TWM638487U - Sampling tube for isolating human mesenchymal stem cells - Google Patents

Abstract

The disclosure is a sampling tube for isolating human mesenchymal stem cells, including a tubular body, an upper cover and reagents. Specifically, the tubular body has an upper opening, the upper cover is arranged at the upper opening of the tubular body, and the reagent is accommodated in the tubular body. In a preferred embodiment, the reagent is a cryopreservative.

Description

Sampling tubes for isolating human mesenchymal stem cells

The present disclosure relates to a sampling tube, especially a sampling tube for separating human mesenchymal stem cells.

Mesenchymal stem cells can be isolated from various tissues including, but not limited to, neonatal tissue (eg, Wharton's jelly of human umbilical cord), bone marrow, peripheral blood, placenta, umbilical cord blood, and adipose tissue. Among these tissues, peripheral blood is the most accessible source of mesenchymal stem cells. However, the content of mesenchymal stem cells in peripheral blood is quite low. Even if mesenchymal stem cells are expanded in vitro, related fields still cannot obtain enough mesenchymal stem cells for subsequent differentiation and related applications.

At present, the peripheral blood sampling tubes provided on the market are mainly used for testing, and different sampling tubes are selected for the purpose of testing, such as blood cell testing, biochemical testing, or immunological testing. However, these sampling tubes are not designed for the isolation of mesenchymal stem cells from peripheral blood and still have their limitations.

Therefore, there is an urgent need in related fields to develop a sampling device that can obtain mesenchymal stem cells from peripheral blood.

In order to provide the reader with a basic understanding, a brief novel summary of the present disclosure is provided below. This summary is not an extensive overview of the disclosure and is not intended to define key/essential aspects of the disclosure. Need elements or outline the scope of the present invention. Its sole purpose is to present some concepts of the disclosure in a simplified form as a prelude to the more detailed description that is presented later.

The inventors of the present invention unexpectedly obtained mesenchymal stem cells (mesenchymal stem cells, MSCs) from the peripheral blood of human individuals, and expanded them in vitro in a culture solution containing glycerol, thereby preparing an isolated, substantially pure, A population of undifferentiated human mesenchymal stem cells (hMSCs), which is suitable for autologous transplantation.

Accordingly, the first aspect of the disclosure relates to a sampling tube for isolating human mesenchymal stem cells. Structurally, the sampling tube includes a tubular body, an upper cover and a cryopreservative. Specifically, the tubular body has an upper opening, the upper cover is arranged on the upper opening of the tubular body, and the cryopreservative is arranged in the tubular body.

In a preferred embodiment, the cryopreservative is glycerin. In an optional embodiment, the glycerin in the sampling tube is at least 10 ml, 20 ml, 30 ml, 40 ml or 50 ml.

In addition, according to a non-limiting embodiment of the present invention, the sampling tube of the present invention further includes an anticoagulant coated on the inner surface of the tubular body.

According to one embodiment of the present invention, the upper cover of the sampling tube is made of elastic material. In a non-limiting embodiment, the elastic material is rubber.

According to another embodiment of the present invention, the sampling tube may further contain a culture solution, and preferably the culture solution does not contain any granulocyte colony stimulating factor (G-CSF) , a mobile agent of the group consisting of granulocyte-macrophage colony stimulating factor (granulocyte-macrophage colony stimulating factor, GM-CSF) and combinations thereof.

It should be noted that the sampling tube proposed by the present invention can be applied to the following method to obtain artificially differentiated human mesenchymal stem cells. The method includes the following steps: (a) obtaining peripheral blood from a human individual into the sampling tube of the present invention (b) isolating human mesenchymal stem cells in the peripheral blood of step (a) relative to other adult stem cells; and (c) culturing the isolated human mesenchymal stem cells of step (b) in a glycerol-containing In a culture medium, the population of human mesenchymal stem cells is prepared; wherein the culture medium does not contain the group selected from granulocyte colony stimulating factor (granulocyte colony stimulating factor, G-CSF), granulocyte-macrophage colony stimulating factor (granulocyte -macrophage colony stimulating factor, GM-CSF) and any mobilization agent of the group consisting of combinations thereof.

According to an embodiment of the present disclosure, in step (a), the peripheral blood is collected in a sampling tube that does not contain any hemolytic agent.

According to an embodiment of the present disclosure, in step (a), the concentration of the cryopreservative (ie, glycerol) in peripheral blood is about 1 to 10 mg/ml.

According to the embodiment of the present disclosure, the prepared undifferentiated human mesenchymal stem cell population is negative for CD34-, CD45- and HLA-DR cell surface markers, and negative for CD73+, CD90+ and CD105+ cell surface markers. positive response.

After referring to the following embodiments, those with ordinary knowledge in the technical field of the present invention can easily understand the basic spirit of the present invention and other novel objectives, as well as the technical means and implementation modes adopted by the present invention.

The main component symbols of the new model are listed as follows:

100, 200: sampling tube

110: tubular body

120: top cover

130: cryopreservative

205: label

In order to make the above and other purposes, features, advantages and embodiments of the present invention more obvious and easy to understand, the accompanying drawings are described as follows: Figure 1A is a sampling for isolating human mesenchymal stem cells according to an embodiment of the present invention Tube 100; FIG. 1B is an exploded view of the sampling tube 100 shown in FIG. 1A; FIG. 2 is a sampling tube 200 shown in another implementation of the present invention.

In order to make the description of the disclosure more detailed and complete, the following provides an illustrative description of the implementation and specific embodiments of the present invention; but this is not the only form of implementing or using the specific embodiments of the present invention. The description covers features of various embodiments as well as method steps and their sequences for constructing and operating those embodiments. However, other embodiments can also be used to achieve the same or equivalent functions and step sequences.

definition

For the convenience of description, specific terms described in this specification, examples, and appended claims are collectively described here. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.

As used herein, the singular forms "a" (a, an) and "the" (the) include plural forms unless the context clearly dictates otherwise.

The term "stem cell" used herein is used in a broad sense, and also includes traditional stem cells, progenitor cells, pre-progenitor cells, etc. The term "stem cell" refers to an undifferentiated cell capable of proliferating and giving rise to further precursor cells capable of giving rise to a large number of differentiated or differentiated daughter cells. Daughter cells can themselves be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types. The term "stem cell" may also refer to a cell that, under certain circumstances, has the ability or potential to differentiate into a more specialized or differentiated phenotype and, in some cases, retains the ability to proliferate in an undifferentiated state Cell.

The term "mesenchymal stem cells" (MSCs) refers to a type of pluripotent stem cells that can differentiate into more than one specific type of connective tissue, such as adipose tissue, bone tissue, stroma tissue, cartilage tissue , elastic tissue and fibrous tissue. For identification purposes, human mesenchymal stem cells can be identified based on the expression of the phenotypic markers CD34-, CD45-, CD73+, CD90+, and CD105+, and can also be based on differentiation into specific life-supporting tissues such as, but not limited to, chondrocytes , cartilage and fat cells) to identify the ability.

With respect to isolated populations of human mesenchymal stem cells (hMSCs), the term "population", "population" or "population" means a population of human mesenchymal stem cells (hMSCs) that has been removed and isolated from a mixed or heterogeneous population of cells. Populations of human mesenchymal stem cells (hMSCs). In some embodiments, the population of hMSCs is a substantially pure population of human mesenchymal stem cells compared to cells isolated or expanded from a heterogeneous population of cells.

The term "substantially pure" in this disclosure with respect to a particular cell population refers to a population that has at least 70%, preferably about 80%, or more, compared to the composition of the total cell population. Preferably about 90%, and optimally about 95% of the cells are pure. The term "substantially pure" with respect to a population of human mesenchymal stem cells prepared according to the methods described in this disclosure means containing less than about 30%, preferably less than about 20%, 15%, 10%, 8%, more preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of the human mesenchymal stem cell population of non-human mesenchymal stem cells. According to some embodiments, the present disclosure comprises a method of expanding a population of human mesenchymal stem cells isolated from peripheral blood, wherein the expanded population of human mesenchymal stem cells derived from peripheral blood is a substantially pure population of human mesenchymal stem cells .

In the context of cellular ontogeny, the adjective "differentiated" is a relative term. A "differentiated cell" refers to a cell that is further along or further down the developmental path than another cell with which it is being compared. Thus, stem cells can differentiate into lineage-restricted precursor cells, which in turn can differentiate into other precursor cell types further downstream. This is followed by redifferentiation into cells of the final stage of differentiation which play a characteristic role in a particular tissue type and which may or may not retain the ability to proliferate further.

As used herein, the term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect, such as tissue regeneration. The aforementioned effects may be prophylactic in terms of total or partial prevention of the disease or its symptoms; and/or therapeutic in terms of total or partial treatment of the disease and/or adverse effects caused by the disease of. As used herein, "Treatment" (Treatment) includes preventive (eg: prophylactic), curative or palliative treatment of a disease in a mammal, especially a human being; and includes: (1) treatment of Preventative (e.g., prophylactic), curative, or palliative treatment of a disease or condition in an individual who may be predisposed to the disease but has not been diagnosed with the disease; (2) inhibiting a disease (eg, by terminating the development of the disease); or (3) ameliorating a disease (eg, reducing symptoms associated with the disease).

The terms "administered, administering," or "transplanting" are used interchangeably herein to refer to any suitable route of delivery, including but not limited to: intravenous, intramuscular, intraperitoneal , intraarterially, intracranially, or subcutaneously administer the population of human mesenchymal stem cells of the present invention to desired parts of the individual, wherein at least a part of the human mesenchymal stem cells are still alive. The period of survival of the cells after administration to an individual can also be as short as a few hours, such as 24 hours, to a few days, and as long as several months.

"An effective amount" as used in this disclosure refers to an effective dose of human mesenchymal stem cells that can achieve the desired results in the treatment of diseases caused by platelet aggregation within a specific required time. For example, in the treatment of wounds, agents that are effective in promoting the regeneration of skin and related soft tissues (ie the human mesenchymal stem cells of the present disclosure). The effective amount of the agent does not necessarily cure the disease or disorder but can provide treatment for a disease or disorder, such as delaying the onset of the disease or disorder, limiting or preventing the onset of the disease or disorder, or improving the symptoms of the disease or disorder. A particular effective or sufficient amount may vary with various factors, such as the particular condition pretreated, the physiological condition of the patient (e.g., the patient's weight, age or sex), the species of mammal or animal being treated, the duration of treatment, concurrent The nature of the course of treatment, if any, and the specific preparations used, etc. The effective amount can be divided into one, two or more doses in a suitable form, whereby it is administered once, two or more times within a specified period of time.

As used herein, the terms "subject" or "patient" are used interchangeably and mean a mammal (including a human) capable of being treated with a compound of the disclosure. The term "mammal" refers to all members of the class Mammalia, which includes humans, primates, domesticated and domestic animals (e.g. rabbits, pigs, sheep, cattle) and animals kept in zoos, used for exercise animals or pets; and rodents (such as mice and rats). Further, the terms "individual" or "subject" or "patient" include males (male) and females (female) unless gender is specifically indicated. Accordingly, the terms "individual, subject" or "patient" include any mammal that would benefit from a method of treatment of the present disclosure. Examples of "individuals, subjects" or "patients" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds and avians. In preferred embodiments, the individual is a human.

The term "medium" refers to a medium containing nutrients that can maintain cell viability and support cell proliferation, and is used to maintain tissues or cell populations, or to culture substrates containing cell populations (eg, culture substrates). Cell culture medium contains any suitable combination of the following substances: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum substitutes, and other ingredients such as growth factors. Those skilled in the art to which the present invention pertains know the cell culture media that a particular cell type will use.

As used herein, the terms "move", "mobilize", and "mobilization" refer to the process by which cells leave their original niche (eg, bone marrow) and enter the blood. The term "mobile agent" (Mobilization agent) is an agent that can make aggregates or populations of stem cells (such as MSCs) residing in peripheral blood and bone marrow stem cell areas lose their adhesion.

The inventors of the present invention unexpectedly obtained mesenchymal stem cells (mesenchymal stem cells, MSCs) from the peripheral blood of human individuals, and expanded them in vitro in a culture solution containing glycerol, thereby preparing an isolated, substantially pure, A population of undifferentiated human mesenchymal stem cells (hMSCs), which is suitable for autologous transplantation.

Accordingly, the first aspect of the disclosure relates to a sampling tube for isolating human mesenchymal stem cells. Please refer to FIG. 1A and FIG. 1B , which illustrate a schematic structural view and an exploded schematic view of the sampling tube 100 .

Structurally, the sampling tube 100 includes a tubular body 110 , an upper cover 120 and a cryopreservative 130 . Specifically, the tubular body 110 has an upper opening 112 , the upper cover 120 is disposed on the upper opening 112 of the tubular body 110 , and the cryopreservative 130 is disposed in the tubular body 110 .

In a specific embodiment, the reagent is a cryopreservative 130 . In a preferred embodiment, the cryopreservative 130 is glycerol. In an optional embodiment, the glycerin in the sampling tube is at least 10 ml, 20 ml, 30 ml, 40 ml or 50 ml.

In a non-limiting embodiment, the upper cover 120 of the sampling tube 100 is made of elastic material. In a non-limiting embodiment, the elastic material is rubber.

The novel sampling tube is suitable for separating human mesenchymal stem cells from peripheral blood. Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells that can generally be obtained from bone marrow. Peripheral blood has low levels of mesenchymal stem cells, and even when these cells are expanded in vitro, mesenchymal stem cells obtained from peripheral blood often do not yield sufficient numbers of cells for therapeutic use. The new type of sampling tube is matched with the method proposed by the author of this case to separate and expand mesenchymal stem cells from peripheral blood, so that A substantially pure population of human mesenchymal stem cells is prepared which can be used alone or in combination with other cells for autologous treatment in regenerative therapies such as wound healing and bone repair and other orthopedic indications.

Accordingly, blood sampling can be performed through the sampling tube 100 of the present invention to provide undifferentiated human mesenchymal stem cell populations. The method includes the following steps: (a) obtaining peripheral blood from a human individual and placing it in the sampling tube of the present invention In 100; (b) isolating human mesenchymal stem cells in the peripheral blood of step (a) relative to other adult stem cells; and (c) culturing the isolated human mesenchymal stem cells of step (b) in a glycerol-containing Human mesenchymal stem cell populations were prepared in this way; wherein the culture medium did not contain anything selected from granulocyte colony stimulating factor (granulocyte colony stimulating factor, G-CSF), granulocyte-macrophage colony stimulating factor ( granulocyte-macrophage colony stimulating factor, GM-CSF) and the medicament of the group formed by its combination.

According to other embodiments, the sampling tube 100 further includes an anticoagulant in the tubular body 110 , which is not shown here. In one embodiment, the anticoagulant is coated inside the tubular body 110 . In an optional embodiment, the anticoagulant is EDTA or heparin.

In a non-limiting embodiment, in the step of preparing a population of human mesenchymal stem cells, step (a) is drawing peripheral blood from a human individual, and collecting the peripheral blood in a sampling tube 100 containing heparin. According to one embodiment of the present disclosure, the individual may be a healthy individual or an individual suffering from a disease or condition requiring human mesenchymal stem cell transplantation.

After culturing, human mesenchymal stem cells can be separated from adult stem cells in peripheral blood containing glycerol by a suitable method, such as a cell sorter (cell sorter) or centrifugal elutriation (centrifugal elutriation) (this Step (b) of the method).

According to certain embodiments of the present disclosure, in step (b) of the method, the human interferon in the peripheral blood is separated by a cell sorter, such as a laser scanning flow cytometer (laser scanning flow cytometer). Mesenchymal stem cells are isolated from adult stem cells. A typical flow cytometer is composed of a laser light, a flow measurement chamber, and an optical system (composed of lenses, filters, and light detectors). Two photomultiplier tubes (one with an angle of 180 degrees and one with a laser angle of 90 degrees) are used to measure forward scattered light (forward scattered light, FSC) and right angle scattered light (side scattered light, SCC) respectively. )). In the forward tube, three fluorescent detectors consisting of filter mirrors and photomultiplier tubes can be used to detect fluorescent light. A fluorescence activated cell sorting (FACS) was set up to select specific forward scatter and/or side scatter cells. FSC is proportional to cell surface area or size. FSCs mostly measure diffracted light and it is detected by a photodiode positioned exactly on the axis of the incident laser beam in the forward direction. FSC is suitable for detecting target particles larger than a given size, independent of the particle's fluorescence. Side-scattered light (SCC) is proportional to granularity or internal complexity. SSC is used to measure light refraction and light reflection occurring at any interface inside a cell that has a change in refractive index. The SSC collects the laser beam through a collection lens at an angle of about 90 degrees to the laser beam, and then redirects it through a beam splitter to a suitable detector. Thereby, cells can be selected by controlling specific FSC and SSC gates. According to an embodiment of the present disclosure, peripheral blood human mesenchymal stem cells are separated from adult stem cells based on the expression of cell surface markers, wherein cell populations are selected by gating for staining with different fluorescence intensities.

In step (b), optionally or additionally, inject the glycerol-containing peripheral blood obtained from step (a) into a cell sorter (such as the COBE Spectra Apheresis System), and optionally add anticoagulation Blood agents are used to keep the blood from clotting during this procedure. The mixture is circulated through a centrifuge to separate peripheral blood into a population of mesenchymal stem cells and monocytes from other blood components and plasma. catch The isolated mesenchymal stem cells are then pumped into a collection bag for storage, while other blood components and plasma are discarded or returned to the individual.

In step (b) of the novel method, alternatively or additionally, human mesenchymal stem cells in peripheral blood containing glycerol are separated from other adult stem cells by centrifugal elutriation, wherein stem cells of smaller size Can be isolated from larger size stem cells. In a specific embodiment, the peripheral blood containing glycerol in step (b) is placed into a conventional funnel-shaped separation chamber located in a spin centrifuge. A stream of liquid elutriation buffer is introduced into the separation chamber containing peripheral blood. As the flow rate of the liquid elutriation buffer solution through the separation chamber is increased, the liquid sweeps smaller, slower-settling cells towards the elutriation boundary of the separation chamber, while larger, faster-settling cells move into the separation chamber The area where the central centrifugal force and the sedimentation force are balanced. Accordingly, peripheral blood can contain many different populations of stem cells, which can then be differentiated into distinct populations by panning, wherein smaller stem cells can be separated from stem cells of larger size. Peripheral blood-derived mesenchymal stem cells can be obtained and/or isolated using any elutriation device.

According to another embodiment of the present invention, the sampling tube 100 may further include a culture solution, and preferably the culture solution does not contain any granulocyte colony stimulating factor (G-CSF) ), granulocyte-macrophage colony stimulating factor (granulocyte-macrophage colony stimulating factor, GM-CSF) and the group consisting of mobile agents.

Accordingly, in the step (c) of the present invention, the human mesenchymal stem cells prepared from step (b) are cultured under culture conditions that can enhance the proliferation of the isolated human mesenchymal stem cells to further expand increase these cells. According to a preferred embodiment of the present disclosure, the human mesenchymal stem cells obtained from step (b) can be cultured in a growth medium containing glycerol in a petri dish (surface area about 10 cm2), and The growth medium lacks any known stem cell mobilizing agents (including, but not limited to: granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and combinations thereof ). It should be understood that when a culture medium is said to be deficient in a particular component, this disclosure also contemplates that the culture medium still contains that component, but at a concentration well below that at which it is minimally active. For example, certain culture fluids may contain minor amounts of the aforementioned stem cell mobilization agents (i.e., G-CSF, GM-CSF, or combinations thereof), however, the methods of the present disclosure pertain to a formulation other than those originally present in commercially available culture fluids In addition to the growth factors produced by the overall adjustment of the concentration of the components of the culture solution, the culture solution does not add growth factors exogenously. In a non-limiting embodiment, the culture dish can also be a sampling tube 100 containing a culture solution.

A typical culture medium suitable for culturing human mesenchymal stem cells according to the present disclosure can be a complete medium (for example: DMEM) containing at least 20% (volume ratio) of serum, preferably at least 30% (volume ratio) of serum , more preferably at least 50% (volume ratio) of serum. The glycerol concentration in the complete culture solution can be about 0.1-1.0 mg per ml (such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mg per ml); more can be about 0.2-1.0 mg per ml; 0.8 mg (eg 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mg per ml); optimally about 0.5 mg per ml. In some embodiments, the growth medium for culturing human mesenchymal stem cells uses 100% (volume ratio) serum, and then adds 0.1-1.0 mg of glycerol per ml. During the cultivation process, supplements required for cell metabolism, such as amino acids, vitamins, minerals, and useful proteins (such as transferrin), can also be added to the culture medium. The culture medium may also contain antibiotics to prevent yeast, bacterial and fungal infections. The antibiotic may be penicillin, streptomycin, gentamicin and the like. According to one embodiment of the present disclosure, the culture medium comprises pituitary gland extract, plasma and fetal bovine serum. The human mesenchymal stem cells can be effectively expanded by culturing for a period of time, so as to prepare a sufficient amount of human mesenchymal stem cells. According to a specific embodiment, the culture can contain 30% (volume ratio) of serum and 0.5 mg of glycerol per ml. liquid, culture the human mesenchymal stem cells obtained in step (c) at 37°C for at least 12 hours, preferably at least 16 hours, more preferably at least 24 hours, so as to prepare a population of human mesenchymal stem cells suitable for autologous transplantation .

According to a specific embodiment of the present disclosure, the human mesenchymal stem cell population prepared by using the novel sampling tube and the disclosed method exhibits negative staining for hematopoietic stem cell markers CD34 and CD45, and is known to mediate graft-versus-host disease ( Graft-versus-host disease (GvHD) human leukocyte antigen-antigen D-related (HLA-DR) cell surface markers also showed negative staining; cell surface markers CD73, CD90 and CD 105 showed positive staining. Methods for determining the expression of cell surface markers are common knowledge in the art. Examples of such methods include immunological methods such as FACS and biochemical methods such as cell surface labeling by radioactivity, fluorescence or avidin-biotin. Another possible way is to use magnetic-activated cell sorting (magnetic-activated cell sorting, MACS) or immunopanning (immunopanning) method to identify cells.

In addition, FIG. 2 shows a sampling tube 200 according to another embodiment of the present invention. The outer surface of the sampling tube 200 is further provided with a label 205 for recording personal information or other labeling information of the sampled individual.

It should be understood that the foregoing descriptions of the embodiments are given only as examples, and those skilled in the art may make various modifications. The above specification, examples and experimental results provide a complete description of the structure and use of exemplary embodiments of the present invention. Although various specific embodiments of the present invention have been disclosed in the above embodiments, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can, without departing from the principle and spirit of the present invention, Various alterations and modifications can be made to it, so the scope of protection of the present invention should be defined by the appended patent scope.

100: sampling tube

110: tubular body

120: top cover

130: cryopreservative

Claims (9)

A sampling tube for separating human mesenchymal stem cells, comprising: a tubular body with an upper opening; an upper cover, arranged at the upper opening of the tubular body; and a low-temperature preservation agent, arranged in the tubular body. The sampling tube according to claim 1, wherein the cryopreservative is glycerol. The sampling tube according to claim 2, wherein the glycerol is at least 10 ml. The sampling tube as claimed in item 2, wherein the glycerol is at least 30 ml. The sampling tube according to claim 2, wherein the glycerol is at least 50 ml. The sampling tube according to claim 1, further comprising an anticoagulant coated on the inner surface of the tubular body. The sampling tube according to claim 1, wherein the upper cover is made of elastic material. The sampling tube as described in claim 1 further includes a culture solution. The sampling tube as described in claim item 8, wherein the culture medium does not contain anything selected from the group consisting of granulocytc colony stimulating factor (granulocytc colony stimulating factor, G-CSF), granulocyte-macrophage colony stimulating factor (granulocyte-macrophage Colony stimulating factor, GM-CSF) and the mobile agent of the group formed by its combination.

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