Monolayer Tissue Culture Research Articles

The Quandary of Cellular Fractionation — Optimizing Ambion™ Paris System to Advance HPV16 Cancer Research

Background and Hypothesis: Human Papillomavirus (HPV) is the causative agent in nearly all cervical cancer cases. It has been shown that the HPV type 16 E6 protein interacts directly with the host protein NFX1-123. Short-term studies have shown that NFX1-123 remains in the cytoplasm; however, it has not been investigated whether NFX1-123 actually translocates to the nucleus in the long term. We hypothesize that over time, NFX1-123 translocates to the nucleus of the cell in long term cultures with 16E6. This present study seeks to optimize the AmbionTM Paris system to allow for pure, proper separation of the cytoplasmic and nuclear cellular compartments.
 Project Methods: Three biologically unique backgrounds of human foreskin keratinocytes (HFKs) were cultured in a monolayer tissue culture dish. Using the AmbionTM Paris system, proteins were isolated as whole cell extracts, or nuclear and cytoplasmic fractions. For one lysis method, only protease inhibitors were added to the lysis buffers of the AmbionTM Paris system. For another lysis method, 1% NP-40 and protease inhibitors were added to the lysis buffers of the Paris system. Protein lysate concentrations were quantified, then purity of subcellular lysates was determined by western blotting. Histone H3 and GAPDH were used to identify nuclear and cytoplasmic compartments, respectfully.
 Results: Western blotting confirmed that adding 1% NP-40 to the lysis reagents of the AmbionTM Paris kit optimized subcellular fractionization.
 Potential Impact: Being able to efficiently separate the cytoplasmic and nuclear compartments will allow for accurate identification of NFX1-123 localization during long-term HPV16 infection. If NFX1-123 is found to move into the nucleus under the influence of HPV 16E6, then this could indicate potential transcriptional regulatory functions of the NFX1-123 protein during HPV infection, which is unique from its function in non-infected cells.

CYTOPATHOGENICITY OF INFLUENZA VIRUS (H4 N3)

Monolayer tissue cultures of chicken embryo fibroblast ( CEF ) cells infected with avian influenza virus isolate were examined by the hematoxylie and eosin (H&E) staining and indirect immunoperoxidase test for studying the cytopathogenic effect of the virus. Cytopathological changes which occurred in the uncleus of infected cells included nuclear and nucleolar hypertropy, chromatin margination and intranuclear inclusions. The most striking cytoplasmic change were the presence of perinuclear. eosinophilic inclusions at 22-36 hours post inoculation ( p. i.). Vacuolization, and granulation were also observed. Indirect immunoperoxidase ( IP ) test demonstrated the localization of influenza virus antigens in infected cells. A positive peroxidase reaction observed in the nucleus and cytoplasm were similar to those shown hematoxyline and eosin staining.

Microfibrous Extracellular Matrix Changes the Liver Hepatocyte Energy Metabolism via Integrins.

Cell line-based liver models are critical tools for liver-related studies. However, the conventional monolayer culture of hepatocytes, the most widely used in vitro model, does not have the extracellular matrix (ECM), which contributes to the three-dimensional (3D) arrangement of the hepatocytes in the liver. As a result, the metabolic properties of the hepatocytes in the monolayer tissue culture may not accurately reflect those of the hepatocytes in the liver. Here, we developed a modular platform for 3D hepatocyte cultures on fibrous ECMs produced by electrospinning, a technique that can turn a polymer solution to the micro/nanofibers and has been widely used to produce scaffolds for 3D cell cultures. Metabolomics quantitation by liquid chromatography-mass spectrometry (LC-MS) indicated that Huh7 hepatocytes grown in microfibers electrospun from silk fibroin exhibited reduced glycolysis and tricarboxylic acid (TCA) cycle, as compared to the cells cultured as a monolayer. Further mechanistic studies suggested that integrins were correlated to the ECM's effects. This is the first time to report how an ECM scaffold could affect the fundamental metabolism of the hepatocytes via integrins.

Methylation Changes of Primary Tumors, Monolayer, and Spheroid Tissue Culture Environments in Malignant Melanoma and Breast Carcinoma.

Epigenetic changes have major role in the normal development and programming of gene expression. Aberrant methylation results in carcinogenesis. The primary objective of our study is to determine whether primary tumor tissue and cultured tumor cells in 2D and 3D tissue culture systems have the same methylation signature for PAX5, TMPRSS2, and SBDS. These findings will play an important role in developing in vitro model system to understand the effect of methylation inhibitors on primary tumor tissue. In a previous study PAX5, TMPRSS2, and SBDS genes that we are investigating were reported to be methylated more than 60% in breast cancer and malignant melanoma cell lines. However, these genes have never been studied in primary tumor tissues. Thus, primary tumor tissues of breast cancer and malignant melanoma were first grown in 2D and 3D cultures. Then these two types of tumor tissues and their 2D and 3D cultures were investigated for changes considering methylation levels in PAX5, TMPRSS2, and SBDS genes using real-time polymerase chain reaction. No differences were observed in the primary tissues and culture systems for both PAX5 and TMPRSS2 in malignant melanoma tissues. We found that PAX5 gene was an efficient marker to measure the effects of methylation inhibitors for in vitro systems for malignant melanoma tissue.

Repurposing Human Osteoarthritic Cartilage as a Bone Graft Substitute in an Athymic Rat Posterolateral Spinal Fusion Model.

Spinal fusion involves both endochondral and intramembranous bone formation. We previously demonstrated that endochondral cartilage grafts that were derived from human osteoarthritic (OA) articular cartilage can be used as a bone graft in mouse models. We hypothesized that OA cartilage could also be recycled and repurposed as a bone graft substitute in a posterolateral lumbar spinal fusion model in athymic rats. OA articular cartilage was obtained from the femoral resection of a healthy 60-year-old man undergoing elective total knee arthroplasty. The chondrocytes recovered from this tissue were dedifferentiated in monolayer tissue culture and then transitioned to culture conditions that promote chondrocyte hypertrophy. The resultant cell pellets were then used as bone graft substitute for single-level posterolateral spinal fusion in 5 athymic rats. Decortication alone was used as the control group. Spinal fusion was assessed with manual palpation, micro-computed tomography, and histologic analysis. In the experimental group, micro-computed tomography at 4 and 8 weeks demonstrated bilateral fusion in 4 of 5 animals and unilateral fusion in 1 animal. At 8 weeks, manual palpation and histologic analysis showed direct correlation with the radiographic findings. Animals undergoing decortication alone failed to generate any spinal fusion. The difference in the fusion rate between groups was statistically significant with respect to both unilateral fusion (P = .047) and bilateral fusion (P = .007). In the absence of additional surgically implanted bone graft, hypertrophic chondrocyte grafts are sufficient for generating single-level posterolateral lumbar fusion in athymic rats. This animal study demonstrates that cartilage harvested from OA knees can be used as a bone graft substitute. Commercially available cell-based bone grafts have previously only used mesenchymal stem cells or osteoblast precursor cells.

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been 'conditioned' by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model.

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been 'conditioned' by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

Varieties of reentrant dynamics

Experiments were carried out in monolayer tissue cultures of embryonic chick heart cells imaged using a calcium sensitive fluorescent dye. The cells were grown in annular geometries and in annular geometries with an isthmus connecting antipodal region of the annulus. We observed a large number of spatially different patterns of propagation consisting of one or more circulating waves. As well, we also observed rhythms in which rotors embedded in the annuli generated propagating pulses. These results demonstrate that many different patterns of excitation can be present in cardiac tissue with simple geometries.

OvAd1, a Novel, Potent, and Selective Chimeric Oncolytic Virus Developed for Ovarian Cancer by 3D-Directed Evolution

Effective therapeutics for ovarian cancer continue to be urgently needed, particularly for chemotherapy-resistant cases. Here we present both a 3D-Matrigel culture-based expansion of our directed evolution method for generation of oncolytic virotherapies and two promising ovarian-cancer targeted oncolytic viruses, OvAd1 and OvAd2. OvAd1 was developed using Matrigel cell cultures, whereas OvAd2 was developed in parallel using traditional monolayer tissue culture methods. Both viruses are potent against a panel of platinum-resistant ovarian cancer cell lines and are attenuated on normal cells in vitro, resulting in therapeutic windows of ∼200-fold. We observed two benefits of the use of Matrigel-based cultures for directed evolution of these oncolytics: (1) use of Matrigel generated a bioselected pool that was more strongly attenuated on normal cells while retaining its potency against ovarian cancer cells, and (2) in an ovarian carcinomatosis model, the Matrigel-derived virus OvAd1 suppressed all tumor growth while the non-Matrigel-derived virus was 50% effective. Neither virus stimulated formation of peritoneal adhesions as seen for Ad5-based therapies. Consequently, these viruses are novel candidates for development as new effective treatments for aggressive ovarian cancer.

"I make efforts, people make comments": Prof. H. Zanyin Gaw-pioneering the world, the trailblazer and founder of China's virology research.

"I make efforts, people make comments": Prof. H. Zanyin Gaw-pioneering the world, the trailblazer and founder of China's virology research.

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction.

To investigate adenoviral transduction in mesenchymal stem cells (MSCs) and effects on stemness in vitro and function as a cell therapy in vivo. Bone marrow-derived adult and fetal MSC were isolated from an equine source and expanded in monolayer tissue culture. Polyethylenimine (PEI)-mediated transfection of pcDNA3-eGFP or adenoviral transduction of green fluorescent protein (GFP) was evaluated in fetal MSCs. Adenoviral-mediated transduction was chosen for subsequent experiments. All experiments were carried out at least in triplicate unless otherwise noted. Outcome assessment was obtained by flow cytometry or immunohystochemistry and included transduction efficiency, cell viability, stemness (i.e., cell proliferation, osteogenic and chondrogenic cell differentiation), and quantification of GFP expression. Fetal and adult MSCs were then transduced with an adenoviral vector containing the gene for the bone morphogenic protein 2 (BMP2). In vitro BMP2 expression was assessed by enzyme linked immunosorbent assay. In addition, MSC-mediated gene delivery of BMP2 was evaluated in vivo in an osteoinduction nude mouse quadriceps model. New bone formation was evaluated by microradiography and histology. PEI provided greater transfection and viability in fetal MSCs than other commercial chemical reagents. Adenoviral transduction efficiency was superior to PEI-mediated transfection of GFP in fetal MSCs (81.3% ± 1.3% vs 35.0% ± 1.6%, P < 0.05) and was similar in adult MSCs (78.1% ± 1.9%). Adenoviral transduction provided significantly greater expression of GFP in fetal than adult MSCs (7.4 ± 0.1 vs 4.4 ± 0.3 millions of mean fluorescence intensity units, P < 0.01) as well as significantly greater in vitro BMP2 expression (0.16 pg/cell-day vs 0.10 pg/cell-day, P < 0.01). Fraction of fetal MSC GFP positive cells decreased significantly faster than adult MSCs (1.15% ± 0.05% vs 11.4% ± 2.1% GFP positive at 2 wk post-transduction, P < 0.05). Cell proliferation and osteogenic differentiation in vitro were not affected by Ad transduction in both fetal and adult MSCs, but fetal MSCs had reduced chondrogenic differentiation in vitro when compared to adult (P < 0.01). Chondrogenic differentiation was also significantly reduced in Ad-GFP transduced cells (P < 0.05). Ad-BMP2 transduced adult MSCs induced new bone formation in more thighs than Ad-BMP2 transduced fetal MSCs (83% vs 17% of the six treated thighs per group, P < 0.05) and resulted in increased femur midshaft diameter due to greater extent of periosteal new bone (1.57 ± 0.35 mm vs 1.27 ± 0.08 mm, P < 0.05). Fetal MSCs may be genetically manipulated ex vivo with adenoviral vectors. Nonetheless, the abbreviated expression of the exogenous gene may limit their applications in vivo.

Tumor cell-targeted delivery of nanoconjugated oligonucleotides in composite spheroids.

Standard tissue culture has often been a poor model for predicting the efficacy of anti-cancer agents including oligonucleotides. In contrast to the simplicity of monolayer tissue cultures, a tumor mass includes tightly packed tumor cells, tortuous blood vessels, high levels of extracellular matrix, and stromal cells that support the tumor. These complexities pose a challenge for delivering therapeutic agents throughout the tumor, with many drugs limited to cells proximal to the vasculature. Multicellular tumor spheroids are superior to traditional monolayer cell culture for the assessment of cancer drug delivery, since they possess many of the characteristics of metastatic tumor foci. However, homogeneous spheroids comprised solely of tumor cells do not account for some of the key aspects of metastatic tumors, particularly the interaction with host cells such as fibroblasts. Further, homogeneous culture does not allow for the assessment of targeted delivery to tumor versus host cells. Here we have evaluated delivery of targeted and untargeted oligonucleotide nanoconjugates and of oligonucleotide polyplexes in both homogeneous and composite tumor spheroids. We find that inclusion of fibroblasts in the spheroids reduces delivery efficacy of the polyplexes. In contrast, targeted multivalent RGD-oligonucleotide nanoconjugates were able to effectively discriminate between melanoma cells and fibroblasts, thus providing tumor-selective uptake and pharmacological effects.

Technical Aspects of 3-Dimentional Culture of Mammalian Cells

Three dimensional-tissue culture systems of human solid tumor cells has been implemented for effective chemotherapeutic screening and to avoid the disadvantages of the over simplified monolayer tissue culture system. The influence of tumor microenvironment to tumor therapeutics and screening of new anticancer drugs has been found to significant. The complex tumor microenvironment is poorly represented in the simple monolayer tissue culture system. The 3D culture models closely imitate several in vivo conditions of solid tumors in terms of geometry and expression of microenvironment. . However, several technical obstacles might discourage the use of this culture model. In the current mini-review, we are presenting the exact protocol for culturing mammalian cell as three dimensional tissue culture systems (multicellular spheroids and multicellular layers) and how to utilize these models for anti-cancer assessment.

Burkholderia mallei and Burkholderia pseudomallei Cluster 1 Type VI Secretion System Gene Expression Is Negatively Regulated by Iron and Zinc

Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM) were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.

Spontaneous Mutation Reveals Influence of Exopolysaccharide on Lactobacillus johnsonii Surface Characteristics

As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.

Abstract 1333: Organ specific biomatrix scaffolds for ex vivo characterization of colorectal cancer metastases

Abstract Colorectal cancer is the second leading cause of cancer-related deaths in the United States with more than 140,000 new cases being diagnosed this year. However, morbidity and mortality is not generally caused by the primary tumor, but by metastases to other organs. There are few models in which to study colorectal cancer metastasis, either in two-dimensional (2D) monolayer tissue culture or in animal xenografts. Both of these models have limitations. Standard 2D tissue culture, though inexpensive and accessible, poorly mimics the metastatic process and xenograft models are costly and technically challenging. Two of the most common sites of colorectal metastasis are the liver and lungs, however, there are few ex vivo models in which to study metastases to these sites. Advances in materials science have led to the development of ex vivo tissue specific models of extracellular matrices (ECM). We have utilized a novel material, biomatrix scaffolds, to engineer a colorectal cancer metastasis ex vivo model. We hypothesized that through the use of liver and lung specific biomatrix, (1) we can better characterize the colorectal cancer metastatic process by mimicking the organ specific microenvironment and (2) more accurately determine the response to therapeutic agents to treat metastases. In this study, we characterized biomatrix derived from rat liver and lung as a growth substrate for human colon cancer cell lines. We used the human colon cancer derived cell lines: HT29, SW480, CaCo2 and Lovo. Chemotherapeutic response to oxaliplatin, irinotecan and 5-fluorouracil for these cell lines grown on biomatrix was examined. We also used primary cultures of colorectal cancer metastases derived from patient samples. The biomatrix scaffolds were generated through decellularization of rat liver or lungs. We preformed mass spectrometry and determined the ECM composition and the associated growth factors found in lysates of liver and lung remain bound to the biomatrix in concentrations similar to those found in vivo. Colorectal cancer cell lines demonstrated different growth rates and morphologies when grown on biomatrix, resulting in organized, 3-dimenasional growth of non-hollow spheroids several hundred microns in diameter. We demonstrated differential responses to chemotherapeutics and observed differential gene expression via microarray analysis when colorectal cancer cell lines were grown on biomatrix compared to plastic, collagen, or matrigel. We also demonstrated increased tumor cell colony formation in primary cultures of patient derived colorectal metastases on biomatrix compared to plastic or collagen. In conclusion, we have developed a decellularized tissue material, biomatrix, that can provide an ex vivo model of colorectal cancer metastasis to aid in therapeutic drug development and genetic characterization of metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1333. doi:1538-7445.AM2012-1333

Cytotoxicity testing of burn wound dressings, ointments and creams: A method using polycarbonate cell culture inserts on a cell culture system

We have developed a method to test the cytotoxicity of wound dressings, ointments, creams and gels used in our Burn Centre, by placing them on a permeable Nunc™ Polycarbonate cell culture insert, incubated with a monolayer of cells (HaCaTs and primary human keratinocytes). Methods We performed two different methods to determine the relative toxicity to cells. (1) Photo visualisation: The dressings or compounds were positioned on the insert's membrane which was placed onto the monolayer tissue culture plate. After 24 h the surviving adherent cells were stained with Toluidine Blue and photos of the plates were taken. The acellular area of non-adherent dead cells which had been washed off with buffer was measured as a percentage of the total area of the plate. (2) Cell count of surviving cells: After 24 h incubation with the test material, the remaining cells were detached with trypsin, spun down and counted in a Haemocytometer with Trypan Blue, which differentiates between live and dead cells. Results Seventeen products were tested. The least cytotoxic products were Melolite™, White soft Paraffin™ and Chlorsig1% Ointment. Some cytotoxicity was shown with Jelonet™, Mepitel ®, PolyMem ®, DuoDerm ® and Xeroform™. The most cytotoxic products included those which contained silver or Chlorhexidine and Paraffin Cream™ a moisturizer which contains the preservative Chlorocresol. Conclusion This in vitro cell culture insert method allows testing of agents without direct cell contact. It is easy and quick to perform, and should help the clinician to determine the relative cytotoxicity of various dressings and the optimal dressing for each individual wound.

Monolayer destruction by leukocytes from patients with multiple sclerosis

Separated lymphoid cells from patients suffering from multiple sclerosis (MS) were co-cultivated with various cell lines. Over 80% of such co-cultivations showed destruction of the tissue-culture monolayers, whereas less than 5% of "normal" blood co-cultivation behaved in the same manner. Because of the possible involvement of virus in the aetiology of MS, many positive co-cultivations were 1) examined electron-microscopically, but no virus particles were seen; and 2) tested for measles and herpes viruses using immunofluorescent techniques, but these also proved negative. Leukocytes from stroke patients showed monolayer destruction in about 50% of cases. Granulocyte contamination was high in the stroke blood samples. Reduction of granulocyte numbers to "normal" levels completely abrogated the effect in the stroke samples, but had no effect on the MS co-cultivations. Monolayer destruction by MS leukocytes also appeared not to be due to lymphotoxin.

Inhibition of Cryptosporidium parvum infection of a mammalian cell culture by recombinant scFv antibodies

Two phage display antibody libraries (Tomlinson I and J) were screened against the whole oocysts of Cryptosporidium parvum to select for scFv (single chain variable fragment) antibodies. Three scFv antibodies were selected that bound to C. parvum oocysts as determined by monoclonal phage ELISA. DNA sequencing revealed that clone A11 lacked the majority of its V (H) chain. Clone B10 had a stop codon in the first framework region of the V (H) chain. We changed this stop codon to Gly by site-directed mutagenesis, and designated the variant mutB10. Clone B9 had a complete scFv gene with no internal stop codons. These antibody genes were individually subcloned into the pET-20b expression vector for soluble scFv antibody production. C. parvum infectivity was determined by infection of HCT-8 tissue culture monolayers and quantified by the foci detection method. By incubating C. parvum oocysts with individual scFv antibodies for 1 h at 37 degrees C prior to infecting the HCT-8 cells with the oocyst-scFv mixture, the infectivity of C. parvum was reduced in a dose-dependent manner. At the highest soluble scFv concentration tested (4 nmol), the mean number of infectious foci was reduced by 82%, 73% and 94% for the A11, B9 and mutB10 scFv, respectively. This inhibition of oocyst infectivity was abolished when the scFvs were exposed to boiling water. The results showed that the 3 selected scFvs bound to C. parvum oocysts, and their ability to neutralize infectivity may have potential therapeutic potential against cryptosporidiosis.