Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets

To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 × 10⁶ cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 × 10³ cells per well. Cells were grown for 1 day in MEMα plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 μM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.     

In vitro culture of human chondrocytes (1): A novel enhancement action of ferrous sulfate on the differentiation of human chondrocytes

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. The aim of our investigation was to characterize the influences of basic fibroblast growth factor (bFGF) and ferroussulfate (FeSO₄) on proliferation and differentiation of human articular chondrocytes (HAC). This is the first report of the effects of FeSO₄ on chondrogenesis of HAC. Multiplied chondrocytes of hip and shoulder joints were cultured in chondrocyte growth medium supplemented with bFGF, FeSO₄, or both bFGF + FeSO₄ for4weeks. A 20 μl aliquot of a cell suspension containing2 × 10⁷ cells ml⁻¹ was delivered onto each well of 24-well tissue culture plates. Cells cultured with the growth medium only was used as a control. Alamar blue and alcian blue staining were done to determine the chondrocyte proliferation and differentiation, respectively, after 4 weeks. The samples exposed to bFGF, FeSO₄, and combination of both indicated sufficient cell proliferation similar to the control level. Differentiations of the HAC exposed to bFGF, FeSO₄,and bFGF + FeSO₄ were 1.2-, 2.0-, and 2.2-fold of the control, respectively. Therefore, chondrocyte differentiation was significantly enhanced by the addition of FeSO₄ andbFGF + FeSO₄. The combined effects of bFGF and FeSO₄ were additive, rather than synergistic. These results suggest that treatment with ferrous sulfate alone or in combination with basic fibroblast growth factor etc, is a powerful tool to promote the differentiation of HAC for the clinical application. This revised version was published online in August 2006 with corrections to the Cover Date.     

A Modified Protocol to Maximize Differentiation of Human Preadipocytes and Improve Metabolic Phenotypes

Adipose stromal cells proliferate and differentiate into adipocytes, providing a valuable model system for studies of adipocyte biology. We compared differentiation protocols for human preadipocytes and report on their metabolic phenotypes. By simply prolonging the adipogenic induction period from the first 3 to 7 days, the proportion of cells acquiring adipocyte morphology increased from 30–70% to over 80% in human subcutaneous preadipocytes (passages 5–6). These morphological changes were accompanied by increases in the adipogenic marker expression and improved adipocyte metabolic phenotypes: enhanced responses to β‐adrenergically stimulated lipolysis and to insulin‐stimulated glucose metabolism into triglyceride (TG). Confirming previous studies, fetal bovine serum (FBS) dose‐dependently inhibited adipogenesis. However, in subcutaneous preadipocytes that differentiate well (donor‐dependant high capacity and subcultured fewer than two times), the use of 7d‐induction protocols in both 3% FBS and serum‐free conditions allowed >80% differentiation. Responsiveness to β‐adrenergically stimulated lipolysis was lower in 3% FBS. Rates of insulin‐stimulated glucose uptake were higher in adipocytes differentiated with 3% FBS, whereas the sensitivity to insulin was almost identical between the two groups. In summary, extending the length of the induction period in adipogenic cocktail improves the degree of differentiation and responses to key metabolic hormones. This protocol permits functional analysis of metabolic phenotypes in valuable primary human adipocyte cultures through multiple passages.     

Establishment and Characterization of a New Muscle Cell Line of Zebrafish (Danio rerio) as an In Vitro Model for Gene Expression Studies

A new continuous fibroblast cell line was established from the muscle tissue of healthy juvenile Danio rerio (Zebrafish) through explant method. Fish cell lines serve as useful tool for investigating basic fish biology, as a model for bioassay of environmental toxicant, toxicity ranking, and for developing molecular biomarkers. The cell line was continuously subcultured for a period of 12 months (61 passages) and maintained at 28 °C in L-15 medium supplemented with 10% FBS and 10 ng/mL of basic fibroblastic growth factor (bFGF) without use of antibiotics. Its growth rate was proportional to the FBS concentration, with optimum growth at 15% FBS. DNA barcoding (16SrRNA and COX1) was used to authenticate the cell line. Cells were incubated with propidium iodide and sorted via flow cytometry to calculate the DNA content to confirm the genetic stability. Significant green fluorescent protein (GFP) signals confirmed the utility of cell line in transgenic and genetic manipulation studies. In vitro assay was performed with MTT to examine the growth potential of the cell line. The muscle cell line would provide a novel invaluable in vitro model to identify important genes to understand regulatory mechanisms that govern the molecular regulation of myogenesis and should be useful in biomedical research.     

TGFβ (transforming growth factor β) and keratocyte motility in 24 h zebrafish explant cultures

Fish keratocytes are used as a model system for the study of the mechanics of cell motility because of their characteristic rapid, smooth gliding motion, but little work has been done on the regulation of fish keratocyte movement. As TGFβ (transforming growth factor β) plays multiple roles in primary human keratinocyte cell migration, we investigated the possible involvement of TGFβ in fish keratocyte migration. Studying the involvement of TGFβ1 in 24 h keratocyte explant allows the examination of the cells before alterations in cellular physiology occur due to extended culture times. During this initial period, TGFβ levels increase 6.2‐fold in SFM (serum‐free medium) and 2.4‐fold in SFM+2% FBS (fetal bovine serum), while TGFβ1 and TGFβRII (TGFβ receptor II) mRNA levels increase ～3‐ and ～5‐fold respectively in each culture condition. Two measures of motility, cell sheet area and migration distance, vary with the amount of exogenous TGFβ1 and culture media. The addition of 100 ng/ml exogenous TGFβ1 in SFM increases both measures [3.3‐fold (P=4.5 × 10^?5) and 26% (P=2.1 × 10^?2) respectively]. In contrast, 100 ng/ml of exogenous TGFβ1 in medium containing 2% FBS decreases migration distance by 2.1‐fold (P=1.7 × 10^?7), but does not affect sheet area. TGFβ1 (10 ng/ml) has little effect on cell sheet area in SFM cultures, but leads to a 1.8‐fold increase (P=1.5 × 10^?2) with 2% FBS. The variable response to TGFβ1 may be, at least in part, explained by the effect of 2% FBS on cell morphology, mode of motility and expression of endogenous TGFβ1 and TGFβRII. Together, these results suggest that expression of TGFβ and its receptor are up‐regulated during zebrafish keratocyte explant culture and that TGFβ promotes fish keratocyte migration.     

Establishment and characterization of stromal cell lines that support differentiation of murine hematopoietic blast cells into osteoclast-like cells

Summary This study aimed to establish and characterize a new stromal cell line that supports the proliferation of hematopoietic blast cells and their differentiation into osteoclast-like cells. Cells isolated from the calvaria of neonatal Balb/c mice were subcultured every 2 to 4 days at 1.2×10⁴ cells/cm². After 18 passages the cells had become immortalized and were designated as MCHT-1. MCHT-1 cells were found to support the proliferation of hematopoietic blast cells and their differentiation into osteoclast-like cells when these two cells were co-cultured in the presence of 1α,25(OH)₂D₃ and dexamethasone. However, because the MCHT-1 cells showed heterogeneity, cloning was performed and each clone was characterized. All the clones obtained supported the proliferation of hematopoietic blast cells and their differentiation into osteoclast-like cells irrespective of their obvious differences in growth capacities and cytochemical characteristics. However, the time-course of the appearance of osteoclast-like cells differed among clones. The supportive effect of these clonal stromal cells on differentiation of hematopoietic blast cells into osteoclast-like cells was completely dependent on the presence of 1α,25(OH)₂D₃ and dexamethasone. These clonal MCHT-1 cells are expected to be useful for precise analysis of the proliferation and differentiation of osteoclasts.     

Cultures of cells from fetal rat brain: Methods to control composition,morphology, and biochemical activity

Fetal tissue transplantation is a promising new approach for the treatment of neurodegenerative diseases, but the optimal conditions for preparing cells for transplantation have not been defined. The growth of a population of septal brain cells, primarily containing cholinergic neurons and glia, was characterized after seeding at densities from 5 × 10⁴ to 6 × 10⁵ cells/cm², on polystyrene‐, collagen‐, laminin‐, and fibronectin‐coated surfaces, in the presence of serum and/or serum‐free medium. Differentiated glial cells were selected by culture on fibronectin or laminin surfaces, in the presence of low amounts of serum (2.5% FBS) and G5, a soluble factor containing EGF and insulin. Differentiated neuronal cells were selected by culture on laminin, in the presence of low amounts of serum (2.5% FBS) and N2, a soluble factor containing supplemental hormones. In each case, a minimum seeding density of 1 × 10⁵ cells/cm² was required. Neuronal growth could be maintained long term (21 days) with high levels of neuronal activity (ChAT activity). © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 461–467, 1999.     

Rapamycin Inhibits Human Adipocyte Differentiation in Primary Culture

Objective: The immunosuppressant drug rapamycin, has been reported to inhibit 3T3‐L1 adipocyte differentiation by interfering with critical postconfluent mitoses that are required early on for successful differentiation of this cell line (clonal expansion phase). In contrast to the murine 3T3‐L1 preadipocyte cell line, human preadipocytes in primary culture do not undergo clonal expansion during differentiation. We investigated whether rapamycin could inhibit human adipocyte differentiation. Research Methods and Procedures: The effect of rapamycin on the induction of differentiation of human preadipocytes in primary culture into adipocytes was measured using Oil Red O staining and glycerol phosphate dehydrogenase activity. Results: We have observed that rapamycin severely curtails human adipocyte differentiation of both omental and abdominal subcutaneous preadipocytes (to 14% and 19% of standard differentiation, respectively). The rapamycin‐mediated inhibition of human adipocyte differentiation could be reversed in the presence of excess amounts of FK‐506, which displaces rapamycin from its intracellular receptor, FKPB12. Measurement of cytosolic protein and [³H]thymidine incorporation into DNA confirmed the absence of proliferation during differentiation of human preadipocytes in primary culture. Discussion: Our data indicate that rapamycin exerts important negative regulatory effects on adipogenesis in human preadipocytes, through a mechanism that does not depend on interruption of clonal expansion.     

Establishment and characterization of a brain‐cell line from kelp grouper Epinephelus moara

A new brain‐cell line, EMB, was developed from kelp grouper Epinephelus moara, a cultured marine fish. The EMB cells were subcultured for more than 60 passages. The cells were cultured in Leibovitz's L‐15 medium (L15) supplemented with antibiotics, foetal bovine serum (FBS), 2‐mercaptoethanol (2‐ME) and basic fibroblast growth factor (bFGF). The cells could grow at 18–30° C, with the maximum growth between 24 and 30° C. The optimum FBS concentration for the cells growth ranged between 15 and 20%. Chromosome analysis indicated that the modal chromosome number was 48 in the cells at passage 45. After being transfected with pEGFP‐N3 plasmid, the cells could successfully express green fluorescence protein (GFP), implying that this cell line can be used for transgenic studies. A significant cytopathic effect (CPE) was observed in the cells after infection with Singapore grouper iridovirus (SGIV) or red spotted grouper nervous necrosis virus (RGNNV) and the viral replication was confirmed by quantitative real‐time PCR (qrt‐PCR) assay, which suggested EMB's application potential for studies of SGIV and RGNNV.     

Optimization of culture conditions for endothelial progenitor cells from porcine bone marrow in vitro

Objectives: The aim of this study was to determine an optimal culture method for porcine bone marrow‐derived endothelial progenitor cells (EPCs). Materials and methods: Mononuclear cells (MNCs) were isolated by density centrifugation and differentiated into EPCs in in vitro. At first‐passage, EPCs were cultured at different cell densities (5 × 10³, 1 × 10⁴, 2 × 10⁴ or 5 × 10⁴/cm²) and in basic medium (EGM, medium 199, DMEM or 1640) supplemented with FBS (2%, 5%, 10% or 20%) and different combinations of cytokines (VEGF, VEGF + bFGF, VEGF + bFGF + EGF, or VEGF + bFGF + EGF + IGF), the experiment being based on L₆₄ (4²¹) orthogonal design. Results and conclusions: This demonstrated that the optimal culture method for our EPCs displayed higher expansion and migration rates as compared to other groups, by analysis of variance; that is, cultured at 1 × 10⁴/cm² in M199 supplemented with 10% FBS and VEGF + bFGF + IGF + EGF. Furthermore, percentage of positive cells stained by Dil‐ac‐LDL and FITC‐UEA‐1 was more than 65%, and as shown by immunohistochemistry, these cells also stained positively for CD133, CD34 and KDR. The present study indicates that the number and function of porcine EPCs significantly increased when using our optimized culture parameters.     

Isolation and long-term serial cultivation of endothelial cells from the microvessels of the adult human dermis

Sumamry A method to isolate and maintain microvascular endothelial cells from the cutaneous vessels of adult human skin in long-term culture has been developed. Endothelial cells lining the microvessels of the papillary dermis are released from surrounding tissue during a brief trypsin incubation (0.3% trypsin, 1% EDTA). Cells are plated onto a fibronectin substrate and maintained in Leibovitz (L15) culture medium containing pooled human serum (50%) and antibiotics. Proliferation is dependent upon the presence of several additional growth factors, cholera enterotoxin (1×10⁻⁹ M), isobutyl methylxanthine (3.3×10⁻⁵ M), and medium conditioned by explant culture of the mouse EHS sarcoma. Using this supplemented medium, cells proliferate readily and can be cultivated serially for more than 6 passages (3 months in vitro). These cells retain their characteristic endothelial cell morphology, stain positively for Factor VIII antigen, and contain Weibel-Palade bodies. This research was supported by grant AG 01312 from the U.S. Public Health Service, Washington, D.C.     

Developing an improved In vitro propagation system for slow-growing species using garcinia mangostana L. (mangosteen)

Summary This investigation disclosed that evaluation of tissue culture parameters of slowly developing species (e.g. Garcinia mangostana) requires monitoring of treatments through two or more successive, relatively long passages. Two 8-wk passages were necessary to observe differences in phytohormone effects. Photoperiod and temperature effects were not clearly evident until tissues had been cultured through three passages; the optimal photoperiod and temperature for shoot proliferation could not be established until after the fifth passage. Our investigation revealed that no auxin supplementation was necessary for bud primordium differentiation in cotyledon explants or proliferation of regenerated shoots. The optimum N⁶-benzyladenine concentration for primordium differentiation was 13.3 μM, and for shoot proliferation ranged from 4.4 to 13.3 μM. Continuous culturing in an 8-h photoperiod at 30°C resulted in progressively intensified degeneration of shoots after three passages. In contrast, successive passages in a 16-h photoperiod/26°C regimen enabled sustained regeneration of shoots. The shoots rooted at a rate of 85% when precultured for 3 d in a medium containing 4921.3 μM indole-3-butyric acid, or 10 d at 492.1 μM, then cultured for two 8-wk passages in phytohormone-free medium. Following acclimatization by gradually lowering the relative humidity in the growth chamber, rooted shoots survived transfer to the greenhouse at a rate of 95%.     

Epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor-bb can substitute for fetal bovine serum and compete with human platelet-rich plasma in the ex vivo expansion of mesenchymal stromal cells derived from adipose tissue

Background aimsHuman mesenchymal stromal cells (MSC) are multipotent cells possessing self-renewal capacity, long-term viability and multilineage potential. We analyzed the effect of four different medium supplements on the expansion and differentiation of adipose tissue-derived MSC (ADSC) in order to avoid the use of xenogeneic serum.MethodsWe compared fetal bovine serum (FBS) with 10% human platelet-rich plasma (hPRP), 3% human platelet-poor plasma (hPPP) and with a cytokine cocktail composed of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor-bb (PDGFbb) added to 3% hPPP. This mixture was developed testing EGF, bFGF, granulocyte–colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF-I), PDGFbb and transforming growth factor (TGF)-β1 added alone or in combination with hPPP.ResultsOur data demonstrate that the addition of EGF, bFGF and PDGFbb, in a medium supplemented with hPPP, obtainable from 150–200 mL whole autologous blood, supports ADSC expansion better than FBS, as confirmed by cumulative population doublings (cPD; 15.0 ± 0.5 versus 9.4 ± 2.8). The addition of human platelet-rich plasma (hPRP) further improved ADSC proliferation (cPD 20.0 ± 1.2), but the achievement of hPRP presented a major drawback, requiring 1000–1200 mL autologous or donor whole blood. The medium supplements did not influence ADSC phenotype: they expressed CD105, CD90 and CD44 lacking hematopoietic antigens. The exposure to the proposed cocktail or to hPRP increased adipogenic and osteogenic differentiation.ConclusionsThe addition of EGF, bFGF and PDGFbb to hPPP could ensure a sufficient number of ADSC for clinical applications, avoiding the use of animal serum and representing a novel approach in regenerative medicine.     

Modulation of Sirt1 by resveratrol and nicotinamide alters proliferation and differentiation of pig preadipocytes

Sirt1, a NAD⁺-dependent histone deacetylase, may regulate senescence, metabolism, and apoptosis. In this study, primary pig preadipocytes were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) with or without reagents affecting Sirt1 activity. The adipocyte differentiation process was visualized by light microscopy after Oil red O staining. Proliferation and differentiation of preadipocytes was measured using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Oil red O extraction. Expression of Sirt1, FoxO1, and adipocyte specific genes was detected with semi-quantitive RT-PCR. The results showed that Sirt1 mRNA was widely expressed in various pig tissues from different developmental stages. Sirt1 mRNA was expressed throughout the entire differentiation process of pig preadipocytes. Resveratrol significantly increased Sirt1 mRNA expression, but decreased the expression of FoxO1 and adipocyte marker gene PPARγ2. Resveratrol significantly inhibited pig preadipocyte proliferation and differentiation. Nicotinamide decreased the expression of Sirt1 mRNA, but increased the expression of FoxO1 and adipocyte specific genes. Nicotinamide greatly stimulated the proliferation and differentiation of pig preadipocytes. In conclusion, these results indicate that Sirt1 may modulate the proliferation and differentiation of pig preadipocytes. Sirt1 may down-regulate pig preadipocytes proliferation and differentiation through repression of adipocyte genes or FoxO1.     

Decreases in local hormone biosynthesis and c-fos gene expression accompany differentiation of porcine preadipocytes

Summary To better understand possible autocrine or paracrine mechanisms involved in adipose tissue development, we have studied the biosynthesis of insulinlike growth factor I (IGF-I) and prostaglandin E₂ (PGE₂) by cultured porcine preadipocytes in response to factors known to modulate cell growth and differentiation. The expression of c-fos was also monitored because of the potential role of that proto-oncogene in coordination of growth and differentiation. Preadipocytes were grown to confluence and then maintained in one of three media treatments: a) standard medium supplemented with 10% fetal bovine serum (FBS), b) FBS supplemented with dexamethasone (Dex), c) FBS supplemented with dibutryladenosine 3′–5′-cyclic monophosphate. Indirect measurements of growth indicated that cell proliferation did not differ due to media type. Histochemical and enzymatic measurements of adipocyte development revealed that differentiation occurred only in those cultures exposed to Dex. The increase in adipocyte differentiation in response to Dex was associated with a decrease in c-fos and actin RNA expression whereas the decrease in c-fos RNA expression in response to Dex was small (approximately 40%); immunocytochemical analysis indicated that induction of Fos protein occurred only in undifferentiated cells. Thus, the cells responsible for the decrease in c-fos RNA expression are possibly those signaled to differentiate into adipocytes. Expression of IGF-I RNA and secretion of IGF-I and PGE₂ were also decreased in response to Dex treatment. These data provide the first demonstration that biosynthesis of IGF-I by preadipocytes can be modulated by a potent inducer of adipocyte differentiation. The combined results indicate that glucocorticoids may stimulate adipocyte differentiation by suppressing intracellular and putative intercellular mitogenic signals. This work was supported in part by grant HD 18447 from the National Institutes of Health, Bethesda, MD (G. J. H.). Mention of a trade mark, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U. S. Department of Agriculture or University of Georgia and does not imply its approval to the exclusion of other products that may be suitable.     

Pooled human platelet lysate versus fetal bovine serum—investigating the proliferation rate,chromosome stability and angiogenic potential of human adipose tissue-derived stem cells intended for clinical use

Background aimsBecause of an increasing focus on the use of adipose-derived stem cells (ASCs) in clinical trials, the culture conditions for these cells are being optimized. We compared the proliferation rates and chromosomal stability of ASCs that had been cultured in Dulbecco's modified Eagle's Medium (DMEM) supplemented with either pooled human platelet lysate (pHPL) or clinical-grade fetal bovine serum (FBS) (DMEM^pHPL versus DMEM^FBS).MethodsASCs from four healthy donors were cultured in either DMEM^pHPL or DMEM^FBS, and the population doubling time (PDT) was calculated. ASCs from two of the donors were expanded in DMEM^pHPL or DMEM^FBS and cultured for the final week before harvesting with or without the addition of vascular endothelial growth factor. We assessed the chromosomal stability (through the use of array comparative genomic hybridization), the expression of ASC and endothelial surface markers and the differentiation and angiogenic potential of these cells.ResultsThe ASCs that were cultured in pHPL exhibited a significantly shorter PDT of 29.6 h (95% confidence interval, 22.3–41.9 h) compared with those cultured in FBS, for which the PDT was 123.9 h (95% confidence interval, 95.6–176.2 h). Comparative genomic hybridization analyses revealed no chromosomal aberrations. Cell differentiation, capillary structure formation and cell-surface marker expression were generally unaffected by the type of medium supplement that was used or by the addition of vascular endothelial growth factor.ConclusionsWe observed that the use of pHPL as a growth supplement for ASCs facilitated a significantly higher proliferation rate compared with FBS without compromising genomic stability or differentiation capacity.     

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this work, the supplementation of Grace’s medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace’s medium supplemented with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra production was comparable to that in Sf900 II. Batch cultivation in Grace’s medium with 2.7 g l⁻¹ glucose, 8 g l⁻¹ YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold (4.7×10⁶ cells ml⁻¹) when compared to Grace’s containing 10% (v/v) FBS (9.5×10⁵ cells ml⁻¹). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6×10⁷ PIBs ml⁻¹) than in Grace’s medium with 10% FBS (0.6×10⁷ PIBs ml⁻¹). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media, keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed for Sf900 II medium. C.A. Pereira is recipient of a CNPq fellowship.     

Observation of production of immunoactive prolactin by normal human connective tissue in cell culture

Summary Data from our in vitro studies indicate a new source of prolactin (PRL)-like activity, normal human connective tissue. Fascial cells from primary culture and subsequent passages produced an extracellular antigen which specifically reacted in a radioimmunoassay RIA developed to detect human pituitary PRL. An initial peak of first surge of fascial PRL-like activity occurred between 4 and 15 d in primary culture. Ibuprofen, cytotoxic levels of 0.01% azide, or 7.5 mM EDTA and medium lacking serum [fetal bovine serum (FBS)] significantly (P≤0.05) reduced PRL-like activity levels, whereas female steroids, 257 to 342 milliosmolarity, 1 to 3.6 mg/ml glucose, 2 to 20% FBS, and dialyzed FBS (MWCO ⊂) 1 kDa) were without effect. Optimum production of PRL-like activity occurred at pH 7.3. A second surge began after 18 d and continued until passage indicating that perhaps two populations of cells produced PRL-like activity in primary culture. Production of PRL-like activity by cells from early passages (1 and 2) became detectable at confluence, was serum-dependent, showed two patterns (tonic, rising to plateau), and averaged 3.2 fg·cell⁻¹·3 d⁻¹ feed interval. Cells from late passages showed morphologic damage from repetitive trypsinization, aging, and reduced production of PRL-like activity with aberrant production pattern. Production of PRL-like activity was maintained in an unusual long-term culture. these in vitro studies demonstrate the most recently recognized and ubiquitous source of human extrapituitary PRL or PRL-like activity, normal connective tissue (fascia). A portion of this paper was presented at the 33rd Annual Meeting of the Society for Gynecologic investigation, Toronto, Ontario, Canada, 19–22 March 1986. This work was supported in part by grant HD21883 (to D. H. R.) from the National Institutes of Health, Bethesda, MD.     

Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro

Background aimsMultipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown.MethodsMSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to “rescue” the proliferative capacity of MSCs.ResultshPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS.ConclusionshPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity.