WO1999043788A1 - Modele in vitro d'infection virale et reaction immunitaire - Google Patents

Modele in vitro d'infection virale et reaction immunitaire Download PDF

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Publication number
WO1999043788A1
WO1999043788A1 PCT/US1999/004425 US9904425W WO9943788A1 WO 1999043788 A1 WO1999043788 A1 WO 1999043788A1 US 9904425 W US9904425 W US 9904425W WO 9943788 A1 WO9943788 A1 WO 9943788A1
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WO
WIPO (PCT)
Prior art keywords
tissue
matrix
histoculture
immunodeficiency
protocol
Prior art date
Application number
PCT/US1999/004425
Other languages
English (en)
Inventor
Leonid B. Margolis
Original Assignee
Anticancer, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Anticancer, Inc. filed Critical Anticancer, Inc.
Priority to EP99908581A priority Critical patent/EP1068298A1/fr
Priority to AU27977/99A priority patent/AU2797799A/en
Publication of WO1999043788A1 publication Critical patent/WO1999043788A1/fr

Links

Classifications

    • 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
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0651—Lymph nodes
    • 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
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the invention relates to a model system for studying the progress of viral diseases and for assessing possible therapeutic and diagnostic protocols. Specifically, the invention concerns three-dimensional histocultures of adult lymph nodes and tonsils which provide substrates for infection, study, and discovering of new therapeutics and diagnostics.
  • the histoculture techniques of the present invention are useful as model systems to study antigen- specific imrnunoregulation in normal lymphoid tissue. Unlike previously described systems, the antigens can be applied directly.
  • the invention is directed to a histoculture system wherein lymph node or tonsil tissue is supported in a three-dimensional, structurally faithful system to serve as a model for viral infection, particularly HIV infection, as well as a model for response to antigen.
  • a histoculture system which is useful as an in vitro model for viral infection or for response to an antigen which system comprises a flexible macromolecular, porous matrix, and supported thereon, an integral macroscopic section of animal lymph node tissue or tonsil tissue, said matrix immersed in a suitable culture medium wherein the surface of the medium is approximately congruent with the interface between the tissue and the matrix. The section of tissue is then infected with an amount of virus effective to maintain growth of the virus or is stimulated with an antigen.
  • the invention is directed to the in vitro histoculture system which is thus infected with a virus.
  • the invention is directed to a method to chart the progress of viral infection using the histoculture system of the invention and to methods to identify therapeutic compounds and protocols effective - 3 - against the infection using the histoculture as a model, or as a diagnostic over the course of treatment administered to a subject.
  • the invention is directed to a method to chart the progress of an immuno logic response to a specific antigen.
  • the histoculture system of this invention can mount an immune response, as well as support infection by a virus, e.g., HIV, the study of the progress of infection is enhanced by tracking the ability of the infection to inhibit the immune response, causing immunodeficiency in vitro.
  • Figure 1 is a schematic representation of the geometric configuration of the histocultures of the invention.
  • Figure 2 shows the response of uninfected histocultures to antigen.
  • the method employs a histoculture system which permits growth of intact tissue samples in three dimensions while providing for adequate supply of nutrients and oxygen.
  • the histoculture system employs a soft, porous matrix which is, preferably, comprised of extracellular material, such as collagen, polysaccharides, and the like. Suitable matrices are described in Leighton, J. (1957), Freeman (1986) and
  • the matrix is soft and flexible and may indent upon placement of the tissue such that the edges of the matrix may contact the vertical edges of the tissue.
  • the matrix provides a trabecular structure with interstices suited for capillary action to deliver nutrients in the aqueous medium to the tissue supported on the matrix.
  • extracellular macromolecules are preferred, other materials that may be appropriate for the matrix include nylon, borosilicate, glass fiber, or polypropylene.
  • solids comprising one or more organic molecules or molecular aggregates which are those produced and secreted by cells into the extracellular space and which serve, in vivo, as support, adhesive and framework for maintaimng three- dimensional tissue organization and function. These molecules include high- - 4 - molecular-weight proteins and glycoproteins, such as collagen, laminin, fibronectin and the like as well as complex polysaccharides.
  • GELFOAMTM gelatinized pork skin
  • Upjohn Company Kalamazoo
  • MI gelatinized pork skin
  • laminin collagen
  • proteoglycan a composition comprising laminin, collagen, proteoglycan and antactin
  • MATRIGELTM Collaborative Research, Inc., Bedford, MA
  • a specialized collagen produced from pig skin by Health Design Industries, Rochester, NY Other materials which can be used are homopolysaccharide sponges, such as those described by Leighton, J. et al, J National Cancer Inst (1951) 12:545-561. A combination of these materials can be used as well, such as a top layer of collagen containing gel and a bottom layer of homopolysaccharide sponge.
  • suitable matrices can be prepared de novo provided that they are capable of conducting medium to the supported material through capillary action and provide flexible support capable of maintaining medium approximately at the interface between the support and the tissue placed thereon.
  • the ratio of the tissue sample size to the support matrix dimensions is variable; preferably the matrix is of larger top supporting surface area than required to cover the entirety of the tissue sample. Multiple samples can be placed on the same matrix; it is preferred that they do not touch.
  • the vertical dimension of the tissue is such that the nutrients of the medium can be transported through the tissue when the medium is maintained at approximately the lower surface of the tissue as placed on the support matrix.
  • the samples are placed on the matrix so that the interface between the tissue and the matrix coincides approximately with the surface of the medium.
  • the matrix with the tissue sample is immersed in a volume of medium sufficient to contact the matrix but not to completely cover the tissue.
  • the support matrix is preferably pretreated with the culture medium before the tissue sample is placed. This serves to equilibrate the matrix with the medium.
  • the matrix is cut to a predetermined size and soaked in the medium in a sterile container for a time sufficient to saturate and equilibrate the matrix, typically 4 hours at 37°C. - 5 -
  • the media employed can vary, but a typical medium would comprise Eagle's Minimum Essential Medium (MEM) with 10% fetal bovine serum, 0.1 mM nonessential amino acids, and the antibiotics gentamycin (100 ⁇ g/ml) and cefotaxime (95 ⁇ g/ml). Other antibiotics may also be used.
  • the histocultures of the invention have been shown to support living tissue for extended periods and can be used as model systems for viral infection, particularly HIV infection.
  • the progress of viral infection can be monitored in a variety of ways as is understood in the art. For example, at various time points after application of an effective amount virus to maintain viral growth, the tissues can be dissociated into single cells and the cells counted. Any method for counting the cells may be used, typically the cells are stained, either with an appropriate dye or with a labeled antibody. The cells can then be counted using any art-recognized techniques, for example, by flow cytometry.
  • the progress of infection can be assessed by other means such as assays designed to monitor surface cell markers or assays designed to distinguish living from dead cells or assays which are relevant to aspects of intracellular metabolism.
  • the histoculture system can mount an immune response to an added antigen.
  • the histoculture system can also serve as a model for specific immune response in normal tissue.
  • a test recall antigen is supplied to the medium over several days and the medium then analyzed for specific immunoglobulins raised against the antigen.
  • Cytokine concentration in the culture medium can also be monitored using matched antibody pairs and cytokine standards (Endogen, Woburn, MA) in an ELIS A sandwich assay. Further, cells can be analyzed by flow cytometry by mechanically isolating them both from control cultures and from cultures treated with antigen, and then staining for cell surface markers using standard techniques.
  • the ability to mount an immune response to added antigen makes the system particularly valuable in the context of monitoring HIV infection, since infection by HIV may inhibit this immune response, and the ability of HIV infection to do so can be monitored.
  • This provides an additional method for screening protocols and compositions useful in treating HIV, since the system can be tested in the presence and absence of the protocol or composition under conditions where an antigen has also been supplied and the effect of the protocol or composition on the development of immunodeficiency can be measured.
  • the histocultures, infected with the virus of interest are cultured in the presence and absence of a candidate drug or protocol and the effect on the growth of the viral cells and on the health of viral-infected cells is assessed.
  • the drug or protocol is a successful candidate for viral treatment.
  • This aspect is particularly important with respect to HIV, in view of the lack of suitable model systems for this virus.
  • this assay system can also be modified when HIV is the infective agent by monitoring the progress of immunodeficiency characterizing a response to an antigen in the presence of HIV infection.
  • the effect of a candidate protocol or composition in controlling this immunodeficiency response can then be assessed.
  • the histocultures may also be used to culture viruses of interest that may be difficult to passage in in vitro culture.
  • the histocultures serve as diagnostic tools in assessing the progress of therapy.
  • biopsies are removed from a patient being treated with a therapeutic regime and the effect on further growth in histoculture is assessed. This can be performed at various stages of the disease.
  • Example 1 Preparation of Tonsil Histocultures Human tonsils, surgically removed during therapeutic tonsillectomy, were dissected and blocks of tissue of about 1-2 mm in diameter were cultured on collagen sponge gel supports at a liquid/air interface for 10-26 days, as described by Freeman, A.E. et al, Proc Natl Acad Sci USA (1986) 83:2694-2698.
  • the histocultures maintained their morphology, and extended networks of follicular dendritic cells were found inside germinal centers by 3D reconstruction of confocal optical sections of histoculture blocks immunostained with FITC-labeled anti-CD21 antibodies.
  • the cultures also produced IgG.
  • Immunohistochemical analysis revealed well-defined germinal centers formed by B cells with T cells concentrated around them. Although in the first 24-36 hours, some depletion of lymphocytes occurred, all of the key elements of tissue architecture were preserved even in the fourth week in culture.
  • Example 2 Virus Production The histocultures prepared as in Example 1 were infected with HIV-1 at a multiplicity of infection (MOI) of 400-900 TCID 50 (median tissue culture infectious dose) per block.
  • MOI multiplicity of infection
  • TCID 50 medium tissue culture infectious dose
  • LAV.04 (SI), a laboratory strain that induces syncytia in various cell lines and peripheral blood cells;
  • virus infectious titers increased from undetectable levels on day - 8 -
  • Example 3 Monitoring Progress of Infection Tissue blocks from the same tonsil were divided in half. One half was inoculated with HIV. The response to infection was monitored by dissociating the tissue blocks into single cells at various time points, staining with fluorescent antibodies and counting cell populations using flow cytometry.
  • the absolute numbers of cell subsets in a block can be estimated by adding an internal standard number of fluorescent beads to the dissociated cell suspensions.
  • the observed decrease in the number of T cells was mainly due to depletion of the CD4 " subset to 4 ⁇ 2% of control. - 9 -
  • Example 4 Monitoring Progress of an Immune Response Tonsil histocultures were prepared as described in Example 1 from 55 human donors. Either tetanus toxoid (TT) or diphtheria toxin (DT) was added to the medium for 2 days; starting at day 2, the medium was changed every 3 days. Control cultures without TT or DT were analyzed along with the test cultures. Total IgG, IgM and anti-TT or anti-DT IgG were assayed in the collected medium samples using standard ELISA techniques with calibrated standards of human IgG and IgM. Human tetanus immunoglobulin (USP-Hyper-Tet, Miles, Inc.) and equine diphtheria antitoxin
  • the response was dose-dependent in the range of 10-100 ng/ml TT.

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  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
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Abstract

La présente invention concerne un système de modèle in vitro d'infection virale et de réaction immunitaire composé d'un bloc de tissu provenant d'une amygdale ou d'un ganglion lymphatique d'adulte placé sur une matrice souple et poreuse, le bloc de tissu sur son support étant cultivé dans un milieu dont la surface se situe au niveau de l'interface bloc de tissu/matrice. Le système de culture tissulaire de l'invention peut être utilisé pour cribler des médicaments antiviraux, pour contrôler l'évolution d'affections virales et pour surveiller une réaction immunitaire provoquée par une stimulation antigénique.
PCT/US1999/004425 1998-02-27 1999-03-01 Modele in vitro d'infection virale et reaction immunitaire WO1999043788A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP99908581A EP1068298A1 (fr) 1998-02-27 1999-03-01 In vitro modele d'infection virale et reaction immunitaire
AU27977/99A AU2797799A (en) 1998-02-27 1999-03-01 (in vitro) model for viral infection and immune response

Applications Claiming Priority (2)

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US7634898P 1998-02-27 1998-02-27
US60/076,348 1998-02-27

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WO1999043788A1 true WO1999043788A1 (fr) 1999-09-02

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001032841A2 (fr) * 1999-10-29 2001-05-10 The Government Of The United States Of America, Represented By The Secretary, Dept. Of Health And Human Services Methode de differentiation in vitro de lymphocytes t generes a partir de cellules souches cd34?+¿
GB2366294A (en) * 2000-09-01 2002-03-06 Fond Jacqueline Beytout Assay for evaluating cell responses to infection
WO2007106559A2 (fr) * 2006-03-15 2007-09-20 Vaxdesign Corporation Équivalent de tissu muqueux in vitro
US7709256B2 (en) * 2004-04-28 2010-05-04 Vaxdesign Corp. Disease model incorporation into an artificial immune system (AIS)
US7709257B2 (en) 2006-06-27 2010-05-04 Vax Design Corp. Models for vaccine assessment
US7771999B2 (en) 2004-04-28 2010-08-10 Vaxdesign Corp. Disease model incorporation into an artificial immune system (AIS)
US7785806B2 (en) 2004-04-28 2010-08-31 Vaxdesign Corporation Method for determining the immunogenicity of an antigen
US7785883B2 (en) * 2004-04-28 2010-08-31 Vax Design Corp. Automatable artificial immune system (AIS)
US7855074B2 (en) * 2004-04-28 2010-12-21 Vaxdesign Corp. Artificial immune system: methods for making and use
US8003387B2 (en) 2005-12-21 2011-08-23 Sanofi Pasteur Vaxdesign Corp. In vitro germinal centers
US8030070B2 (en) 2004-04-28 2011-10-04 Sanofi Pasteur Vaxdesign Corp. Artificial lymphoid tissue equivalent
US8071373B2 (en) 2004-04-28 2011-12-06 Sanofi Pasteur Vaxdesign Corp. Co-culture lymphoid tissue equivalent (LTE) for an artificial immune system (AIS)
AU2010212355B2 (en) * 2004-04-28 2012-01-12 Massachusetts Institute Of Technology Artificial immune system: methods for making and use
US8298824B2 (en) 2004-04-28 2012-10-30 Sanofi Pasteur Vaxdesign Corporation Methods of evaluating a test agent in a diseased cell model
US8647837B2 (en) 2007-07-16 2014-02-11 Sanofi Pasteur Vaxdesign Corp. Artificial tissue constructs comprising alveolar cells and methods for using the same
EP4060022A4 (fr) * 2019-10-11 2024-02-21 Organoidsciences Ltd Procédé de préparation d'organoïdes tonsillaires et son utilisation

Citations (1)

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WO1998023729A1 (fr) * 1996-11-27 1998-06-04 Anticancer, Inc. Modele in vitro destine au vih et a d'autres maladies virales

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WO1998023729A1 (fr) * 1996-11-27 1998-06-04 Anticancer, Inc. Modele in vitro destine au vih et a d'autres maladies virales

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Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001032841A2 (fr) * 1999-10-29 2001-05-10 The Government Of The United States Of America, Represented By The Secretary, Dept. Of Health And Human Services Methode de differentiation in vitro de lymphocytes t generes a partir de cellules souches cd34?+¿
WO2001032841A3 (fr) * 1999-10-29 2002-02-07 Us Health Methode de differentiation in vitro de lymphocytes t generes a partir de cellules souches cd34?+¿
GB2366294A (en) * 2000-09-01 2002-03-06 Fond Jacqueline Beytout Assay for evaluating cell responses to infection
US8062889B2 (en) 2004-04-28 2011-11-22 Sanofi Pasteur Vaxdesign Corp. Methods of evaluating a test agent in a diseased cell model
US8080416B2 (en) 2004-04-28 2011-12-20 Sanofi Pasteur Vaxdesign Corp. Method for determining the immunogenicity of an antigen
US7709256B2 (en) * 2004-04-28 2010-05-04 Vaxdesign Corp. Disease model incorporation into an artificial immune system (AIS)
US9625444B2 (en) 2004-04-28 2017-04-18 Sanofi Pasteur Vaxdesign Corporation Artificial immune system: methods of use
US7771999B2 (en) 2004-04-28 2010-08-10 Vaxdesign Corp. Disease model incorporation into an artificial immune system (AIS)
US7785806B2 (en) 2004-04-28 2010-08-31 Vaxdesign Corporation Method for determining the immunogenicity of an antigen
US7785883B2 (en) * 2004-04-28 2010-08-31 Vax Design Corp. Automatable artificial immune system (AIS)
US7855074B2 (en) * 2004-04-28 2010-12-21 Vaxdesign Corp. Artificial immune system: methods for making and use
US8962319B2 (en) 2004-04-28 2015-02-24 Sanofi Pasteur Vaxdesign Corp. Methods for testing an immune response using cultures of T cells, B cells, dendritic cells and follicular dendritic cells
US8722402B2 (en) 2004-04-28 2014-05-13 Sanofi Pasteur Vaxdesign Corporation Artificial immune system: methods of use
US8030070B2 (en) 2004-04-28 2011-10-04 Sanofi Pasteur Vaxdesign Corp. Artificial lymphoid tissue equivalent
US8697371B2 (en) 2004-04-28 2014-04-15 Sanofi Pasteur Vaxdesign Corp. Methods for testing an immune response using cultures of T cells, B cells and dendritic cells
US8071373B2 (en) 2004-04-28 2011-12-06 Sanofi Pasteur Vaxdesign Corp. Co-culture lymphoid tissue equivalent (LTE) for an artificial immune system (AIS)
US8298824B2 (en) 2004-04-28 2012-10-30 Sanofi Pasteur Vaxdesign Corporation Methods of evaluating a test agent in a diseased cell model
AU2010212355B2 (en) * 2004-04-28 2012-01-12 Massachusetts Institute Of Technology Artificial immune system: methods for making and use
US8119403B2 (en) 2004-04-28 2012-02-21 Sanofi Pasteur Vaxdesign Corp. Disease model incorporation into an artificial immune system (AIS)
US8298823B2 (en) 2004-04-28 2012-10-30 Sanofi Pasteur Vaxdesign Corporation Methods for antibody production
US8288159B2 (en) 2004-04-28 2012-10-16 Sanofi Pasteur Vaxdesign Corp. et al. Artificial immune system: methods for making and use
US8247226B2 (en) 2005-12-21 2012-08-21 Sanofi Pasteur Vaxdesign Corp. Methods of evaluating an immune response to an antigen
US8669105B2 (en) 2005-12-21 2014-03-11 Sanofi Pasteur Vaxdesign Corp. Methods for assaying responses to vaccines
US8003385B2 (en) 2005-12-21 2011-08-23 Sanofi Pasteur Vax Design Corp. In vitro germinal centers
US8003387B2 (en) 2005-12-21 2011-08-23 Sanofi Pasteur Vaxdesign Corp. In vitro germinal centers
WO2007106559A3 (fr) * 2006-03-15 2008-04-03 Vaxdesign Corp Équivalent de tissu muqueux in vitro
WO2007106559A2 (fr) * 2006-03-15 2007-09-20 Vaxdesign Corporation Équivalent de tissu muqueux in vitro
US7709257B2 (en) 2006-06-27 2010-05-04 Vax Design Corp. Models for vaccine assessment
US8647837B2 (en) 2007-07-16 2014-02-11 Sanofi Pasteur Vaxdesign Corp. Artificial tissue constructs comprising alveolar cells and methods for using the same
US9566250B2 (en) 2007-07-16 2017-02-14 Sanofi Pasteur Vaxdesign Corporation Artificial tissue constructs comprising alveolar cells and methods for using the same
EP4060022A4 (fr) * 2019-10-11 2024-02-21 Organoidsciences Ltd Procédé de préparation d'organoïdes tonsillaires et son utilisation

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EP1068298A1 (fr) 2001-01-17

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