of a standard 35 mm plastic ceil culture dish has a hole of ca. 10 mm drilled into it and a coverslip glued to its downside to cover the hole. For a glue we usually use Sylgard, an inert silicone elastomer (Dow Corning) or melted paraffin if we intend to take off the coverslip again.

Two alternative procedures for isolating adipofibroblasts from sheep skeletal muscle

Methods to isolate cells used in the study of adipocyte differentiation are lengthy, potentially damaging to the cells collected, and usually result in a mixed group of cells which are difficult to define clearly. Additionally, much work done on the differentiation of fibroblasts to preadipocytes or preadipocytes to adipocytes has relied on the use of populations of cells which are either embryonic in origin or from genetically unique animals. We therefore report two simple and rapid protocols for obtaining relatively pure populations of preadipocytes from perimuscular fat and from intramuscular fat depots of normal sheep skeletal muscle. In the first procedure, a finely minced preparation of perimuscular adipose tissue is placed directly into flasks for ceiling culture. During the second procedure, free-floating adipocytes, resulting from the enzymatic preparation of satellite cells from skeletal muscle, are placed into ceiling culture. Cells from both isolation procedures attach, grow, and are later harvested for use. These cells demonstrate proliferation and differentiation abilities of normal preadipocytes. Cell populations may be expanded for use in cloning pure populations of adipocytes or used directly in studies to identify mechanisms of adipocyte development and intercellular communication with myogenic cells.

Evaluación de tres técnicas citogenéticas diferentes en los estudios morfométricos del carioti po de Aedes taeniorhynchus (Di ptera: culicidae

Morphometric studies of the Aedes taeniorhynchus mosquito karyotype are of veterinary medical interest because this mosquito is a vector of epidemo-epizootic Venezuelan equine encephalitis. Three different cytogenetic techniques were used for chromosome preparation: squash, air-drying and cell culture. These three techniques were compared to each other to evaluate the obtained metaphases' chromosome length and morphology. The diploid number of the species was 6 in al1 the cariological preparations when any one of the three procedures was used. However, with the squash technique, brain-cell mytotic chromosomes were shorter, discontinuous and had less apparent grades of chromatin compaction. When air-drying was used, better cytological extensions were obtained, the chromosomes being more separated and of a slightly greater length. The best resolution was obtained using ceil culture because of the structure and separation of chromosome homology and length.

Folliculostellate cells of the pituitary.

In addition to the hormone-producing granulated cells, the so-called folliculostellate (FS) cells of the adenohypophysis represent a population of nongranulated cells extensively described in a large number of species. They show distinctive morphological features including a star shape with thin cytoplasmic projections extending between granulated cells and well-developed junctional complexes. FS cells are joined together surrounding irregular microcavities and project microvilli into the lumina. The immunocytochemical localization of S-100 protein, glial fibrillary acidic protein, and vimentin constitutes a reliable and easy method for investigating their presence and distribution in the normal pituitary gland and in pituitary adenomas. Although the expression of glial cell markers raised the hypothesis of a neuroectodermal origin of FS cells, most evidence supports that they derive from the epithelium of the Rathke's pouch, as do granulated adenohypophyseal cells. Morphological studies indicate that FS cells are involved in phagocytosis and possess sustentacular functions. Investigations using cell cultures show that FS cells play important roles in the paracrine regulation of adenohypophy-seal secretion by their ability to liberate several growth factors and regulate the ionic composition of the extracellular fluid. Further research using novel immunocytochemical markers and ceil culture techniques may clarify the origin and the role of this enigmatic cell type in the normal and pathological pituitary gland.

Susceptibility of various ceil culture systems to pseudorabies virus

A comparative study was carried out to determine the susceptibility of five different cell lines to pseudorabies virus (PRV), a herpes virus of pigs. The cell systems tested were swine testicle (ST), mink lung (ML), equine dermal (ED), porcine kidney (PK 15), and bovine turbinate (BT) cells. Virus titers obtained were 10 4.88, 10 4.38, 10 3.75, 10 2.63, and 10 0.25 for ML, ST, PK 15, BT and ED cells, respectively indicating that ML, ST, and PK 15 are optimal cell lines for the growth of PRV whereas BT and ED are not very sensitive.

The effects of androgens and estrogens on human prostatic cells in culture.

A ceil culture system has been developed in order to assess the response of human prostatic carcinoma cells to the presence of androgens and estrogens. A single cell suspension of these cells was incubated in minimum essential medium, in the absence of added sex steroids (standard culture), for 24 h. Equal aliquots of these cultured cells were incubated for a further 24–72 h in standard medium or in medium to which testosterone (T, 17.4 nM), dihydrotestosterone (DHT, 1.7 nM) or estradiol (E2, 0.36 nM) had been added. At the end of the incubations, the levels of tartrate-inhibitable acid phosphatase were compared in the standard cultures and the cultures to which sex steroids had been added.There were three patterns of response of patients' cells to the presence of androgens. From a total of 91 cell cultures, obtained from 85 patients, androgens were associated with an increased acid phosphatase production in 58. In 22 incubations, there was no androgenic effect on acid phosphatase production, and in II incubations, androgens were associated with lower enzyme levels than in the standard cultures. The qualitative effects of T and DHT were the same, but the quantitative effect of DHT was greater than that of T in those experiments in which there was a positive androgenic effect on acid phosphatase production. In 10 of 46 incubations with E2, acid phosphatase levels were lower than the standard incubations, while in 28 there was no difference in enzyme levels. In eight incubations, E2 was associated with a greater acid phosphatase production than that found in the standard cultures.In cultures of the cells from 180 men with benign prostatic hyperplasia and 5 men with normal prostatic tissue, androgens and estrogens had no effect on acid production by these cells, even at DHT concentrations of 1.7 × 10−7 M.It is concluded that prostatic carcinoma cells behave differently, in culture, in response to sex steroids, than do cells from benign hyperplastic or normal prostatic tissue.