Showing papers on "Tissue culture published in 1995"

Three-dimensional cell and tissue culture system

Abstract: The present invention relates to a three-dimensional cell culture system which can be used to culture a variety of different cells and tissues in vitro for prolonged periods of time. In accordance with the invention, cells derived from a desired tissue are inoculated and grown on a pre-established stromal support matrix. The stromal support matrix comprises stromal cells, such as fibroblasts actively growing on a three-dimensional matrix. Stromal cells may also include other cells found in loose connective tissue such as endothelial cells, macrophages/monocytes, adipocytes, pericytes, reticular cells found in bone marrow stroma, etc. The stromal matrix provides the support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of cells in culture. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts found in vivo.

Plant Cell, Tissue and Organ Culture

Abstract: The origin of plant cell and tissue culture can be found in a treatise published during the mid-18th century, entitled La Physique des Arbes, that describes the formation of callus tissue following the for mation of a ring of cortex from elm trees. Over the next two centuries, the discovery of plant growth hormones, in particular auxins and cytokinins, and detailed analyses on the nutritional requirements of plants, led to the formulation of media that could maintain actively dividing cultures derived from gymnosperms, and both dicotyledon ous and monocotyledonous angiosperms. However, much of the prog ress and technological development in the in vitro propagation of plant cells, tissues, and organs has occurred during the last 25 years. Recently, plant tissue culture techniques have been used as basic tools in the rapidly expanding field of plant biotechnology for the development and clonal propagation of new and/or improved plant varieties. Plant tissue culture is used for the micropropagation of commercially valuable cultivars that include ornamentals, oil palm, Glycyrrhiza, Pyrethrum, pine, Eucalyptus, sugar cane, and potatoes. Cultured plant tissue is also used for the selection of cells and, ul timately, the regeneration of plants that are tolerant to physical stresses such as pathogens, drought, and temperature extremes, and to chemical stress agents such as salinity, herbicides, proteins, and pyrethrins. In addition, new plants have been produced by the fusion of protoplasts prepared from cultured cells of different species in cluding sunflower and french bean, tomato and potato, and various cultivars of Datura. Finally, bacterial vectors and various mechanical methods have been used to introduce foreign genes into cultured plant tissues. Genetic transformation can result in profound changes in the phenotype and/or biochemical profile of the regenerated trans genic plants that are not characteristic of the wild type. An impressive variety of technologies in tissue culture, genetic manipulation, and molecular biology have been developed for nu merous plant species. Many of these techniques, sometimes referred to as plant biotechnology, have been extensively summarized and compiled in a well-balanced collection of easy-to-follow protocols presented in three separate, but complimentary, volumes. Plant Cell, Tissue and Organ Culture consists of 22 chapters (with 86 figures) and 5 appendices. The chapters cover critical aspects of (a) the es sential requirements for the operation of a plant tissue culture lab oratory; (b) the basic procedures of micropropagation, regeneration, and somatic embryogenesis; (c) some specific applications of organ culture systems such as embryo rescue and culture, and anther and microspore culture for haploid and double haploid production; (d) elementary transformation technology; and (e) useful microtechnique and analytical protocols specifically adapted to cultured tissues and cells. The appendices provide a convenient summary of media for mulations and commercial suppliers for the materials described in the text.

Culturing satellite cells from living single muscle fiber explants

Abstract: Conventional methods for isolating myogenic (satellite) cells are inadequate when only small quantities of muscle, the tissue in which satellite cells reside, are available. We have developed a tissue culture system that reliably permits isolation of intact, living, single muscle fibers with associated satellite cells from predominantly fast and slow muscles of rat and mouse; maintenance of the isolated fibers in vitro; dissociation, proliferation, and differentiation of satellite cells from each fiber; and removal of the fiber from culture for analysis.

Expression of the Rabies Virus Glycoprotein in Transgenic Tomatoes

Abstract: We have engineered tomato plants (Lycopersicon esculentum Mill var. UC82b) to express a gene for the glycoprotein (G-protein), which coats the outer surface of the rabies virus. The recombinant constructs contained the G-protein gene from the ERA strain of rabies virus, including the signal peptide, under the control of the 35S promoter of cauliflower mosaic virus. Plants were transformed by Agrobacterium tumefaciens-mediated transformation of cotyledons and tissue culture on selective media. PCR confirmed the presence of the G-protein gene in plants surviving selection. Northern blot analysis indicated that RNA of the appropriate molecular weight was produced in both leaves and fruit of the transgenic plants. The recombinant G-protein was immunoprecipitated and detected by Western blot from leaves and fruit using different antisera. The G-protein expressed in tomato appeared as two distinct bands with apparent molecular mass of 62 and 60 kDa as compared to the 66 kDa observed for G-protein from virus grown in BHK cells. Electron microscopy of leaf tissue using immunogold-labeling and antisera specific for rabies G-protein showed localization of the G-protein to the Golgi bodies, vesicles, plasmalemma and cell walls of vascular parenchyma cells. In light of our previous demonstration that orally administered rabies G-protein from the same ERA strain elicits protective immunity in animals, these transgenic plants should provide a valuable tool for the development of edible oral vaccines.

Transdifferentiation of retinal pigment epithelial cells from epithelial to mesenchymal phenotype.

Abstract: PURPOSE To describe and evaluate retinal pigment epithelial (RPE) cell transdifferentiation in vitro and to determine its importance to the development of proliferative vitreoretinal disorders. METHODS Porcine RPE cells from single animals were examined at different passages in culture. The authors examined cellular morphology, contraction of a collagenous matrix, and adhesion to fibronectin and type I collagen-coated substrata. These activities were correlated with loss of epithelial characteristics, redistribution of the actin cytoskeleton, and expression of alpha-smooth muscle actin (alpha-SMA), a marker of myoid differentiation. RESULTS During routine culture on tissue culture plastic, porcine RPE cells lose epithelial characteristics and acquire a mesenchymal cell-like phenotype. The ability of cultured porcine RPE cells to adhere to and exert tractional forces on an extracellular matrix increases with continued passage in vitro and transdifferentiation. This correlates with the loss of the differentiated epithelial morphology, decreased expression of the epithelial marker cytokeratin 18, redistribution of the actin cytoskeleton, and de novo expression of alpha-SMA. CONCLUSION Results indicate that RPE transdifferentiate in culture and that this transition is accompanied by a shift in biologic activities. Therefore, morphologic and behavioral transdifferentiation of these cells in culture are influencing factors in experimental pathology. The potential relevance of these extensive changes to the biology of proliferative vitreoretinal disorders is discussed.

Growth Factor/Matrix-Induced Proliferation of Human Adult β-Cells

Abstract: Proliferation of human β-cells in vitro is desirable for both transplantation and biological studies. In this study, human pancreatic islets obtained from cadavers were kept in tissue culture plates that favored cell attachment. When the cells attached to the matrix produced by the rat-bladder carcinoma cell line 804G, 5′-bromo-2′-deoxyuridine (BrdU) labeling increased from 4.7 ± 2.5 to 13.2 ± 2.2%, while cells simultaneously labeled for insulin and BrdU increased from 0 to 32%. Addition of the growth factor hepatocyte growth factor/scatter (HGF/SF) increased BrdU labeling to 17.5 ± 1.8 and the percentage of double positive (BrdU + insulin) cells to 69%. This is the first in vitro demonstration that human β-cells grown in monolayer culture are able to replicate when exposed to selected matrices and growth factors. These experiments add further evidence that HGF/SF is an important mitogenic agent for human β-cells.

Massive programmed cell death in intestinal epithelial cells induced by three-dimensional growth conditions: suppression by mutant c-H-ras oncogene expression.

Abstract: Deregulation of molecular pathways controlling cell survival and death, including programmed cell death, are thought to be important factors in tumor formation, disease progression, and response to therapy. Studies devoted to analyzing the role of programmed cell death in cancer have been carried out primarily using conventional monolayer cell culture systems. However the majority of cancers grow as three-dimensional solid tumors. Because gene expression, and possibly function, can be significantly altered under such conditions, we decided to analyze the control and characteristics of cell death using a compatible three-dimensional tissue culture system (multicellular spheroids) and compare the results obtained to those using two-dimensional monolayer cell culture. To do so we selected for study an immortalized, but nontumorigenic line of rat intestinal epithelial cells, called IEC-18, and several tumorigenic variants of IEC-18 obtained by transfection with a mutant (activated) c-H-ras oncogene. The rationale for choosing these cell lines was based in part on the fact that intestinal epithelial cells grow in vivo in a monolayer-like manner and form solid tumors only after sustaining certain genetic mutations, including those involving the ras gene family. We found that the IEC-18 cells, which grow readily and survive in monolayer cell culture, undergo massive cell death within 48-72 h when cultured as multicellular spheroids on a nonadhesive surface. This process was accompanied by a number of features associated with programmed cell death including chromatin condensation (Hoechst 33258 staining) apoptotic morphology, DNA degradation, and a virtual complete loss of colony forming (clonogenic) ability in the absence of apparent membrane damage as well as accumulation of lipid containing vacuoles in the cytoplasm. Moreover, enforced over-expression of a transfected bcl-2 gene could prevent this cell death process from taking place. In marked contrast, three different stably transfected ras clones of IEC-18 survived when grown as multicellular spheroids. In addition, an IEC cell line (called clone 25) carrying its mutant transfected ras under a glucocorticoid inducible promoter survived in three-dimensional culture only when the cells were exposed to dexamethasone. If exposure to dexamethasone was delayed for as long as 48 h the cells nevertheless survived, whereas the cells became irreversibly committed to programmed cell death (PCD) if exposed to dexamethasone after 72 h. These results suggest that intestinal epithelial cells may be programmed to activate a PCD pathway upon detachment from a physiologic two-dimensional monolayer configuration, and that this process of adhesion regulated programmed cell death (ARPCD) can be substantially suppressed by expression of a mutant ras oncogene.(ABSTRACT TRUNCATED AT 400 WORDS)

IL-10 mediates immunosuppression following primary infection with Toxoplasma gondii in mice.

Abstract: Summary Suppression of the host immune response by Toxoplasma gondii has been observed in both human and experimental murine infection. In this study, inbred mice were infected with T. gondii. At day 7 post-infection, the lymphoproli-ferative response to both mitogen and superantigen as well as parasite antigen were found to be significantly depressed. Using a transwell system, it was determined that the reduced proliferative response was due to soluble factor (s) being expressed by splenocytes from the infected mice. Isolation of the splenocytes into an adherent and nonadherent population suggested that both macrophages and T cells were able to produce at least one soluble factor. Tissue culture supernatant derived from the splenocytes of the infected mice contain increased levels of IL-10, whereas measurable IL-2 levels could not be quantitated. At day 7 post-infection, both a biologic assay for IFN-γ in culture supernatant and the expression of IFN-γ mRNA in the splenocytes were reduced. Antibody to IL-10 was able to partially neutralize (almost 50%) the in vitro immune downregulation of the tissue culture supernatant. Anti-IL-10 in combination with a nitric oxide (NO) antagonist was able to reverse the inhibitory activity of the culture supernatant by 85%. Since IL-10 is a potent antagonist of IFN-γ, it may represent a critical cytokine involved in mediating T. gondii induced immunosuppression in the infected host.

Dopaminergic neuronal survival and the effects of bFGF in explant, three dimensional and monolayer cultures of embryonic rat ventral mesencephalon

Abstract: Embryonic substantia nigra cells when transplanted into the striatum can reverse many of the defects of Parkinson's disease. The efficacy of such grafts is compromised by the poor survival of grafted dopaminergic neurones; typically, 3–10% survive transplantation. We used three tissue culture models to identify stages in the procedure for the preparation and insertion of grafts which might be responsible for this cell death and to identify environments in which survival is optimised. (1) The ventral mesencephalon was dissected from the donor brain, then placed immediately into culture contained in a collagen gel. (2) The dissected tissue fragments were enzymatically dissociated, then the cells placed into monolayer culture. (3) Enzymatically dissociated tissue was packed into 0.5-mm-diameter porous tubes, to simulate the compaction of cells into a graft deposit in the host brain. Dissociation of the tissue by itself caused the death of approximately 30% of dopaminergic neurones, as judged by the difference in cell counts between the intact embryonic day 14 (E14) mesencephalon, and cells dissociated then packed into tubes. Of the dissociated neurones approximately 60% died during the first 24 h and 87% during the first 3 days in monolayer culture, while only 7% of dopaminergic neurones in three-dimensional cultures and 11% of neurones in explant cultures died over the first 3 days. Embryonic dopaminergic neurones are clearly very vulnerable to adverse conditions during the first days after their removal from the donor brain. The excellent survival of neurones in three-dimensional and explant cultures indicates that close association with other cells, which may provide greatly improved access to trophic factors, can enable the cells to survive this period of vulnerability. In contrast to its effects in monolayer cultures, bFGF had no effect on dopaminergic neuronal survival in either explant or three-dimensional cultures.

Mineral and carbohydrate nutrition of plant cell and tissue cultures

Abstract: Recent advances in our understanding of the mineral (and carbohydrate) nutrition of cultured plant cells and tissues are reviewed. The methods used for empirical selection of nutrient composition of culture media for different plant genera/species and types of culture are critically evaluated. The acquisition of nutrients is discussed in terms of their physical availability in the culture medium and uptake from the medium. The effect on uptake of factors such as pH and water potential and the relationship with growth rates and medium depletion are examined in detail. Finally, some effects of nutrients on morphogenesis of plants are reviewed.

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

Abstract: The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with 3H and auxin was found to promote conversion of zeatin-type cytokinins to 3H-labelled adenine derivatives. When the very rapid metabolism of exogenous [3H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.

Screening of Tissue Cultures and Thalli of Lichens and Some of Their Active Constituents for Inhibition of Tumor Promoter-Induced Epstein-Barr Virus Activation.

Abstract: Inhibition of tumor promoter-induced Epstein-Barr virus (EBV) activation was screened using tissue culture and thallus extracts of lichens. Usnea longissima ACH. thallus and Cetraria ornata MULL. ARG. tissue culture showed strong inhibitory activity. We identified (+)-usnic acid (1), barbatic acid (2), diffractaic acid (3), 4-O-demethylbarbatic acid (4), and evernic acid (5) as inhibitors of EBV activation from the U. longissima thallus. Of these compounds, (+)-usnic acid exhibited the highest inhibitory activity (IC50 = 1.0 microM).

Isolation and characterization of a syncytium-inducing, macrophage/T-cell line-tropic human immunodeficiency virus type 1 isolate that readily infects chimpanzee cells in vitro and in vivo.

Abstract: Fresh human immunodeficiency virus type 1 (HIV-1) isolates from patients with AIDS were screened for infectivity in chimpanzee peripheral blood mononuclear cells (PBMC) to identify strains potentially able to generate high virus loads in an inoculated animal. Only 3 of 23 isolates obtained were infectious in chimpanzee cells. Of these three, only one (HIV-1DH12) was able to initiate a productive infection in PBMC samples from all 25 chimpanzees tested. HIV-1DH12 tissue culture infections were characterized by extremely rapid replication kinetics, profound cytopathicity, and tropism for chimp and human PBMC, primary human macrophage, and several human T-cell lines. An infection was established within 1 week of inoculating a chimpanzee with 50 50% tissue culture infective doses of HIV-1DH12; cell-free virus was recovered from the plasma at weeks 1, 2, and 4 and was associated with the development of lymphadenopathy. Virus loads during the primary infection and at 6 months postinoculation were comparable to those reported in HIV-1-seropositive individuals.

Thymic selection and cell division.

Abstract: Cell division during thymic selection was studied with a system in which purified populations of T cell antigen receptor (TCR)- CD4+8+ (double-positive [DP]) cells and fetal thymic epithelial cells (TEC) were reaggregated in tissue culture. In this system, immature DP cells differentiate into mature single-positive (SP) CD4+8- and CD4-8+ TCRhi cells within 3-4 d, indicative of positive selection. By adding the DNA precursor, bromodeoxyuridine, to the cultures and staining cells for bromodeoxyuridine incorporation, T cell division in reaggregation cultures was found to be high on day 1, low on day 2, and high on days 4-5. Cell separation studies established that cell division on day 1 was restricted to DP blast cells. In the absence of blast cells, small DP cells failed to proliferate and differentiated into SP cells without cell division, thus indicating that proliferation is not an essential component of positive selection. This applied to SP cells generated within the first 2-3 d. Surprisingly, the SP cells generated later in culture showed a high rate of cell division; the proliferating SP cells were TCRhi and included both CD4+8- and CD4-8+ cells. Turnover of TCRhi SP cells was also prominent in the normal neonatal thymus and in TEC reaggregation cultures prepared with adult lymph node T cells. We speculate that division of mature SP cells in the perinatal thymic microenvironment is driven by stimulatory cytokines released from TEC. Such proliferation could be a device to expand the mature T cell repertoire before export to the periphery.

Expression of gamma-glutamyl transpeptidase provides tumor cells with a selective growth advantage at physiologic concentrations of cyst(e)ine

Abstract: Cells from the GGT-negative mouse hepatoma cell line, Hepa 1-6, were transfected with a human GGT cDNA and stably transformed clones were isolated. In standard tissue culture medium the GGT-positive cells and GGT-negative controls grew equally well. However, when the cysteine concentration of the medium was reduced to physiologic levels the GGT-positive cells had a growth advantage. Further investigation revealed that the medium of the GGT-negative Hepa 1-6 cells contained glutathione that had been excreted by the cells, but no glutathione was present in the medium of the GGT-positive cells. We have previously shown that expression of GGT enables cells to use extracellular glutathione as a source of cysteine (Hanigan and Ricketts, Biochem., 32:6302, 1993). These new data reveal that physiologic levels of cysteine can be limiting for cell growth and expression of GGT can provide the cells with a selective growth advantage. These data explain the observation that cells transfected with GGT grow at the same rate as the GGT-negative controls in tissue culture medium which contains a high level of cysteine, but the GGT-positive cells grow more rapidly than the GGT-negative cells when transplanted into animals (Warren et al., Proc. Soc. Exp. Biol. Med., 202:9, 1993). GGT-positive tumor cells have a selective growth advantage in vivo in comparison to GGT-negative tumor cells because they are able to use serum glutathione as a secondary source of cysteine thereby overcoming the growth restriction imposed by serum levels of cysteine.

A tissue culture bilayer model to study the passage of Neisseria meningitidis.

Abstract: A tissue culture bilayer system has been developed as a model to study the mechanisms of attachment and invasion involved in the pathogenesis of Neisseria meningitidis. The model incorporates epithelial and endothelial cell layers separated by a microporous membrane and makes it possible to observe and quantify the passage of bacteria through the multiple layers and to study the mechanisms by which they make this passage. This model is adaptable to a wide variety of microbial pathogens and can be modified by substituting any physiologically relevant eucaryotic cells for the component layers. The system's makeup of cells of human origin and its reproducibility give it advantages over animal and primary organ culture models, while the added complexity of multiple layers allowing cell-to-cell communication makes it a more realistic human tissue model than standard cell monolayers.

Genetic preservation of plant cells in vitro

Abstract: The preservation of different plant varieties and valuable species is an important prerequisite for successful plant breeding. All relevant techniques required to maintain viable plant cells or organs in vitro over extended periods of time are presented in this manual. A maximum level of genetic stability during storage is taken as an overriding priority in each of the techniques. Each chapter is written as a "hands-on" practical guide and presents procedures that can be adapted to suit individual laboratory conditions and modified to best suit the particular material being preserved.

Expression of 5-lipoxygenase in differentiating human skin keratinocytes.

Abstract: We studied the expression of arachidonate 5-lipoxygenase (5-LO) in a cell line of human keratinocytes (HaCaT) and in normal human skin keratinocytes in tissue culture. In undifferentiated keratinocytes 5-LO gene expression was low or undetectable as determined by 5-LO mRNA, protein, cell-free enzyme activity, and leukotriene production in intact cells. However, after shift to culture conditions that promote conversion of prokeratinocytes into a more differentiated phenotype, 5-LO gene expression was markedly induced in HaCaT cells and, to a lesser extent, in normal keratinocytes. These results show that 5-LO gene expression is an intrinsic property of human skin keratinocytes.

Effect of TGF-β1, TGF-α, and EGF on Cell Proliferation and Cell Death in Rat Ventral Prostatic Epithelial Cells in Culture

Abstract: A tissue culture system for rat prostatic epithelial cells was developed, and the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and transforming growth factor beta 1 (TGF-beta 1) on these cells was evaluated. The primary culture was prepared by DNAse/collagenase dissociation of minced ventral prostates. Cells were initially plated in RPMI-1640 medium containing 10% fetal bovine serum to allow the preferential attachment of stromal cells. Twenty-four hours later, the unattached epithelial cells were replated in WAJC-404 medium supplemented with insulin (5 micrograms/ml), transferrin (5 micrograms/ml), and selenious acid (5 ng/ml). Bovine pituitary extract (BPE) (30 micrograms/ml), EGF (10 ng/ml), and TGF-beta 1 (0, 0.1, and 1.0 ng/ml) were added either alone or in combination according to experimental requirements. The rate of cell proliferation was assessed by counting the total cell number and by [3H]thymidine incorporation. Prostatic epithelial cells exhibited a bell-shaped growth curve in a span of 7-8 days, with a growth peak at day 3 or 4 of culture. Treatment of cells with EGF or TGF-alpha resulted in a concentration-dependent increase in cell growth, whereas addition of TGF-beta 1 into the culture resulted in an inhibition of cell proliferation that could be reversed with increasing concentrations of EGF. Cell death was assessed using the terminal deoxynucleotidyl transferase (TdT)-mediated immunoperoxidase-digoxigenin nick end labeling technique and the trypan blue exclusion test. Epithelial cells cultured in media containing EGF had the lowest incidence of cell death. Cells cultured in the absence of EGF demonstrated a marked increase in cells undergoing cell death. The addition of TGF-beta 1 into the EGF-depleted medium caused a further increase of cell death. These results indicated that cell proliferation and cell death in rat prostatic epithelial cells in culture could be modulated by EGF and TGF-beta 1. The former stimulated cell proliferation and prevented cell death, whereas the latter inhibited proliferation in the presence or absence of EGF and induced cell death.

Secretion of biologically active interferon tau by in vitro-derived bovine trophoblastic tissue

Abstract: Secretion of interferon tau (IFN tau) by trophoblastic tissue has been shown to be the first embryonic signal for pregnancy recognition. Therefore we tried to derive biologically active trophoblastic tissue by in vitro techniques. Since conventional in vitro conditions for bovine embryo development were not sufficient for long-term culture, we tested more complex culture conditions, including Menezo B2 or Buffalo rat liver (BRL) cell-conditioned medium, for their ability to support proliferation and IFN tau secretion by in vitro-derived trophoblastic tissue. IFN tau activity was determined by using a biological assay based on the inhibition of the cytopathic effect of vesicular stomatitis virus on Madin-Darby bovine kidney cells. When cultures of individual hatched blastocysts were started in 60-microliters drops of BRL cell-conditioned medium, mean IFN tau secretion (antiviral units/ml/48 h) corresponded to 1200 on Day 11 and to 5000 on Day 13 (p 10(5) antiviral units/ml/48 h on Day 23m, stayed high for about 1 wk, and then slowly declined to levels below 10(3) antiviral U/ml/48 h. The specificity of the cytoprotective effect of IFN tau was tested by Western blot analysis and by immunoneutralization with use of a polyclonal antiserum specific to IFN tau. Our results demonstrate that viable trophoblastic tissue can be maintained entirely in vitro and secretes high amounts of IFN tau.

Dialyzed multiple well tissue culture plate

Abstract: A multiple well tissue culture plate (10) having a plurality of wells (36) with a semi-permeable membrane (20) closing the bottom of each well (36). The multiple well tissue culture plate (10) is placed in contact with a basal medium (26) contained within a basal medium reservoir (12) to allow for exchange of nutrients and waste products between the basal medium (26) and the culture medium (22) across the semi-permeable membrane (20). The dialyzed multiple well tissue culture plate (10) provides dialysis of each individual well (36) within the multiple well tissue culture plate (10) thus eliminating traditional exchange of culture medium to maintain proper culture conditions.

Method of enhancing wound healing by stimulating fibroblast and keratinocyte growth in vivo, utilizing amphipathic peptides

Abstract: A method of treating a wound of a mammalian subject in need of such treatment, to promote healing thereof, comprising administering to the subject, e.g., to the wound locus, a composition comprising a fibroblast and keratinocyte proliferatingly effective amount of an amphipathic peptide, preferably an amphipathic peptide which is antimicrobially effective at such locus. A method is also disclosed of stimulating the accelerated growth of dermal tissue in a tissue culture containing same, comprising applying to the tissue culture a fibroblast and keratinocyte proliferatingly effective amount of an amphipathic peptide, by which the dermal tissue may be grown to produce skin for skin grafting purposes, utilizing a dermal tissue culture containing dermal tissue material of a skin graft recipient of such skin. Novel amphipathic peptides suitable for use in such methods are disclosed.