CN117178980A - Immune cell low-temperature frozen stock solution and application thereof - Google Patents
Immune cell low-temperature frozen stock solution and application thereof Download PDFInfo
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Abstract
The invention discloses an immune cell low-temperature cryopreservation liquid and application thereof, wherein the low-temperature cryopreservation liquid comprises the following components: basic frozen stock solution, dextran, isoleucine, mannitol, resveratrol and hydroxypropyl cellulose. The immune cell low-temperature frozen stock solution supplementing system has simple and definite components, no exogenous components are introduced, and the clinical risks and toxic effects on human bodies in the subsequent immunotherapy are reduced; meanwhile, the frozen immune cells do not need to be frozen in a gradient way by using the frozen immune cell, and the delayed apoptosis phenomenon of the frozen immune cells after 24 hours and 48 hours after resuscitation is obviously reduced.
Description
Technical Field
The invention relates to the field of cell cryopreservation liquid, in particular to immune cell low-temperature cryopreservation liquid and application thereof.
Background
Immunotherapeutic techniques represent a great potential in the future medical direction, where mammalian immune cells, such as T cells, γδ T cells, NK cells or 293T cells, have a low risk of developing anti-host disease in an allogeneic setting, with great advantage in clinical treatment. However, the cells used therein still have objective delayed apoptosis phenomenon in terms of long-term preservation, so that the cells show delayed apoptosis after cryopreservation and resuscitation, and the apoptosis phenomenon causes that nearly half of immune cells cannot be used in subsequent clinical treatment.
The existing immune cell cryopreservation solution utilizes high-concentration (10%) DMSO and serum (FBS) to cryopreserve cells, thereby achieving the purpose of reducing the apoptosis proportion after cell resuscitation. However, the addition ratio of the substances introduces exogenous serum, so that the clinical risk existing in the subsequent immunotherapy is increased, and simultaneously, the toxic effect on cells and human bodies is also increased due to high concentration of DMSO. In addition, when the existing frozen stock solution freezes immune cells, the phenomenon of high apoptosis rate of delayed cells after the cells are recovered, and the cell viability of the immune cells after the frozen stock solution is recovered for 24 hours and 48 hours is low. Therefore, there is an urgent need to develop a cell cryopreservation solution in which the delayed apoptosis phenomenon of immune cells is significantly reduced 24 hours and 48 hours after resuscitation.
Disclosure of Invention
Aiming at the problems, the invention develops an immune cell low-temperature frozen stock solution and application thereof, so as to reduce the phenomenon of delayed apoptosis of immune cells. The invention aims to provide immune cell low-temperature cryopreservation liquid and application thereof, so as to solve the problems in the background technology.
In order to solve the technical problems, the invention provides the following technical scheme:
an immunocyte low-temperature frozen stock solution comprises basic frozen stock solution and additive components,
the basic frozen stock solution is the frozen stock solution of the UC000-N056 cells of the Ikesai;
the additive components are specifically as follows: 60g/L dextran, 3g/L mannitol, 5g/L isoleucine, 20 μg/L resveratrol, 0.2g/L ascorbyl phosphate half magnesium salt, 0.5g/L hydroxypropyl cellulose.
Preferably, the immune cell cryopreservation solution is used for cryopreservation of mammalian immune cells.
Preferably, the mammalian immune cell is any one of a T cell, a gamma delta T cell, an NK cell or a 293T cell.
Compared with the prior art, the invention has the following beneficial effects:
compared with the existing cryopreservation liquid in the market, the immune cell low-temperature cryopreservation liquid has the advantages of simple and clear components, low cost, no exogenous foreign body component, clinical safety, obvious reduction of delayed apoptosis phenomenon after cryopreservation and recovery of immune cells, and maintenance of higher proliferation capacity of cells.
The low-temperature frozen stock solution can reduce the coagulation state of ice crystals in the process of cooling cells, and eliminates excessive active oxygen generated in the process of freezing cells at low temperature, so that immune cells are protected in the cooling process and the low-temperature storage process from two aspects, and the phenomenon of delayed apoptosis after resuscitating is improved.
The low-temperature frozen stock solution capable of preserving immune cells at low temperature (-196 ℃) provided by the invention can also be preserved at-80 ℃, has simple and definite components, does not introduce any animal or human components, is clinically safe and low in cost, can reduce the delayed apoptosis proportion of immune cells after freezing and recovering, and can maintain good cell proliferation capability after recovering.
The cell freezing solution of the invention is based on the cell freezing solution of Ixel (EXCELL) UC000-N056, and other design components are added: dextran, isoleucine, mannitol and the like, the interaction of which can change the coagulation state of ice crystals in cells, and reduce the damage of ice crystal formation to immune cells during freezing; resveratrol and ascorbyl phosphate half magnesium salt are added to be used as antioxidants, so that excessive free radicals generated in cells can be eliminated, and certain protection effect on organelles in immune cells is achieved; hydroxypropyl cellulose is added as a tackifier in the freezing process, so that the formation of ice crystals in cells in the freezing process is reduced, and the freezing damage to the cells is reduced. Through the proportion combination of the raw materials, the cell state of the immune cells frozen at low temperature can be maintained to the greatest extent, and the proportion of delayed apoptosis after the immune cells are resuscitated is reduced. Compared with other existing commercial products, the immune cell frozen stock solution provided by the invention obviously reduces the survival rate and the apoptosis delay phenomenon after the immune cells are resuscitated.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a graph showing comparison of cell viability and cell number of the immunocytes frozen in the low-temperature frozen solutions of example 1 and the control group according to the present invention, 0 hours, 24 hours, and 48 hours after resuscitation.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the following examples, dextran (cat# D872032), isoleucine (cat# I6280), resveratrol (cat# R817263) were all purchased from milin, mannitol was purchased from Allatin (cat# D433338), ascorbyl phosphate hemi-magnesium salt (cat# A8960), hydroxypropyl cellulose (cat# 435007) were all purchased from Sigma (Sigma), ixelUC 000-N056 cell cryopreservation was purchased from IxelSide (EXCELL) (cat# UC000-N056, corresponding to patent publication No. CN 115777693A), and FBS was purchased from IxelSide (EXCELL Bio): FND500, DMSO from aladine (aladin) accession number: d103280, T cell serum-free medium amplification kit was purchased from Ekesai (cat# BA 0042).
Example 1: an immune cell cryopreservation solution comprises the following components: basic frozen stock solution, 60g/L dextran, 3g/L mannitol, 5g/L isoleucine, 20 mug/L resveratrol, 0.2g/L ascorbyl phosphate half magnesium salt and 0.5g/L hydroxypropyl cellulose;
wherein, the basic frozen stock solution adopts the UC000-N056 cell frozen stock solution of the Ikesai.
The preparation method of the immune cell low-temperature frozen stock solution comprises the following steps: taking 100mL of the solution as an example,
A. weighing 0.2g of resveratrol powder, and adding the resveratrol powder into 10mL of absolute ethyl alcohol to prepare resveratrol mother liquor 1; diluting the basic frozen stock solution by 1000 times to prepare resveratrol mother liquor 2;
weighing 0.4g of ascorbic acid half magnesium phosphate, and adding the obtained product into 10mL of injection water to prepare ascorbic acid half magnesium phosphate mother liquor;
B. 6g of glucan, 100 mu L of resveratrol mother liquor 2, 500 mu L of ascorbic acid phosphoric acid half magnesium salt mother liquor, 0.3g of mannitol, 0.5g of isoleucine and 0.05g of hydroxypropyl cellulose are sequentially added into the basic frozen stock solution, and the mixture is fully vortexed until the mixture is completely dissolved to obtain a mixed solution;
C. and (3) adopting a 0.2 mu m filter to perform sterile filtration on the mixed solution to obtain the immune cell low-temperature frozen stock solution.
Example 2: an immune cell cryopreservation solution comprises the following components: basic frozen stock solution, 20g/L dextran, 3g/L mannitol, 5g/L isoleucine, 20 mug/L resveratrol, 0.2g/L ascorbyl phosphate half magnesium salt and 1g/L hydroxypropyl cellulose;
wherein, the basic frozen stock solution adopts the UC000-N056 cell frozen stock solution of the Ikesai.
The preparation method of the immune cell low-temperature frozen stock solution comprises the following steps: taking 100mL of the solution as an example,
A. weighing 0.2g of resveratrol powder, and adding the resveratrol powder into 10mL of absolute ethyl alcohol to prepare resveratrol mother liquor 1; diluting the basic frozen stock solution by 1000 times to prepare resveratrol mother liquor 2;
weighing 0.4g of ascorbic acid half magnesium phosphate, and adding the obtained product into 10mL of injection water to prepare ascorbic acid half magnesium phosphate mother liquor;
B. sequentially adding 2g of glucan, 100 mu L of resveratrol mother liquor 2, 500 mu L of ascorbic acid phosphoric acid half magnesium salt mother liquor, 0.3g of mannitol, 0.5g of isoleucine and 0.1g of hydroxypropyl cellulose into the basic frozen stock solution, and fully vortex until the glucan, the resveratrol mother liquor and the ascorbic acid half magnesium salt mother liquor are completely dissolved to obtain a mixed solution;
C. and (3) adopting a 0.2 mu m filter to perform sterile filtration on the mixed solution to obtain the immune cell low-temperature frozen stock solution.
Example 3: an immune cell cryopreservation solution comprises the following components: basic frozen stock solution, 60g/L dextran, 1g/L mannitol, 2g/L isoleucine, 20 mug/L resveratrol, 0.2g/L ascorbyl phosphate half magnesium salt and 0.5g/L hydroxypropyl cellulose;
wherein, the basic frozen stock solution adopts the UC000-N056 cell frozen stock solution of the Ikesai.
The preparation method of the immune cell low-temperature frozen stock solution comprises the following steps: taking 100mL of the solution as an example,
A. weighing 0.2g of resveratrol powder, and adding the resveratrol powder into 10mL of absolute ethyl alcohol to prepare resveratrol mother liquor 1; diluting the basic frozen stock solution by 1000 times to prepare resveratrol mother liquor 2;
weighing 0.4g of ascorbic acid half magnesium phosphate, and adding the obtained product into 10mL of injection water to prepare ascorbic acid half magnesium phosphate mother liquor;
B. 6g of glucan, 100 mu L of resveratrol mother liquor 2, 500 mu L of ascorbic acid phosphoric acid half magnesium salt mother liquor, 0.1g of mannitol, 0.2g of isoleucine and 0.05g of hydroxypropyl cellulose are sequentially added into the basic frozen stock solution, and the mixture is fully vortexed until the mixture is completely dissolved to obtain a mixed solution;
C. and (3) adopting a 0.2 mu m filter to perform sterile filtration on the mixed solution to obtain the immune cell low-temperature frozen stock solution.
Example 4: an immune cell cryopreservation solution comprises the following components: basic frozen stock solution, 60g/L dextran, 3g/L mannitol, 5g/L isoleucine, 10 mug/L resveratrol, 0.5g/L ascorbyl phosphate half magnesium salt and 0.5g/L hydroxypropyl cellulose;
wherein, the basic frozen stock solution adopts the UC000-N056 cell frozen stock solution of the Ikesai.
The preparation method of the immune cell low-temperature frozen stock solution comprises the following steps: taking 100mL of the solution as an example,
A. weighing 0.2g of resveratrol powder, and adding the resveratrol powder into 10mL of absolute ethyl alcohol to prepare resveratrol mother liquor 1; diluting the basic frozen stock solution by 1000 times to prepare resveratrol mother liquor 2;
weighing 0.4g of ascorbic acid half magnesium phosphate, and adding the obtained product into 10mL of injection water to prepare ascorbic acid half magnesium phosphate mother liquor;
B. 6g of glucan, 50 mu L of resveratrol mother liquor 2, 1250 mu L of ascorbic acid phosphoric acid half magnesium salt mother liquor, 0.3g of mannitol, 0.5g of isoleucine and 0.05g of hydroxypropyl cellulose are sequentially added into the basic frozen stock solution, and the mixture is fully vortexed until the mixture is completely dissolved to obtain a mixed solution;
C. and (3) adopting a 0.2 mu m filter to perform sterile filtration on the mixed solution to obtain the immune cell low-temperature frozen stock solution.
Example 5: an immune cell cryopreservation solution comprises the following components: basic frozen stock solution, 60g/L dextran, 3g/L mannitol, 5g/L isoleucine, 20 mug/L resveratrol, 0.5g/L ascorbyl phosphate half magnesium salt and 1g/L hydroxypropyl cellulose;
wherein, the basic frozen stock solution adopts the UC000-N056 cell frozen stock solution of the Ikesai.
The preparation method of the immune cell low-temperature frozen stock solution comprises the following steps: taking 100mL of the solution as an example,
A. weighing 0.2g of resveratrol powder, and adding the resveratrol powder into 10mL of absolute ethyl alcohol to prepare resveratrol mother liquor 1; diluting the basic frozen stock solution by 1000 times to prepare resveratrol mother liquor 2;
weighing 0.4g of ascorbic acid half magnesium phosphate, and adding the obtained product into 10mL of injection water to prepare ascorbic acid half magnesium phosphate mother liquor;
B. 6g of glucan, 100 mu L of resveratrol mother liquor 2, 1250 mu L of ascorbic acid phosphoric acid half magnesium salt mother liquor, 0.3g of mannitol, 0.5g of isoleucine and 0.1g of hydroxypropyl cellulose are sequentially added into the basic frozen stock solution, and the mixture is fully vortexed until the mixture is completely dissolved to obtain a mixed solution;
C. and (3) adopting a 0.2 mu m filter to perform sterile filtration on the mixed solution to obtain the immune cell low-temperature frozen stock solution.
Example 6: an immune cell cryopreservation solution comprises the following components: basic frozen stock solution, 80g/L dextran, 3g/L mannitol, 5g/L isoleucine, 50 mug/L resveratrol, 0.2g/L ascorbyl phosphate half magnesium salt and 0.5g/L hydroxypropyl cellulose;
wherein, the basic frozen stock solution adopts the UC000-N056 cell frozen stock solution of the Ikesai.
The preparation method of the immune cell low-temperature frozen stock solution comprises the following steps: taking 100mL of the solution as an example,
A. weighing 0.2g of resveratrol powder, and adding the resveratrol powder into 10mL of absolute ethyl alcohol to prepare resveratrol mother liquor 1; diluting the basic frozen stock solution by 1000 times to prepare resveratrol mother liquor 2;
weighing 0.4g of ascorbic acid half magnesium phosphate, and adding the obtained product into 10mL of injection water to prepare ascorbic acid half magnesium phosphate mother liquor;
B. 8g of glucan, 250 mu L of resveratrol mother liquor 2, 500 mu L of ascorbic acid phosphoric acid half magnesium salt mother liquor, 0.3g of mannitol, 0.5g of isoleucine and 0.05g of hydroxypropyl cellulose are sequentially added into the basic frozen stock solution, and the mixture is fully vortexed until the mixture is completely dissolved to obtain a mixed solution;
C. and (3) adopting a 0.2 mu m filter to perform sterile filtration on the mixed solution to obtain the immune cell low-temperature frozen stock solution.
Example 7: an immune cell cryopreservation solution comprises the following components: basic frozen stock solution, 80g/L dextran, 3g/L mannitol, 5g/L isoleucine, 20 mug/L resveratrol, 0.5g/L ascorbyl phosphate half magnesium salt and 0.2g/L hydroxypropyl cellulose;
wherein, the basic frozen stock solution adopts the UC000-N056 cell frozen stock solution of the Ikesai.
The preparation method of the immune cell low-temperature frozen stock solution comprises the following steps: taking 100mL of the solution as an example,
A. weighing 0.2g of resveratrol powder, and adding the resveratrol powder into 10mL of absolute ethyl alcohol to prepare resveratrol mother liquor 1; diluting the basic frozen stock solution by 1000 times to prepare resveratrol mother liquor 2;
weighing 0.4g of ascorbic acid half magnesium phosphate, and adding the obtained product into 10mL of injection water to prepare ascorbic acid half magnesium phosphate mother liquor;
B. 8g of glucan, 100 mu L of resveratrol mother liquor 2, 1250 mu L of ascorbic acid phosphoric acid half magnesium salt mother liquor, 0.3g of mannitol, 0.5g of isoleucine and 0.02g of hydroxypropyl cellulose are sequentially added into the basic frozen stock solution, and the mixture is fully vortexed until the mixture is completely dissolved to obtain a mixed solution;
C. and (3) adopting a 0.2 mu m filter to perform sterile filtration on the mixed solution to obtain the immune cell low-temperature frozen stock solution.
Control group: the low temperature cryopreservation solution in the control group was conventional cell cryopreservation solution (90% fbs+10% dmso).
Comparative example 1: compared with example 1, the adding amount of hydroxypropyl cellulose is 5g/L, and the other preparation steps and the component proportions are the same as those of example 1, so as to obtain the low-temperature frozen stock solution.
Comparative example 2: compared with example 1, the dextran addition amount is 100g/L, and the rest preparation steps and the component proportions are the same as those of example 1, so as to obtain the low-temperature frozen stock solution.
Comparative example 3: compared with example 1, the mannitol is added in an amount of 10g/L, and the other preparation steps and the component proportions are the same as those of example 1, so as to obtain the low-temperature frozen stock solution.
Comparative example 4: compared with example 1, the addition amount of isoleucine was 10g/L, and the other preparation steps and the component ratios were the same as those of example 1, thereby obtaining a low-temperature frozen stock solution.
Comparative example 5: compared with example 1, the resveratrol addition amount is 100 mug/L, and the rest preparation steps and the component proportions are the same as those of example 1, so as to obtain the low-temperature frozen stock solution.
Comparative example 6: compared with example 1, the addition amount of the magnesium ascorbyl phosphate hemi-magnesium phosphate is 3g/L, and the other preparation steps and the component proportions are the same as those of example 1, so as to obtain the low-temperature frozen stock solution.
The application of the immune cell low-temperature cryopreservation solution of examples 1-7, the control group and the comparative examples 1-6 comprises the following steps:
performing effect verification of immune cell low-temperature cryopreservation liquid by adopting gamma delta T cells, wherein the gamma delta T cells are amplified on human Peripheral Blood Mononuclear Cells (PBMC), and the gamma delta T cells are obtained by using a T cell serum-free culture medium amplification kit;
s1, collecting cells: centrifuging to collect immune cells with cell activity rate stabilized over 90%;
s2, preserving cells at low temperature: the immunocytes obtained in step S1 were then frozen in the low-temperature frozen stock solution obtained in example 1 and the low-temperature frozen stock solution in the control group, respectively, at 3X 10 6 After resuspension of the tube density, transferring into a freezing tube, placing into a program freezing box, storing in a refrigerator at-80 ℃ for more than 6 hours (3 tubes for storing low-temperature preservation liquid respectively), and transferring into a liquid nitrogen tank at-196 ℃ for storage;
s3, cell recovery: after 3 days, the frozen cells of the low-temperature frozen stock solution are taken out from the liquid nitrogen tank, the frozen immune cells are recovered, sampling, cell activity rate and cell number detection are carried out, subsequent cell inoculation is carried out, and cell sampling, activity rate and cell number detection are respectively carried out 24 hours and 48 hours after recovery, and the result is shown in figure 1.
Results and analysis
Taking the low-temperature preservation solutions obtained in examples 1-7, the control group and the comparative examples 1-6, simultaneously testing immune cells added with the low-temperature preservation solution, performing cell cryopreservation and recovery detection according to the experimental operation, and recording the cell viability after different recovery times, wherein the results are shown in Table 1.
TABLE 1
From the data in table 1, it is clear that the following conclusions are drawn:
compared with the comparative example, the low-temperature cryopreserved liquid for immune cells provided by the invention is utilized in the example 1, the unique formula combination design obtains lower delayed apoptosis proportion after resuscitating, the survival rate of the cryopreserved immune cells and tissues after resuscitating is obviously improved, the lower apoptosis proportion is shown in 24 hours and 48 hours after resuscitating, the cell viability rate of cells after resuscitating for 48 hours is still more than 50%, and the cell viability rate is obviously superior to that of the cryopreserved liquid for a control group.
The immune cell cryopreservation effect is closely related to the component design, the dosage concentration and the component interaction of the cryopreservation liquid, and the cell cryopreservation with higher efficiency is realized under the component combination and the dosage concentration in the embodiment 1; examples 2 to 7 respectively perform dose adjustment tests within the application range on various concentrations of single or multiple materials, and comparative examples 1 to 6 respectively perform dose tests beyond the application range on additive materials, and compared with example 1, the cell freezing survival rate has larger fluctuation under the condition of different dose concentrations, and the large dose or overdose use of individual materials cannot generate protective effect in the freezing process of immune cells, so that the importance of the composition design and the dose concentration of immune cell freezing solution is further verified.
Experimental results fully demonstrate that the immune cell low-temperature frozen stock solution provided by the invention has the advantages that: the components are clear, safe and effective, the preserved cell has high activity and high yield, and the delayed apoptosis phenomenon after the immune cells are resuscitated is obviously reduced.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. An immunocyte low-temperature frozen stock solution is characterized by comprising a basic frozen stock solution and additive components,
the basic frozen stock solution is the frozen stock solution of the UC000-N056 cells of the Ikesai;
the additive components are specifically as follows: 60g/L dextran, 3g/L mannitol, 5g/L isoleucine, 20 μg/L resveratrol, 0.2g/L ascorbyl phosphate half magnesium salt, 0.5g/L hydroxypropyl cellulose.
2. The use of an immune cell cryopreservation solution according to claim 1, wherein the immune cell cryopreservation solution is used for cryopreservation of immune cells of a mammal.
3. The use of an immune cell cryopreservation solution according to claim 2, wherein the mammalian immune cell is any one of T cell, γδ T cell, NK cell or 293T cell.
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