JP2005229906A - Method for storing cell-seeded base material - Google Patents
Method for storing cell-seeded base materialInfo
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Abstract
Description
本発明は、患者から採取した骨髄細胞、又は、骨髄細胞から分化若しくは誘導させてできた細胞を組織再生用基材に播種した細胞播種基材を、高い活性を維持したまま一時的に保管する細胞播種基材の保管方法に関する。 The present invention temporarily stores bone marrow cells collected from a patient or a cell seeding base material in which cells obtained by differentiation or induction from bone marrow cells are seeded on a tissue regeneration base material while maintaining high activity. The present invention relates to a cell seeding substrate storage method.
近年の細胞工学技術の進展によって、数々の動物細胞の単離や培養が可能となり、また、それらの細胞を用いてヒトの組織や器官を再構築しようとする、いわゆる再生医療の研究が急速に進んでいる。再生医療においては、播種した細胞が増殖分化して三次元的な生体組織様の構造物を構築できるかがポイントであり、そのためには、細胞を効率よく三次元的に培養できる組織再生用基材に播種して細胞播種基材を作製し、その後直ちに又は一定期間培養した後、該細胞播種基材を患者に移植することが行われている。 Recent advances in cell engineering technology have enabled the isolation and culture of numerous animal cells, and rapid research into so-called regenerative medicine that uses these cells to reconstruct human tissues and organs. Progressing. In regenerative medicine, the point is that the seeded cells can proliferate and differentiate to construct a three-dimensional biological tissue-like structure. For this purpose, a tissue regeneration substrate that can efficiently cultivate cells in three dimensions. A cell seeding base material is prepared by seeding on a material, and then the cell seeding base material is transplanted to a patient immediately or after culturing for a certain period of time.
再生医療においては、ヒトの免疫反応による拒絶をいかに防ぐかが大きな課題となっているが、選択肢の一つとして患者自身の細胞（自家細胞）を用いる検討がなされている。骨髄細胞は、患者の骨髄液から比較的容易に採取できることに加え、血管内皮細胞、軟骨細胞、心筋細胞、皮膚細胞等の種々の細胞に分化できることから、その応用が試みられている。なかでも、先天性心疾患の治療、又は、狭窄や血栓等により閉塞してしまった血管の置換等に用いる再生血管は、近年の再生医療の重要なテーマの１つとして注目されており、極めて実用化に近いといえる。 In regenerative medicine, how to prevent rejection due to human immune reaction has become a major issue, but studies using patient's own cells (autologous cells) as an option have been made. Bone marrow cells can be collected from a patient's bone marrow fluid relatively easily, and can be differentiated into various cells such as vascular endothelial cells, chondrocytes, cardiomyocytes, skin cells, and so on, so that application has been attempted. Among these, regenerative blood vessels used for the treatment of congenital heart disease or the replacement of blood vessels that have been occluded due to stenosis or thrombus have attracted attention as one of the important themes of regenerative medicine in recent years. It can be said that it is close to practical use.
再生血管等の細胞播種基材を用いた治療では、通常、まず患者の腸骨から骨髄液を採取し、得られた骨髄液を遠心分離することにより骨髄細胞を含む細胞分画、血清、赤血球及び勾配液に分離することを行う。次いで、単離した細胞分画を組織再生用基材上に播種して細胞播種基材を作製する。 In treatment using a cell seeding substrate such as regenerative blood vessels, usually, bone marrow fluid is first collected from the iliac bone of the patient, and the resulting bone marrow fluid is centrifuged to obtain a cell fraction containing bone marrow cells, serum, red blood cells And separating into a gradient liquid. Next, the isolated cell fraction is seeded on a tissue regeneration substrate to produce a cell seeding substrate.
ここで、得られた細胞播種基材を手術に供するまで保管しておく必要が生じる。この保管時間は、手術の進行状況によっても異なるが、通常は１〜３時間程度、場合によっては６時間程度に達することもある。しかしながら、この間の細胞播種基材の保管方法については特に検討されたこともなく、せいぜい表面が乾燥しないように、生理的食塩水を染み込ませたガーゼにくるむか、バッファー溶液や市販の培養液中に浸漬した状態で静置しているのが現状であった。
しかしながら、本発明者らが検討したところ、この保管時間中における細胞播種基材の保管方法が、治療の成功率に極めて重大な影響を与えることがわかった。
Here, it is necessary to store the obtained cell seeding substrate until it is used for surgery. This storage time varies depending on the progress of the operation, but is usually about 1 to 3 hours, and in some cases may reach about 6 hours. However, the method for storing the cell seeding substrate during this period has not been particularly studied, and it can be wrapped in gauze soaked with physiological saline so that the surface does not dry at all, or in a buffer solution or a commercially available culture solution. It was the present condition that it was allowed to stand in a state immersed in water.
However, when the present inventors examined, it turned out that the storage method of the cell seeding base material during this storage time has a very serious influence on the success rate of treatment.
本発明は、上記現状に鑑み、組織再生用基材上に患者から採取した骨髄細胞、又は、骨髄細胞から分化若しくは誘導させてできた細胞、例えば、間葉系幹細胞、造血幹細胞等を播種した細胞播種基材を、高い活性を維持したまま一時的に保管する細胞播種基材の保管方法を提供することを目的とする。 In view of the current situation, the present invention seeded bone marrow cells collected from a patient on a tissue regeneration substrate, or cells obtained by differentiation or induction from bone marrow cells, such as mesenchymal stem cells, hematopoietic stem cells, and the like. It is an object of the present invention to provide a cell seeding substrate storage method for temporarily storing a cell seeding substrate while maintaining high activity.
本発明は、患者から採取した骨髄細胞、又は、骨髄細胞から分化若しくは誘導させてできた細胞を組織再生用基材に播種した細胞播種基材を保管する方法であって、前記患者から採取した骨髄液由来の血清中に前記細胞播種基材を浸漬する細胞播種基材の保管方法である。
以下に本発明を詳述する。
The present invention is a method for storing a bone marrow cell collected from a patient, or a cell seeding substrate obtained by seeding a cell obtained by differentiation or induction from a bone marrow cell on a tissue regeneration substrate, which is collected from the patient. This is a method for storing a cell seeding substrate, wherein the cell seeding substrate is immersed in serum derived from bone marrow fluid.
The present invention is described in detail below.
本発明者らは、検討の結果、細胞播種基材を手術に供するまでの保管期間中に、播種して接着させた細胞の多くが組織再生用基材から剥離してしまうことを見出した。患者に移植した細胞播種基材が生着し、正常な組織、器官を再構築するためには、充分な数の細胞が細胞播種基材中に存在することが重要であり、これが治療の成功を左右するといっても過言ではない。ましてや、患者から採取可能な骨髄液は限られており、そこから分離した骨髄細胞をできる限り有効に利用すべきであることはいうまでもない。
本発明者らは、更に鋭意検討の結果、患者から採取した骨髄液に由来する血清中に細胞播種基材を浸漬しておけば、通常の手術にかかる１〜６時間程度の間、細胞の組織再生用基材からの剥離を最小限に抑制し、治療効果の高い組織を供することが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 the study, the present inventors have found that many of the cells seeded and adhered during the storage period until the cell seeding substrate is subjected to surgery are detached from the tissue regeneration substrate. In order for the cell seeding substrate transplanted to the patient to engraft and reconstruct normal tissues and organs, it is important that a sufficient number of cells be present in the cell seeding substrate, which is a successful treatment. It's not an exaggeration to say that it affects Needless to say, bone marrow fluids that can be collected from patients are limited, and bone marrow cells separated therefrom should be used as effectively as possible.
As a result of further intensive studies, the present inventors have soaked the cell seeding substrate in serum derived from bone marrow fluid collected from a patient, and the cells have been subjected to normal surgery for about 1 to 6 hours. It has been found that peeling from the tissue regeneration substrate can be suppressed to a minimum and a tissue having a high therapeutic effect can be provided, and the present invention has been completed.
本発明の保管方法では、細胞播種基材を、患者から採取した骨髄液由来の血清中に浸漬する。従来から、細胞保存成分としては、各種の合成培養液をそのまま用いたり、ウシ胎児をはじめとする異種動物の血清や、ＶＥＧＦ、ＨＧＦ、ｂＦＧＦ等のタンパク質性の細胞成長因子を添加した培養液を用いたりすることが知られているが、患者自身の骨髄液由来の血清を用いれば、これらの細胞保存成分を用いた場合と比較しても顕著に高い細胞の維持率を達成できる。従来、骨髄細胞を分離した後の骨髄液は廃棄する以外に使い道がなかったが、本発明では、かかる廃棄される骨髄液に含まれる血清を有効利用して、細胞播種基材を保存する。この方法によれば、患者から別に血液を採取する必要がなく、患者に与える負担を最小限に抑えることができる。更に、他種又は他家由来の生体成分やタンパク質性成分を用いた場合のような免疫反応による拒絶や、プリオン等のタンパク成分やウィルス等による感染等の生物汚染を防止することができる。 In the storage method of the present invention, the cell seeding substrate is immersed in serum derived from bone marrow fluid collected from a patient. Conventionally, as a cell preservation component, various synthetic culture solutions can be used as they are, or a culture solution to which serum from a heterologous animal such as a fetal bovine or a proteinous cell growth factor such as VEGF, HGF, or bFGF is added. Although it is known that a serum derived from the bone marrow of the patient's own is used, a remarkably high cell maintenance rate can be achieved even when these cell preservation components are used. Conventionally, the bone marrow fluid after separating the bone marrow cells had no use other than discarding. In the present invention, the cell seeding base material is preserved by effectively using the serum contained in the discarded bone marrow fluid. According to this method, it is not necessary to separately collect blood from the patient, and the burden on the patient can be minimized. Further, it is possible to prevent biological contamination such as rejection due to an immune reaction as in the case of using biological components or proteinaceous components derived from other species or other families, and infection by protein components such as prions or viruses.
上記細胞播種基材を骨髄液由来の血清中に浸漬する方法としては特に限定されないが、例えば、保管中の汚染等を防げることから、適当な蓋付きの容器中に血清を満たし、この中に浸漬する方法が好ましい。
図１に細胞播種基材の保存容器の一例を示した。図１に示した保存容器３は、本体部３２と蓋部３１とからなり、支持棒３４で支持した状態で筒状の細胞播種基材１を血清２中に浸漬して保存することができる。本体３２の蓋側の縁部には切欠３３が設けられていることから、血清には気液交換により酸素が供給される。かかる構成において、支持捧３４の使用により、筒状の細胞播種基材１を変形しないよう支持すると共に、血清の容量を極限まで少なくする利点がある。かかる構成のものは血管用等、筒状のものを対象としたときに好適である。
また、図２に細胞播種基材の保存容器の別の一例の断面図を示した。図２に示した保存容器４は、本体部４２と蓋部４１とからなり、蓋部４１に設けられた押板４３により押さえつけることにより細胞播種基材１を血清２中に浸漬して保存することができ、この場合も血清の使用量を少なくできる。かかる発想は、他に容器全体の大きさを細胞播種基材１に対して適当なものとすることにより、より少量の血清でも保存が可能になる。また、気液界面を大きくとれることから、気液交換による血清への酸素供給効率にも優れる。
上記容器の材質としては特に限定されないが、滅菌や温度管理が容易であることから、ステンレス等からなるものが好適である。
The method for immersing the cell seeding substrate in bone marrow fluid-derived serum is not particularly limited.For example, in order to prevent contamination during storage, the container is filled with serum in an appropriate lid, A dipping method is preferred.
FIG. 1 shows an example of a cell seeding substrate storage container. The storage container 3 shown in FIG. 1 includes a main body portion 32 and a lid portion 31, and can be stored by immersing the tubular cell seeding substrate 1 in the serum 2 while being supported by a support rod 34. . Since the notch 33 is provided on the lid side edge of the main body 32, oxygen is supplied to the serum by gas-liquid exchange. In such a configuration, the use of the support plate 34 has the advantages of supporting the cylindrical cell seeding substrate 1 so as not to be deformed and reducing the volume of serum to the limit. Such a configuration is suitable for a cylindrical object such as a blood vessel.
FIG. 2 shows a cross-sectional view of another example of the cell seeding substrate storage container. The storage container 4 shown in FIG. 2 includes a main body portion 42 and a lid portion 41, and is stored by immersing the cell seeding substrate 1 in serum 2 by being pressed by a pressing plate 43 provided on the lid portion 41. In this case as well, the amount of serum used can be reduced. Such an idea can be preserved even with a smaller amount of serum by making the size of the entire container appropriate for the cell seeding substrate 1. Moreover, since the gas-liquid interface can be made large, the oxygen supply efficiency to serum by gas-liquid exchange is also excellent.
Although the material of the said container is not specifically limited, Since sterilization and temperature control are easy, what consists of stainless steel etc. is suitable.
本発明の細胞播種基材の保管方法では、細胞播種基材を骨髄由来血清中に浸漬した状態で３７℃程度の温度に保温することが好ましい。保温の方法としては特に限定されず、例えば、上記保存容器ごとインキュベータ中（５％ＣＯ２、３７℃、湿度１００％）に静置する方法等が挙げられる。 In the cell seeding substrate storage method of the present invention, it is preferable that the cell seeding substrate is kept at a temperature of about 37 ° C. while being immersed in bone marrow-derived serum. The method for keeping the temperature is not particularly limited, and examples thereof include a method in which the storage container is left in an incubator (5% CO 2 , 37 ° C., humidity 100%).
次に、本発明の保管方法により保管する細胞播種基材について説明する。
上記細胞播種基材としては、組織再生用基材上に患者から採取した骨髄細胞、又は、骨髄細胞から分化若しくは誘導させてできた細胞を播種することにより作製されたものであれば特に限定されず、その目的により、血管、心筋、軟骨、骨、消化管、皮膚等を再生するのに用いることができる。
上記骨髄細胞は、通常、患者の腸骨から骨髄液を採取し、得られた骨髄液を遠心分離することにより、血清、赤血球及び勾配液等の他の成分から分離したものを用いることができる。なお、上述のように、この際に分離された血清は本発明の保管方法に用いる。
また、上記骨髄細胞から分化若しくは誘導させてできた細胞としては、例えば、間葉系幹細胞、造血幹細胞等が挙げられる。
Next, the cell seeding substrate stored by the storage method of the present invention will be described.
The cell seeding substrate is not particularly limited as long as it is prepared by seeding bone marrow cells collected from a patient on a tissue regeneration substrate, or cells obtained by differentiation or induction from bone marrow cells. It can be used to regenerate blood vessels, myocardium, cartilage, bone, digestive tract, skin, etc.
As the bone marrow cells, those separated from other components such as serum, erythrocytes and gradient fluid by collecting bone marrow fluid from the iliac of the patient and centrifuging the obtained bone marrow fluid can be used. . As described above, the serum separated at this time is used in the storage method of the present invention.
Examples of the cells obtained by differentiation or induction from the bone marrow cells include mesenchymal stem cells and hematopoietic stem cells.
上記組織再生用基材としては特に限定されないが、近年では、生体内吸収性高分子材料からなるものが好適に用いられている。生体内吸収性高分子材料を用いた組織再生用基材は、播種した骨髄細胞が増殖し分化して組織を再生するまで基材としての機能を果たし、組織を再生した後には、分解され生体内に吸収される性質を有することから、再手術等により取り出す必要がない。
上記生体内吸収性高分子材料としては特に限定されないが、例えば、ポリラクチド、ポリグリコリド、ラクチド−ε−カプロラクトン共重合体等が好適であり、なかでも、Ｌ−ラクチド−ε−カプロラクトン共重合体は、Ｌ−ラクチドとε−カプロラクトンとの配合比を調整することにより、強度や柔軟性、生体内に吸収されるまでの期間を調整できることから好ましい。
Although it does not specifically limit as said base material for tissue reproduction | regeneration, In recent years, what consists of a bioabsorbable polymer material is used suitably. A tissue regeneration base material using a bioabsorbable polymer material functions as a base material until the seeded bone marrow cells proliferate and differentiate to regenerate the tissue. Since it has the property of being absorbed into the body, it is not necessary to remove it by re-operation.
The bioabsorbable polymer material is not particularly limited. For example, polylactide, polyglycolide, lactide-ε-caprolactone copolymer, and the like are preferable. Among them, L-lactide-ε-caprolactone copolymer is preferable. By adjusting the blending ratio of L-lactide and ε-caprolactone, it is preferable because the strength and flexibility and the period until absorption into the living body can be adjusted.
上記組織再生用基材は、１０〜５００μｍ程度の孔径の多数の微細小孔を有することが好ましい。このような微細小孔を多数有することにより、播種した骨髄細胞が上記微細小孔内に接着して三次元的に伸展することが可能となり、また、接着した骨髄細胞へ充分な栄養を供給することが可能となり、骨髄細胞を正常に増殖、分化させることができる。上記組織再生用基材の微細小孔は、貫通した孔であってもよいし、貫通しない孔であってもよい。
上記組織再生用基材の形状としては特に限定されず、例えば、シート状、チューブ状等が挙げられる。
The tissue regeneration substrate preferably has a large number of fine pores having a pore diameter of about 10 to 500 μm. By having a large number of such micropores, it becomes possible for the seeded bone marrow cells to adhere to the micropores and extend three-dimensionally, and to supply sufficient nutrients to the adhered bone marrow cells. Bone marrow cells can be normally proliferated and differentiated. The fine pores of the tissue regeneration substrate may be through holes or non-through holes.
The shape of the tissue regeneration substrate is not particularly limited, and examples thereof include a sheet shape and a tube shape.
本発明の細胞播種基材の保管方法によれば、組織再生用基材上に患者から採取した骨髄細胞等を播種した細胞播種基材を、手術に供するまでの間、細胞の剥離を最小限に抑えながら保管できる。また、他種又は他家由来の生体成分やタンパク質性成分を用いないことから、免疫反応による拒絶や、ウィルス等による生物汚染の心配もない。本発明の細胞播種基材の保管方法により保管した細胞播種基材を用いれば、高い治療効果を得ることができる。 According to the cell seeding substrate storage method of the present invention, the cell seeding substrate in which bone marrow cells collected from a patient are seeded on a tissue regeneration substrate is minimally detached until it is subjected to surgery. It can be stored while holding down. In addition, since biological components and proteinaceous components derived from other species or other families are not used, there is no concern about rejection due to an immune reaction or biological contamination due to viruses or the like. By using the cell seeding substrate stored by the cell seeding substrate storage method of the present invention, a high therapeutic effect can be obtained.
本発明によれば、患者から採取した骨髄細胞、又は、骨髄細胞から分化若しくは誘導させてできた細胞を組織再生用基材に播種した細胞播種基材を、高い活性を維持したまま一時的に保管する細胞播種基材の保管方法を提供できる。 According to the present invention, a bone marrow cell collected from a patient, or a cell seeding substrate obtained by seeding a cell obtained by differentiation or induction from a bone marrow cell on a tissue regeneration substrate is temporarily maintained while maintaining high activity. A method for storing a cell seeding substrate to be stored can be provided.
以下に実施例を掲げて本発明を更に詳しく説明するが、本発明はこれら実施例のみに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
（実施例１）
２００デシテックスのポリグリコリド糸から編成した直径１６ｍｍの筒状平編地をテフロンロッドにかぶせ、真空下で１２０℃、３時間熱処理した。これを−８０℃に冷却し、次いでＬ−ラクチド−ε−カプロラクトン共重合体（重量平均分子量４０万、ラクチド含有量５０モル％）をジオキサンに溶解して得た４重量％の溶液にディップした。これを−８０℃で凍結し、真空度０．１Ｔｏｒｒの条件で凍結乾燥した後、７０℃、１２時間真空乾燥した。更に、ロッドから取り外して、反転してテフロンロッドにかぶせた後、−８０℃に冷却し、再度ディップし、−８０℃で凍結し、凍結乾燥した後、７０℃、１２時間真空乾燥し、ポリグリコリド繊維で補強されたＬ−ラクチド−ε−カプロラクトン共重合体からなる内径１６ｍｍ、長さ１３０ｍｍのチューブ状の血管用の組織再生用基材を得た。
(Example 1)
A cylindrical flat knitted fabric with a diameter of 16 mm knitted from 200 decitex polyglycolide yarn was placed on a Teflon rod and heat treated under vacuum at 120 ° C. for 3 hours. This was cooled to −80 ° C., and then dipped into a 4 wt% solution obtained by dissolving L-lactide-ε-caprolactone copolymer (weight average molecular weight 400,000, lactide content 50 mol%) in dioxane. . This was frozen at −80 ° C., freeze-dried under a vacuum degree of 0.1 Torr, and then vacuum-dried at 70 ° C. for 12 hours. Furthermore, after removing from the rod, inverting and placing it on the Teflon rod, cooling to -80 ° C, dipping again, freezing at -80 ° C, freeze-drying, vacuum drying at 70 ° C for 12 hours, A tissue regeneration base material for a tubular blood vessel having an inner diameter of 16 mm and a length of 130 mm made of an L-lactide-ε-caprolactone copolymer reinforced with glycolide fiber was obtained.
体重１０ｋｇの患児の腸骨から骨髄液１００ｍＬを採取し、得られた骨髄液を遠心分離することにより骨髄細胞を含む細胞分画、血清、赤血球及び勾配液に分離した。次いで、単離した細胞分画（骨髄細胞）を５ｍＬのリン酸バッファーで希釈し、組織再生用基材に播種して、細胞播種基材を作製した。 Bone marrow fluid (100 mL) was collected from the iliac bone of a patient weighing 10 kg, and the resulting bone marrow fluid was centrifuged to separate it into cell fractions containing bone marrow cells, serum, erythrocytes and gradient fluid. Next, the isolated cell fraction (bone marrow cells) was diluted with 5 mL of phosphate buffer and seeded on a tissue regeneration substrate to prepare a cell seeding substrate.
図１に示した形状の保存容器中に上記の骨髄液から分離した血清を満たし、この中に得られた細胞播種基材を入れ、３７℃に保温したインキュベータ中に静置して１、３及び６時間保管した。 The storage container having the shape shown in FIG. 1 is filled with the serum separated from the above bone marrow fluid, and the obtained cell seeding substrate is placed in the storage container and left in an incubator kept at 37 ° C. for 1, 3 And stored for 6 hours.
（比較例１）
骨髄液由来の血清の代わりに、リン酸緩衝食塩水（シグマ社製；ＰＢＳ）、細胞培養液（シグマ社製；ＲＰＭＩ１６４０）、成長因子（Ｒ＆Ｄシステムズ社製；ＶＥＧＦ、Ｒ＆Ｄシステムズ社製；ＨＧＦ、シグマ社製；ｂＦＧＦ：ＧＦ）を１．２５μｇ／５００ｍＬの濃度で含有する培養液（ＲＰＭＩ１６４０＋ＧＦ）、成長因子ＧＦとウシ胎児血清（モーゲイトエクスポーツプテイ社製；ＦＢＳ）をそれぞれ１．２５μｇ／５００ｍＬ及び１０重量％の濃度で含有する培養液（ＲＰＭＩ１６４０＋ＧＦ＋ＦＢＳ）を用いて保管を行った以外は実施例１と同様の方法により、細胞播種基材を作製し、これを保管した。
(Comparative Example 1)
Instead of serum derived from bone marrow fluid, phosphate buffered saline (Sigma; PBS), cell culture (Sigma; RPMI1640), growth factor (R & D Systems; VEGF, R & D Systems; HGF, A culture solution (RPMI1640 + GF) containing Sigma: bFGF: GF) at a concentration of 1.25 μg / 500 mL, 1.25 μg / 500 mL of growth factor GF and fetal calf serum (Morgate Exsporttei; FBS), respectively. A cell seeding substrate was prepared and stored in the same manner as in Example 1 except that the culture medium (RPMI1640 + GF + FBS) contained at a concentration of 10% by weight was used.
（評価）
実施例１及び比較例１において保管した後、細胞播種基材を直径６ｍｍに打ち抜き、細胞播種基材中の細胞数を、ＤＮＡアッセイ法により計数した。即ち、凍結融解法にて細胞膜を破壊した後、流出した細胞核内ＤＮＡに蛍光Ｈｏｅｃｈｓｔを結合させ、蛍光量を測定し、検量線から細胞数を算出した。実験は、各区についてｎ＝３で行い、その平均値を求めた。
結果を表１に示した。
また、６時間保管後の細胞播種基材の一部を採取し、その走査型電子顕微鏡像を、実施例１につていは図３に、比較例１でＰＢＳを用いた場合を図４に、比較例１でＲＰＭＩ１６４０を用いた場合を図５に、比較例１でＲＰＭＩ１６４０＋ＧＦを用いた場合を図６に、比較例１でＲＰＭＩ１６４０＋ＧＦ＋ＦＢＳを用いた場合を図７にそれぞれ示した。
(Evaluation)
After storage in Example 1 and Comparative Example 1, the cell seeding substrate was punched out to a diameter of 6 mm, and the number of cells in the cell seeding substrate was counted by the DNA assay method. That is, after disrupting the cell membrane by the freeze-thaw method, fluorescent Hoechst was bound to the outflowing cell nucleus DNA, the amount of fluorescence was measured, and the number of cells was calculated from the calibration curve. The experiment was performed with n = 3 for each section, and the average value was obtained.
The results are shown in Table 1.
Further, a part of the cell seeding substrate after storage for 6 hours is collected, and the scanning electron microscope image thereof is shown in FIG. 3 for Example 1 and in FIG. 4 when PBS is used in Comparative Example 1. FIG. 5 shows the case of using RPMI1640 in Comparative Example 1, FIG. 6 shows the case of using RPMI1640 + GF in Comparative Example 1, and FIG. 7 shows the case of using RPMI1640 + GF + FBS in Comparative Example 1.
本発明によれば、患者から採取した骨髄細胞、又は、骨髄細胞から分化若しくは誘導させてできた細胞を組織再生用基材に播種した細胞播種基材を、高い活性を維持したまま一時的に保管する細胞播種基材の保管方法を提供することができる。 According to the present invention, a bone marrow cell collected from a patient, or a cell seeding substrate obtained by seeding a cell obtained by differentiation or induction from a bone marrow cell on a tissue regeneration substrate is temporarily maintained while maintaining high activity. A method for storing a cell seeding substrate to be stored can be provided.
１ 細胞播種基材
２ 血清
３ 細胞播種基材保存容器
３１ 蓋部
３２ 本体部
３３ 切欠
３４ 支持棒
４ 細胞播種基材保存容器
４１ 蓋部
４２ 本体部
４３ 押板
DESCRIPTION OF SYMBOLS 1 Cell seeding base material 2 Serum 3 Cell seeding base material storage container 31 Lid part 32 Main body part 33 Notch 34 Support rod 4 Cell seeding base material storage container 41 Lid part 42 Main body part 43 Press plate
Claims (2)
The storage method according to claim 1, wherein the cell seeding substrate regenerates blood vessels, heart muscle, cartilage, bone, digestive tract, or skin.
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