WO2002010345A2

WO2002010345A2 - Autologous bio-matrix from mammalian liver

Info

Publication number: WO2002010345A2
Application number: PCT/EP2001/008646
Authority: WO
Inventors: Giovanni Ambrosino
Original assignee: Giovanni Ambrosino
Priority date: 2000-07-28
Filing date: 2001-07-26
Publication date: 2002-02-07
Also published as: IT1318655B1; ITMI20001744A0; ITMI20001744A1; AU2001289759A1; WO2002010345A3

Abstract

The invention concerns an autologous bio-matrix derived from liver of porcine or human origin, which is able to constitute a functional bio-artificial support of hepatic cells homologous with its origin. In fact, the bio-matrix allows the anchoring of the hepatocytes, taken from porcine or human liver and cultivated in vitro, according to a very precise polar arrangement and lets the same arrange themselves in culture in a functional and specific way. This important biological property makes the bio-matrix particularly useful in the support of hepatic cells, also human, used in extracorporeal type apparatuses, such as the bio-artificial liver, or in the transplantation of hepatic cells, both of which may be used for the treatment of acute or chronic hepatic pathologies. The bio-matrix can also be used for experimental purposes for in vitro studies on the function of hepatocytes.

Description

AUTOLOGOUS BIO-MATRIX FROM MAMMALIAN LIVER AND METHOD FOR THE OBTAINMENT OF THE SAME.

FIELD OF THE INVENTION

The present invention concerns an autologous bio-matrix derived from liver of porcine or human origin, the method for its obtainment and its use as a bio- artificial support of hepatocytes of homologous origin in the treatment of acute or chronic hepatic pathologies. PRIOR ART It is known that the liver performs particularly important and sophisticated physiological functions of synthesis and detoxification and that the impairment of such functions account for the seriously disabling clinical evolution, when not fatal in the mid-short period from their onset, of both acute and chronic hepatic diseases of degenerative or metabolic nature. The complexity and centrality of the liver in the functions of synthesis are immediately evident if it is considered that this organ is involved in the following metabolic processes: the synthesis and excretion of bilirubin and porphyrins, production and excretion of bile and biliary acids, excretion of urobilinogen, detoxification of endogenous, such as hormones, and exogenous substances (chemicals or drugs), energy production by mitochondria through biological processes of oxidation, the synthesis, use and elimination of amino acids in the metabolism, the synthesis of urea and catabolism of ammonia, the synthesis and secretion of proteins, the metabolism of carbohydrates, the synthesis and degradation of lipids, the storage of substances (e.g. pigments, copper, carbohydrates, proteins - αι - antitrypsin and fibrinogen, triglycerides, cholesterol, phospholipids, amino acids, collagen, vitamin A and K). The varied etiology hepatitides, also in the form of cirrhotic evolution, both benign and malignant hepatic tumours, the metabolic diseases on a genetic basis and the hepatic diseases of infectious origin have, so far, been pathologies which, despite the progress in the field of therapeutic medicine, seldom find decisive therapies. In many cases it would in fact be necessary to be able to have therapeutic means, above all, which support the liver in its metabolic functions or that stimulate its functional recovery. For operations which are not of pharmaco-therapeutic type, research for the setting-up of extracorporeal apparatuses, the so-called bio- artificial liver, for the temporary substitution of the liver, affected by forms of fulminating hepatitides, in its vital functions, has been in progress for a long time, as well as research aimed at optimizing the transplantation technique of just hepatocytes, this technique also providing useful support both in the event of postoperative hepatic failure and genie corrections.

Artificial liver research, started some years ago, has led to very significant results during the last few years. In fact, it has seen the rapid passage from totally artificial type systems to biological type supports or, even better, bio-artificial ones. The difference between these two categories is substantial and must be stressed: the first kinds do not contain functioning biological units and, therefore, can only have a detoxicant action; instead the bioartificial systems carry out more complex functions than detoxification such as biosynthesis and biotransformation, since they contain a functional unit made up of animal cells. The first scientific news on the use of an artificial support system dates back to 1950, when haemodialysis originated. Since then increasingly sophisticated methods have been set-up: haemoperfusion, haemoadsorption, up to plasmapheresis, the latter method nowadays of widespread use in many hepatic diseases. However, being totally artificial, these systems, especially plasmapheresis, are able to perform, as already said, just one of the hepatic functions: that of detoxification. However, in reality the liver performs other complex actions as we have seen, such as those of synthesis. In the case in which a patient, for various reasons, is affected by acute hepatic failure, it is indeed these synthesis functions, undoubtedly associated with those of detoxification, which are seriously impaired. Then a bio-artificial liver, indeed capable of functioning as a bridge for the functional support of the insufficient organ, must be able to perform both these functions; a third can be added to this, i.e. that of a stimulus for the diseased liver so that it regenerates, i.e. recovers, its full functionality, in a self-sufficient manner. Since such cellular activities are still not reproducible in the laboratory, the idea was put forward of using animal hepatic cells instead of the artificial biological filters, therefore capable of performing all the required complex functions. This then opened up the era of the bio-artificial liver, as a useful support, in cases in which the liver, affected by disease, quickly becomes impaired in its primary functions. A bio-artificial liver, is therefore an extracorporeal system, consisting of a similar apparatus to that of renal dialysis but which, unlike this, has a so-called "bioreactor" heart inside it. The bioreactor, the functional unit of the instrument, is generally made up of pig's liver cells fixed on a glass fibre support. The hepatocytes, once inserted in a bioreactor, expressly built and connected to an apparatus for extracorporeal use, and kept vital inside the system by sophisticated culture techniques, by coming into contact with the plasma of the patient affected by fulminating hepatitis, carry out their synthesis and detoxification action, on one hand temporarily replacing the impaired liver and, on the other, helping it to autonomously recover and to function under critical conditions. The rapid passage of the plasma within the bioreactor does not, in any event, cause rejection reactions by the patient, something which instead would happen if a pig's liver were transplanted in man in toto. Also the grafting of hepatocytes, nowadays commonly called hepatocyte transplantation, is a method widely studied in experimental models to create an auxiliary liver if needed, both as hepatic support in fulminating hepatitides as well as potential treatment of metabolic deficiencies still of hepatic origin. Indeed, during the '30s Hans Popper carried out the first transplantations of liver fragments in the anterior chamber of the eye. Some studies were then carried out in the '60s, but it was only at the start of the 70s that, due to the experimental successes achieved with the transplantation of insulae in treating diabetes mellitus, the hepatocyte studies took shape. Nevertheless, numerous difficulties were met during these years and can be schematically summarized in problems connected with the choice of the cell type to be transplanted - human or animal hepatocyte - and the organ or tissue in which to graft the hepatocytes. To these then another fundamental problem is closely linked: how to prolong the functionality of the cell, once grafted, without it coming up against processes of dedifferentiation also in a neoplastic sense. As for the first problem, which is that of greater importance for the aims of the invention, the answer may appear straightforward and immediate: use human hepatocytes obtained from livers unfit for transplantation, so as to avoid all the problems connected with the animal cell and, not least, its xenogenicity. All that however, in practice, does not prove so simple, since the human hepatocyte is a cell which, when isolated, is very sensitive to the damage induced by the current isolation methods, and even more to the effects of the cryoconservation and defrosting procedures, as well as quite quickly coming up against dedifferentiation. This makes it particularly difficult to keep in culture for long periods without losing its functionality, essential, not so much for the detoxification processes for which it could be replaced by means, also completely artificial, but especially for its functions of synthesis. Instead the animal hepatocyte, that of pigs in particular, is endowed with a greater resistance compared to the human one, which obviously translates into a prolonged functional ability of this cell. The problem connected with its xenogenicity remains and, secondly, that linked to the presence of retrovirus. However the passage from experimental studies to controlled clinical applications has already occurred. In 1992 in Japan, Mito presented a series of 10 cases in which the hepatocytes were isolated from autologous hepatic segments obtained with extensive resections. Further studies were then carried out in America, with moderate success. However, despite all this, the problems connected with hepatocyte use in the bio-artificial liver, or in the transplantation have still not been resolved and such use is limited by the support methods of the same cell. In fact, the maintenance of the functionality of the hepatic cell, once isolated from the liver, both in an artificial as well as biological system is made all but impossible for prolonged periods, because of the hepatocyte's rapid loss of its more sophisticated synthesis functions and a progressive morphological and structural alteration. This is because the isolated hepatocyte does not find, in the standard culture systems, a similar support to the one existing in the liver in toto.

The cellular orientation and, therefore, polarity conservation, are essential factors in the obtainment of those best favourable culture conditions, able to support the cellular vitality and functionality for prolonged periods, also under clinical conditions. Many studies have been carried out by various groups of researchers in order to find supports capable of prolonging the vitality and functionality of the hepatic cell. Microencapsulations in alginate, factors stimulating regeneration, collagene, synthetic matrices, hormones: all these methods did not give satisfactory results. The anchorage of the hepatocytes (generically of pig origin) to the support systems prepared so far, being totally synthetic, do not allow appropriate orientation of the cells; for this reason the hepatocytes quickly lose their biosynthesis properties under critical conditions. Therefore only temporary (up to a maximum of 6 hours) and extracorporeal treatments are possible in which only detoxification and mild synthesis action is eminently conducted, i.e. incapable of fully replacing the insufficient functions of the pathologic liver. Totally implantable systems, on the other hand, do not exist. The transplantation of human hepatocytes is carried out through the standard isolation of the cells and their insertion into the spleen or portal vein.

A support which is capable of keeping the hepatocytes alive and functioning once implanted is not found on the market. These spleen or portal vein grafts have shown positive activity in the first 20 days after grafting, but then quickly lose their functions or, even become detached from the implant site to then disperse in the blood system.

The standard culture media leave the hepatocytes free and without orientation. Therefore, despite the nutritional contribution offered by these, the hepatocyte quickly loses its functions. Moreover, the cells of human origin come up against harmful processes of dedifferentiation. SUMMARY OF THE INVENTION

In order to overcome the above described problems, concerning the survival and functionality of the isolated hepatocyte as a biological unit of detoxification and synthesis in extracorporeal bio-artificial liver bioreactors and in transplantations of hepatocytes -implantable biological liver-, the applicant has now surprisingly found that a matrix consisting of an autologous bio-matrix derived from liver of porcine or human origin can be effectively used as a bio-artificial support of hepatocytes of autologous origin. Therefore the object of the present invention is the autologous bio-matrix originating from suitable mammalian livers, including human ones unfit for transplantation, usable as bio-artificial support of hepatocytes homologous with its origin and particularly of human or porcine origin. Additional objects of the invention are the method for the preparation of the same and its use to support the hepatocytes of homologous origin in bioreactors for extracorporeal bio-artificial liver, and in the hepatocyte transplantations in the implantable biological liver both for the treatment of acute or chronic hepatic pathologies. Furthermore, the bio-matrix is able to be used for experimental purposes for in vitro studies on the function of the hepatocytes. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1a and 1 are photographs of the macroscopic aspect of the autologous bio-matrix. Figure 2a and 2b are photographs of the microscopic aspect of the autologous bio-matrix.

Figure 3 is a photograph of the electronic aspect of the autologous bio-matrix with hepatocytes. DETAILED DESCRIPTION In the field of biotechnologies applied to medicine, one of the parts presently of major interest undoubtedly concerns artificial organs and particularly the bioartificial liver which has already been widely discussed. However, the definition of "artificial organ" is rather generic, since a real organ which is wholly implantable does not exist at present (even if one has witnessed enormous progress especially on the heart). In reality, these apparatuses recreate "artificial organ functions" since they are nothing but temporary supports of the functionality of the organ itself.

In the case of the bio-artificial liver, there is, on the other hand, the need and real difficulty of replacing or combining complex synthesis functions, which only the hepatic cell under appropriate conditions (such as polarity) is capable of facing. It is therefore clear how important it is to be able to have in vitro hepatocytes in culture kept in the most physiological way possible, and for more prolonged periods of time. The instruments nowadays available (solutions, culture media, enzymes, extracts of extracellular matrix), are all artificial or, in any case, products derived from sophisticated isolation techniques that lead to a loss of the starting biological properties of these means. The autologous bio-matrix derived from mammalian liver and particularly of porcine or human origin, object of the present invention, is instead able to advantageously constitute a support, biologically and above all functionally compatible with hepatocytes isolated in vitro of homologous origin, maintaining its structural configuration. In fact, the bio-matrix allows the anchoring of the hepatocytes, taken from porcine or human liver and cultivated in vitro, according to a very precise polar arrangement and lets the same arrange themselves in culture in a functional and specific way. In fact, on this point, it has to be pointed out that the bio-matrix obtained with this method is in actual fact the "framework" indeed of the hepatic parenchyma, isolated using a low toxicity method and able to restore the physiological conditions indispensable to the hepatic cells.

The hepatocytes isolated in vitro and cultivated on the bio-matrix preserve their characteristics and functions for a long time, allowing the applications as already mentioned previously. In order to obtain the biological support of porcine origin (Porcine Liver Autologous Bio-Matrix - PLABM) or human origin (HLABM), object of the invention, able to fix the hepatic cells according to polarity, the applicant has prepared an original enzymatic method and has shown that the hepatocytes thus supported can perform their functions in vitro for extended times also under critical conditions, both if that is when used in extracorporeal systems, such as the bio-artificial liver, and in totally implantable ones, such as the implantable biological liver.

The method for the preparation of the bio-matrix is hereafter described in detail for illustrative and non-limiting purposes, of the present invention, and modifications arising from the standard and present knowledge of the state of the art and from its natural advancement in the field will be able to be made without leading away from the nature and scope of the present invention. Preparation method of the autologous bio-matrix

The preparation method of the autologous bio-matrix is identical for both cases in which it is prepared from pig's liver as well as human liver. The liver, once withdrawn in toto, is placed in a freezer at -20°C. The liver is brought to room temperature one hour before the bio-matrix preparation procedure and sections of 500-1000 micron thickness, cut manually or with a cold blade microtome, are prepared from the withdrawn liver. The sections thus obtained, are treated for 2 hours in distilled water under agitation, changing the distilled water every 30 minutes. The sections are then immersed in an aqueous solution at a concentration of 4% sodium desoxycholate and kept at rest for 2 hours. After such time, the sections are treated with Type I Deoxyribonuclease (DNase) in salt solution (e.g. 2000 Kunitz Units of a solution with 1 M of Sodium Chloride) under agitation. This procedure can be repeated until the macroscopic and microscopic disappearance of cellular traces of hepatocytes. The autologous bio-matrix obtained with the method described is depicted in Figures 1 and 2. From the point of view of its use it should again be mentioned that the bio-matrix must not necessarily be prepared at the moment of its use, but that this bio-matrix can be, once prepared, preserved both in its pure or lyophilized form under appropriate conditions of temperature and sterility. The lyophilized form simply requires rehydration with physiological solutions. In this step the bio-matrix keeps its structural characteristics and biological properties essential for a functional support to hepatocytes completely intact. Similar matrix preparation methods have already been described for the preparation of matrices from other organs, namely bladder and stomach, but not in the case of liver presumably due to this organ's functional and structural peculiarities. In fact, to obtain the bio-matrix at issue and above all to obtain a bio- matrix suitable for the purposes of the present invention, certain technical devices are necessary which the applicant has found as a result of its research in the field, and particularly such devices are necessary to obtain a bio-matrix with the characteristics fit for the purpose, i.e. elasticity and absence of traces of hepatocytes. Such substantial modifications lie in the not obvious fact that the withdrawn liver is frozen to -20°C, with its haematic content, without further treatment. In other words, the withdrawn liver must not be perfused at the moment of its withdrawal from the animal or human, this instead being an essential step for the obtainment of cells or matrices coming from other organs. The preparation of the sections is also an absolutely critical point, since the organ sections must be between 500 and 1000 micron thickness to eliminate the hepatocytic cellular part. It is also found that the washing step in distilled water is of paramount importance for the elimination of hepatocytes. In fact, hepatocyte elimination must take place mostly under agitation in distilled water and not in DNase, to avoid toxic effects of the latter (on the contrary treatment with DNase is instead always required for other organs). In order to examine the actual capacity of the bio-matrix, object of the invention, to function as a support of the hepatocytes isolated according to the technique described, both when used in bioreactors for extracorporeal bio-artificial liver, and when used in the transplantations of hepatocytes in the implantable biological liver for the treatment of acute or chronic hepatic pathologies, tests were carried out, hereafter reported, using isolated hepatocytes supported on the bio-matrix in experimental models of acute hepatic failure. Experimental methods and results A- Method of hepatocyte isolation Known techniques were used for both human and porcine hepatocyte isolation that envisaged, after liver withdrawal, a subsequent series of perfusions, the latter of which with a solution added with type IV° Collagenase at a concentration of 0.05% (w/v) and 5 mmol of calcium chloride at a temperature of 37°C at a flow of 50ml/min. The liver thus perfused was shred for 4 min. and the product obtained was decanted in an erlenmeyer flask and maintained under agitation for 10 min. in a bath at 37°C always under oxygenation. The suspension was filtered twice, first using the 210 micrometre mesh filters and afterwards with the 65 micrometre ones. Thus one proceeded with successive washings with cold buffer at 4°C containing 0.2% V° fraction bovine albumin and 10% foetal bovine serum before purification of the hepatocytes by centrifugation gradient with a 1065g/ml Percoll solution, at a gravity of 76g for 5 min. The resultant pellet of cells was washed twice in a buffer solution and resuspended in DMEM added with 0.2% bovine albumin. The cells were thus counted using a selective dye such as Tripan Bleu. By means of this hepatocyte separation procedure the maximum number of cells obtained was of the order of 10 million per gram of tissue, corresponding to a final product of 2 billion cells with 90% vitality. Cellular vitality and functional activity The cellular vitality was tested by the Tripan Bleu test and with staining with MTT. The functionality of the hepatocytes in the culture medium was tested with laboratory trials: the secreted albumin of the hepatocytes was measured immunometrically; the P450 cytochrome was assessed using molecular biology techniques; the glycogen synthetic activity was histochemically tested. B- Experimental models of acute hepatic failure

Induction of acute hepatic failure, through the administration of exogenous substances such as galactosamine and carbon tetrachloride, although much used, are not particularly suitable in the case in which pigs are used as the experimental animal owing to uncontrollable variables, such as the dose-weight ratio, pretreatment of animals and the metabolism capacity of the drug typical of each animal.

The surgical model was chosen on account of these reasons of experimental nature, despite it proving more complex from a technical point of view and having the disadvantage of being unreversable. The surgical model is in fact always perfectly reproducible and not tied to any factor (weight, type of anaesthesia, age, etc.). Animal mortality always occurs at the same time period, around the 18^th hour in pigs, but this technique allows the study of hepatocyte synthesis activity both in the extracorporeal systems as well as in the transplantation of the same. Experimental methods Female pigs weighing 30 Kg, without food from the previous day but with water ad libitum were used. Surgery was carried out under general anaesthesia by means of oro-tracheal intubation. Surgical technique Stage One: Median incision. Isolation of the portal vein and the vena cava. Execution of portacaval, termino-lateral (portacaval shunt) anastomosis. The pig was closed and stabled for 7 days. A liver biopsy was performed at this first stage. Stage Two: Under general anesthesia the abdomen was reopened following the line of the previous laparotomic incision and the liver was totally devascularized. During this stage it was possible to choose the anhepatic experimental model, performing a total hepatectomy with the restoration of continuity by means of caval-caval anastomosis with PTF prosthesis, or the experimental model with liver in situ, leaving just the liver in situ after devascularization. The biological activity of the hepatocytes isolated as previously described supported on the autologous bio-matrix was tested using the serum of pigs kept anhepatic for 12 hours. The cellular vitality and functionality were measured by: laboratory tests (AST, ALT; Urea, Albumin, PT, Glycemia, Bilirubin); Glycogenic load; MTT. In vitro results

The pig serum kept anhepatic for 12 hours showed a reduction in all the values studied, except that of bilirubin which instead increased. After 30 days of culture of this serum with hepatocytes sown on autologous bio-matrix, albumin and urea were still present; transaminase was steady but showing high values, bilirubin decreased and PT showed a distinct increase (Tab. 1 ); the glycogenic load of the hepatocytes was high and MTT staining was positive. TAB. 1:

TIMES 0 1d. 2d. 15d. 30d.

Albumin mg % 1.1 2.5 2.9 3.1 1.7

Transaminase 700 300 500 500 600

PT 10 70 75 70 60

Bilirubin 5 1.5 1.7 2 2.5

Urea 0.5 0.5 0.61 0.8 0.8 T Thhee vvaalluueess aarree eex xpprreessssed as tl

In vivo results

20 pigs, 10 of which treated and 10 control animals, were used for the experiment. All the pigs were made anhepatic, as described, and connected through the femoral vein and artery to an extracorporeal apparatus thermostatically set at 37°C, consisting of an apparatus for plasmaseparation, an oxygenator and a peristaltic pump, with appended a bioreactor. The bioreactor used was a module consisting of a chamber for cellular cultures, containing, only in the case of treated animals, some plates with hepatocytes sown on autologous bio-matrix. From the plasmapheresis apparatus, the oxygenated plasma was propelled by the peristaltic pump towards the bioreactor. The plasma of the anhepatic treated animal came into contact with the hepatocytes in the bioreactor through a fixed porosity membrane. The plasma thus treated was reinfused into the animal after recovery with its corpuscular part by the same apparatus for plasmapheresis. The treatment was extended for 12 hours.

The parameters measured were: AST, ALT, Glycemia, PT, MEGx (Lidocaine) at to, at 3, 6, 9, 12 hours of treatment. The hepatocytes and the autologous bio-matrix were controlled by optical and electronic microscopy (Figure 3) at the end of treatment. In addition, MTT and the glycogenic load were assessed. The control group, in which the pig was kept anahepatic for 12 hours without treatment with hepatocyted supported on autologous bio-matrix showed: a significant drop in glycemia, an absence of metabolization of lidocaine, a progressive decrease in transaminase until its disappearance, a significant reduction in PT (Tab. 2).

TAB.2 (Group 1)

Times 3h 6h 9h 12h

MEGx 5 2 1 1

Glyc. 100 79 40 20

PT 100 60 45 20

Trans. 40 30 30 5

Instead, in the treated group 2, was highlighted a constant and progressive presence of MEGx, the absence of hypoglycemia and constant values of PT (Tab. 3). Instead transaminase was of little significance.

TAB.3 (Group 2)

Times 3h 6h 9h 12h

MEGx 20 27.4 25 24

Glyc. 70 60 55 50

PT 60 50 50 43.9

Trans. 30 10 160 120

Upon treatment termination, the microscopic examination of the hepatocytes sown on bio-matrix (Group 2) showed a high glycogenic load and a positive MTT, a positive P-450 cytochrome, normal morphology and 85% vitality. The experimental results obtained show that the presence of the autologous bio- matrix undoubtedly gives a functional biological support to the hepatocytes that allows these cells to remain functionally vital and efficient, both concerning detoxification as well as synthesis for a long period. This is because the cell rearranges itself within the same according to a particular polarity and orientation. The latter two are conclusive factors for the purpose of cellular activity, above all for long periods. In fact, the in vitro results obtained show that the hepatocytes sown on autologous bio-matrix are able to perform biosynthetic activities up to 30 days of culture with just 20% mortality. This important biological property makes the bio-matrix particularly useful in the support of hepatic cells, also human, used in extracorporeal type apparatuses, such as the bio-artificial liver, or in the transplantation of hepatic cells in the implantable biological liver. In addition, the bio-matrix can be used for experimental purposes for in vitro studies on the function of the hepatocytes, a function at present still largely unknown in its basic mechanisms.

The method for the obtainment of the bio-matrix, though requiring sufficient manual ability and expertise, proves industrial applicability since particular treatment steps which are difficult to transfer to the industrial field are unnecessary. In addition, the possibility of lyophilizing the autologous bio-matrix gives a further advantage to the purposes of industrial applicability, not posing problems concerning the conservation and use of third parties in the clinical or experimental field.

Claims

1. Autologous bio-matrix prepared from mammalian hepatic tissue usable as a bio- artificial support of autologous hepatocytes;

2. Autologous bio-matrix according to claim 1 of human origin;

3. Autologous bio-matrix according to claim 1 of porcine origin;

4. Autologous bio-matrix according to claim 1 usable as a bio-artificial support of hepatocytes in bioreactors in the bio-artificial liver;

5. Autologous bio-matrix according to claim 1 usable as a bio-artificial support of hepatocytes in the implantable biological liver;

6. Autologous bio-matrix according to claim 1 usable as a bio-artificial support of hepatocytes in the transplantation or grafting of hepatic cells;

7. Autologous bio-matrix according to claim 1 usable as a bio-artificial support of in vitro hepatocytes cultures for experimental purposes;

8. Autologous bio-matrix according to claim 1 obtained with a process characterized in that it includes the following steps:

- freezing of the hepatic tissue to -20°C

- preparation of sections of between 500-1000 microns thickness

- washings up to 2 hours in distilled water under agitation

- treatment with an aqueous solution of 4% sodium desoxycholate - treatment with DNAse in salt solution under agitation until the disappearance of hepatocytes;

9. Use of the autologous bio-matrix according to claim 1 as a bio-artificial support of autologous hepatocytes;

10. Use according to claim 9 in bioreactors in the bio-artificial liver for the treatment of acute or chronic hepatic pathologies;

11. Use according to claim 9 in the implantable biological liver for the treatment of acute or chronic hepatic pathologies;

12. Use according to claim 9 in the transplantation or grafting of hepatocytes for the treatment of acute or chronic hepatic pathologies and in genie therapy; 13. Use according to claim 9 of in vitro hepatocytes in cultures for experimental purposes; 14. Process to obtain an autologous bio-matrix according to claim 1 characterized in that it includes the following steps:

- freezing of the hepatic tissue to -20°C

- preparation of sections of between 500-1000 microns thickness

- washings up to 2 hours in distilled water under agitation

- treatment with an aqueous solution of 4% sodium desoxycholate

- treatment with DNase in salt solution under agitation until the disappearance of hepatocytes.