Showing papers on "Tissue culture published in 2004"

Type I collagen promotes the malignant phenotype of pancreatic ductal adenocarcinoma.

Abstract: Purpose: The purpose of this study was to determine the role of functional interactions between pancreatic cancer cells and pancreatic stellate cells (PSCs) in the formation of the desmoplastic reaction (DR) in pancreatic cancer and to characterize the effect of type I collagen (the predominant component of the DR) on pancreatic cancer cell phenotype. Experimental Design: PSCs and type I collagen were identified in sections of pancreatic cancer using immunohistochemistry, and their anatomic relationship was studied. Interactions among pancreatic cancer cell lines (MIA PaCa-2, Panc-1, and AsPC-1), primary cultures of human PSCs, and type I collagen were investigated in a series of tissue culture models. Results: In vivo, the DR causes gross distortion of normal pancreas, bringing cancer cells into close contact with numerous PSCs and abundant type I collagen. In tissue culture models of pancreatic cancer, conditioned media from each cell line increased PSC [ 3 H]thymidine incorporation up to 6.3-fold that of controls, and AsPC-1 cells also increased PSC collagen synthesis 1.3-fold. Type I collagen was observed to increase long-term survival of pancreatic cancer cells treated with 5-fluorouracil, by up to 62% in clonogenic assays. This was because type I collagen increased the proliferation of cancer cells ([ 3 H]thymidine incorporation was up to 2.8-fold that of cells cultured on tissue culture plastic) and reduced apoptosis of AsPC-1 cells in response to 5-fluorouracil (by regulating mcl-1). Conclusions: These experiments elucidate a mechanism by which the DR in pancreatic cancer may form and, via the collagen within it, promote the malignant phenotype of pancreatic cancer cells, suggesting significant detriment to the host.

MMP‐2 plays an essential role in producing epithelial‐mesenchymal transformations in the avian embryo

Abstract: To investigate the roles that matrix-degrading proteases may have in development of the chicken embryo, we documented the expression pattern of matrix metalloprotease-2 (MMP-2, 72-kDa type IV collagenase or gelatinase A) and perturbed its function in vitro and in vivo. MMP-2 is expressed as neural crest cells detach from the neural epithelium during an epithelial-mesenchymal transformation (EMT) but is rapidly extinguished as they disperse. It is also expressed in the sclerotome and in the dermis at the time that the EMT is initiated, and also as these cells migrate, and is down-regulated once motility has ceased. These patterns suggest that MMP-2 plays a role in cell motility during the EMT and during later morphogenesis. Inhibitors of MMPs, including BB-94 and TIMP-2 (tissue inhibitor of metalloprotease-2), prevent the EMT that generates neural crest cells, both in tissue culture and in vivo, but do not affect migration of the cells that have already detached from the neural tube. Similarly, knockdown of MMP-2 expression in the dorsal neural tube using antisense morpholino oligos perturbs the EMT, but also does not affect migration of neural crest cells after they have detached from the neural tube. On the other hand, when somites in culture are treated with TIMP-2, some mesenchymal cells are produced, suggesting that they undergo the EMT, but show greatly reduced migration through the collagen gel. MMP-2 is also expressed in mesenchyme where tissue remodeling is in progress, such as in the developing feather germs, in the head mesenchyme, in the lateral plate mesoderm, and in the limb dermis, especially in the regions where tendons are developing. Comparisons of these expression patterns in multiple embryonic tissues suggest a probable role for MMP-2 in the migration phase of the EMT, in addition to mesenchyme dispersion and tissue remodeling. Developmental Dynamics 229:42-53, 2004.

Bud endophytes of Scots pine produce adenine derivatives and other compounds that affect morphology and mitigate browning of callus cultures.

Abstract: Endophytes are found in meristematic bud tissues of Scots pine (Pinus sylvestris L.) especially prior to growth, which would suggest their involvement in growth of the bud. To test this hypothesis, production of phytohormones by two bacterial (Methylobacterium extorquens, Pseudomonas synxantha) and one fungal endophyte (Rhodotorula minuta) was studied by mass spectrometry. The most common gibberellins, auxins, or cytokinins were not detected in the fractions studied. Instead, M. extorquens and R. minuta produced adenine derivatives that may be used as precursors in cytokinin biosynthesis. A plant tissue culture medium was conditioned with the endophytes, and pine tissue cultures were started on the media. Tetracycline inhibited callus production, which was restored on the endophyte-conditioned media. In addition, conditioning mitigated browning of the Scots pine explants. However, a decrease in tissue size was observed on the endophyte-conditioned media. Addition of adenosine monophosphate in the plant culture medium restored callus production and increased growth of the tissues, but had no effect on browning. Therefore, production of adenine ribosides by endophytes may play some role in the morphological effect observed in the pine tissues.

Activation of a rice endogenous retrotransposon Tos17 in tissue culture is accompanied by cytosine demethylation and causes heritable alteration in methylation pattern of flanking genomic regions

Abstract: Tos17 is a copia-like, cryptic retrotransposon of rice, but can be activated by tissue culture. To study possible epigenetic mechanism controlling activity of Tos17, we subjected three rice lines (the parental line cv. Matsumae and two introgression lines, RZ2 and RZ35) that harbor different copies of the element to tissue culture. For each line, we investigated transcription and transposition of Tos17 in seed plants, calli and regenerated plants, cytosine-methylation status at CG and CNG positions within Tos17, effect of 5-azacytidine on methylation status and activity of Tos17, and cytosine-methylation states in genomic regions flanking original and some newly transposed copies of Tos17 in calli and regenerated plants. We found that only in introgression line RZ35 wasTos17 transcriptionally activated and temporarily mobilized by tissue culture, which was followed by repression before or upon plant regeneration. The activity and inactivity of Tos17 in calli and regenerated plants of RZ35 are accompanied by hypo- and hyper-CG methylation and hemi- and full CNG methylation, respectively, within the element, whereas immobilization of the element in the other two lines is concomitant with near-constant, full hypermethylation. Treatment with 5-azacytidine induced both CG and CNG partial hypomethylation of Tos17 in two lines (Matsumae and RZ35), which, however, was not accompanied by activation of Tos17 in any line. Heritable alteration in cytosine-methylation patterns occurred in three of seven genomic regions flanking Tos17 in calli and regenerated plants of RZ35, but in none of the five regions flanking dormant Tos17 in the other two lines.

β-Criptoxanthin stimulates bone formation and inhibits bone resorption in tissue culture in vitro

Abstract: The effect of β-cryptoxanthin, which is greatly present in fruits, has not been clarified so far on bone metabolism. The effect of β-cryptoxanthin on bone formation and bone resorption was investigated in tissue culture in vitro. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10–8–10–5 M β-cryptoxanthin. The presence of β-cryptoxanthin (10–6 or 10–5 M) caused a significant increase in calcium content, alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal and metaphyseal tissues. These increases were completely prevented in the presence of cycloheximide (10–6 M), an inhibitor of protein synthesis. β-Carotene (10–6 or 10–5 M) or xantine (10–6 or 10–5 M) had no effect on the diaphyseal and metaphyseal calcium contents. The bone-resorbing factors parathyroid hormone (1–34) (PTH; 10–7 M) or prostaglandin E2 (PGE2; 10–5 M) caused a significant decrease in calcium content in the diaphyseal and metaphyseal tissues. The decrease in bone calcium content induced by PTH or PGE2 was completely inhibited by β-cryptoxanthin (10–8−10–6 M). In addition, β-cryptoxanthin (10–8−10–6 M) completely inhibited the PTH (10–7 M)- or PGE2 (10–5 M)-induced increase in medium glucose consumption and lactic acid production by diaphyseal and metaphyseal tissues. The inhibitory effect of β-cryptoxanthin (10–7 M) on PTH (10–7 M)- or PGE2 (10–5 M)-stimulated decrease in the diaphyseal calcium content was significantly prevented in the presence of 10–3 M vanadate, an inhibitor of protein tyrosine phosphatase. Vanadate (10–3 M) did not have a significant effect on calcium content and lactic acid production in control bone tissues. The present study demonstrates that β-cryptoxanthin has a direct stimulatory effect on bone formation and an inhibitory effect on bone resorption in tissue culture in vitro.

Factors affecting somatic embryogenesis and plant regeneration from a range of recalcitrant genotypes of chinese cottons (gossypium hirsutum l)

Abstract: A new protocol has been developed for the highly efficient somatic embryogenesis and plant regeneration of 10 recalcitrant Chinese cotton cultivars. Calluses and embryogenic calluses were induced on MSB1 medium containing the optimal combination of indolebutyric acid (IBA; 2.46 μM) and kinetin (KT; 2.32 μM). Up to 86.7% of embryogenic calluses differentiated into globular somatic embryos 2 mo. after culture on MSB2 medium containing double KNO3 and free of growth regultors. Up to 38.3% of the somatic embryos were converted into complete plants in 8 wk on MSB3 medium with l-asparagine (Asn)/l-glutamine (Gln) (7.6/13.6 mM). The plants were successfully transferred to soil and grew to maturity. With the protocol described here, we have obtained hundreds of regenerating plantlets from 10 recalcitrant cultivars, which is important for the application of tissue culture to cotton breeding and biotechnology.

Developments in xenobiotic-free culture of human keratinocytes for clinical use

Abstract: We have recently reported that irradiated human fibroblasts can be used as a feeder layer, to expand keratinocytes under serum-free conditions, on a chemically defined plasma polymer surface developed for the culture and transfer of keratinocytes for clinical use. While this is a significant advance in developing a serum-free keratinocyte culture approach, the need to irradiate fibroblasts to growth-arrest them and prevent them from overgrowing the keratinocytes introduces another small, but potential, risk for the patient. The aim of the present study was to develop conditions for the coculture of normal human keratinocytes with nonirradiated normal human fibroblasts under serum-free conditions. We examined the fibroblast/keratinocyte relationship on three separate surfaces: tissue culture plastic, non-tissue culture plastic, and a plasma polymer surface designed for clinical use. We report that it is possible to achieve rapid and successful expansion of human keratinocytes under serum-free conditions on all three surfaces providing one uses a keratinocyte-friendly media, a minimum seeding density of keratinocytes, and a ratio of fibroblasts to keratinocytes that does not exceed 1 : 1. These results provide us with a rapid laboratory expansion of proliferative human keratinocytes, under completely defined culture conditions, without any xenobiotic cells (mouse fibroblasts) or material (bovine serum), for the treatment of patients with extensive skin loss.

Effect of parent genotype on somatic embryogenesis in Scots pine (Pinus sylvestris).

Abstract: Controlled crosses of seven Scots pine (Pinus sylvestris L.) trees produced 49 families that included both reciprocals and selfings. Embryogenic cultures were initiated from immature megagametophytes and after 6 months in maintenance culture, mature somatic embryos were produced from the surviving 166 lines. The effect of parent genotypes on the cultures was evaluated at initiation of the tissue culture period, after 6 months in maintenance culture and at embryo maturation. The effect of the maternal parent was most pronounced at culture initiation. After 6 months in tissue culture, the maternal effect had decreased and the effects of both parents were significant. By the somatic embryo maturation stage, the maternal effect was still considerable but the paternal effect was no longer detectable. There was little correlation between the ranking of mothers and fathers, indicating that the maternal effect was caused by factors other than the paternal effect. No mother × father interaction was found, indicating that mothers successful at initiation and after 6 months in tissue culture, pollinated by any of the successful fathers, produced somatic lines and mature somatic embryos.

Osteogenic Cells Derived From Embryonic Stem Cells Produced Bone Nodules in Three-Dimensional Scaffolds.

Abstract: An approach for 3D bone tissue generation from embryonic stem (ES) cells was investigated. The ES cells were induced to differentiate into osteogenic precursors, capable of proliferating and subsequently differentiating into bone-forming cells. The differentiated cells and the seeded scaffolds were characterized using von Kossa and Alizarin Red staining, electron microscopy, and RT-PCR analysis. The results demonstrated that ES-derived bone-forming cells attached to and colonized the biocompatible and biodegradable scaffolds. Furthermore, these cells produced bone nodules when grown for 3–4 weeks in mineralization medium containing ascorbic acid and beta-glycerophosphate both in tissue culture plates and in scaffolds. The differentiated cells also expressed osteospecific markers when grown both in the culture plates and in 3D scaffolds. Osteogenic cells expressed alkaline phosphatase, osteocalcin, and osteopontin, but not an ES cell-specific marker, oct-4. These findings suggest that ES cell can be used for in vitro tissue engineering and cultivation of graftable skeletal structures.

Agrobacterium tumefaciens-mediated transformation of chickpea (Cicer arietinum L.): gene integration, expression and inheritance

Abstract: A reproducible method of Agrobacterium-mediated transformation was developed for Cicer arietinum (chickpea). Initial explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. The plasmid contained a bi-functional fusion gene conferring both β-glucuronidase and neomycin phosphotransferase activities, under the control of a 35S35SAMV promoter. Using a series of tissue culture media for co-cultivation, shoot initiation and rooting, we recovered transgenic plants from approximately 1.3% of the sliced embryo axes. The addition of a shoot elongation medium to the protocol improved the success rate to 3.1% but increased the time in tissue culture. Inheritance of the gus gene was followed through four generations, both through expression and Southern hybridization assays, and showed the expected Mendelian inheritance pattern.

Callus induction and regeneration in Spirodela and Lemna.

Abstract: The development of tissue culture systems in duckweeds has, to date, been limited to species of the genus Lemna. We report here the establishment of an efficient tissue culture cycle (callus induction, callus growth and plant regeneration) for Spirodela oligorrhiza Hegelm SP, Spirodela punctata 8717 and Lemna gibba var. Hurfeish. Significant differences were found among the three duckweed species pertaining to carbohydrate and phytohormone requirements for callus induction, callus growth and frond regeneration. In vitro incubation with poorly assimilated carbohydrates such as galactose (S. oligorrhiza SP and L. gibba var. Hurfeish) and sorbitol (S. punctata 8717) as sole carbon source yielded high levels of callus induction on phytohormone-supplemented medium. Sorbitol is required for optimal callus growth of S. oligorrhiza SP and S. punctata 8717, while sucrose is required for callus growth of L. gibba var. Hurfeish. Sucrose either alone (S. oligorrhiza SP, L. gibba var. Hurfeish) or in addition to sorbitol (S. punctata 8717) is required for frond regeneration.

Microbial Superantigens Induce Glucocorticoid Receptor β and Steroid Resistance in a Nasal Explant Model

Abstract: Objective To study the role of superantigen (SAg) in inducing glucocorticoid (GC) receptor beta and steroid resistance in an explant model of nasal tissue. Methods Nasal tissue was obtained from inferior turbinates of controls and ragweed (RW)-sensitive patients. Tissue samples were incubated with SAg of staphylococcal enterotoxin B. In addition, tissue samples from RW-sensitive patients were incubated with RW allergen in the presence and absence of both SAg and dexamethasone (DEX). The expression of GC receptor beta was assessed by immunocytochemistry. The expression of interleukin (IL)-2 and IL-4 mRNA was assessed by in situ hybridization. Results SAg induced an increase in the expression of GC receptor beta in atopic tissue and to a lesser extent in nonatopic tissue. The most significant induction of GC receptor beta was observed in response to SAg and RW in atopic tissue. Stimulation of atopic tissue with RW alone and SAg alone induced IL-4 and IL-2 mRNA, respectively. Incubation of atopic tissue with both SAg and RW induced both IL-2 and IL-4 mRNA. The increase in IL-4 mRNA expression was blunted by the addition of DEX to atopic tissue stimulated with RW alone but not to tissue stimulated by both RW and SAg. Conclusion Our results demonstrate that SAgs induce steroid resistance in atopic nasal explant tissue by up-regulating the expression of GC receptor beta. Furthermore, we have shown that the up-regulation of GC receptor beta is a local event that is associated with the coexpression of IL-2 and IL-4 mRNA.

Benefits of 8-bromo-guanosine 3′, 5′-cyclic monophosphate (8-br-cGMP) in human ovarian cortical tissue culture

Abstract: Guanosine 3',5'-cyclic monophosphate (cGMP) is an important intracellular second messenger in many cells of the body; however, its importance in the ovary is only now being realized. The effects of the cGMP analogue 8-bromo-guanosine 3',5'-cyclic monophosphate (8-br-cGMP) were tested on human ovarian follicle development and survival using an established tissue culture method. Ovarian biopsies were collected from 27 women (mean age 32 years) undergoing Caesarean sections or gynaecological operations. Tissue was cut into small pieces and cultured in the presence or absence of 5 mmol/l 8-br-cGMP for 7 and 14 days. 8-br-cGMP enhanced the rate of follicle growth to the secondary stage after both 7 and 14 days of culture. Furthermore, it significantly improved the proportion of viable follicles when compared with control cultures. All cultured follicles reaching the secondary stage were smaller than those uncultured. This suggests that although granulosa cells were stimulated to proliferate and form a double layer, the cells themselves did not become larger as usually occurs with maturation and differentiation. Oestradiol production was greater in the 8-br-cGMP-containing cultures after 12 days compared with control cultures, presumably due to the concurrent increase in the proportion of secondary follicles. In the early stages of human ovarian tissue culture, 8-br-cGMP may be a necessary component as both a growth enhancer and survival factor.

Floral induction in tissue culture: a system for the analysis of LEAFY-dependent gene regulation

Abstract: We have developed a versatile floral induction system that is based on ectopic overexpression of the transcription factor LEAFY (LFY) in callus. During shoot regeneration, flowers or floral organs are formed directly from root explants without prior formation of rosette leaves. Morphological and reporter gene analyses show that leaf-like structures are converted to floral organs in response to LFY activity. Thus, increased levels of LFY activity are sufficient to bypass normal vegetative development and to direct formation of flowers in tissue culture. We found that about half of the cultured cells respond to inducible LFY activity with a rapid upregulation of the known direct target gene of LFY, APETALA1 (AP1). This dramatic increase in the number of LFY-responsive cells compared to whole plants suggested that the tissue culture system could greatly facilitate the analysis of LFY-dependent gene regulation by genomic approaches. To test this, we monitored the gene expression changes that occur in tissue culture after activation of LFY using a flower-specific cDNA microarray. Induction of known LFY target genes was readily detected in these experiments. In addition, several other genes were identified that had not been implicated in signaling downstream of LFY before. Thus, the floral induction system is suitable for the detection of low abundance transcripts whose expression is controlled in an LFY-dependent manner.

Contribution of culture media and chemical properties of polystyrene tissue culture plates to biofilm development by Staphylococcus aureus.

Abstract: Staphylococcal biofilm development on implanted biomaterials represents an important virulence determinant in the pathogenesis of device-related infections. The prevailing environmental milieu and growth media supplements such as NaCl, ethanol, glucose and sub-inhibitory concentrations of antibiotics strongly influence biofilm development. In the laboratory, the microtitre plate assay is one of the commonestmethods used tomeasure the biofilm-forming capability of Staphylococcus aureus isolates. This semiquantitative measurement of biofilm formation involves growing bacterial cultures in individual wells of 96-well polystyrene plates according to the method of Christensen et al. (1985). In our experiments bacteria were grown at 37 8C for 24 h before being vigorously washed three times with distilled H2O and dried for 1 h at 56 8C as recommended by Gelosia et al. (2001) prior to staining with a 0.4% crystal violet solution. The absorbance of adhered, stained cells was measured at 492 nm using a Multiskan plate reader (Flow Laboratories).

Tobacco (Nicotiana tabacum L.)-A model system for tissue culture interventions and genetic engineering

Abstract: Tobacco (Nicotiana tabacum L.) has become a model system for tissue culture and genetic engineering over the past several decades and continues to remain the 'Cinderella of Plant Biotechnology', An ill vitro culture medium (Murashige and Skoog, 1962), based on the studies with tobacco tissue cultures, has now been widely used as culture medium formulation for hundreds of plant species. Studies with tobacco tissue culture have shed light on the control of ill vitro growth and differentiation. Further, induction of haploids, microspore derived embryos and selection of mutant cell lines, have been achieved successfully. Tobacco has also been employed for the culture and fusion of plant protoplasts, providing invaluable information on way to explore the potential of somatic hybridization in other crops. Optimization of genetic transformation, using Agrobacterium tumefaciens and A. rhizogenes, has been central to the cascade of advances in the area of transgenic plants. Developments in the field of molecular farming for the expression and/or production of recombinant proteins, vaccines and antibodies are gaining significance for industrial use and human healthcare.

In vivo and in vitro development of mouse pancreatic β-cells in organotypic slices

Abstract: Taking tissue slices of the embryonic and newborn pancreas is a novel approach for the study of the perinatal development of this gland. The aim of this study was to describe the morphology and physiology of in vivo and in vitro developing β-cells. In addition, we wanted to lay a foundation for the functional analysis of other pancreatic cells, either alone or as part of an integrative pancreatic physiology approach. We used cytochemistry and light microscopy to detect specific markers and the whole-cell patch-clamp to assess the function of single β-cells. The insulin signal in the embryonic β-cells was condensed to a subcellular compartment and redistributed throughout the cytosol during the first 2 days after birth. The hormone distribution correlated well with the development of membrane excitability and hormone release competence in β-cells. Endocrine cells survived in the organotypic tissue culture and maintained their physiological properties for weeks. We conclude that our preparation fulfills the criteria for a method of choice to characterize the function of developing pancreas in wild-type and genetically modified mice that die at birth. We suggest organotypic culture for in vitro studies of the development and regeneration of β-cells.

Evaluation of a continuous quantification method of apoptosis and necrosis in tissue cultures.

Abstract: In tissue-engineering and other life sciences, there is a growing need for real-time, non-destructive information on apoptosis and necrosis in 2D and 3D tissue cultures. Previously, propidium iodide was applied as a fluorescent marker for monitoring necrosis. In the current study this technique was extended with a fluorescent apoptosis marker, YO-PRO-1, to discriminate between both stages of cell death. The main goal was to evaluate the performance of YO-PRO-1 and propidium iodide during monitoring periods of up to 3 days. Apoptosis was induced in C2C12 cultures and the numbers of YP-positive and PI-positive nuclei were counted in time. The performance of the dual staining was evaluated with a metabolic measure and a probe intensity study. Cell metabolism was unaffected during the first 24 h of testing. In conclusion, the YP/PI dual staining method was found to be a powerful tool in obtaining real-time spatial information on viability in cell and tissue culture without culture disruption.

Growth of Piscirickettsia salmonis to High Titers in Insect Tissue Culture Cells

Abstract: Piscirickettsia salmonis was grown in established insect, frog, and fish tissue culture cells. The yield of P. salmonis in Sf21 cells was up to 100 times that obtained in CHSE-214 cells, and virulence for Atlantic salmon was retained. The ceiling temperature for growth of P. salmonis in Sf21 cells was 24°C.

β-Carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells

Abstract: Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat-storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of beta-carotene and retinoic acid (RA), although the direct biogenesis of RA from beta-carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of beta-carotene in HSCs. GRX cells were plated in 25 cm(2) tissue culture flasks, treated during 10 days with 3 micromol/L beta-carotene and subsequently transferred into the standard culture medium. beta-Carotene induced a full cell conversion into the fat-storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high-performance liquid chromatography for beta-carotene and retinoids. Cells accumulated 27.48 +/- 6.5 pmol/L beta-carotene/10(6) cells, but could not convert it to ROH nor produced retinyl esters (RE). beta-Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 +/- 1.23 pmol/L/10(6) cells), promoting the phenotype conversion. After 24-h chase, cells contained 20.15 +/- 1.12 pmol/L beta-carotene/10(6) cells and steadily released beta-carotene into the medium (6.69 +/- 1.75 pmol/ml). We conclude that HSC are the site of the liver beta-carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.

Synthesis and Secretion of ApoC-I and ApoE during Maturation of Human SW872 Liposarcoma Cells

Abstract: Little is known about the regulation of apolipoprotein (apo) C-I production by human adipocytes. The aim of the present study, therefore, was to investigate the effect of different tissue culture conditions on the synthesis and secretion of apoC-I and apoE in human SW872 liposarcoma cells. After 3-4 d in culture (0.5 x 10(6) cells/well, DMEM/F-12 medium with 10% fetal calf serum), cells reached confluence and became growth arrested. The molar ratio of apoE:apoC-I in the cell was 8.9 +/- 0.6 and in the medium was 6.6 +/- 0.5. After 17 d in culture, SW872 cells contained significantly more cholesterol (100%) and triglyceride (3-fold) and secreted more apoC-I [4 vs. 17 d: 0.11 +/- 0.01 vs. 0.23 +/- 0.01 pmol/(10(6) cells . 24 h), P < 0.001] and apoE [0.7 +/- 0.1 vs. 3.1 +/- 0.3 pmol/(10(6) cells . 24 h), P < 0.001]. Cellular apoC-I increased 7-fold and apoE increased 16-fold. Cell maturation was associated with significantly higher levels of apoE mRNA but not apoC-I mRNA. Increases in cell lipids, apoC-I, and apoE were not dependent on the presence of extracellular lipids because similar changes occurred in cells incubated with lipoprotein-deficient serum or in cells incubated without serum. Treatment (7 d) of cells during maturation with insulin (10 or 1000 nmol/L) significantly reduced the secretion of apoC-I and apoE. These results demonstrate that in maturing SW872 cells, cholesterol and triglyceride accumulation in the presence or absence of extracellular lipids, is associated with increased apoC-I and apoE production. Furthermore, apoC-I and apoE production are differentially regulated at the transcriptional level, and long-term treatment with insulin has an inhibitory rather than stimulatory effect on apoC-I and apoE production.