JP2010130929A - Culture container for granular cultured bone - Google Patents

Culture container for granular cultured bone Download PDF

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JP2010130929A
JP2010130929A JP2008309065A JP2008309065A JP2010130929A JP 2010130929 A JP2010130929 A JP 2010130929A JP 2008309065 A JP2008309065 A JP 2008309065A JP 2008309065 A JP2008309065 A JP 2008309065A JP 2010130929 A JP2010130929 A JP 2010130929A
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JP5099781B2 (en
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Hideaki Kagami
秀明 各務
Hideki Agata
秀樹 縣
Yusuke Hori
祐輔 堀
Satoshi Oshima
聡志 大島
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TES Holdings Co Ltd
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
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    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/48—Holding appliances; Racks; Supports
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/16—Particles; Beads; Granular material; Encapsulation

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Abstract

【課題】 本発明は顆粒型培養骨を効率的に製造することのできる顆粒型培養骨用培養容器を提供する。
【解決手段】 顆粒型培養骨を培養する培養チューブが、ぐらつかないようにチューブ立てに立てられている培養容器であって、該培養チューブは高さが７０ｍｍ以上の深底容器であり、かつ、底部より上部開口に至る誘導溝を備え、チューブの中に滅菌された多孔質擬似骨顆粒が入っていることを特徴とする顆粒型培養骨用培養容器。
【選択図】 図２PROBLEM TO BE SOLVED: To provide a culture vessel for granule type cultured bone capable of efficiently producing granule type cultured bone.
A culture tube for culturing granular-type cultured bone is set up on a tube stand so as not to wobble, and the culture tube is a deep bottom container having a height of 70 mm or more, and A culture vessel for granule-type cultured bone, comprising a guide groove extending from the bottom to the upper opening and containing a sterilized porous pseudo-bone granule in the tube.
[Selection] Figure 2

Description

本発明は、顆粒型培養骨用培養容器、特に顆粒型培養骨を効率的に製造することのできる培養容器に関する。   The present invention relates to a culture container for granulated cultured bone, and more particularly to a culture container capable of efficiently producing granulated cultured bone.

歯科口腔外科領域などで認められる、比較的小さく複雑な形態の骨欠損を回復する際には、ブロックタイプの培養骨よりも顆粒型培養骨の方が骨再生材料として適切である。この顆粒型培養骨を製造する際には、シャーレ等の中に顆粒を手作業で入れて、その上に細胞を播種、培養する方法が一般的である。さらに、１５ｍｌ程度の容量の遠心用チューブに手作業で擬似骨顆粒を入れ、その上に細胞を播種し、遠心を行う方法も用いられており、このチューブは汎用されているチューブラックに立てかけて使用されている。（非特許文献１）。
しかしながら、前記のチューブおよびチューブ立ては顆粒型人工骨用に設計されておらず、静電気等による顆粒の飛散、取り出し時の顆粒の分散など操作性の悪さと、作業中のコンタミネーションの可能性など、実際に医療現場で培養し、施術に用いるには不適当であった。
Matsubara et al. J Bone Miner Res. 20,3(2005), 399-409 When recovering relatively small and complicated bone defects found in the field of dental and oral surgery, granular cultured bone is more suitable as a bone regeneration material than block-type cultured bone. When producing this granule-type cultured bone, a method is generally employed in which granules are manually placed in a petri dish or the like, and cells are seeded and cultured thereon. Furthermore, a method is also used in which pseudo bone granules are manually placed in a centrifuge tube having a capacity of about 15 ml, cells are seeded thereon, and centrifugation is performed. This tube is placed against a commonly used tube rack. in use. (Non-Patent Document 1).
However, the above tubes and tube stands are not designed for granule-type artificial bones, and are poor in operability such as scattering of granules due to static electricity, dispersion of granules during removal, and possibility of contamination during work, etc. In fact, it was unsuitable for culturing at a medical site and using it for treatment.
Matsubara et al. J Bone Miner Res. 20,3 (2005), 399-409

本発明は上記従来技術に鑑み行われたものであり、その解決すべき課題は顆粒型培養骨を効率的に製造することのできる顆粒型培養骨用培養容器を提供することにある。   The present invention has been made in view of the above prior art, and a problem to be solved is to provide a culture vessel for granulated cultured bone which can efficiently produce granulated cultured bone.

前記課題を解決するために本発明者らが鋭意研究を行った結果、深底培養容器に誘導溝を備えた培養チューブおよびチューブ立てを備え、培養チューブ内に滅菌された多孔質擬似骨顆粒を入れることにより、培養チューブに均等に振動を与えられ、かつ静置を行うことができ、効率的に顆粒型培養骨を製造できることを見出し、本発明を完成するに至った。   As a result of intensive studies conducted by the present inventors to solve the above-mentioned problems, a porous pseudo-bone granule having a culture tube and a tube stand provided with a guide groove in a deep-bottom culture vessel and sterilized in the culture tube is obtained. It has been found that, by inserting, the culture tube can be evenly vibrated and can be allowed to stand, and granule-type cultured bone can be efficiently produced, and the present invention has been completed.

すなわち本発明の顆粒型培養骨用培養容器は、顆粒型培養骨を培養する培養チューブが、顆粒型培養骨を培養する培養チューブが、ぐらつかないようにチューブ立てに立てられている培養容器であって、該培養チューブは高さが７０ｍｍ以上の深底容器であり、かつ、底部より上部開口に至る誘導溝を備え、チューブの中に滅菌された多孔質擬似骨顆粒が入っていることを特徴とする。
前記多孔質擬似骨顆粒は乾燥時みかけ比重が培養液よりも小さく、培養液中で該培養液を吸液し、沈降する多孔質体であることが好適である。
前記多孔質擬似骨顆粒は培養チューブの培養液中を１分以上、３０分以下で沈降することが好適である。
前記多孔質擬似骨顆粒がβ−リン酸三カルシウム（β−ＴＣＰ）、ハイドロキシアパタイト、乾燥骨粉を含む多孔質生体吸収性顆粒より選択される一種または二種以上であることが好適である。
前記容器は、細胞を播種する工程において、培養液投入の際に２０〜１２０Ｈｚでの振動播種に用いられることが好適である。 That is, the culture vessel for granule-type cultured bone of the present invention is a culture vessel in which a culture tube for culturing granule-type cultured bone is erected on a tube stand so that the culture tube for culturing granule-type cultured bone does not wobble. The culture tube is a deep-bottomed container having a height of 70 mm or more, has a guide groove extending from the bottom to the upper opening, and contains a sterilized porous pseudo-bone granule in the tube. And
The porous pseudo-bone granule is preferably a porous body that has an apparent specific gravity when dried that is smaller than that of the culture solution and absorbs the culture solution in the culture solution and settles.
It is preferable that the porous pseudo-bone granule settles in the culture solution of the culture tube in 1 minute or more and 30 minutes or less.
The porous pseudo bone granule is preferably one or more selected from porous bioabsorbable granules including β-tricalcium phosphate (β-TCP), hydroxyapatite, and dry bone powder.
In the step of seeding cells, the container is preferably used for vibration seeding at 20 to 120 Hz when the culture solution is added.

本発明によれば、すべてのチューブに均等に振動および静置を与えられる顆粒型培養骨用培養容器を得ることができる。また、滅菌された多孔質擬似骨顆粒が入っている該培養容器により、顆粒型培養骨を効率的に製造することができる。   According to the present invention, it is possible to obtain a granule-type cultured bone culture container in which all tubes are equally vibrated and allowed to stand. Moreover, granule-type cultured bone can be efficiently produced by the culture container containing the sterilized porous pseudo-bone granule.

以下、本発明の好適な実施形態について説明する。はじめに、本発明の培養容器により効率的に製造が可能である顆粒型培養骨について説明する。顆粒型培養骨は、骨髄液の採取、骨髄由来間葉系幹細胞の培養、間葉系幹細胞の播種、培養骨芽細胞様細胞への分化誘導を行うことにより製造することができる。
なお、以下の工程は、骨髄液を用いた形態を示しているが、本発明の培養容器は、骨髄液以外にも、骨膜、脂肪、末梢血等から分離培養し、同様の方法で培養骨芽細胞様細胞へと分化誘導することによって作製される顆粒型培養骨においても、好適に使用することができる。 Hereinafter, preferred embodiments of the present invention will be described. First, the granular cultured bone that can be efficiently produced by the culture container of the present invention will be described. Granular cultured bone can be produced by collecting bone marrow fluid, culturing bone marrow-derived mesenchymal stem cells, seeding mesenchymal stem cells, and inducing differentiation into cultured osteoblast-like cells.
The following steps show a form using bone marrow fluid, but the culture container of the present invention is cultured separately from periosteum, fat, peripheral blood, etc. in addition to bone marrow fluid, and cultured bones in the same manner. It can also be suitably used in granulated cultured bone produced by inducing differentiation into blast-like cells.

・骨髄液の採取
骨髄液は、手術の約１ヶ月前に採取する。まず、骨髄液採取部に麻酔を行い、後上腸骨陵より無菌的に吸引して回収する。 • Bone marrow fluid collection Bone marrow fluid should be collected approximately one month before surgery. First, the bone marrow fluid collection part is anesthetized and aseptically sucked and collected from the upper superior iliac crest.

・骨髄由来間葉系幹細胞の培養
細胞培養用培地で４倍希釈した骨髄液１０を細胞培養用フラスコ１２に播いて、図１（Ａ）のように炭酸ガス培養を始め、培養開始後４日目に全量培地替えを行う。なお、細胞培養用培地には、一次培養、分化培養ともに、血清入りαＭＥＭまたは無血清培地のいずれも使用できる。
培養過程における一般細菌および真菌に感染していないことの確認は、培地交換毎の培地の観察（感染していれば培地が濁る）により行うとともに、培養開始時、分化誘導前および培養骨芽細胞様細胞の回収時に無菌試験（日本薬局方の基準を満たすかどうか）で確認する。
その後、週２回全量培地交換を行う。継代のタイミングは細胞の状態を見て判断するが、培養開始から約２１〜２８日後に行う。継代時には生細胞数および細胞数を計測する。 -Cultivation of bone marrow-derived mesenchymal stem cells Bone marrow fluid 10 diluted 4-fold with cell culture medium is seeded in cell culture flask 12, and carbon dioxide culture is started as shown in FIG. Change the whole medium to the eyes. As the cell culture medium, either serum-containing αMEM or serum-free medium can be used for both primary culture and differentiation culture.
Confirmation that there is no infection with general bacteria and fungi during the culturing process is made by observing the medium every time the medium is exchanged (if the medium is infected, the medium becomes cloudy), and at the start of culture, before differentiation induction, and cultured osteoblasts Confirm the sterility test (whether it meets the standards of the Japanese Pharmacopoeia) at the time of recovery of the cells.
Thereafter, the whole medium is changed twice a week. The subculture timing is determined based on the state of the cells, but is about 21 to 28 days after the start of culture. At the time of passage, the number of living cells and the number of cells are counted.

・間葉系幹細胞の播種
継代は以下のように行う。フラスコの培地を吸い取った後、ダルベコリン酸バッファー（Ｄ−ＰＢＳ）にて洗浄し、その後、Ｄ−ＰＢＳを吸い取り、細胞解離剤を加え、３７℃で１０分間培養する。細胞がはがれていることを確認したのち、Ｄ−ＰＢＳまたは培地を加え細胞を回収した後、遠心する。培地に再サスペンドして細胞数を計測する。計測後、図１（Ｂ）のような多孔質擬似骨顆粒１４があらかじめ入っている培養チューブ１６に、培養した骨髄液１０を投入する。５０ｍｇの多孔質擬似骨顆粒１４に対し、100,000〜1,000,000の細胞を播種するが、500,000程度の細胞を播種することが好ましい。多孔質擬似骨顆粒１４の入った培養チューブ１６に、培養した骨髄液１０を投入すると、図１（Ｃ）のように細胞は浮遊し、多孔質擬似骨顆粒１４は浮揚する。
培養した骨髄液１０の投入の際には、培養チューブ１６に緩やかな振動を与えることが好適である。培養チューブ１６に加える振動数を調整することにより、細胞と擬似骨顆粒１４との沈降速度を適合させ、擬似骨顆粒１４上に細胞が適切に播種することができる。特に２０〜１２０Ｈｚ程度の振動をバイオシェーカー等により与えることが好適であるが、その他の機器を用いて同等の振幅を与えてもよい。全く振動を与えないと、擬似骨顆粒が培養液と分離してしまい、沈降に長い時間を有する場合がある。 ・ Seeding of mesenchymal stem cells Passage is performed as follows. After sucking up the culture medium in the flask, it is washed with dulbecolinic acid buffer (D-PBS), and then D-PBS is sucked out and a cell dissociating agent is added, followed by incubation at 37 ° C. for 10 minutes. After confirming that the cells are peeled off, add D-PBS or a medium to collect the cells, and then centrifuge. Resuspend in medium and count cells. After the measurement, the cultured bone marrow fluid 10 is put into a culture tube 16 in which a porous pseudo bone granule 14 as shown in FIG. Although 100,000 to 1,000,000 cells are seeded on 50 mg of the porous pseudo-bone granule 14, it is preferable to seed approximately 500,000 cells. When the cultured bone marrow fluid 10 is put into the culture tube 16 containing the porous pseudo bone granules 14, the cells float as shown in FIG. 1C, and the porous pseudo bone granules 14 float.
When the cultured bone marrow fluid 10 is introduced, it is preferable to give a gentle vibration to the culture tube 16. By adjusting the frequency applied to the culture tube 16, the sedimentation speed of the cells and the pseudo bone granules 14 can be adjusted, and the cells can be appropriately seeded on the pseudo bone granules 14. In particular, it is preferable to give a vibration of about 20 to 120 Hz by a bioshaker or the like, but an equivalent amplitude may be given using other equipment. If no vibration is applied at all, the pseudo-bone granule separates from the culture solution and may have a long time for sedimentation.

・培養骨芽細胞様細胞への分化誘導
多孔質擬似骨顆粒１４に細胞を播種してから細胞の機能回復のために一晩静置する。１日後には、図１（Ｄ）のように骨形成に関与する細胞等の細胞および多孔質擬似骨顆粒１４は培養チューブ１６の下部に沈んでいる。このことを確認した後、培地を分化誘導培地に交換する。分化誘導期間は１〜３週間で、培地交換は週２回程度行う。分化誘導が進んでいくと、図１（Ｅ）のように、擬似骨顆粒１４のまわりに培養骨芽細胞様細胞が集まっていく。
分化確認試験（ＡＬＰ活性測定）を行い、分化していることを確認して、培養骨芽細胞様細胞を得る。 -Differentiation induction into cultured osteoblast-like cells Cells are seeded on the porous pseudo-bone granule 14 and then allowed to stand overnight to recover the function of the cells. After one day, as shown in FIG. 1D, cells such as cells involved in bone formation and the porous pseudo-bone granule 14 are submerged in the lower part of the culture tube 16. After confirming this, the medium is replaced with a differentiation-inducing medium. The differentiation induction period is 1 to 3 weeks, and the medium is changed about twice a week. As differentiation induction proceeds, cultured osteoblast-like cells gather around the pseudo-bone granule 14 as shown in FIG.
A differentiation confirmation test (ALP activity measurement) is performed to confirm differentiation, and cultured osteoblast-like cells are obtained.

本発明の顆粒型培養骨用培養容器は、多孔質擬似骨顆粒に培養した間葉系幹細胞を播種し、分化誘導を行い、培養骨芽細胞様細胞を得る際に使用される。図２、図３は本発明の顆粒型培養骨用培養容器１８を示した図である。本発明における顆粒型培養骨用培養容器１８は、培養チューブ１６およびチューブ立て２０から構成される。   The granule-type cultured bone culture container of the present invention is used when seeding mesenchymal stem cells cultured on porous pseudo-bone granules, inducing differentiation, and obtaining cultured osteoblast-like cells. 2 and 3 are views showing the granule-type cultured bone culture vessel 18 of the present invention. The culture vessel 18 for granulated bone according to the present invention is composed of a culture tube 16 and a tube stand 20.

培養チューブ
本発明の培養チューブ１６は、樹脂、ガラスなど通常チューブに用いられる材料からなる深底容器である。
すなわち、本実施形態にかかる培養チューブ１６は、図４に示すように、円筒状深底容器であり、その容量は１０〜２０ｍｌ、高さは７０ｍｍ〜１５０ｍｍであることが好適である。高さがあまりに低いと擬似骨顆粒による細胞担持が十分に行われない場合がある。また、高さがあまりに高いと取り扱い性が悪く、特に培養終了時に培養骨の取り出しが困難となる場合がある。 Culture Tube The culture tube 16 of the present invention is a deep bottom container made of a material usually used for tubes such as resin and glass.
That is, as shown in FIG. 4, the culture tube 16 according to the present embodiment is a cylindrical deep bottom container, and preferably has a capacity of 10 to 20 ml and a height of 70 mm to 150 mm. If the height is too low, cell support by pseudo bone granules may not be performed sufficiently. Further, if the height is too high, the handleability is poor, and it may be difficult to take out the cultured bone particularly at the end of the culture.

また、本実施形態にかかる培養チューブ１６は、その側壁内面に底部より上部開口に至る誘導溝２２を有している。誘導溝２２の幅は擬似骨顆粒の量により左右されるが、５０ｍｇの擬似骨顆粒では３〜５ｍｍであることが好適である。
培養終了時には、培養骨は骨芽細胞等が出す細胞間物質（膠原線維等）により粘着性を有しており、過度の撹拌は細胞にストレスを与えることとなる。また、取り出し後の培養骨塊は、そのまま施術に用いられるよう、塊状を維持していることが好ましい。このため、培養骨塊をばらけさせることなく取り出しやすいように、誘導溝２２を設けているのである。 Moreover, the culture tube 16 concerning this embodiment has the induction | guidance | derivation groove | channel 22 which reaches the upper opening from the bottom part in the side wall inner surface. The width of the guide groove 22 depends on the amount of pseudo bone granules, but it is preferably 3 to 5 mm for 50 mg pseudo bone granules.
At the end of the culture, the cultured bones are sticky due to intercellular substances (collagen fibers etc.) produced by osteoblasts and the like, and excessive stirring gives stress to the cells. Moreover, it is preferable that the cultured bone lump after taking out maintains the lump shape so that it may be used for the treatment as it is. For this reason, the guide groove 22 is provided so that the cultured bone mass can be easily taken out without being separated.

また、本発明の培養チューブ１６の中には、あらかじめ高圧蒸気により滅菌された多孔質擬似骨顆粒１４が入っている。この多孔質擬似骨顆粒１４は、乾燥時みかけ比重が培養液よりも小さい擬似骨顆粒を好適に使用することができる。すなわち、多孔質擬似骨顆粒１４は、β−リン酸三カルシウム（β−ＴＣＰ）、ハイドロキシアパタイト、乾燥骨粉ないしこれらの混合物等であることが好適であり、β−リン酸三カルシウムであることが特に好適である。β−リン酸三カルシウムは市販品としてアパセラム、ボーンセラム、ボーンフィル、セラタイト、骨補填剤オスフェリオン（オリンパステルモバイオマテリアル社）などとして入手することができる。本発明の培養容器で製造する培養骨は、β−リン酸三カルシウムを骨補填剤としてではなく細胞の足場として検討している。β−リン酸三カルシウムの顆粒径は、０．１〜１．５ｍｍであることが好適である。   The culture tube 16 of the present invention contains porous pseudo-bone granules 14 sterilized by high-pressure steam in advance. As the porous pseudo-bone granule 14, a pseudo-bone granule having an apparent specific gravity when dried that is smaller than that of the culture solution can be preferably used. That is, the porous pseudo-bone granule 14 is preferably β-tricalcium phosphate (β-TCP), hydroxyapatite, dry bone meal or a mixture thereof, and is β-tricalcium phosphate. Particularly preferred. β-tricalcium phosphate can be obtained as a commercially available product as apaterum, bone serum, bone fill, seratite, bone filler Osferion (Olympel Termo Biomaterials), and the like. The cultured bone produced in the culture container of the present invention is examined using β-tricalcium phosphate as a cell scaffold, not as a bone filler. The granule diameter of β-tricalcium phosphate is preferably 0.1 to 1.5 mm.

本発明にかかる顆粒型培養骨用培養容器１８は、顆粒型培養骨製造の際の間葉系幹細胞を播種する工程において使用されるが、多孔質擬似骨顆粒１４は、培養チューブ１６内で滅菌された乾燥状態にある。ここに培養した骨髄液１０を投入すると、図１（Ｃ）に示すように、多孔質擬似骨顆粒１４が培養液中に浮揚する。その後、多孔質擬似骨顆粒１４は培養液を吸液し、徐々に沈降し、図１（Ｄ）に示すように沈殿する。このように、多孔質擬似骨顆粒１４を一時的に浮揚状態とし、その後数分間かけて沈降させることにより、多孔質擬似骨顆粒１４への細胞付着が極めて効率的に行われ、培養骨の収率および骨再生確率の大幅な向上が可能となる。   The culture vessel 18 for granule-type cultured bone according to the present invention is used in the step of seeding mesenchymal stem cells in the production of granule-type cultured bone, but the porous pseudo-bone granule 14 is sterilized in the culture tube 16. In a dry state. When the cultured bone marrow fluid 10 is added here, as shown in FIG. 1 (C), the porous pseudo-bone granule 14 floats in the culture fluid. Thereafter, the porous pseudo-bone granule 14 absorbs the culture solution, gradually settles, and settles as shown in FIG. As described above, the porous pseudo-bone granule 14 is temporarily floated and then allowed to settle for several minutes, so that the cell attachment to the porous pseudo-bone granule 14 is performed extremely efficiently, and the cultured bone is collected. The rate and bone regeneration probability can be greatly improved.

多孔質擬似骨顆粒１４に浮遊状態の幹細胞を高密度で担持させるため、培養チューブ１６は７０ｍｍ以上の深さを有することが好適であり、また、多孔質擬似骨顆粒１４は、培養液中を１〜３０分程度で沈降することが好ましい。沈降速度があまりに速いと、細胞担持が十分に行われない場合があり、沈降速度があまりに遅いと、長時間の浮遊による細胞活性の低下が大きくなる場合がある。   The culture tube 16 preferably has a depth of 70 mm or more in order to carry the suspended stem cells at a high density in the porous pseudo-bone granule 14, and the porous pseudo-bone granule 14 is contained in the culture solution. It is preferable to settle in about 1 to 30 minutes. If the sedimentation rate is too high, cell loading may not be performed sufficiently, and if the sedimentation rate is too slow, the decrease in cell activity due to floating for a long time may increase.

本発明にかかる顆粒型培養骨用培養容器１８には、培養チューブ１６にあらかじめ滅菌された多孔質擬似骨顆粒１４が入っていることにより、培養骨を製造する際に、容器および顆粒を滅菌する手間がかからなくなり、顆粒型培養骨を効率的に製造することができる。また、チューブ用キャップ２４を備えることにより、容易に顆粒型培養骨を移動および保存することができる。   The granule-type cultured bone culture container 18 according to the present invention contains a porous pseudo-bone granule 14 sterilized in advance in a culture tube 16 so that the container and the granule are sterilized when the cultured bone is manufactured. Eliminates time and effort, and can efficiently produce granule-type cultured bone. In addition, by providing the tube cap 24, the granular cultured bone can be easily moved and stored.

チューブ立て
本発明のチューブ立て２０は、チューブ立て下部２６、チューブ立て用フタ２８からなり、各種プラスチックなど通常チューブ立てに用いられる材料からなっている。チューブ立て２０に収容できる培養チューブ１６の数は特に限定はないが１〜２４本収容できることが好適である。
チューブ立て下部２６は本発明の培養チューブ１６の形状に合わせて作られており、培養チューブ１６を固定することができる。このため、培養液投入の際の振動を均等に与えることも可能であるし、静置も容易にすることができる。 Tube Stand The tube stand 20 of the present invention includes a tube stand lower portion 26 and a tube stand lid 28, and is made of a material normally used for tube stands such as various plastics. The number of the culture tubes 16 that can be accommodated in the tube stand 20 is not particularly limited, but it is preferable that 1 to 24 culture tubes 16 can be accommodated.
The tube stand lower part 26 is made according to the shape of the culture tube 16 of the present invention, and can fix the culture tube 16. For this reason, it is possible to give the vibration at the time of culture | cultivation liquid addition equally, and can also leave still easily.

顆粒型培養骨製造方法の概略工程図。The schematic process drawing of a granule type cultured bone manufacturing method. 本発明の顆粒型培養骨用培養容器の構成図。The block diagram of the culture container for granule type culture bones of this invention. 顆粒型培養骨用培養容器を横から見た図。The figure which looked at the culture container for granule type culture bones from the side.

符号の説明Explanation of symbols

１０…骨髄液
１２…細胞培養用フラスコ
１４…多孔質擬似骨顆粒
１６…培養チューブ
１８…顆粒型培養骨用培養容器
２０…チューブ立て
２２…誘導溝
２４…チューブ用キャップ
２６…チューブ立て下部
２８…チューブ立て用フタ DESCRIPTION OF SYMBOLS 10 ... Bone marrow fluid 12 ... Cell culture flask 14 ... Porous pseudo-bone granule 16 ... Culture tube 18 ... Granule type culture bone culture container 20 ... Tube stand 22 ... Guide groove 24 ... Tube cap 26 ... Tube stand lower part 28 ... Tube stand lid

Claims (5)

顆粒型培養骨を培養する培養チューブが、ぐらつかないようにチューブ立てに立てられている培養容器であって、
該培養チューブは高さが７０ｍｍ以上の深底容器であり、かつ、底部より上部開口に至る誘導溝を備え、チューブの中に滅菌された多孔質擬似骨顆粒が入っていることを特徴とする顆粒型培養骨用培養容器。 A culture tube for culturing granule-type cultured bone is a culture container that is set up on a tube stand so as not to wobble,
The culture tube is a deep-bottomed container having a height of 70 mm or more, has a guide groove extending from the bottom to the upper opening, and contains sterilized porous pseudo-bone granule in the tube. Granule-type cultured bone culture container. 多孔質擬似骨顆粒は乾燥時みかけ比重が培養液よりも小さく、培養液中で該培養液を吸液し、沈降する多孔質体であることを特徴とする請求項１に記載の顆粒型培養骨用培養容器。   2. The granule-type culture according to claim 1, wherein the porous pseudo-bone granule is a porous body that has an apparent specific gravity when dried that is smaller than that of the culture solution, and absorbs and precipitates the culture solution in the culture solution. Bone culture container. 多孔質擬似骨顆粒は培養チューブの培養液中を１分以上、３０分以下で沈降することを特徴とする請求項１または２に記載の顆粒型培養骨用培養容器。   The culture vessel for granule-type cultured bone according to claim 1 or 2, wherein the porous pseudo-bone granule settles in the culture solution of the culture tube in 1 minute or more and 30 minutes or less. 多孔質擬似骨顆粒がβ−リン酸三カルシウム（β−ＴＣＰ）、ハイドロキシアパタイト、乾燥骨粉を含む多孔質生体吸収性顆粒より選択される一種または二種以上であることを特徴とする請求項１〜３のいずれかに記載の顆粒型培養骨用培養容器。   The porous pseudo bone granule is one or more selected from porous bioabsorbable granules including β-tricalcium phosphate (β-TCP), hydroxyapatite, and dry bone powder. The culture vessel for granule-type cultured bone according to any one of to 3. 細胞を播種する工程において、培養液投入の際に２０〜１２０Ｈｚでの振動播種に用いられることを特徴とする請求項１〜４のいずれかに記載の顆粒型培養骨用培養容器。   5. The granule-cultured bone culture container according to any one of claims 1 to 4, which is used for vibration seeding at 20 to 120 Hz when the culture solution is introduced in the step of seeding cells.
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JPH0956369A (en) * 1995-08-24 1997-03-04 Millennium Biologics Inc Multiwell bone cell culture device
JP2004129512A (en) * 2002-10-08 2004-04-30 Olympus Corp Vessel
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JPH0956369A (en) * 1995-08-24 1997-03-04 Millennium Biologics Inc Multiwell bone cell culture device
JP2004129512A (en) * 2002-10-08 2004-04-30 Olympus Corp Vessel
JP2005160596A (en) * 2003-11-28 2005-06-23 National Institute Of Advanced Industrial & Technology Biomaterial pretreatment method and application
JP2006006546A (en) * 2004-06-24 2006-01-12 Olympus Corp Bone prosthetic material, bone prosthesis and method of manufacturing the same
JP2007135503A (en) * 2005-11-21 2007-06-07 Olympus Corp Cultured bone transport container

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014236673A (en) * 2013-06-06 2014-12-18 オリンパス株式会社 Osteogenesis promoter and production method thereof

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