Plasma solution, platelet lysate, and method for producing said plasma solution and method for producing said platelet lysate

Abstract

Disclosed are: a plasma solution containing a plasma coming from a first leukocyte removal filter through which human whole blood has been passed and a plasma coming from a second leukocyte removal filter through which the human whole blood has been passed, wherein the plasma coming from the second leukocyte removal filter is a plasma contained in a solution obtained by passing, through the second leukocyte removal filter, a solution containing the plasma coming from the first leukocyte removal filter; a platelet lysate; a method for producing the plasma solution; and a method for producing the platelet lysate.

Description

Plasma solution and platelet lysate and methods for producing them

[0001] The present disclosure relates to plasma solutions and platelet lysates containing components derived from leukocyte depletion filters through which human whole blood has been passed, and methods for their production.

[0002] Blood products are products manufactured using blood collected from humans as a raw material, and in addition to whole blood products, which are prepared from blood itself, known products include plasma products, platelet products, and red blood cell products. These are usually manufactured using human blood collected through blood donations, and are primarily used for transfusions in medical procedures.

[0003] On the other hand, blood components contained in these blood products are known to have uses other than transfusion, such as plasma solution, which is a solution containing plasma derived from human blood, and platelet lysate (PL), which is obtained by dissolving platelets derived from human blood by ultrasonic treatment or freeze-thawing. Among these, platelet lysate contains many components that promote cell proliferation and is known to be an alternative to fetal bovine serum (FBS) as a source of cell growth factors in cell culture (Non-Patent Document 1). In particular, when culturing stem cells, such as mesenchymal stem cells, for regenerative medicine purposes, it is considered desirable to use human blood-derived platelet lysate instead of animal-derived components such as FBS, in order to reduce the risk of transmission of prion diseases and from the perspective of animal bioethics.

[0004] As a platelet lysate for use in cell culture, for example, Patent Document 1 describes a composition characterized by containing heparin, a growth factor stabilized with heparin, and a platelet lysate component having a fibrinogen concentration of less than 4 μg / mL.

[0005] Japanese Patent No. 6179955

[0006] Doucet et al., "Platelet lysates promote mesenchymal stem cell expansion: A safety substitute for animal serum in cell-based therapy applications", J. Cell Phys. 205:228-236 (2005).Thierry Burnouf et al., "Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?", Biomaterials 76, 371-387 (2016).

[0007] Plasma solutions and platelet lysates are typically produced using human blood obtained through whole blood donations or human blood components obtained through component donations. However, such human blood and human blood components are typically used for transfusions, and their supply is limited. Therefore, it is not easy to obtain human blood and human blood components on an industrial scale for uses other than transfusions. While human whole blood is available for purchase in some countries, it is extremely expensive and can pose a risk of infection.

[0008] The present disclosure aims to provide a plasma solution and a platelet lysate, as well as methods for producing them. It also aims to provide a plasma solution and a platelet lysate that are produced without using human blood or human blood components obtained through blood donations as raw materials, as well as methods for producing them.

[0009] Leukocyte removal filters are filters used to remove leukocytes when converting human whole blood obtained through whole blood donations into blood products, and are usually discarded as medical waste after passing through them. The present inventors have discovered that plasma solutions and platelet lysates can be produced from used leukocyte removal filters that were previously discarded.

[0010] Furthermore, the present inventors have found that in addition to platelet lysates, platelet lysates produced using plasma solutions prepared from used leukocyte removal filters exhibit excellent cell proliferation-promoting effects.

[0011] The present disclosure relates to, for example, the following: [1] A plasma solution containing non-blood cell components derived from a first leukocyte removal filter through which human whole blood has been passed and non-blood cell components derived from a second leukocyte removal filter through which human whole blood has been passed, wherein the non-blood cell components derived from the second leukocyte removal filter are non-blood cell components contained in a solution obtained by passing a solution containing non-blood cell components derived from the first leukocyte removal filter through the second leukocyte removal filter. [2] A plasma solution containing a supernatant obtained from an (n+1)th filter-passed solution, wherein the (n+1)th filter-passed solution is a solution obtained by passing the supernatant obtained from the nth filter-passed solution through an (n+1)th leukocyte removal filter through which human whole blood has been passed, and the first filter-passed solution is a solution obtained by passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed. (where n is an integer of 1 or more, and the second paragraph is repeated n times by decreasing the number substituted for n by 1.) [3] A method for producing a plasma solution, comprising: a first passing step: a step of passing an aqueous solution through a first leukocyte removal filter that has passed human whole blood, and recovering the solution after passing to obtain a first filter-passed solution; an i-th plasma recovery step: a step of recovering a supernatant from the i-th filter-passed solution; and an i+1-th passing step: a step of passing the supernatant obtained in the i-th plasma recovery step through an i+1-th leukocyte removal filter that has passed human whole blood, and recovering the solution after passing to obtain an i+1-th filter-passed solution; (where n is an integer of 1 or more, i is an integer of 1 to n, and the i-th plasma recovery step and the i+1-th passing step are repeated n times in this order by increasing the number substituted for i by 1 in succession, starting from 1.) [4] A plasma solution produced by the production method described in [3]. [5] A plasma solution produced by the manufacturing method according to [3], wherein the platelet lysate produced using the plasma solution has a higher stem cell proliferation activity than a mixture of the fresh frozen plasma prepared from human blood and the supernatant recovered in the first plasma recovery step, in which the fresh frozen plasma prepared from human blood accounts for 20% by volume or more. [6] A method for producing a platelet lysate, comprising a step of mixing the plasma solution produced by the manufacturing method according to [3] with platelets or a lysate thereof.[7] First passing step: a step of passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed, and recovering the solution after passing to obtain a first filter-passed solution; First red blood cell removal step: a step of removing red blood cells from the first filter-passed solution obtained in the first passing step; i-th recovery step: a step of centrifuging the solution obtained in the i-th red blood cell removal step, recovering the supernatant, and recovering the residue after supernatant recovery as a platelet concentrate; i+1-th passing step: a step of passing the supernatant obtained in the i-th recovery step through the i+1-th leukocyte removal filter through which human whole blood has been passed, and recovering the solution after passing to obtain an i+1-th filter-passed solution; i+1-th red blood cell removal step: a step of removing red blood cells from the i+1-th filter-passed solution obtained in the i+1-th passing step; (where n is an integer of 1 or more, i is an integer of 1 or more and n or less, and the i-th collection step, the i+1-th passing step, and the i+1-th red blood cell removal step are repeated n times in this order while the number substituted for i is increased by 1 in succession starting from 1.) A method for producing a platelet lysate, comprising: a mixing step: a step of mixing at least one platelet concentrate selected from the group consisting of the platelet concentrates obtained in the first to n-th collection steps with the solution obtained in the n+1-th red blood cell removal step; and a platelet dissolving step: a step of dissolving platelets in the solution obtained in the mixing step.[8] First passing step: a step of passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed, and recovering the solution after passing to obtain a first filter-passed solution; First red blood cell removal step: a step of removing red blood cells from the first filter-passed solution obtained in the first passing step; i-th recovery step: a step of centrifuging the solution obtained in the i-th red blood cell removal step, recovering the supernatant, and recovering the residue after supernatant recovery as a platelet concentrate; i+1-th passing step: a step of passing the supernatant obtained in the i-th recovery step through the i+1-th leukocyte removal filter through which human whole blood has been passed, and recovering the solution after passing to obtain an i+1-th filter-passed solution; i+1-th red blood cell removal step: a step of removing red blood cells from the i+1-th filter-passed solution obtained in the i+1-th passing step; (where n is an integer of 1 or more, i is an integer of 1 or more and n or less, and the i-th collection step, the (i+1)th passing step, and the (i+1)th red blood cell removal step are repeated n times in this order while the number substituted for i is increased by 1 in succession, starting from 1.) A method for producing a platelet lysate, comprising: a platelet dissolving step: a step of dissolving platelets in at least one platelet concentrate selected from the group consisting of the platelet concentrates obtained in the first to n-th collection steps; and a mixing step: a step of mixing the at least one platelet concentrate after platelet lysis obtained in the platelet dissolving step with the solution obtained in the (n+1)th red blood cell removal step. [9] A platelet lysate produced according to the production method described in any one of [6] to [8].

[10] A platelet lysate containing a platelet lysate, non-blood cell components derived from a first leukocyte removal filter through which human whole blood has been passed, and non-blood cell components derived from a second leukocyte removal filter through which human whole blood has been passed, wherein the non-blood cell components derived from the second leukocyte removal filter are non-blood cell components contained in a solution obtained by passing a solution containing non-blood cell components derived from the first leukocyte removal filter through the second leukocyte removal filter.

[11] A platelet lysate comprising a platelet lysate and a supernatant obtained from a second filter-passed solution, wherein the second filter-passed solution is a solution obtained by passing the supernatant obtained from the first filter-passed solution through a second leukocyte removal filter through which human whole blood has been passed, and the first filter-passed solution is a solution obtained by passing an aqueous solution through the first leukocyte removal filter through which human whole blood has been passed.

[12] The platelet lysate according to

[10] or

[11] , wherein the platelet lysate is a lysate of platelets derived from the first leukocyte removal filter through which human whole blood has been passed.

[13] A platelet lysate produced by the production method according to any one of [6] to [8] or the platelet lysate according to any one of [9] to

[12] , wherein the cell proliferation activity of stem cells is higher than that of a plasma solution which is a mixture of fresh frozen plasma prepared from human blood and the supernatant recovered in the first plasma recovery step, wherein the fresh frozen plasma prepared from human blood accounts for 20% by volume or more.

[14] A platelet lysate produced by the production method according to any one of [6] to [8] or the platelet lysate according to any one of [9] to

[13] , wherein the fibrinogen concentration is 100 μg / mL or more and 1500 μg / mL or less.

[15] A platelet lysate produced by the production method according to any one of [6] to [8] or the platelet lysate according to any one of [9] to

[14] , wherein the stem cell proliferation-promoting activity is three times or more that of fetal bovine serum.

[16] A platelet lysate produced by the production method according to any one of [6] to [8] or the platelet lysate according to any one of [9] to

[15] , wherein the plasma-derived protein is 6 mg / mL or more.

[17] A platelet lysate produced by the production method according to any one of [6] to [8] or the platelet lysate according to any one of [9] to

[15] , wherein the plasma-derived protein is 18 mg / mL or more.

[18] A platelet lysate produced by the production method according to any one of [6] to [8] or the platelet lysate according to any one of [9] to

[15] , containing 27 mg / mL or more of plasma-derived protein.

[19] A platelet lysate containing 6 mg / mL or more of plasma-derived protein.

[20] A platelet lysate containing 18 mg / mL or more of plasma-derived protein.

[21] A platelet lysate containing 27 mg / mL or more of plasma-derived protein.

[22] The platelet lysate according to any one of

[19] to

[21] , produced according to the production method according to any one of [6] to [8].

[23] The platelet lysate according to any one of

[19] to

[22] , having a fibrinogen concentration of 100 μg / mL or more and 1500 μg / mL or less.

[24] The platelet lysate according to any one of

[19] to

[23] , having a stem cell proliferation-promoting activity three times or more that of fetal bovine serum.

[0012] According to one embodiment of the present disclosure, a plasma solution can be produced from a leukocyte removal filter after human whole blood has been passed through it. That is, according to one embodiment of the present disclosure, a plasma solution containing plasma derived from a leukocyte removal filter after human whole blood has been passed through it, and a method for producing the same can be provided. Furthermore, when using a plasma solution produced from such a leukocyte removal filter after human whole blood has been passed through it, a platelet lysate having a superior cell proliferation-promoting effect can be obtained compared to when an expired human plasma solution is used.

[0013] According to one embodiment of the present disclosure, a plasma solution having a protein concentration equal to or greater than a predetermined ratio relative to the protein concentration in fresh frozen plasma prepared from human blood can be produced from a leukocyte removal filter after passing human whole blood through it. Such a plasma solution can be produced animal-free and inexpensively because it uses a used leukocyte removal filter that is normally discarded as medical waste.

[0014] According to one embodiment of the present disclosure, a platelet lysate can be produced from a leukocyte removal filter after passing human whole blood. The platelet lysate contains platelet lysates and plasma. That is, according to one embodiment of the present disclosure, a platelet lysate containing platelet lysates and plasma derived from a leukocyte removal filter after passing human whole blood, and a method for producing the same, can be provided. Such a platelet lysate can be produced animal-free and inexpensively because it uses a used leukocyte removal filter that is normally discarded as medical waste.

[0015] According to one embodiment of the present disclosure, a platelet lysate having excellent cell proliferation-promoting activity can be produced from a leukocyte removal filter through which human whole blood has been passed. This platelet lysate contains platelet lysate and plasma. That is, according to one embodiment of the present disclosure, a platelet lysate having excellent cell proliferation-promoting activity and containing platelet lysate and plasma derived from a leukocyte removal filter through which human whole blood has been passed, and a method for producing the same, can be provided. For example, such a platelet lysate has a stem cell proliferation-promoting activity that is at least three times that of fetal bovine serum.

[0016] Fig. 1 is a graph showing the cell proliferation rate of mesenchymal stem cells in Test Example 2. Fig. 2 is a graph showing a transmitted light image of mesenchymal stem cells cultured in Test Example 2. Fig. 3 is a graph showing the cell proliferation rate of mesenchymal stem cells in Test Example 3. Fig. 4 is a graph showing a transmitted light image of mesenchymal stem cells cultured in Test Example 3. Fig. 5 is a graph showing the cell proliferation rate of mesenchymal stem cells in Test Example 4. Fig. 6 is a graph showing the cell proliferation rate and transmitted light image of mesenchymal stem cells in the test of Test Example 5. Fig. 7 is a graph showing the cell proliferation rate of mesenchymal stem cells in Test Example 6. Fig. 8 is a graph showing the results of statistical processing of the cell proliferation rate of mesenchymal stem cells in Test Example 6.

[0017] Hereinafter, embodiments for carrying out the present disclosure will be described, but the present disclosure is not limited to the following embodiments.

[0018] <Plasma Solution> A first aspect of the present disclosure is a plasma solution containing non-blood cell components derived from a first leukocyte removal filter through which human whole blood has been passed and non-blood cell components derived from a second leukocyte removal filter through which human whole blood has been passed, wherein the non-blood cell components derived from the second leukocyte removal filter are non-blood cell components contained in a solution obtained by passing a solution containing non-blood cell components derived from the first leukocyte removal filter through the second leukocyte removal filter. A first aspect of the present disclosure may also be the following: "A plasma solution comprising a supernatant obtained from an (n+1)th filter-passed solution, wherein the (n+1)th filter-passed solution is a solution obtained by passing the supernatant obtained from the nth filter-passed solution through an (n+1)th leukocyte removal filter through which human whole blood has been passed, and the first filter-passed solution is a solution obtained by passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed. (Here, n is an integer of 1 or more, and the second paragraph is repeated n times by decreasing the number substituted for n by 1.)" In other words, the first aspect of the present disclosure may also be the following: "A plasma solution comprising a supernatant obtained from the (n+1)th filter-passed solution, wherein the (i+1)th filter-passed solution is a solution obtained by passing the supernatant obtained from the i-th filter-passed solution through the (i+1)th leukocyte removal filter through which human whole blood has been passed, and the first filter-passed solution is a solution obtained by passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed. (Here, i is an integer of 1 or more, and the second paragraph is repeated n times by decreasing the number substituted for i from n by 1.)" In further other words, the first aspect of the present disclosure can also be: "A plasma solution comprising a supernatant obtained from the (n+1)th filter-passed solution, wherein the (i+1)th filter-passed solution is a solution obtained by passing the supernatant obtained from the i-th filter-passed solution through the (i+1)th leukocyte removal filter through which human whole blood has been passed, and the first filter-passed solution is a solution obtained by passing an aqueous solution through the first leukocyte removal filter through which human whole blood has been passed, wherein n is an integer of 1 or more, and i represents all integers from 1 to n, inclusive."In these cases, n may be, for example, 1, 2, 3, 4, or 5. Furthermore, in one embodiment, the first aspect of the present disclosure may be a plasma solution produced by the method for producing a plasma solution according to the second aspect of the present disclosure described below.

[0019] For example, one embodiment (where n=1) of the first aspect of the present disclosure is as follows: "A plasma solution comprising a supernatant obtained from a second filtered solution, wherein the second filtered solution is a solution obtained by passing the supernatant obtained from the first filtered solution through a second leukocyte removal filter through which human whole blood has been passed, and the first filtered solution is a solution obtained by passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed." Furthermore, for example, one embodiment (where n=2) of the first aspect of the present disclosure is as follows: "A plasma solution comprising a supernatant obtained from a third filter-passed solution, wherein the third filter-passed solution is a solution obtained by passing the supernatant obtained from the second filter-passed solution through a third leukocyte removal filter through which human whole blood has been passed, the second filter-passed solution is a solution obtained by passing the supernatant obtained from the first filter-passed solution through a second leukocyte removal filter through which human whole blood has been passed, and the first filter-passed solution is a solution obtained by passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed." Furthermore, for example, one embodiment (where n = 3) of the first aspect of the present disclosure is as follows: "A plasma solution comprising a supernatant obtained from a fourth filter-passed solution, wherein the fourth filter-passed solution is a solution obtained by passing the supernatant obtained from the third filter-passed solution through a fourth leukocyte removal filter through which human whole blood has been passed, the third filter-passed solution is a solution obtained by passing the supernatant obtained from the second filter-passed solution through a third leukocyte removal filter through which human whole blood has been passed, the second filter-passed solution is a solution obtained by passing the supernatant obtained from the first filter-passed solution through a second leukocyte removal filter through which human whole blood has been passed, and the first filter-passed solution is a solution obtained by passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed."

[0020] In the present disclosure, the i in "ith filter-passed solution" (i is an integer of 1 or greater) refers to the number of leukocyte removal filters through which human whole blood has been passed, through which the solution has passed. Also, in the present disclosure, the i in "ith leukocyte removal filter" (i is an integer of 1 or greater) refers to the i-th filter through which the aqueous solution has passed. For example, the second filter-passed solution is a solution obtained by passing an aqueous solution through a first leukocyte removal filter, and then subjecting the solution to any necessary treatment (e.g., centrifugation and supernatant recovery) (first filter-passed solution), and then passing the resulting solution through a second leukocyte removal filter.

[0021] In the present disclosure, the terms "used leukocyte removal filter" and "leukocyte removal filter through which human whole blood has been passed" refer to a leukocyte removal filter that has not been subjected to any other treatment (e.g., passage of a solution) after human whole blood has been passed through it.

[0022] The human whole blood according to the present disclosure is not particularly limited as long as it is collected from a human, and is not particularly limited, for example, by its blood type or the race, sex, age, etc. of the person from whom the human whole blood is collected. Furthermore, the method for obtaining the human whole blood according to the present disclosure is not particularly limited, and may be, for example, human whole blood collected at a whole blood donation by Red Cross societies of various countries, such as the Japanese Red Cross Society and the American Red Cross, or human whole blood sold in a country where blood can be bought and sold. However, from the viewpoints of stable quality, stable supply, and prevention of infectious diseases when the plasma solution is used directly or indirectly for regenerative medicine purposes, human whole blood collected at a whole blood donation is preferred.

[0023] A leukocyte reduction filter is a filter used to remove leukocytes from human whole blood collected from a human being, in order to prevent immune side effects such as nonhemolytic febrile fever (NHFTR), platelet transfusion inability, and human T-cell leukemia virus (HTLV-1) infection caused by the entry of different types of leukocytes into the body when the collected whole blood is used for transfusion or the production of blood products. The leukocyte reduction filter according to the present disclosure can be a commercially available filter used for removing leukocytes from human whole blood, such as the Sepacel series (Asahi Kasei Medical Co., Ltd.) and the Imguard III series (Terumo Corporation). The leukocyte reduction filter is not limited to the following description, but is typically a water-repellent, hard container or syringe made of polycarbonate or the like with two openings, the interior of which is filled with porous fibers capable of trapping leukocytes. With such a configuration, when human whole blood is passed from one opening to the other opening, leukocytes in the human whole blood are removed.

[0024] Non-blood cell components are components contained in blood excluding cellular components, including blood cells such as red blood cells, white blood cells, and platelets. A liquid containing non-blood cell components (plasma) can be obtained as the supernatant obtained by centrifuging blood. Non-blood cell components include proteins such as albumin, globulin, and fibrinogen, as well as various metal ions and vitamins. A plasma solution, which is a solution containing non-blood cell components, can be obtained as the supernatant obtained by centrifuging a solution containing blood.

[0025] The plasma solution according to the first aspect of the present disclosure contains non-blood cell components derived from a leukocyte removal filter through which human whole blood has been passed. That is, the plasma solution according to the first aspect of the present disclosure contains non-blood cell components contained in human whole blood that remain in the leukocyte removal filter through which human whole blood has been passed to remove leukocytes. The non-blood cell components derived from the leukocyte removal filter through which human whole blood has been passed can be recovered from the leukocyte removal filter by passing an aqueous solution (e.g., physiological saline) through the leukocyte removal filter. The plasma solution according to one embodiment of the first aspect of the present disclosure may contain, as non-blood cell components, all blood-derived components other than blood cell components contained in the solution (filter-passing solution) recovered by passing an aqueous solution through the leukocyte removal filter after passing human whole blood through it.

[0026] A plasma solution according to one embodiment of the first aspect of the present disclosure contains non-blood cell components derived from a first leukocyte removal filter through which human whole blood has been passed and non-blood cell components derived from a second leukocyte removal filter through which human whole blood has been passed, and the non-blood cell components derived from the second leukocyte removal filter are non-blood cell components contained in a solution obtained by passing a solution containing non-blood cell components derived from the first leukocyte removal filter through the second leukocyte removal filter. That is, the plasma solution according to the first aspect of the present disclosure can be prepared from a solution containing non-blood cell components obtained by passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed, or a non-blood cell component-containing liquid prepared from such a solution, which is then passed through a second leukocyte removal filter through which human whole blood has been passed. For example, the (i+1)th filtered solution (i is an integer of 1 or greater) according to one embodiment of the first aspect of the present disclosure may be a solution obtained by passing a supernatant containing non-blood cell components obtained from the i)th filtered solution, or a liquid containing non-blood cell components prepared from the supernatant, through the (i+1)th leukocyte removal filter through which human whole blood has been passed.

[0027] The solvent in the plasma solution according to the first aspect of the present disclosure is an aqueous solvent, preferably a solvent containing 80% or more by volume of water, more preferably water, and preferably physiological saline, for example.

[0028] The plasma solution according to one embodiment of the first aspect of the present disclosure has a protein concentration of 20% or more of that of fresh frozen plasma prepared from human blood. Fresh frozen plasma (FFP) prepared from human blood is obtained by removing leukocytes from human whole blood obtained by whole blood donation using a leukocyte removal filter, followed by centrifugation to remove red blood cells, and then freezing the supernatant. Examples of such plasma include fresh frozen plasma-LR "Nisseki" 120 and fresh frozen plasma-LR "Nisseki" 240 (both from the Japanese Red Cross Society). The protein concentration in the plasma solution according to one embodiment of the first aspect of the present disclosure may be 20% or more, 25% or more, 30% or more, 35% or more, or 40% or more of that of fresh frozen plasma prepared from human blood (e.g., fresh frozen plasma-LR "Nisseki" 480), and may be less than 100%, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, or 45% or less.

[0029] In one embodiment of the first aspect of the present disclosure, the plasma solution may be for use in the production of a platelet lysate. That is, in one embodiment of the first aspect of the present disclosure, the plasma solution may be for use in the production of a platelet lysate. Also, in one embodiment of the first aspect of the present disclosure, the plasma solution may be for use in the production of a platelet lysate for use in promoting cell proliferation.

[0030] The cell proliferation activity of stem cells in a platelet lysate produced using a plasma solution according to one embodiment of the first aspect of the present disclosure is higher, e.g., significantly higher, specifically 1.5 times higher, than that of a platelet lysate produced using a mixture of fresh frozen plasma prepared from human blood (e.g., fresh frozen plasma - LR "Nisseki" 120 or fresh frozen plasma - LR "Nisseki" 240), in which a predetermined proportion of fresh frozen plasma prepared from the human blood is present, and a plasma solution produced from a solution passed only once through a used leukocyte reduction filter (corresponding to the supernatant recovered in the first plasma recovery step described below). In these cases, the predetermined proportion may be 20% by volume or more, 25% by volume or more, 30% by volume or more, 35% by volume or more, or 40% by volume or more, or may be less than 100% by volume, 90% by volume or less, 80% by volume or less, 70% by volume or less, 60% by volume or less, or 50% by volume or less, and may be, for example, 40% by volume.

[0031] The cell proliferation activity of stem cells in a platelet lysate produced using the plasma solution according to one embodiment of the first aspect of the present disclosure is higher, for example, significantly higher, specifically 1.5 times higher, than that of a platelet lysate produced using a plasma solution prepared by diluting fresh frozen plasma prepared from human blood with an aqueous solution (e.g., physiological saline) at a predetermined ratio, where the predetermined ratio may be, for example, 45% by volume.

[0032] The plasma solution according to one embodiment of the first aspect of the present disclosure may have a protein concentration of 10,000 μg / mL or more, 15,000 μg / mL or more, 20,000 μg / mL or more, 22,000 μg / mL or more, 24,000 μg / mL or more, or 25,000 μg / mL or more, or 60,000 μg / mL or less, 50,000 μg / mL or less, 40,000 μg / mL or less, 36,000 μg / mL or less, 33,000 μg / mL or less, or 30,000 μg / mL or less.

[0033] The plasma solution according to one embodiment of the first aspect of the present disclosure may have an RNA concentration of 100 ng / mL or more, 200 ng / mL or more, 300 ng / mL or more, 400 ng / mL or more, 450 ng / mL or more, 500 ng / mL or more, 505 ng / mL or more, 510 ng / mL or more, or 512 ng / mL or more, or 1000 ng / mL or less, 900 ng / mL or less, 800 ng / mL or less, 700 ng / mL or less, 600 ng / mL or less, 590 ng / mL or less, 580 ng / mL or less, 570 ng / mL or less, 560 ng / mL or less, 550 ng / mL or less, 540 ng / mL or less, 535 ng / mL or less, 530 ng / mL or less, or 525 ng / mL or less. When the RNA concentration in the plasma solution according to one embodiment of the first aspect of the present disclosure is within the above range, the platelet lysate produced using the plasma solution according to one embodiment of the first aspect of the present disclosure has a high cell proliferation-promoting ability. The RNA concentration may be measured, for example, by absorbance spectrometry (wavelength 260 nm).

[0034] The plasma solution according to one embodiment of the first aspect of the present disclosure may have a fibrinogen concentration of 100 μg / mL to 1500 μg / mL, 150 μg / mL to 1000 μg / mL, or 200 μg / mL to 500 μg / mL. When the fibrinogen concentration is within the above range, the risk of blood coagulation can be reduced when cells cultured using a platelet lysate produced using the plasma solution according to one embodiment of the first aspect of the present disclosure are used for regenerative medicine purposes.

[0035] The plasma solution according to one embodiment of the first aspect of the present disclosure may have a heparin concentration of 2 U / mL or less, 1.5 U / mL or less, 1.0 U / mL or less, 0.6 U / mL or less, 0.3 U / mL or less, or 0.1 U / mL or less. Furthermore, the plasma solution according to one embodiment of the first aspect of the present disclosure may be free of heparin derived from sources other than the leukocyte reduction filter.

[0036] The plasma solution according to one embodiment of the first aspect of the present disclosure may contain, as more specific components, for example, platelet-derived exosomes and microvesicles, RNA, platelet-derived growth factor isoforms (PDGF-AA, -AB, and -BB), transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblasts, etc. The composition may contain growth factors (bFGF, FGF-2), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein-4 (BMP-4), bone morphogenetic protein-6 (BMP-6), platelet factor 4 (PF4), osteonectin, and the like.

[0037] The plasma solution according to one embodiment of the first aspect of the present disclosure may have a contaminating red blood cell count of, for example, 10,000 or less, 7,000 or less, 5,500 or less, or 4,500 or less per mL. The contaminating red blood cell count can be measured, for example, by flow cytometry. The plasma solution according to one embodiment of the first aspect of the present disclosure may have a contaminating white blood cell count of, for example, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, 100 or less, 40 or less, or 20 or less per mL. The contaminating white blood cell count can be measured, for example, by flow cytometry. For such measurements, a multi-parameter automated hematology analyzer XN-1000 (Sysmex Corporation) or the like can be used.

[0038] In one embodiment of the first aspect, the conditions for centrifugation in preparing a supernatant from the ith filter-passed solution (i is an integer of 1 or greater) are not particularly limited as long as they are conditions that can precipitate red blood cells, platelets, aggregates of biopolymers, and remaining white blood cells without precipitating non-blood cell components. The centrifugal force in the centrifugation may be, for example, 1,000 g or more and 6,000 g or less, or 1,500 g or more and 5,000 g or less, and specific examples thereof may be 2,000 g, 2,500 g, 3,000 g, 3,500 g, or 4,000 g. The centrifugation time may be, for example, 1 minute or more and 180 minutes or less, 5 minutes or more and 60 minutes or less, or 9 minutes or more and 45 minutes or less, and specific examples thereof may be 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes. Such centrifugation may be repeated two, three, four or more times, and the supernatant may be recovered after each centrifugation, and only that supernatant may be centrifuged again. For example, it is preferable to perform centrifugation once, recover the supernatant, and then perform centrifugation once more.

[0039] In one embodiment of the first aspect, the supernatant in preparing the i-th filter-passed solution (i is an integer of 1 or more) may be, for example, a solution of 50 vol% or more, 70 vol% or more, 75 vol% or more, 80 vol% or more, 85 vol% or more, 90 vol% or more, 95 vol% or more, 97 vol% or more, 98 vol% or more, 99 vol% or more, or 100 vol% of the liquid component (supernatant fraction) after centrifugation, or may be a solution of 95 vol% or less, 90 vol% or less, or 80 vol% or less.

[0040] <Method for producing a plasma solution> A second aspect of the present disclosure is a method for producing a plasma solution, comprising: a step of passing an aqueous solution through a first leukocyte removal filter that has had human whole blood passed through it, and recovering the passed solution to obtain a first filter-passed solution (first passing step); a step of recovering a supernatant from the i-th filter-passed solution (i-th plasma recovering step); and a step of passing the supernatant obtained in the i-th plasma recovering step through an i+1-th leukocyte removal filter, and recovering the passed solution to obtain an i+1-th filter-passed solution (i+1-th passing step); (where n is an integer of 1 or more, i is an integer of 1 to n, and the i-th plasma recovering step and the i+1-th passing step are repeated n times in this order, while the number substituted for i is increased by 1 in succession, starting from 1.) One embodiment of the second aspect of the present disclosure is a method for producing a plasma solution, where n is 1, including the steps of passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed and recovering the solution after passing to obtain a first filter-passed solution (first passing step), recovering a supernatant from the first filter-passed solution (first plasma recovery step), and passing the supernatant obtained in the first plasma recovery step through a second leukocyte removal filter through which human whole blood has been passed and recovering the solution after passing to obtain a second filter-passed solution (second passing step), and will be described below using this embodiment as an example. One embodiment of the second aspect of the present disclosure includes the first passing step, first plasma recovery step, and second passing step, in this order.

[0041] The leukocyte removal filter through which human whole blood has passed, used in the manufacturing method according to the second aspect of the present disclosure, may be, for example, a leukocyte removal filter (a used leukocyte removal filter) used to remove leukocytes when converting human whole blood obtained by whole blood donation into a blood product, or may be a leukocyte removal filter prepared in a step of preparing at least n+1 leukocyte removal filters through which human whole blood has passed (a preparation step). That is, in one embodiment, the manufacturing method according to the second aspect of the present disclosure may or may not include a preparation step prior to the first passing step. In the preparation step, a first leukocyte removal filter through which human whole blood has passed and a second leukocyte removal filter through which human whole blood has passed are prepared. Human whole blood collected by whole blood donation or the like is usually contained in a blood bag. Therefore, in one embodiment of the preparation step, one end of a tube, such as a water-repellent resin transfusion tube, is connected to a blood outlet hole (communication piece), such as a spigot or rubber stopper, provided on the blood bag. The other end of the tube is connected to one opening of the leukocyte removal filter, and another tube is connected to the other opening. An empty blood bag is attached to the other end of the tube. These tubes may be equipped with attachments (e.g., clamps or clips) that can control the flow of blood.

[0042] In a closed system including a blood bag containing human whole blood, a leukocyte removal filter, and an empty blood bag thus prepared, the human whole blood in the blood bag containing human whole blood can be passed through a leukocyte removal filter by flowing the human whole blood from the blood bag containing human whole blood toward the empty blood bag. Flowing the human whole blood can be achieved, for example, by placing the blood bag containing human whole blood vertically upward and allowing the blood to flow by gravity. Such a placement can be achieved, for example, by suspending the blood bag containing human whole blood on a stand (e.g., a transfusion stand) capable of suspending the blood bag, while holding a portion (hanging port) of the blood bag configured to allow blood to flow by gravity. Flowing the human whole blood can also be achieved, for example, by physically compressing the blood bag containing human whole blood. Flowing the human whole blood can also be achieved, for example, by connecting a pump between the leukocyte removal filter and the empty blood bag in the closed system and using the pump to suck up blood from the blood bag containing human whole blood and deliver it to the empty blood bag.

[0043] In the first passing step, an aqueous solution is passed through a first leukocyte removal filter through which human whole blood has been passed, and the passed solution is recovered to obtain a first filter-passed solution. The aqueous solution is not particularly limited as long as it is capable of recovering blood components (non-blood cell components and platelets) from the leukocyte removal filter, and may be, for example, physiological saline. The method for passing the aqueous solvent through the first leukocyte removal filter is not particularly limited, and may be, for example, a method in which the aqueous solvent is passed by gravity through a suspension, a method in which the aqueous solvent is forced out by physically compressing (compressing) a container containing the aqueous solvent, or a method in which the aqueous solvent is passed through using a pump, as in the method for passing human whole blood through the first leukocyte removal filter in the preparation step.

[0044] In a preferred embodiment, in the first passing step, the aqueous solvent may be passed through the leukocyte removal filter in either the same direction as the human whole blood passed through in the preparation step or the opposite direction. For example, the first passing step can be easily performed by connecting a bag containing the aqueous solvent to the closed system used in the preparation step in place of the blood bag into which the human whole blood flowed in the preparation step. In this case, in a more preferred embodiment, the first filter-passed solution may be recovered in the blood bag that originally contained the human whole blood in the preparation step. This allows the first filter-passed solution to be recovered while maintaining the closed system, thereby reducing the risk of infection when cells cultured using a platelet lysate produced using a plasma solution according to one embodiment of the first aspect of the present disclosure are used for regenerative medicine purposes.

[0045] The amount of aqueous solvent passed through the leukocyte removal filter in the first passing step is not particularly limited, and may be, for example, 0.1 times or more, 0.3 times or more, 0.5 times or more, 0.7 times or more, 0.9 times or more, 1.0 times or more, 1.5 times or more, 2.0 times or more, or 5.0 times or more the volume of a predetermined volume of human whole blood determined as the dose when the leukocyte removal filter is used for removing leukocytes or the volume of human whole blood passed through in the preparation step, or may be 10 times or less, 8.0 times or less, 6.0 times or less, 5.0 times or less, 4.0 times or less, 3.0 times or less, 2.0 times or less, 1.7 times or less, 1.2 times or less, 1.1 times or less, 1.0 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, or 0.3 times or less.

[0046] In the first plasma recovery step, a supernatant is recovered from the first filter-passed solution obtained in the first passing step. Methods for obtaining the supernatant in the first plasma recovery step include centrifugation and filtration, which are methods for obtaining a solution containing non-blood cell components from the first filter-passed solution. Centrifugation is preferred. For example, in the first plasma recovery step, red blood cells, platelets, aggregates of biopolymers, and remaining white blood cells in the first filter-passed solution are precipitated by centrifugation, and a solution containing non-blood cell components is obtained as a supernatant.

[0047] The conditions for centrifugation in the first plasma recovery step are not particularly limited as long as they allow the precipitation of red blood cells, platelets, biopolymer aggregates, and remaining white blood cells without precipitating non-blood cell components. The centrifugal force in the centrifugation may be, for example, 1,000 g to 6,000 g or 1,500 g to 5,000 g, specifically 2,000 g, 2,500 g, 3,000 g, 3,500 g, or 4,000 g. The centrifugation time may be, for example, 1 minute to 180 minutes, 5 minutes to 60 minutes, or 9 minutes to 45 minutes, specifically 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes. In the first plasma recovery step, such centrifugation may be repeated two, three, four, or more times. After each centrifugation, the supernatant may be recovered and the supernatant may be centrifuged again. For example, it is preferable to perform centrifugation once, recover the supernatant, and then perform centrifugation once more.

[0048] When the method for obtaining the supernatant in the first plasma recovery step is centrifugation, recovering the supernatant may mean, for example, recovering 50% by volume or more, 70% by volume or more, 75% by volume or more, 80% by volume or more, 85% by volume or more, 90% by volume or more, 95% by volume or more, 97% by volume or more, 98% by volume or more, 99% by volume or more, or 100% by volume of the supernatant after centrifugation, or may mean recovering 95% by volume or less, 90% by volume or less, or 80% by volume or less.

[0049] In the second passing step, the supernatant obtained in the first plasma recovery step is passed through a second leukocyte removal filter through which human whole blood has been passed, and the solution after passing through is recovered to obtain a second filter-passed solution. The second passing step can be performed by the same method as described in the first passing step, except that a bag containing the supernatant obtained in the first plasma recovery step is used instead of a blood bag containing an aqueous solvent.

[0050] In one embodiment, the production method according to the second aspect of the present disclosure may further include an (n+1)th plasma recovery step (a second plasma recovery step in the production method according to one embodiment of the second aspect of the present disclosure described above). In the (n+1)th plasma recovery step, red blood cell, platelet, and biopolymer aggregates in the (n+1)th filtered solution are precipitated by a method similar to that in the first plasma recovery step, and a solution containing non-blood cell components, i.e., a plasma solution, is obtained as the supernatant. The plasma solution thus obtained may be stored frozen.

[0051] Although the production method according to the second aspect of the present disclosure has been described above using an example in which n is 1 in one embodiment, n in the production method according to the second aspect of the present disclosure is not limited to 1 and may be an integer of 2 or greater (e.g., 2, 3, 4, or 5). In these cases, the i-th plasma recovery step and the (i+1)-passing step may be performed in the same manner as the first plasma recovery step and second pass-through step according to the embodiment described above. For example, in one embodiment, the production method according to the second aspect of the present disclosure may include, in this order, a first pass-through step, a first plasma recovery step, a second pass-through step, a second plasma recovery step, and a third pass-through step. Furthermore, in one embodiment, the production method according to the second aspect of the present disclosure may include, in this order, a first pass-through step, a first plasma recovery step, a second pass-through step, a second plasma recovery step, a third pass-through step, a third plasma recovery step, and a fourth pass-through step.

[0052] <Platelet Lysate> A third aspect of the present disclosure provides a platelet lysate containing a platelet lysate, non-blood cell components derived from a first leukocyte removal filter through which human whole blood has been passed, and non-blood cell components derived from a second leukocyte removal filter through which human whole blood has been passed, wherein the non-blood cell components derived from the second leukocyte removal filter are non-blood cell components contained in a solution obtained by passing a solution containing non-blood cell components derived from the first leukocyte removal filter through the second leukocyte removal filter. That is, in one embodiment, the third aspect of the present disclosure may be a platelet lysate containing a platelet lysate and a plasma solution according to one embodiment of the first aspect of the present disclosure. In these cases, the platelet lysate may be a platelet lysate derived from a leukocyte removal filter through which human whole blood has been passed, such as a platelet lysate derived from the first leukocyte removal filter through which human whole blood has been passed. Furthermore, in one embodiment, the third aspect of the present disclosure may be a platelet lysate produced by the method for producing a platelet lysate according to the fourth aspect of the present disclosure, which will be described later.

[0053] Platelet lysate (PL) is a solution containing the contents of platelets, obtained by lysing platelets derived from human blood using ultrasonic treatment, freeze-thawing, or the like. Platelet lysate contains many components that promote cell proliferation and is known to be an alternative to fetal bovine serum (FBS) as a source of cell growth factors in cell culture (Non-Patent Document 1). In this disclosure, platelet lysate refers to a collection of biomolecules that were the contents of platelets and are released outside the platelets by lysing the platelets.

[0054] The platelet lysate according to one embodiment of the third aspect of the present disclosure may contain components that the plasma solution according to the first aspect of the present disclosure contains or may contain, and the amount of the components may be within the same ranges as described in the first aspect of the present disclosure.

[0055] The solvent in the platelet lysate according to the third aspect of the present disclosure is an aqueous solvent, preferably a solvent in which water accounts for 80% by volume or more, and more preferably water.

[0056] In one embodiment of the third aspect of the present disclosure, the platelet lysate has a platelet concentration of 10×10 before the dissolution treatment (platelet dissolution step) in the manufacturing process. ４ pcs / μL or more 1000×10 ４ pcs / μL or less, 20×10 ４ pcs / μL or more 500×10 ４ pcs / μL or less or 50×10 ４ pcs / μL or more 200×10 ４ In other words, the platelet lysate according to one embodiment of the third aspect of the present disclosure can be prepared by dissolving platelet lysates (intracellular proteins and the like that were contained in platelets) in a concentration of 10 × 10 ４ pcs / μL or more 1000×10 ４ pcs / μL or less, 20×10 ４ pcs / μL or more 500×10 ４ pcs / μL or less or 50×10 ４ pcs / μL or more 200×10 ４ It may be contained at a concentration contained in platelets of not more than 1 / μL.

[0057] In a platelet lysate according to an embodiment of the third aspect of the present disclosure, the number of platelets before the lysis treatment (platelet lysis step) in the manufacturing process is 1500 × 10 per 400 mL of human whole blood passed through a first leukocyte removal filter. ７ 5000×10 pieces or more ７ Less than or equal to 2000×10 ７ 4500×10 pieces or more ７ Less than or equal to 2200 x 10 ７ 4000×10 pieces or more ７ pcs or less or 2500 x 10 ７ pcs or more 3500×10 ７ In other words, the platelet lysate according to one embodiment of the third aspect of the present disclosure is a platelet lysate (intracellular proteins and the like that were contained in platelets) that has been filtered through a first leukocyte removal filter and has a concentration of 10 x 10 or less per 400 mL of human whole blood. ４ pcs / μL or more 1000×10 ４ pcs / μL or less, 20×10 ４ pcs / μL or more 500×10 ４ pcs / μL or less or 50×10 ４ pcs / μL or more 200×10４ It may be contained at a concentration contained in platelets of not more than 1 / μL.

[0058] Specifically, the platelet lysate according to one embodiment of the third aspect of the present disclosure may have a plasma-derived protein concentration of 20% or more, 25% or more, 30% or more, 35% or more, or 40% or more, or may be less than 100%, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, or 45% or less, of that of fresh frozen plasma prepared from human blood (e.g., fresh frozen plasma-LR "Nisseki" 480).

[0059] The platelet lysate according to one embodiment of the third aspect of the present disclosure may have a plasma-derived protein concentration of, for example, 6 mg / mL or more, and from the viewpoint of excellent cell proliferation-promoting ability, may be preferably 18 mg / mL or more or 27 mg / mL or more. When the platelet lysate according to one embodiment of the third aspect of the present disclosure has a plasma-derived protein concentration of 18 mg / mL or more, it exhibits a cell proliferation-promoting ability comparable to that of a platelet lysate produced from platelets obtained from human blood or human blood components. When the platelet lysate according to one embodiment of the third aspect of the present disclosure has a plasma-derived protein concentration of 27 mg / mL or more, it exhibits a cell proliferation-promoting ability superior to that of a platelet lysate produced from platelets obtained from human blood or human blood components. Based on these facts, another aspect of the present disclosure may also be a platelet lysate containing plasma-derived proteins at a concentration equal to or higher than a predetermined lower limit, the predetermined lower limit being, for example, 6 mg / mL, preferably 18 mg / mL or 27 mg / mL. In this case, the plasma-derived proteins are not particularly limited to those in a used leukocyte reduction filter.

[0060] The platelet lysate according to one embodiment of the third aspect of the present disclosure may be used to promote cell proliferation. The cell proliferation activity of stem cells in the platelet lysate according to one embodiment of the third aspect of the present disclosure is higher, for example, significantly higher, specifically 1.5 times higher, than that of a platelet lysate produced using a mixture of fresh frozen plasma prepared from human blood (e.g., fresh frozen plasma-LR "Nisseki" 120 or fresh frozen plasma-LR "Nisseki" 240) containing a predetermined proportion of the fresh frozen plasma prepared from the human blood and a plasma solution (corresponding to the supernatant recovered in the first plasma recovery step) produced from a solution passed only once through a used leukocyte reduction filter. In these cases, the predetermined proportion may be 20% by volume or more, 25% by volume or more, 30% by volume or more, 35% by volume or more, or 40% by volume or more, or may be less than 100% by volume, 90% by volume or less, 80% by volume or less, 70% by volume or less, 60% by volume or less, or 50% by volume or less, and may be, for example, 40% by volume.

[0061] In one embodiment of the platelet lysate according to the third aspect of the present disclosure, the plasma-derived RNA concentration may be 100 ng / mL or more, 200 ng / mL or more, 300 ng / mL or more, 400 ng / mL or more, 450 ng / mL or more, 500 ng / mL or more, 505 ng / mL or more, 510 ng / mL or more, or 512 ng / mL or more, or 1000 ng / mL or less, 900 ng / mL or less, 800 ng / mL or less, 700 ng / mL or less, 600 ng / mL or less, 590 ng / mL or less, 580 ng / mL or less, 570 ng / mL or less, 560 ng / mL or less, 550 ng / mL or less, 540 ng / mL or less, 535 ng / mL or less, 530 ng / mL or less, or 525 ng / mL or less. When the plasma-derived RNA concentration in the platelet lysate according to an embodiment of the third aspect of the present disclosure is within the above range, the platelet lysate according to an embodiment of the third aspect of the present disclosure has a high cell proliferation-promoting ability. The above RNA concentration may be measured, for example, by absorbance spectrometry (wavelength 260 nm).

[0062] Specifically, the platelet lysate according to an embodiment of the third aspect of the present disclosure may have a fibrinogen concentration of 100 μg / mL to 1500 μg / mL, 150 μg / mL to 1000 μg / mL, or 200 μg / mL to 500 μg / mL. When the fibrinogen concentration is within the above range, the risk of blood coagulation can be reduced when the platelet lysate is used for regenerative medicine purposes.

[0063] Specifically, the platelet lysate according to an embodiment of the third aspect of the present disclosure may have a heparin concentration of 2.0 U / mL or less, 1.5 U / mL or less, 1.0 U / mL or less, 0.6 U / mL or less, 0.3 U / mL or less, or 0.1 U / mL or less. The plasma solution according to an embodiment of the third aspect of the present disclosure may be free of heparin derived from sources other than the leukocyte reduction filter.

[0064] The platelet lysate according to one embodiment of the third aspect of the present disclosure may contain, as more specific components, for example, platelet-derived exosomes and microvesicles, RNA, platelet-derived growth factor isoforms (PDGF-AA, -AB, and -BB), transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblasts, etc. The composition may contain growth factors (bFGF, FGF-2), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein-4 (BMP-4), bone morphogenetic protein-6 (BMP-6), platelet factor 4 (PF4), osteonectin, and the like.

[0065] The platelet lysate according to an embodiment of the third aspect of the present disclosure may have a contaminating red blood cell count of, for example, 10,000 or less, 7,000 or less, 5,500 or less, or 4,500 or less per mL. The contaminating red blood cell count can be measured, for example, by sheath flow electrical resistance spectroscopy. The platelet lysate according to an embodiment of the third aspect of the present disclosure may have a contaminating white blood cell count of, for example, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, 100 or less, 40 or less, or 20 or less per mL. The contaminating white blood cell count can be measured, for example, by flow cytometry. For such measurements, a multi-parameter automated hematology analyzer XN-1000 (Sysmex Corporation) or the like can be used.

[0066] The platelet lysate according to one embodiment of the third aspect of the present disclosure may have a stem cell proliferation-promoting effect that is 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, or 3.8 times or more that of fetal bovine serum. The fetal bovine serum may be, for example, non-inactivated Fetal Bovine Serum, Qualified, United States (Gibco, Thermo Fisher Scientific). In this case, the stem cell proliferation-promoting effect of the platelet lysate and fetal bovine serum can be evaluated, for example, based on the fold increase in cell number after a predetermined period (e.g., 120 hours) when stem cells are cultured in a culture medium (e.g., MEMα containing gentamicin (40 μg / mL) and heparin (2 U / mL)) to which the platelet lysate or fetal bovine serum has been added at a ratio of 10% by volume.

[0067] <Method for producing platelet lysate> A fourth aspect of the present disclosure relates to a method for producing a platelet lysate, comprising: a step of passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed, and recovering the solution after passing to obtain a first filter-passed solution (first passing step); a step of removing red blood cells from the first filter-passed solution obtained in the first passing step (first red blood cell removal step); a step of centrifuging the solution obtained in the i-th red blood cell removal step, recovering the supernatant, and recovering the residue after supernatant recovery as a platelet concentrate (i-th recovery step); a step of passing the supernatant obtained in the i-th recovery step through an i+1-th leukocyte removal filter through which human whole blood has been passed, and recovering the solution after passing to obtain the i+1-th filter-passed solution (i+1-th passing step); a step of removing red blood cells from the (i+1)th filter-passed solution obtained in the (i+1)-passing step (i+1) (i+1)-passing step); (where n is an integer of 1 or more, i is an integer of 1 to n, and the (i+1)-th collecting step, the (i+1)-th passing step, and the (i+1)-th red blood cell removing step are repeated n times in this order while the number substituted for i is increased by 1 in succession starting from 1); a step of mixing at least one platelet concentrate selected from the group consisting of the platelet concentrates obtained in the first to n-th collecting steps with the solution obtained in the (n+1)-th red blood cell removing step (mixing step), and a step of dissolving platelets in the solution obtained in the mixing step (platelet dissolving step). In one embodiment, the fourth aspect of the present disclosure may include, in this order, a first passing step, a first red blood cell removal step, an i-th collection step, an i+1-th passing step, an i+1-th red blood cell removal step (where n is an integer of 1 or more, i is an integer of 1 to n, and the i-th collection step, the i+1-th passing step, and the i+1-th red blood cell removal step are repeated n times in this order while the number substituted for i is increased by 1 in succession, starting from 1), a mixing step, and a platelet dissolving step. Furthermore, in one embodiment, the fourth aspect of the present disclosure may include, in this order, a first passing step, a first red blood cell removal step, an i-th collection step, an i+1-th passing step, an i+1-th red blood cell removal step (where n is an integer of 1 or more, i is an integer of 1 to n, and the i-th collection step, the i+1-th passing step, and the i+1-th red blood cell removal step are repeated n times in this order while the number substituted for i is increased by 1 in succession, starting from 1), a platelet dissolving step, and a mixing step.In one embodiment, the fourth aspect of the present disclosure includes a first passing step, an i-th collection step, an (i+1)-th passing step (where n is an integer of 1 or more, i is an integer of 1 to n inclusive, and the i-th collection step and the (i+1)-th passing step are repeated n times in this order while the number substituted for i is increased by 1 in succession, starting from 1), a mixing step, and a platelet dissolving step, in this order, and may further include a first platelet removal step and / or a j+1-th platelet removal step (where j is at least one integer selected from the group consisting of integers of 1 to n inclusive, and is performed after the j+1-th passing step). In one embodiment, the fourth aspect of the present disclosure includes a first passing step, an i-th collection step, an i+1-th passing step (where n is an integer of 1 or more, i is an integer of 1 to n, and the i-th collection step and the i+1-th passing step are repeated n times in this order while the number substituted for i is increased by 1 in succession, starting from 1), a platelet dissolving step, and a mixing step, and may further include a first platelet removal step and / or a j+1-th platelet removal step (where j is at least one integer selected from the group consisting of integers of 1 to n, and is performed after the j+1-th passing step). In these cases, n is an integer of 1 or more, and may be, for example, 1, 2, 3, 4, or 5.

[0068] In one embodiment, the production method according to the fourth aspect of the present disclosure may be such that n=1, and this embodiment will be described below. In one embodiment, the production method according to the fourth aspect of the present disclosure includes the steps of: passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed, and recovering the solution after passing to obtain a first filter-passed solution (first passing step); removing red blood cells from the first filter-passed solution obtained in the first passing step (first red blood cell removal step); centrifuging the solution obtained in the first red blood cell removal step to recover the supernatant, and recovering the residue after supernatant recovery as a platelet concentrate (first recovery step); The method for producing a platelet lysate may include the steps of: passing the supernatant obtained in the first collection step through a blood cell removal filter and collecting the solution after the filter-passage to obtain a second filter-passage solution (second pass step); removing red blood cells from the second filter-passage solution obtained in the second pass step (second red blood cell removal step); mixing the platelet concentrate obtained in the first collection step with the solution obtained in the second red blood cell removal step (mixing step); and dissolving platelets in the solution obtained in the mixing step (platelet dissolution step). One embodiment of the fourth aspect of the present disclosure may include the first pass step, first red blood cell removal step, first collection step, second pass step, second red blood cell removal step, mixing step, and platelet dissolution step, in this order. Another embodiment of the fourth aspect of the present disclosure may include the first pass step, first red blood cell removal step, first collection step, second pass step, second red blood cell removal step, platelet dissolution step, and mixing step, in this order. An embodiment of the fourth aspect of the present disclosure includes a first passing step, a first collecting step, a second passing step, a mixing step, and a platelet dissolving step, in this order, and may further include a first platelet removing step and a second platelet removing step.An embodiment of the fourth aspect of the present disclosure includes a first passing step, a first collecting step, a second passing step, a platelet dissolving step, and a mixing step, in this order, and may further include a first platelet removing step and a second platelet removing step.

[0069] In the production method according to the fourth aspect of the present disclosure, the leukocyte removal filter through which human whole blood has passed can be the same as that described in the production method according to the second aspect. In one embodiment, the production method according to the fourth aspect may include a preparation step prior to the first passing step, and the preparation step can be carried out in the same manner as the preparation step in the second aspect of the present disclosure.

[0070] The first pass step can be carried out in the same manner as the first pass step in the second aspect of the present disclosure.

[0071] In the first red blood cell removal step, red blood cells are removed from the first filter-passed solution obtained in the first passing step. In one embodiment, in the first red blood cell removal step, the first filter-passed solution is centrifuged to precipitate red blood cells and remaining white blood cells in the first filter-passed solution, and a solution containing non-blood cell components and platelets is obtained as a supernatant.

[0072] The conditions for centrifugation in the first red blood cell removal step are not particularly limited as long as they allow the precipitation of red blood cells and remaining white blood cells without precipitating non-blood cell components and platelets. The centrifugal force in the centrifugation may be, for example, 50 g to 500 g, 80 g to 300 g, or 100 g to 200 g, specifically 100 g, 120 g, 140 g, 150 g, 160 g, 180 g, or 200 g. The centrifugation time may be, for example, 1 minute to 180 minutes, 5 minutes to 60 minutes, or 9 minutes to 45 minutes, specifically 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes. In the first red blood cell removal step, such centrifugation may be repeated two, three, four, or more times. For example, it is preferable to perform centrifugation once, recover the supernatant, and then perform centrifugation again.

[0073] In the first red blood cell removal step, recovering the supernatant may mean recovering, for example, 50 vol% or more, 70 vol% or more, 75 vol% or more, 80 vol% or more, 85 vol% or more, 90 vol% or more, 95 vol% or more, 97 vol% or more, 98 vol% or more, 99 vol% or more, or 100 vol% of the supernatant after centrifugation, or may mean recovering 95 vol% or less, 90 vol% or less, or 80 vol% or less.

[0074] In the first red blood cell removal step, after the supernatant is recovered, the number of red blood cells and the number of white blood cells contaminating the recovered supernatant may be measured. The number of contaminating red blood cells is 5.00 × 10 per leukocyte removal filter for 400 mL of used blood. ７ The number of contaminating leukocytes is preferably 3.20 × 10 per leukocyte removal filter for 400 mL of used blood. ５ If the number of contaminating red blood cells and / or white blood cells exceeds the above-mentioned number, the number of contaminating cells can be reduced to the above-mentioned number or less by repeating the centrifugation in the first red blood cell removal step.

[0075] In the first recovery step, the solution obtained in the first red blood cell removal step is centrifuged to recover the supernatant, and the residue after the supernatant recovery is recovered as a platelet concentrate. The residue (platelet concentrate) may be a solid precipitate (pellet), or may be a liquid containing a high concentration of platelets, including the liquid that was not recovered as the supernatant. In a preferred embodiment, in the first recovery step, the solution obtained in the first red blood cell removal step is centrifuged to recover the supernatant, and the precipitate (pellet) is recovered as the platelet concentrate. The recovered supernatant contains non-blood cell components, and the recovered platelet concentrate contains platelets. Note that the recovery of the supernatant in the first recovery step corresponds to the recovery of the supernatant in the first plasma recovery step according to one embodiment of the first aspect of the present disclosure.

[0076] The conditions for centrifugation in the first recovery step are not particularly limited as long as they allow platelets to precipitate without precipitating non-blood cell components. The centrifugal force in the centrifugation may be, for example, 1,000 g to 6,000 g or 1,500 g to 5,000 g, specifically 2,000 g, 2,500 g, 3,000 g, 3,500 g, or 4,000 g. The centrifugation time may be, for example, 1 minute to 180 minutes, 5 minutes to 60 minutes, or 9 minutes to 45 minutes, specifically 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes. In the first recovery step, such centrifugation may be repeated two, three, four, or more times, and the supernatant may be recovered after each centrifugation, and only the supernatant may be centrifuged again.

[0077] In the first recovery step, recovering the supernatant may mean, for example, recovering 50% by volume or more, 70% by volume or more, 75% by volume or more, 80% by volume or more, 85% by volume or more, 90% by volume or more, 95% by volume or more, 97% by volume or more, 98% by volume or more, 99% by volume or more, or 100% by volume of the supernatant after centrifugation, or may mean recovering 95% by volume or less, 90% by volume or less, or 80% by volume or less.

[0078] The supernatant and platelet concentrate collected in the first collection step may be frozen and stored until use in the subsequent step.

[0079] The second pass step can be carried out in the same manner as the second pass step in the first aspect of the present disclosure, except that the supernatant recovered in the first plasma recovery step is replaced with the supernatant recovered in the first plasma recovery step.

[0080] In the second red blood cell removal step, red blood cells are removed from the second filter-passed solution obtained in the second passing step. In one embodiment, in the second red blood cell removal step, the second filter-passed solution is centrifuged to precipitate at least red blood cells, and a supernatant containing at least non-blood cell components is recovered. The centrifugation conditions and the recovery of the supernatant in the second red blood cell removal step may be the same as those described in the first recovery step.

[0081] In the second red blood cell removal step, after the supernatant is recovered, the number of red blood cells and the number of white blood cells contaminating the recovered supernatant may be measured. The number of contaminating red blood cells is 1.00 × 10 per leukocyte removal filter for 400 mL of used blood. ６ The number of contaminating leukocytes is preferably 5.00 × 10 per leukocyte removal filter for 400 mL of used blood. ４ If the number of contaminating red blood cells and / or white blood cells exceeds the above-mentioned value, the number of contaminating cells can be reduced to the above-mentioned value or less by repeating centrifugation in the second red blood cell removal step, and for example, it is preferable to perform centrifugation once, recover the supernatant, and then perform centrifugation once more.

[0082] In the second red blood cell removal step, after the supernatant is recovered, the protein concentration in the recovered supernatant may be measured, and the protein concentration may be 20% or more, 25% or more, 30% or more, 35% or more, or 40% or more, or may be less than 100%, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, or 45% or less, of fresh frozen plasma prepared from human blood (e.g., fresh frozen plasma-LR "Nisseki" 120 or fresh frozen plasma-LR "Nisseki" 240). Proteins contained in the supernatant recovered in such a second red blood cell removal step are usually plasma-derived proteins. That is, in one embodiment, the supernatant recovered in the second red blood cell removal step may have a plasma-derived protein concentration of 20% or more, 25% or more, 30% or more, 35% or more, or 40% or more, or may be less than 100%, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, or 45% or less, of that of fresh frozen plasma prepared from human blood. The protein content in the above may be measured by, for example, the BCA method, the Bradford method, the Lowry method, or the like, and a specific example may be the BCA method.

[0083] In the mixing step, the platelet concentrate or its lysate obtained in the first collection step is mixed with the solution obtained in the second red blood cell removal step. The method of mixing is not particularly limited, and can be carried out, for example, by adding the solution obtained in the second red blood cell removal step to a bag containing the platelet concentrate or its lysate obtained in the first collection step.

[0084] In the platelet dissolution step, platelets in the platelet concentrate obtained in the first collection step or the mixture obtained in the mixing step are dissolved. Examples of platelet dissolution treatments that can be used include freeze-thawing, ultrasonic treatment, platelet activation with calcium or thrombin, and solvent / detergent (S / D) treatment (Non-Patent Document 2). When the platelet dissolution treatment is freeze-thawing, the freezing conditions are not particularly limited as long as they freeze the solvent in the platelet concentrate or the mixture obtained in the mixing step, and may be, for example, standing in a freezer. Furthermore, the thawing conditions may be any conditions that allow the frozen solvent in the platelet concentrate or the mixture obtained in the mixing step to thaw. However, from the viewpoint of preventing protein denaturation and inactivation, temperature conditions of 0°C or higher and room temperature or lower are preferred, and cooling conditions of approximately 0 to 10°C (e.g., ice bath conditions or standing in a refrigerator at approximately 4°C) are preferred, and the thawing time may be, for example, 5 minutes to 48 hours. Furthermore, when the platelet lysis step involves freezing and thawing, the cycle of freezing and thawing may be repeated two, three, four or more times, and in a specific example, may be repeated three times. The mixture obtained in the platelet lysis step can be used as a platelet lysate.

[0085] In one embodiment, the production method according to the fourth aspect of the present disclosure may further include a cellular component / debris removal step after both the mixing step and the platelet dissolving step are completed. In the cellular component / debris removal step, the mixture containing the platelet lysate obtained by the mixing step and the platelet dissolving step is centrifuged to precipitate cellular components remaining in the mixture and insufficiently lysed cell parts (cell debris), and the supernatant is recovered. The centrifugation conditions and supernatant recovery conditions in the cellular component / debris removal step may be the same as those described for the first recovery step. The mixture obtained in the cellular component / debris removal step may be used as a platelet lysate.

[0086] In one embodiment, the production method according to the fourth aspect of the present disclosure may include a filtration step after both the mixing step and the platelet lysis step if the production method does not include a cellular component / debris removal step, or after the cellular component / debris removal step if the production method includes a cellular component / debris removal step. When the production method according to the fourth aspect of the present disclosure includes a filtration step, bacteria, undissolved cellular components, and protein aggregates can be removed. That is, the filtration step can also remove bacteria (sterilize). The filter used in the filtration step is not particularly limited as long as it can remove undissolved cellular components and protein aggregates. For example, the filter may be configured to trap objects having a maximum width of 2.0 μm or more, 1.5 μm or more, 1.0 μm or more, 0.8 μm or more, 0.5 μm or more, 0.4 μm or more, 0.3 μm or more, 0.2 μm or more, or 0.1 μm or more, or a combination of these filters may be used. As a specific example, the mixture may be first filtered through a membrane filter having a pore size of 0.50 μm, 0.45 μm, or 0.40 μm, and the filtrate may then be further filtered through a membrane filter having a pore size of 0.25 μm, 0.20 μm, or 0.15 μm. The filtrate obtained from the filtration step may be used as the platelet lysate.

[0087] The platelet lysate produced as described above may be stored frozen until use, for example, at about -80°C.

[0088] The method of production according to one embodiment of the fourth aspect of the present disclosure may not include adding heparin at a concentration of 1 U / mL or more based on the volume of the platelet lysate obtained. The method of production according to a preferred embodiment of the fourth aspect of the present disclosure may not include adding heparin.

[0089] Although the manufacturing method according to the fourth aspect of the present disclosure has been described above using an example in which n is 1 in one embodiment, n in the manufacturing method according to the fourth aspect of the present disclosure is not limited to 1 and may be an integer of 2 or greater (e.g., 2, 3, 4, or 5). In these cases, the i-th collection step, the (i+1)-th passing step, and the (i+1)-th red blood cell removal step may be performed in the same manner as the first collection step, the second passing step, and the second red blood cell removal step in the embodiment described above. For example, in one embodiment, the manufacturing method according to the fourth aspect of the present disclosure may include, in this order, the first passing step, the first red blood cell removal step, the first collection step, the second passing step, the second red blood cell removal step, the second collection step, the third passing step, the third red blood cell removal step, the mixing step, and the platelet lysis step. Furthermore, for example, in one embodiment, the production method according to the fourth aspect of the present disclosure may include, in this order, a first passing step, a first red blood cell removal step, a first recovery step, a second passing step, a second red blood cell removal step, a second recovery step, a third passing step, a third red blood cell removal step, a platelet dissolution step, and a mixing step. Furthermore, for example, in one embodiment, the production method according to the fourth aspect of the present disclosure may include, in this order, a first passing step, a first red blood cell removal step, a first recovery step, a second passing step, a second red blood cell removal step, a second recovery step, a third passing step, a third red blood cell removal step, a third recovery step, a fourth passing step, a fourth red blood cell removal step, a mixing step, and a platelet dissolution step. Furthermore, for example, in one embodiment, the manufacturing method according to the fourth aspect of the present disclosure may include, in this order, a first passing step, a first red blood cell removal step, a first recovery step, a second passing step, a second red blood cell removal step, a second recovery step, a third passing step, a third red blood cell removal step, a third recovery step, a fourth passing step, a fourth red blood cell removal step, a platelet dissolving step, and a mixing step.

[0090] According to one embodiment of the first or second aspect of the present disclosure, a non-blood cell solution containing non-blood cell components derived from a leukocyte removal filter that has been subjected to passage of human whole blood, and a method for producing the same, can be provided. The plasma solution produced from the leukocyte removal filter after passage of such human whole blood can be used, for example, to produce a platelet lysate.

[0091] According to one embodiment of the first or second aspect of the present disclosure, a plasma solution having a protein concentration equal to or greater than a predetermined ratio relative to the protein concentration in fresh frozen plasma prepared from human blood can be provided from a leukocyte removal filter after passing human whole blood through it. Such a plasma solution can be produced animal-free and inexpensively because it uses a used leukocyte removal filter that would normally be discarded as medical waste. When the plasma solution is animal-free, immune responses due to different animal species can be suppressed when the plasma solution is used directly or indirectly for regenerative medicine purposes, and this is also preferable from the standpoint of animal welfare.

[0092] According to one embodiment of the third or fourth aspect of the present disclosure, a platelet lysate containing a lysate of platelets and non-blood cell components derived from a leukocyte removal filter that has been passed through human whole blood, and a method for producing the same, can be provided. Such a platelet lysate can be produced animal-free and inexpensively. Such a platelet lysate can be produced animal-free and inexpensively because it uses a used leukocyte removal filter that is normally discarded as medical waste. If the plasma solution is animal-free, immune responses due to different animal species can be suppressed when the plasma solution is used directly or indirectly for regenerative medicine purposes, and this is also preferable from the standpoint of animal welfare.

[0093] According to one embodiment of the third or fourth aspect of the present disclosure, there can be provided a platelet lysate having an excellent cell proliferation-promoting effect, which contains a lysate of platelets and non-blood cell components derived from a leukocyte removal filter that has passed human whole blood, and a method for producing the same, and the plasma solution according to one embodiment of the first or second aspect can be used as a source of the non-blood cell components. Such a platelet lysate has, for example, a stem cell proliferation-promoting effect that is three times or more that of fetal bovine serum.

[0094] Such a platelet lysate can be used, for example, for cell culture, and in a preferred embodiment, it may be for use in culturing stem cells, which may be pluripotent stem cells, such as mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPS cells), or embryonic stem cells (ES). In a more preferred embodiment, the platelet lysate may be for use in culturing mesenchymal stem cells. Mesenchymal stem cells have the advantages of being less susceptible to immune rejection and being easier to culture than other pluripotent stem cells, particularly when aiming at applications in regenerative medicine.

[0095] One application of multipotent stem cells is in regenerative medicine targeting cerebral infarction. During cerebral infarction, nerve cells die in areas deprived of blood supply. Even if the infarction itself improves and blood flow is restored, the damaged nerves do not recover, often resulting in severe side effects such as motor and cognitive impairment. In response to this, it has recently been reported that mesenchymal stem cells collected from the bone marrow of human subjects with severe motor impairments due to nerve damage caused by cerebral infarction, cultured, and then injected into the brain of the human subjects at the site of nerve damage, improved the motor impairment. In such regenerative medicine applications, the time it takes for multipotent stem cells, such as mesenchymal stem cells collected from human subjects, to proliferate to the amount required for administration to the treatment site can be a rate-limiting step in treatment. Therefore, if stem cells can be efficiently cultured and expanded using the cell proliferation-promoting ability of the platelet lysate prepared from the plasma solution according to one embodiment of the first or second aspect of the present disclosure or the platelet lysate according to one embodiment of the third or fourth aspect of the present disclosure, regenerative medicine aimed at restoring nerves damaged after cerebral infarction can be applied promptly, before the symptoms progress over time after the onset of the disease.

[0096] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to the following examples.

[0097] Production Example 1: Preparation of Plasma Solution from a Leukocyte Removal Filter After Passage of Human Whole Blood A blood component separation bag with a filter containing 400 mL of human whole blood collected by whole blood donation was prepared. Similar to the leukocyte removal process before storage of whole blood that is performed in the production of blood products, 400 mL of human whole blood in the blood collection bag was passed through a leukocyte removal filter for 400 mL of human whole blood (Sepacel RZ-2000N, Asahi Kasei Medical Corporation). More specifically, after inverting the blood collection bag at least five times to mix the blood, the hanging opening of the blood collection bag was hung on a bag hanger, the connecting piece of the blood collection bag was opened, and the blood was passed through the leukocyte removal filter, where filtration was performed by head (passage of the blood through the leukocyte removal filter). The filtration head was approximately 140 cm. After all the blood in the blood collection bag was filtered (passed through the leukocyte removal filter), the tube below the leukocyte removal filter was sealed. The above steps were carried out for 15 sets of blood bags and leukocyte removal filters (preparation step).

[0098] Next, for eight of the 15 leukocyte removal filters used in the preparation step, the tubing at the bottom of the filter was aseptically connected to a bag containing 2 L of physiological saline. The bag containing the physiological saline was then hung on a stand so that the side where the tubing at the bottom of the filter was connected to the bag containing the physiological saline was vertically downward, and the physiological saline was passed through the filter by gravity in the opposite direction to that of the human whole blood. The physiological saline that passed through the filter was collected as a first filter-passing solution in the eight blood bags that originally contained the human whole blood (first pass step).

[0099] For the first filtered solution collected in the first pass step, two blood bags were combined into one bag. The four bags containing the first filtered solution thus obtained were centrifuged at 180 g for 10 minutes at 22°C to precipitate red blood cells, and the supernatant was then collected and transferred to a new bag. The resulting supernatant was further centrifuged at 120 g for 10 minutes at 22°C to precipitate red blood cells, and the supernatant was then collected and transferred to a new bag (first red blood cell removal step).

[0100] The supernatants from two bags collected in the first red blood cell removal step were combined into one bag. The contents of blood components in the solutions in these two bags were measured, and the remaining white blood cell count was found to be 1.29 x 10 ６ The remaining red blood cell count is 1.97 x 10 ８ The bags were then centrifuged at 4,000 g for 20 minutes at 22°C to precipitate the platelets, and the supernatant was collected in a new bag as a first plasma solution, and the residue after the supernatant collection was collected as a platelet concentrate (first collection step). The obtained first plasma solution and platelet concentrate were frozen in a freezer.

[0101] The frozen first plasma solution was thawed with shaking at 37°C using a thawing machine FP-40 (manufactured by Hokuyo Denki Co., Ltd.). For seven of the systems used in the preparation process that were not used in the first pass step, the tube at the bottom of the leukocyte removal filter was aseptically connected to a bag containing approximately 600 mL of the first plasma solution obtained in the first recovery step. The bag containing the first plasma solution was then hung on a stand so that the side where the bag containing the first plasma solution and the tube at the bottom of the leukocyte removal filter were connected was vertically downward, and the first plasma solution was passed through the second leukocyte removal filter by gravity in the opposite direction to that in which the human whole blood passed. The first plasma solution that had passed through the leukocyte removal filter was collected as a second filter-passed solution in the blood bags (seven) that originally contained the first plasma solution (second pass step).

[0102] The second filtered solutions from the seven blood bags were combined into one bag. The bag was centrifuged at 2000 g for 20 minutes at 22°C to precipitate cellular components including red blood cells, white blood cells, and platelets, and the supernatant was collected and transferred to a new bag. The bag was then centrifuged at 2000 g for 20 minutes at 22°C to precipitate cellular components including red blood cells, white blood cells, and platelets, and the supernatant was collected and transferred to a new bag (second red blood cell removal step). The blood component content of the thus obtained supernatant, the second plasma solution, was measured, and the residual white blood cell count was 2.0 x 10５ The remaining red blood cell count is 3.5 x 10 ６ After confirming that the number of particles was less than 1, the mixture was frozen in a freezer.

[0103] Production Example 2: Preparation of platelet lysate from a leukocyte removal filter after passage of human whole blood The second plasma solution obtained in the second red blood cell removal step of Production Example 1 was left standing at 4°C for 20 hours or more to thaw. This solution was added to the frozen platelet concentrate obtained in Production Example 1 (mixing step). The mixture was left standing at 4°C for 20 hours or more to thaw the platelet concentrate in the mixture (first platelet lysis step). The mixture was then left standing in a freezer for 4.5 hours or more, and then left standing at 4°C for 20 hours or more to thaw. This process was repeated twice (second and third platelet lysis steps). The mixture was then centrifuged at 4°C for 20 minutes at 4000 g to precipitate unlysed cellular components, and the supernatant was collected. The supernatant was filtered through a 0.45 μm membrane filter, and the filtrate was further filtered through a 0.20 μm membrane filter to collect the platelet lysate.

[0104] The platelet count of the platelet lysate thus obtained was measured using a multi-parameter automated hematology analyzer XS-1000i (Sysmex Corporation) and was 1.95 × 10 １１ The number of contaminating red blood cells and white blood cells in the platelet lysate thus obtained was measured using the Blood Bank mode of a multi-parameter automated blood cell analyzer XN-1000 (Sysmex Corporation), and the number of contaminating red blood cells and white blood cells was 2 × 10 or more relative to the total amount of the platelet lysate obtained. ８ 1.49 x 10 or less ６ There were less than one.

[0105] Furthermore, when the same preparation as above was carried out using 15 leukocyte removal filters, 200 mL of platelet lysate was obtained. Furthermore, when the same preparation as above was carried out using 284 leukocyte removal filters, 4000 mL of platelet lysate was obtained.

[0106] Production Example 3: Preparation of platelet lysate using platelets derived from leukocyte reduction filters and expired fresh frozen plasma. Expired fresh frozen plasma (trade name: Fresh Frozen Plasma-LR "Nisseki" 120, Fresh Frozen Plasma, FFP) and / or the first plasma solution obtained in the first recovery step of Production Example 1 was added to a frozen platelet concentrate obtained in the first recovery step of a production performed using 48 leukocyte reduction filters in the same manner as in Production Example 1, so that the ratio of FFP to the added liquid was 0 vol%, 20 vol%, 35 vol%, 40 vol%, 70 vol%, or 100 vol% (note that "adding so that the ratio of FFP is 0 vol%" means that the first plasma solution was added, and "adding so that the ratio of FFP is 100 vol%" means that FFP itself was added). The platelet concentration in the mixture obtained by the addition was 112.2±1.2×10 ４ The platelet density was 1 / μL (n=6, Mean±SD). The mixture was subjected to freeze-thawing, centrifugation and filtration in the same manner as in Production Example 2 to obtain a platelet lysate.

[0107] Test Example 1: Measurement of protein concentration in plasma solution The protein concentrations of the second plasma solution (F-plasma) and expired fresh frozen plasma (FFP) obtained by the same preparation method as in Production Example 1 were measured. The protein concentration was measured by the BCA method using a BCA Protein Assay Reagent Kit (Thermo Scientific, 23225). As a result, the protein concentration of the FFP was 66,616.6 ± 833.3 μg / mL (n = 3, Mean ± S.D.). The protein concentrations of the four lots of F-plasma obtained by the same preparation method as in Production Example 1 were the values ​​shown in the table below.

[0108]

[0109] Test Example 2: Comparison of cell proliferation promoting ability between platelet lysate prepared from platelets derived from a leukocyte removal filter and platelet lysate containing no blood cell components. Human-derived mesenchymal stem cells (PT-2501, MSC, manufactured by Lonza) were cultured at a density of 5,000 cells / cm. ２The cells were seeded into 24-well plates at a density of 1000 x g / ml. The cells were cultured for 5 days in a culture medium (MEMα containing gentamicin (40 μg / mL) and heparin (2 U / mL)) containing 10% by volume of either (I) platelet lysate (Filter-PL FFP) prepared in Production Example 3 by adding FFP and the first plasma solution obtained in the first recovery step of Production Example 1 so that the ratio of FFP to the added liquid was 0%, 40%, or 100% by volume, (II) platelet lysate (Apheresis Platelet Lysate, Aph-PL) prepared from expired platelets (irradiated platelet concentrate - LR "Nisseki"); or (III) two types of fetal bovine serum (FBS#1: Gibco, MSC FBS, 12662-029; FBS#2: EQUITECH-BIO). Three lots of platelet lysate containing 0% FFP by volume were tested, and two lots of FBS were tested. A portion of the cell solution was then sampled and stained with acridine orange and propidium iodide. The cells were then counted using a Luna FL fluorescent / bright-field automatic cell counter (Logos Biosystems), and the cell proliferation rate from the start of culture was calculated.

[0110] The results of measuring the cell proliferation rate are shown in Figure 1. In addition to the cell proliferation rate, Figure 1 also shows the platelet count (×10) before freezing and thawing in the platelet lysate. ４The cell density (cells / mL) is also shown. Figure 2 shows transmitted light images of MSCs obtained from platelet lysates or Aph-PL prepared in Production Example 3 by adding FFP at a ratio of 0 vol. %, 40 vol. %, or 100 vol. % to the added liquid. Of the three lots containing 0 vol. % FFP, in one lot, cells detached from the culture dish on day 3 of culture. Figures 1 and 2 show that platelet lysates containing 40 vol. % or 100 vol. % FFP exhibited a higher cell proliferation-promoting ability than platelet lysates containing 0 vol. % FFP. This indicates that a certain amount of plasma is required to prepare PL with good cell proliferation-promoting ability. Figure 1 also shows that platelet lysates containing 40 vol. % or 100 vol. % FFP exhibited a cell proliferation-promoting ability three times higher than that of FBS.

[0111] Test Example 3: Comparison of the cell proliferation-promoting ability of a platelet lysate prepared from platelets derived from a leukocyte removal filter and a plasma solution A test similar to that in Test Example 2 was carried out using FBS, expired fresh frozen plasma (FFP), the platelet lysate prepared by adding FFP as prepared in Production Example 3 (Filter-PL FFP 100%), and a platelet lysate prepared from expired platelets (Aph-PL).

[0112] The results of measuring the cell proliferation rate are shown in Figure 3. The results in Figure 3 are shown as mean ± standard deviation (Mean ± S.D.) for n = 5. Figure 4 shows a transmitted light image of MSCs obtained under conditions in which FBS, FFP, and Aph-PL (PL) were added. Figures 3 and 4 show that the cell proliferation-promoting ability of 100% Filter-PL FFP was higher than that of expired fresh frozen plasma (FFP). The results of Test Examples 2 and 3 revealed that the inclusion of non-blood cell components in the platelet lysate further enhances the cell proliferation-promoting ability.

[0113] Test Example 4: Examination of the dependency of the cell proliferation-promoting ability of platelet lysates containing non-blood cell components on the concentration of non-blood cell components A test similar to that in Test Example 2 was performed using: (I) a platelet lysate (Filter-PL FFP) prepared by adding the FFP prepared in Production Example 3 and the first plasma solution obtained in the first recovery step of Production Example 1 so that the ratio of FFP to the added liquid was 0 vol%, 20 vol%, 35 vol%, 40 vol%, 70 vol%, or 100 vol%, (II) a platelet lysate (Aph-PL) prepared from expired platelets, (III) a commercially available platelet lysate (nLivenPR, PL-PR-100, commercially available PL, manufactured by Cook Pharmaceuticals), and (IV) two different lots of fetal bovine serum (FBS) similar to those in Test Example 2. The platelet lysate prepared in Production Example 3 using the first plasma solution (i.e., by adding FFP so that the proportion of FFP was 0% by volume) contains platelet lysate and non-blood cell components derived from the first leukocyte removal filter, and therefore corresponds to a platelet lysate produced using only one used leukocyte removal filter.

[0114] The results of the cell proliferation rate are shown in Figure 5. In Figure 5, Protein Con. indicates the plasma-derived protein concentration before the addition of each platelet lysate-derived protein. Figure 5 shows that, compared with a platelet lysate with an FFP content of 0% by volume, i.e., no FFP, the cell proliferation rate was significantly higher when a platelet lysate containing 20% ​​or more by volume of FFP was added, and the cell proliferation rate was even higher when a platelet lysate containing more than 20% by volume of FFP was added.

[0115] Test Example 5: Cell proliferation-promoting ability of platelet lysates prepared from platelets derived from a leukocyte removal filter and from a plasma solution derived from a leukocyte removal filter A test similar to that in Test Example 2 was carried out using (I) the platelet lysate prepared in Production Example 2 (filter platelets / plasma PL), (II) the platelet lysate prepared in Production Example 3 by adding FFP so that the FFP accounted for 40% by volume or 100% by volume (Filter-PL FFP), (III) a platelet lysate prepared from expired platelets (Aph-PL), and (IV) the same two lots of fetal bovine serum (FBS) as in Test Example 2.

[0116] The test results are shown in Figure 6. Figure 6 reveals that the platelet lysate prepared in Production Example 2 has a high cell proliferation-promoting ability, exhibiting a cell proliferation-promoting ability three times greater than that of FBS. Furthermore, Figure 6 shows that mesenchymal stem cells in the presence of the platelet lysate prepared in Production Example 2 exhibited a morphology similar to that in the presence of the platelet lysate prepared from expired platelets. The results in Figures 1 and 6 demonstrate that the PL prepared from the first filter-passed solution has insufficient cell proliferation-promoting ability, whereas the PL prepared from the second filter-passed solution has sufficient cell proliferation-promoting ability, confirming the usefulness of plasma obtained by passing through a filter two or more times.

[0117] Test Example 6: Cell proliferation-promoting ability of platelets derived from a leukocyte removal filter and platelet lysates prepared from a plasma solution derived from a leukocyte removal filter A test similar to Test Example 2 was performed using a platelet lysate (F-PL) prepared from the second plasma solution prepared in Production Example 1 and platelets derived from a leukocyte removal filter, a solution obtained by diluting FFP with physiological saline to 45% by volume and a platelet lysate (45% FFP-PL) prepared from platelets derived from a leukocyte removal filter, a platelet lysate (Aph-PL) prepared from expired platelets, and two lots of fetal bovine serum (FBS) similar to those in Test Example 2. Specifically, F-PL and 45% FFP-PL were prepared by first mixing 13.5 mL of expired fresh frozen plasma (FFP) with 16.5 mL of physiological saline to prepare a 45% FFP diluted solution. The supernatant (platelet concentration 19.0 × 10) obtained in the first red blood cell removal step in Production Example 1 was diluted with 45% FFP. ４ The platelet concentrate was then centrifuged at 4000 g for 20 minutes at room temperature to precipitate the platelets, and the supernatant was removed. Each of the three tubes containing the platelet concentrate thus prepared was filled with 110 x 10 platelets at a final concentration of 110 x 10 ４Eight mL of the plasma solution or 45% FFP diluted solution prepared in Production Example 1 was added so that the platelet concentration became 8 / μL, and then the platelets were lysed by freeze-thawing in the same manner as in Production Example 2. Then, by filtration using a syringe filter with a pore size of 0.2 μm, three samples each of F-PL and 45% FFP-PL (#1 to #3) were obtained, and these samples were subjected to the same test as in Test Example 2.

[0118] The results are shown in Figure 7. Statistical analysis of the results for F-PL and 45% FFP-PL in Figure 7 is shown in Figure 8. Figures 7 and 8 show that the cell proliferation-promoting ability of the plasma solution prepared in Production Example 1 and the platelet lysate (F-PL) prepared from platelets derived from the leukocyte removal filter was significantly higher than that of 45% FFP-PL.

[0119] Furthermore, the protein and RNA concentrations of F-PL ("PL" in the F-PL column in Table 2), 45% FFP-PL ("PL" in the 45% FFP-PL column in Table 2), the second plasma solution prepared in Production Example 1 ("Plasma" in the F-PL column in Table 2), and the 45% FFP diluted solution ("Plasma" in the 45% FFP-PL column in Table 2) were measured. Protein concentration was measured by the BCA method using a BCA Protein Assay Reagent Kit (Thermo Scientific, 23225). RNA concentration was measured at a wavelength of 260 nm (extinction coefficient 40) using a NanoDropLite (Thermo). The results of measuring the protein concentration and plasma-derived RNA concentration are shown in Table 2, and the results of statistical processing (n=3, Mean±SD) of the results in Table 2 are shown in Table 3. No significant differences were observed in the protein concentration and RNA concentration between the two.

[0120]

[0121]

Claims

1. A plasma solution containing non-blood cell components derived from a first leukocyte removal filter through which human whole blood has been passed and non-blood cell components derived from a second leukocyte removal filter through which human whole blood has been passed, wherein the non-blood cell components derived from the second leukocyte removal filter are non-blood cell components contained in a solution obtained by passing a solution containing the non-blood cell components derived from the first leukocyte removal filter through the second leukocyte removal filter.

2. A plasma solution containing a supernatant obtained from the n+1th filter-passed solution, wherein the n+1th filter-passed solution is a solution obtained by passing the supernatant obtained from the nth filter-passed solution through the n+1th leukocyte removal filter through which human whole blood has been passed, and the first filter-passed solution is a solution obtained by passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed. (Note that n is an integer of 1 or more, and the second paragraph is repeated n times by decreasing the number substituted for n by 1.) 3. A method for producing a plasma solution, comprising: a first passing step: a step of passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed, and recovering the solution after passing to obtain a first filter-passed solution; an i-th plasma recovery step: a step of recovering a supernatant from the i-th filter-passed solution; and an i+1-th passing step: a step of passing the supernatant obtained in the i-th plasma recovery step through an i+1-th leukocyte removal filter through which human whole blood has been passed, and recovering the solution after passing to obtain an i+1-th filter-passed solution; (wherein n is an integer of 1 or more, i is an integer of 1 to n, and the i-th plasma recovery step and the i+1-th passing step are repeated n times in this order while the number substituted for i is increased by 1 in succession, starting from 1.) 4. A plasma solution produced by the method of claim 3.

5. A method for producing a platelet lysate, comprising the step of mixing the plasma solution produced by the method according to claim 3 with platelets or a lysate thereof.

6. first passing step: a step of passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed, and recovering the solution after passing to obtain a first filter-passed solution; first red blood cell removal step: a step of removing red blood cells from the first filter-passed solution obtained in the first passing step; ith recovery step: a step of centrifuging the solution obtained in the ith red blood cell removal step, recovering the supernatant, and recovering the residue after the supernatant recovery as a platelet concentrate; i+1th passing step: a step of passing the supernatant obtained in the i+1th leukocyte removal filter through which human whole blood has been passed, and recovering the solution after passing to obtain an i+1th filter-passed solution; i+1th red blood cell removal step: a step of removing red blood cells from the i+1th filter-passed solution obtained in the i+1th passing step; (wherein n is an integer of 1 or more, i is an integer of 1 or more and n or less, and the i-th recovery step, the i+1th passing step, and the i+1th red blood cell removal step are repeated n times in this order while the number substituted for i is increased by 1 in sequence starting from 1.) A method for producing a platelet lysate, comprising: a mixing step: a step of mixing at least one platelet concentrate selected from the group consisting of the platelet concentrates obtained in the 1st to nth recovery steps with a solution obtained in the n+1th red blood cell removal step; and a platelet dissolving step: a step of dissolving platelets in the solution obtained in the mixing step.

7. First passing step: a step of passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed, and recovering the solution after passing to obtain a first filter-passed solution; First red blood cell removal step: a step of removing red blood cells from the first filter-passed solution obtained in the first passing step; ith recovery step: a step of centrifuging the solution obtained in the ith red blood cell removal step, recovering the supernatant, and recovering the residue after the supernatant recovery as a platelet concentrate; i+1th passing step: a step of passing the supernatant obtained in the i+1th leukocyte removal filter through which human whole blood has been passed, and recovering the solution after passing to obtain an i+1th filter-passed solution; i+1th red blood cell removal step: a step of removing red blood cells from the i+1th filter-passed solution obtained in the i+1th passing step; (wherein n is an integer of 1 or more, i is an integer of 1 or more and n or less, and the i-th recovery step, the i+1th passing step, and the i+1th red blood cell removal step are repeated n times in this order while the number substituted for i is increased by 1 in sequence starting from 1.) A platelet dissolving step: a step of dissolving platelets in at least one platelet concentrate selected from the group consisting of the platelet concentrates obtained in the first to n-th recovery steps; and a mixing step: a step of mixing the at least one platelet concentrate after platelet lysis obtained in the platelet dissolving step with a solution obtained in the n+1th red blood cell removal step.

8. A platelet lysate produced according to the method of any one of claims 5 to 7.

9. A platelet lysate comprising a platelet lysate, non-blood cell components derived from a first leukocyte removal filter through which human whole blood has been passed, and non-blood cell components derived from a second leukocyte removal filter through which human whole blood has been passed, wherein the non-blood cell components derived from the second leukocyte removal filter are non-blood cell components contained in a solution obtained by passing a solution containing non-blood cell components derived from the first leukocyte removal filter through the second leukocyte removal filter.

10. A platelet lysate comprising a platelet lysate and a supernatant obtained from a second filtered solution, wherein the second filtered solution is a solution obtained by passing the supernatant obtained from the first filtered solution through a second leukocyte removal filter through which human whole blood has been passed, and the first filtered solution is a solution obtained by passing an aqueous solution through a first leukocyte removal filter through which human whole blood has been passed.

11. The platelet lysate according to claim 9 or 10, wherein the platelet lysate is a lysate of platelets derived from a first leukocyte reduction filter through which human whole blood has been passed.

12. A platelet lysate produced according to the production method described in any one of claims 5 to 7 or the platelet lysate described in claim 9 or 10, having a fibrinogen concentration of 100 μg / mL or more and 1500 μg / mL or less.

13. A platelet lysate produced according to the production method described in any one of claims 5 to 7 or the platelet lysate described in claim 9 or 10, which has a stem cell proliferation-promoting effect that is three times or more greater than that of fetal bovine serum.

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