Effects of serum and serum-derived factors on growth and differentiation of mouse keratinocytes

Summary Mouse epidermal keratinocytes (MK cells) were grown as replicating subcultures at clonal density, in a serum-free, low calcium basal medium supplemented with seven different growth factors (Bertolero et al., Exp. Cell. Res. 155:64–80, 1984). This serum-free system was used to investigate the activity of cells. bovine serum (FBS) and of serum-derived factors on the growth and differentiation of MK cells. Unfractionated, whole FBS inhibited growth and induced terminal differentiation of normal MK cells. The growth inhibitory activity was considerably reduced by passing whole FBS over a resin (Chelex) to remove Ca²⁺ and other di- and trivalent cations. It is not known whether this treatment removed other factors. Addition of individual serum components either stimulated or inhibited cell-growth and differentiation. Fetuin, a major α-globulin of FBS, and high density lipoprotein strongly inhibited the colony forming efficiency (CFE) of MK cells, whereas bovine serum albumin increased the CFE 4.5-fold and stimulated the growth rate as well. The addition of impure commercial preparations of platelet-derived growth factor inhibited the CFE and induced the morphological features of squamous terminally-differentiating keratinocytes. As reported in other systems, transforming growth factor beta (TGF-β) inhibited the growth of secondary keratinocytes in a dose-dependent manner. Thus, at least three factors present in FBS inhibited growth whereas others were stimulatory. These observations explain the difficulties in obtaining replicating subcultures of mouse keratinocytes in serum-supplemented media and emphasize the importance of a serum-free system for studies on growth control and carcinogenesis in keratinocytes. Editor’s Statement This report contributes significantly to our knowledge of keratinocyte cell biology in two ways. First, a serum-free medium has been developed that can now be used by many investigators to define growth versus differentiation factors for these cells. This is important since several impure or relatively crude preparations of factors are known to influence these cells. Second, the finding that TGF-Beta is an inhibitor of keratinocyte growth opens new avenues to investigate the biochemical events leading to differentiation. David A. Sirbasku     

Characterization of proliferation and differentiation of opossum kidney cells in a serum-free defined medium

Summary Proliferation and differentiation of opossum kidney cells in a serum-free defined medium was investigated and compared to that under conditions in which fetal bovine serum FBS (10%) was employed. Monolayers were grown in Dulbecco's modified Eagle's medium-Ham's F12 nutrient mixture containing insulin (10 μg/ml), bovine serum albumin fraction V (1 mg/ml) and fetuin (1 mg/ml). Cells in serum-free medium seeded at 1×10⁴ per cm² grew to confluency within 6 to 8 d and formed hemicysts or domes at a frequency equivalent to those in serum-containing medium. Electron microscopy of cultures grown in serum-free medium revealed polarized monolayers with the presence of microvilli and tight junctions. The differentiated characteristics, including sodium-dependent phosphate transport, the inhibition of this transport by parathyroid hormone (PTH), and the generation of cyclic AMP in response to PTH, were preserved in opossum kidney cells grown in serum-free medium.     

A serum-free medium that supports the growth of piscine cell cultures

Summary An undefined, serum-free medium was developed for use with fish cell cultures. Lactalbumin hydrolyzate, trypticase-soy broth, Bacto-peptone, dextrose, yeastolate, and polyvinylpyrrolidone were initially combined in 100 ml of distilled H₂O, autoclaved, and added to 5% of the final volume of Medium 199. In addition, filter sterilized bovine pancreatic insulin, glutamine, and nonessential amino acids were added to the medium. The addition of insulin was observed to be unnecessary. Five fish cell lines [goldfish-derived CAR cells, fathead minnow (FHM) cells, epithelioma papillosum cyprini (EPC) cells, chinook salmon embryo (CHSE-214) cells, and a new cell line from goldfish air bladders (ABIII)] were all capable of growth in the serum-free medium at rates equivalent to cells grown in fetal bovine serum (FBS). The morphology of all cell lines, except CHSE-214 cells, was identical to cells grown in FBS. All cell lines were capable of long-term growth in the serum-free medium. The CAR, ABIII, EPC, and CHSE-214 cells in the serum-free medium supported the replication of goldfish virus-2 at levels equivalent to cells grown in FBS.     

Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content.

The growth and antibody production of the SP2/0-derived hybridoma HB124 (ATCC) grown in media containing varying amounts of fetal bovine serum (FBS) were monitored using biochemical and flow cytometric methods. Hybridomas grown in 100 ml spinner flasks with RPMI-1640 containing varying amounts of serum demonstrated that cell growth, viability and IgG production show significant changes when serum content is decreased from 10.0 to 5.5 to 1.0 and 0.5%. A longer lag phase resulted when the lower serum content media were used. Cellular rates of glucose uptake showed a significant increase as serum levels were lowered. Similarly, exponential phase IgG production rates increased as the amount of serum was decreased, probably as a result of the decreased rate of exponential growth. Flow cytometric analysis showed a similar increase in cellular IgG content as medium serum levels declined. In contrast, the maximum IgG concentrations were found in flasks containing 1% FBS or above with the lowest concentration in the 0.5% FBS flask being due to the lower numbers of viable cells. Cells grown in microporous hollow fiber reactors were fed with medium containing serum which was decreased stepwise with time. Decreasing medium serum content stepwise from 10 to 2.5% resulted in increased antibody production. However, complete removal of serum from the medium resulted in a significant drop in antibody productivity. Cumulative antibody production was equivalent for cells grown entirely in medium containing 10% FBS and for those which experienced a drop to 2.5% FBS. To compare a defined serum-free medium preparation with medium containing 10% FBS, cells were again grown in batch suspension culture and analyzed. The growth rates were similar but there was a significant difference in IgG production rates. The serum-free culture exhibited both higher cellular production rates and higher IgG concentrations. These results indicate that decreasing medium serum content can adversely affect antibody yield because of lower cell viabilities, not because of lower production rates. Use of a defined serum-free medium, as done in this study, results in higher yields because of a higher IgG production rate as well as good cell growth and viability.     

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.     

Influence of culture conditions on monoamine oxidase A and B activity in rat astrocytes

Astroglial cells dispersed from newborn rat hemispheres were established in medium supplemented with 20 per cent fetal calf serum (FBS) and then grown to a confluent monolayer in the presence of 10 per cent FBS or charcoal-stripped FBS (CS). Type 1 astrocytes were subcultured and either maintained under the same conditions of the primary cultures or converted to serum-free chemically defined medium (CDM). No differences were found in either MAO A or MAO B activity of astrocytes grown in the presence of FBS or CS after 15 and 21 days in vitro (day 1 and 6 of subculture). In contrast, on day 21 both MAO A and MAO B activities were markedly higher in astrocytes subcultured in CDM compared with cells maintained in serum-supplemented medium. This difference appeared to be due to increased number of enzyme molecules, since kinetic analysis showed an increase in V_max of both MAO isoenzymes in serum-free medium, but no change in K_m. Consistently, the recovery of MAO A and MAO B activity after irreversible enzyme inhibition by clorgyline and deprenyl was faster in CDM than in FBS-supplemented medium, indicating enhanced enzyme synthesis under serum-free condition. Estimates of half-lives for the recovery of MAO A and MAO B activity indicated that, under both culture conditions, type A activity had a higher turnover rate than type B. The effect of CDM on astrocyte MAO does not appear to be due to selection of a subpopulation of cells, but rather linked to a morphological change (differentiation) with increased synthesis of both MAO isoenzymes.     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

Growth and development of rabbit oocytes in vitro: Effect of fetal bovine serum concentration on culture medium

The objective was to develop a culture system that produced blastocyst stage embryos from rabbit oocytes grown in vitro. Two experiments were performed. First, various concentrations of fetal bovine serum (FBS, 0, 0.05, 0.5 and 5%) were used in the culture medium for in vitro growth (IVG) of oocytes recovered from follicles 200 to 299 μm in diameter. Intracytoplasmic sperm injection (ICSI) was performed on mature oocytes obtained after IVG for 8 days and in vitro maturation for 14 to 16 h. Rates of survival and pronuclear formation after ICSI were higher for oocytes grown in a medium with 0.05% FBS compared to oocytes grown in a medium lacking FBS (97.6 vs. 76.9%, 97.5 vs. 70%, P < 0.1). The rate of development to the blastocyst stage was also higher in the medium containing 0.05% FBS than in the medium lacking FBS (9.5 vs. 17.9%, P < 0.05). Next, using oocytes recovered from follicles 200 to 399 μm in diameter which were cultured in 0.05% FBS, oxygen consumption and the number of cells were analyzed. Blastocysts from oocytes grown in vitro with 0.05% FBS had reduced oxygen consumption and number of cells compared with those from ovulated oocytes (21.66 ± 4.54 × 10¹⁴ vs. 50.19 ± 4.61 × 10¹⁴ mol/sec, 244 ± 25 vs. 398 ± 24, P < 0.05). Rabbit oocytes grown in vitro with 0.05% FBS achieved pregnancy, but pregnancies were not maintained to term. In conclusion, the addition of 0.05% FBS to the culture medium for IVG improved developmental competence of rabbit oocytes grown in vitro.     

Comparison of embryonic developmental competence of mouse oocytes grown with and without serum.

The first objective of this study was to determine whether oocyte growth in serum-free medium affects the solubility of the zona pellucida to alpha-chymotrypsin digestion, which is an index of zona pellucida "hardening" and reflects the potential penetrability of the zona pellucida by sperm. Oocyte-granulosa cell complexes were isolated from the preantral follicles of 12-day-old mice and cultured for 10 days in medium containing 5% fetal bovine serum (FBS) or in serum-free medium. The zonae pellucidae of oocytes grown in serum-free medium were four times as hard as freshly isolated germinal vesicle (GV)-stage oocytes grown in vivo or oocytes grown in vitro in FBS-containing medium. The hardening of the zonae pellucidae of oocytes grown in serum-free medium was prevented by addition of fetuin. The second objective was to compare the competence to undergo embryogenesis of oocytes that grew in serum-free vs. FBS-containing medium. Approximately 70% of the oocytes underwent maturation regardless of whether the medium was serum-free or contained FBS. Of the mature ova grown in medium containing FBS, 53% cleaved to the two-cell stage after insemination compared with only 6% of the ova grown in serum-free medium. Addition of fetuin to the serum-free medium used for oocyte growth increased the frequency of cleavage to the two-cell stage. Of the embryos derived from oocytes that grew in FBS-containing medium, 70% completed the two-cell stage to blastocyst transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of reduced serum and serum-free media for the cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV)

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2% fetal bovine serum (FBS), SF-900, and a serum-free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum-supplemented medium. (c) 1992 John Wiley & Sons, Inc.     

Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum-free medium

Growth and differentiation of keratinocytes in a serum-free medium (keratinocyte growth medium or KGM) was studied and compared to that under conditions in which serum and feeder cell layers were used. Cells were grown in KGM containing 0.1 mM calcium (KGM/low calcium), KGM containing 1.2 mM calcium (KGM/normal calcium), or Dulbecco's modified Eagles medium containing 5% fetal calf serum and 1.8 mM calcium in presence of mitomycin treated 3T3 M cells (DMEM/5% FCS). Plating efficiency and rate of growth were similar in the three media till confluence. In postconfluent cultures, protein and DNA content of cells attached to the plate in KGM/low-calcium dishes decreased as an increased number of cells were shed into the medium. Cell shedding was much less evident in the presence of normal calcium. Cells grown in KGM/low calcium had a higher rate of cell proliferation (3H-thymidine incorporation into cellular DNA) than cells grown in normal calcium. Transglutaminase activity, involucrin content, and cornified envelope formation were greatest in cells grown in KGM/normal calcium, intermediate in cells grown in DMEM/5% FCS, and least in cells grown in KGM/low calcium. Keratin profiles from cells grown in KGM/low calcium showed a lower percentage of high molecular weight bands compared to the keratin profiles from cells grown in the presence of normal calcium. Keratinocytes in KGM/low calcium grew as a monolayer of cuboidal cells with few features of differentiation, whereas cells grown in KGM/normal calcium stratified into multilayered islands (3-5 layers) surmounted by 2-4 layers of enucleated cells with thickened cornified envelopes. Cells grown in KGM/normal calcium also contained tonofilaments and lamellar bodies unlike cells grown in KGM/low calcium. Cells grown in DMEM/5% FCS also formed stratified layers comparable to cells grown in KGM/normal calcium but lacked cornified cells, keratohyalin granules, tonofilament bundles, and lamellar bodies. These studies indicate the usefulness of serum-free conditions for the culture of human keratinocytes and confirm the importance of extracellular calcium in keratinocyte differentiation.     

Expansion of human bone marrow-derived mesenchymal stromal cells: serum-reduced medium is better than conventional medium

Background aimsHuman mesenchymal stromal cells (hMSC) are of enormous interest for various clinical applications. For the expansion of isolated hMSC to relevant numbers for clinical applications, 10% fetal bovine serum (FBS)-supplemented medium is commonly used. The main critical disadvantage of FBS is the possibility of transmission of infectious agents as well as the possibility of immune rejection of the transplanted cells in response to the bovine serum. Therefore, we tested a commercially available medium, Panserin 401, that was specifically developed for serum-free cell cultivation.MethodshMSC were isolated from bone marrow (BM) and expanded in either Dulbecco's modified Eagle medium (DMEM) or Panserin 401 alone, or combined with FBS (2% or 10%), with or without supplementary growth factors. Cell proliferation and cytotoxicity were monitored twice a week for 3 weeks.Results and ConclusionsNo proliferation was observed in any of the serum-free media. However, DMEM/10% FBS (the conventional culture medium for hMSC) and DMEM/2% FBS with growth factors revealed moderate proliferation. Interestingly, the best proliferation was obtained using Panserin 401 supplemented with 2% FBS and growth factors (as well as with 10% FBS). Analysis of cell growth in Panserin 401 supplemented with 2% FBS only or with growth factors only revealed no proliferation, demonstrating the necessity of the combination of 2% FBS and growth factors. Efficient isolation and expansion of hMSC from cancellous bone could also be performed using Panserin 401 with 2% FBS and growth factors. Furthermore, these isolated cultures maintained multipotency, as demonstrated by adipogenic and osteogenic differentiation.     

Evaluation of methods for cultivating limbal mesenchymal stromal cells

Background aimsMesenchymal stromal cells (MSC) with similar properties to bone marrow-derived mesenchymal stromal cells (BM-MSC) have recently been grown from the limbus of the human cornea. We have evaluated methods for culturing human limbal MSC (L-MSC).MethodsFour basic strategies were compared: serum-supplemented medium (10% fetal bovine serum; FBS), standard serum-free medium supplemented with B-27, epidermal growth factor and fibroblast growth factor 2, or one of two commercial serum-free media, defined keratinocyte serum-free medium (Invitrogen) and MesenCult-XF® (Stem Cell Technologies). The resulting cultures were examined using photography, flow cytometry (for CD34, CD45, CD73, CD90, CD105, CD141 and CD271), immunocytochemistry (alpha-smooth muscle actin; α-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis) and co-culture experiments with human limbal epithelial (HLE) cells.ResultsWhile all techniques supported the establishment of cultures to varying degrees, sustained growth and serial propagation were only achieved in 10% FBS medium or MesenCult-XF medium. Cultures established in 10% FBS medium were 70–80% CD34^? CD45^? CD90⁺ CD73⁺ CD105⁺, approximately 25% α-sma⁺ and displayed multipotency. Cultures established in MesenCult-XF were > 95% CD34^? CD45^? CD90⁺ CD73⁺ CD105⁺, 40% CD141⁺, rarely expressed α-sma, and displayed multipotency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of MesenCult-XF-grown L-MSC. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker ?Np63, along with the corneal differentiation marker cytokeratin 3.ConclusionsMesenCult-XF is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells.     

Production of human immunodeficiency virus from T-lymphoblastoid cells using serum-free medium

Summary A serum-free medium was constructed for the suspension culture of mammalian cells. Effects of the serum-free medium on the cultivation of T-lymphoblastoid cells secreting human immunodeficiency virus (HIV) were observed. The cell growth and HIV production in the serum-free medium were comparable to those in the medium supplemented with 10% fetal bovine serum (FBS), which showed a possibility of economical industrial application.     

The in vitro response of human tumour cells to desferrioxamine is growth medium dependent

Abstract. Iron chelating agents have been demonstrated to inhibit tumour cell growth. However, in vitro and in vivo results using desferrioxamine a hexadentate iron chelating agent, for anti-cancer treatment are not always in agreement. Therefore, we have studied the response of three human tumour cell lines (HL-60 promyelocytic leukaemia, MCF-7 breast cancer and HepG2 hepatoma), grown in culture medium supplemented with either human pooled (HPS) or fet al bovine serum (FBS), to desferrioxamine. Desferrioxamine, at micromolar concentrations, induced severe cytotoxicity in all tumour cell lines grown in FBS medium. When grown in HPS medium, comparable desferrioxamine cytotoxicity was observed in the millimolar range. The addition of 50% saturated human transferrin to FBS medium resulted in protection against desferrioxamine cytotoxicity. HL-60 cells were further studied for iron metabolism characteristics. HL-60 cells, grown in medium with FBS, were found to have an 8.4 fold increase in surface transferrin receptor (TfR) expression ( P < 0.001) as compared with HL-60 cells grown in medium with HPS. However, iron uptake of HPS cultured HL-60 cells, after incubation with saturated human transferrin, was higher, resulting in a higher concentration of iron in HPS cultured HL-60 cells as compared with FBS cultured cells (1.72 ± 0.02 μmol/g protein v. 1.32 ± 0.14 μmol/g protein; P < 0.001). Using desferrioxamine it was shown that TfR expression is dependent on the biological availability of iron in the cell. Consistent with the lower iron content in FBS cultured cells, we conclude that the cytotoxicity of desferrioxamine is dependent on the ability of cells to replenish cellular iron stores from the culture medium. Cells grown in FBS medium lack this ability and are therefore more susceptible to desferrioxamine.     

Hydrogen peroxide-induced neurotoxicity in cultured cortical cells grown in serum-free and serum-containing media

To compare different culture conditions for neuroprotection assays in cultured cortic neurons, we evaluated cell viability after H₂O₂ exposure in cells cultured with standard N2 and with the enriched B-27 developed by GIBCO, both serum-free supplements. The following additives/associations were compared: N2 (+N2), B-27 (+B-27), 10% FBS (+FBS), 1% FBS in combination with N2 (FBS/N2) or N2 supplement preceded by an 1 hour precoating with 10% FBS (N2 + precoated). Our data demonstrated that B-27 is as efficient as 10% FBS to support neuronal growth for more than a week. As shown by phase-contrast optics cells grown in N2 started degenerating within 24-48 hours although measurable absorbance was seen with MTT. The precoating procedure failed to modify substantially cell viability as compared with N2 alone. Dose-response curves for H₂O₂ to induce neuronal apoptosis were almost identical for B-27 and serum supplemented samples. Catalase (100 U/ml) or vitamin E (200 M) prevented cell death in both culture conditions. Our results indicate that DMEM/B-27 provides a serum-free cell culture environment that allows neurons to grow with optimal cell viability, comparable to that obtained with serum. We conclude that this culture condition reveals as a useful tool to test the efficacy of neuroprotectants when a serum free medium is required.     

Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: Effect of transferrin,insulin and cell density

Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells. This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes of Health grants CA 24604-09 and CA 16463-14.     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

Polypeptide growth factors augment interleukin 1-induced release of prostaglandin E2 by rheumatoid arthritis synovial cells in vitro

When stimulated with increasing amounts of interleukin 1 beta (IL 1 beta) rheumatoid arthritis (RA), as compared with osteoarthritis (OA), synovial cells grown in RPMI plus fetal bovine serum (FBS), released significantly more prostaglandin E2 (PGE2) (p less than 0.05; paired t test, two-tailed). PGE2 release by IL 1 beta-stimulated RA synovial cells grown for 14 days in serum-free RPMI was significantly less than that released by the same cells grown in medium plus 10% FBS (p less than 0.03; two-tailed). Since these data suggest that growth factors present in FBS may augment the effects of IL 1 beta, experiments were conducted to study the influence of four polypeptide growth factors--transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), on IL 1 beta-induced release of PGE2 by cultured RA synovial cells. Both EGF and bFGF significantly enhanced IL 1 beta-induced release of PGE2 (p less than 0.05; paired t test, one-tailed), while PDGF was synergistic with IL 1 beta, significantly increasing release of PGE2 by these cultured cells (p less than 0.02; two-tailed). No such effect was seen when TGF-beta was added to the culture medium. Taken together, these data lend support to the concept that within the synovial micro-environment small quantities of individual growth factors may potentiate the effects of IL 1 beta to amplify intra-articular inflammation.