US20090075355A1 - Human serum for cell culture - Google Patents

Human serum for cell culture Download PDF

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Publication number
US20090075355A1
US20090075355A1 US11/719,392 US71939205A US2009075355A1 US 20090075355 A1 US20090075355 A1 US 20090075355A1 US 71939205 A US71939205 A US 71939205A US 2009075355 A1 US2009075355 A1 US 2009075355A1
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blood
serum
human serum
platelets
cell culture
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US11/719,392
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English (en)
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Koji Suzuki
Seishin Tanaka
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JMS Co Ltd
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Individual
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Assigned to JMS CO., LTD. reassignment JMS CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SUZUKI, KOJI, TANAKA, SEISHIN
Publication of US20090075355A1 publication Critical patent/US20090075355A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/135—Platelet-derived growth factor [PDGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)

Definitions

  • the present invention relates to a human serum for cell culture, and more particularly to a human serum for cell culture including a large amount of cell growth factors.
  • stem cells are multipotent and can differentiate into a variety of tissues and organs, and they have been attracting attention as cells which are the key to regenerative medicine.
  • Patent Document 1 As a method of preparing such a serum, a method in which a blood collection tube is used that contains a blood coagulation accelerating solid, such as glass powder, is disclosed (see Patent Document 1). Furthermore, a method of facilitating collection of a serum having a large amount and many kinds of growth factors by bringing the blood into contact with glass powder so as to rapidly separate a coagulation substance such as fibrin mixed in the serum is disclosed (see Patent Document 2). Moreover, a method of producing a serum as a raw material with human plasma is disclosed (see Patent Document 3). In addition, a method of obtaining growth factors by adding a calcium compound and glass beads to plasma is disclosed (see patent document 4).
  • Patent Document 1 Japanese Unexamined Patent Application Publication No. 2000-000228
  • Patent Document 2 Japanese Unexamined Patent Application Publication No. Hei 04-83165
  • Patent Document 3 Japanese Unexamined Patent Application Publication No. 2001-275662
  • Patent Document 4 Japanese Unexamined Patent Application Publication No. 2004-269409
  • the method disclosed in patent document 1 or 2 uses a low capacity blood collection tube designed for blood tests, and therefore, preparatory procedures must be repeated many times to prepare a serum in an amount required for a culture of stem cells. Hence, this method is not suited for practical applications.
  • the method disclosed in patent document 3 uses thrombin as an anticoagulant, and therefore, is not desirable because it entails the infection risk caused by a substance of biologic origin.
  • the method disclosed in patent document 4 cannot efficiently culture stem cells because it uses plasma having only a small amount of cell growth factors as a raw material, and accordingly, the resulting serum does not contain sufficient cell growth factors.
  • the present invention was made in view of the foregoing problems, and it is an object of the present invention to provide a serum which contains a large amount of a growth factor capable of efficiently promoting the growth of stem cells.
  • a human serum for cell culture obtained from a fluid comprising humoral components derived from blood having blood coagulation factors and platelets, and wherein a residual ratio of the platelets remaining within 20 minutes after blood collection in relation to the whole amount of the platelets is 0% to 20%, and a release ratio of cell growth factors is 20% to 100%.
  • the release of the cell growth factors can be quickly promoted, resulting from the residual ratio of the platelets remaining within 20 minutes after collection of the blood in relation to the whole amount of the platelets is 0% to 20%. Furthermore, stem cells can be efficiently proliferated, resulting from the release ratio of cell growth factors being 20% to 100%.
  • the fluid including at least humoral components and platelets derived from blood is used, a serum having a larger amount of growth factors can be produced in comparison with the case where the serum is prepared from the plasma, thereby enabling the production of a serum having a cell growth effect equivalent to a fetal bovine serum.
  • blood used herein indicates whole blood including hemocytes (erythrocytes, leucocytes, platelets) and plasma (serum) as a liquid component, and a liquid containing at least one of these (for example, blood collected by apheresis).
  • hemocytes erythrocytes, leucocytes, platelets
  • plasma plasma
  • serum a liquid containing at least one of these (for example, blood collected by apheresis).
  • serum used herein means a pale yellow liquid obtained by allowing collected blood to stand, resulting in a reduction in fluidity, followed by separation from the red coagulated block (clot).
  • the meaning of “serum” according to the present invention is different from common serums in terms of the production process not including separation from the clot, but it means a humoral component in the blood that is useful in cell culture and that includes coagulation factors and growth factors substantially equivalent to those in common serums.
  • the term “humoral component derived from blood” used herein indicates “blood components other than hemocytes” or “mixture of blood components other than hemocytes and an agent such as an anticoagulant added thereto”.
  • the term “cell growth factor” used herein means a platelet-derived growth factor (PDGF), a transforming growth factor (TGF ⁇ 1), a vascular endothelial growth factor (VEGF), an insulin-like growth factor (IGF), a hepatocellular growth factor (HGF), a brain-derived neurotrophic factor (BDNF), a basic fibroblast growth factor(bFGF), or the like.
  • PDGF platelet-derived growth factor
  • TGF ⁇ 1 transforming growth factor
  • VEGF vascular endothelial growth factor
  • IGF insulin-like growth factor
  • HGF hepatocellular growth factor
  • BDNF brain-derived neurotrophic factor
  • bFGF basic fibroblast growth factor
  • release ratio of cell growth factors indicates a ratio of an amount of cell growth factors to a potential amount, where the amount of cell growth factors contained in a serum prepared from a predetermined amount of blood collected in a vacuum collection tube is assumed to be the potential amount (100% of cell growth factors are released).
  • a human serum for cell culture obtained from a fluid comprising humoral components derived from blood having blood coagulation factors and platelets, and wherein a content of the cell growth factors is greater than that of a human serum prepared from plasma.
  • stem cells can be efficiently proliferated, due to the content of the cell growth factors of the human serum being greater than that of a human serum which is prepared from plasma.
  • plasma used herein indicates a supernatant liquid obtained by adding an anticoagulant, such as heparin, CPD, or the like, to the collected blood, followed by centrifugal separation.
  • human serum prepared from plasma used herein indicates a human serum obtained by centrifuging blood collected from the subject under conditions where the platelets are completely precipitated (for example, 4,400 (g) ⁇ 5 (min.) or greater) to prepare plasma, followed by separating coagulation factors from the plasma.
  • a human serum for cell culture obtained from a fluid comprising humoral components derived from blood having blood coagulation factors and platelets, and wherein a content of the cell growth factors is greater than that of a human serum prepared by allowing the blood to coagulate.
  • stem cells can be efficiently proliferated, due to the content of the cell growth factors of the human serum being greater than that of a human serum prepared by allowing the blood to coagulate.
  • human serum prepared by allowing the blood to coagulate indicates letting human serum obtained from blood collected from the subject coagulate in a flexible vessel at room temperature for approximately one hour, followed by centrifugal separation.
  • the human serum for cell culture according to any one of the first to third aspects, the cell growth factors comprise at least one of PDGF-BB and TGF- ⁇ 1.
  • stem cells can be efficiently proliferated, due to the cell growth factors comprising at least one of PDGF-BB and TGF- ⁇ 1, which are higher among the growth factors in terms of cellular proliferative potential.
  • PDGF-BB indicates one of three types of PDGF (dimer) (PDGF-AA,PDGF-BB,PDGF-AB).
  • the human serum for cell culture according to any one of the first to fourth aspects, the cell growth factors are obtained by bringing the fluid into contact with a processed glass body.
  • the fluid In order to activate the platelets in the fluid to have the cell growth factors released, the fluid is required to be brought into contact with foreign matter.
  • cell growth factors can be more efficiently released in comparison with the case of a polyethylene pellet, as a result of the processed glass body being used as the foreign matter.
  • the “processed glass body” used herein indicates glass powder, glass beads, or the like.
  • the shape of the processed glass body is preferably formed to be nearly spherical.
  • the surface of the processed glass body is preferably formed with a layer comprising a silicon dioxide compound.
  • a silicon dioxide compound which may be used include at least one selected from glass, silica, diatomaceous earth, kaolin and the like, but are not limited thereto.
  • the human serum for cell culture according to the fifth aspect is comprised of glass beads.
  • the platelets in the fluid can be further activated, due the processed glass body being comprised of glass beads.
  • porous glass beads are more preferable because they have a large contact area with the fluid.
  • the surface area of the glass beads per 1 ml of the fluid is preferably in the range of 0.1 to 25 (mm 2 /ml).
  • the human serum for cell culture according to any one of the first to sixth aspects, the human serum is prepared without being exposed to the atmosphere.
  • the risk of contamination by bacteria, microorganisms, or the like can be reduced, resulting from the human serum being prepared without being exposed to the atmosphere. Accordingly, a serum of high safety can be produced in large quantities, thereby ensuring high safety. Furthermore, the human serum for cell culture according to the present invention is preferably prepared by such an apparatus, which will prevent it from being exposed to the atmosphere.
  • the human serum for cell culture according to any one of the first to seventh aspects, the human serum is usable for a regenerative medicine method.
  • use of the serum as a medium for cell culture upon culturing by inoculating stem cells collected from a subject to this medium can culture the cells faster. Furthermore, because of the fact that the cells can be cultured using an autologous serum, the probability of adverse reaction or the like will be reduced, thereby being superior in safety.
  • the human serum for cell culture according to the present invention contains a large amount of a growth factor, and accordingly, can more efficiently culture stem cells in comparison with a conventional serum. Accordingly, the use of this human serum for regenerative medicine ensures that tissues and functions of the subject can be regenerated safely and certainly.
  • FIG. 1 illustrates a view of a serum preparation apparatus for preparing a human serum for cell culture according to the present invention
  • FIG. 2 illustrates a view of a procedure of preparing a human serum for cell culture according to the present invention
  • FIG. 3 illustrates a view of a state of shaking a blood reservoir 10 of the serum preparation apparatus according to the present invention
  • FIG. 4 illustrates a graph of a relationship between elapsed time following the shaking and the residual ratio of platelets in each specimen
  • FIG. 5 illustrates a graph of a relationship between elapsed time following the shaking and the release ratio of PDGF-BB.
  • FIG. 6 illustrates a graph of a relationship between elapsed time following the shaking and release ratio of TGF- ⁇ 1.
  • a human serum for cell culture according to the present invention is preferably prepared with a serum preparation apparatus for a cell culture for preparing a serum for a cell culturing method that includes a serum preparing step of preparing a serum containing a cell growth factor and a culturing step of culturing cells in the presence of the prepared serum.
  • the serum preparation apparatus is usable for cell culture, and includes a blood reservoir for holding a fluid including at least humoral components and platelets derived from the blood having blood coagulation factors, and the blood reservoir has a serum producing function which produces a serum suitable for a cell culturing step.
  • the serum preparation apparatus is an apparatus of a closed system, which can produce a serum without being exposed thereof to the atmosphere.
  • the serum producing function is intended to indicate a function of activating the platelets in the fluids, and accordingly, increasing the content of the growth factors in a serum so that the recovery of the serum is facilitated. This function is provided by a blood coagulation accelerating solid whose specific gravity is greater than that of the fluids.
  • the serum is prepared with the serum preparation apparatus described below, but this is not limited thereto.
  • the serum preparation apparatus 1 comprises a blood reservoir 10 , and a component storage member 20 as main elements.
  • the blood reservoir 10 and the component storage member 20 are constituted from a main body part 11 formed with two sheets of a flexible resin material, for example, soft polyvinylchloride, fused to each other at the external marginal part 11 a to give a bag shape, and a processed glass body 12 disposed inside of the main body part 11 .
  • the glass processed bodies 12 serving as a blood coagulation accelerating solid in the main body part 11 , is freely-movable in the main body part 11 , each of the glass processed bodies 12 having a substantially spherical shape composed of, for example, soda glass. It is preferred to define the surface area of the processed glass body 12 to satisfy a relationship to the volume of reservable blood to be at least 0.1 mm 2 /ml, so that activation of platelets and coagulation factors in the blood is promoted.
  • both a suspension of hemolysis and activation promotion of platelets and coagulation factors during the activation promoting step, and the centrifugal separation step are made possible when the surface area of the processed glass body 12 is defined to satisfy the relationship of 0.1 mm 2/ ml to 25 mm 2 /ml in the volume of the blood which can be reserved in the blood reservoir.
  • Two tubes 41 and 42 are connected in an air-tight manner at the upper edge end of the main body part 11 of the blood reservoir 10 to the connection ports thereof, respectively.
  • the tube 41 among them serves as an introducing path for introducing the blood, and accordingly, a needle for collecting blood 30 or a junction, which can be connected to a needle for collecting blood, is connected at the other end thereof.
  • the serum preparation apparatus thus constructed enables for the serum to be prepared from the collected blood without atmospheric exposure.
  • the other tube 42 connected in an air-tight manner to the blood reservoir 10 is connected to each of the bags 21 to 26 via tubes 43 to 46 and 51 to 56 , and Branches 61 to 65 . These serve as a discharging path for discharging separated blood components.
  • These tubes 41 to 46 and 51 to 56 are constituted from a resin material having flexibility, for example, a material such as soft polyvinylchloride or the like.
  • the bags 21 to 26 and each tube 51 to 56 of the component storage member 20 are also connected in an air-tight manner by, for example, solvent adhesion, thermal welding, ultrasound welding or the like.
  • the serum preparation step using the serum preparation apparatus 1 having the constitution as described above is explained with reference to FIG. 2 and FIG. 3 .
  • the blood separation process using the aforementioned serum preparation apparatus 1 is includes seven steps (S 1 to S 7 ) as generally classified.
  • the needle for collecting blood 30 shown in FIG. 1 is inserted into a subject (patient), and blood is collected.
  • the blood collected from the needle for collecting blood 30 is stored in the blood reservoir 10 , which is positioned lower, via the tube 41 (reservation step S 1 ).
  • the channel of the tube 42 is closed on the blood reservoir 10 side by way of a clamp or the like such that the collected blood in the blood reservoir 10 does not flow into the component storage member 20 .
  • the reservation step S 1 is terminated after the required amount has been collected, taking into account the patient's physical condition upon collecting the blood.
  • the required amount referred to herein may be approximately 200 to 600 ml when the physical constitution and physical condition of the patient are problem free.
  • the blood reservoir 10 is shaken (activation promoting step S 2 ) while the reservation step S 1 is being carried out.
  • the blood reservoir 10 storing the collected blood is gently agitated by a shaking apparatus 100 brought into contact with the glass processed bodies 12 stored inside. Then, the platelets and coagulation factors consisted in the blood are coagulated on the surface of the processed glass body 12 , and from the platelets activated during the coagulation, growth factors derived therefrom are released.
  • this activation promoting step carried out at a low temperature is effective in the acceleration of platelet agglutination.
  • the needle for collecting blood 30 is removed from the subject of the blood collection, and then a part of the tube 41 connecting the needle for collecting blood 30 with the blood reservoir 10 is cut and fused, sealing its cut edge (fusion cutting step S 3 ) at the same time.
  • the fusion cutting step S 3 and the centrifugal separation step S 4 may be carried out prior to the activation promoting step S 2 .
  • the centrifugal separation may be conducted under the following conditions:
  • the factors to be activated including hemocyte components, which were activated in the activation promoting step S 2 through the centrifugal separation step S 4 , form a block shape and are separated from the blood (separation step S 5 ). Furthermore, the serum 71 separated and extracted in the blood reservoir 10 in the separation step S 5 is sequentially divided into all or some of the bags 21 to 26 by compressing the blood reservoir 10 (discharging step S 6 ).
  • the tube 51 is cut and sealed (fusion cutting step S 7 ).
  • This cutting and sealing may be performed using a method that is similar to the cutting and sealing of the tube 42 prior to the aforementioned centrifugal separation step S 4 .
  • the bag 21 having the serum contained therein is subjected to a storage treatment such as frozen storage.
  • the discharging step S 6 and fusion cutting step S 7 are carried out sequentially on each of the bags 21 to 26 , and the operation for serum preparation is stopped when the serum is contained in all or some of the bags 21 to 26 .
  • the erythrocytes may be washed and diluted, as needed, with physiological saline, or an anticoagulant such as CPD, or ACD-A liquid, or a liquid for preserving blood such as MAP, and can be stored as blood for transfusion.
  • Fresh human blood 20 (ml) was added to a vessel in which five glass beads ( ⁇ : 4 mm, 50 mm 2 ) were stored, and shaken by a Multi Shaker (MMS-300, manufactured by Tokyo Rikakikai Co., Ltd.). At 10, 20, 30 and 60 minutes after shaking was initiated, 1 (ml) of blood was collected in a sampling tube in which an anticoagulant was stored, and the blood was centrifuged to isolate a serum. In addition, CPD-added blood was collected from the same subject to isolate plasma. Calcium chloride was added to the isolated plasma so that fibrin was deposited at 37° C. and a serum was prepared.
  • the growth factors (PDGF-BB, TGF- ⁇ 1) were measured for both the serum prepared from the vessel in which glass beads were stored, and the serum prepared from the CPD-added blood. The results are illustrated in Table 1.
  • the amounts of the growth factors (TGF- ⁇ 1, PDGF-BB) were measured by a commercially available test kit manufactured by R&D SYSTEMS, Inc. using a microplate reader (Multiskan BICHROMATIC manufactured by Labsystem).
  • PDGF-BB in the serum prepared from the vessel in which five glass beads were contained, the content of PDGF-BB increased as time elapsed following the shaking, and 1677.2 (pg) of PDGF-BB was released per 1 ml of the serum after 60 minutes elapsed. In the serum prepared from the plasma of the same subject, the amount of PDGF-BB then was less than 32.5 (pg/ml) of the detection limit.
  • TGF- ⁇ 1 in the serum prepared from the vessel in which five glass beads were contained, 21.2 (ng) of TGF- ⁇ 1 had already been released per 1 (ml) after 10 minutes of shaking.
  • TGF- ⁇ 1 The amount released thereafter gradually increased in a time dependent manner, and 29.6 (ng) of TGF- ⁇ 1 was released after 60 minutes.
  • the amount of TGF- ⁇ 1 was 1.5 (ng) per 1 (ml) of the serum.
  • the following three types of vessels for collecting blood were prepared.
  • the first type of vessel for collecting blood (specimen 3 ) contained no glass beads therein
  • the second type of vessel for collecting blood (specimen 1 ) contained five glass beads therein
  • the third type of vessel for collecting blood (specimen 2 ) contained 20 polyethylene pellets therein.
  • 20 ml of fresh blood derived from the same subject was added to each of the vessels, and was shaken by a Multi Shaker (MMS-300, manufactured by Tokyo Rikakikai Co., Ltd.).
  • FIG. 4 illustrates a graph of a relationship between elapsed time following the shaking and the residual ratio of platelets in each specimen.
  • the residual ratio of platelets in specimen 1 was reduced to approximately 2% within 20 minutes after shaking had been initiated.
  • the residual ratio of platelets in specimen 2 was reduced within 30 minutes after shaking had been initiated, slightly behind specimen 1 .
  • the residual ratio of platelets in specimen 3 was reduced to less than 2% in 60 minutes after shaking had been initiated, although it was rapidly reduced immediately after shaking had been initiated.
  • FIG. 5 illustrates a graph of a relationship between elapsed time following the shaking and the release ratio of PDGF-BB in each of the specimens. It has been discovered that, as for specimen 1 , PDGF-BB was rapidly released in a time dependent manner, so that 90% of the potential amount in the serum was released within an hour after the shaking. As for the other two specimens, less than 20% of the amount of PDGF-BB to the potential amount in the serum was released, although the number of platelets had been reduced.
  • FIG. 6 illustrates a graph of a relationship between elapsed time following the shaking and the release ratio of TGF- ⁇ 1 in each of the specimens.
  • the serum in specimen 1 nearly 70% of the amount of TGF ⁇ 1 to the potential amount in the serum was released within ten minutes after the shaking.
  • the release ratio of the amount of TGF ⁇ 1 to the potential amount in the serum had remained approximately 40% within sixty minutes after the shaking.
  • the polyethylene pellet plays a role similar to the glass beads in terms of eliminating the platelets in the blood, but cannot activate the platelets.

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US20100254953A1 (en) * 2007-09-11 2010-10-07 Sapporo Medical University Cell growth method and pharmaceutical preparation for tissue repair and regeneration
US20120108455A1 (en) * 2010-09-08 2012-05-03 Lalitha Kodandapani Methods for assessing and identifying or evolving conditionally active therapeutic proteins

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ES2360782B1 (es) 2009-07-28 2012-03-12 Grifols, S.A. Medios para cultivo de células de mamíferos que comprenden sobrenadante de etapas del fraccionamiento de Cohn y uso de los mismos.
JP6452271B2 (ja) * 2012-12-13 2019-01-16 株式会社ジェイ・エム・エス 血清の調製方法
WO2016006782A1 (ko) * 2014-07-08 2016-01-14 라정찬 줄기세포의 보관 안정성 증진용 조성물
CN105769909A (zh) * 2016-05-13 2016-07-20 云南舜喜再生医学工程有限公司 一种直接获得富含细胞因子血清的采血器及方法
CN114146096B (zh) * 2021-11-25 2024-04-02 成都清科生物科技有限公司 一种富含细胞因子的条件血清的制备方法及应用

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US20100167402A1 (en) 2010-07-01
KR101016504B1 (ko) 2011-02-24
KR20100024522A (ko) 2010-03-05
EP1820852A1 (en) 2007-08-22
WO2006054448A1 (ja) 2006-05-26
JPWO2006054448A1 (ja) 2008-05-29
US8679838B2 (en) 2014-03-25
KR20070060148A (ko) 2007-06-12
CN101061218A (zh) 2007-10-24
CA2587885C (en) 2013-05-21
EP1820852A4 (en) 2007-11-14

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