WO2017160147A1 - Système de culture organotypique - Google Patents

Système de culture organotypique Download PDF

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Publication number
WO2017160147A1
WO2017160147A1 PCT/NL2017/050163 NL2017050163W WO2017160147A1 WO 2017160147 A1 WO2017160147 A1 WO 2017160147A1 NL 2017050163 W NL2017050163 W NL 2017050163W WO 2017160147 A1 WO2017160147 A1 WO 2017160147A1
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Prior art keywords
slice
tissue
culture
gas mixture
tumor
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PCT/NL2017/050163
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English (en)
Inventor
Marianna Kruithof-De Julio
Boudewijn KRUITHOF
Gabri Van Der Pluijm
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Academisch Ziekenhuis Leiden H.O.D.N. Lumc
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Publication of WO2017160147A1 publication Critical patent/WO2017160147A1/fr

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    • 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
    • C12N5/0693—Tumour cells; Cancer cells
    • 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
    • G01N33/5011—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 for testing antineoplastic activity
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • 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
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • 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
    • C12N2533/70—Polysaccharides
    • C12N2533/78—Cellulose

Definitions

  • the invention relates to a tissue culture system for the culturing of slices of various tissues.
  • the invention relates in particular to organotypic cultures with regulated high oxygen level gas mixtures.
  • the invention in particular relates to organotypic culture systems wherein the response to exposure to different compounds, treatments and gene delivery vehicles and the like is determined.
  • the culture system is particularly useful in determining the response of tumor slices.
  • OCS organotypic culture system
  • Organotypic culture systems typically involve growing cells in a three- dimensional (3-D) environment and two-dimensional (-D) micro-environment as opposed to single cells originating cell lines cultures in culture dishes.
  • This 3D and even 2D culture system is biochemically and physiologically more similar to in vivo tissue.
  • the invention provides a method for culturing a tissue ex vivo.
  • the method comprises providing a slice of explanted tissue comprising hyperplastic, tumorigenic, bone or bone cells or a combination thereof, applying the slice to a porous supporting surface, applying culture medium to the bottom of the supporting surface and incubating the slice at the selected appropriate culture temperature, the method characterized in that a gas mixture is applied above the slice comprising between 25-70% O2, wherein the percentage indicates volume with respect to the total volume of the gas mixture applied.
  • the slice of explanted tissue comprises hyperplastic, tumorigenic, bone or bone cells or a combination thereof.
  • slice cultures comprising such cells typically respond to drugs and treatments in ways more akin the in vivo situation by providing such cultures with a gas mixture with a higher percentage of oxygen when compared to normal air (all percentages of oxygen and other gases are indicated by volume with respect to the total volume of the gases in the mixture).
  • the gas mixture preferably comprises 25-70% O2. In a preferred embodiment the gas mixture comprises 30-60% O2. In a particularly preferred embodiment it comprises 35-55% O2. In a particularly preferred embodiment it comprises 40-50% 02. In a preferred embodiment the gas mixture comprises about 40% O2 (volume with respect to the total volume of the gas mixture). For buffering purposes most presently available culture media require the gas mixture to have 3- 7% CO2. For media that rely on a different buffer system, the gas mixture does not have to comprise significant amounts of CO2. In the context of the present invention it is preferred that the gas mixture comprises CO2. Media comprising buffering systems not based on CO2 are considered equivalent. Typically the mixture comprises 4-6% CO2, the percentage of CO2 is normally set to about 5%.
  • the remainder of the gas in the mixture is typically a gas (or mixture thereof) that is inert under the conditions used.
  • the inert gas comprises N2.
  • other inert gases are within the scope of the claims.
  • a typical inert gas as used herein is a noble gas such as but not limited to helium or argon.
  • CO2 is not an inert gas as the pH in the culture can change depending on the amount of CO2 in the mixture.
  • the invention relates to means and methods for culturing a tissue ex vivo and describes the use of a gas mixture of 25-70% 02 (hyperoxia) in organotypic cultures on porous supporting surface, and optionally 3- 7% C02, oxygen levels.
  • the gas mixture comprises 3-7% CO2, 25- 70% O2 and N2 optionally further comprising other, preferably inert, gases to make up 100%.
  • the gas mixture comprises 3-7% C02, 30-60% 02 and N2 optionally further comprising other, preferably inert, gases to make up 100%.
  • the gas mixture comprises 3-7% C02, 35-55% 02 and N2 optionally further comprising other, preferably inert, gases to make up 100%.
  • the gas mixture comprises 3-7% C02, 40-50% 02 and N2 optionally further comprising other, preferably inert, gases to make up
  • the gas mixture comprises 3-7% C02, about 40% 02 and N2 optionally further comprising other, preferably inert, gases to make up 100%.
  • Gases that can or might have an effect on the tissue culture slice may be added to the culture as a drug or candidate drug. Such gases are typically not regarded as inert gases. However, the amount of such a gas drug or candidate drug is calculated when calculating the total volume of the gas mixture in the culture. All indicated percentages are percentages with respect to the total volume of the gas mixture in the culture.
  • the term "slice” and “slice of explanted tissue” are used interchangeably herein.
  • the slice preferably comprises hyperplastic, tumorigenic, bone or bone cells or a combination thereof.
  • the hyperplastic cells are cells of a hyperplasia.
  • Hyperplasia or hypergenesis is an increase in the amount of organic tissue that results from cell proliferation. It may lead to the gross enlargement of an organ and the term is sometimes confused with benign neoplasia or benign tumor.
  • Hyperplasia is a common preneoplastic response to stimulus. Microscopically, cells resemble normal cells but are increased in numbers. Sometimes cells may also be increased in size (hypertrophy). Hyperplasia is different from hypertrophy in that the adaptive cell change in hypertrophy is an increase in the size of cells, whereas hyperplasia involves an increase in the number of cells. Hyperplasias are typically regarded as pre-neoplastic. They may progress to become malignant or benign growths but not necessarily so. A neoplasm is an abnormal growth of tissue, and when also forming a mass is commonly referred to as a tumor or tumour. This abnormal growth (neoplasia) usually but not always forms a mass.
  • neoplasms The World Health Organization (WHO) classifies neoplasms into four main groups: benign neoplasms, in situ neoplasms, malignant neoplasms, and neoplasms of uncertain or unknown behavior. Malignant neoplasms are also simply known as cancers. Prior to the abnormal growth of tissue, as neoplasia, cells often undergo an abnormal pattern of growth, such as hyperplasia, but these do not always progress to neoplasia. The term tumor is used herein as a synonym of neoplasm. Tumor is not synonymous with cancer. While cancer is by definition malignant, a tumor can be benign, precancerous, or malignant.
  • the tissue slice preferably comprises cells of a solid tumor.
  • a solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (not cancerous), or malignant (cancerous). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas.
  • tumor does not always imply cancer.
  • solid tumor is used to describe tumors that are malignant (cancerous).
  • Leukemia is a type of tumor that typically takes on the fluid properties of the organ it affects - e.g. the blood.
  • the first sign of leukemia may be the development of a solid leukemic mass or tumor outside of the bone marrow. Such a solid mass is typically not considered a solid tumor.
  • the tumor is preferably a cancer.
  • the cancer is a sarcoma, carcinoma, or lymphoma.
  • the cancer is a sarcoma or a carcinoma.
  • the cancer is a carcinoma.
  • a squamous-cell carcinoma or an adenocarcinoma Preferably a squamous-cell carcinoma or an adenocarcinoma.
  • the cancer is ovarian cancer, colorectal cancer, kidney cancer such as renal cell carcinoma, liver cancer, a lung carcinoma, a breast cancer, a bladder cancer, head and neck cancer, pancreatic cancer, prostate cancer or metastasis thereof.
  • the cancer is a prostate cancer, a bladder cancer, a breast cancer or metastasis thereof.
  • Ovarian cancer, colorectal cancer, kidney cancer, liver cancer, a lung carcinoma, a breast cancer, a bladder cancer, head and neck cancer, pancreatic cancer, prostate cancer or metastasis thereof are in the present invention considered to be solid tumors. I.e. the term "solid tumor" in a claim can be exchanged with one or more of the mentioned cancers with extending the scope of the claim.
  • the tumor in a method of the invention is a solid tumor.
  • the tumor is prostate cancer, bladder cancer, breast cancer or metastasis thereof.
  • Hyperplastic cells are preferably prostate hyperplastic cells.
  • An explant or explanted tissue is a piece of tissue that has been removed from a body.
  • the explant or explanted tissue is of animal origin, preferably a mammal, more preferably a primate, more preferably a human origin.
  • the explant or explanted tissue is provided to the artisan performing a method of the invention. Preferably in the form of a tissue biopsy. The artisan does not him/herself remove the explant or explanted tissue from the body of the animal.
  • explanted tissue obtaining a slice from a tissue that has already, prior to preparing the slice, been removed from the body of the animal.
  • explant is also used for single cells obtained from an intact tissue.
  • the definition does not extend to single cell preparation but rather means tissues with intact 3D or 2D-structure.
  • the tissue is harvested in an aseptic manner, the slice is prepared by a cutting tool that can be prepare slices of even thickness directly, or by trimming certain parts from the prepared slice.
  • the slice is prepared and placed typically placed in a holding liquid until use.
  • the slice is typically transferred to culture conditions as quickly as possible. However, slices may be stored for an amount of time on ice prior to initiating the culture.
  • Explant culture refers to the culturing of the tissue pieces themselves, where cells are left in their surrounding extracellular matrix to more accurately mimic the in vivo environment e.g. cartilage explant culture.
  • the slice is applied to a porous supporting surface.
  • the supporting surface may allow adherence of cells of the slice to the surface or not.
  • the supporting surface is typically a membrane, a porous membrane.
  • the porous membrane is preferably a polycarbonate membrane, a nitrocellulose membrane or a Teflon membrane (Vaira et al (2010) PNAS Vol. 107 pp 8352-8356 "Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors").
  • the porous supporting surface comprises preferably comprises pores of between 0.01-20 pm, preferably 0.04-5 pm, more preferably 0.1-2 pm, preferably 0.4 pm.
  • the porous solid support is preferably a culture membrane, preferably a nitrocellulose membrane.
  • the slice is incubated at an appropriate culture temperature and the gas mixture is applied.
  • the temperature may change depending for instance on the type of animal the slice is prepared from.
  • the temperature is typically chosen to best suit the conditions for the particular animal, however, temperature may also be set or changed during the culture to test an effect thereof of the (cells) of the slice, for instance to determine a heat shock response.
  • Culture medium is typically applied to the bottom of the supporting surface and the gas mixture is typically applied above the supporting surface or the side of the tissue slice that is not in contact with the supporting surface.
  • the cultures can be reversed and have the medium on top of the membrane and the tissue slice applied to the bottom of the membrane.
  • the membrane separates the medium side from the gas mixture side and that the slice is on the gas mixture side of the membrane.
  • the culture system is setup such that the slice does not dry out on the gas mixture side.
  • Methods to maintain sufficient wetness of the slice are known to the skilled person.
  • the culture systems contain a system that inherently maintains the slice and supporting surface sufficiently level to maintain sufficient wetness and equal culture conditions over the membrane. Excess fluid is typically removed from the slice prior to applying them to the supporting surface. Hydration, transport of nutrients and waste is typically achieved via the medium that touches the medium side of the supporting surface.
  • Such cultures are sometimes referred to as "interface cultures”.
  • the tissue slice may be disposed upon a first surface of the support facing the gaseous phase (gas mixture). A second surface of the support faces and is in contact with the culture medium.
  • the culture medium is retained in contact with the support and with the tissue slice, typically by means of surface tension and/or capillarity.
  • the support preferably has a porosity that allows permeation of the support by medium which can then reach the tissue slice on the opposite surface of the support.
  • the tissue slice on the support is typically not immersed in the medium.
  • the culture method, or culture of the invention is particularly suited to test the effect of treatments or drugs on the cells in the slice.
  • the response of the cells is more akin the natural situation in vivo than corresponding classical cell cultures that do not maintain 3D or even 2D-structure.
  • a method of the invention further comprises providing the culture with a drug, candidate drug, treatment or combination thereof .
  • the drug is preferably a cancer drug.
  • the drug can be a chemical compound, a protein (such as an antibody) a nucleic acid molecule or a combination thereof. Many of the present day drugs are chemical compounds. The structure of these compounds can vary significantly. Relevant is only that the drug is applied to the culture whereupon the effect of the drug at the conditions used (if any) is measured.
  • a candidate drug can be a chemical compound, a protein (such as an antibody) a nucleic acid molecule or a combination thereof.
  • the chemical structure of candidate drug can vary significantly. Relevant is only that the candidate drug is applied to the culture whereupon the effect of the candidate drug at the conditions used (if any) is measured.
  • the treatment can be a heat (cold) treatment, light treatment, radiation treatment. Combinations of drugs, candidate drugs and treatments are also possible. For instance, presently many different combinations of drug are being developed for a specific purpose. Also a treatment can be combined with another treatment or with a (candidate) drug.
  • proteins are erythropoietin, G-CSF, Various blood-clotting factors, various proteins for enzyme treatments such as ADA, Glucocerebrosidase and the like.
  • Other proteins are ligands, receptors, solubilized ligands or solubilized receptors (typically via attaching an Fc tail to an extra-cellular domain of the ligand or receptor), antibodies, peptide of up to 50 amino acids, MHC molecules and the like.
  • the protein can be provided as such or in the form of one or more nucleic acid molecule(s) that encode the protein.
  • the nucleic acid molecule can also be a drug by itself.
  • RNAi molecules are known to interfere with the expression of a protein in a cell.
  • Non-limiting examples are RNAi molecules, shRNA molecules, miRNA, other small RNAs, various chemical modifications of RNA or DNA, such as morpholino modifications, LNA modifications, 2'-0-methyl or 2'-0-methoxyethyl modifications, phosphorothioate and other modification.
  • morpholino modifications such as LNA modifications, 2'-0-methyl or 2'-0-methoxyethyl modifications, phosphorothioate and other modification.
  • nucleic acid molecules can/are linked to peptides or other molecules to target them to specific cells or to promote entry of the nucleic acid.
  • a drug can be administered as such or in the context of a drug delivery devise such as a liposome.
  • Nucleic acid molecules can be added as such, for instance for oligonucleotides or in the context of a nucleic acid or gene delivery vehicle.
  • Preferred nucleic acid or gene delivery vehicles are viral vectors such as adenoviral, adeno-associated viral, lentiviral or retroviral vectors. Other nucleic acid or gene delivery vehicles are also possible. Many types indeed exist today, for instance (gold) particle mediated transfer, electroporation and transfection to name just a few.
  • the culture is typically maintained for several days. Slices can be maintained such cultures for a prolonged amount of time. For prolonged culture it is preferred that medium and/or gas is exchanged for fresh medium or gas. As a non-limiting example, for a culture of 20 days it is preferred to exchange the gas in the gas chamber every 5 days.
  • the method further comprises incubating the slice of explanted tissue for a period of at least one day. In a preferred embodiment the incubation is for at least two, more preferably at least three days. In a particularly preferred embodiment the culture is for at least one week, more preferably at least two, three or four weeks. Cultures for more than one day typically, though not necessarily, require the provision of fresh nutrients and the disposal of waste. Typically this is achieved by changing the medium for fresh medium.
  • the method of the invention thus preferably further comprises comparing the incubated slice of explanted tissue with a reference.
  • the slice of explanted tissue can have varying thicknesses.
  • the thickness of the slice is not particularly critical as long as diffusion of nutrients, gases and waste is sufficient. Thickness can typically vary between one cell thick and 15 cells thick.
  • Two-dimensional representation of tissue and/or tumor micro-environment can be provided by cultures of tissue slices of one cells thick. Two or more cells thick slices are typically suited for three dimensional representations of tissue and/or tumor micro-environment.
  • a slice is typically 100-200 uM thick although thicker and thinner slices are also possible.
  • ranges include the values of x and xx and the range spanned by x and xx.
  • integers are intended the range does not include fractions and where fractions are intended to be included the range includes the values x and xx and integers and fractions with the range spanned by x and xx.
  • Tissue (derived from transurethral resection of either prostate, bladder carcinoma or bone metastasis i.e. waste material) is equally sliced and grown in defined culture conditions 10 days compared to the tissue prior to culturing (direct fixation)
  • FIG. 1 Tissue (derived from transurethral resection of either prostate, bladder carcinoma or bone metastaes i.e. waste material) is equally sliced and grown in defined culture conditions for 3 and 7 days compared to the tissue prior to culturing (direct fixation)
  • Figure 3 Effects of compound in our patented ex vivo 'near-patient' tumor slice models. Tumor tissue is cultured for 7 days in the presence of compound and stained for HE (upper level) and cytokeratin 5 (TURP) or pan-cytokeratins (TURB).
  • Figure 4 Effects of oxygen in our patented ex vivo 'near-patient' tumor slice models. Tumor tissue is cultured for 7 days in the presence of different range of oxygenation. The condition non-oxygen is with an oxygen level of normal air (approx.. 20%). This results in proliferation being highest in the 40-50% range. Examples
  • Prostate and bladder tumor slices from transurethral resection of prostate cancer tissue (TURP), bladder cancer tissue (TURB) and bone metastasis are collected after resection.
  • the tissue is placed in media (DMEM with P/S and 1% serum) and transported at room temperature to the tissue culture. In sterile conditions the tissue is placed in a petri dish. The tissue is then sectioned in 100-200 micron slices using a curved spring scissor and a dented forceps. The tissue slices are placed semi dry in the petri dish and sequentially numbered. 6 well plates, 12 or 24 well plates are opened in a sterile way and inserts with nitrocellulose support are placed in the wells.
  • the sliced tissue is place in on top of the membrane with the larges surface in contact with the membrane itself.
  • Media is added to the bottom of the well with particular care in not creating air bubbles under the nitrocellulose membrane.
  • Place the culture dish in the plastic container and perfuse for 10 minutes with air mix we use is composed of Carbon dioxide (CO2) 5%, Oxygen (O2) 40%, and balance Nitrogen (N2)), a so-called Certified Standard Biological
  • tissue slice model To provide sufficient material for optimization of the tissue slice model, we initially used tumor material from mouse xenografts. We have generated xenografts of human PC3, PC3-DR, PC-3M-Pro4Luc2 cells, UMUC, T24 and RT4 and cut 100- 200 ⁇ thick tissue slices. The tissue was treated with doxetaxel and/or cabazitaxel which are the first line treatment in clinic for prostate and bladder cancer.
  • Marianne is this added to the medium or to the top of the slice?
  • TURP and TURB treated it with a compound that effectively inhibits prostate and bladder cancer growth both in vitro (2D cell models) and in vivo (xenograft and zebrafish models).
  • PDX model were al generated from human bone metastases (HCC, RCC, Prostate, Urothelial, Breast,). Tissue culture slice model was set up as described above and slices were treated with cancer specific treatment: addition to the culture media or intra-tumoral injection.
  • Prostate cancer tissue was collected during a standard transurethral resection of the prostate (TURP), a type of prostate surgery done to relieve moderate to severe urinary symptoms caused by an enlarged prostate, a condition known as benign prostatic hyperplasia (BPH).
  • Transurethral resection of the bladder serves primarily for pathological staging. In case of non-muscle invasive bladder cancer the TURB is in itself the treatment, but in case of muscle invasive cancer, the procedure is insufficient for final treatment.
  • the tissue specimen was divided into equal pieces, one was immediately fixed in PFA 4% and sub sequentially processed for paraffin embedding. The rest was used for 3D culture. Oral consent for removal of the tissue for research purposes was obtained from the patients. Confirmation that the Medical Research Involving Human Subjects Act (WMO) does not apply to the present study since the research was performed on 'waste' material.
  • WMO Medical Research Involving Human Subjects Act
  • Hematoxylin and Eosin (H&E) staining were performed using standard protocols on 10- ⁇ cryosections. Immunofluorescence staining was performed on 10- ⁇ cryosections, which were incubated in 3% H2O2 and Antigen Unmasking Solution (Vector Labs). Primary antibodies and dilutions used are as follows: anti- CK18 1:500 (DAKO) and anti- CK8 1:500 (Univ. Iowa). Sections were blocked with 1% bovine serum albumin (BSA)- PBS-0.1% v/v Tween 20) and incubated with primary antibodies diluted in the blocking solution, overnight at 4°C or room temperature.
  • BSA bovine serum albumin
  • Sections then were incubated with secondary antibodies labeled with Alexa Fluor 488, 555, or 647 (Invitrogen Molecular Probes, 1:250 in PBS-0.1% Tween 20). Sections were counterstained with TO-PR03 (1: 1000 in PBS-0.1% Tween20) (Invitrogen/Molecular Probes) to visualize nuclei, and mounted with Prolong G mounting medium (Invitrogen/Molecular Probes), which contains DAPI. Immunofluorescence staining were imaged using a Leica TC-SP8 spectral confocal microscope.
  • the ex vivo model recapitulates the in vivo situation and 2. the model is predictive for drug efficacy; we tested a known compounds and monitored their effect in the ex vivo model.
  • Our data shows that the overall morphological tumor aspect is preserved upon 10 day culture and longer and the tissue responds to compound stimulation (Figure 2,3). In particular the compound shown in Figure 3 effectively eradicated prostate cancer cells as in the mouse in vivo model. Discussion

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Abstract

L'invention concerne des moyens et des procédés de culture tissulaire ex vivo. Le procédé consiste à prendre une tranche d'un tissu explanté comprenant des cellules hyperplasiques, des cellules tumorales, un os ou un cartilage ou une combinaison correspondante, à appliquer la tranche sur une surface support poreuse, à appliquer un milieu de culture sur le fond de la surface support et à incuber la tranche à la température de culture appropriée sélectionnée, le procédé étant caractérisé en ce qu'un mélange gazeux est appliqué au-dessus de la tranche, comprenant entre 25 et 70 % d'O2 et éventuellement de 3 à 7 % de CO2, le pourcentage indiquant le volume par rapport au volume total du mélange gazeux appliqué.
PCT/NL2017/050163 2016-03-15 2017-03-15 Système de culture organotypique WO2017160147A1 (fr)

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CA2922079A1 (fr) * 2013-08-23 2015-02-26 Sumitomo Chemical Company, Limited Procede de production de tissu retinien et de cellules associees a la retine

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