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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
  • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
  • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
  • A61K8/985—Skin or skin outgrowth, e.g. hair, nails
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/08—Anti-ageing preparations
• • 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
  • C12N5/0608—Germ cells
  • C12N5/0609—Oocytes, oogonia
• • 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
  • C12N5/10—Cells modified by introduction of foreign genetic material
  • C12N5/12—Fused cells, e.g. hybridomas
  • C12N5/16—Animal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
  • A61K2800/86—Products or compounds obtained by genetic engineering
• • 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
  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2517/00—Cells related to new breeds of animals
  • C12N2517/04—Cells produced using nuclear transfer

Definitions

• the present invention provides systems and methods for genetically and epigenetically treating cells.
• Such systems and methods are particularly useful in the field of cellular treatments, in particular for producing autografts from differentiated cells or embryonic or fetal strains.
• Cellular treatments are currently practiced to repair damaged tissue suffering from a disease, cell deficit or necrosis.
• This technique generally involves taking a few healthy cells of the tissue concerned, to cell culture these cells to form a cell stock or tissue and to re-implant these cells in the tissue to be treated. These reprogrammed cells can then allow the tissue concerned to recover its original morphological and functional capabilities.
• this technique is used for the repair of articular cartilage.
• Joint cartilage has limited repair potential and lesions of a certain volume rarely heal well.
• chondrocytes bathed in an extracellular matrix, are removed, removed from the matrix, for example by enzymatic digestion and then cultured, generally on sera of fetal calf or preferably in the patient's serum, and in three-dimensional matrices (for example an agarose, collagen or globin matrix).
• three-dimensional matrices for example an agarose, collagen or globin matrix.
• the disadvantage of these multiplication techniques is that generally the cells removed are cells that have already undergone numerous mitotic divisions.
• cell culture for their multiplication causes a slight decrease in telomeres at each mitosis and this multiplication is often done on cells already aged, close to their end of life and their end of functions, and whose DNA is in besides likely to be altered.
• cellular aging results in a gradual gnawing of telomeres (end of chromosomes). Now these telomeres condition the remaining number of mitotic divisions.
• culturing mother cells that have already undergone numerous mitoses may result in a large colony of aged daughter cells with shortened survival that may also exhibit alterations in gene functionality.
• the object of the present invention is to provide systems and methods for genetic and epigenetic treatment overcoming the aforementioned drawbacks.
• the present invention aims to provide systems and methods of cell processing for rapid mass production of healthy cells with improved gene functionality and / or capable of being genetically and epigenetically rejuvenated, aged and / or repaired to a desired degree.
• Still another object of the present invention is to provide systems and methods for cell processing resulting in both reconstitution of missing, failing, enhancing or modifying autologous tissue, and genetic rejuvenation of tissue in which cells have been implanted.
• the subject of the present invention is therefore a system for the genetic and epigenetic treatment of cells to be treated, comprising: at least one cell to be treated,
• MRG genetic reprogramming medium
• CCG genetic reprogramming cell
• the subject of the present invention is also a method of genetic and epigenetic treatment of cells to be treated, comprising the following steps:
• MRG genetic reprogramming medium
• the invention relates in particular to the modification of the environment of a cell nucleus with or without extracellular or intra-oocyte transfer, so as to put the nucleus under the influence of a medium inducing its partial genetic reprogramming, but without causing a return of the nucleus to the development of embryonic cells.
• This medium will cause better repair of cellular DNA during division and aggression and / or genetic rejuvenation by the action of an inverting medium of biological time, such as an oocyte.
• the present invention relates in particular to systems and methods applicable to the field of treatment and / or repair and / or functional and / or morphological cellular enhancement intended to open many perspectives for the fight against a large number of diseases but also against Tissue senescence is largely due to the loss of their functional and morphological capacity for proliferation, regeneration and repair.
• the present invention relates to a system and methods capable of treating cells of a tissue, in particular for rejuvenating, aging and / or repairing these cells.
• the cells are then cultured in a suitable medium so as to create a stock or tissue of genetically and epigenetically treated cells capable of being implanted in the tissue in question, or at a distance from it, where these cells can emit particular proteins and and / or signaling peptides and / or stimulation of metabolism, and / or DNA repair enzymes, for the tissue in question.
• the systems and methods of the invention comprise contacting at least a portion of a nucleus of at least one cell to be treated with a genetic reprogramming medium (MRG).
• MRG genetic reprogramming medium
• This MRG comprises at least the natural cytoplasm of at least one genetic reprogramming cell (CRG) and / or synthetic cytoplasm.
• a reconstituted and / or synthetic cytoplasm can be composed in particular of embryonic serum extracts, cicatrization and / or cells subjected to metabolic activation.
• a completely synthetic cytoplasm for example made by physical and chemical reconstitution of active substances, is possible. It is also conceivable to produce a MRG in the form of a "slurry" of CRG, with or without cores.
• cell extracts or cytoplasm and / or other substances known to be capable of activating nuclear metabolism such as cells or cell extracts appearing during healing and / or signaling or metabolism-stimulating proteins or peptides and / or growth factors and / or cells or extracts of cancer cells.
• the cell or cytoplasmic extracts can be obtained by well-known physical or chemical treatments.
• the advantage of using cancer cells, and in particular the cytoplasm thereof, is explained by the fact that metabolic activation, signaling and mitosis factors are particularly intense and may induce a temporary metabolic or nuclear reactivation of a cell to be treated. Cancer contagion is unlikely because cancers are usually not transmissible to a different tissue, and their cytoplasm remains normal.
• the use of cytoplasm of selected cancer cells is possible to temporarily treat nuclei or cells, adult or otherwise, that are insufficiently capable of dividing spontaneously or in culture, to repair their poorly copied DNA, or which are functionally deficient.
• cancer Since cancer is not directly contagious for non-cancer cells, even if they are ill, the selective regenerative application of cancer cells can be done, for example, in two ways: intracellular treatment: a nucleus is taken from a cancer cell and introduced into the cytoplasm of a tissue cell to treat; this nucleus preferably remains separated from the normal nucleus by a tongue or membrane of a porous biocompatible fabric possibly impregnated with antibodies, said tissue permitting the signaling proteins but preventing any passage of genes or chromosomes. Under these conditions, the signaling proteins emitted by the cancerous nucleus will cause a partial genetic reprogramming of the cell nucleus to be treated, especially in its defective functions.
• intracellular treatment a nucleus is taken from a cancer cell and introduced into the cytoplasm of a tissue cell to treat; this nucleus preferably remains separated from the normal nucleus by a tongue or membrane of a porous biocompatible fabric possibly impregnated with antibodies, said tissue permitting the signaling proteins but preventing
• the cancerous nucleus will be removed, and the reprogrammed cell may be multiplied in a suitable cell culture.
• extracellular treatment selected cancer cells are approximated with a few cells to be treated in a cell culture bath adapted to include at least one factor capable of increasing the permeability of cell membranes, particularly to signaling proteins. This can be done until the observation or genetic, proteomic, biochemical or biophysical tests allow to note the induction of a functional reactivation of the nuclear deficiency (s) to be corrected.
• selective biochips will make it possible to target the signaling proteins emitted from the cancerous nucleus and selectively reprogram the nucleus to be treated in its intended and programmed functions.
• Three main types of treatment can be considered, namely the rejuvenation of the biological age of a cell, the aging of the biological age of a cell and the repair of a cell.
• Rejuvenation of the biological age of a cell also promotes self-repair capacity of this cell, especially at the level of its DNA.
• MRG includes all or part of one or more CRGs.
• a CRG is advantageously an oocyte, an embryonic cell, an embryonic or adult stem cell, a fetal cell, or a cellular container reconstituted from these cells, or synthesized.
• the systems, methods and applications for achieving such rejuvenation will be described in more detail below.
• the aging of a cell is particularly conceivable for treating fetal diseases or in the newborn, due in particular to cancers of embryonic origin, such as glioblastoma.
• these cancerous cells can be artificially aged by replacing the cancerous nucleus with a healthy nucleus of the same autologous or homologous but older tissue, preferably HLA (Human Lymphocyte Antigen) compatible.
• HLA Human Lymphocyte Antigen
• the interaction between the older nucleus and the young cytoplasm promotes some aging temporarily accelerated at least the cytoplasm of the young cell. Aging can then be increased by multiplication of cells in a culture bath, the young cytoplasm causing accelerated mitoses of the older nucleus, inducing a decrease in telomeres.
• healthy cell sorting is possible to re-implant healthy tissue in place of the original diseased tissue.
• Another possible application is to repair a cell, in particular in its chromosomal constitution, by treating only a part of a nucleus, for example a chromosome.
• the chromosome particularly the "Philadelphia" chromosome
• the chromosomes may be destroyed during the metaphase where the chromosomes are deployed, for example by means of an ultrathin laser beam, preferably diameter equal to or less than 1 micron.
• a healthy equivalent chromosome in the metaphase of an equivalent cell of the patient or a compatible HLA donor and is implanted in the cancer cell, especially during its mitosis.
• the treatment could be considered in particular for a large number of cancers, for example glioblastoma, cancer of the breast or rectum.
• Another possible application is to repair only part of the chromosome.
• a specific part of a chromosome for example the part whose genes are responsible for graft rejection. This can be done using an ultrathin laser beam.
• the equivalent part of the equivalent chromosome in the recipient of the graft which can also be done by laser cutting using an ultrathin laser beam.
• This part of the chromosome is then reintroduced into the original chromosome, which can be done using plasmids, phagemids, synthetic vectors, or micromanipulations in nanotechnology. Synthetic vectors are made in the laboratory and consist of structures called "copolymer blocks" that bind to DNA or RNA. More generally, this type of repair is conceivable to repair any deficiency or dysfunction of a cell part, especially due to age.
• Various embodiments and applications of cellular rejuvenation will now be described in more detail.
• nucleus in order to rejuvenate or regenerate a differentiated cell, its nucleus (with or without its attached cytoplasm) is removed and transferred to the MRG, advantageously in a CRG of the oocyte, embryonic, fetal cell or cancerous. This core is left in the MRG for a predetermined time and then removed.
• the nucleus is removed before the end of the nucleus telophase, that is to say that the nucleus is extracted from the MRG before its division into two cells, that is to say before the end of its first mitosis.
• the inventor It has been observed that this temporary introduction of a nucleus into an MRG, especially in a CRG, results in a rapid and important lengthening of the telomeres, often synonymous with rejuvenation of the chromosomal material.
• the regenerated nucleus can then be introduced into a differentiated recipient cell, strain or embryo, preferably enucleated, preferably autologous with respect to said nucleus, preferably of identical tissue, advantageously in the cell of origin of the nucleus or a neighboring cell, in which mitotic division can continue and thus lead to the birth of two daughter cells whose nucleic material is regenerated.
• these cells may be subjected to a multiplication culture and reach such quantities that at least millions of cells sufficiently differentiated to be functionally and morphologically able to be implanted in the original tissue concerned.
• the nucleus can be removed from the MRG after one or more mitoses, then one (or more) of the rejuvenated nuclei thus obtained is (are) reintroduced into a differentiated receptor cell, advantageously autologous with respect to the nucleus. preferably in the cell of origin of the nucleus or a neighboring cell.
• the step of collecting and transferring the nucleus of the differentiated cell comprises taking, in addition to the nucleus, at least a part of the cytoplasm contained in the differentiated cell, in order to find in the MRG, particularly in CRG, some cytoplasmic components initially present in the cell differentiated, such as endoplasmic reticulum, golgy apparatus, ribosomes and / or mitochondria.
• the bringing into contact of at least the nucleus of a differentiated cell with said MRG may consist of transferring MRG into a differentiated cell, for example by means of a pipette or by induced transfer. by a pressure difference. This can be accomplished by creating at least one slit or aperture in the membrane of the differentiated cell, and transferring MRG into said differentiated cell through said at least one slit or aperture.
• a CRG and a differentiated cell side by side, to make an opening in the CRG membrane and an opening in the membrane of the differentiated cell, then to compress the CRG to at least partially transfer the cytoplasm of CRG in the differentiated cell.
• This compression can be obtained by placing a pipette or the like above the membrane of the cell to be compressed, preferably locked against a wall, and exerting a suitable pressure.
• This pressure could also be exerted by means of a fluid, preferably viscous, which can overflow the pipette without detaching it.
• MAF can be removed before or after the first mitosis of the differentiated cell nucleus.
• means may be provided for closing the differentiated cell with at least a portion of the remaining MRG included therein.
• the oocyte used may possibly be a mammalian oocyte.
• a rabbit or sheep oocyte could be used.
• Oocytes derived from differentiation induced from embryonic stem cells (OPCE) can also be created in vitro. These OPCEs, obtained for example by cloning, can come from the recipient of the grafts and the treated nuclei thus become particularly autologous because the cytoplasm of the OPCs will comprise only a part of its foreign DNA and / or RNA, especially in the mitochondria and ribosomes.
• nucleus which is, for example, in early, spontaneous or induced mitosis, or else chromosomes or genes or parts of nuclei to be treated in an embryonic-type cell.
• Embryo-like cells that may be artificially activated by proteins or peptides of genetic signaling or cell activation or regulation may also be used, providing an environment capable of inducing some neighborhood genetic reprogramming.
• removal of the nucleus from the differentiated cell can be advantageously done in anaphase or during telophase depending on the degree of genetic rejuvenation desired.
• optical means such as a microscope, may be used.
• a CRG will then preferably come from the same tissue, for example, cartilage, myocardial, etc., preferably partially pitted and cultivable in vitro, in vivo or in situ.
• This or these cell (s) will be cultured for propagation preferably a sufficient time in vivo in embryonic tissues to obtain a partial dedifferentiation.
• the nuclei thus treated can be left either in the embryonic-type cells to constitute a graftable tissue in the organism of origin of the nucleus, or extracted from their receptor cells to become inducer of local cellular or trans-membrane local regeneration at the level of the nucleus.
• a differentiated tissue preferably autologous and identical.
• the implantation of the nucleus or nuclear part can also be carried out inside a stem cell, preferably of embryonic or fetal type.
• Such partially and selectively dedifferentiated cells can then be introduced into differentiated cells, such as chondrocytes, immune function, endocrine, cardiac cells, tissue cells that have undergone anti-cancer treatment, ⁇ cells and ⁇ islets of Langerhans, cells of the same origin as a graft to be transplanted, hepatocytes, etc., in order to regenerate the corresponding tissue.
• differentiated cells such as chondrocytes, immune function, endocrine, cardiac cells, tissue cells that have undergone anti-cancer treatment, ⁇ cells and ⁇ islets of Langerhans, cells of the same origin as a graft to be transplanted, hepatocytes, etc.
• This function also comprises the ability of these genetically activated cells to act remotely by secretion, release or induction of peptides and / or genetic signaling proteins, in particular, by specific biochemical molecules. This trans-membrane and / or trans-humoral genetic activation gives these cells the ability to actively and continuously stimulate other senescent deficient cells or to inhibit carcinogenic factors.
• the system according to the invention and the cell regeneration methods used preferably comprise four successive stages, namely a preparation of the nuclear material, a genetic reprogramming, a multiplication in culture and a reimplantation in the organism of origin of the nucleus.
• the preparation of the nuclear material consists in taking the nucleus of the cell, preferably differentiated with more or less cytoplasm, in order to preserve, if possible, the cytoplasmic components, such as the mitochondria, the ribosomes, the endoplasmic reticulum, the Golgi apparatus, lysosomes, peroxisomes, etc., of the initial differentiated cell at the level of the oocyte hosting this sample.
• this step ensures synchronous reprogramming of the various vital structures around the nucleus and a probable conservation of the cellular "morpho-temporal field". Moreover, it is possible that certain constituents of the nuclear material such as chromosomes, a set of genes, one or more isolated genes (natural, recombined, semi-synthetic or synthetic) have previously been subjected to such a regeneration process. In this way, some elements of the preparation will have a different biological age.
• a segment including plant DNA or synthetic vectors such as copolymer blocks, coding for example for vitamin C, E, folic acid, essential amino acids, unsaturated lipids essential peptides such as natriuretic (BNP) or atrial natriuretic (ANP) brain peptides, peptides C and Y, glutathione, peptide hormones such as glucagon, insulin, ACTH, peptides antibiotics, proteins such as globulins, immunoglobulins and albumins, DNA and RNA repair, restriction, replication and transcription enzymes, as well as cytochromes, cytokines, etc.
• BNP natriuretic
• ADP atrial natriuretic
• a gene or a chromosome expressing for example erythropoietin or various albumin for example during metaphase, or the nuclear membrane to anaphase, telophase or a corresponding interphase.
• the inventor estimates that the increase in cell biological age results in a decrease in the high elasticity of chromosomes, in particular metaphase chromosomes. This, in turn, leads in particular to a decrease in the accessibility of DNA polymerases, endo- and exonucleases and chromosomal DNA enzymes and thus constitutes an epigenetic and genetic cause of aging which can be combated by the genetic rejuvenation of the genome. 'invention.
• the membranocytoplasmic container (oocyte) for treating the cytoplasmic reprogramming elements may sometimes be too small for the cellular elements to be treated.
• oocyte membranocytoplasmic container
• the membranocytoplasmic container (oocyte) for treating the cytoplasmic reprogramming elements may sometimes be too small for the cellular elements to be treated.
• nucleus with a part of its cytoplasm, or of several sometimes different nuclei such as in a nephron, a muscle cell, a myocardial autorythmic cell, a hair follicle, a unit epidermal melanization, an epidermal-dermal unit, a glandular unit, a hepatobiliary unit, a retinal functional unit (such as a pigmented epithelium: cones, rods, bipolar cells, horizontal cells and M ⁇ ller cells), a vascular unit (cell endothelial and myoarterial), a hematopoietic
• RAF membranocytoplasmic vessel with preserved oocyte function
• Such an RAF can be made by gluing the membranes respective mammalian autologous or autologous oocytes, for example by manual or robotic micromanipulations, preferably preserving each respective cytoplasm within their respective membrane and creating a spherical, ovoid or cylindrical type volume. Such manipulation requires preserving the vital environment for each oocyte.
• This membrane bonding may for example be achieved by means of a micro laser beam, a small heating light ray, a biological adhesive, etc.
• the in vitro multiplication is preceded by the introduction of the nucleo-cytoplasmic material into a preferably enucleated cell, identical to that from which the nucleus originates, and with at least recoverable vitality.
• Current multiplication techniques allow, for example, in two weeks to obtain about half a billion cells from tens.
• the regeneration method according to the invention ensures a rapid and significant increase in the length of the telomeres in less than a day, thus making it possible to counterbalance their shortening resulting from so many successive replications.
• This multiplication can also be done in vivo but is generally much slower and often requires sufficient initiation in vitro. This decreases the amount of cells required as well as the significant elongation of the telomeres and probably allows for better functional adaptation as well as greater genetic influence at a distance.
• a cellular micromanipulation can be envisaged to produce an enlarged enclosure with a genetic and epigenetic reprogramming function capable of reversing temporally the evolution of the biological age of the nuclei and / or multiple cytoplasm that are introduced there.
• cells with an oocyte function are, for example, cut into two parts, preferably by a cold-light laser micro-ray.
• Both of these portions are open, and their membranes may be attached to a protein layer, such as globin, which has preferably been applied to a flexible surface.
• This carpet of oocyte or embryonic membranes presents the cytoplasm upwards.
• a sufficient surface of such a cytoplasmic velvet (VC) is formed, several differentiated cell nuclei can be placed there, with or without their cytoplasm, and then roll the VC around them as close as possible.
• This interactive cell sandwich will preferably remain in the conventional cell culture nutrient liquid for the desired time to achieve the desired mitosis phase.
• This cell regeneration is applicable to many cell types and can therefore create controlled regeneration tissues to treat many organic and tissue lesions.
• this remote inductive cellular rejuvenation in particular by signaling proteins, may, in some cases, oppose the development of a local cancer, slow down its extension or even destroy all its metastases.
• an autologous or even homologous ophthalmic retina including a functional cellular unit, for example consisting of a few cells of the pigmented epithelium, cones, rods, bi-polar cells and / or Muller cells, which has taken and regenerated, may be of great interest in the case of AMD.
• Severe renal insufficiency can be combated by the implantation of partially dedifferentiated cells obtained, for example after transfer into and out of oocytes, of nuclei of different nephron cells.
• Osteoarthritis can be treated by implantation of chondrocytes from cell regeneration.
• oocytes preferably one or more nuclei or parts of cell nuclei.
• hair follicle, melanocytes and keratinocytes for regeneration of the hair and / or its color.
• Yet another application of the invention may consist in reinforcing or recreating the thymic functions by genetic rejuvenation of homologous or, if possible autologous, sufficiently dedifferentiated thymic cells to actively resuscitate the immunoprotective functions of the body.
• a kidney can be regenerated in vitro from the various nephron cells (CNE) obtained for example by surgical or endoscopic renal biopsy under visual control.
• CNE nephron cells
• the CNEs will be treated by the treatment of the invention in order, for example, to reduce their biological age by three quarters, and the CNEs thus obtained will be amplified.
• BNE nephrons
• this BNE in function makes it possible to detect the different diseased cell segments that will be sampled and replaced, by in vitro microsurgery by rejuvenated and geometrically reconstituted identical cell segments.
• this part of the kidney (which may even be a whole kidney) will be reimplanted into the patient with resumption of the vascular and urinary connections.
• a particularly advantageous biomedical application for this method of cell regeneration generally concerns degenerative diseases of the joints (osteoarthritis).
• Cartilage chondrocytes which often degenerate with age, can be removed by biopsy, endoscopy, local surgery, or arthroscopy. separated from their surrounding cartilage.
• the reimplantation of the regenerated cells in the original joint should preferably be done in the vicinity, but outside the surfaces of the mobile articular cavity that supports the mechanical loads in order to avoid any crevices in its moving surfaces.
• a defect of indirect blood supply of chondrocytes which is mainly by imbibition, must be corrected.
• an autologous vascular functional tissue comprising small arteries - arterioles - capillaries - venules - and small veins, these vascular functional units can advantageously come from a post-regeneration autologous cell culture according to the invention.
• This implantation of rejuvenated cells may be in the form of preformed three-dimensional lamellae layers according to the local geometry of the previously measured articular cavity, or in the form of a swarming in order to cause the durable emission in particular of signaling proteins.
• the post-regeneration chondrocytes will progressively reconstitute a thicker, smooth, well lubricated cartilage.
• Osteoporosis is a degenerative disease of bone tissue that occurs with age. To fight against this disease, it is advisable to regenerate autologous osteoblasts (and possibly osteocytes) and to re-implant them, preferably, at several levels of the bone. Osteoblasts are preferably propagated in culture with artificial geometric solicitations, in particular by imposing mechanical stresses, for example using a support frame.
• This support frame may comprise at least one mobile side for movements in a plane.
• two moving sides are used in the culture support frame and / or the possibility of performing motor rotations. dimensional.
• the present invention can also be applied to subjects who have undergone serious inflammation, in particular by reaction weakenings of the various antibody-producing lymphocytes and pro-cytokines. anti-inflammatories.
• the regeneration method according to the invention can then allow to revive these lymphocytes in number and function.
• these lymphocytes can be subjected to the method according to the invention by placing a lymphocyte nucleus in an oocyte in the presence or absence of traces of antigens created by the infection in question in the oocyte cytoplasm.
• the lymphocytes will be rejuvenated and multiplied and then reimplanted into the organism where they will have already "memorized” the dangerous antigens and produce or cause to be made to produce the corresponding antibodies. in large quantity.
• the presence of specific antigens during the cell regeneration process may allow to "memorize” or exteriorize the antigens on the cell membranes and to optimize the reaction of production of the antigens. antibodies by their immediate appearance as rejuvenated lymphocyte functions recur.
• the present invention also applies to cancer control.
• the treatment of the invention makes it possible to create a method of personalizing cancer control. It is possible to proceed in the following manner: hematopoietic, lymphocyte, dendritic cells are collected at the level of the bone marrow or at the periphery, and different categories are isolated which are subjected to the treatment of the invention. After amplification of these cells in vitro, they are cultured in a nutrient bath in the vicinity of cancer cells taken from the tumor of the patient. It may then be useful to limit the nutrients and oxygen in the culture bath to stimulate competition and survival struggle between the two categories of cells.
• the genetically rejuvenated lymphocytes of the patient will naturally develop specific antibodies against the antigens cancer cells and against certain substances and biological factors necessary for the metabolism and secretion of cancer cells. It is for example anti-sense or guide RNAs, often small, previously transfected in the DNA, in particular lymphocyte, by plasmids carrying the genes selected or constructed for this purpose. If the lymphocytes succeed in destroying the cancerous cells, they can be reinjected, preferably after their multiplication, into the organism of the patient from which they come. If, on the other hand, the lymphocyte cells fail in the destruction of the cancerous cells, the former must be reinforced, in particular by the injection of plasmids and / or cosmids and / or selected synthetic vectors.
• DNA polymerases or DNA segments comprising synthetic or natural genes producing new antibodies or specific toxic substances against the cells to be combated.
• a greater capacity is thus provided to produce antibodies and / or to stimulate lymphocyte metabolisms and mitoses, either by the selection of preferably highly immunogenic cells, such as so-called "memory effectors" with enhanced anti-tumor potential.
• memory effectors for example by strengthening the genetic rejuvenation treatment of lymphocytes using the treatment of the invention.
• cancer cells are autologous, genotypic and differential epigenotypic examination reveals the small part of the genome of cancer cells that differs from autologous normal cells of the same tissue (PGD).
• PGD autologous normal cells of the same tissue
• the genes involved can produce different m RNAs including editing (editing) or differential splicing. It is therefore necessary to know the biological and biochemical behaviors of the specific PGD each patient may, in part, vary in response to a therapy, for example biological, of the type of the present invention. In vitro, one can then try to find known innocuous viruses or bacteria such as certain bacteriophages and colifacts selected and / or genetically manipulated.
• an adult homologous foreign cytoplasm EH
• genetic reprogramming of the mitochondrial DNAs and ribosomal RNAs of the CEH is possible. This possibility can be used to protect an adult nucleus placed in an active oocyte against subsequent transfections by the oocyte cytoplasm as they occur during conventional cloning. Partial cloning removes the nucleus to be treated (NT) from the oocyte before its first cell division and places this reprogrammed nucleus in a cell preferably enucleated identical to its original cell.
• NT nucleus to be treated
• the oocyte cytoplasm is distant from the NT nucleus at the time of division of the nucleus, and this division takes place within an original cytoplasm (CO) of the NT nucleus. It is necessary to reprogram the CO genetically and to increase the quantity. To this end, we can take a second oocyte identical to the first and suck part of its cytoplasm and replace it with a cytoplasm of a cell identical to that of the NT core (CCINT).
• CO original cytoplasm
• the CCINT is removed from this oocyte and surrounded by the NT nucleus, which has kept some of its cytoplasm of CO origin, with the, or if necessary, several CCINT reprogrammed and recovered, before introducing the NT nucleus, thus re-packaged in a cell of origin of the NT nucleus, preferably enucleated and emptied of a part of its cytoplasm. If necessary, this cell can be enlarged according to one of the membrane manipulations previously described.
• the present invention may advantageously also be applied to ulcers. Chronic ulcers, especially in the legs, often very long before healing, and this healing often leaves a significant cutaneous and subcutaneous sequelae. Other ulcers never heal.
• the CRG (s) intended to accommodate this unit can be quite bulky and can therefore be for example artificially enlarged by the method previously described.
• the cell may for example be chosen from a keratinocyte, Langerhans, Merkel and / or melanocyte cell, taken alone or in combination, while at the dermal level, cutaneous fibroblasts may be taken.
• the cells of the epidermis and dermis can be placed in separate oocytes.
• the epidermodermal cells collected after regeneration should as far as possible be positioned and fixed in the culture bath in a reciprocal conformation close to that naturally observed, so as to promote functional cell growth and simplify the implantation of the layer.
• tissue regenerated on the receiving skin When culturing the regenerated cells, it may be possible to rearrange the respective position of the different categories of cells, or even to cultivate several variants of assembly intended for grafts at different locations or having a different morphology or function.
• a device of the invention can provide for locally removing the epidermis and / or the damaged dermis, treating it with the regeneration according to the invention, and then manufacturing from this regenerated tissue.
• genetically an extract of these cells which may be fixed for example in a cream, solution or the like for an external cutaneous application.
• This extract can also be used to create a solution for injection in subcutaneous, intradermal or intra epidermal. It thus becomes possible to quickly and temporarily restore the repair functions of the epidermal and / or dermal DNA.
• the invention makes it possible to fight genetically against the senescence of the skin, by modifying the collagens, in particular by rejuvenating them, to restore elasticity to the skin.
• necrotic, fibrotic or inactive tissue areas are also an application of the present invention.
• Such damaged tissue areas appear, for example, at the level of a myocardium, particularly following an infarction or heart failure, or for example at a body that has developed a tumor targeted by a destructive anticancer treatment.
• the invention also applies to heart valves. These can be organic, with a limited lifespan (around 15 years). They can also be artificial, with a longer lifespan
• the invention makes it possible to create a biological, artificial, mixed, or repaired cardiac valve, and to coat the surface of this substrate which is in contact with the blood of the patient. less a regenerated autologous cell layer.
• This coating can be made from a treatment of cardiovascular endothelial autologous cells previously removed by cardiac or vascular catheterization, treated according to the present invention, and then implanted on the valve. In the case of a plasty, this implantation can be done intraoperatively, that is to say during the operation, covering at least a portion of the valve and the valve ring. In this way, anticoagulant therapy may become unnecessary.
• the present invention also makes it possible to perform cardiac autografting instead of homologous grafts and mechanical artificial hearts with pneumatic or electrical transcutaneous supply.
• the inventor was the first in the fifties to implant a totally artificial heart in a dog, allowing him to survive a short time while his natural heart was in a jar (R. Monod, F. Zacouto, E. Coraboeuf and R. Saumont: "Cardiopulmonary bypass allowing the temporary exclusion of the heart and its replacement by an intrathoracic mechanical device", Compt Rend Soc Biol., 150, No. l, 48 (1956)).
• 3D echocardiography can advantageously be used to accurately reconstruct the geometries of the heart in order to best adapt to the thoracic volume specific to each patient (N. Mirochnik, A. Hagège, F. Zacouto and C. Guérot, "Physical reproduction of cardiac structures: a new avenue of exploration in cardiology.” Heart and Vessel Diseases Archives, Volume 93, No. 10, October 2000, pp. 1203-1209).
• the present invention can also assist in the determination of the mechanism responsible for a disorder of the health status of a mammal. Indeed, the root cause of an impairment of the vital balance is sometimes difficult to find.
• Langerhans taken for example by endoscope, will show an induced secretion of insulin or normal glucagon in contrast to the equivalent cells not subject to cell regeneration.
• the origin of disease states occurring after a certain age is likely to be revealed by the functional comparison of existing or present suspicious tissue with respect to its now genetically rejuvenated tissue ancestor at a given biological age.
• the present invention is also useful for diseases characterized by cell deficiency.
• the present invention may allow regeneration and multiplication of ⁇ and ⁇ cells of islets of
• Langerhans that can be re-implanted in the pancreas or elsewhere in order to restore the secretion of insulin or glucagon in an organism.
• the implantation of regenerated hepatocytes can ensure healing of liver disorders.
• Still another application of the present invention is, for example, the maintenance of an implant in a bone using a simple envelope or framework or support of regenerated cells.
• This application comprises regenerating bone cells, in particular osteoblasts, preferably taken at an early stage of their spontaneous or induced mitosis in a CRG and then placing these regenerated osteoblasts before the end of their mitosis in a receptor cell preferably of osteoblasts, to cultivate osteoblasts in a suitable culture medium in order to obtain appropriate numbers and mechanical behavior and then to distribute the osteoblasts in the form of a sleeve, pedestal, framework, or envelope between an artificial bone implant and the bone.
• the genetically rejuvenated layer of osteoblasts then ensures a good bonding of the bone implant and the bone by the osteoblastic thrust, and thus reinforces the maintenance of the implant in the bone and strengthens the bone structure itself.
• the use of such regenerated cells ensures the long-term maintenance of the implant in the bone and can have a lasting, increased and curative efficacy compared to protein creams based on different BMPs (Bone Morphogenic Proteins) usually implemented.
• Another possible use of the present invention is to ensure good histocompatibility between a donor graft and the immune system of a recipient.
• a healthy cell of the graft member of the recipient can be removed and regenerated according to the described process and then transferred to a suitable receptor cell so as to cause the proliferation of these cells.
• These cells can then be placed around the donor graft so that the recipient's immune system recognizes the critical molecules on the graft surface as self molecules and thus does not generate a strong immune response in the presence of the graft. .
• the creation of histocompatibility can in particular be carried out in the following ways:
• the present invention is also of particular interest in the field of dental stomatology.
• the absence of teeth must often be compensated by ceramic, plastic, etc. metal implants. These implants require a sufficient maxillary bone support base to ensure strong fixation of the implant.
• this bone regeneration is combined with the maxillary bone, which can be done by local injections of regenerated cells into, in contact with, or near the bone, a coating of the implant with at least one layer of bone cells. and / or regenerated periodontal, which will improve the fixation, the viscoelastic strength and the corresponding strength of the implant in the bone, as well as the strength of the bone itself.
• Another example of application of the cell regeneration method according to the invention relates to fractures and bone surgery.
• Some bone fractures and malformations require surgery that sometimes requires extra bone mass graftable and solid. This can be achieved by genetic rejuvenation of local cells with multiplication each time the final surgery can be delayed by at least two weeks. This is particularly the case for interventions for pseudarthrosis, vertebral bone deformities of children or degenerative, arthritis or deforming arthrosis.
• In vitro cell multiplication of osteoblast cells should preferably be carried out taking into account, as soon as they are cultured, the mechanical stresses they must withstand, for example after implantation in the femur, maxillary, vertebrae, etc.
• the inventor has developed an original adjustable vertebral fixator (US-6,835,207) and an original adjustable vertebral disc (US-6,692,495), both of which can advantageously be combined with vertebral tissue resulting from such cellular regeneration, and for example serve as a pedestal base for fixation pedicle screws or for filling crowded or fractured vertebrae, or as a supporting framework.
• Another application of the invention relates to non-autologous grafts. Indeed, a major problem concerns the rejection of transplants by the recipient. However, it is known that the fetal cells or near embryonic cells are less rejected. Sufficiently rejuvenated cells, for example by several successive treatments according to the invention, would make it possible to reduce the problem of rejections of non-autologous grafts. It should be noted that changing the biological age of a cell
• the nucleus of a cell is transferred into an oocyte, will divide and become able to reproduce in utero or artificially in vitro the original tissue of said nucleus.
• this means that the nucleus is in contact with a cytoplasm containing mitochondria and ribosomes capable of influencing the function and / or evolution of the nucleus, in particular via foreign DNAs and / or RNAs, because oocytes they contain.
• a cytoplasm containing mitochondria and ribosomes capable of influencing the function and / or evolution of the nucleus, in particular via foreign DNAs and / or RNAs, because oocytes they contain.
• the oocyte comes from the mother, it is possible that it has different characteristics due in particular to the influence of the environment, therapies, diseases, age, etc. To obtain a purely autologous tissue, it would be necessary to use an oocyte of the mother obtained at the time of the birth of origin, which is rarely feasible.
• partial cloning which consists in temporarily introducing a nucleus into an oocyte for a short time, then retransferring it to an autologous recipient cell, preferably identical to its cell of origin
• the tissue obtained is purely autologous, whatever the oocyte (or CRG or equivalent MRG cell) used.
• This can make it possible to use an oocyte of a mammal not necessarily identical while ensuring maximum genetic purity.
• the methods according to the invention can also be used to obtain embryonic cells from an adult nucleus. For this, one surrounds the previously reprogrammed nucleus preferably with additional cytoplasm autologous to the nucleus, as previously described.
• the treated nucleus thus packaged is placed in an oocyte (or MRG), enlarged if necessary according to the invention, and the embryonic cell divisions are allowed to develop.
• oocyte or MRG
• embryonic or fetal cells from a younger nucleus are created and the differences in biological age between this nucleus and the embryonic or fetal cells created are reduced.

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