WO2007009981A1 - Support et procede destines a la culture de tissus - Google Patents

Support et procede destines a la culture de tissus Download PDF

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Publication number
WO2007009981A1
WO2007009981A1 PCT/EP2006/064342 EP2006064342W WO2007009981A1 WO 2007009981 A1 WO2007009981 A1 WO 2007009981A1 EP 2006064342 W EP2006064342 W EP 2006064342W WO 2007009981 A1 WO2007009981 A1 WO 2007009981A1
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tissue
cat
available
medium
biochrom
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PCT/EP2006/064342
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English (en)
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Peter Vogt
Christina Allmeling
Kerstin Reimers
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Medizinische Hochschule Hannover
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Publication of WO2007009981A1 publication Critical patent/WO2007009981A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids

Definitions

  • the present invention relates to an apparatus and a process for the cultivation of tissue pieces and organs.
  • the cultivation can be used for long-term preservation of tissue and organs without decay or significant morphological changes, e.g. providing a method for preservation for tissue and organs in a viable state, e.g. suitable for subsequent transplantation.
  • the present invention relates to a process for the generation of differentiated tissue types obtainable from original adipose tissue.
  • the differentiated tissue types preferably originate from a fat tissue obtained from a biopsy and therefore are immunologically compatible with the donor organism because they maintain the immunological properties of the original fat tissue.
  • differentiated tissue generated from fat tissue by the cultivation process of the invention is characterized by a vascular system that is sufficient for anastomosis in a transplant recipient, allowing the connection of the differentiated tissue to the blood circulation of the transplant recipient.
  • differentiated tissue encompass tissue types that differ from the original tissue, which preferably is adipose tissue, e.g. differentiated to muscle, nerve, skin, cartilage, and bone, or differentiated to adipose tissue having a vascular system differing from the original vascular system.
  • adipose tissue e.g. differentiated to muscle, nerve, skin, cartilage, and bone, or differentiated to adipose tissue having a vascular system differing from the original vascular system.
  • the present invention relates to the process for cultivation of adipose tissue and the generation of differentiated tissue, to a medium suitable for the cultivation process, both for long-term preservation of tissue and organs and for tissue differentiation, and to a bio- reactor that is suitable for the cultivation process.
  • the invention offers the specific advantage that the medium is adapted to induce the transdifferentiation in e.g. adipose tissue during cultivation, e.g. using the bio-reactor according to the invention, resulting in the generation of vascularized adipose tissue.
  • Adipose tissue vascularized in this way can be used to generate desired differentiated tissue types, cellular aggregates and/or organs in vitro, for example bone tissue, muscle tissue, skin tissue, nerve tissue, liver or kidney tissue.
  • growth factors or differentiation factors can be added to the cultivation medium, which can for example be the medium according to the invention.
  • the present invention relates to the storage or preservation of tissue, for example in the form of organs, because the medium according to the invention is suitable for preserving cell aggregates and tissue viable over a very extended period of time from days over several weeks up to several months, preferably for one to twelve months or longer.
  • the tissue is maintained in the medium according to the invention at temperatures from 1 to 37 °C, preferably at 5 to 25 °C, more preferably at 10 to 15 °C and is viable and is in a state suitable for subsequent transplantation.
  • the preservation process of the invention offers the specific advantage that during the preservation in the medium, essentially no cell division is occurring.
  • a regular change of medium is also dispensable during the long-term cultivation of the preservation process, but a regular change of medium is also desirable, e.g. for extending cultivation processes for differentiation of tissue and for long-term preservation of tissue or organs.
  • the present invention provides a medium for cell culture which is free of serum components and other complex additives, which is characterized by supporting the long-term cultivation process of the invention for the preservation of cells, tissue and organs, or, preferably with differentiation inducing agents added, characterized by supporting the cultivation process of the invention for differentiation of one type of vascularized tissue into another type of vascularized tissue.
  • the use of medium for the cultivation process allows both to maintain cultivated cells or tissue, especially primary cultures or the first passage thereof, as well as cellular aggregates, especially organs, viable for a longer period of time, especially for more than twelve months in so-called permanent long-term cultures.
  • permanent long-term cultures no morphological changes, like apoptotic cell changes, are observable.
  • the process for preservation maintains cells, tissue and organs viable and essentially without morphological, histological or immunological alterations at cultivation temperatures of 1 to 37°C, without cell division being initiated.
  • the present invention also relates to a serum- free medium, which is suitable for maintaining viable animal cells or organs for a long period of time, with no significant morphological alterations occurring, as well as to a process for the preservation of animal cells, tissues and organs, which is characterized by the use of the medium according to the invention. Accordingly, the present invention also relates to cells, tissue and organs preserved by the preservation process of long-term cultivation.
  • cells have been found to be essentially in the G0-phase.
  • the medium of the invention if applicable supplemented with additives necessary for differentiation.
  • the invention is based on the property of the novel medium to maintain the cell cycle of eucaryotic cells, especially of animal cells, within the G0-phase. Therefore, in addition to the preservation process by long-term cultivation also the use of the medium for a synchronization process of cells into the G0-phase is part of the invention, for instance in a synchronization of primary cells or organs.
  • the invention in a second embodiment, relates to a bio-reactor that is suitable for the differentiation process of tissue, e.g. adipose tissue, by cultivation.
  • tissue e.g. adipose tissue
  • This differentiation process leads to the generation of differentiated tissue, differing from the cell type of the originally used tissue, for example naturally grown vascularized adipose tissue, e.g. obtained from a biopsy, such that the adipose tissue is further differentiated into more intensely vascularized adipose tissue.
  • the serum- free medium of the invention for sustaining the long-term cultivation of the adipose tissue in the bio-reactor and to allow and foster its vascularization, respectively.
  • the invention relates to a process for the generation of differentiated tissue in vitro, as well as to the tissue generated by the process, which is preferably finally differentiated tissue having the same immunological properties as the originally used tissue, e.g. obtained by biopsy.
  • the process for cultivation is based on the property of the medium according to the invention to maintain and/or support the vascularization of adipose tissue during the cultivation process within the bio-reactor such that on this basis a further differentiation to specific tissue types is possible while maintaining or further differentiating the vascular system generated during the cultivation process.
  • the cultivation medium for example tissue specific factors and/or hormones.
  • the serum- free medium according to the invention is preferably employed until attaining the differentiation to specific cell types and beyond the state of terminal differentiation into a tissue type differing from that of the originally used tissue of a biopsy for preservation in long-term culture.
  • complex medium additives e.g. horse serum or the active component thereof, may be added as differentiation initiating agents.
  • the medium according to the invention has the following composition:
  • - ad 1 L DMEM/F12 medium (available from Biochrom, Germany, Cat. -No. F4815), - 10 mL: IST-A supplement (available from Gibco, Cat. -No. 51300-044), containing 1.0 g/L insulin, 0.67 g/L Na-selenite, 0.55 g/L transferrin and 11.0 g/L Na-pyruvate,
  • HEPES available from Biochrom, Cat.-No. F4815
  • hyaluronic acid available from Sigma, St. Louis, Cat.-No. Hl 876
  • single components can be replaced by components with the same properties, for example the antibiotics added can be replaced by different antibiotics for cell culture or can be omitted at sufficiently sterile cultivation conditions.
  • a total of about 400 mL of medium can be used for circulation, of which about 174 th are present in the bio-reactor for engulfing the piece of tissue supported in the reactor.
  • the medium for use of the medium according to the invention for synchronizing cells into the G0-phase and for the preservation of cells, tissue and organs, respectively, can be utilized in conventional cultivation dishes or in culture vessels or in containers, in which larger cell aggregates, for example tissue pieces or organs are supported, for example mechanically fixed to a support.
  • Such a support can for example be present in the form of a pin, onto which a piece of tissue or an organ is fixed, for example by skewering, clamping, tying with threads or by glueing.
  • a piece of tissue or organ it is especially preferred to let the piece of tissue or organ to be cultivated for preservation and/or for differentiation hang within the medium according to the invention from the support, for example from the lower end of a stick or a thread, which is fixed to the upper portion of the vessel.
  • the piece of tissue and organ, respectively is also to be fixed to the support, for example by skewering, clamping or tying by tying by means of threads.
  • the bio-reactor according to the invention is schematically shown in Figure 1 and comprises a support for supporting a piece of tissue or an organ.
  • the preferred embodiment is schematically represented, wherein the piece of tissue is fixed to the support, realized in the form of a thread.
  • the piece of tissue or organ fixed to the support is arranged within the fluid stream of medium.
  • the medium can stream towards and around the piece of tissue or organ as it is delivered by and exiting from a pipe, ending above the piece of tissue or organ.
  • the piece of tissue or organ is positioned within medium flowing around it.
  • the support in the form of a pipe realized here by a nozzle or a pipette tip, also conducts medium onto the piece of tissue or organ that is fixed to the support.
  • an exit is provided, which is for example arranged within the reactor bottom, within a wall or within the lid, with the opening of the exit preferably arranged within the vicinity of the reactor bottom.
  • the exit in the form of a pipe which is represented by a cannula or pipette tip as an example, is carried through the lid of the bio-reactor and its opening is arranged at a small distance above the reactor bottom. In this manner, the medium can be withdrawn from the reactor bottom.
  • cultivation medium is withdrawn at low flow rate, for example in the range of 0.1 to 10 mL/min by the exit pipe and introduced by means of the entrance pipe to the piece of tissue and organ, respectively, to let the medium flow around the piece of tissue and organ, respectively, at low flow rate.
  • the medium can also be partially replaced by fresh medium.
  • the cultivation according to the present invention is especially suitable for the preservation of tissue or organs, especially for maintaining tissue or organs over an extended period of time in a state suitable for transplantation without significant impairments or alterations.
  • the cultivation process according to the present invention preferably originally using adipose tissue, e.g. adipose tissue originating from the epifascial adipose tissue region by biopsy, is suitable for generating a perforating vascular system within the cultivated tissue, which vascular system is characterized by a vascular connection that is accessible e.g. by microsurgical anastomosis.
  • the cultivation process is capable of generating in the cultivated tissue an essentially complete microcapillary network, preferably as well providing a process for acellularization and/or colonization of the cultivated and differentiated tissue by heterologous epithelial cells.
  • tissue obtainable according to the present invention iulfils the prerequisites for a vascular and capillary network generated within the tissue by the cultivation process, including the differentiation over the original tissue, which can be produced until now only by methods of tissue engineering.
  • vascular systems produced by tissue engineering according to the present state of scientific investigations cannot be produced with the structure that is necessary for transplantation, especially not with a vascular connection for anastomozation, i.e. a vascular connection that is accessible by anastomosis.
  • the present invention provides a method for producing adipose tissue and other terminally differentiated tissue types on the basis of adipose tissue, preferably including a natural vascular system, characterized in that the tissues have a capillary network and a vascular connection accessible by anastomosis.
  • Transplants for use in surgery having the required vascular system, e.g. for restorative and plastic surgery, are obtainable according to the invention , especially vascularized tissue having a vascular connection accessible by anastomosis.
  • Transplants obtainable by the cultivation process according to the present invention e.g. originating from biopsied adipose tissue are for example comprised in the group of vascularized adipose transplants, fat-skin- transplants, muscle transplants, bone and/or cartilage transplants, nerve transplants, as well as bio-artificial transplants, especially liver transplants, pancreatic transplants, kidney transplants and endocrine transplants, e.g. parahyroideal transplants.
  • vascularized transplants obtained by microsurgery can be cultivated within the bio-reactor according to the invention using medium according to the invention, for maintaining the vitality and perfusability of the vascular system of the transplant, as well as for providing for modification, e.g. by differentiation of the vascularized transplant and, accordingly, for providing modified and/or differentiated vascularized transplants for transplantation.
  • the bio-reactor containing the medium can also be used for the following variations of cultivation and modification, respectively, of cells or tissue: addition of growth factors and/or cytokines, addition of other cell types, colonization of a supported piece of tissue, the addition of other types of tissue to the tissue cultivated in a preliminary separate cultivation process, as well as for generating a vascular connection, for preservation and/or for modification of differentiated organs.
  • Figure 1 schematically depicts a lab-scale bioreactor for use in the cultivation processes of the invention
  • Figure 2 shows a comparison of cultivated fat tissue A) without differentiation and B) with osteogenic differentiation in Alizarin red staining
  • FIG. 3 shows the course of cultivation with osteogenic differentiation of fat tissue in Alizarin red staining A) at day 10 of cultivation, B) at day 18 of cultivation, C) and D) at day 34 of cultivation,
  • Figure 4 shows different stains of a cultivated rat fat lobe after cultivation over 34 days with osteogenic differentiation, under A) in hemalum staining, B) Alizarin red staining, C) Masson Goldner Tri chrom staining, and D) Sudan black staining,
  • Figure 5 shows Alcian blue stains of a rat fat lobe A) at non-differentiating cultivation in comparison to B) after cultivation with chondrogenic differentiation
  • Figure 6 shows different stains of a cultivated rat groin fat lobe after cultivation over 4 weeks with chondrogenic differentiation, under A) with Alcian blue staining for cartilage tissue, and B) with hemalum staining, under C) with Alizarin red staining for bone tissue, and under D) with Sudan black for remaining fat cells staining,
  • Figure 7 shows Alcian blue and Alizarin red stains of a cultivated rat groin fat lobe after cultivation over 4 weeks with chondrogenic differentiation, under A) with Alcian blue staining, and B) of a control cartilage tissue (rat ear) with Alcian blue staining, under C) of cartilage tissue generated after 4 weeks differenting cultivation with Alizarin red staining, and under D) of the control rat ear with Alizarin red staining,
  • Figure 8 shows a hemalum stain of rat groin fat lobe, namely of an entrance vascular region after cultivation over 18 days with myogenic differentiation at 10 x magnification
  • Figure 9 shows a light micrograph of a rat groin fat lobe after cultivation for 2 months A) with non-differentiating culture conditions (control) in comparison to B) with myogenic differentiation, at 10 x magnification each,
  • Figure 10 shows a light micrograph of a rat groin fat lobe after cultivation for 2 months with myogenic differentiation
  • Figure 11 shows an inverse light micrograph of a rat groin fat lobe after cultivation for 2 months with myogenic differentiation at 10Ox magnification.
  • the serum- free medium of the invention was used, whereas for differentiating cultivation, the following media were employed, containing per 100 mL
  • PeniStrep Penicillin/Streptomycin mixture (Biochrom) in concentration suitable for cell culture
  • A-2-P ascorbate-2-phosphate
  • IST+ supplement (Invitrogen) containing insulin, selenium, transferrin
  • Dexa Dexamethason
  • TGF Transforming Growth Factor
  • FBS fetal bovine serum
  • IBMX isobutyl-1-methyl-xanthine
  • HS horse serum
  • Example 1 Generation and maintenance of primary cell cultures and of tissue by preservation using cultivation in medium according to the invention
  • Primary cell cultures obtained for example from human skin or from fibroblasts, e..g. according to Dupuytrene, were covered with medium according to the invention and kept at a temperature of 37 °C.
  • a standard cell culture dish (9 cm diameter) was used, in which the cell culture was kept without passaging for more than 12 months.
  • the medium volume was 10 mL, which was exchanged regularly.
  • a cultivation vessel was used, having an inlet nozzle for medium, arranged opposite an outlet nozzle, and an apparatus for circulating the medium at a constant filling level of the inner vessel of 50 mL total volume at a flow rate of 0.1 to 1 mL /min.
  • the total volume of the medium was 500 mL.
  • Morphological examinations of the cultivated cells showed that at durations of the preservation cultivation process of two weeks up to twelve months, no morphological changes, especially essentially no apoptosis of the cells were detectable.
  • the preservation of pieces of tissue was tested on a human skin biopsy, a human fat tissue biopsy, and a human bone biopsy, optionally containing hemopoietic stem cells as examples of tissue, as well as on a pig kidney, optionally containing hematopoietic stem cells as an example for an organ.
  • the pieces of tissue and the organ, respectively, were suspended from a thread serving as the support into the lab scale bio-reactor according to the invention according to Figure 1, such that they were arranged in the centre of the filling level of the medium.
  • Medium was flowing against and around the piece of tissue and the organ, respectively, from an inlet nozzle, having the form of a sterile cannula, the exit opening of which was arranged above the tissue and organ, at a flow rate from the cannula set at a range of 0.1 mL /min to 1 mL /min.
  • the outlet nozzle was arranged above the reactor bottom and both the pipe connected to the outlet nozzle as well as the pipe connected to the inlet nozzle were led through the lid of the bio-reactor.
  • Medium was circulated between outlet nozzle and inlet nozzle by pumping at the flow rate of 0.1 mL /min to 1 mL /min.
  • kidneys Like the pig kidney, also rat kidneys could also be preserved in the bio-reactor by permanent cultivation, wherein both kidneys showed a good catabolic function. Excretion from the kidney could be demonstrated by urethral catheterization. Further, the cortex of kidney showed a normal cellular structure. Using the same volume of medium without any intermediate treatment or additions/replacements outside the bio-reactor, the function of the kidneys could be maintained without problems over four days, without any defects or degenerations observable.
  • the cultivation temperature was 37 °C, in a 5% CO 2 atmosphere.
  • the perfusion rate through the bio-reactor can be set to about 880 ⁇ L /min.
  • the atmosphere preferably contains no additional CO 2 ; the cultivation and incubation temperatures, respectively, are preferably set to about 37 °C.
  • rat and human livers perfused in the bio-reactor for 1 week were found to show a normal, essentially unchanged histology, with cellular associations and vascular structures well maintained.
  • the lobe could be re-transplantated after this cultivation over two to four months in the medium according to the invention and adhered to the donor organism without any reactions of rejection.
  • Example 3 Generation of vascularized adipose tissue
  • adipose tissue As a precursor for the generation of differentiated cells and differentiated tissue having a vascular connection that is accessible by anastomosis, respectively, according to the invention it is preferred as a first step to generate and cultivate vascularized adipose tissue.
  • adipose tissue designated in Figure 1 as fat lobe, is supported in the bio-reactor by suspension from a support according to the invention and positioned in a stream of medium according to the invention.
  • tissue e.g. an adipose lobe
  • a vascular system that can be connected to the inlet nozzle of the medium for perfusion, more preferably in connection with a surrounding flow of medium.
  • the pieces of tissue have dimensions of about 1 to 5 mm each and were supported in a bio- reactor according to example 1 by fixation to a thread and arranged within medium according to the invention.
  • Medium according to the invention was flowing at a circulating speed of 0.01 mL/min to 0.2 mL/min, the cultivation temperature was 37 °C, the atmosphere contained 5% CO 2 .
  • Pre-adipocytes cultivated in the bio-reactor according to the invention could differentiate into adipogenic or osteogenic tissue. Differentiation was reached at a duration of the cultivation process of about 5 days, respectively, using the process, the bio-reactor, and the medium according to the invention.
  • Example 4 Differentiation of adipose tissue
  • a biopsy was taken by dissecting in parallel to the median level of the groin. Skin was prepared subcutanously and separated into kaudal, cranial, lateral and medial sections. The fascia was lifted to get access to the fascial adipose lobe, which was biopsied including its accessory vascularization. In a sterile petridish, the prepared artery of the lobe is connected to a micro-cannula by a suture. The micro-cannula was suspended into the bio-reactor. As a control, the control lateral adipose lobe of the groin was prepared. Both groin lobes were perfused in parallel in the bio- reactor using the serum- free medium for seven days according to Example 1. Sequently, the lobes were transferred into differentiating media, which are known in the state of art. In detail, the following additives of Table 1 can be used for specific modification and differentiation, respectively:
  • tissue was sampled and prepared for histology. Samples were cryoconserved and cut using a microtome at freezing conditions into layers of 40 ⁇ m. Prior to histological staining, the microtome sections were airdried at room temperature for 3 to 4 hours.
  • Chondrogenically differentiated cells were identified by staining with alcian blue. Alcian blue is used for selective staining of glycosamino glycanes, which are present in the extracellular matrix of cartilage.
  • Sudan black was used for staining fat vacuola as well as Masson trichrome staining of connective tissue.
  • acidic hemalum and eosin was used according to P. Mayer.
  • Ig eosin was dissolved in 100 mL distilled water, which was further diluted 1/100 in destilled water for staining applications.
  • staining nuclei in the microtome sections the sections were incubated in hemalum solution for 10 minutes. Strongly acidic hemalum solution first stains nuclei slightly red. Then, sections were washed under flowing tap water. By incubation for 10 minutes, a more neutral pH value was obtained, resulting in a change of colour to blue. Staining of the cytoplasm was achieved by dipping the sections into the eosin solution for 1 minute. Excess eosin was rinsed off with distilled water and the sections were dried at room temperature before observation under the microscope.
  • Staining with alizarin red has a sensitivity of about 0.004 mg calcium 2+ /mm 2 .
  • Calcium ions are stained intensively orange to yellow - orange with the alizarin red forming chelate compounds with divalent cations like calcium 2+ .
  • samples were fixed in alcohol.
  • a staining solution at pH 9 was used. Although staining at pH 9 only effects a slow release of calcium ions from calcium deposits, staining is true to the location of the calcium deposits.
  • Ig alizarin red S was dissolved in 200 ml distilled water, with the pH 9 adjusted using sodium hydroxide. Prior to staining, cells were fixed with 70% ethano I/water, rinsed with distilled water and incubated for 10 minutes in the solution of alizarin red. After 10 minutes, cells were rinsed with distilled water and incubated for 10 seconds in acidic alcohol (1 volumetric part concentrated HCl to 10.000 volumetric parts 96% ethanol) for differentiation.
  • alcian blue was used for visualisation of glycosamino glycanes, without differentiation between carboxyl groups and sulfate groups.
  • tissue differentiated chondrogenically by the cultivation process of the invention was fixed in a 4% formalin solution and embedded in paraffin. Sections were prepared by microtomy. For staining, paraffin was extracted from the sections before incubation in water. Sections were then transferred into 3% acetic acid, followed by a thirty minute incubation in a solution of 1% alcian blue in 3% acetic acid, followed by rinsing in 3% acetic acid. Finally, sections were washed with distilled water and observed by microscopy.
  • results of differentiation of tissue are shown on the example of a human groin fat lobe differentiated by the cultivation process of the invention.
  • cultivation was as described in this example at 37 °C in a 5 % CO 2 atmosphere.
  • the groin fat lobe that was obtained by biopsy showed increased vascularization that was accessible by anastomosis for connection to the blood vessel system of a recipient receiving the differentiated tissue as a modified autologous or syngeneic transplant.
  • the fat tissue vascularized in vitro by a first cultivation process was differentiated into specific tissue types by subsequent secondary cultivation using specific differentiation additives.
  • a groin fat lobe is shown in a comparison of the originally non-differentiated tissue of the biopsy and following differentiation by cultivation for 1 month in the medium supplemented with FCS (fetal calf serum), ascorbate-2-phosphate, Dexamethason, ⁇ -glycerol for inducing differentiation.
  • FCS fetal calf serum
  • ascorbate-2-phosphate ascorbate-2-phosphate
  • Dexamethason Dexamethason
  • ⁇ -glycerol for inducing differentiation.
  • the incorporation of mineral salts is demonstrated by increased staining intensity (orange-red) for the differentiated tissue, indicating the beginning of osteogenesis.
  • FIG 3 staining of samples of the differentiated tissue taken at the indicated duration of the cultivation process are shown for tissue differentiated by cultivation in medium supplemented with FCS (fetal calf serum), ascorbate-2-phosphate, Dexamethason, ⁇ -glycerol: Staining shows a reduction of fat vacuola in the differentiated tissue and, accordingly, the increase in density of the cellular and of the extracellular matrices occuring in parallel.
  • FCS fetal calf serum
  • ascorbate-2-phosphate ascorbate-2-phosphate
  • Dexamethason Dexamethason
  • ⁇ -glycerol Staining shows a reduction of fat vacuola in the differentiated tissue and, accordingly, the increase in density of the cellular and of the extracellular matrices occuring in parallel.
  • the differentiated fat lobe was generated by cultivation using the differentiating additive ascorbate-2-phosphate, IST+, sodium pyruvate, proline, Dexamethason, TGF ⁇ l (transforming growth factor).
  • the differentiating additive ascorbate-2-phosphate, IST+, sodium pyruvate, proline, Dexamethason, TGF ⁇ l (transforming growth factor).
  • light blue staining for glycosamino glycane indicates typical cartilage tissue, generated by differentiating cultivation of the original fat tissue lobe.
  • a fat lobe generated to differentiated cartilage tissue by cultivation with ascorbate-2-phosphate, IST+, sodium pyruvate, proline, Dexamethason, TGF ⁇ l as the differentiating additive is shown in histological staining with hemalum-eosin.

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Abstract

La présente invention concerne un dispositif et un procédé destinés à la culture de tissus et d'organes. La culture peut être utilisée pour la conservation longue durée de tissus et d'organes sans dégradations ou variations morphologiques importantes, fournissant ainsi un procédé de conservation de tissus et d'organes dans un état viable, par exemple, pour une transplantation ultérieure, par exemple. La présente invention concerne également un procédé destiné à la production de types de tissus différenciés à partir de tissus adipeux. Ces types de tissus différenciés proviennent, de préférence, de tissus adipeux prélevés par biopsie et sont, par conséquent, compatibles sur le plan immunologique avec l'organisme donneur, car ils conservent les propriétés immunologiques du tissu adipeux de départ. Par ailleurs, le tissu différencié produit à partir du tissu adipeux par ledit procédé de culture se caractérise par un système vasculaire suffisant pour une anastomose chez un hôte receveur, ce qui permet de raccorder le tissu différencié au système vasculaire de l'hôte receveur.
PCT/EP2006/064342 2005-07-15 2006-07-17 Support et procede destines a la culture de tissus WO2007009981A1 (fr)

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CN101984051B (zh) * 2010-11-19 2012-08-29 中山大学 适合肿瘤干细胞富集与培养的无血清细胞培养液
US10751246B2 (en) 2017-12-26 2020-08-25 Sanjeev Kaila Acoustic shock wave therapeutic methods

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