Method for producing pharmaceutical containing culture of stem cell

Abstract

To provide a method for quickly producing a pharmaceutical that uses a culture of a self cell that has been cultured with self serum.SOLUTION: A method for producing a pharmaceutical containing a culture of a stem cell includes the steps of: preparing first serum, using blood taken from a subject, from which to take a biological sample, at a first timing before the biological sample is taken; preparing second serum, using blood taken from the subject at a second timing after taking the biological sample; culturing a stem cell which can be included in the biological sample, in a first medium containing the first serum; and culturing the stem cell cultured in the first medium, in a second medium containing the second serum.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing a medicament containing a culture of stem cells.

Background Art

[0002] In recent years, research and development of regenerative medicine using cultures of stem cells have been actively conducted. As regenerative medicine using stem cells, there is cell therapy for replenishing damaged cells. When using cell therapy to treat acute injuries such as spinal cord injuries caused by accidents or the like, it is important to rapidly and massively proliferate cells provided from a donor (donor cells). Regarding in vitro cell proliferation, it has been reported that a growth promoting factor (for example, serum) is added to the medium when culturing donor cells in order to improve the cell survival rate and increase the proliferation rate.

[0003] In the field of cell therapy, serum is often used to efficiently proliferate desired cells. Serum used in the production of a medicament for cell therapy enters the body together when applying a medicament containing cultured cells to the body of a subject, and thus it is desirable that the serum be autologous serum derived from the subject that has little influence on the subject. However, human serum alone does not provide the desired cell growth promoting activity, and fetal bovine serum (FBS) or other growth factors are added (Patent Document 1 and Patent Document 2).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] When applying cell therapy, it is desirable from the perspective of safety such as reducing the risk of graft-versus-host disease to use a culture obtained by culturing one's own cells using autologous serum. In the case of treating disorders where rapid cell therapy is desired, in order to rapidly and massively proliferate one's own cells, a large amount of blood is required in a short period after collecting one's own cells for producing autologous serum. However, collecting a large amount of blood from the same subject in a short period is restricted from the perspective of maintaining the health of the subject. Therefore, there is a demand in this field for a method for preparing autologous serum to rapidly provide a medicament using a culture of autologous cells while considering the maintenance of the health of the subject.

Means for Solving the Problems

[0006] The present inventors have found that by collecting a predetermined amount or less of blood from the same subject as the subject from whom the biological sample is collected within a predetermined period before and after collecting the biological sample, it is possible to rapidly provide a medicament using a culture of autologous cells while reducing the health burden on the subject, and thus completed the present invention.

[0007] Specifically, the present invention includes preparing a first serum using blood collected from a subject from whom a biological sample is collected at a first time before collecting the biological sample, preparing a second serum using blood collected from the subject at a second time after collecting the biological sample, culturing stem cells that may be contained in the biological sample in a first medium containing the first serum, and culturing the stem cells cultured in the first medium in a second medium containing the second serum, wherein the total amount of the blood collected at the first time and the amount of the blood collected at the second time is more than 0.8 L and 1.1 L or less, and the amount of the blood collected is 0.4 L or less per blood collection, and provides a method for manufacturing a medicament containing a culture of stem cells.

Modes for Carrying Out the Invention

[0008] As used herein, "blood" refers to a body fluid flowing in the body of a subject, which means a body fluid containing blood cell components such as red blood cells, white blood cells, platelets, and plasma. The blood collected from the subject may be, but is not limited to, venous blood, arterial blood, or peripheral blood. In one embodiment, the blood collected from the subject is peripheral blood. The blood can be collected from the subject according to known methods, which are not limited. In one example, the blood can be collected from the subject using a syringe needle and a blood collection container. In one embodiment, the blood for preparing serum does not contain a preservative.

[0009] As used herein, "blood collection container" refers to a container that can aseptically hold the blood collected from the subject. The blood collection container may be, but is not limited to, a container known in the blood collection field and commercially available. The blood collection container may be, for example, a blood collection bag or a blood collection tube. In one example, the blood collection container may be the blood-containing device described in US Patent Application Publication No. US2013 / 0130382 (the content of which is incorporated herein by reference).

[0010] As used herein, "blood clot" refers to a structure formed during the process of blood coagulation of a subject. The blood clot contains, but is not limited to, blood cell components and fibrin (also referred to as "fibrin"). In one example, the blood clot can be formed by aseptically leaving the subject's blood in a container at room temperature (e.g., 22°C to 27°C) without an anticoagulant according to a known method.

[0011] As used herein, "serum" refers to the liquid part separated from the blood clot formed by coagulation of the subject's blood. The serum can be prepared from the blood collected from the subject using known methods, which are not limited. The serum can be used by adding it to a medium for culturing stem cells using a dispenser or the like, which is not limited.

[0012] As used herein, the "first period" means the period from the day when blood is first collected from a subject from whom a biological sample is taken to the day when the biological sample is taken from the subject, for the purpose of preparing serum used in the manufacture of the medicament described in this specification. In one embodiment, the first period is within 2 weeks before the collection of the biological sample.

[0013] The blood collected during the first period is, but not limited to, at least one volume of blood collected from the subject during the first period. The blood collected during the first period is, for example, the blood collected once, the blood collected twice, or the blood collected three times. In one example, the blood collected for the first time during the first period is collected on any day from 6 to 14 days before the collection of the biological sample, and the blood collected for the second time during the first period may be the blood collected on any day from 2 to 7 days before the collection of the biological sample. The blood collected for the second time or later is, but not limited to, collected with a 3- to 5-day interval from the day of the previous blood collection, for the purpose of restoring the blood volume of the subject from whom the blood is collected.

[0014] The blood collected during the first period is, but not limited to, in an amount of 0.3 L or more and 0.6 L or less. The blood collected during the first period is, for example, in an amount of 0.4 L, 0.48 L, or 0.56 L. In one embodiment, the blood collected during the first period is in an amount of 0.48 L.

[0015] As used herein, the "second period" means the period from the day when a biological sample is taken from a subject to the day when blood is last collected from the subject, for the purpose of preparing serum used in the manufacture of the medicament described in this specification. In one embodiment, the second period is within 3 weeks after the collection of the biological sample.

[0016] The blood collected in the second period is, but not limited to, at least one aliquot of blood collected from the subject in the second period. The blood collected in the second period is, for example, the blood collected once, the blood collected twice, or the blood collected three times. In one example, the blood collected for the first time in the second period is collected on any day from 2 days to 8 days after the collection of the biological sample, and the blood collected for the second time in the second period may be the blood collected on any day from 4 days to 14 days after the collection of the biological sample. The blood collected for the second time or later is, but not limited to, collected between the day after the previous blood collection and 10 days, preferably with a 3-day to 10-day interval from the day of the previous blood collection, for the purpose of recovery of the blood volume of the subject from whom the blood was collected.

[0017] The blood collected in the second period is, but not limited to, in an amount of 0.3 L or more and 0.6 L or less. The blood collected in the second period is, for example, in an amount of 0.4 L, 0.48 L, or 0.56 L. In one embodiment, the blood collected in the second period is in an amount of 0.48 L.

[0018] The total amount of the blood collected in the first period and the blood collected in the second period is, but not limited to, 0.8 L or more and less than 1.1 L. The total amount of the blood is, for example, 0.85 L to 1.0 L. In one embodiment, the total amount of the blood is 0.88 L to 0.96 L or 0.96 L.

[0019] The amount of blood collected per collection in the first period and / or the second period is, but not limited to, 0.4 L or less. In one example, when the subject suffers from a disease or disorder (e.g., spinal cord injury) and is prone to anemia, the amount of blood collected per collection is preferably, for example, 0.3 L or less. When the blood for preparing serum is collected twice in the first period and the second period, the amount of the blood is, but not limited to, 0.15 to 0.40 L, 0.15 L to 0.30 L, 0.2 L to 0.25 L, or 0.24 L per collection.

[0020] The total number of times of blood collection in the first period and the number of times of blood collection in the second period is at least three times, although not limited thereto. The total number of times of blood collection is, for example, three times, four times, five times or six times. In one example, the number of times of blood collection in the first period is two times, the number of times of blood collection in the second period is two times, and the total number of times of blood collection is four times. In another example, the number of times of blood collection in the first period is two times, the number of times of blood collection in the second period is three times, and the total number of times of blood collection is five times.

[0021] The amount of blood collected in the first period may be the same as or more than the amount of blood collected in the second period, although not limited thereto. In one example, the amount of blood collected in the first period may be 0.48 L, and the amount of blood collected in the second period may be the same amount of 0.48 L, and the total amount of the blood is 0.96 L. In another example, when the subject suffers from a disease or disorder (for example, spinal cord injury) that is prone to anemia, the amount of blood collected in the first period may be 1.5 times or more of the amount of blood collected in the second period. Thereby, it becomes possible to prevent the subject from becoming anemic after the biological sample collection and delay the administration time of the prepared medicine. In this example, the blood may be collected three times at 0.2 L per time in the first period, and 0.2 L of blood at the first time and 0.16 L of blood at the second time in the second period. Thereby, the amount of blood collected in the first period is 1.66 times the amount of blood collected in the second period, and the total amount of the blood can be 0.96 L.

[0022] As used herein, "the first serum" means each serum prepared from each blood collected in the first period. As used herein, "the second serum" means each serum prepared from each blood collected in the second period. In one example, the first serum or the second serum may be cryopreserved (for example, at -20°C) after its preparation.

[0023] As used herein, "medium" means a composition that enables the maintenance of the survival of stem cells and cell division in vitro. The medium contains serum prepared by the method for producing serum described herein. The medium can be produced using a basal medium for cell culture mainly composed of amino acids, vitamins, and electrolytes. The basal medium that can be used includes, but is not limited to, Dulbecco's Modified Eagle Medium (DMEM), NPBM, and αMEM. In one embodiment, the medium is DMEM. The medium can be prepared according to known methods and is also commercially available.

[0024] The medium contains, but is not limited to, serum prepared by the method for producing serum described herein in an amount of 1 to 20% by volume, preferably 5 to 15% by volume, more preferably 8 to 12% by volume per volume of the medium in the basal medium. The medium may contain, but is not limited to, antibiotics (such as penicillin, streptomycin, etc.) commonly used in the field of cell culture, either alone or in combination. In one example, the medium may contain penicillin and streptomycin at 0.5 to 2% by volume, preferably 0.8 to 1.2% by volume, respectively, based on the medium.

[0025] As used herein, the medium containing the first serum is also referred to as the first medium. As used herein, the medium containing the second serum is also referred to as the second medium. The characteristics described herein for the medium are respectively applicable to the first medium and the second medium. In one embodiment, the first medium containing the first serum contains penicillin and streptomycin, and the second medium containing the second serum does not contain antibiotics.

[0026] Growth and growth factors and / or differentiation-inducing factors may be added to the above-described basal medium as necessary. The growth and growth factors and differentiation-inducing factors are selected according to the desired direction and level of differentiation, the required growth rate, and the like. Examples include, but are not limited to, vitamins such as ascorbic acid and nicotinamide, neurotrophic factors such as NGF and BDNF, bone morphogenetic factors such as BMP, epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor, and cytokines such as IL-2. In one embodiment, the medium does not contain serum prepared from blood from a different species (heterologous) or another individual of the same species that is different from the serum prepared by the method for producing serum described herein.

[0027] The culture of stem cells can be carried out under conditions known in the field of cell culture. The culture of stem cells can be carried out, but is not limited to, under the conditions of 37°C and 5% CO2. The culture of stem cells can be carried out using a culture vessel of any size and shape. The size or shape of the culture of stem cells can be adjusted by the size or shape of the cell attachment surface in the culture vessel. The culture of mesenchymal stem cells is disclosed, for example, in JP-A-2012-100662 (the content of which is incorporated herein by reference).

[0028] As used herein, "subject" means a human in need of treatment with the medicament described herein. The subject is, but is not limited to, a human suffering from spinal cord injury, stroke, dementia, or spinal cord infarction.

[0029] As used herein, "biological sample" means a part of a subject's tissue or body fluid that may contain stem cells. The biological sample may be, but is not limited to, the tissue or body fluid itself collected from the subject, or may be subjected to appropriate treatments (such as removal of unnecessary components, purification of specific cell fractions, enzymatic treatment, preservation treatment) as necessary. The body fluid may be, for example, bone marrow fluid, blood (peripheral blood or cord blood), or lymph fluid. The tissue may be, for example, muscle tissue, bone tissue, skin, lymphoid tissue, blood vessels, or tissues such as the digestive tract. In one embodiment, the biological sample is bone marrow fluid.

[0030] In one example, when the biological sample is bone marrow fluid, the collection of the biological sample can be performed, for example, by anesthetizing (local or general anesthesia) the subject from whom the bone marrow fluid is to be collected, inserting a needle into the sternum or ilium, and aspirating with a syringe. In another example, when the biological sample is cord blood, the collection of the biological sample can be performed, for example, by directly inserting a needle into the umbilical cord and aspirating with a syringe.

[0031] The amount of the biological sample to be collected may be determined depending on, but is not limited to, the amount of stem cells expected to be contained in the biological sample. In one example, when the biological sample is bone marrow fluid, although not limited, the biological sample is collected in an amount of 20 mL to 50 mL.

[0032] The timing of collecting the biological sample may be appropriately determined based on, but is not limited to, the type of disease, or the physical condition or wishes of the subject. In one example, when the disease is acute spinal cord injury caused by factors such as an accident, although not limited, the biological sample (preferably bone marrow fluid) is collected within 31 days, for example, 10 days to 25 days, from the day when the subject received the factor causing the acute spinal cord injury.

[0033] As used herein, "stem cell" means a cell having both self-renewal ability and differentiation ability. As used herein, "self-renewal ability" means the ability to continuously produce stem cells with the same ability as itself as at least one daughter cell through cell division. As used herein, "differentiation ability" means the ability to divide into specialized cells different from itself.

[0034] Stem cells may be, but are not limited to, pluripotent stem cells that can differentiate into multiple types of cells belonging to each of the three germ layers of endoderm, ectoderm, and mesoderm, multipotent stem cells that can differentiate into multiple types of cells limited to one germ layer, and unipotent stem cells that can only differentiate into one type of cell. Pluripotent stem cells may be, for example, embryonic stem cells or recombinant cells (induced pluripotent stem cells) in which pluripotency is imparted to somatic cells by genetic engineering. Multipotent stem cells may be, for example, hematopoietic stem cells or mesenchymal stem cells. Unipotent stem cells may be, for example, germ stem cells. Stem cells can be obtained from a biological sample derived from a subject using known methods.

[0035] As used herein, "mesenchymal stem cell" has non-hematopoietic adhesion and self-renewal ability, and can differentiate not only into connective tissue cells such as osteocytes, chondrocytes, and adipocytes, but also into nerve cells, cardiomyocytes, and liver cells. Mesenchymal stem cells are present only in very small amounts (0.001 - 0.01% in the cell population having nuclei) in bone marrow fluid. Stem cells or mesenchymal stem cells may be, for example, cells that can be attached to a culture vessel and cultured, or cells that can be cultured in a suspended state. In one embodiment, stem cells or mesenchymal stem cells are cells that can be attached to a culture vessel and cultured.

[0036] In one embodiment, the stem cells include one or more types of stem cells. In one embodiment, the stem cells include at least one type of mesenchymal stem cell. In one embodiment, the stem cells are human mesenchymal stem cells derived from bone marrow. Mesenchymal stem cells derived from bone marrow can be obtained by proliferating mesenchymal stem cells present in bone marrow fluid collected from a human by cell culture, although not limited thereto.

[0037] As used herein, a "pharmaceutical" containing a culture of stem cells means a composition used for treating a disease or disorder in which cell therapy can be effective. The pharmaceutical meets various criteria required for medical products, such as criteria regarding impurities derived from the manufacturing process. The pharmaceutical may contain, although not limited to, pharmaceutically acceptable diluents, excipients, and / or substrates depending on the use. The pharmaceutical may be in an injection form (liquid) and a transplantation form (e.g., sheet-like), although not limited to these. The method of delivering the pharmaceutical to the treatment site is appropriately selected according to the form of the pharmaceutical. The delivery method of the pharmaceutical may be, for example, local transplantation by surgical means, intravenous administration, lumbar puncture administration, local injection administration, subcutaneous administration, intradermal administration, intraperitoneal administration, intramuscular administration, intracerebral administration, intraventricular administration, or intravenous administration. In one embodiment, the pharmaceutical is in an injection form and is administered intravenously.

[0038] The pharmaceutical can be used, although not limited to, for tissue repair or tissue regeneration. The pharmaceutical is used for treating nerve diseases. Nerve diseases include, although not limited to, central and peripheral demyelinating diseases, central and peripheral degenerative diseases, stroke (including cerebral infarction, cerebral hemorrhage, and subarachnoid hemorrhage), brain tumors, higher-order functional disorders including dementia, mental diseases, epilepsy, traumatic nerve diseases (including head injury, brain contusion, and spinal cord injury), and spinal cord infarction. The pharmaceutical is not limited to the treatment of nerve diseases and can be used, for example, for treating diseases related to abnormalities in tissues such as myocardium, joints, cartilage, liver, and bone marrow.

[0039] In one embodiment, the medicament can be used, for example, to treat spinal cord injury, stroke, dementia, or spinal cord infarction. In one embodiment, the medicament can be used to treat acute diseases among the above-mentioned diseases. In one example, the medicament can be used to treat, for example, acute spinal cord injury, acute stroke, or spinal cord infarction.

[0040] In one embodiment, the medicament can be used to treat spinal cord injury. In this example, the medicament can be used in subjects who meet appropriate criteria, such as subjects with grade A (complete: complete paralysis of both sensation and movement at S4 - S5), B (incomplete: only sensory function remains below the neurological level including S4 - S5), or C (incomplete: motor function remains below the neurological level, but more than half of the major muscle groups have a muscle strength of less than grade 3) according to the ASIA impairment scale (Clin Orthop Relate Res (2017) 475: 1499 - 1504).

[0041] In one embodiment, the medicament is administered at a maximum of 3.34×10 6 stem cells per kg of the subject's body weight. In other examples, the medicament is administered at 0.5×10 8 to 2.0×10 8 stem cells per administration to subjects weighing 15 kg to 60 kg. In the case of acute spinal cord injury caused by an accident or the like, it is preferable to administer the medicament to the subject from whom the biological sample was collected promptly after the preparation of the medicament.

[0042] Embodiments of the present invention may be, for example, those described below, but are not limited thereto: [Item 1] A method for manufacturing a medicament containing a culture of stem cells, comprising: preparing a first serum using blood collected from a subject from whom a biological sample is taken at a first time before the collection of the biological sample; preparing a second serum using blood collected from the subject at a second time after the collection of the biological sample; culturing stem cells that may be contained in the biological sample in a first medium containing the first serum; and culturing the stem cells cultured in the first medium in a second medium containing the second serum, wherein the total amount of blood collected at the first time and the amount of blood collected at the second time is more than 0.8 L and not more than 1.1 L, and the amount of blood collected is not more than 0.4 L per blood collection. [Item 2] The manufacturing method according to Item 1, wherein the first time is within 2 weeks before the collection of the biological sample, and the second time is within 3 weeks after the collection of the biological sample. [Item 3] The manufacturing method according to Item 1 or 2, wherein the amount of blood collected is from 0.15 L to 0.40 L per blood collection. [Item 4] The manufacturing method according to any one of Items 1 to 3, wherein the first medium and the second medium each contain the first serum and the second serum at 1 to 20% per medium volume. [Item 5] The manufacturing method according to any one of Items 1 to 4, wherein the biological sample is bone marrow fluid. [Item 6] The manufacturing method according to any one of Items 1 to 5, wherein the stem cells are mesenchymal stem cells. [Item 7] The manufacturing method according to any one of Items 1 to 6, wherein the medicament is for treating spinal cord injury, stroke, dementia, or spinal cord infarction. [Item 8] The manufacturing method according to any one of Items 1 to 7, further comprising collecting a biological sample from the subject. [Item 9] The manufacturing method according to any one of Items 1 to 8, further comprising recovering the stem cells cultured in the second medium and manufacturing the medicament.

[0043] Specific examples are described below, but they show preferred embodiments of the present invention and do not limit the invention described in the appended claims in any way.

[0044] [Example] Peripheral blood and bone marrow fluid were collected from 13 subjects suffering from spinal cord injury, including 6 subjects with an ASIA impairment scale of A, 2 subjects with a scale of B, and 5 subjects with a scale of C. Stem cells in the bone marrow fluid were cultured in a medium containing serum prepared from the peripheral blood to prepare a medicament for cell therapy. The amount of peripheral blood collected was approximately 0.48 L for up to two times before the collection of bone marrow fluid, and the total amount of blood collected before and after the collection of bone marrow fluid was approximately 0.96 L as a guideline.

[0045] In one case, the bone marrow fluid of subject A was collected on the 11th day after spinal cord injury. Subject A had peripheral blood collected twice before the collection of bone marrow fluid (the first period). The first peripheral blood collection was on the 5th day after spinal cord injury (6 days before the bone marrow fluid collection), and the second peripheral blood collection was on the 8th day after spinal cord injury (3 days before the bone marrow fluid collection). Subject A also had peripheral blood collected twice after the collection of bone marrow fluid (the second period). The first peripheral blood collection was on the 13th day after spinal cord injury (2 days after the bone marrow fluid collection), and the second peripheral blood collection was on the 15th day after spinal cord injury (4 days after the bone marrow fluid collection). Subject A was administered a medicament for cell therapy containing a culture obtained by culturing stem cells in the bone marrow fluid in a medium containing serum prepared from the peripheral blood on the 47th day after spinal cord injury (36 days after the bone marrow fluid collection).

[0046] Subjects B - M were also treated in the same manner as subject A. Peripheral blood and bone marrow fluid were collected, and a medicament for cell therapy containing a culture obtained by culturing stem cells in the bone marrow fluid in a medium containing serum prepared from the peripheral blood was administered. The schedule of peripheral blood collection, bone marrow fluid collection, and medicament administration for the 13 subjects A - M is summarized in Table 1.

Table 1

[0047] All 13 subjects received 0.5×10 8 ~2×10 8They were administered once by intravenous injection. The proportion of cases in which the ASIA impairment scale improved by one or more levels from immediately before administration (within 7 days of administration) on the 220th day after spinal cord injury was evaluated. Improvement was observed in 12 out of 13 cases (92.3%), including 5 out of 6 subjects (83.3%) with ASIA A, 2 out of 2 subjects (100%) with ASIA B, and 5 out of 5 subjects (100%) with ASIA C.

[0048] In addition to the above 13 subjects, 17 subjects including 4 additional subjects were evaluated for safety. Among the 17 subjects, anemia occurred in 2 subjects during peripheral blood collection, but neither was severe.

[0049] The above test results indicate that when treating spinal cord injury prone to anemia with cell therapy, it was possible to rapidly provide a medicament using a culture of autologous cells while reducing the health burden on subjects (especially anemia related to blood collection).

Claims

1. A method for manufacturing a medicament containing a culture of stem cells, comprising: preparing a first serum using blood collected from a subject from whom a biological sample is taken at a first time before the collection of the biological sample; preparing a second serum using blood collected from the subject at a second time after the collection of the biological sample; culturing stem cells that may be contained in the biological sample in a first medium containing the first serum; and culturing the stem cells cultured in the first medium in a second medium containing the second serum, wherein the total amount of blood collected at the first time and the amount of blood collected at the second time is more than 0.8 L and at most 1.1 L, and the amount of blood collected is at most 0.4 L per blood collection.

2. The first time is within 2 weeks before the collection of the biological sample, and the second time is within 3 weeks after the collection of the biological sample. The manufacturing method according to Claim 1.

3. The amount of blood collected is 0.15 L to 0.40 L per blood collection. The manufacturing method according to Claim 1 or 2.

4. The first medium and the second medium each contain the first serum and the second serum at 1 to 20% per medium volume. The manufacturing method according to any one of Claims 1 to 3.

5. The biological sample is bone marrow fluid. The manufacturing method according to any one of Claims 1 to 4.

6. The stem cells are mesenchymal stem cells. The manufacturing method according to any one of Claims 1 to 5.

7. The medicament is for treating spinal cord injury, stroke, dementia, or spinal cord infarction. The manufacturing method according to any one of Claims 1 to 6.

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