Progress in the development of shrimp cell cultures in Thailand

Primary shrimp cell cultures were developed from lymphoid organ and ovaries of black tiger shrimp, Penaeus monodon, in double-strength Leibovitz's L-15 medium supplemented with 15% fetal bovine serum, 1% glucose, 5 g/L NaCl, 15% shrimp meat extract. The optimum conditions for primary culture in vitro were obtained in L-15 medium with an osmolality of approximately 730 ± 10 mmol/kg, a temperature range of 25--28 °C and incubation in a normal atmosphere. However, basal medium supplemented with 0.01% cholesterol could enhance good growth and cells performance initiated from lymphoid organ. Both epithelial-like and fibroblastic-like cells were observed from those organs within 2 days incubation. Within 3 days, 80% confluent monolayers were obtained from the lymphoid organ while cultures from other tissues required 5 days. Cultures were maintained for at least 43 days. Only cells from lymphoid organ could be subcultured and confluent monolayers achieved within 10 days post-spilt. Healthy cultures of the lymphoid cells did not persist beyond the third passage. Application of these primary shrimp cell cultures for studying pathogenic viruses of shrimp in vitro will be discussed.     

Culture media variation as related to in vitro aging of human fibroblasts: II. Effects on nucleolar number/cell, volume/nucleolus and total nucleolar volume/cell

The relative effect of five commonly used culture media (MEM, BME, McCoy's 5A, M199 and HMEM) on the average nucleolar number/cell, the average volume/nucleolus and the total nucleolar volume/cell was examined during in vitro senescence of WI-38 human fetal fibroblasts. Statistical analyses show that cells aging in MEM show a higher number of nucleoli/cell than that of cells aging in any other medium. For cells aging in the other four media, there are no significant differences in the average number of nucleoli/cell. Linear regression analysis shows that in all cases there is a linear decrease in the average number of nucleoli/cell as a function of PDL. Statistical analyses show that the average volume/nucleolus is significantly greater for cells aging in M199 than in any other medium. Cells aging in HMEM show smaller average nucleolar volume than cells aging in M199, but display larger volumes than that of cells aged in BME, McCoy's 5A, or MEM. Cells aging in BME and McCoy's 5A media show no significant difference among each other in terms of average nucleolar volume, but a difference in this parameter is noted in cells aging in BME and MEM. A linear regression analysis shows that the average volume/nucleolus increases linearly as a function of age for cells grown in all five media. Analysis of the total nucleolar volume/cell in the five media shows that cells aging in M199 and HMEM are not significantly different from each other in terms of this variable, but show significantly larger volumes than those of cells aging in BME, McCoy's 5A and MEM. Cells aging in BME, McCoy's 5A and MEM display no significant difference with regard to this parameter. Linear regression analysis shows a positive linear relationship between the PDL and the total nucleolar volume/cell. The relative effects of all five media are not the same on the three cellular variables studied during in vitro aging of WI-38 cells. We, therefore, suggest that one should note this medium differential in order to allow meaningful comparison of results on possible changes in various morphological parameters during in vitro senescence of diploid human fibroblasts.     

Recovery of live virus after storage at ambient temperature using ViveST™

BackgroundA major impediment to performing virological field studies in developing nations is the lack of ultra-low freezers as well as the expense and difficulty of shipping frozen samples. A commercially available product, ViveST?, was developed to preserve nucleic acids at ambient temperature for use in specimen storage and transportation. However, its applications as a viral storage, transport and recovery device have not been evaluated.ObjectiveTo examine the ability of ViveST to preserve live virus following storage at ambient temperature.Study designA panel of six viruses was stored at ambient temperature (～22 °C) in ViveST with fetal bovine serum (FBS), or ViveST with minimal essential media (MEM) and compared with virus stored in universal transport media (M4RT), MEM, and FBS alone. Stored viruses included: human adenovirus (14p), dengue virus 2 (16608), echovirus 3 (Morrisey), human rhinovirus 15 (1734), Coxsackie virus B5 (Faulkner), and herpes simplex virus 1 (HF). After 7 days storage at ambient temperature, virus recovery was measured via titration using viral plaque assays or focus-forming unit assays.ResultsViral titer studies indicate that ViveST with either FBS or M4RT preserved/recovered 5 different viruses for 1 week at ambient temperature. MEM preserved 4 viruses while FBS and ViveST with MEM preserved 3 viruses each. Statistical analyses indicate that M4RT and ViveST with FBS preserved significantly more virus than the other treatments.ConclusionsThese data suggest that ViveST with either FBS or M4RT may be useful in field specimen collection scenarios where ultra-cold storage is not available.     

Establishment of primary cell cultures: Experiences with 155 cell strains

Summary Cell culture systems allow the examination of cell populations in a functional state. To simulate in vivo conditions as closely as possible freshly established cell strains are superior to permanent cell lines. Different aspects for the establishment of primary cell cultures obtained from various tissues are compared: (1) Disintegration, (2) culture media supplemented with basal additions, (3) special supplements (growth factors, hormones), and (4) attachment factors. The proliferation rates of the attained cell strains were evaluated by determination of cell doubling times. Procedures for how to obtain a relatively high plating efficiency (approx. 70% in our series of 219 attempts) of primary growth in vitro are described: (1) Mechanical disintegration is superior to enzymatic digestion. If mechanical treatment alone did not produce a sufficient number of viable cells, additional digestion with collagenase/dispase revealed a higher number of proliferating primary cultures than with trypsin. (2) Proliferation of cell cultures from normal and tumorous tissues of epithelial origin was superior in Leibovitz L 15 medium (58 of 87 (67%) cases). Cultures from mesenchymal tissues and tumors were found to have shortest cell doubling times in MEM and RPMI 1640 (16 of 23 (70%) cases). The media were supplemented with the basal additions indicated. (3) In approx. 30% of the cases special supplements like growth factors or hormones increased cell replication, although they were almost always not essential for cell growth. (4) Attachment factors only rarely contributed to the initiation of primary monolayer cultures. The application of various culture conditions does not lead to a protocol optimal for all tissues, for all probes of the same type of tumor, or for all tumor specimens of unique differentiation.Abbreviations EGF epidermal growth factor - FGF fibroblast growth factor - GHL glycyl-L-histidyl-L-lysine - PAN pancreozymin - E estrogen - PR progesterone Supported by the Deutsche Forschungsgemeinschaft, grant Di 276/1–2, SFB 232, Hamburger Landesverband zur Krebsbekämpfung und Krebsforschung, and Hamburger Stiftung zur Förderung der Krebsbekämpfung     

Culture media variation as related to in vitro aging of human fibroblasts: I. Effects on population doubling, nuclear volume and nuclear morphology

The relative effect of five commonly used culture media (MEM, BME, McCoy's 5A, M199 and HMEM) on the population doubling level (PDL), nuclear volume and nuclear morphology was examined during in vitro senescence of WI-38 human fetal fibroblasts. Statistical analyses showed that cells grown in M199 had a significantly lower PDL than that of cells cultured in any other medium. The PDL in McCoy's 5A was significantly lower compared to that in BME, MEM and HMEM. Cells grown in BME, MEM and HMEM showed similar PDL. It was found that the nuclei of aged cells grown in M199 were significantly larger in volume than cells aged in any other medium. The average increases in nuclear volume of cells during aging in BME, MEM and McCoy's 5A were statistically equivalent. The increase in nuclear volume in HMEM was significantly smaller than that of cells aging in M199 and was longer than that of cells aging in BME or MEM. Linear regression analysis showed that there was a linear increase in nuclear volume as a function of PDL for cells aged in all five media. However, the rate of increase in nuclear volume with increasing PDL varied from medium to medium. There was no significant difference between media on induction of abnormal nuclear morphology as related to PDL. The relative effects of all five media were not uniform on the three cellular parameters investigated during in vitro aging of WI-38 cells. It is, therefore, suggested that one should keep this medium differential in mind to allow meaningful comparison of possible changes in various morphological parameters during in vitro senescence of diploid human fibroblasts such as WI-38.     

Use of serum substitutes for the growth of four cell lines commonly used for virus isolation

Four serum extenders (Opti-MEM I, BioRich, Excyte I and Excyte III) and two serum substitutes (Omni Serum and Serum Plus) were evaluated for the reduction or replacement of FBS for the growth and maintenance of four representative cell lines used for virus isolation. MRC-5, human fetal foreskin fibroblast (HF), A549 and Hep-2 cells were seeded into culture flasks containing MEM with 10% FBS or with the serum extenders or substitutes and subcultured into 16 x 125 mm glass tubes and 1-dram shell vials. Only cells cultured with Opti-MEM I and Omni Serum grew consistently in tubes and vials and these reagents were compared to FBS for viral isolation and detection. Laboratory stocks of CMV, HSV, VZV, adenovirus (Ad) and RSV were tested. In HF and MRC-5 cells, CMV was isolated in cells cultured in either Opti-MEM I or Omni Serum before CPE appeared in cultures containing FBS. Ad and VZV CPE were observed first in HF and MRC-5 cells containing Omni Serum. HSV CPE was seen at the same time in HF and MRC-5 cells with all 3 media. HSV and Ad CPE in A549 cells were also seen at the same time in all 3 media. RSV and Ad CPE in Hep-2 cells were observed first in FBS containing media. Both Opti-MEM I and Omni Serum performed well as substitutes for FBS for growth of stock viruses without loss of cell sensitivity.     

Establishment of cell culture systems from penaeid shrimp and their susceptibility to white spot disease and yellow head viruses

Monolayer cultures were established from ovary, heart, lymphoid tissue and peripheral hemocytes of penaeid shrimps including Penaeus monodon, P. japonicus and P. penicillatus. The most favorable conditions for the culture of penaeid shrimp cells in vitro was in CMRL and L-15 tissue culture media when used within an osmolarity range of 620--760 mmol/kg. The optimal maintenance temperature was 25 °C for tissues of P. japonicus and 28 °C for tissues of P. monodon and P. penicillatus. Among the four tissues tested, lymphoid tissue, or 'Oka organ', was superior to the other tissues for the formation of confluent cell monolayers. Cell cultures from lymphoid tissue and ovary have been subcultured up to three times. When peripheral hemocytes and heart were cultured, a maximum survival of 4 days was obtained. In contrast, cell cultures derived from ovary and lymphoid tissue were maintained alive for at least 20 days in appropriate culture systems. Neither confluent cell sheet nor adherence of cells was obtained in cultivation of hepatopancreas using the present culture systems. The results obtained from the present study also revealed that ovary extract, muscle extract and lobster hemolymph enhanced the survival of the cultured cells of penaeid shrimp in vitro. When the 'Oka organ' cell monolayer was incubated with either white spot disease virus (WSDV) or yellow head virus (YHV), no cytopathic effect (CPE) was obtained. However, at 5--7 days after establishment, significant CPE (a few foci) was observed in cell monolayers derived from WSDV- and YHV-infected Oka tissue. By electron microscopy, virions of WSDV and YHV were observed in the nuclei and cytoplasm of cultured cells. The CPE foci developed further with increased incubation time.     

Establishment of embryonic cell line from sea bass (Lates calcarifer) for virus isolation

A continuous cell line was established from blastula stage embryos of sea bass (Lates calcarifer). The sea bass embryonic cells were maintained in Leibovitz's L-15 supplemented with 15% fetal bovine serum. The embryonic cell line was sub-cultured more than 70 passages over a period of 1.5 years and is designated as Sahul Indian sea bass embryonic (SISE) cell line. The cells were able to grow at temperatures between 25 and 32 degrees C with an optimum temperature of 28 degrees C. The growth rate of sea bass embryonic cells increased as the FBS proportion increased from 2 to 20% at 28 degrees C with optimum growth at the concentration of 15 or 20%. Polymerase chain reaction products were obtained from embryonic cells and blastula of sea bass with primer sets of microsatellite markers of sea bass. Four fish viruses were tested on this cell line to determine its susceptibility to these viruses and this cell line was found to be susceptible to IPNV VR-299 and nodavirus, and the infection was confirmed by cytopathic effect (CPE) and RT-PCR. Further, this cell line was characterized by immunocytochemistry using confocal-laser-scanning microscopy (CFLSM), transfection with pEGFP-N1, proliferate marker (BrdU).     

影响家蝇胚胎细胞系建立的相关因素研究

目的探讨家蝇胚胎细胞系建立的影响因素。方法取不同发育时间的家蝇卵（胚胎），用不同培养基将细胞培养于玻璃培养瓶和塑料培养瓶中，观察细胞生长。结果产出约6h的家蝇胚胎细胞能生长增殖，并成功建立贴壁生长细胞。其它时间段产出卵的家蝇胚胎不能建立贴壁细胞：家蝇胚胎细胞在TC-199培养基中不能生长，在TC-199加酵母提取液和水解乳蛋白的培养基中，家蝇胚胎细胞生长缓慢。在M3培养基中迅速生长，其中在15％胎牛血清（FBS）、20％FBS的M3培养基中，细胞生长迅速并可传代。细胞在玻璃培养瓶中不能贴壁生长，而在塑料培养瓶中能迅速贴壁生长。结论取产出后约6h的家蝇胚胎细胞接种于含15％～20％FBS的M3培养基的塑料培养瓶中，家蝇胚胎细胞能贴壁生长．成功传代。     

Bluetongue virus propagation and plaque assay: variation due to medium and serum supplement

Two base media, minimal essential medium (MEM) and RPMI 1640, were supplemented with a variety of serum extenders and/or substitutes for the purpose of defining a medium formulation capable of supporting good cell (VERO) growth and virologic assays. Bluetongue virus (BTV), the prototype Orbivirus in the Reoviridae, was used in all studies. In general, VERO cells grown in RPMI performed better than those grown in MEM relative to cell growth, virus production and plaque assay. RPMI was better for supporting cell growth when serum extenders (NuSerum or SerXtend) were employed as supplements. Relative to virologic techniques, cells grown in RPMI produced higher virus titers in both propagation studies and plaque assays. The interval from infection to greater than 90% cytopathic effect (CPE) was consistently shorter with RPMI as the base medium. Cell cultures supported with RPMI base medium, supplemented with 3.5% FBS with SerXtend, provided the best overall performance relative to: (a) amount of virus produced by infected cell monolayers, (b) sensitivity to productive infection under overlay conditions (revealed the highest titer of a standard virus stock) and (c) plaque assay quality including cell quality, plaque size and plaque clarity.     

Pathogenicity of Spiroplasma sabaudiense (mollicute) for the cells (c6/36) of Aedes albopictus (Insecta: Diptera) in vitro

An initial stock of Spiroplasma sabaudiense had been maintained in cell-free medium for 2-3 years. Subsequent passages in Leibovitz's medium L-15 in the presence of Aedes albopictus (C6/36) cells dramatically increased the pathogenicity of S. sabaudiense towards these cells. Cytopathogenicity included production of syncytia, an increase in the number of mitochondria and alteration of their morphology, vacuolisation and a reduction in the rate of growth. Cell lysis was associated with cytadsorption by spiroplasmas and was apparent following the third passage. At six days post-inoculation during the fourth and fifth passages, cell cultures had been totally destroyed by S. sabaudiense. The intra-cellular stage of S. sabaudiense is described and the literature pertaining to the pathogenicity of spiroplasmas for insects is reviewed.     

Comparison of a serum replacement (Omni Serum) and fetal bovine serum in cell cultures used to isolate herpes simplex virus from clinical specimens.

Traditionally, fetal bovine serum (FBS) has been the principal component in media used in the growth and maintenance of cell cultures. Recent shortages have affected the cost and availability of FBS to clinical laboratories. Furthermore, lot-to-lot variability can affect cell culture performance and growth. We evaluated a commercially available serum replacement (Omni Serum; Advanced Biotechnologies Inc., Columbia, Md.) for use in the growth of cell cultures and for use in maintenance media used for the isolation of herpes simplex virus from clinical specimens. Cells (rhabdomyosarcoma and mink lung) raised on 5% Omni Serum grew as well as those grown on 10% FBS. The sensitivity of the Omni-raised cells to herpes simplex virus that had been isolated from 111 clinical specimens was equal to that of the cells raised and maintained with FBS. Cells grown with 10% FBS and maintained with 2% Omni Serum displayed the same sensitivity and integrity in tubes (rhabdomyosarcoma and mink lung) and vials (MRC-5 cells) as cells grown with 10% FBS and maintained with 5% FBS. This study indicates that Omni Serum is an acceptable substitute for FBS in maintenance media for cell culture tubes and vials used for viral isolation from clinical specimens.     

The morphological and karyological characteristics of MDCK and vero (B) cells cultures on plant hydrolyzate-based nutrient media

MDCK and Vero (B) cell cultures were propagated during 10 passages in the experimental nutrient media containing the soybean powder hydrolyzate prepared using trypsin and bromelain enzymes and the rice powder hydrolysate prepared with trypsin and in the control DMEM and SFM4 MegaVir media. The karyological, morphological, and proliferative characteristics of continuous cultures were examined and compared. The experimental media supplied with 3% fetal bovine serum (FBS) (Gibco, U.S.A.) showed high growth-enhancing properties and failed to affect their morphology. After propagated during 10 passages in the experimental media preserved a stable karyotype. MDCK cell cultures in the nutrient media based on rice and soybean powder hydrolyzates low (2%) in FBS caused no substantial changes in the proliferation indices and morphological and karyological characteristics of cell cultures.     

Improved technique for short-term culture and cytogenetic analysis of human breast cancer.

Various growth media and procedures for tissue disaggregation and culturing were tested with regard to cell attachment, the type of cells to grow out, and the emergence of cytogenetically abnormal clones in cultures of 20 primary breast carcinomas. Clonal chromosome abnormalities were detected in 16 cases (80%). Our findings allow us to suggest a series of modifications of existing culturing and chromosome preparation techniques for breast cancer cytogenetic analysis. The improvements include: (1) combined mechanical and enzymatic disaggregation of the tumor samples, (2) initiation of short-term cultures in plastic flasks that have a Primaria-modified tissue culture surface or have been coated with Vitrogen 100, (3) use of serum-free growth medium, CDM-5, but with temporary (24 hours) enrichment with 20% FBS if rapid cell attachment is not achieved, (4) partial and sequential harvesting of the cultures, and (5) use of minimal volumes of hypotonic and fixative solutions during harvesting.     

Effect of polyoxyethylene sorbate compounds (Tweens) on colonial morphology, growth, and ultrastructure of Mycobacterium paratuberculosis

Polyoxyethylene sorbate (Tween) compounds were tested to compare their growth stimulation effects on Mycobacterium paratuberculosis. Three low passage and three high passage clinical isolates and ATCC strain 19698 were used. Tween 20, 40, 60, and 80 were tested at concentrations of 0, 0.001, 0.01, 1.0, and 3.0% (w/v) in radiometric broth culture media and in Middlebrook 7H9 agar plates. Scanning and transmission electron microscopy were used to examine cell wall appearance and ultrastructure, respectively. In broth culture, 0.1% (w/v) Tween 60 most dramatically enhanced growth of M. paratuberculosis ATCC strain 19698. The effects of Tween 40 and 80 on growth took a bimodal form, enhancing growth at concentration ranges of 0-0.01% and 0.1-1.0% (w/v) but suppressing growth at concentrations of 0.01-0.1% (w/v). Two of three high passage clinical isolates grew optimally in the presence of 1.0% (w/v) Tween 80, while the remaining high passage isolate and all three low passage isolates grew best in media containing 0.1% (w/v) Tween 80. Colonial morphology of all strains grown on Middlebrook 7H9 agar without Tween 80 was irregular and granular whereas colonies on plate media containing greater than 0.01% (w/v) Tween 80 were entire, smooth, and domed. Scanning electron microscopy also revealed a transition from rough to smooth cell walls with increasing Tween 80 concentration. Transmission electron microscopy showed the presence of low electron dense intracellular vacuoles in Tween 80 grown M. paratuberculosis cells. Thus, Tweens altered colonial morphology, the cell wall surface, and ultrastructure of M. paratuberculosis and stimulated its growth in vitro in a concentration-dependent, and often bimodal, fashion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of selected picornaviruses by high hydrostatic pressure

The potential of high hydrostatic pressure processing (HPP) to inactivate Aichi virus (AiV), human parechovirus-1 (HPeV-1), and the coxsackievirus strains A9 and B5 was investigated. For coxsackievirus A9 (CAV9), a 5-min HPP treatment in minimum essential growth medium (MEM) supplemented with 10% fetal bovine sera (FBS) resulted in 3.4-, 6.5-, and 7.6-log(10) tissue culture infectious dose (50%) (TCID(50)) reductions at 400, 500, and 600megaPascals (MPa), respectively. For HPeV-1, a 5-min treatment in MEM with 10% FBS resulted in reductions of 1.3-, 4.3-, and 4.6-log(10) TCID(50) at 400, 500, and 600MPa, respectively; however, AiV and coxsackievirus B5 (CBV5) in MEM supplemented with 2 and 10% FBS, respectively, remained fully infectious after a 5-min treatment at 600MPa. These data establish that different picornaviruses have widely variable pressure inactivation thresholds in response to HPP.     

大鼠角朊细胞导电聚吡咯膜原代培养法的建立

采用正交实验设计方法，对影响大鼠朊细胞生长的细胞培养液中四种主要组成成分及导电聚吡咯膜促细胞生长条件进行了研究。结果表明，每1000ml DMEM中胎牛血、NEAA、胰岛素、氢化可的松的含量分别为10％、1％，6ml、10mg时，对角朊细胞的培养效果较佳；而浓度为0.1M的吡咯单体的电流为10mA、通电100s条件下制备的聚吡咯膜，在电刺激电压100mV，刺激时间为2h的条件下对角朊细胞生长作用最显著。且在此培养条件下检测的细胞数是常规培养的1.30倍。     

Studies on primary cell cultures derived from ovarian tissue of Penaeus monodon

As part of a bioassay approach to investigate ovarian development and function, primary cell cultures were derived from Penaeus monodon ovaries at various stages of maturation. These cultures were established in modified Grace's or modified 2× L-15 media. Various supplements including growth factors, vitamins and minerals were trailed. Four morphologically different types of cells (epithelioid, fibroblastic, rounded, and epithelioid with large nuclei) were maintained for up to 17 months. Epithelioid cells grew best in modified Grace's medium but were generally short-lived (less than two months). Fibroblast-like cells formed confluent monolayers in modified 2× L-15 medium, were passaged three times and survived for 17 months. In other cultures, millions of rounded cells migrated from tissue. They survived for prolonged periods (up to ten months), either loosely attached to the flask or suspended in the medium. A change in dominant cell type from fibroblastic to epithelioid was observed in some cultures after three or nine months incubation. These epithelioid cells which had very large nuclei, grew to confluence but could not be sub-cultured. It is noteworthy that the rounded cells and the epithelioid cells with the large nuclei both produced vitellogenin in protein-free media.     

Composition of surface oxide film of titanium with culturing murine fibroblasts L929

Changes in the composition of surface oxide film on titanium specimens in the presence of amino acids, serum proteins, and cells were characterized using X-ray photoelectron spectroscopy. The surface oxide film on titanium formed in the air is so protective that the further oxidation of titanium is prevented in various circumstances. During immersion of the specimen in Hanks' solution, Eagle's minimum essential medium (MEM), and MEM with the addition of fetal bovine serum (MEM+FBS), calcium phosphate precipitated, causing the increase in thickness of the surface oxide film. Calcium phosphate was also precipitated with culturing murine fibroblast L929, but the amount of the calcium phosphate was smaller than those in Hanks' solution, MEM, and MEM+FBS. The relative concentration ratio of calcium to phosphorous, [Ca]/[P], increased with proteins charging negatively, while the ratio decreased with the cells whose extracellular matrix charging positively. In addition, sulfur precipitated as S(0) and/or S(2-) only with culturing the cells. Sulfate ions in the MEM+FBS are reduced at the interface between titanium and the solution with the existence of cells.     

Fetal bovine serum contains an inhibitor of interleukin-1

The keratinocyte cell line COLO-16 constitutively produces factors with interleukin-1 (IL-1) activity including IL-1 alpha and IL-1 beta. IL-1 activity assayed by thymocyte proliferation from cell supernatants was 20-50% less if cells were maintained in media containing 10% fetal bovine serum (FBS) compared to media without serum 24 h prior to harvest. The increased IL-1 activity in supernatants from cells in serum free media was not due to increased cellular levels of IL-1 alpha or IL-1 beta mRNA. Similarly, IL-1 activity recovered from conditioned supernatants of COS cells transfected with expression vectors containing IL-1 beta cDNA was approximately 22-45% less in cells grown in 20% FBS medium compared to similar cultures grown for 3 days post transfection in 1% FBS. When serial dilutions of recombinant IL-1 were made in buffer containing 10% FBS and assayed by a thymocyte proliferation method, a 30-50% decrease in activity was observed. IL-1 activity was also measured by its ability to induce prostaglandin E2 synthesis by fibroblasts. When COS conditioned supernatants were applied to fibroblast cultures there was 30% less prostaglandin E2 activity from fibroblasts treated with COS supernatants containing 20% FBS, compared to supernatants containing 1% FBS. The inhibitor molecule was partially purified by gel filtration and found to have a molecular weight of approximately 85,000. The presence of FBS in cell-conditioned media significantly reduces the sensitivity of IL-1 detection by bioassay techniques.