RU2460786C1 - Method for preparing blood serum of grown cattle for animal and human cell culture - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for preparing blood serum involves two stages. At the first stage, a native blood serum of grown cattle is produced. Blood is sampled in the aseptic environment exclusively from male calve's hearts in polycarbonate bottles pre-filled with 5% sodium chloride 100-200 ml. The bottles are filled with blood approximately in half and placed in a cold storage at temperature +1-+2°C, herewith being inclined at 35° for 72 hours. Then serum is drained off and pre-purified by EKS filters at temperature +2-+6°C. Thereafter polyethylene cans are filled with serum and kept at temperature -(15-20)°C for one week or more. The second stage consists in removing the cell growth inhibiting substances from native blood serum by single-stage freezing and thawing. Upper light serum layers are collected starting from above to a boundary line with hemolysed layers; it is followed by pre-purification through EKS deep-bed filters. Sterilisation filtration is conducted through membrane filters at temperature +2-+6°C.

EFFECT: method enables producing serum applicable for animal and human cell culture, including hybridomas, enhancing serum yield in relation to blood volume taken, improving its quality.

6 tbl, 5 ex

Description

The invention relates to biotechnology and can be used in the cultivation of cell lines of animals, humans, as well as hybridomas.

In well-known literature, the technology for producing this blood serum is described very superficially (Methods of cell culture for biochemists / R.A. Adams: Transl. From English. - M .: Mir. - 1983. - 264 p .; Animal cell in culture / Under Edited by Prof. L.P. Dyakonov. - M.: Sputnik + Publishing House. - 2009. - 656 p.), and an Internet source (http: wwwleitran.ru/page.php? al = sivorotka) generally does not carry any information about its production.

There is a known method for producing blood serum of adult cattle, which we took as a close analogue: by taking blood from animals from the heart or jugular vein, sludge, collecting and sterilizing filtration of the final product, described in the reference manual edited by B.I. Antonov (Laboratory research in veterinary medicine: Reference book / B.I. Antonov, V. Borisov, P.M. Volkov and others, under the editorship of B.I. Antonov. - M .: Agropromizdat. - 1986. - 352 p.). But in this analogue there are significant disadvantages:

1. Blood is taken from both cows and gobies (it is well known that the blood serum of animals differs significantly from their gender);

2. Blood sampling is carried out in two ways: from the heart without observing aseptic and from the jugular vein with observing aseptic, but the duration of filling 10-liter bottles to 5-liter mark is 6-10 minutes or more;

3. Blood is filled only in glass bottles, which are often beaten and are a potential source of injury to employees working with them, in addition, they are expensive;

4. Serum sediment is carried out in a refrigerating chamber for 24-48 hours at a temperature of + 6- + 10 ° С, and at such a high temperature there is a rapid reproduction of vulgar microflora, leading to a decrease in the quality of the final product;

5. The yield of serum is insignificant (20-25% of the volume of blood taken) due to the use of physiological NaCl solution to wet the walls of the bottles and take blood from the jugular vein;

6. The collected serum is heated to + 30- + 37 ° C, for pre-cleaning through EKS depth filters, and then sterilizing filtration through millipore membrane filters. Taking into account that whey filtration is a long process, at high temperature there is a rapid accumulation of microflora in it, which negatively affects its quality;

7. Due to the loss of proteins and for more reliable sterilization, 2-3-fold filtration of whey is recommended, which increases the preparation time of the final product and the cost of consumables;

8. The serum obtained by this technology does not correspond to a number of physicochemical parameters and is unsuitable for culturing animal cells.

Another analogue, but only in terms of quality characteristics, we see fetal blood serum of cow fruits, which is still considered the best biological additive in nutrient media for growing animal and human cell cultures (TU FS 42-7BC-85), approved by the USSR Ministry of Health 4 April 1985

For a clear idea of the purpose of the invention, Table 1 shows the concentrations of cell growth inhibitors and other quality indicators in natural blood serum of adult cattle according to TU 49.24 b-82 and in native blood serum of cow fruits according to TU FS 42-7BC-85, approved by the Ministry of Health USSR April 4, 1985.

Table 1 Indicator Blood serum of adult cattle according to TU 49.24 b-82 Blood serum of cow embryos according to TU FS 42-7BC-85 Transparency (535 nm) Not more than 0.450 units. optical density Not more than 0.300 units. optical density PH 7.0-7.8 7.6 ± 0.2 Free hemoglobin No more than 60 mg / dl No more than 50 mg / dl Common lipids 40-200 mg / dl 30-50 mg / dl Cholesterol 90-170 mg / dl 10-20 mg / dl Total protein, including: 6.3-7.7 g / dl 4.5 ± 2.5 g / dl albumin 55 ± 5% α-globulins 35 ± 5% β-globulins 10 ± 5% gamma globulins Below 1% Growth-stimulating activity on transplantable cell lines by proliferation index Not less than 4.0-5.0 No less than 6.0-7.0

The closest in technical solution is a method for producing blood serum of adult cattle, which is described in RF patent No. 1813783 "Method for producing animal blood serum for culturing animal tissue cells" dated March 5, 1994, which we have taken as a prototype of the present invention.

The technological process of this patent includes freezing native animal blood serums at - (15-20) ° C for 24-48 hours, followed by thawing them at room temperature, collecting light layers of serum, starting from the top, to the interface between them and hemolyzed fractions, sterilizing filtration and quality control of the preparations obtained.

The objective of the invention is to significantly increase the yield of serum to the volume of blood taken, improve its quality, as well as obtain the blood serum of adult cattle suitable for culturing animal and human cells, including hybridomas, by removing individual inhibitors from the native blood serum of bovine cell growth.

The problem is solved by us by the fact that in the method of obtaining blood serum from adult cattle, significant, directly opposite to the existing literature sources, changes in the technology of its production were carried out.

In contrast to this invention, in our project, the percentage of gobies removed during thawing of blood serum is reduced to 5-10%, in addition, blood serum is pre-filtered at + 2- + 6 ° C through EKS filters, and then through sterilizing membrane filters of any reliable company, for example: Millipore Intertec USA, its branch in Ireland, or Vladisart Russia. This method of obtaining blood serum of adult cattle for the cultivation of cells of animal origin includes two stages:

The 1st stage consists in a radical change in the production of native blood serum of adult cattle, which is a very significant moment in the change in the technological process: blood is taken only from bulls and only from the heart by aseptic special systems in glass or polycarbonate bottles with a capacity of 10-20 liters into which a 5% solution of sodium chloride was previously poured in a volume of 100-200 ml. The bottles are filled with blood, approximately half their volume, left to stand upright for 2-3 hours in the slaughterhouse, and then transferred to a refrigerator with a temperature of + 1- + 2 ° C and placed at an angle of 35 ° for 72 hours, after why a special system collects the settled serum (its output to the volume of blood taken exceeds 40%), its preliminary cleaning with EKS filters at a temperature of + 2- + 6 ° C, which is filled with polyethylene canisters with a capacity of 20 liters and kept at a temperature of - (15 20) ° C for at least a week.

The 2nd stage consists in the removal of substances that inhibit cell growth (residual hemoglobin, total lipids, γ-globulins, etc.) from the native blood serum of calves by freezing and thawing, collecting the upper light layers of serum, starting from the top, to its border with hemolized fractions pre-cleaning through EKS depth filters, and then sterilizing filtration through membrane filters with 400-450 ml packaging in 500 ml bottles, monitoring sterility and biological activity.

A significant difference between the method of obtaining blood serum of cattle is that we use the technology of its production directly opposite to existing methods, the rationale of which is given below by a number of examples.

Example 1. Taking blood from gobies and separating serum.

Blood sampling from bulls, in order to obtain serum, was carried out with aseptic observance using two aseptic methods: from the jugular vein and heart into 10-liter glass or 20-liter polycarbonate bottles, into which 100-200 ml of a 5% sodium chloride solution is poured .

We found that the time for filling 10-liter bottles to the 5-liter mark when taking blood from the heart of bulls was 1-2 minutes, and when taking from the jugular vein - 8-10 minutes.

After taking blood in a bottle, they were left to stand upright at room temperature for 2 hours, and then placed in a refrigerator on racks at an angle of 35 ° for 72 hours at a temperature of + 1- + 2 ° С. The location of the bottles with blood at an angle of 35 ° promotes better retraction of the clots, and the temperature of the serum sludge + 1- + 2 ° C prevents the multiplication of microflora that accidentally enters the bloodstream.

Example 2. A comparative study of the separation of serum of calves, depending on the place of blood collection and the percentage of sodium chloride in solution.

The results of this experiment are shown in table 2. In the experiment, three options. In each version, 20 bottles with a blood volume of 4.65 to 5.10 liters. In option 1, blood was taken from the jugular vein into 10-liter bottles, in which there were 100 ml of a 1% NaCl solution. In option 2, blood was taken from the heart in bottles with 1% NaCl solution of 100 ml, and in option 3 blood was also taken from the heart, but a 5% solution of 100 ml of NaCl was poured into the bottle.

table 2 Experience option Blood volume in bottles, l Suctioned whey liters % reliability, P No. M ± m M ± m M ± m one 4.65 ± 0.32 1.17 ± 0.13 25.06 ± 1.28 2 5.10 ± 0.24 1.80 ± 0.15 35.30 ± 1.62 ≤0.01 3 4.95 ± 0.20 2.07 ± 0.19 41.82 ± 1.70 ≤0.001

As follows from table 2, the output of serum from blood taken from the heart of bulls in a bottle with a 1% solution of NaCl is significantly higher than from blood taken from the jugular vein. The use of a hypertonic 5% NaCl solution, poured into a bottle to wet their walls, contributes to an even greater output of serum from blood taken from the heart of gobies. A high yield of serum by the proposed method takes place in a production environment. So, for example, 150 liters of blood were taken in 30 bottles, and after sedimentation 63 liters of serum or 42% of the blood volume were aspirated.

Example 3. Qualitative indicators of blood serum of gobies and cows.

The study of blood serum of gobies and cows was carried out according to the following qualitative indicators: optical density, total protein and residual hemoglobin content, growth-stimulating activity on transplanted cell cultures. In gobies weighing 300-400 kg and cows 400-450 kg, blood was taken from the heart into 10-liter bottles containing 100 ml of a 5% NaCl solution; up to a 5-liter mark, the serum was defended for 72 hours in a refrigerator at a temperature + 1- + 2 ° С, after which serum samples were taken, which were frozen, thawed and sterilized through membrane filters.

The transparency (optical density), total protein and residual hemoglobin of the blood serum of individual animals were established immediately after taking serum samples, as well as after centrifuging it at 1500 rpm for 10 minutes. Total protein was determined by a biuret reaction, and residual hemoglobin was determined by the hemiglobin cyanide method after centrifugation of serum. Growth-stimulating activity of blood serum was evaluated on SPEV and MDBK cell cultures by proliferation index after 72-hour growth at a seeding density of 40,000 cells / cm ² and by the multiplicity of increase in the cell population after 5-day growth at a seeding density of 20,000 cells / cm ² . Cells were cultured in Carrel vials with an inoculated area of 8 cm ² in Eagle MEM medium with glutamine, kanamycin and 10% blood serum.

Blood was taken from 40 bulls and 40 cows. The results of these experiments are shown in table 3. After a 72-hour sludge, the optical density of the blood serum of cows exceeded that of bulls by more than 2 times. Almost the same ratio was noted for the variability of this indicator. After centrifugation, the optical density of the blood serum of cows decreased, although it remained slightly higher than in gobies. In the blood serum of cows, higher concentrations of total protein and residual hemoglobin were recorded compared with these indicators of blood serum of gobies. The coefficient of variation in residual hemoglobin of blood serum of cows exceeded 54%. The high optical density and concentration of residual hemoglobin of blood serum of cows, apparently, are associated with less resistance to destruction of their red blood cells. The growth-promoting activity of cow serum (according to the proliferation index and the rate of increase in the cell population) in SPEV and MDBK cell cultures is inferior to the biological activity of that obtained from the blood of gobies.

Table 3 Indicator Blood serum gobies
M ± m cows
M ± m Transparency, units optical density, after: serum collection: 0.258 ± 0.015 0.538 ± 0.035 the coefficient of variation, % 22.02 38.07 credibility P <0.001 serum centrifugation: 0.152 ± 0.006 0.201 ± 0.009 the coefficient of variation, % 20.30 22.52 credibility P <0.001 Total protein, g / dl: 7.04 ± 0.12 7.62 ± 0.17 the coefficient of variation, % 6.30 9.24 credibility P <0.001 Residual hemoglobin, mg / dl: 54.1 ± 3.2 101.2 ± 10.8 the coefficient of variation, % 27.25 54.03 credibility P <0.001 Growth-stimulating activity of blood serum on cell cultures by: proliferation index 6.40-6.96 5.85-6.47 the multiplication factor of the cell population 10.3-11.2 9.5-10.2

Example 4. The advantage of taking blood from the heart of bulls to obtain serum intended for cell cultivation.

The purpose of the study was to identify the features of the biochemical composition of sera obtained from blood taken from the jugular vein, carotid artery and heart of gobies, as well as to evaluate their growth properties on transplanted cell cultures. To obtain serum from 18 calves weighing 300-400 kg, 300 ml of blood was taken aseptically from the jugular vein, carotid artery and heart into 500 ml vials with 5 ml of 5% NaCl solution (based on 2 vials per animal) and placed them at an angle of 35 ° into the refrigerator with a temperature of + 2 ° C for 48 hours. Settled serum from each pair of vials was collected separately, centrifuged and frozen at -15 ° C, after taking samples for physico-biochemical studies. Before sterilizing filtration through membrane filters, blood serum obtained from the heart, carotid artery and jugular vein was mixed separately in 200 ml.

Determination of pH and optical density, control on sterility, as well as assessment of growth-promoting properties of blood serum was carried out by conventional methods. Total protein was determined by a biuret reaction, residual hemoglobin was determined by the hemiglobin cyanide method, cholesterol was measured using Konelab reagents (Finland), in addition, low molecular weight nitrogenous substances (creatinine, urea and uric acid), pigments (direct and total bilirubin), enzymes were controlled (LDH, CPK, glutamine transferase, amylase, AcAT, alkaline phosphatase, AlAT), inorganic substances (calcium, iron, phosphorus), glucose using reagents from Rendox on a Daytona Rendox instrument (Great Britain) and serum electrophoresis was performed proteins on filters atsetattselyuloznyh Vladisart type - FMATS-0,45- (80 · 100) -11106 with a pore size of 0.45 microns. Digital material was subjected to computer static processing with calculation of arithmetic mean (M), arithmetic mean error (m), standard deviation (σ), coefficient of variation (C _V ), and significance of differences (P) of the results using Microsoft Excel. The results of the listed studies of blood serum of calves are shown in table 4.

Table 4 Indicators Heart Artery Vein M ± m C _V,% M ± m C _V,% R M ± m C _V,% R pH 7.6 ± 0.02 0.8 7.71 ± 0.02 0.68 ≤0.01 7.56 ± 0.02 0.58 ≤0.05 Transparency, units opt. pl. 0.267 ± 0.01 4.9 0.279 ± 0.01 4.16 ≤0.01 0.310 ± 0.01 5.52 ≤0.01 Inorganic substances: phosphorus, mmol / l 1.64 ± 0.09 15.4 1.40 ± 0.11 22.39 ≤0.01 1.63 ± 0.14 23.51 > 0.05 N.D calcium, mmol / l 2.01 ± 0.06 9.0 1.82 ± 0.07 9.97 ≤0.05 1.99 ± 0.07 9.53 > 0.05 N.D iron, μm / l 9.60 ± 0.53 15.6 9.75 ± 0.43 12.37 N.D. 10.91 ± 0.52 13.5 ≤0.05 Residual hemoglobin, mg / dl 54.8 ± 1.4 7.2 61.2 ± 2.07 9.57 ≤0.05 70.4 ± 1.80 7.21 ≤0.05 Total lipids, g / l
including: 1.98 ± 0.08 10.8 2.26 ± 0.09 10.29 ≤0.01 1.84 ± 0.07 10.47 ≤0.01 cholesterol, mmol / l 2.64 ± 0.24 25.6 2.97 ± 0.25 23.49 ≤0.01 3.18 ± 0.26 23.54 ≤0.05 triglycerides, mmol / l 0.34 ± 0.03 23,2 0.40 ± 0.03 21.10 ≤0.05 0.47 ± 0.04 22.75 ≤0.05 Total protein, g / l
including protein fractions,% 73.4 ± 2.85 10.9 82.3 ± 3.07 10.54 ≤0.05 79.99 ± 2.91 9.48 ≤0.05 Albumin 44.47 ± 1.12 7.1 42.99 ± 1.19 7.85 ≤0.01 42.40 ± 1.11 7.41 ≤0.05 globulins: α- 8.63 ± 0.18 5.8 9.40 ± 0.22 6.69 ≤0.01 9.04 ± 0.29 8.93 ≤0.05 β- 10.56 ± 0.22 5.8 10.51 ± 0.34 8.39 ≤0.05 10.22 ± 0.16 4.42 ≤0.05 γ- 36.4 ± 1.12 8.9 37.1 ± 1.23 9.66 ≤0.05 38.30 ± 1.21 8.89 ≤0.05 Enzymes, U / L: LDH 2191 ± 119 15.4 2250 ± 47 10.63 ≤0.01 1918 ± 261 38.52 ≤0.05 KFK 170 ± 6 9.2 179 ± 12 19.03 ≤0.05 169 ± 11 17.89 > 0.05 N.D. glutamine transferase 21.02 ± 1.1 15,5 21.80 ± 1.46 18.96 ≤0.05 22.17 ± 1.31 16.72 ≤0.05 amylase 17.58 ± 1.4 23.0 19.87 ± 1.26 17.15 ≤0.05 21.58 ± 1.15 15.10 ≤0.05 AsAT 91.88 ± 6.69 20.6 103.90 ± 9.13 24.86 ≤0.05 99.13 ± 7.06 20.15 ≤0.01 alkaline phosphatase 96.92 ± 10.06 29.4 98.53 ± 9.15 26.27 ≤0.01 106.91 ± 10.4 27.53 ≤0.01 AlAT 34.66 ± 1.97 16.1 39.73 ± 1.93 13.75 ≤0.05 36.67 ± 1.86 14.35 ≤0.01 Bilirubin, μM / L: straight 0.63 ± 0.07 33,2 0.55 ± 0.07 38.24 ≤0.05 0.48 ± 0.09 53.0 ≤0.01 common 3.42 ± 0.29 24.2 3.71 ± 0.17 13.13 ≤0.01 3.19 ± 0.27 24.16 ≤0.05 Glucose, mmol / l 27.58 ± 1.41 14,4 11.28 ± 0.91 22.73 ≤0.01 12.38 ± 1.06 24.32 ≤0.01 Low molecular weight nitrogenous substances: creatinine, μM / L 138.11 ± 8.66 17.7 150.79 ± 8.3 15,56 ≤0.01 161.71 ± 6.5 11.36 ≤0.05 urea, units / l 4.78 ± 0.25 14.5 4.56 ± 0.24 14.93 ≤0.01 4.95 ± 0.23 13.05 ≤0.01 uric acid, m / l 220.0 ± 14.8 9.1 170.0 ± 16.9 28.18 ≤0.05 156.0 ± 10.5 18.96 ≤0.05

To assess the growth-promoting properties of blood serum of gobies, transplantable renal lines of SPEV (pig embryo) and MDBK (cow embryo) were used as cell models. Sterile blood serum from the jugular vein, carotid artery, and heart of gobies was added at 10% concentration to Eagle's MEM medium with glutamine and kanamycin. To calculate the proliferation index (PI), the cell suspension was dispersed in 2 ml each with a sowing area of 3 cm ² at a seed density of 42,000 cells / cm ² . After 72 hours of growth, the number of cells in the resulting monolayer was counted. Eight experiments were carried out with each culture; in the experiments, the number of cells was counted in the Goryaev chamber 4 times. The results of the proliferative activity of SPEV and MDBK cells on media by blood serum of gobies from different parts of the circulatory system are shown in table 5.

Table 5 Blood out Cell culture SPEV MDBK IP C _V,% R IP C _V,% R Hearts 4.83 ± 0.15 3.3 - 5.05 ± 0.10 1,6 - Carotid artery 4.42 ± 0.18 4,5 ≤0.05 4.71 ± 0.13 2.2 ≤0.05 Jugular vein 4.01 ± 0.14 5.2 ≤0.05 4.16 ± 0.15 2.5 ≤0.05

The proliferation of cell cultures grown on media with sera obtained from blood taken from the carotid artery, jugular vein was lower than that of cells grown on media with serum from the heart, which is undoubtedly due to their different biochemical composition.

Example 5. Removing from the native blood serum of bull calves substances that inhibit cell growth, by freezing and thawing. Individual serum samples have different biochemical composition, depending on a number of factors: feeding and keeping animals, season of the year, breed, gender, etc. Freezing and thawing can produce blood serum for cell cultures with a predicted biochemical composition.

Of the whole variety of obtaining serum by the method of freezing and thawing, we have preferred the option that includes: one freezing of the native blood serum of bulls at - (15-20) ° С for at least 7 days, followed by thawing them at + 20- + 40 ° С, collecting light layers of serums, starting from the top, to the interface between them and hemolized layers, preliminary, and then sterilizing filtration and quality control of the preparations obtained.

The main physical and chemical indicators of bovine serum in comparison with native serum are lower, namely: protein concentration - 1.19-1.94; free hemoglobin - in 1.32-2.94; total lipids - 1.56-2.64, including total cholesterol - 1.37-2.53 times.

According to the results of electrophoresis of serum proteins in a polyacrylamide gel, in blood serum obtained by freezing and thawing, the relative concentration of albumin increases in comparison with native blood serum and the concentration of γ-globulins decreases, while the concentration of α- and β-globulins remains almost unchanged.

The indicators of the composition of bovine serum obtained by the method of freezing and thawing are presented in table 6.

Table 6 Indicators Bovine serum Appearance Transparent straw yellow liquid Transparency, units opt. density 0.183 ± 0.027 pH 7.82 ± 0.04 Residual hemoglobin, mg / dl 39.2 ± 5.1 Total lipids, mg / dl 78.1 ± 10.1 Cholesterol, mg / dl 34.0 ± 3.3 Total protein, g / dl 4.94 ± 0.43 including: albumin 1.87 ± 0.28 α-globulins 1,60 ± 0,13 β-globulins 0.67 ± 0.18 gamma globulins 1.80 ± 0.20 Growth stimulating activity by

transplantable cell line proliferation index 7.32-7.81 Possible contaminants: bacteria are absent mycoplasmas are absent viruses are absent

It should be noted that the proliferation index of transplantable cultures of PK-15, NGUK-1, VERO, MDBK, LEK cells grown on GLA and MEM Needle with bovine blood serum obtained by freezing and thawing by 0.50-2.40 (more often 1.5-2.0) more than with the native blood serum of gobies.

Claims (1)

A method for producing blood serum of adult cattle for culturing animal and human cells, including blood sampling, separation, collection and 2-3-fold sterilizing filtering of serum, characterized in that blood sampling is carried out aseptically only in gobies and only from the heart into polycarbonate bottles with a hypertonic NaCl solution, the whey is separated for 72 hours at + 1 ... + 2 ° С, the serum is collected and cleaned with EKS filters, followed by freezing in polyethylene canisters at -15 ... -20 ° С for at least 7 And then thawed and removed by 5% lower hemolyzed layers and light-1 serum was subjected to sterilizing filtration times at 2 + ... + 6 ° C.