Method for sequential assessment of recipient's state following cadaveric liver transplantation

FIELD: medicine.

SUBSTANCE: patient's peripheral blood is simultaneously examined for the percentage of circulating stem hemopoietic cell CD34+ (SHC) and the volume fraction of lymphocytes (Lph, mln/ml); they are individually related as R by formula R=LPH/SHC, and an R (y axis) to SHC (x axis) law curve is drawn to be used as a calibration curve within the log-log grid, while the current recipient's state is assessed by examining the peripheral blood for LPH and SHC to be related as Rp and found in the calibration system of axis. If Rp is found either on the calibration curve, or above the same, the current recipient's state is considered to be favourable, otherwise the unfavourable state is shown by Rp found under the calibration curve.

EFFECT: using the declared method enables improving the accuracy of assessing the post-transplantation recipient's state.

4 tbl, 4 dwg, 3 ex

 

The invention relates to medicine, more specifically, to organ transplantation and can be used for the transplant post-mortem liver.

A steady increase in the number of patients with liver disease, including resulting in terminal liver failure, causes an increase in the demand for treatment by the method of transplantation of the organ. Liver transplantation is associated with the possibility of post-transplantation complications, the most dangerous of which are varying degrees of severity of dysfunction and rejection of the donor liver, and infection.

Currently it is accepted that the survival of the graft is determined largely by the degree of immune reaction of the recipient against the foreign tissue. Therefore, to maintain graft function use special drugs are immunosuppressants, which are constantly evolving in the direction of improving the selectivity of their action from "non-specific" to "specific".

Predicting the risk of transplant rejection, including donor liver is an unsolved problem. Search unbiased predictors of the possibility of rejection is an urgent problem, the solution of which can improve the effectiveness of post-transplant treatment.

A known method for predicting the risk of posttransplantation complications on concentric is in the recipient's blood T-lymphocytes. The authors recommend to maintain the concentration of T-lymphocytes to prevent rejection of the transplanted liver within 50/μl and below [T. Muller et al. Transplantation Proc. - 1999. - Vol.3B - P.12S-15S].

Expresses the relationship of severity of immunological reactions with the number of circulating lymphocytes of the recipient. Questionable recommendation to maintain the level of T-lymphocytes in the peripheral blood within 50/µl. In our experience, successful transplantation cases was accompanied by the concentration of T-lymphocytes more than 50/ál and, conversely, when the level is below this value were reported complications early post-transplant period. Moreover, successful retransplantation liver after a failed first was accompanied by a gradual rise in the concentration of T-lymphocytes and exceeded 50/µl.

Clinical observations indicate a lack of strict correlation between the quantity of T-lymphocytes in the blood of the recipient liver with the nature of the flow of the early post-transplant period.

Available data from the literature that the success of transplantation of cadaveric liver is not determined only by the degree of compatibility on leukocyte complex antigens human (HLA) [Liver Transplantation. . P.80-90, P.1405-1413], led to the fact that we need to search for other factors that affect the functional viability of transplantation.

We have previously monitored the subpopulation composition of the mononuclear fraction of blood in patients after transplantation they cadaveric liver for 7 months after surgery [Medical immunology. 2009. No. 4-5 (C). S] and noted the oscillations of different subpopulations of mononuclear cells and at different times after surgery. On the basis of the received data to reach a conclusion on the diagnostic advantages of an indicator has failed.

We have identified beneficial effects of transplantation of autologous bone marrow on the functional activity of the kidney with glomerulonephritis [Ryabov C., Rakityansky I.A., Shutko A.N. In the book "the Kidneys and the immune system". Leningrad: Nauka. 1989. P.131]. Based on these data, we have suggested the possible involvement of progenitor cells of bone marrow origin in maintaining the viability of the graft liver [Granov A.M., Shutko A.N. Paradoxes of malignant growth and tissue incompatibility. Ed. Hippocrates, 2002].

Later the positive results of the transplantation of progenitor cells from the bone marrow registered with liver cirrhosis [Medical immunology. 2009. No. 4-5 (C). S].

Along with fluctuations in the content of different fractions of Mature lymphocytes in the blood, we have found substantial kalopanagiotis as hematopoietic stem cells (SGK), and precursor cells of lymphocytopenia in the mononuclear fraction of peripheral blood of the recipients of the liver in different periods of time after transplantation, i.e. the condition of turbulent blood.

According to several studies, SGK able to migrate into various tissues and organs, liver, lungs, gastrointestinal tract, heart and vessels, supporting them in proliferative processes [Nat.Med. 2000. Vol.6. P.1229-1234, Stem Cells. 2001. Vol.19. P.304-312]. It is important to note that some of the circulating SGK carries markers precursors of endothelial cells and may thus influence the condition of the capillary network as a critical target in ischemic damage or rejection of the transplanted organ. As SGK are parental for all types of circulating cells, fluctuations in the content of stem cells in the blood are determined by fluctuations of the other subpopulations. We have shown [European Radiation Research. (University of Leicester UK). 2005. P.166]that the concentration of the main lymphocyte subpopulations in the blood of apparently healthy people are quantitative and temporal dependencies from SGK, the contents of which are also very variable in terms of turbulent blood. It follows that SGK are fundamental reference marker system infoproducts, making them a priority and when is terpretation received immuno-hematological results.

Analyzing the sometimes contradictory literature data and the results of their own observations, we found it necessary to conduct a study to elucidate the dynamics of the number of circulating SGK in the early post-transplantation period in recipients of cadaveric liver (during the first month after the operation).

The study included 36 patients after liver transplantation (study group) and 23 (control group) without liver disease, including after major surgical interventions performed on other organs and systems. Completed more than 200 tests. All members of the control and main groups, with an average frequency of 1 time per week, were used to define different cellular markers of mononuclear cells from blood, such as CD3+, CD4+, CD8+, CD11b+, CD34+, and others. The number of cells having a particular marker, was assessed as a percentage of the total number of mononuclear cells.

The results were analyzed to identify specific features of the dynamics of each of the indicators during the first month after transplantation. Month period was divided into 2 intervals: up to 10 and after 10 days. For these time intervals have been calculated average concentrations of the analyzed subpopulations. On the basis of consideration of the average values of concentrations, taking into account fluctua the AI of their separate values, was not found in a single type of kinetics indicators except SGK (CD34+). Only on the basis of this parameter were able to divide the main group of patients statistically.

This feature formed the basis of the developed method for predicting the risk of complications in recipients after transplantation of cadaveric liver, which is taken as a prototype of the present invention [the Patent of the Russian Federation 2424529].

The method consists in the fact that the recipient 2 times per week during the first month after transplantation of cadaveric liver measured in the blood concentration of circulating SGK (CD34+)find the average value for the first 10 and the next 20 days. If the magnitude of these values is 0.1% or more in both time intervals, the recipient is referred to the group of patients with a favorable outcome of transplantation. If these values are below 0.1% in the second, or both time intervals, the recipient predict the risk of complications in the postoperative period.

However, using this method the average criterion further showed that in some cases it is not possible to correctly estimate the state of a particular recipient. This is because individual state itself may be changed periodically. In our opinion, such fluctuations in the degree of "health" recipients caused by a periodic change not only what to SGK, but the total number of circulating blood lymphocytes derived SGK and related processes graft/transplant rejection, containment of infectious processes, etc. This fact was not taken into account in our previous work, resulting method was not sufficiently accurate for personal, not group forecast, especially in conditions of turbulent blood, typical of the early post-transplant period.

The technical result of the present invention is to improve the accuracy assessment of the status of the recipient in the post-transplant period due to additional definitions in his peripheral blood content of circulating lymphocytes.

This result is achieved by the known method of assessing the status of the recipient after transplantation of cadaveric liver by the determination in his peripheral blood, the percentage of circulating hematopoietic stem cells CD34+ (SGK) according to the invention previously in the peripheral blood of not less than 20 recipients retrospective group weekly for 1-2 months after transplantation they cadaveric liver simultaneously determine the percentage of SGK and volumetric content of circulating lymphocytes (LF) in million/ml, find their individual relation R by the formula R=LF/SGK and streetcrime power-law dependence R (y-axis) from SGC (x-axis), using it as a calibration curve in the double logarithmic coordinates, and to assess the condition of the recipient at the current moment in his peripheral blood simultaneously determine the values of LF and SGK, find their attitude Rp, position it in the calibration coordinate system and the location of the Rp at or above the calibration curve the current status of the recipient is assessed as favourable, lower curve - negative.

Long-term monitoring of recipients after transplantation of them cadaveric liver showed that changes in their condition over time is not random. After verifying the actual information opportunities indicators LF and SGK separately, we decided to clarify the nature of the interdependence between these values. As their changes over time were opposite in direction, i.e. a maximum of one indicator corresponded to the minimum of the other, and Vice versa, their relationship, we decided to characterize the most simple way, namely, the relation R is the number of the volume content of lymphocytes in the blood to the percentage of CH: R=LF/SGK. Registering the parameter R, which unites the real possibilities of each indicator, we hoped to obtain a more complete assessment of the condition of the patient at different periods of his treatment.

So, in 2002 we reported about the possibility in which adsene state recipients of liver, until his death, when a too low number of circulating lymphoid cells [Granov A.M., Shutko A.N. Paradoxes of malignant growth and tissue incompatibility. Ed. Hippocrates, 2002]. Rate R based on these data is presented as more promising, combining predictive capability as SGK, and LF. It was found that the ratio of the two indices R=LF/SGK is more variable than the variability of each of them separately. We have shown that the prognostic meaning of the individual values of R can be estimated reliably, if you explore diversity in the retrospective group of typical recipients during the interest period.

For this purpose with the research we had taken a group of recipients of the liver in the number of 20 people. For each of these 20 recipients retrospective of the group during the 2-month period after transplantation, we measured 6-7 times values (more than 140) SGK,%, and LF, million/ml, and then calculating their individual relationship: R=LF/SGK. The data obtained are shown in table 1. Next was built by the graph of the values of R (on the y-axis y) on the values of SGK (along the axis x-x). The obtained dependence was approximatively in the "l", choosing the approximating function according to the criterion of the maximum of a hundred is sticheskogo match the location of the points, ensuring that the correlation coefficient was reliable with maximum likelihood (at least p≤0,01). This condition ensures reliability further use dependency as a reference. In our case, the most accurate function was exponential function: y=ax-bwhere a and b are constants. For the convenience of practical use from the reference dependence of R=f (SGK) it is presented in double logarithmic coordinates, in which it becomes almost a straight line (figure 1). The figure shows that the specific point in the vicinity of the approximation straight line is located so that a part of them is on it or above it, and the other part is on the bottom. To assess the condition of the recipient at the current time it is necessary to determine the position of its values R calculated according to the data of simultaneous measurements of LF and SGK, relative to the reference line in the figure. The position of the R values on or above the line corresponds to, as shown by our preliminary studies, a compensated state of the recipient. Position R under the line requires more attention to the condition of the recipient until a decision about the weakening of the intensity of immunosuppressive therapy.

T the blitz 1
The way dynamic assessment of the recipient after transplantation of cadaveric liver
Patient No.12345.......20
Data and calculations
LF, million/ml0,341,750,740,770,28.......3,16
SGK, %0,160,160,180,0310,11.......0,26
R3,410,94,12,472,63.......12,2
LF, million/ml1360,551,89 1,21,1.......2,45
SGK, %0,080,460,060,340,27.......0,097
R171,1931,53,534,1.......253
LF, million/ml.................................................

The essence of the method is illustrated by example.

Example 1. Patient M., born in 1956 (and a/b No. 474), was admitted to the hospital MCRT 04.04.2008 to perform orthotopic liver transplantation (OTP).

Diagnosis: Chronic viral hepatitis, cirrhotic stage decompensation.

The diagnosis was established according to the preliminary survey, made on the basis MCRT.

Ultrasound examination of the abdominal cavity (ultrasound) 08.02.2008: Atrophic cirrhosis of the liver. CNC g is the hypertension. Ascites. Echo-cardiography 08.02.2008: moderate Signs of connective tissue dysplasia cardiac structures. Mitral valve prolapse 1 tbsp.

Fibrogastroduodenoscopy 01.02.2008: Varicose veins of the esophagus Art. III

Multislice spiral computed tomography 08.02.2008: cirrhosis of the liver with symptoms of atrophy, portal hypertension. Splenomegaly. Ascites. OTP made 04.04.2008.

The patient received triple immunosuppression: tacrolimus 5 mg daily under the control of drug concentration in the blood, the target concentration in the first month believed 10-15 ng/ml, in a remote period of 3-5 ng/ml, azathioprine 50 to 100 mg, prednisolone descending to the scheme from the initial dose of 200 mg per day intravenously to 10 mg per day orally for discharge. Immunosuppressive therapy, beginning with the first day after transplantation, next lifetime.

The study SGK blood was carried out in the first month at 5, 12,19, 26, 32 and 40 days after the OTP.

In figure 2 the dynamics of the ratio of the volume concentration of circulating lymphocytes to the percentage of lymphocytes in the mononuclear fraction of blood. Numerical values of the indicators are presented in table 2. R values in bold correspond to the points above the curve.

Table 2
The day after the OTPLF (million/ml)SGK (%)R
50,270,3130,85
120,940,146,7
191,130,1110
261,67is 0.1355
321,230,10312
400,730,0957,7

These changes SGK in the peripheral blood corresponded to the period of recovery, during which liver function recovered after 1 month. The content of bilirubin 30 days after the OTP 20, 5 µmol/l, ALT - 24 Ed., ACT - 15 Ed. The patient was discharged to outpatient treatment at 32 days after the OTP (06.05.12). Constantly take immunosuppressive drugs prograf 3 mg / day, prednisolone 10 mg per day, after 3 months prednisolone was cancelled and adipren 100 mg per day. In July 2008, marked with signs of cholangitis, according to the U.S. 22.07.2008: in both lobes of the liver is determined by the minor to 1-2 mm extension sub-segmental and segmental bile ducts. Equity ducts are not expanded, around the area of fibrosis.

Conclusion: Cholangitis the blood 22.07.08 Hemoglobin 117 g/l, erythrocytes of 3.8×1012/l, leukocyte count of 4.3×109/l, ESR 28 mm/h, bilirubin 32 µmol/l, ALT 56 Ed./l, ACT 45 Units./l, GGT 129 Ed./l, creatinine 100 µmol/l, total protein 62 g/l, albumin 30 g/l, glucose 5.2 mmol/L. Given the infection, the number of LF, equal to 0.36 million/ml, SGK, or 0.27%, hence R=1,33 (the point is below the curve in figure 3), reduced immunosuppression: prograf 2 mg / day, azathioprine cancelled, prednisolone was canceled.

For the treatment of cholangitis patient was hospitalized MCRT 01.08.08 (and a/b No. 2402). On the background of reduction of immunosuppressive therapy and antibiotic therapy with ciprofloxacin 1 g / day, then marinemom 2 g per day condition has stabilized. This corresponded to the values R=6,6, 32,6 from table 3, the corresponding points in figure 3 are located above the curve. After 5 months, the patient noted the signs of rejection in the form of the increasing rate of cytolysis (ALT 134 Ed., ACT 89 Ed.), the growth in the concentration of bilirubin to 47 µmol/l dated 05.09.08. The indicators in these terms LF 0.53 mln/ml, SGK 0,15%, R=3,5 (point 3 is located below the curve). All uh what about the required change of tactics of treatment of the patient. Held pulse therapy solumedrol 500 mg, increased doses of immunosuppressants, resumed prednisone 10 mg per day. Against this background, developed a relapse of severe cholangitis, which led to a deterioration of the function of the graft, a sharp increase in cholestasis. The patient was given large doses of antibiotics (Meronem, doxycycline), protivogribkovye drugs (Cancidas).

According to ultrasound in the dynamics (13.08.08, 10.09.08): extended Stored segmental and sub-segmental bile ducts. In the area around the portal vein infiltration, ducts are not visible. Focal formations not lazerette. In the analysis of blood 11.09.08: Hemoglobin 119 g/l, erythrocytes of 3.2×1012/l, leukocyte count of 6.5×109/l, ESR 95 mm/h, bilirubin 230 µmol/l, direct of 167.2 µmol/l, ALT 108 Units/l, ACT 326 Ed./l, GGS 1076 Ed./l, creatinine 65 µmol/l, total protein 51 g/l, albumin 34 g/l, glucose of 4.8 mmol/l, LDH 519 Ed./L.

Presents the clinical picture and the corresponding data R (see table 3, figures R 3,5, 3,66, 0,6) demanded the necessity of surgical intervention.

12.09.2008 conducted relaparotomy, the revision of the abdominal cavity, choledochotomy, cholangiography. Resection hepathology, separate external drainage of the equity of the bile ducts, drainage of the abdominal cavity.

23.09.2008 a relaparotomy, sanitation and drainage of the abdominal cavity. Conducted anti-Christ. arterially therapy taking into account the sensitivity sown bacterial flora (Meronem, zyvox diflucan).

Table 3
The day after the OTPLFSGKR
22.07.08 (3,5 months).0,360,271,33
18.08.08 (4,5 months).0,730,116,6
25.08.08 (4.8 months).0,790,02432,8
10.09.08 (5.3 months).0,530,153,5
19.09.08 (5,5 months)0,510,143,66
30.09.08 (5.8 months).0,30,50,6

30.09.08 in blood hemoglobin 78 g/l, erythrocytes of 1.3×1012/l leukocytes of 4.8×10%, chromacity 32×109/l, ESR 155 mm/h, bilirubin 249,8 µmol/l, direct of 182.2 mmol/l, ALT 17th Ed./l, ACT 23 Ed./l, GGT 798 Ed./l, creatinine 166 μmol/l, total protein 51 g/l, albumin 38 g/l, g is ucose 4.8 mmol/l, LDH 163 Ed./l, LF 0.3 million/ml, SGK 0,5%, R=0,6 (below the curve, see figure 3, table 3). These data were consistent with severe, irreversible dysfunction of the liver transplant that has identified the need for re-transplantation of the liver.

Re OTP made 13.10.08 (6 months postoperatively). Within 5 months the patient was in the hospital. The duration of stay was due to recurrent cholangitis, recurrent dresirovaniye biliary tract. Despite ongoing antibiotic therapy, minimization of immunosuppression, the patient was formed chronostasis, she always received antibacterial therapy. Developed stricture of the common bile duct and multiple reanastomosis stricture of the bile duct.

According to table 4 and figure 4 all figures R were low, which resulted in a poor prognosis for the patient.

In the blood remained anemia, inflammatory changes moderate cholestasis.

Blood 13.11.08 (30 days after re-OTP): Hemoglobin 81 g/l erythrocytes of 2.4×1012/l, leukocytes to 4.1×109/l, erythrocyte sedimentation rate of 50 mm/h, bilirubin and 36.2 μmol/l, direct 6.7 µmol/l, ALT 143 Ed./l ACT 61 Ed./l, GGT 609 Ed./l creatinine 56 µmol/l, total protein 53 g/l, albumin 28 g/l, glucose 6: mmol/l, LDH 149 Ed./L.

Immunosuppression: prograf 1 mg per day, Myfortic 360 mg per day prednisolone was nananatili.

Despite ongoing antibiotic treatment, remained phenomena cholangitis violation of the outflow of bile. Stenting of the bile duct is made 06.12.2008. Upon further observation of the patient's condition remained difficult despite a moderate increase in the concentration of bilirubin in the serum remained phenomena chronic sepsis.

In further performed multiple percutaneous drainage and balloon dilatation of the bile ducts (07.05.2009. 19.05.2009, 10.09.2009, 27.01.2010, 27.02.2010 (and a/b No. 2402, No. 2644). The function of the transplanted liver progressively worsened. Later the patient died from erosivnogo bleeding from ulcers of the gastric cardia in the background of severe sepsis.

Table 4
The day after the re-OTPLFSGKR
15.10.08 (2 day)0,40,361,11
23.10.08 (10 days)0,220,096of 2.26
30.10.08 (17 days)0,30,097,09

To date, using the proposed method was monitoring more than 20 patients after liver transplantation. In all cases, values of R were consistent with the clinical condition of the patients and taking into account, if necessary, changed tactics of treatment.

The proposed method is compared with the known has several advantages, the main of which is to improve the accuracy of determining the current status of the recipient, ensuring timely detection of deviations in its General status portability immunosuppressive therapy.

The method developed in the group of clinical transplantation joint laboratory methods of immunological research and clinically tested with 20 recipients of cadaveric transplant liver with a positive result.

The way dynamic assessment of the recipient after transplantation of cadaveric liver, including the definition in the peripheral blood of the recipient of the percentage of circulating hematopoietic stem cells CD 34+(SGK), wherein the pre in the peripheral blood of not less than 20 recipients retrospective group weekly for 1-2 months after transplantation they cadaveric liver simultaneously determine the percentage of SGK and volumetric content of C is kulinowski lymphocytes (LF) in million/ml, find their individual relation R by the formula R=LF/SGK curve and build a power-law dependence R (y-axis) from SGC (x-axis), using it as a calibration curve in the double logarithmic coordinates, and to assess the condition of the recipient at the current moment in his peripheral blood define LF and SGK, find their attitude Rp, position it in the calibration coordinate system and the location of the Rp at or above the calibration curve the current status of the recipient is assessed as favourable, lower curve - adverse.



 

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4 dwg, 7 ex

FIELD: biotechnology.

SUBSTANCE: method comprises adding to the part of the wells with lymphocytes of mitogens or test substances, incubation of the contents of the wells, selection of the contents of the wells, cytolysis and isolation of nucleic acids. The reaction of reverse transcription (RT) is carried out with primers for genes tpa and cdc2: primer for RT with mRNA of gene tpa 5'-CATCTTCATCTCACTCTTC-3', primer for RT with mRNA of gene cdc2 5'-CTGGAGTTGAGTAACGAG-3'. Real-time PCR is carried out with the obtained cDNA on the specific primers and with use of the labeled oligonucleotides for genes tpa and cdc2: for gene tpa the forward primer is 5'-TGTCGTGTCAGACCTTGAAGC-3', the reverse primer is 5'-CCTTGGATTTCTTGCTTGTGAC-3', the probe (FAM)-5'-TGTACCACTGTCTCAAGCCCTCCTGC-3'-(BHQ1), for cdc2 gene the forward primer is 5'-CTTCACTTGTTAAGAGTTATTTATAC-3', the reverse primer is 5'-CCAGAGTGTTACTACCTCATGTG-3', the probe (FAM)-5'-TGCCTTGCCAGAGC(FdT)TTTGGAATAC-3'-(BHQ1). The degree of proliferation of lymphocytes is expressed in proliferation index which is the number indicating how many times the amount of mRNA of gene tpa and/or cdc2 increases during culturing lymphocytes with/without addition of PHA or the test substance as compared with the amount of mRNA of the genes in lymphocytes freshly isolated from the blood of healthy donor.

EFFECT: invention enables to monitor directly the number of dividing cells, assessing the degree of expression of the genes involved and regulating the cell division.

4 dwg, 4 ex

FIELD: biotechnology.

SUBSTANCE: population of Tr1-cells directed against an antigen associated with disseminated sclerosis and having quiescent phenotype CD4+CD25-FoxP3- is isolated. The resulting population of Tr1-cells in combination with one or more pharmaceutically acceptable carriers, or in combination with one or more pharmaceutical agents used for treating disseminated sclerosis, selected from the group comprising interferon-beta, glatimer acetate, mitoxantrone, cyclophosphamide, methotrexate, asitropine, or natalizumab is used in pharmaceutical compositions for treating disseminated sclerosis.

EFFECT: invention enables to obtain an effective remedy for treating disseminated sclerosis.

11 cl, 2 dwg

FIELD: biotechnology.

SUBSTANCE: method of production of induced pluripotent stem cells (IPS cells) from skin fibroblasts of patients with Down syndrome is proposed, which includes preliminary induction using lentiviral transfection with gene hTERT, further transfection of the resulting cells with three lentiviral constructs with genes Oct4, Sox2, Nanog, with subsequent cultivation for 14-21 days with valproic acid under conditions of low oxygen content 5% O2.

EFFECT: improvement of the method.

2 ex

FIELD: biotechnology.

SUBSTANCE: method provides for culturing mammalian cells expressing the recombinant protein of interest in a culture medium containing anti-aging compound selected from carnosine, acetyl-carnosine, homo-carnosine, anserine, and K-alanine and their combinations, under conditions and for a time sufficient for expression of the protein.

EFFECT: invention enables to improve the performance of the cell culture selected from higher titer, of increased specific cell productivity, increased cell viability, increased integrated viable cell density, decreased accumulation of high molecular aggregates, decreased accumulation of acidic molecules, and their combinations.

44 cl, 14 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to field of biochemistry. Claimed is preparation for preservation of culture of stem and differentiated human and animal cells in cultivation, storage and cryoconservation. Preparation represents gel, which contains emulsion of perfluororganic compounds (PFOC) in their stabilisation in emulsified state with water solution of non-ionogenic surface active compounds (SAS) based on block-copolymer of polyethylene oxide (PEO) and polypropylene oxide (PPO) with ratio PEO/PPO from 9:1 to 7:3. SAS concentration in preparation constitutes from 20 to 90% with average molecular weight of SAS from 5000 Da to 21000 Da. Content of PFOC in emulsion constitutes from 5 to 70 vol.%. Size of PFOC emulsion particles lies in the interval from 15 nm to 2000 nm. Water phase of gel contains osmolytes of organic and/or inorganic nature for supporting in it osmotic pressure from 250 to 350 mOsm. As PFOC emulsion, emulsion of perfluorodecalin, perfluoro methylcyclohexyl piperidine and perfluorotributylamine mixture can be used. Also claimed is method of obtaining said preparation. Extrusion of coarse mixture of water SAS solution and liquid PFOCs or PFOC mixture is performed under pressure 400-700 atm until emulsion is obtained. After that, it is mixed in hot condition with concentrated water solution of inorganic salts or osmolytes are added into it until osmotic pressure equal from 250 to 350 mOsm is obtained. Then obtained composition is cooled. After that, partial freeze-drying or centrifugation of gel with observation of temperature gradient and acceleration from 15000 g to 35000 g is performed. Methods of preservation of culture of stem and differentiated human and animal cells in storage, cryoconservation and cultivation are claimed. In storage of cells preparation with PFOC content from 15 to 59 vol.% is added to culture medium with controlled value pH=7.0-7.4, temperature 37°C in CO2 incubator atmosphere or with low pH<6.0, temperature 20°C in air atmosphere. In cryoconservation resuspended in fresh culture medium cells are placed into medium, which contains preparation with PFOC content 59-70 vol.%. After that, cooling is carried out in two stages: first at rate 1 degree per min to -80°C, then with maximally possibly rate to temperature -196°C. In cultivation of cells as medium component preparation with PFOC content from 15 to 70 vol.% is used.

EFFECT: cell cultures after cryoconservation, storage or cultivation in presence of PFOC-containing gels have on average 90% of viable cells, are easily separated from gel, re-inoculated in standard conditions, divided and fully preserve their functions and ability to differentiate.

16 cl, 23 dwg, 1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to field of biochemistry. Claimed is preparation for preservation of culture of stem and differentiated human and animal cells in cultivation, storage and cryoconservation. Preparation represents gel, which contains emulsion of perfluororganic compounds (PFOC) in their stabilisation in emulsified state with water solution of non-ionogenic surface active compounds (SAS) based on block-copolymer of polyethylene oxide (PEO) and polypropylene oxide (PPO) with ratio PEO/PPO from 9:1 to 7:3. SAS concentration in preparation constitutes from 20 to 90% with average molecular weight of SAS from 5000 Da to 21000 Da. Content of PFOC in emulsion constitutes from 5 to 70 vol.%. Size of PFOC emulsion particles lies in the interval from 15 nm to 2000 nm. Water phase of gel contains osmolytes of organic and/or inorganic nature for supporting in it osmotic pressure from 250 to 350 mOsm. As PFOC emulsion, emulsion of perfluorodecalin, perfluoro methylcyclohexyl piperidine and perfluorotributylamine mixture can be used. Also claimed is method of obtaining said preparation. Extrusion of coarse mixture of water SAS solution and liquid PFOCs or PFOC mixture is performed under pressure 400-700 atm until emulsion is obtained. After that, it is mixed in hot condition with concentrated water solution of inorganic salts or osmolytes are added into it until osmotic pressure equal from 250 to 350 mOsm is obtained. Then obtained composition is cooled. After that, partial freeze-drying or centrifugation of gel with observation of temperature gradient and acceleration from 15000 g to 35000 g is performed. Methods of preservation of culture of stem and differentiated human and animal cells in storage, cryoconservation and cultivation are claimed. In storage of cells preparation with PFOC content from 15 to 59 vol.% is added to culture medium with controlled value pH=7.0-7.4, temperature 37°C in CO2 incubator atmosphere or with low pH<6.0, temperature 20°C in air atmosphere. In cryoconservation resuspended in fresh culture medium cells are placed into medium, which contains preparation with PFOC content 59-70 vol.%. After that, cooling is carried out in two stages: first at rate 1 degree per min to -80°C, then with maximally possibly rate to temperature -196°C. In cultivation of cells as medium component preparation with PFOC content from 15 to 70 vol.% is used.

EFFECT: cell cultures after cryoconservation, storage or cultivation in presence of PFOC-containing gels have on average 90% of viable cells, are easily separated from gel, re-inoculated in standard conditions, divided and fully preserve their functions and ability to differentiate.

16 cl, 23 dwg, 1 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: presented cell line is produced from primarily trypsinized tissue of Siberial sturgeon fins by means of long-term passivation in the medium 199 with Hanks salts with 10% embryonal serum and is deposited in the Russian Academy of Agricultural Sciences (agricultural animals) under the No.76. The line may be used in laboratory research in production of virus vaccines and diagnostic preparations for extraction, accumulation, titration and study of a virus haemorrhagic septicemia virus (VHSV) of salmon fishes, infectious haemopoetic necrosis (IHNV) of salmon fish tissue, spring viremia of carps (SVCV), carp iridovirus (CCIV) and herpes of sturgeon virus (SbSHV).

EFFECT: cell line has a high coefficient of passage, which makes it possible to accumulate high number of biomaterial for a short period of time, and is a promising heterogenerous system for cultivation of viruses from fishes of non-sturgeon breeds, preventing development of non-specific reactions due to absence of antibodies to cell proteins.

FIELD: biotechnologies.

SUBSTANCE: permanent cell line of calf kidney Bos taurus RBT, deposited in 2010 into the Russian Collection of Cell Cultures (V) (Russian Academy of Agricultural Sciences, agricultural animals) under the No.74. The RBT line is produced from primarily trypsinized kidney cells of a 2-month calf in process of 65 serial passages in a single layer in the medium of 0.4 GLA on Earl with 10% foetal serum of cattle. Forms a population of epithelial cells, which is homogeneous by morphology, with considerable polymorphism in karyotype. At the same time population of cells with 51 chromosomes makes 32%, in contrast to normal karyotype Bos taurus (60 chromosomes), in RBT there are 9 metacentric and 1 submetacentric chromosomes, the number of polyploids is not more than 2%.

EFFECT: RBT line has high sensitivity to a corona virus, a virus of infectious rhinotracheitis, a plague virus and virus diarrhoea, and also high proliferative activity and ability for growth in rotary vessels.

4 dwg, 5 tbl, 4 ex

FIELD: organic chemistry, natural compounds, medicine, oncology.

SUBSTANCE: invention represents new saponin mixtures used for inhibition of initiation and activation of mammalian epithelial cell in pre-malignant or malignant state, for stimulation of apoptosis of mammalian malignant cell, prophylaxis of anomalous proliferation of mammalian epithelial cell, for treatment of inflammatory and regulation of angiogenesis in mammal. These mixtures are isolated form plants of species Acacia victoriae. Also, invention relates to methods for their applying. These compounds can comprise triterpene component, such as acacic or oleanolic acid to which oligosaccharides and monoterpenoid components are joined. Mixtures and compounds elicit properties associated with regulation of apoptosis and cytotoxicity of cells and strong anti-tumor effect with respect to different tumor cells.

EFFECT: valuable medicinal properties of compositions.

43 cl, 53 tbl, 50 dwg, 44 ex

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