RU204435U1 - Biocomposite filter for the biological circuit of the extracorporeal hemoperfusion system - Google Patents

Abstract

The utility model relates to medical technology, namely to devices for the exchange of blood processing in order to compensate for the liver functions of patients with irreversible hepatic insufficiency, for example, during the waiting period for donor organ transplantation. The technical result is to create an extracorporeal hemoperfusion system based on a flow-through incubator with functional hepatocytes. human, providing the body with detoxification, metabolic and synthetic functions. A filter for the biological circuit of the extracorporeal hemoperfusion system, composed of 8 plasma filters with hepatocytes 7 connected in series and sealed in a sealed case, connected in series to each other by means of tubes and external means of supply and outflow without the formation of air spaces, characterized in that the device is used as a plasma filter flow incubator 9 with a modular design, each of which is made of a biocompatible polymer and consists of a cover 1, a cavity 3 for plasma transport with a spiral arrangement of channels and outlet openings for plasma inlet and outlet, a cavity 2 for populating hepatocytes, repeating the course of the channels of cavity 3 for plasma transport, blocked by a track membrane 5 with a pore size sufficient to prevent hepatocyte cells from entering the plasma circulation, for example, not more than 5.0 μm, cavity 4 for the filtrate containing a nutrient medium for hepatocyte cells, with outlet openings for input and output, moreover, between the cavity 2 of the colonization of hepatocytes and the cavity 4 for the filtrate there is a track membrane 6 with pore sizes sufficient to prevent the entry of hepatocyte cells into the circulation of the nutrient medium, for example, not less than 0.2 μm. 5 dwg, 3 tbl

Description

The utility model relates to medical technology, namely, devices for the exchange of blood processing in order to compensate for the liver functions of patients with irreversible liver failure, for example, while waiting for a donor organ transplant.

The liver is the largest gland in the human body. The functions of the liver are numerous and significant: detoxification, excretory, storage, synthetic, immune, homeostatic, storage, hematopoietic (in embryogenesis), etc., which define it as a vital organ.

Liver transplantation is the only option for patients with irreversible and severe organ destruction. According to statistics based on data from the register of the Russian Transplant Society, in Russia in 2012, 488 people were awaiting liver transplantation, in 2019 the number was already 1900, and the average waiting time is 3.6 years. Despite the increase in the number of liver transplant operations, there is still a problem of performing this operation for those in need due to the lack of donor organs, living donors. In this regard, methods and devices for extracorporeal liver support during the waiting period for a transplant are of great practical importance.

Known artificial systems MARS and Prometheus, which are acellular 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 / / Blood purification. - 2005. - T. 23. - No. 5. - P. 349-358; 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. - S. 276-284).

The known systems use a complex device of a rather complex design, combining the processes of dialysis, selective apheresis and sorption technologies. At the same time, the data do not have synthetic and metabolic functions, for example, the synthesis of albumin, which can only be provided by living functional liver cells.

There are a number of bio-artificial 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 into the system. For example, hiHep-BAL is based on induced human hepatocytes (see Shi XL 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), HepatAssist and SRBAL are based on pig hepatocytes (see Demetriou AA 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 CT et al. Concise review: liver regenerative medicine: from hepatocyte transplantation to bioartificial livers and bioengineered grafts // Stem Cells. - 2017. - T. 35. - No. 1. - S. 42-50).

Despite the presence of certain hepatocytes in the above systems, each system has its own drawbacks. For example, ELAD contains human hepatocarcinoma HepG2 / C3A cells, which exhibit reduced functional activity compared to native human hepatocytes, cannot excrete ammonia, and there is a risk of tumor cells entering the patient's bloodstream (see Shi XL 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; Nicolas CT 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; Lee KCL, Stadlbauer V., Jalan R. Extracorporeal liver support devices for listed patients // Liver Transplantation. - 2016. - T. 22. - No. 6. - S. 839-848). HepatAssist and SRBAL have disadvantages due to the risk of xenozoonosis, since they contain pig hepatocytes, and are severely restricted in some religious countries (see Shi XL 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). AMC-BAL was developed on the basis of porcine hepatocytes, however, in the first clinical trial, DNA of an endogenous pig retrovirus was found in one of the patients, which is why testing of this device was limited (see Lee KCL, Stadlbauer V., Jalan R. Extracorporeal liver support devices for listed patients // Liver Transplantation. - 2016. - T. 22. - No. 6. - P. 839-848; Van de Kerkhove MP et al. Liver support therapy: an overview of the AMC-bioartificial liver research // Digestive Surgery. - 2005. - T. 22. - No. 4. - S. 254-264). As for hiHep-BAL, this device is safe from the point of view of transmigration of tumor cells and xenozoonosis, but it has not yet been tested in humans (see Shi XL 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).

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 can only be provided by artificial devices for extracorporeal liver support), but also with synthetic, metabolic functions of the liver. In addition to supporting function while waiting for a donor organ, such systems are also capable of inducing 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 disease (see Thompson J. et al. Extracorporeal cellular therapy (ELAD) in severe alcoholic hepatitis: a multinational, prospective, controlled, randomized trial // Liver Transplantation. - 2018. - T. 24. - No. 3. - P. 380-393).

The apparatus of biological artificial liver (see RU No. 2163148, class A61M 1/36, publ. 20.02.2001) contains a housing containing tissue fragments of the liver, filled with a homogeneous mixture of particles of a neutral carrier, namely agarose gel. Plasma processing is as follows: heparinized blood or plasma from a 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 volume 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 liver tissue.

Known apparatus for extracorporeal blood purification (see RU No. 93276, CL A61M 1/14, A61M 1/36, publ. 04/27/2010), containing a dialyzer, divided by a semipermeable membrane into a blood section and connected to the patient's circulatory system through a plasma filter with regulating distributors and a pump, and a section for dialysis fluid connected to a cleaning circuit filled with dialysis fluid, which is a 10-12% solution of human albumin or liver cytosol, equipped with at least one column with a carbon sorbent, inlet which is the input of the spent dialysis fluid, and the output is communicated directly or through the intermediate elements of the cleaning circuit with the container for the dialysis fluid or the inlet of the dialysis fluid section of the dialyzer.

The widespread use of this apparatus for detoxification purposes is limited by the complexity of the design and the high cost of the equipment.

There is a known method of hemocorrection in the treatment of liver failure (see RU No. 2693677, class A61M 1/16, publ. 03.07.2019), which includes the flow of the patient's blood into the device, filtration of blood plasma from blood cells, plasma perfusion through cartridges containing directionally differentiated hepatocytes, mixing the processed plasma with blood cells and returning the blood to the patient. In this case, differentiated stem cells are autocells, which were obtained by reprogramming peripheral blood mononuclear cells.

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

The problem to be solved by the claimed solution is to create a device for the normalization of the biochemical parameters of the blood of patients with irreversible liver failure, characterized by ease of use.

The technical effect obtained when solving the problem is expressed in the creation of an extracorporeal hemoperfusion system containing a filter with functional human hepatocytes, which provides the body with detoxification, metabolic and synthetic functions.

To achieve the set task, the filter for the biological circuit of the extracorporeal hemoperfusion system, made up of plasma filters with hepatocytes connected in series and sealed in a sealed case, connected in series to each other by means of tubes and external means of supply and outflow without the formation of air spaces, differs in that as plasma filters use a flow-through incubator device with a modular design, each of which is made of a biocompatible polymer and consists of layer-by-layer superimposed elements, namely, a lid, 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 hepatocytes, repeating channel paths cavities for plasma transport, blocked by a track membrane with a pore size sufficient to prevent hepatocyte cells from entering the plasma circulation, for example, not more than 5.0 μm, cavities for filtrate containing pitate a natural environment for hepatocyte cells, with outlet openings for input and output, moreover, a track membrane with pore sizes sufficient to prevent the entry of hepatocyte cells into the circulation of the nutrient medium, for example, not less than 0.2 μm, is placed between the cavity of hepatocyte colonization and the cavity for the filtrate ...

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

The set of new features provides a solution to the stated technical problem, namely, due to the system of extracorporeal support, 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 of not only detoxification, but also metabolic, synthetic function; the use of a biological circuit that facilitates the rapid production and cultivation of cells; increasing 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 semipermeable membranes placed in an environment with cultured hepatocytes, metabolic processes occur between 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 a maintenance therapy for patients with severe forms of liver failure.

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

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

In the cavity of the filtrate 4 for the culture medium, toxins and metabolic products will be removed, therefore, separate cavities and tubes are provided for the flow of these liquids for blood plasma and culture medium in order to prevent their mixing.

For the prototype, the diameter of the flow-through incubator structure was 84 mm, while the layered elements were made of a synthetic polymer polylactide by 3D printing (see Figs. 1, 4). The choice of polymer material was determined by the following properties: biocompatibility, which is necessary for working with patients; low degree of bioresorption; thermoplasticity; material availability (see D. Belov 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 the membranes were selected track membranes "Reatrek" of a known design made of polyethylene terephthalate with pore diameters of 5.0 and 0.2 μm, respectively. The advantage of track membranes is their correct geometry of pores, the ability to control their number, as well as uniformity in pore sizes, which prevents the risk of penetration of both cell units and their constituents in the event of destruction (see Volkov V.V. et al. Membranes and nanotechnologies // Russian nanotechnologies. - 2008. - T. 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 hepatocyte cells entering the patient's bloodstream, since the average diameter of hepatocytes varies from 20 to 30 μm.

To form a biological contour, composite cartridges are pre-populated with human hepatocytes, for which purpose, hepatocytes are inoculated in cavity 2, the bottom of which is made in the form of a semipermeable track membrane 6. Further, layer 2 with track membrane 6, on which hepatocyte cells are adhered, are transferred into the filtrate cavity 4 scaffolds designed for the culture medium. Then, on top of the hepatocytes, a porous-permeable membrane 5 is applied to prevent the entry of hepatocytes into the patient's bloodstream. After that, the remaining layers are applied: cavity 3 for the flow of plasma through the network of branched channels and cover 1. All layers of the cartridge are placed relative to each other in the "sandwich" type (see Figs. 2, 3).

Flow-through incubators 9 with hepatocytes are connected in series with medical tubes and sealed in a sealed case 8. Thus, the manufactured biological circuit is supplied to a specialized medical institution, where for use it is connected through outlet tubes to external means of supply and outflow of substances without the formation of air spaces, for example, to infusion pump, which supplies 10 blood to the biological circuit, and through the other line provides the outflow 11 of bile and used culture medium (see Fig. 5).

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 enters the plasma filter, where blood plasma is separated from the formed elements through the membrane. For example, in 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 SC et al. Treatment of severe liver failure with a bioartificial liver // Annals of the New York Academy of Sciences. - 1997. - T. 831. - S. 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 bilirubins (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 . Human hepatocyte transplantation: state of the art / E. Fitzpatrick, RR Mitry, A. Dhawan // J. Intern. Med. - 2009. - Vol. 266. - P. 339-357; Nahmias Y, Berthiaume F, Yarmush 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; Afanasyeva Yu.I. Histology, embryol ogia, cytology. Textbook / Yu.I. Afanasyeva, 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 medico-biological bulletin named after V.I. 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 inflow rate of fresh medium should be from 1.2 to 2.0 ml / h (see Basok Y.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 - 2018. - T. 20. - No. 1. - P. 70-78; 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. The stability of the reactor at a calculated oxygenator concentration of at least 0.3 nmol / s 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 blood flow.

For experimental work, the 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, ВР 7290-34184 (FSZ 2010/07857), OKP 94 4300 using colorimetric, photometric technologies to determine the concentration of individual chemicals and exchange products. The levels of the following biochemical parameters were determined: albumin, aspartate aminotransferase, alanine aminotransferase, sodium, total and unbound bilirubin.

Plasma of conventionally 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 pre-fabricated cartridge with inoculated hepatocytes on the surface of the filtration membrane of the cell placement layer (incubation layer), which is simultaneously the upper wall of the dialysate cavity with nutrient medium. At the same time, within the framework of the experiment, the plasma was retained on one cartridge 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 liver function.

At the exit, there was a significant increase in all samples in the levels of aspartate aminotransferase, sodium and a moderate rise in the level of albumin, as well as an obvious decrease in the concentration of unbound bilirubins (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 polymer 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 was found that the system of extracorporeal liver support with the claimed device of the biocomposite filter is able to provide a supporting function in liver failure, compensate for the violation of detoxification, metabolic and synthetic functions of the liver, and therefore, increase the survival rate of patients who are expecting a donor liver, and reduce the likelihood of complications. as well as spontaneous liver regeneration of patients with a milder course of liver disease, which will allow targeting the pool of donor organs to patients with more severe stages of the disease.

Table 1

Results of biochemical analysis of blood plasma after plasma processing in the flow system

Sample no. Albumin (g / L) Alanine aminotransferase (U / L) Aspartate aminotransferase (U / L) Control After 30 minutes After 1 h Control After 30 minutes After 1 h Control After 30 minutes After 1 h 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 processing in the flow system

Glucose (mmol / L) Urea (mmol / l) Sodium (mmol / L) Sample no. Control After 30 minutes After 1 h Control After 30 minutes After 1 h Control After 30 minutes After 1 h 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 processing in the flow system

Direct bilirubin (μmol / l) Indirect bilirubin (μmol / l) Sample no. Control After 30 minutes After 1 h Control After 30 minutes After 1 h 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 filter for the circuit of an extracorporeal hemoperfusion system, including plasma filters with hepatocytes connected in series and sealed in a sealed case, connected by means of tubes to each other and to external means of supply and outflow without the formation of air spaces, characterized in that the plasma filters are flow-through incubators of modular design, each made made of a biocompatible polymer and includes a lid, a cavity for plasma transport with a spiral arrangement of channels and outlet openings for plasma inlet and outlet, a cavity for populating hepatocytes, repeating the course of the channels of the cavity for plasma transport, and a cavity for the filtrate containing a nutrient medium for hepatocyte cells, when a track membrane with pore sizes is located between the cavity for plasma transport and the cavity for populating hepatocytes to prevent hepatocyte cells from entering the plasma circulation, and between the cavity for populating hepatocytes and the cavity for filtrate p A track membrane with pore sizes was placed to prevent the entry of hepatocyte cells into the circulation of the nutrient medium.

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