CN111154710A - Bovine serum freezing high-dose irradiation method - Google Patents

Abstract

The invention provides a frozen high dose>25kGy)Co⁶⁰A method of irradiating bovine serum starting material or finished serum. The method uses the sterilization effect of viruses (BVDV), mycoplasma (mycoplasma oralis) and bacteria (escherichia coli and staphylococcus aureus) before and after irradiation through biological safety, and evaluates the culture effect of serum on a plurality of cells before and after irradiation. The applicant integrates the results of cell culture and microorganism inactivation experiments, determines a high-dose irradiation (25-30kGy) process according to the regulations of Chinese pharmacopoeia, American FDA and European Union, and selects proper serum package and outer package to ensure that the serum is at a freezing temperature during irradiation.

Description

Bovine serum freezing high-dose irradiation method

Technical Field

The invention relates to the technical field of serum, and particularly discloses a frozen high-dose (A)>25kGy)Co⁶⁰A method of irradiating bovine serum starting material or finished serum.

Background

The blood contains blood plasma and blood cells, the blood is serum after removing the blood cells and fibrin, the main function of the serum is to carry the blood cells, transport substances required for maintaining the life activities of human bodies, waste products generated in vivo and the like, the serum is equivalent to the intercellular substance of connective tissue, the serum is an important component of the blood and is yellowish liquid (containing bilirubin), the water content of the chemical components of the serum is 90-92%, the other 10% of the chemical components are mainly solute plasma protein, and the content of the newborn bovine serum is about 3.5-5%. Serum also contains electrolytes, nutrients, enzymes, hormones, cholesterol and other important components.

Bovine serum is an important raw material in vaccine production and life science research. The stable production and supply of bovine serum relate to the safety and stability of vaccine production. Also relates to important reagents for successfully completing life science research.

Co is mostly adopted for bovine serum irradiation in the current market⁶⁰The irradiation process of (1). Co⁶⁰The irradiation of (2) is easy to design because of its strong penetration ability. Co⁶⁰The irradiation time is generally longer, and according to the energy of the cobalt source of most irradiation companies,>the irradiation dose of 25kGy needs more than 10 hours, the protein concentration is reduced after bovine serum is irradiated, the turbidity and the hydrophobicity are increased, and a large amount of effective components such as protein are damaged and lost under the damage of gamma rays and the like. Serum melting in the irradiation process easily causes degradation loss of growth factors and adherence factors in the serum. If Co is present⁶⁰The high-dose irradiation process is not suitable, the quality of serum is easily reduced, and finally a comprehensive cell evaluation system cannot be passed. In order to meet the requirement of cell culture and subsequent application of bovine serum, the domestic irradiation dose adopted before is generally below 15 kGy. However, according to the Chinese pharmacopoeia, the United states FDA and the pharmacopoeia of the European Union, the irradiation dose in the sterilization level irradiation process is mentioned>25kGy can be required. Since the 0.1 μm filtration used for bovine serum production did not remove the virus, only the high Co dose was used⁶⁰Irradiation ensures that bovine serum, the animal-derived material, is free of viral risk.

Disclosure of Invention

To overcome the above problems, the present invention adopts the following embodiments:

the invention provides a bovine serum irradiation method which is characterized in that the method uses high-dose Co under refrigeration⁶⁰Irradiation is carried out.

Preferably, the high dose is >25 kGy.

Preferably, the high dose is 25-30 kGy.

Preferably, the irradiation is performed at full-time freezing.

Preferably, the method comprises the steps of properly placing the serum bottle and filling the periphery of the serum bottle with an ice bag.

Preferably, the method comprises customizing a suitable foam box, the thickness of the foam box being >2.8 cm.

Preferably, the one foam box bottles 20 bottles, and the bottling position and the ice bag position and number are fixed.

Preferably, the number of the ice bags is 30.

Preferably, the irradiation is performed after bovine serum packaging, cold chain transportation.

The invention also provides bovine serum, which is characterized in that the bovine serum is irradiated by the method.

The process adopts bovine serum frozen high-dose Co after packaging and cold chain transportation⁶⁰Irradiation (>25kGy), frozen serum Co⁶⁰In the high-dose irradiation process, a serum bottle is reasonably placed (the periphery of the serum bottle is filled with an ice bag), and a proper foam box (with the thickness being equal to that of the foam box) is customized>2.8cm), one foam box is filled with 20 bottles, and the bottling position and the ice bag position and number are fixed (30 ice bags). The foam box is verified that frozen and compacted serum in a certain bottle number can not be dissolved within 48 hours. Can ensure the whole course refrigeration of the bovine serum in the irradiation process and ensure the loss of the effective components of the bovine serum in the irradiation process as little as possible. And placing an irradiation indicator at the monitoring point to observe whether the irradiation dose is proper or not at each time, and controlling the stability of the irradiation process.

Co of 0-40kGy is used for process verification⁶⁰And carrying out irradiation treatment on the bovine serum finished product by irradiation dose. Adding excess BVDV, mycoplasma and bacteria (Escherichia coli, Staphylococcus aureus) before irradiationCocci) and after a certain irradiation dose (15-20kGy), various verification contaminated microorganisms are added into the serum after the irradiation is detected and the removal state of the contaminated microorganisms is achieved. 25-30kGy high-dose Co for serum cell culture comprehensive system verification and evaluation before and after irradiation with different irradiation doses⁶⁰Irradiation has no negative influence on Vero and MRC5 cell morphology, adherence, continuous passage and the like in cell culture, and has no influence on SP2/0 cell cloning rate and cell growth curve. Irradiation doses of 30-35kGy have an effect on MRC5 and Vero cell morphology, proliferation and serial passaging.

The invention has the following remarkable technical effects:

high freezing dose of the final product (>25 kGy; usually 25 to 30kGy) Co⁶⁰Irradiation can ensure the safety of serum and the use requirement on the aspect of cell culture. Meanwhile, the irradiation dose meets the pharmacopoeia safety standards in China and abroad.

Drawings

FIG. 1 is a photograph of MRC5 cells serially passaged for the third generation.

FIG. 2 is a picture of Vero cells serially passaged for the third generation.

FIG. 3 is a graph showing the growth of SP2/0-Ag14 cells.

FIG. 4 shows Co⁶⁰And (3) verifying the effect of the BVDV virus in the irradiation inactivated cattle serum.

FIG. 5 is a photograph of Escherichia coli phage.

FIG. 6 shows the results of a cell culture staining experiment.

FIG. 7 shows the detection of Co by the culture method⁶⁰And (5) irradiating to inactivate the Escherichia coli.

FIG. 8 shows the detection of Co by the culture method⁶⁰Results of irradiation inactivation of staphylococcus aureus.

Detailed Description

The present invention is further illustrated by the following examples in which the starting materials, reagents, apparatus and equipment are well known to those skilled in the art and are either commercially available or readily available or synthesized.

Example 1: the bovine serum finished product or raw material is processed by Co with different dosages⁶⁰After irradiation, cell culturesThe lines (MRC5 cells, Vero cells and SP2/0 cells) were evaluated for the suitability of the irradiating agent.

Three batches of newborn bovine serum which is not irradiated and has no BVDV infection and good cell growth after being checked are selected, mixed, filtered and then subpackaged into 500ml PET bottles. Number of samples per batch: bottle 21, filling: 500 ml/bottle, respectively numbered: no irradiation (FZ-001, FZ-002 and FZ-003); the irradiation dose is 10kGy (FZ-101, FZ-102 and FZ-103); the irradiation dose is 15kGy (FZ-151, FZ-152 and FZ-153); the irradiation dose is 20kGy (FZ-201, FZ-202 and FZ-203); the irradiation dose is 25kGy (FZ-251, FZ-252 and FZ-253); the irradiation dose is 30kGy (FZ-301, FZ-302 and FZ-303); the irradiation dose is 35kGy (FZ-351, FZ-352 and FZ-353); the irradiation dose is 40kGy (FZ-401, FZ-402 and FZ-403); and sticking an irradiation mark on each serum bottle. The dose of the irradiation station can be controlled to be +5kGy, namely the required irradiation dose is 25kGy, the irradiation dose can be ensured to be 25-30kGy, and the actual irradiation dose is detected by using the indicator in each irradiation. The following demonstrates that the sample design for the experimental irradiation dose is consistent with the experiment.

Co according to our design⁶⁰The finished product bovine serum and the raw material bovine serum placing device of the irradiated bovine serum have the cell verification group Co designed by experiments⁶⁰And (5) irradiating the cells under different doses to control the culture effect.

Cell culture experiments: the serum without irradiation is used as a control, and the serum after irradiation is used as an experimental sample. And performing continuous subculture on VERO and MRC5 cells, and observing cell adherence, cell proliferation and cell morphology.

Serial passaging evaluation of MRC5 cells

The operation process comprises the following steps: the cells are passed for 3 generations in total, the first 2 generation is passed by using a T25 glass square bottle for 1:2 minutes, 2 bottles/sample and 10% serum; the 3 rd generation was seeded in corning T25 culture flask with 5X 10 cell concentration⁴The number of the cells per ml, 2 bottles per sample, 10% serum, digestion count after growing a monolayer, and calculating the 3 rd generation relative growth rate according to the result of the control serum; the cell morphology and growth were observed daily and recorded by photography.

The third generation picture is shown in fig. 1.

From cell culture data, Co of 25-30kGy⁶⁰Irradiation is the highest irradiation dose suitable for cell culture, and the serum has the influence on continuous passage of the MRC5 diploid cells under the irradiation dose of 30-35kGy, the cell morphology is poor, and the cell proliferation speed is slow.

Continuous passage of Vero cells to the third generation

The operation process comprises the following steps: the cells are passed for 3 generations in total, the first 2 generation is passed by using a T25 glass square bottle for 1:2 minutes, 2 bottles/sample and 10% serum; the 3 rd generation is planted in a Corning T25 culture bottle, the concentration of inoculated cells is 5 multiplied by 104/ml, 2 bottles/sample and 10% serum are digested and counted after a monolayer is full, and the 3 rd generation relative growth rate is calculated according to the result of the control serum; the cell morphology and growth were observed daily, photographed and recorded.

See fig. 2 for third generation pictures.

From cell culture data, Co of 25-30kGy⁶⁰Irradiation is the highest suitable irradiation dose for cell culture, exceeding this dose (30-35kGy) has an effect on Vero cell morphology, adherence and serial passage.

3. Cell cloning efficiency, doubling experiments:

using SP2/0-Ag14 cells to carry out growth curve and cloning rate test experiments, and calculating doubling time and cloning rate;

cell cloning efficiency, doubling experiments: the cell culture irradiation sample FZ251-253 is cultured by SP2/0 cell suspension culture with the same control sample, the average clone rate of each test sample cell is more than 85%, the maximum proliferation concentration of the cell is 1.46-1.52 multiplied by 106 cells/ml, the cell doubling time is 16.4-16.8 h, and no obvious difference is found. This validation was performed three times to check reproducibility. FZ301-303, the cell cloning rate is reduced, the average cell cloning rate is about 76%, and the cell doubling time is increased to 17.1-18.2 h. The irradiation dose lower than 25kGy had no influence on the cloning efficiency and proliferation in cell culture.

1) Cloning efficiency of SP2/0-Ag14 cells

The operation is as follows:

a. culture solution: RPMI1640 culture medium, serum ratio: 10 percent;

b. diluting the cells by a limiting dilution method, inoculating the cells to a 96-well cell culture plate according to the concentration of 5-15 cells per well, inoculating at least 48 wells per plate, culturing at 37 ℃ for 24 hours by 5% carbon dioxide, recording the number of wells with only 1 cell colony, observing after one week, calculating the cloning rate to be not lower than 70%.

c. The cloning efficiency is A/B × 100%

In the formula, A represents the number of positive wells for single cell growth (green part (+);

b is the total number of wells of 24H single cells (yellow part (upper half A-H) in the table below).

The cloning efficiency was (52 ÷ 56) × 100%

And (3) counting the cloning rate of different doses:

the results are statistical data of the cloning efficiency after 3 irradiations.

From the results, the samples with the irradiation dose of 25-30kGy were acceptable, and 3 samples with the irradiation dose of 30-35kGy were acceptable in 2 batches and not acceptable in 1 batch. 25-30kGy is the irradiation dose which is qualified by the cell cloning rate.

2) SP2/0-Ag14 cell growth curve

SP2/0-Ag14 was inoculated into 8 flasks per sample, 1 flask was removed every 24 hours, and cell counts were performed

The growth curve is shown in figure 3.

The growth curve results are given in the following table:

	irradiation of 0kGy	25-30kGy irradiation	30-35kGy irradiation
				Maximum cell proliferation concentration	1.52×106Per ml	1.50×106Per ml	1.41×106Per ml
Cell doubling time (h)	16.4	16.5	17.6

The experimental result shows that when the irradiation dose reaches 30-35kGy, the maximum proliferation concentration of SP2/0-Ag14 is slightly lower than that of a control, and the doubling time is slightly longer. This check passed at a radiation dose of 30-35 kGy.

Example 2: co⁶⁰Effect verification experiment of BVDV virus or phage in irradiation inactivated cattle serum

1.Co⁶⁰Effect verification experiment of BVDV virus in irradiation inactivated cattle serum

Positive sample: inoculation 10^-6TCID50BVDV to bovine serum bottles (negative serum was detected when no BVDV virus was inoculated). With different doses of Co⁶⁰After bovine serum is irradiated, whether the dose of irradiation can be detected by MDBK cell culture lesion experiment sensitive to BVDV virusAnd inactivating the BVDV virus. The negative sample is bovine serum without BVDV, and the bovine serum without BVDV virus is detected.

BVDV infectivity assay: BVDV number: NADL, serial 10-fold dilution of virus solution in penicillin bottles, from 10^-1-10^-10. Inoculating the diluted virus into a 96-hole micro-culture plate, wherein each dilution is inoculated into a vertical row of 8 holes, and each hole is inoculated with 100 mu l; adding 100 μ l of bovine testis secondary cell suspension into each well to make cell amount reach 1 × 10⁵One per ml. The normal cell control was set as two vertical rows. (100. mu.l growth medium + 100. mu.l cell suspension) the results were observed and recorded day by day, typically for 5-7 days. TCID50 was calculated according to the Reed-Muench method. The BVDV TCID50 was calculated to be 10^-60.1ml, i.e. diluting the virus 10^-6Cells were diseased by seeding 100 ul.

The results of the experiment are shown in FIG. 4.

According to the results of the cell lesion method, the result shows that the BVDV virus can be completely inactivated when the irradiation dose is 15-20kGy, cells continuously pass 3 generations, and lesions do not appear. Irradiation dose of 10-15kGy, complete inactivation of BVDV is not achieved in 1 out of 3 experiments, and Co is detected⁶⁰The dose for irradiating BVDV in serum to reach biological safety is more than 15 kGy.

2.Co⁶⁰Effect verification experiment of BVDV virus in irradiation inactivated cattle serum

Taking the escherichia coli, and placing the escherichia coli on a constant temperature shaking table at 36 ℃ for 2 hours for later use.

And (3) proliferation culture: 16ml of nutrient broth +4ml of sample to be tested +1ml of E.coli (cultivated) were added to 100ml of glass.

Sealing the bottle, and then putting the bottle into a constant temperature shaker at 36 +/-1 ℃ for culturing for 18-24 hours. The same method is used for positive and negative control. Phage of about 10 in positive control⁶CFU/ml。

Sample application: spreading Escherichia coli (5ml) on nutrient agar plate, sucking and air drying after 20 min, and adding into the air dried nutrient

And (3) adding negative and positive controls and samples on the agar plate according to the divided region points, then putting the agar plate into a constant-temperature incubator at 36 +/-1 ℃ for culture, observing after 24 hours, and judging after 48 hours.

And (4) judging a result: when the test is invalid, the test is rechecked according to the steps, and the test is judged to be in accordance with the condition if no phage spot appears.

And (3) record filling: after the test is completed, the E.coli phage test record should be filled in and the photograph attached in time, see FIG. 5.

In the figure, 9 is a positive control (two samples are dotted) and 10 is a negative control. 5 and 6 were irradiated at 10-15kGy (4 samples spotted, 1 positive, 3 negative); 7 and 8 are irradiated at 15-20kGy (4 are negative); 1 and 2 are 25-35kGy irradiation (4 are negative); 3 and 4 are 35-40kGy irradiation (4 are negative)

Co of 15-20kGy obtained from the results of the experiment⁶⁰Irradiation on inactivated serum 10⁶CFU/ml phage is the effective dose.

Example 3: co⁶⁰Effect verification experiment of radiation inactivated mycoplasma in cattle serum

Positive sample: inoculation 2X 10⁶Bovine serum of mycoplasma (negative serum detected without mycoplasma inoculation). With different doses of Co⁶⁰After serum irradiation, Co detection by direct culture and cell culture staining⁶⁰The effect of irradiating to kill mycoplasma.

1. Direct mycoplasma culture method

Taking 4 mycoplasma liquid culture mediums with the volume of 10ml each, inoculating 0.5-1.0ml serum sample to each mycoplasma semifluid culture medium with 2 mycoplasma semifluid culture mediums (cooled to 35-37 ℃), placing in a constant temperature incubator with the temperature of 35-37 ℃ for 21 days, taking 2 mycoplasma in the 4 mycoplasma liquid culture mediums on the 7 th day after inoculation for secondary culture (transfer), transferring each culture medium to the corresponding mycoplasma semifluid culture medium and 2 mycoplasma semifluid culture mediums respectively, placing in the constant temperature incubator with the temperature of 37 ℃ for 21 days, observing once every three days, and recording the result.

	Irradiation of 0kGy	5-10kGy irradiation	Negative control	Positive control
					Liquid culture medium	+	-	-	+
Semi-solid culture medium	+	-	-	+

Direct culture method shows that 5-10kGy of Co⁶⁰Irradiation dose can kill serum 2X 10⁶Mycoplasma.

2. Cell culture staining method

Culturing Vero cells in antibiotic-free DMEM medium, forming cell monolayer, digesting to obtain 10 per 1ml⁵The sterilized glass slides are placed in a 6-well plate, then 0.5ml of the cell suspension is inoculated on the glass slides in the 6-well cell culture plate, 3ml of antibiotic-free culture medium is added in each well, and the cell suspension is cultured overnight at 36 +/-1 ℃ in a 5% carbon dioxide incubator for later use.

2ml of serum sample (cell culture supernatant) was added to the prepared plate, and the plate was cultured in a 5% carbon dioxide incubator at 36. + -. 1 ℃ for 3 to 5 days. Sucking out the culture solution in the culture hole, adding 5ml of fixing solution, standing for 5 minutes, sucking out the fixing solution, adding 5ml of fixing solution for fixing for 10 minutes, sucking out the fixing solution, drying the glass slide in the air, adding 5ml of working solution of the dibenzoyl amide fluorescent dye, covering, standing at room temperature for 30 minutes, sucking out the dye, washing each hole with 5ml of water for 3 times, sucking out water, drying the glass slide in the air, taking out a clean glass cover, adding one drop of sealing solution, and respectively covering the glass cover on the glass slide with the surface facing downwards to prepare sealing sheets. Observed with a fluorescence microscope. The test sample was replaced with 2ml of antibiotic-free medium, and the procedure was the same as that used for negative control. Serum irradiated with 0kGy (added with Mycoplasma sample) was used as a sample, and the procedure was the same as the above, and used as a positive control.

The results of the cell culture staining experiments (see FIG. 6) show that 5-10kGy of Co⁶⁰Irradiation dose can kill serum 2X 10⁶Mycoplasma, results were consistent with the direct culture method.

Example 4: co⁶⁰Effect verification experiment of irradiating inactivated bovine serum with escherichia coli

Positive sample: inoculation 3X 10⁶Bovine serum from E.coli (negative serum was detected without inoculation of Mycoplasma virus). With different doses of Co⁶⁰After serum irradiation, 10% serum sample is added into LB culture medium, cultured overnight, plated, and Co is detected by culture method⁶⁰The effect of inactivating escherichia coli by irradiation.

The results of the culture method (see FIG. 7) show that Co is 10 to 15kGy⁶⁰Irradiation dose can kill 3X 10 in serum⁶A quantity of E.coli.

Example 5: co⁶⁰Effect verification experiment of irradiation inactivated staphylococcus aureus in cattle serum

Positive sample: inoculation 1.6X 10⁶Bovine serum of staphylococcus aureus (negative serum detected without staphylococcus aureus). With different doses of Co⁶⁰After serum irradiation, 10% serum sample is added into LB culture medium, cultured overnight, plated, and Co is detected by culture method⁶⁰Effect of irradiation to inactivate Staphylococcus aureus.

The results of the culture method (FIG. 8) show that Co is 10 to 15kGy⁶⁰The irradiation dose can deactivate 10 in blood serum⁶Golden yellow in quantityA staphylococcal bacterium.

It is finally necessary to point out here: the above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Claims (10)

1. A bovine serum irradiation method, characterized in that the method uses high-dose Co under refrigeration⁶⁰Irradiation is carried out.

2. The method of claim 1, wherein the high dose is >25 kGy.

3. The method of claim 2, wherein the high dose is 25-30 kGy.

4. The method according to claim 1 or 2, wherein the irradiation is performed at full freeze.

5. A method according to claim 1 or 2, characterized in that the method comprises placing the serum bottle properly and filling the serum bottle with an ice bag around it.

6. A method according to claim 1 or 2, characterized in that the method comprises customizing a suitable foam box, the thickness of which is >2.8 cm.

7. The method of claim 6, wherein said one foam box bottles 20 bottles, fixed bottle filling location and ice pack location and number.

8. The method of claim 7, wherein the number of ice packs is 30.

9. The method according to claim 1 or 2, wherein the irradiation is performed after bovine serum packaging, cold chain transportation.

10. A bovine serum that has been irradiated by the method of any of claims 1-8.

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