WO2007009981A1

WO2007009981A1 - Medium and process for tissue cultivation

Info

Publication number: WO2007009981A1
Application number: PCT/EP2006/064342
Authority: WO
Inventors: Peter Vogt; Christina Allmeling; Kerstin Reimers
Original assignee: Medizinische Hochschule Hannover
Priority date: 2005-07-15
Filing date: 2006-07-17
Publication date: 2007-01-25

Abstract

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. Further, 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. Further, 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 connnection of the differentiated tissue to the blood circulation of the transplant recipient.

Description

TAR.UTTIS

Patentanwaltskanzlei

TARUmS - Vahrenwalder Str. 7 - D-3016₅ Hannover Dr. rer. nat. Stefan Taruttis

Diplom-Ingenieur

Europaisches Patentamt Patentanwalt

European Patent Attorney

ErhardtstraBe 27 European Trademark Attorney D-80331 Mϋnchen D-30165 Hannover, Vahrenwalder Str. 7 Tel: ++49 511 93 57 22 0 Fax: ++49 511 93 57 222 www.taruttis-patent.de in Kooperation mit

Dr. -Ing. Hartmut Schϋtte

Patentanwalt

D-59302 Oelde, Beethovenstr. 34

Your Ref: Our Ref: N1028PCT 17. July 2006

Novel PCT application,

"Apparatus and process for tissue cultivation", Medizinische Hochschulc Hannover,

Prof. Dr. Vogt, Allmeling, Dr. Reimers

MEDIUM AND PROCESS FOR TISSUE CULTIVATION

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. Further, 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. Further, 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.

For the purposes of the invention, 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.

In addition, 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. For differentiation of the vascularized adipose tissue into differing and finally differentiated tissue types, growth factors or differentiation factors can be added to the cultivation medium, which can for example be the medium according to the invention.

Further, 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. In the preservation process 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. Preferably, 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. General description of the invention

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.

In a first embodiment, 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. In these permanent long-term cultures, no morphological changes, like apoptotic cell changes, are observable. In greater detail, 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.

Accordingly, 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.

In the long-term cultivation process for preservation of the first embodiment, cells have been found to be essentially in the G0-phase. For the long-term cultivation, it is preferred to use the medium of the invention, if applicable supplemented with additives necessary for differentiation.

Accordingly, it is assumed that 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.

In a second embodiment, the invention relates to a bio-reactor that is suitable for the differentiation process of tissue, e.g. adipose tissue, by cultivation. 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. Upon use of the bio-reactor according to the invention for the cultivation process of adipose tissue, promoting its vascularization, it is preferred to use 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.

In a third embodiment, 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.

For supporting the differentiation to specific tissue types, it is a preferred option to add specific growth factors to the cultivation medium, for example tissue specific factors and/or hormones. For the cultivation process for the generation of differentiated tissue, 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. However, in this embodiment, 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,

- 5 mL 100 x concentrated penicillin/streptomycin, (available from Biochrome, Cat.- No. A2412),

- 250 μL Amphotericin (C₄₆H₇₃Nθ2₀) (available from Biochrom, Cat. -No. 2612),

- 20 mL HEPES (available from Biochrom, Cat.-No. F4815),

- 50 mg hyaluronic acid (available from Sigma, St. Louis, Cat.-No. Hl 876),

- 1000 μL human insulin (400 IU), 3,24 μL glucagone hydrochloride (108 mg/mL) and 456 μL solu-decortin (prednisolon-21-hydrogensuccinat, 10 mg/mL), (known as "Waymouth supplement")

Within this medium composition, 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.

Further, the quantitative proportions can be varied as long as the effect according to the invention is obtained.

For a piece of tissue (e.g. an adipose lobe, preferably vascularized fat tissue, obtained by biopsy), 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.

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, the medium 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. However, 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. In this embodiment, 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. Thereat, it is preferred that the piece of tissue or organ fixed to the support is arranged within the fluid stream of medium. As an example, 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. In this way, the piece of tissue or organ is positioned within medium flowing around it. In Figure 1, one such embodiment is shown, wherein 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.

For withdrawal of medium from the bio-reactor, 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. One possible embodiment is shown in Figure 1, wherein 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.

According to the invention, for operation of the bio-reactor, 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. In a simple embodiment it is possible to let the medium circulate without change. Alternatively, it is also possible to treat the medium outside of the bio-reactor, for example to separate floating matter from the circulating medium by ultra- filtration, or for disposal of catabolic products by dialysis and/or for introduction of fresh medium components. Outside of the bio-reactor, the medium can also be partially replaced by fresh medium. Detailed description of the invention

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.

Further, 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. Further, 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.

Accordingly, 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. However, 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.

In contrast to the present state of art tissue engineering, 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.

For generation of transplants according to the invention, 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.

In a further embodiment of the invention, 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.

The present invention will now be described in greater detail by way of examples with reference to the figures, wherein

• 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,

• Figure 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, and

• Figure 11 shows an inverse light micrograph of a rat groin fat lobe after cultivation for 2 months with myogenic differentiation at 10Ox magnification.

For non-differentiating cultures, corresponding to long-term preservation culture process, the serum- free medium of the invention was used, whereas for differentiating cultivation, the following media were employed, containing per 100 mL

for chondrogenic differentiation: for osteogenic differentiation:

DMEM/F12 88.7 mL

FBS 1O mL

PeniStrep I mL

A-2-P 100 μL

Dexa 196.3μl β-glycerol 75.6 mg

for adipogenic differentiation: and for myogenic differentiation:

DMEM/F12 85.6 mL DMEM/F12 88.7 mL

FBS 1O mL FBS 1O mL

PeniStrep I mL HS 5 mL

A-2-P 100 μL PeniStrep I mL

Dexa 1.963 mL Dexa 196.3 μL

IBMX l.l l l mL hydrocortisone 10 μL

Indomethacine 179 μL (50mg/ml) insuline 10 μL HEPES (IM) 4 mL

wherein

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.

For this preservation process, 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. Alternatively, 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.

Examples for fat tissue preserved by long term cultivation are given in Figures 2 A), 5 B), and 9 A).

Example 2: Preservation of organs by permanent cultivation

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% CO₂ atmosphere. Alternatively, the perfusion rate through the bio-reactor can be set to about 880 μL /min. The atmosphere preferably contains no additional CO₂; the cultivation and incubation temperatures, respectively, are preferably set to about 37 °C.

Using the same experimental setup, 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. In the case of a skin lobe from a human or rat biopsy preserved by the long-term cultivation process for preservation according to the invention, 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

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. For this purpose, 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.

For the tissue, e.g. an adipose lobe, it is preferred to have 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% CO₂.

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:

Table 1: Additives for modification and differentiation of adipose tissue during cultivation

Following cultivation for periods indicated in the appended figures, the tissue was sampled and prepared for histology. Samples were cryoconserved and cut using a microtome at freezing conditions into layers of 40 μm. Prior to histological staining, the microtome sections were airdried at room temperature for 3 to 4 hours.

Cells and nuclei were stained according to common protocols using hemalum-eosin. For osteogenically differentiated tissue, alizarin red was used, the affinity of which to calcium forming the basis for its use as an intravital marker for bone in light microscopy.

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. For control purposes, Sudan black was used for staining fat vacuola as well as Masson trichrome staining of connective tissue.

In detail, acidic hemalum and eosin was used according to P. Mayer. For a 1% stock solution, Ig eosin was dissolved in 100 mL distilled water, which was further diluted 1/100 in destilled water for staining applications. For 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⁺ /mm². Calcium ions are stained intensively orange to yellow - orange with the alizarin red forming chelate compounds with divalent cations like calcium ²⁺. Before staining, samples were fixed in alcohol. In order to avoid diffusion artifacts that arise from acid solution containing dissolved calcium ²⁺, 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.

For the staining solution, 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.

For visualisation of glycosamino glycanes, without differentiation between carboxyl groups and sulfate groups, alcian blue was used. First, 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. For these results, cultivation was as described in this example at 37 °C in a 5 % CO₂ atmosphere. In general, 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. For generation of differentiated tissue, 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.

In Figure 2, 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, β-glycerol for inducing differentiation. In addition to the vascularization, the incorporation of mineral salts is demonstrated by increased staining intensity (orange-red) for the differentiated tissue, indicating the beginning of osteogenesis.

In Figure 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, β-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.

In Figure 4, histological staining with hemalum-eosin is shown, also demonstrating the reduction of fat vacuola in the differentiated tissue and, accordingly, the increase in density of the cellular and of the extracellular matrices. Alizarin red shown incorporation of mineral salts, Masson TriChrome indicates connective type tissue structures in blue, and Sudan black stains fat vacuola deep black. In the comparative fat tissue lobe, Sudan black is so intense that it could not be photographed.

In Figure 5, the differentiated fat lobe was generated by cultivation using the differentiating additive ascorbate-2-phosphate, IST+, sodium pyruvate, proline, Dexamethason, TGFβl (transforming growth factor). As as result, light blue staining for glycosamino glycane indicates typical cartilage tissue, generated by differentiating cultivation of the original fat tissue lobe. In Figure 6, a fat lobe generated to differentiated cartilage tissue by cultivation with ascorbate-2-phosphate, IST+, sodium pyruvate, proline, Dexamethason, TGFβl as the differentiating additive is shown in histological staining with hemalum-eosin. The decrease in fat vacuola is shown, alizarin red staining demonstrates incorporation of mineral salts, alcian blue staining demonstrates the generation of glycosamino glycane, and Sudan black indicates a reduction of fat vacuola.

In Figure 7, natural cartilage tissue is shown in the same staining examinations as for Figure 6 as a further comparison to the cartilage tissue generated from iat lobe shown in Figure 6. The comparison demonstrates that the cultivation process according to the invention is capable of generating differentiated tissue having an essentially natural histology and structure, exemplified here by cartilage tissue.

In Figure 8, the generation of muscle tissue from the fat lobe is demonstrated by myogenic differentiation, using FCS, horse serum, Dexamethason, and hydrocortisone as the myogenic differentiating additive in the secondary cultivation process. In hemalum-eosin staining, maintenance of the vascular system that was generated in the first cultivation process of the fat lobe without differentiating additive during secondary myogenic differentiation is shown.

In Figures 9 and 10, the tissue of Figure 8 is shown in hemalum-eosin staining in comparison to the original fat lobe as a control. It can be seen clearly that cell elements are arranged in bundles corresponding to muscle fibrils, whereas no fat vacuola can be detected.

In Figure 11, a higher enlargement (100Ox) of the sample of Figure 9 is shown, indicating the fusion of cells to poly-nuclear syncytia.

Claims

1. Cultivated tissue, obtainable by in vitro cultivation of natural mammalian tissue using a medium comprising 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,

5 mL 100 x concentrated penicillin/streptomycin, (available from Biochrom,

Cat. -No. A2412),

250 μL Ampho (available from Biochrom, Cat. -No. 2612),

20 mL HEPES (available from Biochrom, Cat.-No. F4815),

50 mg hyaluronic acid (available from Sigma, St. Louis, Cat.-No. Hl 876),

1000 μL human insulin (400 IU), 3,24 μL glucagone hydrochloride (108 mg/mL) and 456 μL solu-decortin (10 mg/mL), (known as "Waymouth supplement") and circulating the medium to completely contact the tissue, which tissue is vascularized and has a vascular connection that is accessible to anastomosis.

2. Cultivated tissue according to claim 1, wherein the natural mammalian tissue is adipose tissue.

3. Cultivated tissue according to one of the preceding claims, wherein the cultivated tissue is differentiated to a tissue type differing from the type of the natural mammalian tissue by cultivation in a medium containing a differentiation inducing component.

4. Cultivated tissue according to claim 3, wherein the differentiatiion inducing component is selected from the group comprising estradiol, ascorbate-2-phosphate, IST+, Dexamethason, TGFβl, FGF2 and horse serum.

5. Process for preserving tissue by cultivation in a medium comprising 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,

5 mL 100 x concentrated penicillin/streptomycin, (available from Biochrom, Cat. -No. A2412),

250 μL Ampho (available from Biochrom, Cat. -No. 2612), 20 mL HEPES (available from Biochrom, Cat.-No. F4815), 50 mg hyaluronic acid (available from Sigma, St. Louis, Cat.-No. Hl 876), 1000 μL human insulin (400 IU), 3,24 μL glucagone hydrochloride (108 mg/mL) and 456 μL solu-decortin (10 mg/mL), (known as "Waymouth supplement") and circulating the medium to completely contact the tissue.

6. Process for differentiating a natural mammalian tissue obtainable by biopsy, comprising the cultivation in a medium comprising ad 1 L: DMEM/D12 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,

5 mL 100 x concentrated penicillin/streptomycin, (available from Biochrome, Cat. -No. A2412),

250 μL Amphotericine (available from Biochrome, Cat.-No. 2612), 20 mL HEPES (available from Biochrome, Cat.-No. F4815), 50 mg hyaluronic acid (available from Sigma, St. Louis, Cat.-No. Hl 876), 1000 μL human insulin (400 IU), 3,24 μL glucagone hydrochloride (108 mg/mL) and 456 μL solu-decortin (10 mg/mL), (known as "Waymouth supplement") and circulating the medium to completely contact the tissue.

7. Process according to claim 5 or 6, wherein the tissue is selected from the group comprising natural mammalian tissue and mammalian organs.

8. Process according to claim 7, comprising a secondary cultivation in a medium comprising ad 1 L: DMEM/F12 medium (available from Biochrome, Germany, Cat. -No.

F4815),

5 mL 100 x concentrated penicillin/streptomycin, (available from Biochrom,

Cat. -No. A2412),

250 μL Amphotericine (available from Biochrom, Cat. -No. 2612),

20 mL HEPES (available from Biochrom, Cat.-No. F4815),

50 mg hyaluronic acid (available from Sigma, St. Louis, Cat.-No. Hl 876),

1000 μL human insulin (400 IU), 3,24 μL glucagone hydrochloride (108 mg/mL) and 456 μL solu-decortin (10 mg/mL), (known as "Waymouth supplement"), supplemented with a differentiation inducing component, and circulating the medium to completely contact the tissue.

9. Process according to claim 8, wherein the differentiation inducing component is selected from the group comprising estradiol, ascorbate-2-phosphate, IST+, Dexamethason, TGFβl, FGF2 and horse serum.

10. Use of a medium comprising ad 1 L: DMEM/F12 medium (available from Biochrome, Germany, Cat.-No.

F4815),

5 mL 100 x concentrated penicillin/streptomycin, (available from Biochrom,

Cat. -No. A2412),

250 μL Amphotericine (available from Biochrom, Cat.-No. 2612),

20 mL HEPES (available from Biochrom, Cat.-No. F4815),

50 mg hyaluronic acid (available from Sigma, St. Louis, Cat.-No. Hl 876), 1000 μL human insulin (400 IU), 3,24 μL glucagone hydrochloride (108 mg/mL) and 456 μL solu-decortin (10 mg/mL), (known as "Waymouth supplement"), in a process according to one of claims 5 to 9.

11. Medium comprising ad 1 L: DMEM/F12 medium (available from Biochrome, Germany, Cat. -No.

F4815),

5 mL 100 x concentrated penicillin/streptomycin, (available from Biochrom,

Cat. -No. A2412),

250 μL Amphotericine (available from Biochrom, Cat. -No. 2612),

20 mL HEPES (available from Biochrom, Cat.-No. F4815),

50 mg hyaluronic acid (available from Sigma, St. Louis, Cat.-No. Hl 876),

1000 μL human insulin (400 IU), 3,24 μL glucagone hydrochloride (108 mg/mL) and 456 μL solu-decortin (10 mg/mL), (known as "Waymouth supplement").