CN113994951A - CIK cell cryopreservation liquid and cryopreservation method - Google Patents
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- 210000004405 cytokine-induced killer cell Anatomy 0.000 title claims abstract description 92
- 238000005138 cryopreservation Methods 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 30
- 239000007788 liquid Substances 0.000 title claims abstract description 19
- 238000001816 cooling Methods 0.000 claims abstract description 26
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 20
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- IHTCCHVMPGDDSL-ZJNDIJRCSA-N Cucurbitacin A Natural products O=C([C@@](O)(C)[C@H]1[C@@H](O)C[C@]2(C)[C@]1(C)CC(=O)[C@]1(CO)[C@H]2CC=C2C(C)(C)C(=O)[C@H](O)C[C@@H]12)/C=C/C(OC(=O)C)(C)C IHTCCHVMPGDDSL-ZJNDIJRCSA-N 0.000 claims abstract description 19
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- 239000000243 solution Substances 0.000 claims abstract description 15
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- 229920002307 Dextran Polymers 0.000 claims abstract description 10
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- 229960001008 heparin sodium Drugs 0.000 claims abstract description 10
- 239000008354 sodium chloride injection Substances 0.000 claims abstract description 10
- 235000019154 vitamin C Nutrition 0.000 claims abstract description 10
- 239000011718 vitamin C Substances 0.000 claims abstract description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 8
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
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- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- RECUKUPTGUEGMW-UHFFFAOYSA-N carvacrol Chemical compound CC(C)C1=CC=C(C)C(O)=C1 RECUKUPTGUEGMW-UHFFFAOYSA-N 0.000 description 1
- HHTWOMMSBMNRKP-UHFFFAOYSA-N carvacrol Natural products CC(=C)C1=CC=C(C)C(O)=C1 HHTWOMMSBMNRKP-UHFFFAOYSA-N 0.000 description 1
- 235000007746 carvacrol Nutrition 0.000 description 1
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- WYXXLXHHWYNKJF-UHFFFAOYSA-N isocarvacrol Natural products CC(C)C1=CC=C(O)C(C)=C1 WYXXLXHHWYNKJF-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a CIK cell cryopreservation solution, which consists of the following components: 0.9% sodium chloride injection, low molecular dextran, cucurbitacin B, carvyl alcohol, leucinerite, vitamin C, and heparin sodium. The crystallized aphenylene peptide in the frozen stock solution provided by the invention helps to maintain the stability of internal and external osmotic pressure of CIK cells, and avoids cell death caused by increased concentration of local electrolytes in the cells in the cooling process. The cucurbitacin B and the carvyl alcohol have a protective effect on the CIK cells, so that on one hand, the damage of ice crystals to the CIK cells is reduced; on the other hand, the CIK cells keep good proliferation activity, the survival rate of the recovered CIK cells is high, the proliferation activity is not reduced, and the CIK cells after being frozen can still be used for clinical immunotherapy. The invention also provides a cryopreservation method of the CIK cells, which comprises the steps of firstly reducing the temperature to-80 ℃ by adopting a program, then transferring the CIK cells to liquid nitrogen for cryopreservation, and effectively ensuring the survival rate of the cells by combining the cryopreservation liquid provided by the invention, wherein the cells after cryopreservation have better proliferation activity.
Description
Technical Field
The invention relates to the field of immune cells, in particular to a CIK cell cryopreservation solution and a CIK cell cryopreservation method.
Background
The CIK cell, namely Cytokine-Induced Killer (CIK), is a novel immunocompetent cell, has the characteristics of high proliferation speed, high tumor killing activity and wide tumor killing spectrum, and plays an important role in improving the anti-tumor immunity of a patient. Can also play a positive role in improving the quality of life and prolonging the life of patients with middle and late stage tumors which can not be operated or are resistant to chemotherapy. However, induction of the CIK cells takes a long time, and multiple times of induction and blood sampling are needed in clinical application, so that great pain and economic burden are caused to patients. In order to solve the problems, the CIK cells can be preserved at ultralow temperature by adopting a cell cryopreservation technology, and then recovered and proliferated for use when needed.
Cell cryopreservation is a technology for storing cells in a low-temperature environment to slow down cell metabolism and achieve long-term storage. The principle is that the cells are placed in liquid nitrogen at the temperature of 196 ℃ below zero for ultralow temperature preservation, so that the cells can be temporarily separated from the growth state to preserve the characteristics of the cells, and the cells can be used by reviving the cells when needed. When the cells are frozen, the protective agent is added into the cell suspension, so that the freezing point of the solution can be lowered, water in the cells permeates out of the cells under the condition of slow freezing, and the formation of ice crystals in the cells is reduced, thereby avoiding the damage of the cells in the freezing process.
And CIK cells have a plurality of problems in cryopreservation. For example, CIK cells are easy to be damaged by ice crystals, and the cell survival rate is low after recovery. The activity of CIK cells after cryopreservation is directly influenced by a cell cryopreservation solution, and at present, the common CIK cell cryopreservation solution mostly adopts dimethyl sulfoxide as a protective agent and is added with fetal calf serum, so that the permeability of cell membranes to water is improved, and the formation of ice crystals in cells is reduced, thereby reducing the cell damage caused by the formation of the ice crystals. However, dimethyl sulfoxide has cytotoxicity, fetal calf serum components are uncertain, and pollution sources such as bacteria and viruses can be introduced, so that the adoptive immunotherapy of the human body can be influenced to a certain extent. Therefore, there is a need to provide a CIK cell cryopreservation solution with safe components, which ensures the cell viability and proliferation activity of CIK cells after cryopreservation.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a CIK cell freezing solution which is not added with DMSO and fetal calf serum, has safe components and does not influence the activity of cells.
The second purpose of the invention is to provide a method for cryopreserving CIK cells.
One of the purposes of the invention is realized by adopting the following technical scheme:
a CIK cell cryopreservation liquid comprises the following components: 0.9% sodium chloride injection, low molecular dextran, cucurbitacin B, carvyl alcohol, leucinerite, vitamin C, and heparin sodium.
Preferably, the CIK cell frozen stock solution consists of the following components: 90-100 parts of 0.9% sodium chloride injection, 1.5-2 parts of low molecular dextran, 0.8-1.2 parts of cucurbitacin B, 1.2-1.8 parts of carvyl alcohol, 2.1-2.4 parts of leucinerin, 0.5-1 part of vitamin C and 0.1-0.5 part of heparin sodium.
Preferably, the CIK cell frozen stock solution consists of the following components: 95 parts of 0.9% sodium chloride injection, 1.7 parts of low molecular dextran, 1.1 parts of cucurbitacin B, 1.4 parts of carvyl alcohol, 2.3 parts of leucinerin, 0.8 part of vitamin C and 0.3 part of heparin sodium.
The second purpose of the invention is realized by adopting the following technical scheme:
a cryopreservation method of CIK cells adopts the cryopreservation liquid.
Preferably, the method for cryopreserving the CIK cells comprises the following steps:
(1) subpackaging the frozen stock solution in an aseptic freezing tube, cooling to 4-6 ℃, adding CIK cells to be frozen and uniformly blowing;
(2) and (3) cooling the freezing tube program in the step (1) to-80 ℃, freezing for 10-12h, and then transferring to liquid nitrogen for freezing.
Preferably, the density of CIK cells in the frozen stock solution is 5-10 x 106one/mL.
Preferably, the program cooling process in the step (2) is as follows: waiting for 1-2min at 4-6 deg.C, cooling to-20 deg.C at 1-2 deg.C/min, and cooling to-80 deg.C at 10-15 deg.C/min.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a CIK cell frozen stock solution, and the leuphanin helps to maintain the stability of internal and external osmotic pressure of CIK cells and avoid cell death caused by increase of local electrolyte concentration of the cells in the cooling process. The cucurbitacin B and the carvyl alcohol have a protective effect on the CIK cells, so that on one hand, the damage of ice crystals to the CIK cells is reduced; on the other hand, the CIK cells keep good proliferation activity, the survival rate of the recovered CIK cells is high, the proliferation activity is not reduced, and the CIK cells after being frozen can still be used for clinical immunotherapy. The frozen stock solution of the invention does not add DMSO and exogenous serum, has definite components and better safety. The mutual matching of all the components in the frozen stock solution reduces the damage to the CIK cells in the freezing stock process.
The invention also provides a method for cryopreserving the CIK cells, which comprises the steps of firstly reducing the temperature to minus 80 ℃ by adopting a program, then transferring the CIK cells to liquid nitrogen for cryopreservation, and effectively ensuring the survival rate of the CIK cells by combining the cryopreservation liquid disclosed by the invention, wherein the cryopreserved CIK cells still have good proliferation activity.
Detailed Description
The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.
Example 1
A CIK cell cryopreservation liquid comprises the following components: 95 parts of 0.9% sodium chloride injection, 1.7 parts of low molecular dextran, 1.1 parts of cucurbitacin B, 1.4 parts of carvyl alcohol, 2.3 parts of leucinerin, 0.8 part of vitamin C and 0.3 part of heparin sodium.
A method for cryopreserving CIK cells comprises the following steps:
(1) subpackaging the frozen stock solution in sterile freezing tubes, each tube is 2mL, cooling to 4 deg.C, adding CIK cells to be frozen, and uniformly blowing to obtain frozen stock solution with CIK cell concentration of 8 × 106Per mL;
(2) and (2) cooling the freezing storage tube in the step (1) to-80 ℃, wherein the procedure cooling process is as follows: waiting for 2min at 4 ℃, cooling to-20 ℃ at 1 ℃/min, cooling to-80 ℃ at 10 ℃/min, freezing and storing for 10h at-80 ℃, and then transferring to liquid nitrogen for freezing and storing.
Example 2
A CIK cell cryopreservation liquid comprises the following components: 90 parts of 0.9% sodium chloride injection, 1.5 parts of low molecular dextran, 0.8 part of cucurbitacin B, 1.2 parts of carvyl alcohol, 2.1 parts of leucinerin, 0.5 part of vitamin C and 0.1 part of heparin sodium.
A method for cryopreserving CIK cells comprises the following steps:
(1) subpackaging the frozen stock solution in sterile freezing tubes, each tube is 2mL, cooling to 4 deg.C, adding CIK cells to be frozen, and uniformly blowing to obtain frozen stock solution with CIK cell concentration of 5 × 106Per mL;
(2) and (2) cooling the freezing storage tube in the step (1) to-80 ℃, wherein the procedure cooling process is as follows: waiting for 2min at 4 ℃, cooling to-20 ℃ at 2 ℃/min, cooling to-80 ℃ at 12 ℃/min, freezing and storing for 11h at-80 ℃, and then transferring to liquid nitrogen for freezing and storing.
Example 3
A CIK cell cryopreservation liquid comprises the following components: 100 parts of 0.9% sodium chloride injection, 2 parts of low molecular dextran, 1.2 parts of cucurbitacin B, 1.8 parts of carvyl alcohol, 2.4 parts of leucinerite, 1 parts of vitamin C and 0.5 part of heparin sodium.
A method for cryopreserving CIK cells comprises the following steps:
(1) subpackaging the frozen stock solution in sterile freezing tubes, each tube is 2mL, cooling to 6 deg.C, adding CIK cells to be frozen, and uniformly blowing to obtain frozen stock solution with CIK cell concentration of 10 × 106Per mL;
(2) and (2) cooling the freezing storage tube in the step (1) to-80 ℃, wherein the procedure cooling process is as follows: waiting for 1min at 6 ℃, cooling to-20 ℃ at 1 ℃/min, cooling to-80 ℃ at 15 ℃/min, freezing and storing for 12h at-80 ℃, and then transferring to liquid nitrogen for freezing and storing.
Comparative example 1
Comparative example 1 provides a CIK cell cryopreservation solution, which is different from example 1 in that: the leuphanin peptide was omitted and the rest was the same as in example 1.
Comparative example 2
Comparative example 1 provides a CIK cell cryopreservation solution, which is different from example 1 in that: cucurbitacin B was omitted and the procedure was as in example 1.
Comparative example 3
Comparative example 3 provides a CIK cell cryopreservation solution, which is different from example 1 in that: carrageenol was omitted and the procedure in example 1 was repeated.
Comparative example 4
Comparative example 4 provides a CIK cell cryopreservation solution, which is different from example 1 in that: the cucurbitacin B was omitted, and the amount of carvacrol was adjusted to 2.5 parts, the remainder being the same as in example 1.
Comparative example 5
Comparative example 5 provides a CIK cell cryopreservation solution, which is different from example 1 in that: carrageenol was omitted and the amount of cucurbitacin B was adjusted to 2.5 parts, the remainder being the same as in example 1.
In the cases of freezing and storing the CIK cells in the embodiments 1 to 3 and the comparative examples 1 to 5 for 1 month, 3 months, 6 months, 9 months and 12 months, three cryovials are taken out each time for rapid resuscitation at 37 ℃, the cells are collected after centrifugation, X-VIVO 15 culture medium is used for preparing suspension, 50 microliter of cell suspension is taken and placed in an EP tube, 6.2 microliter of 0.4% trypan blue is added for staining, the trypan blue dye rejection rate is calculated to obtain the survival rate of the CIK cells, and the detection results of the three cryovials are averaged for each group of data, and the results are shown in Table 1.
TABLE 1
| Sample (I) | Survival rate of frozen 1 month (%) | Survival rate of frozen 3 months (%) | Survival rate of frozen 6 months (%) | Survival rate of frozen 9 months (%) | Survival rate (%) -12 months after frozen storage |
| Example 1 | 98.02 | 97.01 | 95.36 | 93.14 | 91.35 |
| Example 2 | 97.74 | 96.43 | 94.22 | 92.07 | 90.89 |
| Example 3 | 97.21 | 96.14 | 94.11 | 92.02 | 90.04 |
| Comparative example 1 | 84.65 | 80.69 | 76.32 | 71.67 | 64.98 |
| Comparative example 2 | 87.19 | 84.27 | 80.49 | 75.66 | 70.91 |
| Comparative example 3 | 81.32 | 77.05 | 72.84 | 66.97 | 61.43 |
| Comparative example 4 | 83.57 | 80.06 | 76.91 | 72.79 | 66.46 |
| Comparative example 5 | 80.44 | 76.21 | 71.85 | 65.37 | 58.19 |
From table 1, it can be seen that the cell viability of the CIK cells in examples 1 to 3 is above 90% after the cells are recovered after being frozen for 12 months. There was a different degree of degradation in each of comparative examples 1 to 5.
In comparative example 1, the leuphine silk peptide is omitted, the survival rate of the CIK cells is reduced remarkably along with the prolongation of the freezing storage time, and the survival rate of the CIK cells after the low-temperature freezing storage for 12 months is only 64.98%, so that the requirement of clinical treatment on the survival rate of the CIK cells is far not met. The reason is that the leuphin silk peptide can help to maintain the stability of the internal and external osmotic pressure of the CIK cells, so that the cell death caused by the increase of the local electrolyte concentration of the cells in the cooling process is avoided, and the survival rate of the CIK cells is further improved.
In comparative examples 2 to 5, cucurbitacin B or carvyl alcohol is omitted respectively, or the amount of the residual raw materials is increased after one of the components is omitted, and it can be seen from Table 1 that the survival rate of CIK cells is in a trend of decreasing obviously along with the prolongation of the freezing storage time, and after 12 months of freezing storage, the survival rate of the recovered CIK cells can not meet the use requirement. The cucurbitacin B and the carvyl alcohol are added into the frozen stock solution to protect the CIK cells, so that the damage of ice crystals to the CIK cells is reduced, the recovered CIK cells have high survival rate and no reduction of proliferation activity, and the frozen CIK cells can still be used for clinical immunotherapy.
CIK cells not frozen are taken as comparative example 6, CIK cells recovered after being frozen for 12 months in example 1 and comparative examples 1 to 5 are taken respectively, AIM-V medium CTS medium is added to the cells of comparative example 6 for re-suspension (IL-2500U/mL and IL-21300U/mL are added to the medium), and the cell density is adjusted to be 1 × 105Per mL, transferring the above cell-inoculated mediumInto a T75 flask, and placing at 37 deg.C and 5% CO2The culture box is used for culturing, and the culture medium is supplemented in time in the culture process to maintain the cell concentration at 1 × 105cell/mL, 10 days after the culture, the number of CIK cells was counted by trypan blue staining, and the proliferation fold of CIK cells was calculated, and the results are shown in Table 2.
TABLE 2
| Group of | Multiplication factor of value added |
| Example 1 | 182 |
| Comparative example 1 | 151 |
| Comparative example 2 | 132 |
| Comparative example 3 | 146 |
| Comparative example 4 | 129 |
| Comparative example 5 | 150 |
| Comparative example 6 | 175 |
It can be seen from Table 2 that the proliferation fold of CIK cells in example 1 is equivalent to that of CIK cells in comparative example 6 which have not been cryopreserved. Comparative examples 1 to 5 are the amplification factors of CIK cells stored after adjusting the composition of the frozen stock solution. As can be seen from Table 2, the decrease was different. In comparative example 1, the leuphinidin peptide was omitted, in comparative examples 2 to 5, the cucurbitacin B or carvyl alcohol was omitted, or the amount of the remaining raw material was increased after omitting one of the components, and the proliferation activity of CIK cells after cryopreservation was affected to various degrees. The invention is proved that the addition of the leuphinidin, the cucurbitacin B and the carvyl alcohol in the frozen stock solution is beneficial to the preservation of the proliferation activity of the CIK cells, and the cells after frozen stock can still quickly recover the activity and proliferate in a large quantity, thereby fully meeting the clinical application requirements.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (7)
1. The CIK cell cryopreservation liquid is characterized by consisting of the following components: 0.9% sodium chloride injection, low molecular dextran, cucurbitacin B, carvyl alcohol, leucinerite, vitamin C, and heparin sodium.
2. The CIK cell cryopreservation solution of claim 1, which consists of the following components: 90-100 parts of 0.9% sodium chloride injection, 1.5-2 parts of low molecular dextran, 0.8-1.2 parts of cucurbitacin B, 1.2-1.8 parts of carvyl alcohol, 2.1-2.4 parts of leucinerin, 0.5-1 part of vitamin C and 0.1-0.5 part of heparin sodium.
3. The CIK cell cryopreservation solution of claim 2, which consists of the following components: 95 parts of 0.9% sodium chloride injection, 1.7 parts of low molecular dextran, 1.1 parts of cucurbitacin B, 1.4 parts of carvyl alcohol, 2.3 parts of leucinerin, 0.8 part of vitamin C and 0.3 part of heparin sodium.
4. A method for cryopreserving CIK cells, which comprises using the cryopreserving liquid according to any one of claims 1 to 3.
5. The cryopreservation method of CIK cells according to claim 4, comprising the following steps:
(1) subpackaging the frozen stock solution in an aseptic freezing tube, cooling to 4-6 ℃, adding CIK cells to be frozen and uniformly blowing;
(2) and (3) cooling the freezing tube program in the step (1) to-80 ℃, freezing for 10-12h, and then transferring to liquid nitrogen for freezing.
6. The method for cryopreserving CIK cells according to claim 5, wherein the density of CIK cells in the cryopreservation solution is 5-10 x 106one/mL.
7. The cryopreservation method of CIK cells according to claim 5, wherein the temperature reduction process in the step (2) is as follows: waiting for 1-2min at 4-6 deg.C, cooling to-20 deg.C at 1-2 deg.C/min, and cooling to-80 deg.C at 10-15 deg.C/min.
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| CN115637254A (en) * | 2022-09-20 | 2023-01-24 | 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心） | Freezing shock neutrophil granulocytes and preparation method thereof |
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