JP6412696B2 - Serum preparation method and instrument - Google Patents

Description

  The present invention relates to a method for separating serum, a device for separating serum, a method for transporting blood for preparing autoserum, a transport container, and the like.

  In recent years, attempts have been made to transplant various cells in order to repair damaged tissues and the like. For example, fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, etc. have been tried to repair myocardial tissue damaged by ischemic heart diseases such as angina pectoris and myocardial infarction. (Non-Patent Document 1).

As part of such attempts, cell structures formed using scaffolds and sheet-shaped cell cultures in which cells are formed into sheets have been developed (Patent Document 1).
For the application of sheet cell culture to the treatment, use of cultured epidermis sheet for skin damage caused by burns, use of corneal epithelial sheet cell culture for corneal injury, oral mucosa sheet for endoscopic resection of esophageal cancer Studies such as the use of cell cultures are ongoing.

  In the production of such a cell culture for medical use, serum containing various useful components in a well-balanced manner is often used for cell survival. Since the serum used for the production of the cell culture enters the body together when the cell culture is applied to the body of the subject, it is desirable that the serum is autologous serum derived from the subject with little influence on the subject. However, medical facilities that collect blood, which is a raw material for autologous serum, from subjects are often provided only with equipment for separating serum for clinical testing. In such facilities, it is difficult to prepare a sufficient amount of serum for the production of medical cell cultures, and there is also a risk of contamination with endotoxins and other harmful substances. It is not suitable for the preparation of autologous serum for use in the production of cell cultures. Accordingly, it is desirable to prepare autologous serum for use in the production of medical cell cultures at a medical cell culture production facility that conforms to predetermined standards. However, such a manufacturing facility is not necessarily located near a medical facility that collects blood, and blood may have to be transported for a long time. There has been no known method for preparing serum suitable for a given situation or a method for transporting blood for serum preparation for a long time.

Special Table 2007-528755

Haraguchi et al., Stem Cells Transl Med. 2012 Feb; 1 (2): 136-41

  An object of the present invention is to provide a serum preparation method suitable for a case where a blood collection place and a serum preparation place are separated, and a method for transporting blood for serum preparation for a long time.

  While the present inventors are diligently researching to solve the above problems, the whole blood collected from the subject is transported in a state of being mixed with a red blood cell preservation solution to prepare a high-quality serum without hemolysis or the like. The present invention has been completed.

That is, the present invention relates to the following.
(1) mixing whole blood and a red blood cell preservative, and causing a blood coagulation reaction in the mixture of the whole blood and red blood cell preservative obtained in the step;
A method for preparing serum, comprising:
(2) The method according to (1), wherein the erythrocyte preservative comprises a sugar alcohol and / or disaccharide, glucose, adenine and / or inosine, sodium chloride, and an anticoagulant.
(3) The method according to (1) or (2), wherein the blood coagulation reaction is performed for 12 to 120 hours.
(4) The method according to any one of (1) to (3), wherein the blood coagulation reaction is controlled by adjusting the temperature of the mixture.

(5) The method according to any one of (1) to (4), wherein the blood coagulation reaction is performed while the mixture is transported from the blood collection site to the serum preparation site.
(6) A serum separation device comprising a blood collection container containing an erythrocyte preservative and a serum storage container airtightly connected to the blood collection container.
(7) mixing the whole blood collected from the first subject and the red blood cell preservative;
Causing a blood coagulation reaction in the mixture of whole blood and red blood cell preservative obtained in the above step;
A method for producing a medical cell culture, comprising: collecting serum from the mixture; and incubating cells collected from a second subject in the presence of the collected serum.
(8) A medical cell culture produced by the method according to (7).

(9) A method for treating a disease in a subject, comprising administering an effective amount of the cell culture according to (8) to a subject in need thereof.
(10) A method for transporting whole blood for serum preparation collected from a subject, the method comprising transporting a mixture of the whole blood and a red blood cell preservative while causing a blood coagulation reaction in the mixture. .
(11) The method according to (10), wherein in the step of transporting, whole blood is transported together with cells that are collected from a subject and are used as a material for a medical cell culture.
(12) A cell culture production material comprising: a first storage unit that stores the serum separation device according to (6); and a second storage unit that stores a container that stores cells collected from the subject. Transport container.

According to the serum preparation method of the present invention, a high-quality serum suitable for the production of a medical cell culture can be prepared by effectively utilizing the transport time from the blood collection site to the serum preparation site. The product can be efficiently manufactured, and a great contribution to regenerative medicine using a medical cell culture can be expected.
In addition, since the blood for autologous serum preparation and the cells and tissues for producing medical cell cultures are transported together by the transport method of the present invention, other target blood is mistakenly combined with the cells and tissues. The risk of being collected is reduced and the management of the collected material from the subject becomes easier. Furthermore, these effects can be further enhanced by using a dedicated transport container in the transport method of the present invention.

FIG. 1 is a view showing an embodiment of the serum separation device of the present invention. FIG. 2 is a view showing an embodiment of the transport container of the present invention.

  Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are hereby incorporated by reference in their entirety.

One aspect of the present invention is to prepare a serum comprising the steps of mixing whole blood and a red blood cell preservative, and causing a blood coagulation reaction in the mixture of whole blood and red blood cell preservative obtained in the previous step. The present invention relates to a method (hereinafter sometimes abbreviated as “serum preparation method”).
In the present invention, the red blood cell preservative refers to any agent that can be added to a liquid containing a red blood cell component separated from whole blood (for example, concentrated red blood cell liquid) to extend the storage time of red blood cells. Those containing / or disaccharide, glucose, adenine and / or inosine, and sodium chloride are preferable. Examples of the sugar alcohol include, but are not limited to, mannitol, sorbitol, maltitol, and the like. Examples of the disaccharide include, but are not limited to, lactose, sucrose, maltose, and the like. In addition to the above components, the erythrocyte preservative in the present invention is citrate (for example, sodium citrate), an anticoagulant such as heparin, a pH adjuster such as citric acid, and ATP production promotion such as sodium dihydrogen phosphate. Components such as agents may be included. The saccharide contained in the erythrocyte preservative in the present invention may be D-form or L-form, but D-form is preferred. The erythrocyte preservative in the present invention may be a liquid erythrocyte storage solution by dissolving the above components in a liquid solvent. The liquid solvent is typically water, but may be other solvents that do not adversely affect red blood cells.

  When the erythrocyte preservative in the present invention contains an anticoagulant such as citrate, it is preferable to contain the same component in a coagulation delay amount. Here, the amount of clotting delay is less than the amount in which coagulation is completely suppressed as in a blood preservation solution such as an ACD solution or a CPD solution. is there. As a non-limiting example of the clotting delay amount in the present invention, for example, the final concentration when sodium citrate is mixed with whole blood is preferably 0.0003 to 0.3 w / v%, more preferably 0.001 to 0. 0.1 w / v%, more preferably 0.003 to 0.06 w / v%, particularly preferably 0.007 to 0.03 w / v%. By containing an anticoagulant with an amount of clotting delay, clot formation is delayed, and when clots are formed early, blood clots collide with the inner wall of the container due to impact during transportation, etc. Blood cells forming sputum are destroyed, and components in the blood cells can be prevented from being mixed into the serum.

A more preferred embodiment of the erythrocyte preservative in the present invention contains mannitol, adenine, glucose, sodium chloride and water. Examples of the erythrocyte preservative include, but are not limited to, for example, MAP solution (in water, D-mannitol 1.457 w / v%, adenine 0.014 w / v%, sodium dihydrogen phosphate 0.094 w / v%, citric acid Sodium hydrate 0.150 w / v%, citric acid hydrate 0.020 w / v%, glucose 0.721 w / v%, sodium chloride 0.497 w / v%), SAG-M (chlorinated in 1 L of water) Sodium 8.77 g, anhydrous glucose 8.2 g, adenine 0.169 g, mannitol 5.25 g), Adsol ^(R) (AS-1) (9.0 g sodium chloride in 1 L water, 22.0 g glucose monohydrate) , adenine 0.27 g, mannitol ^{7.5g), OPTISOL (R) (} AS-5) ( in water 111 mL, sodium chloride 974 mg, glucose monohydrate 1.0 g, adenine 33.3 mg, mannitol 586 mg), PAGGSM (in water, mannitol 55 mM, adenine 1.4 mM, guanosine 1.4 mM, sodium dihydrogen phosphate 8 mM, disodium hydrogen phosphate 16 mM, glucose 47 mM, sodium chloride 72 mM ), A known product such as RAS2 may be used, but an erythrocyte preservative having other components containing the above components and having an erythrocyte protective effect may be appropriately prepared and used. The above products are intended to be added to a liquid containing red blood cell components separated from whole blood (eg, concentrated red blood cell liquid), and are not intended to be added to whole blood. Further, as the erythrocyte preservative in the present invention, other known erythrocyte preservatives such as Nutricel ^(R) (AS-3), SAGP-maltose, and Erythrol-4 may be used.

  The erythrocyte preservative in the present invention preferably does not contain an anticoagulant having an anticoagulation amount or more. Here, the anticoagulation amount refers to an amount that completely suppresses the blood coagulation reaction, which corresponds to the amount of sodium citrate contained in a blood preservation solution such as an ACD solution or a CPD solution. The final concentration of sodium citrate when the ACD solution is mixed with whole blood is 0.33 w / v%, and the final concentration of sodium citrate when the ACD solution is mixed with whole blood is about 0.37 w / v%. It is. Therefore, a preferred erythrocyte preservative in the present invention does not contain an anticoagulant, or even if it contains an anticoagulant, it does not contain an amount greater than the above anticoagulant amount.

  Mixing of whole blood and a red blood cell preservative can be performed by any method that does not damage blood cell components contained in whole blood. As such a technique, for example, a method of adding a red blood cell preservative to a container (for example, a blood collection container) containing whole blood and mixing gently, or a method of gently injecting whole blood into a container (for example, a blood collection container) containing a red blood cell preservative And the like.

  The blood coagulation reaction can be caused by leaving a mixture of whole blood and a red blood cell preservative for a predetermined time. The time during which the blood coagulation reaction is performed is not particularly limited, and may be, for example, 12 to 120 hours, 18 to 96 hours, 24 to 72 hours, and the like. In addition, since the reaction can be suppressed by cooling the mixture, the progress of the blood coagulation reaction can be controlled by adjusting the cooling timing, the cooling time, and / or the cooling rate. Cooling is typically done by refrigeration of the mixture at about 2-8 ° C.

  The step of generating a blood clotting reaction can be performed while the whole blood is being transported from the blood collection site to the serum preparation site. Serum production by blood clotting reaction takes a certain amount of time, but by carrying out this process during transportation from the blood collection site to the serum preparation site, it is possible to save the trouble of performing the blood coagulation reaction at the serum preparation site, Time loss can be greatly reduced.

  In the mixture in which the blood coagulation reaction has been completed, the clot and the serum are separated, from which the serum can be collected. Therefore, the serum preparation method of the present invention may further include a step of recovering serum from the mixture. The serum can be collected, for example, by transferring the separated serum portion as it is to another container, by centrifuging the clot, and transferring the serum obtained as a supernatant to another container, or by removing the clot in the mixture. The serum obtained as a filtrate after removal by a filter can be transferred to another container, and further can be performed by other known serum recovery methods. In order to prevent contamination with microorganisms, the collected serum is preferably transferred aseptically to another container. Moreover, in order to prevent contamination of impurities such as endotoxin, it is preferable to move the collected serum to another container in a liquid-tight and / or air-tight manner. The technique for transferring the collected serum to another container in a liquid-tight and / or air-tight manner is not limited, and for example, at least two containers connected in an air-tight manner, such as the serum separation instrument of the present invention described in detail below. In one of these, a mixture of whole blood and a red blood cell preservative is added to produce serum, and the separated serum is transferred to another container through a connecting portion.

Another aspect of the present invention relates to a serum separation device including a blood collection container containing an erythrocyte preservative and a serum storage container airtightly connected to the blood collection container.
The blood collection container and the serum storage container in the present invention are not particularly limited as long as they can accommodate blood. For example, soft thermoplastic resins such as soft vinyl chloride resin and ethylene-vinyl acetate copolymer, glass, polystyrene Those formed of polypropylene or the like can be used. Various forms such as bags and tubes can be used depending on the material. For example, a soft thermoplastic resin is suitable for a bag-type container, and glass, polystyrene, and polypropylene are suitable for a tube-type container.

  The connection between the blood collection container and the serum storage container is not particularly limited as long as an airtight connection is achieved. For example, a thermoplastic resin such as a soft vinyl chloride resin, an ethylene-vinyl acetate copolymer, polycarbonate, or polyethylene terephthalate is used. It can be performed through a formed connecting means, for example, a connecting tube or a connecting tool. In the connecting means for connecting the serum storage container or the blood collection container, clot components remaining in the blood collection container after transferring the serum to the serum storage container are mixed into the serum storage container, or conversely A closing device such as a clamp, a clip or a stopcock may be provided to prevent the serum stored in the container from flowing back into the blood collection container.

  The components and types of the erythrocyte preservative contained in the blood collection container are as described above for the serum preparation method. The content of the erythrocyte preservative varies depending on the amount of blood to be collected in the blood collection container, for example, 10-50 v / v%, 15-40 v / v%, 20-30 v / v%, It can be 23-25 v / v%. More specifically, for example, when the planned blood collection amount is 200 mL, 50 mL of the red blood cell preservative can be placed in a blood collection container.

  A blood collection needle may be connected to the blood collection container. The blood collection needle is preferably liquid-tight and / or air-tightly connected to the blood collection container, and is not limited. For example, thermoplastic resins such as soft vinyl chloride resin, ethylene-vinyl acetate copolymer, polycarbonate, and polyethylene terephthalate It may be connected via a blood introduction connecting means formed by, for example, a blood collection tube.

  The serum separation instrument of the present invention can be used in the serum preparation method of the present invention. Specifically, for example, blood collected from a subject is placed in a blood collection container containing a red blood cell preservative, mixed in the container to cause a blood coagulation reaction, and the produced serum is connected to the blood collection container Serum can be prepared by transferring to a serum storage container.

  A specific embodiment of the serum separation instrument of the present invention is shown in FIG. The serum separating device 1 includes a blood collection container (blood collection bag) 11 and a serum storage container (serum bag) 12. A blood collection needle 14 is connected to one end of the blood introduction connecting means (blood collection tube) 15, and the other end is connected to the blood collection bag 11. The blood collection needle 14 may be covered with a cap 18. The blood collection container 11 is connected to the serum bag 12 via a serum circulation connecting means (connecting tube) 13. The blood collection bag 11 and / or the serum bag 12 are provided with one or two or more co-infusion ports 16 and / or a flow path closing means 17 capable of communicating as required. As the mixed injection port and the channel closing means capable of communicating, for example, those described in JP-A-62-258322 can be used. The blood collection bag 11 contains a red blood cell preservative in an amount corresponding to the planned blood collection amount.

The serum separation instrument of FIG. 1 can be used as follows. Blood collection is started, and blood is introduced from the blood collection tube 15 into the blood collection bag 11. During blood collection, the blood collection bag 11 is periodically rocked gently in order to mix the inflowed blood with the red blood cell preservative contained in the blood collection bag 11. When the blood collection is completed, the blood collection bag 11 is turned over and the blood and the red blood cell preservative are mixed carefully. Thereafter, serum is generated in the blood collection bag 11. If necessary, the blood collection bag 11 may be cooled to adjust the serum production time. When the serum is generated, the blood collection bag 11 is centrifuged to separate the serum, and the separated serum is transferred to the serum bag 12 through the connecting tube 13. When the serum has been transferred to the serum bag 12, the connecting tube 13 is closed by heat sealing, a clamp, or the like to prevent backflow of serum or contamination of components remaining in the blood bag 11.
The said aspect is only an example of the serum separation instrument of the present invention, and does not limit the scope of the present invention. In the present invention, each configuration can be replaced with any other configuration capable of exhibiting the same function, and any configuration can be added.

  Another aspect of the present invention is the step of mixing the whole blood collected from the first subject and the red blood cell preservative, the step of causing a blood coagulation reaction in the mixture of the whole blood and red blood cell preservative obtained in the step, Recovering serum from the mixture, and incubating cells collected from a second subject in the presence of the recovered serum (hereinafter referred to as “manufacturing method”). And a medical cell culture produced by the production method (hereinafter sometimes abbreviated as “cell culture”).

In the production method of the present invention, the steps of mixing the whole blood and the red blood cell preservative, causing the blood coagulation reaction, and recovering the serum are as described above with respect to the serum preparation method of the present invention.
In the present invention, the term “subject” refers to any individual organism, preferably a multicellular organism that can be treated with cell culture, more preferably an animal, even more preferably a mammal (eg, human, non-human primate, dog). , Cats, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), pigs, horses, goats, sheep, rabbits, etc.), more preferably human individuals.

  In the present invention, the “first subject” for collecting whole blood and the “second subject” for collecting cells may be the same or different. When the first object and the second object are different, the first object and the second object may be the same species (same species) or different species (heterogeneous species). In the present invention, from the viewpoint of providing the produced cell culture for medical use, the first object and the second object are preferably the same type, and more preferably the same.

  In the present invention, the term “medical use” means that it is used for medical purposes, for example, for treatment of diseases, transplantation medicine, regenerative medicine and the like. Therefore, the “medical cell culture” of the present invention satisfies various standards required for medical products, for example, standards related to manufacturing process-derived impurities. Examples of such standards include those set by administrative authorities. Medical care includes both human medicine and veterinary medicine.

  The cells in the production method of the present invention include any type used for medical applications. Examples of such cells include, but are not limited to, for example, hematopoietic stem cells used for treatment of blood diseases, lymphocytes used for immunotherapy and the like, immune cells such as dendritic cells, and cells that can form a sheet-like cell culture, such as , Myoblasts (eg, skeletal myoblasts, etc.), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair root, muscle tissue, endometrium, placenta, umbilical cord blood, etc.), Cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synoviocytes, chondrocytes, epithelial cells (eg, oral mucosal epithelial cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (eg, , Endothelial cells, etc.), hepatocytes (eg, liver parenchymal cells, etc.), pancreatic cells (eg, islet cells, etc.), kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, etc. .

  The step of incubating in the production method of the present invention can be performed under any conditions suitable for production of a cell culture. The amount of the collected serum used for the incubation is not particularly limited as long as it is suitable for the production of the cell culture, but when the incubation is performed in a liquid medium such as a medium, the final concentration is, for example, 1 to 50 v / v%. , 5 to 40 v / v%, 10 to 30 v / v%, and the like.

  Only one type of cell may be incubated, but two or more types of cells can also be used. In a preferred embodiment of the present invention, when there are two or more types of cells forming a cell culture, the ratio (purity) of the most cells is 60% or more, preferably 70% or more at the end of cell culture production. Preferably it is 75% or more. As the cells to be incubated, the cells collected from the subject may be used as they are. However, before and / or after the incubation, treatments such as proliferation, freezing / thawing, purification, and differentiation may be performed as necessary. Moreover, the cell extract | collected from the object contains the cell isolate | separated from the tissue extract | collected from the object.

  The cell culture in the present invention can take various forms suitable for medical use, such as a cell suspension and a cell structure. The cell structure may have various shapes such as a sheet shape, a column shape, a block shape, and a plug shape. In the present invention, a sheet-like cell structure, that is, a sheet-like cell culture is preferable from the viewpoint of high therapeutic effect. “Sheet cell culture” refers to a sheet in which cells are connected to each other and is typically composed of one cell layer, but may also be composed of two or more cell layers. Including. The cells may be linked to each other directly (including those via cell elements such as adhesion molecules) and / or via intervening substances. The intervening substance is not particularly limited as long as it is a substance that can connect cells at least physically (mechanically), and examples thereof include an extracellular matrix. The intervening substance is preferably derived from cells, in particular, derived from the cells constituting the cell culture. The cells are at least physically (mechanically) connected, but may be further functionally, for example, chemically or electrically connected.

  Although the cell culture of the present invention may or may not contain a scaffold (support), it is preferable not to contain a scaffold (support) from the viewpoint of biocompatibility and high therapeutic effect. Scaffolds may be used in the art to attach cells on and / or within its surface and maintain the physical integrity of the cell culture, eg, polyvinylidene difluoride (PVDF) Although preferred membranes of the present invention are known, the physical integrity thereof can be maintained without such a scaffold. In addition, the cell culture of the present invention preferably consists only of substances derived from the cells constituting the cell culture and does not contain any other substances.

  The cell culture of the present invention is useful for the treatment of various diseases, particularly diseases associated with tissue abnormalities. Accordingly, in one aspect, the cell culture of the present invention is for use in the treatment of diseases associated with tissue abnormalities. Examples of the tissue to be treated include, but are not limited to, myocardium, cornea, retina, esophagus, skin, joint, cartilage, liver, pancreas, gingiva, kidney, thyroid, skeletal muscle, middle ear, bone marrow, and the like. It is done. In addition, the disease to be treated is not limited, and for example, heart disease (eg, myocardial injury (myocardial infarction, cardiac injury), cardiomyopathy, etc.), corneal disease (eg, corneal epithelial stem cell exhaustion, cornea) Injury (heat / chemical corrosion), corneal ulcer, corneal opacity, corneal perforation, corneal scar, Stevens-Johnson syndrome, pemphigoid, etc.), retinal diseases (eg retinitis pigmentosa, age-related macular degeneration) , Esophageal diseases (eg, prevention of esophageal inflammation / stenosis after esophageal surgery (eg, after removal of esophageal cancer)), skin diseases (eg, skin damage (trauma, burns), etc.), joint diseases (eg, , Osteoarthritis, etc.), cartilage disease (eg, cartilage damage), liver disease (eg, chronic liver disease), pancreatic disease (eg, diabetes), dental disease (eg, periodontal disease), kidney Disease (eg, Failure, renal anemia, renal osteodystrophy, etc., thyroid disease (eg, hypothyroidism), muscle disease (eg, muscle damage, myositis), middle ear disease (eg, otitis media), bone marrow Diseases (eg, leukemia, aplastic anemia, immunodeficiency diseases, etc.) can be mentioned.

  The usefulness of the cell culture of the present invention for the above-mentioned diseases is, for example, Patent Document 1, Non-Patent Document 1, Arauchi et al., Tissue Eng Part A. 2009 Dec; 15 (12): 3943-9, Ito et al., Tissue Eng. 2005 Mar-Apr; 11 (3-4): 489-96, Yaji et al., Biomaterials. 2009 Feb; 30 (5): 797-803, Yaguchi et al., Acta Otolaryngol. 2007 Oct; 127 (10): 1038-44, Watanabe et al., Transplantation. 2011 Apr 15; 91 (7): 700-6, Shimizu et al., Biomaterials. 2009 Oct; 30 (30): 5943-9 Ebihara et al., Biomaterials. 2012 May; 33 (15): 3846-51, Takagi et al., World J Gastroenterol. 2012 Oct 7; 18 (37): 5145-50, and the like.

  Although the cell culture of the present invention can be applied to a tissue to be treated and used to repair and regenerate it, it can be used as a source of a physiologically active substance such as a hormone other than the tissue to be treated. (For example, Arauchi et al., Tissue Eng Part A. 2009 Dec; 15 (12): 3943-9, Shimizu et al., Biomaterials. 2009 Oct; 30 (30): 5943-9). It is also possible to fragment the sheet-like cell culture of the present invention into an injectable size and inject it into a site requiring treatment (Wang et al., Cardiovasc Res. 2008 Feb 1; 77 (3) : 515-24).

  The cell culture of the present invention is useful for treating various additional components such as a pharmaceutically acceptable carrier, a component that enhances the viability, engraftment and / or function of the cell culture, and the target disease. It may further contain other active ingredients. Any known additional components can be used, and those skilled in the art are familiar with these additional components. In addition, the cell culture of the present invention can be used in combination with components that enhance the viability, engraftment and / or function of the cell culture, and other active ingredients useful for treating the target disease.

  The cell culture of the present invention can be produced by any known method using the serum prepared by the serum preparation method of the present invention. For example, a method for producing a sheet-shaped cell culture is disclosed in Patent Document 1, JP 2010-081829, JP 2010-226962, JP 2010-226991, JP 2011-110368, JP 2011-115058, and JP 2011-150. 172925 and the like.

A particularly preferred embodiment of the production method of the present invention comprises the following steps:
(1) a step of mixing whole blood collected from a subject with a red blood cell preservative;
(2) causing a blood coagulation reaction in the mixture of whole blood and red blood cell preservative obtained in the above step;
(3) recovering serum from the mixture;
(4) freezing cells collected from the subject;
(5) thawing frozen cells;
(6) using the cells thawed in step (5) to form a sheet-like cell culture in the presence of the serum recovered in step (3);
(7) A step of collecting the formed sheet-shaped cell culture.
Steps (1) to (3) have already been described above. In this embodiment, the subject from which the whole blood is collected and the subject from which the cells are collected are the same.

Step (4) can be performed by any known method. Such techniques include, but are not limited to, for example, subjecting the cells in the container to a freezing means such as a freezer, a deep freezer, or a low-temperature medium (for example, liquid nitrogen). The temperature of the freezing means is not particularly limited as long as it is a temperature at which a part of the cell population in the container, preferably the whole can be frozen, but is typically 0 ° C. or lower, preferably −20 ° C. or lower, more preferably − 40 ° C. or lower, more preferably −80 ° C. or lower. The cooling rate in the freezing operation is not particularly limited as long as it does not significantly impair the viability and function of the cells after freezing and thawing. Typically, the cooling rate is from 1 to 5 until cooling begins at 4 ° C and reaches -80 ° C. The cooling rate is on the order of time, preferably 2 to 4 hours, especially about 3 hours. Specifically, for example, cooling can be performed at a rate of 0.46 ° C./min. Such a cooling rate can be achieved by providing the container containing the cells directly or in a freezing treatment container in a freezing means set to a desired temperature. The freezing treatment container may have a function of controlling the temperature lowering speed in the container to a predetermined speed. As such a freezing container, any known container such as BICELL ^(R) (Japan Freezer) can be used.

  The freezing operation may be performed while the cells are immersed in a culture solution or physiological buffer solution, but a cryoprotectant for protecting the cells from freezing and thawing operations is added to the culture solution, or the culture solution is cryoprotected. You may perform after performing the process of replacing with the cryopreservation liquid containing an agent. Therefore, the production method of the present invention may further include a step of adding a cryoprotectant to the culture solution or a step of replacing the culture solution with a cryopreservation solution. When replacing the culture solution with a cryopreservation solution, if the solution in which cells are immersed during freezing contains an effective concentration of cryoprotectant, remove the culture solution before adding the cryopreservation solution. Alternatively, the cryopreservation solution may be added while leaving a part of the culture solution. Here, the “effective concentration” means that the cryoprotectant exhibits a cryoprotective effect without exhibiting toxicity, for example, the viability, vitality, and function of the cell after freeze-thawing compared to the case where the cryoprotectant is not used. This means a concentration that exhibits a decrease-suppressing effect. Such a concentration is known to those skilled in the art or can be appropriately determined by routine experimentation.

  The cryoprotectant is not particularly limited as long as it exhibits a cryoprotective action on cells, for example, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sericin, propanediol, dextran, polyvinylpyrrolidone, Polyvinyl alcohol, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, formamide, acetamide, adonitol, perseitol, raffinose, lactose, trehalose, sucrose, mannitol and the like. Cryoprotectants may be used alone or in combination of two or more.

  The concentration of the cryoprotectant added to the culture solution or the concentration of the cryoprotectant in the cryopreservation solution is not particularly limited as long as it is an effective concentration as defined above. It is 2 to 20% (v / v) with respect to the whole preservation solution. However, although outside this concentration range, alternative use concentrations known or experimentally determined for each cryoprotectant may be employed, and such concentrations are within the scope of the present invention.

  Step (5) can be performed by any known cell thawing technique, typically involving, for example, freezing cells from a thawing means, eg, a solid, liquid or gaseous medium at a temperature above the freezing temperature. (For example, water), water baths, incubators, incubators, etc., or by immersing frozen cells in a medium (for example, a culture medium) at a temperature higher than the freezing temperature. It is not limited. The temperature of the thawing means or the immersion medium is not particularly limited as long as the cells can be thawed within a desired time, but typically 4 to 50 ° C, preferably 30 to 40 ° C, more preferably 36 to 38. ° C. The thawing time is not particularly limited as long as it does not significantly impair the viability and function of the cells after thawing, but it is typically within 2 minutes, and in particular within 20 seconds can reduce the viability. It can be greatly suppressed. The thawing time can be adjusted, for example, by changing the temperature of the thawing means or the immersion medium, the volume or composition of the culture solution or cryopreservation solution at the time of freezing.

  Step (6) can be performed by any known method. Such a method is not limited and is, for example, Patent Document 1, JP 2010-081829, JP 2010-226962, JP 2010-226991, JP 2011-110368, JP 2011-115058, JP 2011-150 172925 etc. are mentioned. Forming the sheet-shaped cell culture may include seeding the cells on a culture substrate and sheeting the seeded cells.

The culture substrate is not particularly limited as long as cells can form a sheet-like cell culture thereon, and includes, for example, containers of various materials, solid or semi-solid surfaces in containers, and the like. The container preferably has a structure / material that does not allow permeation of a liquid such as a culture solution. Examples of such materials include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl. Examples include acrylamide and metals (for example, iron, stainless steel, aluminum, copper, brass). The container preferably has at least one flat surface. Examples of such containers include, but are not limited to, cell culture dishes and cell culture bottles. Further, the container may have a solid or semi-solid surface therein. Examples of solid surfaces include plates and containers of various materials as described above, and examples of semi-solid surfaces include gels and soft polymer matrices. The culture substrate may be prepared using the above materials, or commercially available materials may be used. Preferable culture substrates include, but are not limited to, substrates having an adhesive surface suitable for the formation of sheet cell cultures. Specifically, a substrate having a hydrophilic surface, for example, a substrate coated with a hydrophilic compound such as polystyrene subjected to corona discharge treatment, collagen gel or hydrophilic polymer, and further, collagen, fibronectin, laminin , Substrates coated with an extracellular matrix such as vitronectin, proteoglycan and glycosaminoglycan, and cell adhesion factors such as cadherin family, selectin family and integrin family. Further, such substrates are commercially available ^{(e.g., Corning (R) TC-Treated} Culture Dish, etc. manufactured by Corning).

The surface of the culture substrate may be coated with a material whose physical properties change in response to stimulation, for example, temperature or light. Examples of such materials include, but are not limited to, (meth) acrylamide compounds, N-alkyl substituted (meth) acrylamide derivatives (for example, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylate Amides), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethyl) Chloramide and the like), (meth) acrylamide derivatives having a cyclic group (for example, 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidine, 4- (1-oxo -2-propenyl) -morpholine, 1- (1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidine, 4- (1-oxo -2-methyl-2-propenyl) -morpholine), or a vinyl ether derivative (for example, methyl vinyl ether) homopolymer or copolymer, a temperature-responsive material, a light-absorbing polymer having an azobenzene group, triphenylmethane leucohydro Copolymer of vinyl derivative of oxide and acrylamide monomer, and spirobenzopyra It can be used to include N- and isopropyl acrylamide gels known, such as photoresponsive materials (e.g., JP-A-2-211865, see JP-2003-33177). By giving a predetermined stimulus to these materials, the physical properties, for example, hydrophilicity and hydrophobicity can be changed, and peeling of the cell culture adhered on the materials can be promoted. Culture dishes coated with a temperature-responsive material are commercially available (e.g. UpCell ^(R), etc. manufactured by Cellseed), it can be used in the production method of the present invention.

  The culture substrate may be coated (coated or coated) with the serum collected in step (3). By using a culture substrate coated with serum, a denser sheet-shaped cell culture can be formed. “Coated with serum” means a state in which serum components are attached to the surface of a culture substrate. Such a state is not limited, and can be obtained, for example, by treating a culture substrate with serum. Treatment with serum includes contacting the serum with a culture substrate and, if necessary, incubating for a predetermined period of time.

  When incubating on a culture substrate, serum may be used as a stock solution or diluted. Dilution can be any medium such as, without limitation, water, saline, various buffers (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, etc.) can be used. The dilution concentration is not particularly limited as long as the serum component can adhere to the culture substrate. For example, 0.5 to 100% (v / v), preferably 1 to 60% (v / v), more preferably Is 5-40% (v / v).

  The incubation time is not particularly limited as long as the serum component can adhere to the culture substrate. For example, the incubation time is 1 to 72 hours, preferably 4 to 48 hours, more preferably 5 to 24 hours, and further preferably 6 to 12 hours. It's time. The incubation temperature is not particularly limited as long as the serum component can adhere to the culture substrate, and is, for example, 0 to 60 ° C, preferably 4 to 45 ° C, more preferably room temperature to 40 ° C.

  Serum may be discarded after incubation. As a method for discarding serum, a conventional liquid disposal method such as suction with a pipette or decantation can be used. In a preferred embodiment of the present invention, the culture substrate may be washed with a serum-free washing solution after serum is discarded. The serum-free washing solution is not particularly limited as long as it is a liquid medium that does not contain serum and does not adversely affect the serum components attached to the culture substrate. For example, without limitation, water, physiological saline, various buffers Liquid (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80 -7, DMEM / F12, etc.). As the washing method, a conventional culture substrate washing method, for example, without limitation, a method of adding a serum-free washing solution on the culture substrate, stirring for a predetermined time (for example, 5 to 60 seconds), and then discarding it is used. be able to.

  Cell seeding on the culture substrate can be performed by any known technique and condition. The seeding of the cells on the culture substrate may be performed, for example, by injecting a cell suspension obtained by suspending the cells in the culture solution into the culture substrate (culture vessel). For the injection of the cell suspension, an apparatus suitable for the operation of injecting the cell suspension such as a dropper or a pipette can be used.

  The step of forming the seeded cells into a sheet can also be performed by any known technique and condition. Non-limiting examples of such methods include, for example, Patent Document 1, JP 2010-081829, JP 2010-226962, JP 2010-226991, JP 2011-110368, JP 2011-115058, JP 2011-172925, and the like. It is described in. It is considered that the formation of a cell sheet is achieved when cells adhere to each other via an adhesion molecule or an intercellular adhesion mechanism such as an extracellular matrix. Therefore, the step of forming the seeded cells into a sheet can be achieved, for example, by culturing the cells under conditions that form cell-cell adhesion. Such conditions may be any as long as cell-cell adhesion can be formed, but cell-cell adhesion can usually be formed under the same conditions as general cell culture conditions. A person skilled in the art can select optimal conditions according to the type of cells to be seeded. In the present specification, the culture for forming the seeded cells into a sheet may be referred to as “sheet culture”.

The cell culture medium used for the culture (sometimes simply referred to as “culture medium” or “medium”) is not particularly limited as long as it can maintain cell survival, but typically, amino acids, vitamins, electrolytes are used. Can be used. A culture solution based on a basal medium for cell culture may be used. Such a basal medium is not limited, for example, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12 and the like are included. Many of these basal media are commercially available, and their compositions are also known.
The basal medium may be used in a standard composition (for example, as it is commercially available), or the composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the basal medium used in the present invention is not limited to those having a known composition, and includes one in which one or more components are added, removed, increased or decreased.
The serum collected in step (3) is added to the culture solution. Serum is added so that the final concentration in the culture solution is 1 to 50 v / v%, preferably 5 to 40 v / v%, more preferably 10 to 30 v / v%.

Cell culture can be performed under conditions usually used in the art. For example, typical culture conditions include culture at 37 ° C. and 5% CO ₂ . Culturing can be performed in containers of any size and shape. The size and shape of the cell culture can be adjusted by adjusting the size and shape of the cell adhesion surface of the culture vessel, or by installing a mold of the desired size and shape on the cell adhesion surface of the culture vessel. It can be arbitrarily adjusted by culturing the cells with, for example.

  The collection of the sheet-shaped cell culture in step (7) is not particularly limited as long as the sheet-shaped cell culture can be released (peeled) from the culture substrate serving as a scaffold while at least partially maintaining the sheet structure. For example, enzymatic treatment with a proteolytic enzyme (such as trypsin) and / or mechanical treatment such as pipetting. In addition, when cells are cultured on a culture substrate whose surface is coated with a material that changes its physical properties in response to stimulation, for example, temperature or light, a predetermined stimulation is applied. It can also be released non-enzymatically.

  Another aspect of the present invention is a method for treating a disease in a subject, comprising the step of administering an effective amount of the cell culture of the present invention to the subject in need thereof (hereinafter abbreviated as “treatment method”). In some cases). The tissues and diseases to be treated by the treatment method of the present invention are as described above for the cell culture of the present invention. Further, in the treatment method of the present invention, components that enhance the viability, engraftment and / or function of the cell culture, and other active ingredients useful for the treatment of the target disease are used. Can be used together.

  The treatment method of the present invention may further comprise a step of producing a cell culture according to the production method of the present invention. The treatment method of the present invention may further include the step of collecting whole blood, cells or tissue serving as a source of cells for producing a cell culture from a subject before the step of producing the cell culture. In one embodiment, the subject from whom whole blood and / or cells or tissue that is the source of cells is collected is the same individual as the subject receiving the cell culture. In another embodiment, the subject from whom whole blood and / or cells or tissue that is the source of cells is collected is a separate species of the same type as the subject receiving the cell culture. In another embodiment, the subject from whom the whole blood and / or cells or tissue that is the source of the cells is collected is a heterogeneous individual from the subject receiving the cell culture.

In the present invention, the subject may be healthy or afflicted with some disease, but when treatment of the disease is intended, the subject is typically afflicted with or affected by the disease. Means a subject at risk.
The term “treatment” is also intended to encompass all types of medically acceptable prophylactic and / or therapeutic interventions intended to cure, temporarily ameliorate or prevent disease. For example, the term “treatment” encompasses medically acceptable interventions for various purposes, including delaying or stopping the progression of the disease, regression or disappearance of the lesion, prevention of the onset of the disease or prevention of recurrence, etc. .

  In the present invention, the effective amount means, for example, an amount that can suppress the onset or recurrence of a disease, reduce symptoms, or delay or stop progression (for example, the number of cells contained in a cell culture, Size, weight, etc.), preferably an amount that prevents the onset and recurrence of the disease or cures the disease. In addition, an amount that does not cause adverse effects exceeding the benefits of administration is preferred. Such an amount can be appropriately determined by, for example, testing in laboratory animals such as mice, rats, dogs or pigs, and disease model animals, and such test methods are well known to those skilled in the art. In addition, the size of the tissue lesion to be treated can be an important index for determining the effective amount.

The cell culture of the present invention can be administered from various routes such as intravenous, intramuscular, subcutaneous, topical, intraarterial, intraportal, intraventricular, and intraperitoneal. In the case of a sheet-like cell culture, the administration method typically includes direct application to a tissue. However, when a fragment of a sheet-like cell culture is used, there are various methods that can be administered by injection. Administration may be via a route such as intravenous, intramuscular, subcutaneous, topical, intraarterial, intraportal, intraventricular, intraperitoneal, and the like.
The frequency of administration is typically once per treatment, but multiple administrations are possible if the desired effect is not obtained.

Another aspect of the present invention is a method for transporting whole blood for serum preparation collected from a subject, the step of transporting a mixture of the whole blood and a red blood cell preservative while causing a blood coagulation reaction in the mixture. (Hereinafter sometimes abbreviated as “transport method”). In the transporting method of the present invention, it is preferable that in the transporting step, the serum-prepared whole blood collected from the subject is transported together with the cells that are collected from the same subject and become the material of the medical cell culture.
The subject, erythrocyte preservative, blood coagulation reaction, medical cell culture and cells in the delivery method of the present invention are as described above for the other aspects of the present invention. Cells used as a material for a medical cell culture may be in an isolated state or contained in a tissue collected from a subject. In the latter case, what is conveyed is apparently tissue taken from the subject that contains cells that are the material of the medical cell culture.

  For transportation in the present invention, any known method suitable for transportation of blood, cells, tissues, etc., for example, a method using walking or a vehicle can be used. At the time of transportation, it is preferable to transport the container containing the whole blood, for example, the serum separation instrument of the present invention in a dedicated transport container. In addition, when whole blood is transported together with cells that are the material of a medical cell culture, it is preferable that the whole blood and the cells are contained in the same transport container and transported.

When cooling the mixture of whole blood and erythrocyte preservative to suppress the blood coagulation reaction, use a transport container having a cooling means, a transport means having a cooling means, for example, a vehicle equipped with a refrigeration device. be able to. Examples of the transporting container having the cooling means include a cooler box containing a cooling agent and a container having a temperature adjusting function.
When transporting whole blood together with cells that are the material of a medical cell culture, whole blood and cells may be subjected to the same temperature environment, but both may be subjected to different temperature environments as required. .

  The transportation method of the present invention is particularly useful when the place for collecting blood for serum preparation is remote from the place for serum preparation. In addition, when the prepared serum is used for the production of a medical cell culture, the whole blood collected from the subject is transported together with the cells that are the material of the medical cell culture, so that the serum and the cell culture It is possible to manage the cells that are the material in a batch, reduce the risk of errors such as using serum from other subjects in the production of the cell culture, and the material for producing the cell culture Management can be made more efficient.

Another aspect of the present invention is a cell culture production comprising a first storage unit that stores the serum separation instrument of the present invention and a second storage unit that stores a container that stores cells collected from a subject. The present invention relates to a material transport container (hereinafter sometimes abbreviated as “transport container”).
The transport container of the present invention is typically used to transport whole blood collected from a subject and cells used as a material for cell culture from the place where the whole blood and cells are collected to the place where the cell culture is manufactured. . Therefore, the transport container of the present invention can be advantageously used in the transport method of the present invention.

  The transport container of the present invention may be formed of any material suitable for transporting blood or cells. However, if it is intended to be stored in a cold place such as a refrigeration apparatus, it is thermally conductive. Can be formed of a high-quality material such as, but not limited to, a metal such as aluminum or a metal alloy, while the cooling means provided in the transport container can cool the serum separating instrument and the container containing the cells. If intended, it can be made of a highly heat-insulating material.

  In order to protect whole blood and cells from impact, the transport container of the present invention may be provided with a cushioning material that absorbs impact during transport. The buffer material of the storage unit can be disposed so as to contact the serum separating instrument or the cell storage container to be stored. For example, when the serum separation instrument includes a blood collection bag as shown in FIG. 1 and is stored upright in a storage portion such that a connection portion such as a connecting tube is on the top, blood collection is performed in order to suppress shaking of the blood collection bag. Fill the gap between the bag and the inner wall of the container (if the first container is equipped with a removable bucket that can be attached to the centrifuge), etc., with buffer material Can do. As the buffer material, any known one can be used.

  The transport container of the present invention is preferably self-supporting and has a structure that does not easily fall over. Moreover, it is preferable that the accommodating part can shield the serum separation instrument and cell accommodation container accommodated from an external environment by shielding members, such as a lid and a door which can be opened and closed, and a drawer structure.

The transport container of the present invention may be provided with a temperature control means for controlling the temperature in the accommodating portion. Any known temperature control means may be used, but one capable of adjusting the temperature in the accommodating portion in a range of about 2 ° C to about 37 ° C is preferable. As the temperature control means, for example, a small cooling device having a Peltier element controlled by a thermostat can be used.
The transport container of the present invention may include various structures and members useful for transport operations, such as a handle for lifting and moving the transport container.

  In the transport container of the present invention, the first storage portion and the second storage portion may be integral with each other or may be separable from each other. In the separable aspect, it is preferable that each housing portion is self-supporting and has a structure that hardly falls. The separable mode is useful when the first storage unit and the second storage unit are transported together and then used in different places.

  The transport container of the present invention may have a structure in which the serum separating device accommodated in the first accommodating portion can be attached to the centrifuge without touching the serum separating device. For example, the 1st accommodating part may be equipped with the detachable bucket which can be mounted | worn with a centrifuge, and may have a structure which accommodates a serum separation instrument in the said bucket. In this case, for example, the first accommodating portion may include a gap adapted to the outer shape of the bucket for fixing the bucket, and a buffer that abuts against the bucket and suppresses the vibration of the bucket. A material may be provided. In the aspect which can isolate | separate a 1st accommodating part and a 2nd accommodating part, the 1st accommodating part may have a structure which can be attached to a centrifuge as it is. By having such a structure, when the serum separating device is installed in the centrifuge, the serum separating device is not directly gripped and operated, the impact applied to the clot formed in the serum separating device is reduced, and hemolysis is performed. Risk can be suppressed.

  Since the whole blood collected from the subject and the cells that are the material of the medical cell culture can be handled as a whole by the transport container of the present invention, either one of the whole blood and the cell is lost, or the cell culture In addition, it is possible to reduce the risk of occurrence of mistakes such as using serum from other subjects in the production of cells, and to improve the management of cell culture production materials.

A specific embodiment of the transport container of the present invention is shown in FIG. The transport container 2 includes a first housing portion 21 and a second housing portion 22. A bucket 213 that accommodates the serum separating device 1 is detachably attached to the first accommodating portion 21. The bucket 213 has an attachment member 214 to the centrifuge, and the bucket 213 can be detached from the first accommodating portion 21 and attached to the centrifuge as it is. The serum separating device 1 is accommodated in the bucket 213 in an upright state with a connection portion such as a connection tube facing up. Inside the bucket 213, a buffer material 212 is filled in a gap between the serum separating device 1 and the bucket inner wall. The first accommodating part 21 is provided with a lid 211, which can shield the inside from the outside. The first accommodating portion 21 is provided with a handle 23 that is gripped during transportation. In addition, a display unit 24 that displays information about the stored whole blood and cells is provided on the side surface of the first storage unit 21. The second accommodating portion 22 has a shape suitable for accommodating the petri dish-type cell accommodating container 3, and a buffer material 222 is formed in the gap between the inner wall of the second accommodating portion 22 and the cell accommodating container 3. Is filled. The second accommodating portion 22 is provided with a lid 221 that can shield the inside from the outside. The 1st accommodating part 21 and the 2nd accommodating part 22 are connected with the connector 25 so that isolation | separation is possible. The transport container 2 is intended to be housed and transported in a cold place such as a refrigeration apparatus, and is not provided with a cooling means and is made of a material having high thermal conductivity.
The said aspect is only an example of the conveyance container of this invention, and does not limit the scope of the present invention. In the present invention, each configuration can be replaced with any other configuration capable of exhibiting the same function, and any configuration can be added.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, this is a specific example of the present invention, and the present invention is not limited thereto.

Example 1 Preparation of Serum 100 mL of whole blood was collected from a human subject and introduced into a blood collection container (Terumo Separation Bag, Terumo). A MAP solution (Terumo blood bag MAP solution, Terumo Corporation) was injected into the blood collection container at a concentration of 4.5 to 19 v / v% with respect to whole blood, and gently mixed by inversion. Blood collection containers were transported on foot and by vehicle for 24-72 hours while refrigerated at 5 ° C. After arriving at the serum preparation site, observing the situation in the blood collection container, it was confirmed that a clot was formed and the serum was separated. Visible hemolysis was not observed. The blood collection container was centrifuged at 1200 rpm for 10 minutes in a centrifuge, and the serum separated as a supernatant was transferred to another container and stored at −30 ° C. until use. The Hb concentration in the serum obtained after refrigerated transportation for 48 hours was compared between blood added with MAP solution at a concentration of 19 v / v% and blood not added with MAP solution. It was confirmed that no hemolysis was observed even in the evaluation by Hb concentration, which was 2.0 mg / dL in the serum derived from MAP solution-added blood, compared with 12.0 mg / dL in the serum of No. 1.

Example 2 Preparation of Sheet-like Cell Culture Skeletal myoblasts isolated from muscle tissue collected from the same subject from which whole blood was collected were cultured at a concentration of 300,000 cells / cm ² or higher (UpCell ^{(R )} , Cell seeds), cultured in DMEM / F12 medium containing 20% of the serum prepared in Example 1 at 37 ° C., 5% CO ₂ for 12 hours to form a monolayer sheet, and then cultured. The temperature of the dish was lowered to 20 ° C., and the sheet-shaped cell culture was detached from the culture dish. The peeled sheet-shaped cell culture was white and round, and was confirmed to be clinically applicable quality by visual inspection without tearing.

1 Serum Separation Container 11 Blood Collection Container (Blood Collection Bag)
12 Serum storage container (serum bag)
13 Connection means (connection tube)
14 Blood collection needle 15 Blood introduction connecting means (blood collection tube)
16 Mixed injection port 17 Flow path closing means 18 Cap 2 Transport container 21 First accommodating portion 211 Lid 212 Buffer material 213 Bucket 214 Mounting member 22 Second accommodating portion 221 Lid 222 Buffer material 23 Handle 24 Display portion 25 Connector 3 Cell Container

Claims (10)

Mixing whole blood and erythrocyte preservative, and causing a blood coagulation reaction in the mixture of whole blood and erythrocyte preservative obtained in the step;
A method of preparing serum, wherein the erythrocyte preservative comprises a clotting delay amount of an anticoagulant.   The method of claim 1, wherein the erythrocyte preservative comprises a sugar alcohol and / or disaccharide, glucose, adenine and / or inosine, sodium chloride, and an anticoagulant.   The method according to claim 1 or 2, wherein the blood coagulation reaction is performed for 12 to 120 hours.   The method according to any one of claims 1 to 3, wherein the blood coagulation reaction is controlled by adjusting the temperature of the mixture.   The method according to any one of claims 1 to 4, wherein the blood clotting reaction is carried out during transport of the mixture from the blood collection site to the serum preparation site.   A serum separation device comprising a blood collection container containing an erythrocyte preservative containing an anticoagulant of a coagulation delay amount, and a serum storage container airtightly connected to the blood collection container. Mixing whole blood collected from the first subject with a red blood cell preservative,
Causing a blood coagulation reaction in the mixture of whole blood and red blood cell preservative obtained in the above step;
A method for producing a medical cell culture, comprising: collecting serum from the mixture; and incubating cells collected from a second subject in the presence of the collected serum. The method comprising a coagulation retarding amount of an anticoagulant.   A method for transporting whole blood for serum preparation collected from a subject, wherein the mixture of the whole blood and an erythrocyte preservative containing an anticoagulant having a coagulation delay amount is transported in the mixture while causing a blood coagulation reaction. Said method comprising the steps. 9. The method of claim 8 , wherein in the step of transporting, whole blood is transported along with cells that are collected from the subject and that serve as the material of the medical cell culture.   A transport container for cell culture production material, comprising: a first container for housing the serum separating instrument according to claim 6; and a second container for housing a container for collecting cells collected from the subject.