JP2022104240A - Cell freezing solution, and cell freezing method - Google Patents

Abstract

To provide cell freezing solution that has a high cell survival rate after freezing, inexpensive with excellent safety, and for which raw materials are easily available.SOLUTION: Cell freezing solution has physiological saline as a solvent, contains trehalose and chondroitin sulfuric acid as an additive, and preferably, the chondroitin sulfuric acid is added to trehalose solution containing the trehalose at the concentration of 200 mM or more and 300 mM or less in the physiological saline. More preferably, cell freezing solution has the chondroitin sulfuric acid added to the trehalose solution at the ratio of 5vol% or more and 10vol% or less.SELECTED DRAWING: Figure 2

Description

The present invention relates to a cell cryopreservation solution and a cell freezing method.

In recent years, a cell freezing technique has been attracting attention, in which cell tissues of various organs, pluripotent cells, and the like are cryopreserved, and the cells cryopreserved at the time of treatment such as transplantation are thawed and used. A general method for freezing cell tissue is, for example, to prepare a cell suspension in which a cryopreservation solution containing various additives added to physiological saline is added to the cell tissue, and a container containing the cell suspension ( (Cryotube, etc.) is placed in a cell freezing container filled with 2-propanol, and the cell freezing container is held in the freezer of the freezing device.

More specifically, it is carried out by cooling to a predetermined temperature (for example, −80 ° C.) at a predetermined temperature lowering rate (for example, 1 ° C./min or the like) using a program freezer or the like. Alternatively, it is carried out by holding the cell freezing container in a freezer set to a predetermined temperature for a predetermined time (for example, 24H). Then, the container containing the frozen cell suspension is immersed in liquid nitrogen to store the frozen cells.

The following Non-Patent Documents 1 and 2 describe a technique for cryopreserving iPS cells. Further, the following Non-Patent Document 3 describes a method for cryopreserving cell tissues such as iPS cells and an outline of a cryopreservation solution for cell tissues. The following Patent Document 1 and Non-Patent Document 4 describe a cell cryopreservation composition which is a cell cryopreservation solution having low cytotoxicity. The following Patent Document 2 describes a freezing device for freezing cells while applying a magnetic field in relation to an embodiment of the present invention.

Japanese Unexamined Patent Publication No. 2006-115837 Japanese Unexamined Patent Publication No. 2017-104061

Kyoto University, "Establishment of effective cryopreservation method for human iPS cells", [online], [Search on December 20, 2nd year of Reiwa], Internet <URL: http://www.kyoto-u.ac.jp /static/ja/news_data/h/h1/news6/2010/101201_3.htm ＞ The Society for Biotechnology, Japan, "The Society for Biotechnology, Primates", [online], [Searched on December 20, 2nd year of Reiwa], Internet <URL: https://www.sbj.or. jp / wp-content / uploads / file / sbj / 9009/9009_tokushu-1_3.pdf ＞ Xenoac Resource Co., Ltd., "STEM-CELLBANKER GMP grade Product Information (" STEM-CELLBANKER "is a registered trademark)", [online], [Search on December 20, 2nd year of Reiwa], Internet <URL: http: // www.zenoaq.jp/zenoaq_resource/stem-cellbanker.html ＞ Nacalai Tesque Co., Ltd., "Cell Reservoir One", [online], [Search on December 20, 2nd year of Reiwa], Internet <URL: https://www.nacalai.co.jp/products/entry/ d001007.html ＞

A general cell cryopreservation solution contains dimethyl sulfoxide (DMSO) as an additive, as described in Non-Patent Document 3. However, DSMO is known to be cytotoxic. Therefore, considering that the cell cryopreservation technology for medical use, which is at the current experimental level and clinical level, will be transferred to the practical level in the future, the cell cryopreservation solution is required to have higher safety. Of course, the cell cryopreservation solution is required to have a high cell viability rate at the time of thawing as well as safety.

The cell cryopreservation solutions described in Patent Document 1 and Non-Patent Document 4 contain an additive mainly containing sericin, which is a silk protein, and these documents also disclose those which do not contain DSMO. .. However, since sericin is obtained by using silk protein derived from eyebrows as a raw material and extracting and purifying it from wastewater generated in the smelting process of the thread making process, it is difficult to produce sericin at a lower cost. Also, the sources of raw materials are limited.

In order to raise the cell freezing technology for medical use to a practical level, a cell cryopreservation solution using raw materials that are safe, have high cell viability, and are inexpensive and easily available is required.

Therefore, the present invention provides a cell cryopreservation solution having a high cell survival rate after thawing, excellent safety, low cost, and easy availability of raw materials, and a cell freezing method using this cell cryopreservation solution. I am aiming.

One aspect of the present invention for achieving the above object is a cell cryopreservation solution containing trehalose and chondroitin sulfate as additives using physiological saline as a solvent.

It is preferable to use a cell cryopreservation solution obtained by adding the chondroitin sulfate to the trehalose solution containing the trehalose at a concentration of 200 mM or more and 300 mM or less in the physiological saline. Further, it is more preferable to prepare a cell cryopreservation solution obtained by adding the chondroitin sulfate to the trehalose solution at a ratio of 5 vol% or more to 10 vol% or less.

Aspects of the present invention also include a method of freezing cells using the above-mentioned cell cryopreservation solution.

A sample preparation step of preparing a cell suspension in which the cell cryopreservation solution is added to cells to be frozen is prepared as a sample.

A sample installation step of installing the first container containing the sample in a second container filled with 2-propanol.

Following the sample installation step, a freezing step in which the second container is placed in a freezer to freeze the sample, and a freezing step.

Including

In the freezing step, a magnetic field is generated in the freezer, the magnetic field is applied to the sample, the temperature is gradually cooled to −50 ° C. or higher and −30 ° C. or lower, and the temperature is maintained at the temperature for a predetermined time.

It is a cell freezing method.

According to the present invention, there is provided a cell cryopreservation solution having a high cell survival rate after thawing, an inexpensive and excellent safety solution, and a cell freezing method using the cell cryopreservation solution. Other effects will be clarified in the following description.

It is a figure which shows an example of the appearance of the magnetic field generation type refrigerating apparatus which can generate a magnetic field in a freezing tank. It is a figure which shows an example of the structure of the said magnetic field generation type refrigeration apparatus. It is a figure which shows the cell viability when the sample obtained by adding the cell cryopreservation solution which added various saccharides to the physiological saline (D-PBS) to HEK293 cells (test cells) is frozen and thawed. .. It is a figure which shows the cell viability when the sample obtained by adding the cell cryopreservation solution which added trehalose and various proteins to the said test cell to the said D-PBS is frozen and thawed. When a sample obtained by adding a cell cryopreservation solution prepared by adding chondroitin sulfate in various amounts to a solution containing trehalose at a predetermined concentration in the above D-PBS to the above test cells was frozen and thawed. It is a figure which shows the cell viability. A sample obtained by adding a cell cryopreservation solution prepared by adding the above trehalose and the above chondroitin sulfate to the above test cells to the above D-PBS was frozen at various temperatures using a magnetic field generation type freezing device and thawed. It is a figure which shows the cell viability at the time.

Examples of the present invention will be described below with reference to the accompanying drawings.

=== First Example ===
<Main additive>

As described above, in order to put the cell cryopreservation technique for medical use into practical use, it is important that it is inexpensive and easily available in addition to high safety and high cell viability. Therefore, the present inventor studied the use of a food-derived substance in a cell cryopreservation solution, and then repeated diligent studies to achieve a higher cell viability. Then, he came up with the present invention.

The cell cryopreservation solution according to the embodiment of the present invention contains a food-derived substance as an additive. As an additive of the cell cryopreservation solution derived from food, there is a saccharide as described in Patent Document 1 above. Therefore, first, a cell suspension using a cryopreservation solution containing saccharides as a main additive was used as a sample, the sample was frozen, and the cell viability after thawing was examined.

<Sample preparation procedure>
Hereinafter, an example of the sample preparation procedure, the sample freezing procedure, and the sample thawing procedure will be specifically described. Here, as the cell tissue to be frozen, HEK293 cells obtained by culturing a human fetal-derived renal cell line (HEK293A) were adopted.

The sample preparation procedure is as follows: First, a human fetal-derived renal cell line (HEK293A) is prepared, and fetal bovine serum (FBS) and penicillin streptomycin are added to DMEM (Dulbecco's Modified Eagle's Medium) as a basal medium, respectively. HEK293 cells (hereinafter, may be referred to as "test cells") are obtained by culturing in an environment of a temperature of 37 ° C. and a CO ₂ concentration of 5% using a medium containing 10 vol% and 1 vol%. Next, a cell suspension prepared by adding a cell cryopreservation solution to the test cells is used as a sample. The cell concentration in the cell suspension was 1 × ¹⁰⁷ (cells / ml).

The cell cryopreservation solution used for the sample was prepared by adding saccharides to D-PBS (Dulbecco phosphate buffered saline) as a main component, and the types of saccharides differ depending on the sample. The concentration of saccharides in each cell cryopreservation solution was uniformly set to 200 mM by changing the amount of D-PBS for each sample.

Next, using the cryotube as the first container, 500 μl of the sample was placed in the first container (hereinafter, may be referred to as “tube”). Next, the tube containing the sample is placed in a freezing storage container which is a second container filled with 2-propanol, and the freezing storage container (hereinafter, may be referred to as “container”) is described later. The sample was frozen using a freezer.

Thawing was carried out by holding a cryopreservation container in which a tube containing a frozen sample was placed in a constant temperature bath at 37 ° C. for 1 minute. Then, for each sample, a freeze-thaw test in which the cells are frozen and thawed according to the above procedure is performed, the number of surviving cells in the sample after thawing is measured, and the number of cells before freezing and the survival after thawing are measured. Cell viability was determined based on the number of cells. The number of viable cells was measured by automatically analyzing with a float cytometer to determine whether the test cells in the sample stained with the microstaining dye (PI) were alive or dead.

<Refrigerator>
The conventional procedure for freezing the cell suspension is to place the sample in the freezer of the program freezer and move the sample from room temperature to about -80 ° C at a predetermined temperature lowering rate (for example, -1 ° C / min). Cool to a very low temperature and freeze. There is also a freezing method in which the sample is placed in a freezer set at a temperature of -80 ° C. In the following, the freezing method according to these procedures will be referred to as a general freezing method.

By the way, the cell cryopreservation solution according to the example contains a saccharide as an additive, unlike the one containing DMSO that permeates into the cell. As is well known, saccharides are non-toxic and not lipophilic, so if the sample is frozen using a general freezing method that freezes at a very low temperature of -80 ° C, the sample may not be evaluated properly.

Specifically, when the cells are frozen, the intracellular ice crystal formation has a greater effect on the cell viability than the extracellular ice crystal formation. Therefore, when the sample is frozen using a cell cryopreservation solution containing DMSO, the DMSO, which is a cryoprotectant, is sufficiently permeated into the cells by a general freezing method, and the free water that becomes the source of the ice inside the cells is used. It is important to replace with a cryoprotectant and suppress damage to intracellular organs (organizers) due to ice crystal formation.

On the other hand, in the cell cryopreservation solution according to the example, since a saccharide having a large molecular weight is used as an additive, it is difficult to penetrate into the cell. Therefore, a method of dehydrating the free water in the cells as much as possible, or freezing the cells at once by maintaining the temperature at an overcooled state (for example, -30 ° C) so that the osmotic pressure in the cells is maintained to some extent. Can be considered suitable. Then, in order to correctly evaluate the performance of the cell cryopreservation solution according to the examples, the sample is frozen by an appropriate method in consideration of the physical characteristics of the additives so that the performances of different additives can be compared. It is necessary.

Therefore, it was decided to use a refrigerating apparatus similar to the refrigerating apparatus described in Patent Document 2 for freezing the sample. This type of freezing device can generate a magnetic field in the freezer, and in Patent Document 2, a sample using a conventional cell cryopreservation solution containing DMSO is used as a freezing device described in the document. Is used to freeze the sample while applying a magnetic field. It is also disclosed that the survival rate and the like are improved. Therefore, various samples prepared for evaluating the cell cryopreservation solution according to the examples basically use this kind of freezing device (hereinafter, may be referred to as "magnetic field generation type freezing device"). It is supposed to be frozen in a magnetic field.

For reference, FIGS. 1 and 2 show an example of the magnetic field generation type refrigerating apparatus 1. FIG. 1 is an external view of the magnetic field generation type refrigeration apparatus 1, and FIG. 2 shows a schematic configuration of the magnetic field generation type refrigeration apparatus 1.

The magnetic field generation type refrigerating apparatus 1 shown in FIG. 1 is a control unit in which a box-shaped refrigerating apparatus main body (hereinafter, also referred to as a main body 10) which is long in the vertical direction in a state of being placed on a horizontal plane, and the main body 10 and a cable 2 are connected to each other. It is composed of 20. The control unit 20 is a device for controlling the operation of the main body 10 and monitoring the operating state of the main body, and as a user interface, an input unit (23, 26) for receiving various setting operations and the main body 10 It is provided with display units (24, 27) for displaying the setting status and the like.

As shown in FIG. 2, the main body 10 has a freezer (hereinafter, may be referred to as a “freezing tank”) 11 inside, a refrigerator 12 such as a sterling refrigerator, a cooling head 13 which is a heat exchanger, and a heater 14. , A temperature sensor 15 for monitoring the temperature in the freezing tank 11, a coil 16 for generating a magnetic field in the freezing tank 11, and the like are included. The conducting wire constituting the coil 16 is wound around the freezing tank 11 in a rectangular shape with the vertical direction as an axis, for example. The upper surface of the cooling head 13 constitutes the bottom surface of the freezing tank, and the heater 14 and the temperature sensor 15 are incorporated in the cooling head 13. As a result, the inside of the freezing tank 11 is directly cooled or heated via the cooling head 13.

<Test results>
Samples using various cell cryopreservation solutions with different types of saccharides were placed in the freezing tank of the magnetic field generation type freezing device (CAS-LAB1, manufactured by Abbey Co., Ltd.), and the temperature was lowered to -30 ° C at a temperature lowering rate of -2 ° C / min. It was cooled to −30 ° C. and maintained at a temperature of −30 ° C. for 10 minutes. During the temperature lowering process and the maintenance period at −30 ° C., a magnetic field having a frequency of 50 Hz and a magnetic flux density of 0.30 to 0.31 mT was applied to the sample. Then, after maintaining the sample at −30 ° C., the sample was transferred to a freezer at −80 ° C. and stored for 24 hours. The sample frozen and stored in this way was thawed by the above method and the cell viability was examined. Unless otherwise noted, each sample shown below was frozen under the same conditions and thawed by the same method.

FIG. 3 shows the cell viability in each of the samples using various cell cryopreservation solutions having different types of saccharides. As shown in FIG. 3, the cell viability exceeded 50% in the sample using trehalose as an additive.

<Sub-ingredients of additives>
As described above, it was found that a cell viability of at least 50% or more can be obtained by using a cell cryopreservation solution containing trehalose, which is a food-derived sugar and does not contain a cytotoxic additive. .. Therefore, in order to further improve the cell viability, a sample was prepared using a cell cryopreservation solution containing trehalose as the main component of the additive and an additive consisting of trehalose and an auxiliary component other than trehalose. Then, a freeze-thaw test was performed on the sample in the same procedure as above to examine the cell viability of the sample.

Here, various proteins were used as subcomponents of the additive. Like sugar, protein is an extremely important polymer in living organisms, and since water is used as a solvent in living organisms, cells containing sugar and protein as additives for D-PBS, which is a physiological saline solution. It was assumed that if the cryopreservation solution is used, the interaction (hydration) between the additive and water suppresses the destruction of cells during freezing and improves the cell viability after thawing. Then, sericin, FBS, and bovine serum albumin (BSA) described in Patent Document 1 and Non-Patent Document 4, which have been proven as additives for cell cryopreservation solutions, were selected as sub-ingredients.

Furthermore, since sericin, FBS, and BSA are not inexpensive, chondroitin sulfate was selected because it is necessary to consider additives that have no track record for cell cryopreservation solutions. As chondroitin sulfate, for example, there is a pig cartilage extract containing a large amount of chondroitin sulfate A from pig cartilage as a raw material. This type of chondroitin sulfate is widely used as a raw material for supplements and the like, is highly safe, is extremely inexpensive, and is easily available.

Therefore, various cell cryopreservation solutions prepared by adding any of the above subcomponents to a trehalose solution having a concentration of 200 mM using D-PBS as a solvent were prepared, and cell suspensions using the respective cell cryopreservation solutions were used as samples. did. The concentration of the auxiliary component in the cell cryopreservation solution was set to 10 vol%.

FIG. 4 shows the cell viability after the freeze-thaw test for samples using various cell cryopreservation solutions having different types of sub-ingredients. As shown in FIG. 4, the cell viability was 89.0% in the sample using the cell cryopreservation solution containing chondroitin sulfate as a sub-component of the additive, and the sample was a sample using other sub-components. The cell viability was specifically high. Therefore, in order to improve the cell viability, it is effective to use trehalose and chondroitin sulfate as additives for the cell cryopreservation solution.

As described above, trehalose and chondroitin sulfate used as additives for the cell cryopreservation solution are food-derived highly safe additives, and are inexpensive and easily available. The cell cryopreservation solution according to the first embodiment is a cell cryopreservation solution containing these additives, and the cell cryopreservation solution according to the first embodiment is for practical use of medical treatment using frozen cells. Is also considered to contribute.

<Optimization of cell cryopreservation solution>
Next, using D-PBS as a solvent, the amount of chondroitin sulfate added to the trehalose solution having a sugar concentration of 300 mM was changed to prepare various cell cryopreservation solutions having different concentrations of chondroitin sulfate in the cell cryopreservation solution. A cell suspension similar to the above was prepared. Then, using these cell suspensions as samples, each sample was subjected to a cryopreservation test in the same procedure as described above, and then the cell viability in each sample was examined.

FIG. 5 shows the cell viability of samples using various cell cryopreservation solutions having different concentrations of chondroitin sulfate in the trehalose solution. From the cell viability of each sample shown in FIG. 5, a cell viability of about 80% was obtained even in the sample containing a trace amount (0.1 vol%) of chondroitin sulfate. When the concentration of chondroitin sulfate was 5.0 vol% or more, an extremely high cell survival rate of about 90% was obtained.

Then, the survival rate of the sample in which the concentration of chondroitin sulfate was 10 vol% using the 200 mM trehalose solution shown in FIG. 4 and the concentration of chondroitin sulfate in 10 vol using the 300 mM trehalose solution shown in FIG. 5 were obtained. Considering that the survival rate of the sample set to% is 89.0%, which is the same as the survival rate of the sample, if the concentration of the trehalose solution is 200 mM or more and 300 mM or less, the high cell survival rate of about 80% is more certain. It is thought that it will be obtained. It was also found that when the concentration of chondroitin sulfate is 5.0 vol% or more, an extremely high cell survival rate of about 90% can be obtained.

The upper limit of the concentration of chondroitin sulfate is not particularly specified, but from the test results shown in FIGS. 4 and 5, stable and high cell viability can be achieved if the concentration is at least 10 vol%. It can be said that it can be obtained. Furthermore, as shown in FIG. 5, although the concentration of chondroitin sulfate and the cell viability seemed to have a threshold value around 5.0 vol%, no clear correlation was found, so the concentration was increased. It is reasonable to think that it is not necessary. Therefore, it is realistic that the upper limit of the concentration of chondroitin sulfate is 10 vol%.

In any case, the technical significance of the cell cryopreservation solution according to the examples is that chondroitin sulfate can be used as an additive for the cell cryopreservation solution, which could not be conceived from conventional applications, and chondroitin sulfate is used as an additive. By doing so, it was confirmed that an unexpectedly high effect is achieved while ensuring high safety.

<Freezing temperature>
In the above freeze-thaw test using the cell cryopreservation solution according to the example, after freezing the sample with a magnetic field generation type freezer, the sample was held for 24 hours in a freezer set to a temperature of -80 ° C. .. That is, by applying a magnetic field using a magnetic field generation type freezing device, the freezing temperature was -30 ° C, which is 50 ° C higher than −80 ° C of the general freezing method.

In the general freezing method, the inside of the freezer is cooled to the freezing temperature of −80 ° C., and it takes a long time for the inside of the freezer to reach the set freezing temperature. Naturally, the power consumption of the refrigerating device also increases. Therefore, in the general freezing method, a large amount of time and operating cost of the freezer are required each time the sample is frozen. Therefore, when the sample is frozen at an extremely low temperature, considering the overall cost including time, the inside of the freezer is cooled rather than slowly cooled to the set freezing temperature using a program freezer at a predetermined lowering rate. It is preferable to maintain the freezing temperature and put the sample in a freezer at the freezing temperature to freeze it.

Therefore, a freezing method was examined in consideration of the overall cost while using the cell cryopreservation solution according to the examples. Specifically, a cell suspension using the above test cells and a cell cryopreservation solution containing 10 vol% chondroitin sulfate in a 300 mM trehalose solution was prepared as a sample. Next, a magnetic field application freezing test in which the sample is frozen by changing the freezing temperature while using a magnetic field generation type freezing device, and a general freezing method in which the sample is placed in a freezer maintained at a temperature of -80 ° C. A freezing test by the method was performed. In the magnetic field application freezing test, the sample is placed in a freezing room at an ambient temperature, and then the temperature inside the freezer is lowered to any of -20 ° C, -30 ° C, and -50 ° C, respectively. The sample was frozen by maintaining it at the freezing temperature for 10 minutes. Then, the sample after the freezing test by the general freezing method and after the magnetic field application freezing test at each temperature was held for 24 hours in a freezer set to a temperature of -80 ° C, and then thawed to examine the cell viability. .. In the magnetic field application freezing test, a magnetic field with a frequency of 50 Hz and a magnetic flux density of 0.30 to 0.31 mT is applied to the sample, cooled to each temperature at a temperature lowering rate of -2 ° C / min, and then maintained at that temperature for 10 minutes. This frozen the sample.

FIG. 6 shows the results of the magnetic field application freezing test. As shown in FIG. 6, by cooling the sample by a freezing method while applying a magnetic field (hereinafter, may be referred to as “magnetic field application freezing method”), the sample is cooled at a high temperature for an extremely short time (10 minutes). ) Could freeze the sample, and the cell viability after thawing was also higher than that of the sample frozen by the general freezing method. In particular, samples frozen at a temperature of −30 ° C. while applying a magnetic field showed high cell viability (89.2%).

As described above, if the cells are frozen using the cell cryopreservation solution according to the example and the magnetic field generation type freezing device, the cell survival rate can be improved and the cell freezing cost can be reduced. It will be possible. That is, the synergistic effect of improving the cell survival rate and reducing the freezing cost can further enhance the practicality of the cell freezing technique for medical use. If the magnetic flux density applied to the sample, the temperature in the freezer, or the cooling procedure is optimized, the survival rate can be expected to be further improved.

=== Supplementary explanation, other examples ===
In each of the above examples, HEK293 cells were used as test cells, but each of the above examples can also be applied to other cells.

The above sample was prepared each time the test was performed. Therefore, the samples used in the examples may have slightly different test results such as cell viability even if the preparation conditions and test methods are the same.

The magnetic field generation type freezing device 1 shown in FIGS. 1 and 2 is for explaining the basic or typical configuration of the magnetic field generation type freezing device, and the magnetic field used in the freezing test of each of the above samples. As a matter of course, the appearance, detailed configuration, specifications, etc. may differ from the generation type freezing device and the magnetic field generation type refrigerating device introduced at the time of practical use.

1 Magnetic field generation type freezer, 10 Refrigerator body, 11 Freezer tank (freezer),
12 freezer, 15 temperature sensor, 16 coil, 20 control unit,
22 Coil control unit, 25 Temperature control unit

Claims (4)

A cell cryopreservation solution containing trehalose and chondroitin sulfate as additives using physiological saline as a solvent. The cell cryopreservation solution according to claim 1, wherein the chondroitin sulfate is added to the trehalose solution containing the trehalose at a concentration of 200 mM or more and 300 mM or less in the physiological saline. The cell cryopreservation solution according to claim 2, wherein the chondroitin sulfate is added to the trehalose solution at a ratio of 5 vol% or more to 10 vol% or less. A method for freezing cells using the cell cryopreservation solution according to any one of claims 1 to 3.
A sample preparation step of preparing a cell suspension in which the cell cryopreservation solution is added to cells to be frozen is prepared as a sample.
A sample installation step of installing the first container containing the sample in a second container filled with 2-propanol.
Following the sample installation step, a freezing step in which the second container is placed in a freezer to freeze the sample, and a freezing step.
Including
In the freezing step, a magnetic field is generated in the freezer, the magnetic field is applied to the sample, the temperature is gradually cooled to −50 ° C. or higher and −30 ° C. or lower, and the temperature is maintained at the temperature for a predetermined time.
Cell freezing method.

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