RU2797291C2 - Method of normalizing biochemical blood parameters in patients with irreversible liver failure - Google Patents

Abstract

FIELD: medicine, hepatology.

SUBSTANCE: invention can be used to normalize the biochemical parameters of the blood of patients with irreversible liver failure in the experiment. Blood hemoperfusion is carried out into the extracorporeal circuit, filtration with separation of blood cells from plasma, blood plasma perfusion through hepatocyte cell cultures, mixing of the treated plasma with blood cells and its return. As a plasma filter, a device of a flow-through incubator of a biological circuit with a modular design is used, a separate module of which is made of a biocompatible polymer and consists of layer-by-layer superimposed elements — a cover, a cavity for plasma transport with a spiral arrangement of channels and outlet openings for the input and output of plasma, a cavity for populating lines hepatocytes, repeating the course of the channels of the overlying layer, blocked by a porous-permeable track membrane with a pore diameter of not more than 5.0 μm, cavities for the filtrate containing a nutrient medium for cells, with outlet openings for the input and output of liquids. A track membrane with a pore diameter of at least 0.2 μm is placed between the cavity for the colonization of hepatocyte lines and the cavity for the filtrate. In the biological circuit, made up of flow modules with hepatocytes connected in series and sealed in a sealed case, the modules are connected in series to each other by means of outlet tubes and then to external means of plasma supply and bile outflow. Then the incoming blood plasma is perfused.

EFFECT: method makes it possible to achieve normalization of blood biochemical parameters in patients with irreversible liver failure, for example, while waiting for donor organ transplantation by creating an extracorporeal hemoperfusion system based on a flow incubator with functional human hepatocytes.

1 cl, 6 dwg, 3 tbl

Description

The invention relates to clinical medicine, namely hepatology, and is intended to compensate for the functions of the liver of patients with irreversible liver failure, for example, while waiting for donor organ transplantation.

The liver is a vital organ with many functions, and the only option for patients with irreversible and serious organ pathologies is liver transplantation. Despite the increase in the number of liver transplants, there is still a problem of providing this operation for those in need due to the lack of donor organs and living donors. In this regard, methods and devices for extracorporeal liver support during the transplant waiting period are of great practical importance.

Artificial systems MARS and Prometheus are known, which are cell-free extracorporeal devices that are aimed only at removing toxins by dialysis of albumin (see Evenepoel P. et al. Detoxifying capacity and kinetics of Prometheus®–a new extracorporeal system for the treatment of liver failure / Evenepoel P. et al Prometheus versus molecular adsorbents recirculating system: comparison of efficiency in two different liver detoxification devices //Artificial organs - 2006. - T. 30. - No. 4. - P. 276-284).

The data do not have the synthetic and metabolic functions, such as albumin synthesis, that can only be provided by living functional liver cells.

There are a number of bioartificial extracorporeal systems such as ELAD, HepatAssist, AMC-BAL, SRBAL, hiHep-BAL, the peculiarity of which is that the functions of the liver are provided by the cells that are part of the system, and the difference is in the type of cells populated in the system. For example, hiHep-BAL is based on induced human hepatocytes (see Shi X. L. et al. Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes // Cell research. - 2016. - T. 26. - No. 2. - P. 206-216), HepatAssist and SRBAL are based on porcine hepatocytes (see Demetriou A. A. et al. Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure // Annals of surgery. - 2004. - T. 239. - No. 5. - P. 660; Nicolas C. T. et al. Concise review: liver regenerative medicine: from hepatocyte transplantation to bioartificial livers and bioengineered grafts // Stem Cells. - 2017. - T. 35. - No. 1. - P. 42-50).

Despite the presence of certain hepatocytes in the above systems, each system has its own drawbacks. For example, ELAD contains HepG2/C3A human hepatocarcinoma cells, which show reduced functional activity compared to native human hepatocytes, cannot excrete ammonia, and there is also a risk of tumor cells entering the patient's bloodstream (see Shi X. L. et al. Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes // Cell research, 2016, vol. 26, no. 2, pp. 206-216; Lee K. C. L., Stadlbauer V., Jalan R. Extracorporeal liver support devices for listed patients //Liver Transplantation. - 2016. - T. 22. - No. 6. - P. 839-848). HepatAssist and SRBAL are disadvantageous due to the risk of xenozoonosis because they contain porcine hepatocytes and are severely restricted in some religious countries (see Shi X. L. et al. Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes // Cell research, 2016, vol. 26, no. 2, pp. 206-216). AMC-BAL was developed on the basis of porcine hepatocytes, however, in the first clinical trial, porcine endogenous retrovirus DNA was found in one of the patients, which is why testing of this device was limited (see Lee K. C. L., Stadlbauer V., Jalan R. Extracorporeal liver support devices for listed patients //Liver Transplantation, 2016, vol.22, no.6, pp. 839-848; Surgery. - 2005. - V. 22. - No. 4. - P. 254-264). As for hiHep-BAL, this device is safe in terms of tumor cell transmigration and xenozoonosis, but it has not yet been tested in humans (see Shi X. L. et al. Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes // Cell research. - 2016. - V. 26. - No. 2. - P. 206-216).

Thus, bioartificial systems are considered the most effective, in which, unlike artificial ones, the main component is a bioreactor containing functional liver cells - hepatocytes. The latter, during therapy, provide the patient's body not only with detoxification (which only artificial devices for extracorporeal liver support can provide), but also with synthetic, metabolic functions of the liver. In addition to supporting the function while waiting for a donor organ, such systems can also induce spontaneous liver regeneration. Studies are known from the literature that prove the beneficial effect of extracorporeal systems on the processes of liver regeneration in young patients with a milder course of liver diseases (see Thompson J. et al. Extracorporeal cellular therapy (ELAD) in severe alcoholic hepatitis: a multinational, prospective, controlled, randomized trial // Liver Transplantation. - 2018. - V. 24. - No. 3. - P. 380-393).

Apparatus for biological artificial liver (see RU No. 2163148, class A61M 1/36, publ. 20.02.2001) contains a body containing tissue fragments of the liver, filled with a homogeneous mixture of particles of a neutral carrier, namely agarose gel. Plasma processing occurs as follows: Heparinized blood or plasma of the patient enters the system using a peristaltic pump, passes through a bioreactor that contains liver fragments, and then returns to the patient's bloodstream.

The disadvantage of the known apparatus is the need for a large amount of biomaterial, the amount of which is limited in practice. In addition, there is a high risk of damage to the integrity of the cell membrane of hepatocytes during fragmentation of the liver tissue.

A known method of hemocorrection in the treatment of liver failure (see RU No. 2693677, class A61M 1/16, publ. hepatocytes, mixing the treated plasma with blood cells, and returning the blood to the patient. At the same time, differentiable stem cells are autocells that were obtained by reprogramming peripheral blood mononuclear cells.

The disadvantage of the known technical solution is the duration of the processes of obtaining, reprogramming, culturing and directed differentiation of stem autocells, in addition, the implementation of the processes is time-consuming and resource-intensive.

The problem to be solved by the claimed invention is to achieve the normalization of biochemical parameters of the blood of patients with irreversible liver failure during the period of waiting for transplantation of a donor organ.

The technical result obtained in solving the problem is expressed in achieving the normalization of biochemical parameters of the blood of patients with irreversible liver failure, for example, during the period of waiting for transplantation of a donor organ, by creating an extracorporeal hemoperfusion system based on a flow incubator with functional human hepatocytes.

To achieve this task, a method for normalizing blood biochemical parameters in patients with irreversible liver failure in the experiment, including hemoperfusion of blood into an extracorporeal circuit, filtration with separation of blood cells from plasma, perfusion of blood plasma through cultures of hepatocyte cells, mixing of the treated plasma with blood cells and its return, differs in that as a plasma filter, a device of a flow-through incubator of a biological circuit with a modular design is used, a separate module of which is made of a biocompatible polymer and consists of layer-by-layer superimposed elements - a cover, a cavity for plasma transport with a spiral arrangement of channels and outlet holes for input and output plasma, cavities for colonization of hepatocyte lines, repeating the course of the channels of the overlying layer, blocked by a porous-permeable track membrane with pore sizes sufficient to prevent cells from entering the circulation, for example, no more than 5.0 μm, filtrate cavities containing a nutrient medium for cells , with outlet holes for input and output of liquids, and, between the cavity of the colonization of hepatocyte lines and the cavity for the filtrate, there is a track membrane with pore sizes sufficient to prevent cells from entering the circulation, for example, at least 0.2 μm, for which the biological circuit, made up of flow modules with hepatocytes connected in series and sealed in a sealed case, are connected in series to each other by means of outlet tubes and then to external means of plasma supply and bile outflow, after which the incoming blood plasma is perfused.

Comparative analysis of the features of the claimed solution with known features indicates the compliance of the claimed solution with the criterion of "novelty".

The set of features of the invention provides a solution to the claimed technical problem through an extracorporeal support system, represented by a network of semipermeable membranes with functional human hepatocytes for subsequent use in extracorporeal hemoperfusion systems as maintenance therapy for the liver of patients with various irreversible liver pathologies with partially or completely lost functions, providing the body with detoxification , metabolic and synthetic functions, for example, while waiting for a donor organ.

The advantages of the claimed solution are: no risk of xenozoonosis, since human cells are used; compensation not only for detoxification, but also for metabolic, synthetic functions; the use of a biological circuit that facilitates the rapid production and cultivation of cells; increase in the regenerative potential of the liver.

The new technical solution uses a flow-through incubator device, which is a structural and functional unit of a biological circuit, where, as a result of hemofiltration through a special network of semi-permeable membranes placed in a medium with cultured hepatocytes, exchange processes occur between the cells, the cultured medium and the patient's plasma.

Thus, the biological circuit is intended for further use in a hemoperfusion blood purification system as maintenance therapy for patients with severe forms of liver failure.

The claimed method is illustrated by a drawing, where the figure 1 shows the frame elements of the flow-through incubator: cover (1), cell placement layer (2), plasma flow layer (plasma cavity) (3), reservoir (filtrate or dialysate cavity) ( 4); figure 2 - assembly diagram of the frame of the flow-through incubator for the biological circuit; figure 3 - layout of the porous-permeable membrane (5) between the layers of plasma flow and cultured cells; figure 4 is a diagram of the design of a flow-through incubator with a semi-permeable membrane (6) and hepatocytes (7); figure 5 - photo of the cell placement layer of the frame of the flow-through incubator in the culture dish; figure 6 shows a diagram of a biological circuit connected to external systems for the supply and removal of substances (10 - the direction of circulation of the blood plasma flow, 11 - the direction of circulation of the bile flow), including sequentially located flow incubators (9) in a sealed housing (8).

A biocompatible polymer is used to make the frame of the flow-through incubator. The frame consists of four layered elements: a cover 1, a cavity 3 for plasma flow with a spiral arrangement of channels, a cavity 2 for populating hepatocyte lines, repeating the course of the channels of the overlying layer 3, a reservoir 4 for the culture medium (see Fig. 1, 2). Since hepatocytes 7 must simultaneously contact with the patient's plasma and culture medium, and the mixing of two liquids is unacceptable, the cells are located in the middle, and track membranes are applied above and below. Thus, between cavity 3 for plasma and cavity 2 for hepatocytes 7, a porous-permeable membrane 5 is placed, and between cavity 2 for hepatocytes 7 and reservoir 4, a semi-permeable membrane 6 is placed (see Fig. 3, 4).

In the culture medium tank 4, toxins and metabolic products will be removed, therefore, separate cavities and tubes are provided for the blood plasma and culture medium for the flow of these fluids in order to prevent them from mixing.

For the prototype, the diameter of the flow-through incubator structure was 84 mm, while the layered elements were made of a synthetic polylactide polymer by 3D printing (see Figs. 1, 5). The choice of polymer material was determined by the following properties: biocompatibility, which is necessary for working with patients; low degree of bioresorption; thermoplasticity; availability of the material (see Belov D. Biodegradable polymer polylactide // Science and innovations. - 2013. - V. 9. - No. 127; Levchenko E.V., Chernysheva N.L. Production of biodegradable polymer polylactide // Bulletin of youth science - 2016. - No. 4 (6)).

As porous-permeable and semi-permeable membranes, Reatrek track membranes of a well-known design made of polyethylene terephthalate with pore diameters of 5.0 and 0.2 μm, respectively, were chosen. The advantage of track membranes is their correct pore geometry, the ability to control their number, as well as uniformity in pore size, which does not allow the risk of penetration of both cell units and their components in case of destruction (see Volkov V. V. et al. Membranes and Nanotechnologies // Russian Nanotechnologies - 2008. - V. 3. - No. 11-12. - P. 67-99).

The pore diameter of the track membrane separating hepatocytes from plasma, equal to 5 μm, was chosen to avoid the risk of cells entering the patient's bloodstream, since the average diameter of hepatocytes varies from 20 to 30 μm.

To form a biological circuit, composite cartridges are pre-populated with human hepatocytes, for which hepatocytes are seeded in cavity 2, the bottom of which is made in the form of a semi-permeable track membrane 6. Further, layer 2 with track membrane 6, on which cells are adhered, is transferred to the reservoir 4 of the frame , intended for the culture medium. Then, a porous-permeable membrane 5 is applied over the hepatocytes to prevent hepatocytes from entering the patient's bloodstream. After that, the remaining layers are applied: cavity 3 for plasma flow through a network of branched channels and cover 1. All layers of the cartridge are placed relative to each other in a "sandwich" manner (see Fig. 2-4).

Flow incubators 9 with hepatocytes are connected in series with medical tubes and sealed in a sealed case 8. Thus, the manufactured biological circuit is delivered to a specialized medical institution, where it is connected for use by means of output tubes to external means of supply and outflow of substances without the formation of air spaces, for example, to an infusion pump that supplies 10 blood to the biological circuit, and through another line provides an outflow 11 of bile and used culture medium (see Fig. 6).

The claimed technical solution is implemented as follows.

At the beginning of therapy, the patient's blood is removed from the vein through a double-lumen catheter using a pumping system, then it enters the plasma filter, where the blood plasma is separated from the blood cells through the membrane. For example, in well-known studies, blood for such purposes was taken from a patient from the right superficial femoral vein at a rate of 90-100 ml / min (see Chen S. C. et al. Treatment of severe liver failure with a bioartificial liver // Annals of the New York Academy of Sciences. - 1997. - T. 831. - P. 350).

After that, the resulting plasma through a separate channel enters the biological circuit, which by this time should contain at least 10 million hepatocyte cells, and circulates in the capillary system, interacting with hepatocytes, which provide a biological effect - the synthesis of substances, the binding of toxins, in particular bilirubin (see Malhi, H. Hepatocyte transplantation: new horizons and challenges / H. Malhf, S. Gupta // J. Hepatobiliary Pancreat. Surg. - 2001. - Vol. 8. - P. 40-50; Fitzpatrick, E Nahmias Y, Berthiaume F, Yarmush, Human hepatocyte transplantation: state of the art / E. Fitzpatrick, R. R. Mitry, A. Dhawan // J. Intern. Med. - 2009. - Vol. 266. - P. 339-357 ML Integration of technologies for hepatic tissue engineering Adv Biochem Eng Biotechnol 2007 103: 309–329 Dolgikh M S Modern technologies for creating an implantable bioartificial liver / M S Dolgikh // Biomedical Chemistry 2010 - v. 56 - issue 4 - pp. 425-442; Afanas'eva YI Histology, embryology, cytology. Textbook / Yu.I. Afanasiev, N.A. Yurina // GEOTAR Media. – 2018. – p. 595; Ponomarev B.L., Morphology of human hepatocytes in the early periods of ontogenesis / B.L. Ponomarev, L.E. Obukhova, Yu.A. Vysotsky, N.I. Barsukova, T.M. Cherdantsev // Russian Medical and Biological Bulletin. acad. I.P. Pavlova. 2013. No. 3).

Metabolic products and toxins of hepatocytes are excreted into the culture medium, which flows out with the help of a pump. The optimal flow rate of fresh medium should be from 1.2 to 2.0 ml/h (see Basok Yu.B. et al. Cultivation of human liver cells and mesenchymal stromal cells of human adipose tissue in a perfusion bioreactor // Bulletin of Transplantology and Artificial Organs Mueller D. et al. Real-time in situ viability assessment in a 3D bioreactor with liver cells using resazurin assay //Cytotechnology.-2013 . - T. 65. - No. 2. - S. 297-305).

Since hepatocytes consume a large amount of oxygen, it is allowed to use an oxygenator to enrich the culture medium with oxygen. Reactor stability at a calculated oxygenator concentration of at least 0.3 nmol/sec per 10 million cells.

At the end, the processed blood plasma is combined with the erythrocyte mass, after which the blood is returned to the patient, and the extracorporeal hemoperfusion procedure is completed, the device is disconnected from the bloodstream.

For experimental work, blood plasma of ten apparently healthy volunteers was used. In order to comply with safety measures, volunteers additionally donated blood for the determination of HIV, hepatitis B and C. The experiment was carried out under aseptic conditions in compliance with biological safety requirements.

The study of the biochemical spectrum of the studied plasma was carried out using an automatic biochemical analyzer Horiba ABX Pentra 400, Horiba ABIX SAS, France, BP 7290-34184 (FSZ 2010/07857), OKP 94 4300 using colorimetric, photometric technologies to determine the concentration of individual chemicals and products of exchange. The levels of the following biochemical indicators were determined: albumin, aspartate aminotransferase, alanine aminotransferase, sodium, total and unbound bilirubin.

Plasma of apparently healthy donors, prepared in a volume of about 50 ml by centrifugation, was transported through the plasma filter channel through the plasma flow layer of a prefabricated cartridge with populated hepatocytes on the surface of the filtration membrane of the cell accommodation layer (incubation layer), which is simultaneously the upper wall of the dialysis solution cavity with nutrient medium. At the same time, within the framework of the experiment, on one cartridge, the plasma was delayed for an average of 30 minutes and was removed through the outlet of the plasma flow cavity. The extracted plasma was examined for changes in the concentration of substances that directly or indirectly reflect the functions of the liver.

At the output, a significant increase in the levels of aspartate aminotransferase, sodium and a moderate increase in the level of albumin, as well as an obvious decrease in the concentration of unbound bilirubins, was observed in all samples (see tables 1-3).

An increase in the level of albumin indicates the presence of a synthetic process in hepatocytes and their functionality. The increase in aminotransferases is explained by the destruction of cells in the presence of a polymeric framework in one medium. The presence of indirect bile-forming signs associated with the metabolism of direct bilirubin can be judged from a decrease in the level of bilirubin after treatment and an increase in the concentration of sodium ions, which are an integral part of bile acids.

Thus, it has been established that the claimed system of extracorporeal liver support is able to provide a supportive function in liver failure, compensate for the violation of the detoxification, metabolic and synthetic functions of the liver, and therefore increase the survival of patients who are waiting for a donor liver, reduce the likelihood of complications, as well as spontaneous liver regeneration patients with a milder course of liver diseases, which will allow targeting the pool of donor organs for patients with more severe stages of the disease.

Table 1

Results of biochemical analysis of blood plasma after plasma treatment in a flow system

sample number Albumin (g/l) Alanine aminotransferase (U/l) Aspartate aminotransferase (U/l) Control After 30 minutes After 1 hour Control After 30 minutes After 1 hour Control After 30 minutes After 1 hour 1.1 45.7 32.6 33.1 40.8 29.2 31.0 24.7 36.3 45.8 1.2 34.4 32.8 31.0 30.2 26.0 31.0 1.3 32.1 31.6 28.2 27.1 25.5 34.2 1.4 36.9 35.6 31.8 32.8 25.5 32.1 2.1 43.9 27.3 27.6 99.5 56.6 58.7 69.2 59.4 57.3 2.2 38.1 38.0 85.0 84.5 65.5 66.6 2.3 22.2 23.8 49.6 54.3 44.2 67.9 2.4 31.4 31.2 70.0 65.6 55.2 57.9

table 2

Results of biochemical analysis of blood plasma after plasma treatment in a flow system

Glucose (mmol/l) Urea (mmol/l) Sodium (mmol/l) sample number Control After 30 minutes After 1 hour Control After 30 minutes After 1 hour Control After 30 minutes After 1 hour 1.1 5.86 3.94 3.87 5.44 3.65 3.77 81.8 277.2 281.8 1.2 4.38 4.10 3.83 4.25 278.4 272.7 1.3 3.92 3.81 3.18 3.36 262.5 280.6 1.4 4.59 4.3 4.07 4.01 234.0 280.6 2.1 6.11 8.65 8.17 4.67 4.13 4.85 95.4 143 128.6 2.2 6.47 6.20 4.67 5.03 127.2 192.8 2.3 3.0 3.06 0.25 2.52 285.2 295.7 2.4 4.14 3.92 3.65 4.01 278.4 285.2

Table 3

Results of biochemical analysis of blood plasma after plasma treatment in a flow system

Bilirubin direct (µmol/l) Bilirubin indirect (µmol/l) sample number Control After 30 minutes After 1 hour Control After 30 minutes After 1 hour 1.1 2.7 2.6 2.0 13.3 12.9 12.7 1.2 2.6 1.9 13.0 12.5 1.3 2.3 2.1 12.6 12.2 1.4 2.7 2.5 13.0 12.5 2.1 4.0 3.9 3.8 16.1 14.9 14.4 2.2 3.5 3.5 15.5 15.3 2.3 3.7 3.3 15.7 15.1 2.4 3.5 3.7 14.1 14.0

Claims (1)

A method for normalizing blood biochemical parameters in patients with irreversible liver failure in an experiment, including hemoperfusion of blood into an extracorporeal circuit, filtration with separation of blood cells from plasma, perfusion of blood plasma through hepatocyte cell cultures, mixing of the treated plasma with blood cells and its return, characterized in that that a device of a flow-through incubator of a biological circuit with a modular design is used as a plasma filter, a separate module of which is made of a biocompatible polymer and consists of layer-by-layer superimposed elements - a cover, a cavity for plasma transport with a spiral arrangement of channels and outlet openings for inlet and outlet of plasma, a cavity for colonization of hepatocyte lines, repeating the course of the channels of the overlying layer, blocked by a porous-permeable track membrane with a pore diameter of not more than 5.0 μm, cavities for the filtrate containing a nutrient medium for cells, with outlet openings for the input and output of liquids, and between the cavity of the colonization of the lines hepatocytes and a cavity for the filtrate, a track membrane with a pore diameter of at least 0.2 μm is placed in the biological circuit, made up of flow modules with hepatocytes connected in series and sealed in a sealed housing, the modules are connected in series to each other by means of output tubes and then to external means plasma supply and bile outflow, after which the incoming blood plasma is perfused.

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