WO1998026291A1 - Tissus gastriques sous-muqueux constituant de nouveaux outils diagnostiques - Google Patents

Tissus gastriques sous-muqueux constituant de nouveaux outils diagnostiques Download PDF

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Publication number
WO1998026291A1
WO1998026291A1 PCT/US1997/022729 US9722729W WO9826291A1 WO 1998026291 A1 WO1998026291 A1 WO 1998026291A1 US 9722729 W US9722729 W US 9722729W WO 9826291 A1 WO9826291 A1 WO 9826291A1
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Prior art keywords
submucosal tissue
stomach
cells
tissue
vitro
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PCT/US1997/022729
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English (en)
Inventor
Stephen F. Badylak
George B. Boder
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Purdue Research Foundation
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Application filed by Purdue Research Foundation filed Critical Purdue Research Foundation
Priority to JP52695198A priority Critical patent/JP4152444B2/ja
Priority to AU53797/98A priority patent/AU735848B2/en
Priority to CA002274082A priority patent/CA2274082A1/fr
Priority to EP97950919A priority patent/EP0946872A1/fr
Publication of WO1998026291A1 publication Critical patent/WO1998026291A1/fr
Priority to US12/503,774 priority patent/US8647677B2/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56922—Campylobacter
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
    • C12N2533/92—Amnion; Decellularised dermis or mucosa

Definitions

  • the present invention relates to a submucosal tissue composition and the use of those compositions to promote growth of fastidious cells. More particularly, the present invention is directed to the use of submucosal tissue cell culture substrates to enhance the in vitro growth, and thus the identification and diagnosis, of vertebrate infections agents.
  • Bacteria are a diverse group of organisms that live in a broad variety of environments. In particular, many bacterial species live both on and in vertebrate hosts. Bacteria that colonize the interior spaces of vertebrates typically exhibit specific interactions with the tissues that comprise the bacteria's optimal habitat in the host organism. Many bacteria express adhesions having fine tuned specificities for interacting with eukaryotic cell-surface proteins or carbohydrate structures so that only a restricted range of hosts and tissue that carry the appropriate receptors are available for bacterial colonization.
  • tissue tropism For example if bacteria cannot adhere to the mucosal layer of the vertebrate digestive system they will be removed rapidly by the local non-specific host- defense mechanisms (peristalsis, ciliary action and turnover of the epithelial cell populations and mucus layer).
  • peripheralsis ciliary action and turnover of the epithelial cell populations and mucus layer.
  • biochemical parameters such as pH and antimicrobial peptides
  • select for bacterial species/strains select for bacterial species/strains that can colonize specific niches. The result of this selective process is often referred to as tissue tropism.
  • New strains of bacteria are continually being discovered as techniques for their detection improve. However, many detected strains have proven to be difficult to culture outside their natural micronenvironment due to the unique culture conditions required by these organisms. Accordingly, researchers attempting to culture microorganisms try to provide an in vitro microenvironment which mimics the in vivo environment in which the microorganisms grow. The ability to propagate microorganisms in vitro is of particular importance for the identification of infectious and pathogenic organisms, and for diagnosis of diseases. In addition, an in vitro microenvironment which mimics the in vivo environment enables the study of such organisms in vitro.
  • H. pylori a gram negative spiral shaped microaerophilic bacteria.
  • H. pylori live in the mucous layer lining the stomach of vertebrate species and are partially protected from the stomach's acid by the mucosal layer.
  • the organisms secrete proteins that interact with the stomach's epithelial cells and attract phagocytic cells, such as macrophages and leukocytes, and those phagocytic cells induce inflammation and gastritis.
  • the bacteria produce urease, an enzyme that helps to break down urea into ammonia and carbon dioxide.
  • H. pylori also secretes toxins that contribute to the formation of stomach ulcers.
  • H.pylori has been suggested to be a causative agent of chronic active gastritis and gastric duodenal ulcers. More recently, H. pylori infections have also been associated with the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma of the stomach.
  • H. pylori are not the only bacteria capable of colonizing the surface of a primate's stomach. Since the discovery of H. pylori bacteria, scientists have isolated 11 other organisms from the stomachs of other primates such as dog, cats, rodents, ferrets and even cheetahs. These bacteria, for now are considered to be members of the Helocobacter family. All are spiral shaped and highly mobile, properties that enable them to resist muscle contractions that regularly empty the stomach. These organisms grow best at oxygen levels of 5%, matching the level found in the stomachs mucus layer (ambient air is 21% oxygen). Surveys using an antibody-based blood test to reveal the presence H.
  • H. pylori have indicated that one-third to one-half of the world's population carry H. pylori. In the United States and Western Europe children rarely become infected, but the bacteria's prevalence rises with age such that more than half of all sixty year olds in those countries have the bacteria. In contrast, sixty to seventy percent of the children in developing countries show positive test results by age 10, and the infection rate remains higher for adults. H. pylori infection is also common in institutionalized children. H. pylori is capable of long term persistence in untreated individuals, and in the absence of treatment H. pylori remains persistent in the gastric mucosa for the lifetime of the host.
  • the blood test is based on detecting antibodies to H. pylori and thus is not a direct test for the presence of viable H. pylori bacteria. Screening for antibodies only provides information on whether the individual has been exposed to H. pylori. Furthermore, the blood test is know to give false positives.
  • the present invention describes a direct assay for the presence of H. pylori that utilizes a unique cell culture matrix to grow H. pylori. In 1983 H. pylori were cultured in vitro for the first time by using a complex media (Walkers media) and extending the culture timeperiods (5 days instead of the normal 2 day culture).
  • the present invention is directed to an extracellular matrix, comprising vertebrate submucosal tissues, that provides the necessary microenvironment to allow the in vitro culture of fastidious organisms.
  • Naturally occurring extracellular matrices have the ability to serve as a substrate for the growth of prokaryotic organisms, fungal agents and less well defined infectious agents of different genus and species which are difficult to grow in standard culture media.
  • the extracellular matrixes of the present invention enhance the growth, and thus the detection of, and diagnosis of disease states caused by, these organisms.
  • cell culture substrates comprising submucosal tissue isolated from the stomach, the urinary tract, or the intestines can be utilized to mimic the natural in vivo environments of those tissue source organs. Thus cells growing on such substrates in vitro will provide more physiologically relevant data regarding the susceptibility of these organisms to various potential therapeutic agents.
  • contacting as used herein with reference to cell culture is intended to include both direct and indirect contact, for example in fluid communication, between the submucosal tissue and the cultured cells.
  • condition conducive to fastidious cell growth and “conditions conducive to prokaryote cell growth” as used herein refer to the environmental conditions, such as sterile technique, temperature and nutrient supply, that are considered optimal for the growth of those cells. Typically those conditions will mimic the conditions the cells are exposed to in their natural habitats.
  • fastidious organism or “fastidious cell” as used herein refers to organisms or cells that fail to grow (or grow very slowly) on standard growth substrates.
  • fastidious organisms include prokaryotes, fungal agents and less well defined infectious agents of different genus and species which are difficult to grow in standard culture media.
  • the method comprises the step of contacting the fastidious cells, in vitro, with a vertebrate submucosa-derived matrix under conditions conducive to the growth of those cells.
  • the submucosa derived matrices for use in accordance with the present invention comprise highly conserved collagens, glycoproteins, proteoglycans, and glycosaminoglycans in their natural configuration and natural concentration.
  • the submucosal tissue for use in this invention can be obtained from various organ sources, including stomach, bladder or intestinal tissue harvested from animals raised for meat production, including, for example, pigs, cattle and sheep or other warm-blooded vertebrates. This tissue is normally a discarded by-product of meat processing.
  • the tissue can be used in either its natural configuration or in a comminuted or partially digested fluidized form. Vertebrate submucosal tissue is a plentiful by-product of commercial meat production operations and is thus a low cost cell growth substrate, especially when the submucosal tissue is used in its native sheet configuration.
  • submucosal tissue is prepared from warm-blooded tissues including the alimentary, respiratory, intestinal, urinary or genital tracts by delaminating the submucosa from both the smooth muscle layers and the mucosal layers.
  • the preparation of intestinal submucosa is described and claimed in U.S. Patent No.
  • the cell culture substrates comprise stomach submucosa derived from stomach tissue of a warm-blooded vertebrate.
  • the wall of the stomach is composed of the following layers: the tunica mucosa (including an epithelium layer, a tunica intestinal layer consisting of reticular or fine areolar tissue, and a glandular layer), the tunica submucosa layer (composed of areolar tissue and lacking glands), the tunica muscularis layer (composed of three layers of muscle), and the serosa (a layer of mesothelium outside the loose connective tissue which invests the muscle layers). Blood vessels, lymphatic tissue and neurological tissue also pervade the stomach tissues including the tunica submucosa.
  • Stomach submucosal tissue in accordance with the present invention comprises stomach submucosa delaminated from the glandular portion of the tunica mucosa and the smooth muscle layers of the muscularis externa.
  • the composition has proven to have the ability to induce cell growth and proliferation in vitro, when used as a growth substrate material.
  • a cell substrate comprising stomach submucosal tissue has been found to enhance the in vitro growth of organisms that naturally inhabit the stomach of primates.
  • the material can serve as a useful tool for evaluating the natural patterns of growth and proliferation of culture of pathogenic organisms thus allowing better characterization of the pathogenesis of the disease process.
  • the submucosal cell culture substrates in accordance with one embodiment of the present invention comprises stomach submucosa of a warm-blooded vertebrate delaminated from adjacent stomach tissue layers.
  • the stomach submucosa is prepared from the stomach tissue of primates or other acid producing tissues of vertebrate digestive tracts.
  • the present submucosal cell culture substrates comprise submucosa delaminated from the smooth muscle layers of the muscularis externa and at least the luminal portion of the mucosal layer of a segment of the stomach of a warm-blooded vertebrate.
  • the submucosal tissue compositions comprise the tunica submucosa and basilar portions of the tunica mucosa of the stomach of a warm blooded vertebrate.
  • the delamination technique described below provides a tissue composition consisting essentially of stomach submucosa. Those compositions are referred to herein generically as stomach submucosal tissue.
  • stomach submucosal tissue from a segment of stomach is similar to the procedure for preparing intestinal submucosal tissue as detailed in U.S. Patent No. 4,902,508, the disclosure of which is expressly incorporated herein by reference.
  • a segment of stomach tissue is first subjected to abrasion using a longitudinal wiping motion to remove the outer layers (particularly the smooth muscle layers) and the luminal portions of the tunica mucosa layers.
  • the resulting submucosa tissue has a thickness of about 100 to about 200 micrometers, and consists primarily (greater than 98%) of acellular, eosinophilic staining (H&E stain) extracellular matrix material. Occasional blood vessels and spindle cells consistent with fibrocytes are scattered randomly throughout the tissue.
  • the submucosa is rinsed with water for approximately 2 hours and optionally stored in a frozen hydrated state until used as described below.
  • Fluidized submucosal tissue can be prepared in a manner similar to the preparation of fluidized intestinal submucosa, as described in U.S. Patent No. 5,275,826 the disclosure of which is expressly incorporated herein by reference.
  • the submucosal tissue is comminuted by tearing, cutting, grinding, shearing and the like. Grinding the submucosal tissue in a frozen or freeze-dried state is preferred although good results can be obtained as well by subjecting a suspension of submucosal tissue pieces to treatment in a high speed (high shear) blender and dewatering by centrifuging and decanting excess water.
  • the comminuted fluidized tissue can be solubilized by enzymatic digestion of the submucosal tissue including the use of proteases, such as trypsin or pepsin, or other appropriate enzymes or mixtures of enzymes, for a period of time sufficient to solubilize said tissue and form a substantially uniform or homogeneous solution.
  • proteases such as trypsin or pepsin, or other appropriate enzymes or mixtures of enzymes
  • the present invention also contemplates the use of powder forms of submucosal tissue.
  • a powder form of submucosal tissue is prepared by pulverizing submucosal tissue under liquid nitrogen to produce particles ranging in size from 0.1 to 1 mm 2 .
  • the paniculate composition is then lyophilized overnight and sterilized to form a solid substantially anhydrous particulate composite.
  • a powder form of submucosal tissue can be formed from fluidized submucosa by drying the suspensions or solutions of comminuted and/or partially digested stomach submucosa.
  • the present submucosal tissue compositions may be sterilized using conventional sterilization techniques including tanning with glutaraldehyde, formaldehyde tanning at acidic pH, ethylene oxide treatment, propylene oxide treatment, gas plasma sterilization, gamma radiation, and peracetic acid sterilization.
  • a sterilization technique which does not significantly weaken the mechanical strength and biotropic properties of the graft is preferably used. For instance, it is believed that strong gamma radiation may cause loss of strength in the graft material. Because one of the most attractive features of the submucosa grafts is their ability to induce host-remodeling responses, it is desirable not to use a sterilization approach which will detract from that property.
  • Preferred sterilization techniques include exposing the graft to peracetic acid, low dose gamma irradiation ( ⁇ 2.5 mRad) and gas plasma sterilization; peracetic acid sterilization being the most preferred method.
  • peracetic acid sterilization being the most preferred method.
  • the composition is wrapped in a non-porous plastic wrap and sterilized again using ethylene oxide or gamma irradiation sterilization techniques.
  • the submucosal tissue compositions of the present invention are used in accordance with this invention in a method and composition for promoting the growth and proliferation of fastidious cell cultured in vitro.
  • the method comprises the step of contacting fastidious cells, in vitro, with a vertebrate submucosa-derived matrix under conditions conducive to cell growth.
  • optimum cell culture conditions used for culturing cells such as prokaryotes, depend somewhat on the particular cell type, cell growth conditions are generally well known in the art.
  • Stomach submucosal tissue of warm blooded vertebrates is one preferred source of the cell culture substrate for use in this invention.
  • compositions comprising stomach submucosal tissue can be used for supporting growth or proliferation of fastidious prokaryotic cells in vitro.
  • Stomach submucosal tissue can be used in accordance with this invention as a cell growth substrate in a variety of forms, including its native sheet-like configuration, as a gel matrix, as a supplemental component in art-recognized cell/tissue culture media, or as coating for cultureware to provide a more physiologically relevant substrate that supports and enhances the proliferation of cells in contact with the submucosal matrix.
  • the submucosal tissue is preferably sterilized prior to use in cell culture applications.
  • fastidious prokaryotic cells such as H. pylori
  • stomach submucosal tissue under conditions conducive to prokaryotic cell proliferation.
  • the porous nature of stomach submucosal tissue allows diffusion of cell nutrients throughout the submucosal matrix.
  • cells can be cultured on either the luminal or abluminal surface of the stomach submucosal tissue.
  • the luminal surface is the submucosal surface facing the lumen of the organ source and typically adjacent to an inner mucosa layer in vivo
  • the abluminal surface is the submucosal surface facing away from the lumen of the organ and typically in contact with smooth muscle tissue in vivo.
  • the cells are seeded on a cell growth substrate comprising stomach submucosal tissue of a warm-blooded vertebrate and provided a culture medium containing nutrients necessary to the proliferation of said cells.
  • the seeded cells are then cultured under a preselected variable cell growth condition for varying lengths of time and then the mucosal tissue substrate and the cell population on the substrate are histologically examined.
  • the selected growth condition can be the presence or concentration of a cell growth modifier compound, such as cytokines or cytotoxic agents, in the nutrient medium.
  • the selected growth condition may be the modification of environmental factors such as temperature, pH, electromagnetic radiation, or nutrient composition.
  • the effect of the selected growth condition on the morphology and growth of the cells can then be assessed by histological analysis of the control (cells cultured in the absence of the selected growth condition) and the test cell cultures.
  • cell growth substrates in accordance with the present invention are formed from fluidized forms of submucosal tissue that have been solubilized by enzymatic digestion.
  • the fluidized, digested submucosal tissue can be gelled to form a solid or semi-solid matrix, for example, stomach submucosal tissue can be fluidized, enzymatically digested and gelled to form a gelled cell culture substrate comprising stomach submucosal tissue.
  • the viscosity of fluidized submucosa for use in accordance with this invention can be manipulated by controlling the concentration of the submucosa component and the degree of hydration.
  • the viscosity can be adjusted to a range of about 2 to about 300,000 cps at 25° C.
  • Higher viscosity formulations for example, gels, can be prepared from the submucosa digest solutions by adjusting the pH of such solutions to about 6.0 to about 7.4. Eukaryotic or prokaryotic cells can then be seeded directly on the surface of the matrix and cultured under conditions conducive to cell proliferation.
  • the cell growth substrate of the present invention can be combined with nutrients, including minerals, amino acids, sugars, peptides, proteins, or glycoproteins that facilitate cellular proliferation.
  • fluidized or powder forms of submucosal tissue can be used to supplement standard culture media to enhance the standard media's capacity for sustaining and inducing the proliferation of fastidious cells cultured in vitro.
  • a cell culture composition for supporting growth, in vitro, of fastidious organisms (including both eukaryotic and prokaryotic organisms), the composition comprising submucosal tissue of a warm-blooded vertebrate.
  • the composition may further comprise added nutrients, and/or growth factors required for optimal growth of the cultured cells.
  • the submucosa substrates of the present invention can be used with commercially available cell culture liquid media (both serum based and serum free).
  • proliferating cells can either be in direct contact with the submucosal tissue or they can simply be in fluid communication with the submucosal tissue.
  • the tissue graft material of this invention is prepared in accordance with the following steps:
  • the stomach is first removed from the animal source by cutting the esophagus and small intestine at their respective entrance and exit points on the stomach. Any excess mesentery tissue or fat is removed from the stomach and the contents of the stomach are emptied and any remaining residues are removed from the inside of the stomach by rinsing with running tap water.
  • the stomach is then everted to expose the inside layers of the stomach.
  • the portions of the stomach that begin to form the entrance or exit points of the stomach are removed.
  • the stomach is typically left whole, however the stomach can also be cut and flattened prior to removal of unwanted tissues.
  • the luminal surface of the stomach is subject to abrasion using the handle portion of a pair of scissors or hemostats to scrape off the inner layers of the stomach including at least the luminal portion of the tunica mucosa. A thin residual layer will remain at this point. If the tissue was left whole, the stomach tissue is everted again to return the luminal surface of the stomach to the interior of the graft construct. A small cut is then made in the exterior muscle fiber layer.
  • the muscle layers are then delaminated from the submucosal tissue through the use of a pair of scissors or hemostat to enlarge the cut in the muscle and scrape off the muscle layers.
  • the remaining tissue is everted again to place the luminal side on the exterior of the tissue graft.
  • the luminal surface is scraped to remove the remaining inside residue which has a brownish color.
  • the stomach tissue is scraped until the tissue appears pinkish-white in color.
  • care is taken to keep the tissue by periodically hydrating the tissue with water.
  • the stomach submucosa tissue is rinsed in running tap water for approximately two hours to remove any blood or loose tissue scrapings.
  • Example 2 After rinsing the tissue should appear white, if the tissue remains pinkish in color the tissue is rubbed under water until the tissue appears white. After rinsing is complete excess water is removed by ringing the tissue by hand or the use of mechanical ringers. The tissue is then stored in liquid nitrogen at -80°C° .
  • stomach submucosa The ability of stomach submucosa to serve as an extracellular matrix to support in-vitro cell growth was tested by applying several cell types to the stomach submucosal tissue surface under standard cell culture conditions.
  • the cell types tested included 3T3 fibroblasts, intestinal epithelium cells, and FR (fetal rat) mesenchymal cells. All three cell types showed the ability to proliferate readily upon this extracellular matrix without the addition of the supplements that would be needed to grow these cells on a plastic surface. Therefore, it can be concluded that the material contains necessary structure and composition "nutrients" to serve as a cell culture substrate for supporting cell growth.
  • Kirby-Bauer test was conducted. Individual colonies of H. pylori were isolated from a chocolate agar plate and used to inoculate a 1 ml. solution of sterile saline in a small tube. This sterile saline solution was then used to inoculate a chocolate agar plate through the use of a sterile cotton swab. A small piece of stomach submucosal tissue (approximately 25-50 mm. in diameter) was placed in the middle of the inoculated chocolate agar plate and pressed onto the surface of the plate to assure that the submucosa tissue sticks to the chocolate agar.
  • the experiment was conducted in duplicate; two plates having the luminal side of the submucosa tissue in contact with the chocolate agar, and two plates having the abluminal surface of the submucosa tissue in contact with the agar.
  • the plates were then incubated in a Campyjar in the 37° C aerobic incubator for 3-4 days. After incubation the plates were removed from the incubator and Campyjar. The plates were observed to determine if there was a zone of inhibition surrounding the submucosal membranes.
  • the Kirby-Bauer results show that stomach submucosal does not inhibit the growth of H. pylori or other organisms.
  • the lids of a separate sterile 24 well tray were marked with the labels of the appropriate controls and samples.
  • the stomach submucosal tissue was cut into ⁇ 1 cm pieces using a scissors sterilized with alcohol and flamed, and the individual pieces were transferred into the appropriate marked wells using forceps sterilized with alcohol and flamed.
  • Stomach submucosal tissue was placed into the wells either with the luminal side facing up or with the luminal side facing down. 400ml of the appropriate concentration (dilution) of media plus bacteria (10 "3 , 10 "4 , 10 "5 ) was added to each of the wells.
  • the cells were isolated from the tissue as follows: The submucosal tissue was removed from the well and cut into 2 or 3 pieces. These pieces were placed into a centrifuge tube containing 400 ⁇ l of fresh Walker's media and the tube was vortexed for -30 seconds. 100 ⁇ l of the vortexed solution was plated onto a chocolate agar plate and spread with an alcohol flamed hockey stick.
  • stomach submucosa tissue is capable of supporting H. pylori growth.
  • the number of colonies present on the chocolate agar plates increased with each serial dilution as expected which indicates growth of H. pylori. Contamination continues to be a problem in these experiments. Accordingly, the optimal conditions for growing H. pylori on stomach submucosa have not yet been determined. However the experiments demonstrate stomach submucosa's ability to support H. pylori growth. It is anticipated that once the optimal conditions have been defined the difficulties with contamination by other organisms will be eliminated.
  • the present experiment analyzed the growth of H. pylori on stomach submucosal tissue substrates in the presence of bacterial cell culture media.
  • the stomach submucosal tissue substrates can be used to culture H. pylori in the presence of eukaryotic cell culture media.
  • the presence of the eukaryotic cell culture media, and most preferably mammalian cell culture media, will provide a more physiological environment and thus optimize the function of the stomach submucosal tissue as a host for microbial residence and pathogenicity.
  • the culture substrates of the present invention can be used not only for detecting the presence of H. pylori from a source tissue, but can also be utilized to investigate the optimal antibiotics and antibiotic concentrations necessary to effectively treat patients infected with H. pylori.

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Abstract

L'invention porte sur un substrat sous-muqueux de culture de cellules consistant en un tissu de vertébrés à sang chaud et sur un procédé de culture d'organismes exigeants. Les tissus utilisés d ans le cadre de cette invention facilitent la prolifération des cellules, lorsque lesdites cellules sont en contact avec des tissus sous-muqueux dans des conditions conduisant à la prolifération cellulaire.
PCT/US1997/022729 1996-12-10 1997-12-10 Tissus gastriques sous-muqueux constituant de nouveaux outils diagnostiques WO1998026291A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP52695198A JP4152444B2 (ja) 1996-12-10 1997-12-10 新規な診断用具としての胃粘膜下組織
AU53797/98A AU735848B2 (en) 1996-12-10 1997-12-10 Gastric submucosal tissue as a novel diagnostic tool
CA002274082A CA2274082A1 (fr) 1996-12-10 1997-12-10 Tissus gastriques sous-muqueux constituant de nouveaux outils diagnostiques
EP97950919A EP0946872A1 (fr) 1996-12-10 1997-12-10 Tissus gastriques sous-muqueux constituant de nouveaux outils diagnostiques
US12/503,774 US8647677B2 (en) 1996-12-10 2009-07-15 Gastric submucosal tissue as a novel diagnostic tool

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US3268696P 1996-12-10 1996-12-10
US60/032,686 1996-12-10

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US09319841 A-371-Of-International 1997-12-10
US09/990,906 Continuation US6696270B2 (en) 1996-12-10 2001-11-14 Gastric submucosal tissue as a novel diagnostic tool

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WO2000032250A1 (fr) * 1998-12-01 2000-06-08 Cook Biotech, Inc. Dispositif medical comprenant un biomateriau collagenique multi-forme
WO2000032112A1 (fr) 1998-12-01 2000-06-08 Washington University Dispositif d"embolisation
WO2001054176A1 (fr) 2000-01-18 2001-07-26 Xros, Inc., Nortel Networks Liage destine a minimiser la contrainte integree des microstructures et des micromiroirs en silicium
US6666892B2 (en) 1996-08-23 2003-12-23 Cook Biotech Incorporated Multi-formed collagenous biomaterial medical device
US7244444B2 (en) 2004-03-31 2007-07-17 Cook Incorporated Graft material, stent graft and method
US7449027B2 (en) 2004-03-29 2008-11-11 Cook Incorporated Modifying fluid flow in a body vessel lumen to promote intraluminal flow-sensitive processes
US7563277B2 (en) 2005-10-24 2009-07-21 Cook Incorporated Removable covering for implantable frame projections
DE20122916U1 (de) 2000-01-31 2009-12-10 Cook Biotech, Inc., West Lafayette Stentventil
US7648527B2 (en) 2006-03-01 2010-01-19 Cook Incorporated Methods of reducing retrograde flow
US7652077B2 (en) 1996-08-23 2010-01-26 Cook Incorporated Graft prosthesis, materials and methods
US7846199B2 (en) 2007-11-19 2010-12-07 Cook Incorporated Remodelable prosthetic valve
US8388643B2 (en) 1999-09-27 2013-03-05 The Regents Of The University Of California Bioabsorbable polymeric implants and a method of using the same to create occlusions
US8647677B2 (en) 1996-12-10 2014-02-11 Purdue Research Foundation Gastric submucosal tissue as a novel diagnostic tool
US8652191B2 (en) 1999-08-06 2014-02-18 Cook Biotech Incorporated Tubular graft construct
US8728144B2 (en) 2005-12-29 2014-05-20 Cook Medical Technologies Llc Endoluminal device including a mechanism for proximal or distal fixation, and sealing and methods of use thereof
US8882850B2 (en) 1998-12-01 2014-11-11 Cook Biotech Incorporated Multi-formed collagenous biomaterial medical device
US10420636B2 (en) 2004-02-09 2019-09-24 Cook Medical Technologies Llc Stent graft devices having collagen coating
CN110499242A (zh) * 2019-09-24 2019-11-26 扬州大学 一种瘤胃上皮组织体外培养的模拟装置及其模拟方法
US10940167B2 (en) 2012-02-10 2021-03-09 Cvdevices, Llc Methods and uses of biological tissues for various stent and other medical applications
US11406495B2 (en) 2013-02-11 2022-08-09 Cook Medical Technologies Llc Expandable support frame and medical device

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US8920515B2 (en) 1996-08-23 2014-12-30 Cook Biotech Incorporated Graft prosthesis, materials and methods
US9138444B2 (en) 1996-08-23 2015-09-22 Cook Biotech Incorporated Dried collagenous biomaterial medical device
US6666892B2 (en) 1996-08-23 2003-12-23 Cook Biotech Incorporated Multi-formed collagenous biomaterial medical device
US8920516B2 (en) 1996-08-23 2014-12-30 Cook Biotech Incorporated Graft prosthesis, material and methods
US7652077B2 (en) 1996-08-23 2010-01-26 Cook Incorporated Graft prosthesis, materials and methods
US8647677B2 (en) 1996-12-10 2014-02-11 Purdue Research Foundation Gastric submucosal tissue as a novel diagnostic tool
WO2000032112A1 (fr) 1998-12-01 2000-06-08 Washington University Dispositif d"embolisation
US9089626B2 (en) 1998-12-01 2015-07-28 Cook Biotech Incorporated Radiopaque implantable collagenous biomaterial device
EP1985320A1 (fr) 1998-12-01 2008-10-29 Cook Biotech, Inc. Dispositif médical à biomatériau de collagène à formations multiples
WO2000032250A1 (fr) * 1998-12-01 2000-06-08 Cook Biotech, Inc. Dispositif medical comprenant un biomateriau collagenique multi-forme
US8882850B2 (en) 1998-12-01 2014-11-11 Cook Biotech Incorporated Multi-formed collagenous biomaterial medical device
US7713552B2 (en) 1998-12-01 2010-05-11 Cook Biotech Incorporated Radiopaque implantable collagenous biomaterial device
US8652191B2 (en) 1999-08-06 2014-02-18 Cook Biotech Incorporated Tubular graft construct
US8388643B2 (en) 1999-09-27 2013-03-05 The Regents Of The University Of California Bioabsorbable polymeric implants and a method of using the same to create occlusions
WO2001054176A1 (fr) 2000-01-18 2001-07-26 Xros, Inc., Nortel Networks Liage destine a minimiser la contrainte integree des microstructures et des micromiroirs en silicium
EP2329796A2 (fr) 2000-01-31 2011-06-08 Cook Biotech, Inc. Valvules d'endoprothèse vasculaire et ses utilisations
DE20122916U1 (de) 2000-01-31 2009-12-10 Cook Biotech, Inc., West Lafayette Stentventil
US8906083B2 (en) 2000-01-31 2014-12-09 Cook Biotech Incorporated Stent valves and uses of same
US10420636B2 (en) 2004-02-09 2019-09-24 Cook Medical Technologies Llc Stent graft devices having collagen coating
US7449027B2 (en) 2004-03-29 2008-11-11 Cook Incorporated Modifying fluid flow in a body vessel lumen to promote intraluminal flow-sensitive processes
US7244444B2 (en) 2004-03-31 2007-07-17 Cook Incorporated Graft material, stent graft and method
US8500796B2 (en) 2005-10-24 2013-08-06 Cook Medical Technologies Llc Removable covering for implantable frame projections
US8252043B2 (en) 2005-10-24 2012-08-28 Cook Medical Technologies Llc Removable covering for implantable frame projections
US7563277B2 (en) 2005-10-24 2009-07-21 Cook Incorporated Removable covering for implantable frame projections
US8728144B2 (en) 2005-12-29 2014-05-20 Cook Medical Technologies Llc Endoluminal device including a mechanism for proximal or distal fixation, and sealing and methods of use thereof
US9101468B2 (en) 2006-03-01 2015-08-11 Cook Medical Technologies Llc Methods of reducing retrograde flow
US7648527B2 (en) 2006-03-01 2010-01-19 Cook Incorporated Methods of reducing retrograde flow
US8323332B2 (en) 2006-03-01 2012-12-04 Cook Medical Technologies Llc Methods of reducing retrograde flow
US7846199B2 (en) 2007-11-19 2010-12-07 Cook Incorporated Remodelable prosthetic valve
US10940167B2 (en) 2012-02-10 2021-03-09 Cvdevices, Llc Methods and uses of biological tissues for various stent and other medical applications
US11406495B2 (en) 2013-02-11 2022-08-09 Cook Medical Technologies Llc Expandable support frame and medical device
CN110499242A (zh) * 2019-09-24 2019-11-26 扬州大学 一种瘤胃上皮组织体外培养的模拟装置及其模拟方法

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EP0946872A1 (fr) 1999-10-06
JP4152444B2 (ja) 2008-09-17
AU5379798A (en) 1998-07-03

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