JP2007053906A - Method for purifying three-dimensional cultured material and purified three-dimensional cultured material - Google Patents
Method for purifying three-dimensional cultured material and purified three-dimensional cultured material Download PDFInfo
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Abstract
Description
本発明は、三次元培養物の精製方法及び精製された三次元培養物に関する。 The present invention relates to a method for purifying a three-dimensional culture and a purified three-dimensional culture.
近年、ヒト細胞をインビトロ（生体外）で培養して増殖させることによって得られた培養物を、例えば、患者の治療を目的として欠損部を補填するための移植用組織として再生医療に利用する試みがなされている。一般に、ヒト細胞を培養する際の培地としては、ヒト細胞の活性を上げ良好な増殖を促すために、例えばウシ胎児血清などの異種動物由来の血清を培地に添加した血清添加培地が利用されている。また、ウシ胎児血清の他に、異なる個体から得られたヒト血清を培地に添加したり、培養時におけるコンタミネーションを防止するために例えばペニシリンなどの抗生物質を培地に添加したりする。しかしながら、このような異種動物由来の血清やヒト血清、抗生物質といった成分（以下、除去対象成分とする）が高濃度で残留した状態の培養物を患者に移植すると、この除去対象成分に基づく免疫系の疾患を引き起こすことが懸念される。このような懸念を払拭する方法として、無血清培地や抗生物質を含まない培地を用いてヒト細胞を培養することも考えられるが、無血清培地を用いた培養では、血清を添加した培養に比べて十分な増殖能が得られにくく、所望の培養物が作製できない場合がある。また、抗生物質を含まない培地を用いた培養では、培養時にコンタミネーションを引き起こす可能性が高くなる。これらの点に鑑み、例えば特許文献１では、非ヒト由来の血清を含む血清添加培地を用いて培養した培養細胞を非ヒト由来の血清を含まない無血清培地中でインキュベートすることによって非ヒト血清由来のタンパク質を培養物から除去する方法が提案されている。すなわち、まず、培養容器の底面に付着させたヒト皮膚線維芽細胞をウシ胎児血清を含む血清添加培地を用いて培養し、培養容器の底面で増殖させる。その後、血清添加培地を無血清培地に置換し、培養した細胞を約３０〜４０℃で１６〜１８時間インキュベートする。これにより、存在すれば被験者に対して免疫系の疾患を引き起こす可能性を否定できないウシ胎児血清由来のタンパク質を被験者に注入する前に培養物から実質的に除去することができる、と記載されている。
さて、近年では、三次元培養物の研究が積極的に行われており、例えば、三次元スキャフォールド（Ｓｃａｆｆｏｌｄ）にヒト細胞を保持させた状態で培養することによって三次元培養物を得ることができる。ヒト細胞で三次元培養物を作製する場合にも、非ヒト由来の血清に含まれるタンパク質などの除去対象成分を含む血清添加培地が用いられており、三次元培養物の場合には、上述の特許文献１の場合以上に除去対象成分が残留する可能性が高い。そこで、三次元培養物から除去対象成分を除去するために、三次元培養物に対し上述した特許文献１の方法を適用したところ、除去対象成分を十分に除去することができなかった。これは、特許文献１の培養細胞が平面構造（二次元構造）であるのに対し三次元培養物は立体構造であるために、三次元培養物内部にトラップされている除去対象成分を十分に除去することができなかったためと考えられる。また、三次元培養物の培養時には、三次元培養物の外周面近傍への培地による栄養供給が内部に対してリッチな状態になり、該外周面近傍の細胞が活性化しやすい。このため、外周面近傍の細胞が産生する細胞外マトリクスによって三次元培養物表面が被覆され、三次元培養物の内部でトラップされている除去対象成分が抜けにくい構造が形成されることも、除去対象成分を十分に除去することができなかった理由の一つと考えられる。このように、三次元培養物では内部にトラップされた除去対象成分が除去されにくく、上述の特許文献１の方法では、三次元培養物において培地由来の除去対象成分を有効に除去できなかった。 In recent years, research on three-dimensional cultures has been actively conducted. For example, it is possible to obtain a three-dimensional culture by culturing human cells in a three-dimensional scaffold (scaffold). it can. When preparing a three-dimensional culture with human cells, a serum-added medium containing components to be removed such as proteins contained in non-human-derived serum is used. In the case of a three-dimensional culture, the above-mentioned There is a high possibility that the component to be removed remains more than in the case of Patent Document 1. Then, in order to remove the removal target component from the three-dimensional culture, when the method of Patent Document 1 described above was applied to the three-dimensional culture, the removal target component could not be sufficiently removed. This is because the cultured cells of Patent Document 1 have a planar structure (two-dimensional structure), whereas a three-dimensional culture has a three-dimensional structure. This is probably because it could not be removed. Further, when culturing a three-dimensional culture, nutrient supply by the medium to the vicinity of the outer peripheral surface of the three-dimensional culture becomes rich with respect to the inside, and cells near the outer peripheral surface are likely to be activated. For this reason, the surface of the 3D culture is covered with an extracellular matrix produced by cells in the vicinity of the outer peripheral surface, and it is also possible to form a structure in which the removal target component trapped inside the 3D culture is difficult to escape. This is considered to be one reason why the target component could not be sufficiently removed. Thus, the removal target component trapped inside is difficult to remove in the three-dimensional culture, and the above-described method of Patent Document 1 cannot effectively remove the removal target component derived from the medium in the three-dimensional culture.
本発明は、このような課題を解決するためになされたものであり、培地に含まれている除去対象成分を三次元培養物から効率よく除去する三次元培養物の精製方法を提供することを目的の一つとする。またこの三次元培養物の精製方法によって精製された三次元培養物を提供することを目的の一つとする。 The present invention has been made to solve such problems, and provides a method for purifying a three-dimensional culture that efficiently removes components to be removed contained in the medium from the three-dimensional culture. One of the purposes. Another object of the present invention is to provide a three-dimensional culture purified by this three-dimensional culture purification method.
本発明は、上述の目的の少なくとも一部を達成するために以下の手段を採用した。 The present invention employs the following means in order to achieve at least a part of the above object.
すなわち、本発明の三次元培養物の精製方法は、
除去対象成分を含む培地を利用して培養対象となる細胞を培養することによって得た三次元培養物から前記培地に由来する前記除去対象成分を除去する三次元培養物の精製方法であって、
（ａ）洗浄容器内で前記三次元培養物を前記除去対象成分を含まない洗浄液に浸漬する工程と、
（ｂ）前記工程（ａ）の前、前記工程（ａ）のあと又は前記工程（ａ）と並行して前記三次元培養物から前記除去対象成分の少なくとも一部が前記洗浄液に溶出するのを促進させる工程と、
を含むものである。
That is, the method for purifying a three-dimensional culture of the present invention comprises:
A three-dimensional culture purification method for removing the removal target component derived from the medium from a three-dimensional culture obtained by culturing cells to be cultured using a medium containing the removal target component,
(A) a step of immersing the three-dimensional culture in a cleaning solution that does not contain the component to be removed in a cleaning container;
(B) Before the step (a), after the step (a) or in parallel with the step (a), at least a part of the removal target component is eluted from the three-dimensional culture into the washing solution. Promoting the process;
Is included.
この三次元培養物の精製方法では、除去対象成分を含む培地を用いて培養対象となる細胞を培養することにより作製された三次元培養物を、除去対象成分を含まない洗浄液に浸漬し、その前、そのあと又はこれと並行して三次元培養物から除去対象成分が洗浄液に溶出するのを促進させる。これにより、三次元培養物に含まれる除去対象成分が洗浄液に溶出しやすくなるため、培地に由来する除去対象成分を三次元培養物から効率よく除去することができる。 In this three-dimensional culture purification method, a three-dimensional culture prepared by culturing cells to be cultured using a medium containing a component to be removed is immersed in a cleaning solution that does not contain the component to be removed. Before, after or in parallel with this, the removal target component is promoted to be eluted from the three-dimensional culture into the washing solution. Thereby, since the removal target component contained in the three-dimensional culture is easily eluted in the washing liquid, the removal target component derived from the medium can be efficiently removed from the three-dimensional culture.
本発明の三次元培養物の精製方法によって精製された三次元培養物は、該三次元培養物に含まれる培地由来の除去対象成分が精製前に比べて極めて少ない。したがって、例えば、細胞を患者に移植したときに患者が免疫系の疾患を引き起こす可能性を低減することができる。 The three-dimensional culture purified by the three-dimensional culture purification method of the present invention has very few components to be removed from the medium contained in the three-dimensional culture as compared to before purification. Thus, for example, the likelihood that a patient will cause an immune system disease when cells are transplanted into the patient can be reduced.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明の三次元培養物の精製方法において、前記工程（ｂ）は、前記所定の洗浄容器を振盪することによって前記三次元培養物から前記除去対象成分の少なくとも一部が前記洗浄液に溶出するのを促進させる工程としてもよい。こうすれば、振盪によって洗浄液が揺動するため、三次元培養物から除去対象成分が洗浄液に溶出しやすくなる。ここで、振盪の条件は、培養対象となる細胞の種類や三次元培養物の構造などによって適宜選定すればよいが、例えば、単なる左右動による振盪でもよいし回転動による振盪でもよいが、回転動による振盪が好ましい。回転動による振盪の場合、その振盪の回転数は、三次元培養物に対して、洗浄液の揺動によるストレスが適度に加わる程度であることが好ましい。 In the three-dimensional culture purification method of the present invention, in the step (b), at least a part of the removal target component is eluted from the three-dimensional culture into the washing solution by shaking the predetermined washing container. It is good also as a process of promoting. By doing so, the washing solution is shaken by shaking, and thus the components to be removed are easily eluted from the three-dimensional culture into the washing solution. Here, the conditions for shaking may be appropriately selected depending on the type of cells to be cultured and the structure of the three-dimensional culture.For example, the shaking may be simple left-right motion or rotational motion. Shaking by motion is preferred. In the case of shaking by rotation, the number of rotations of shaking is preferably such that a stress due to shaking of the washing solution is moderately applied to the three-dimensional culture.
本発明の三次元培養物の精製方法において、前記工程（ｂ）は、前記三次元培養物のうち培養時に前記培地と境界をなしていた面以外の部分を前記洗浄液に露出させることによって前記三次元培養物から前記除去対象成分の少なくとも一部が前記洗浄液に溶出するのを促進させる工程としてもよい。こうすれば、三次元培養物が洗浄液に接触する面積が大きくなるため、より効果的に除去対象成分を除去することができる。殊に、三次元培養物のうち培養時に培地と境界をなしていた面、すなわち三次元培養物の外周面は、培地による栄養供給がリッチであるため細胞が活性化しやすく、これらの細胞により密に産生された細胞外マトリクスと増殖により増加した細胞とによって被覆されていることが考えられるため、該面からは除去対象成分が溶出しにくいところ、こうすれば、細胞外マトリクスによって被覆されていない部分を洗浄液に露出することができ、該部分から三次元培養物内の除去対象成分を効率的に除去することができる。ここで、三次元培養物のうち培養時に培地と境界をなしていた面以外の部分を洗浄液に露出させるには、例えば前記三次元培養物を作製した培養面から前記三次元培養物を剥離し、該剥離した面を前記洗浄液に露出させることによって前記三次元培養物から前記除去対象成分の少なくとも一部が前記洗浄液に溶出するのを促進させてもよい。この培養面とは、培養容器を用いて三次元培養物を培養している場合には、三次元培養物が培養容器に接触している面をいい、具体的には培養容器の底面をいう。三次元培養物のうちこの培養面に接触していた面を培養面、すなわち培養容器の底面から剥離するには、三次元培養物を培養容器から剥離可能であればその剥離方法は限定されない。例えば、ピンセットなどを用いて物理的に剥離してもよいし、タンパク質分解酵素などを用いた化学的な処理によって剥離してもよい。また、培養容器の底面に敷いた半透膜の上に設置した三次元培養物を半透膜の下方から培地が供給されないように培養した後、半透膜を持ち上げることによって三次元培養物の下方面を洗浄液に露出させるとしてもよいし、感温性ポリマーなどを敷設した特殊な培養容器を用いて三次元培養物を培養したあと温度制御などにより剥離して三次元培養物の下方面を洗浄液に露出させるとしてもよい。あるいは、前記三次元培養物に切断面又は穿孔を設け該切断面又は穿孔に前記洗浄液を露出させることによって前記三次元培養物から前記除去対象成分の少なくとも一部が前記洗浄液に溶出するのを促進させてもよい。また、上述の促進工程に、前述した振盪による促進工程を組み合わせて実施してもよい。 In the method for purifying a three-dimensional culture of the present invention, the step (b) includes the step of exposing the portion of the three-dimensional culture other than the surface that has bounded the medium at the time of culture to the cleaning solution. It is good also as a process of accelerating that at least a part of the removal object ingredient elutes from the original culture into the washing solution. By doing so, the area where the three-dimensional culture comes into contact with the washing liquid is increased, so that the component to be removed can be more effectively removed. In particular, the surface of the three-dimensional culture that bounds the medium at the time of culturing, that is, the outer peripheral surface of the three-dimensional culture, is rich in nutrient supply by the medium, so that the cells are likely to be activated. It is considered that the component to be removed is difficult to elute from the surface because it is covered with the extracellular matrix produced in the cell and the cells increased by proliferation. In this way, it is not covered with the extracellular matrix. A part can be exposed to a washing | cleaning liquid, and the removal target component in a three-dimensional culture can be efficiently removed from this part. Here, in order to expose a portion of the three-dimensional culture other than the surface that has bounded the medium during culture to the washing liquid, for example, the three-dimensional culture is peeled off from the culture surface on which the three-dimensional culture is prepared. Further, by exposing the peeled surface to the cleaning liquid, it may be promoted that at least a part of the component to be removed is eluted from the three-dimensional culture into the cleaning liquid. When the three-dimensional culture is cultured using a culture vessel, the culture surface means a surface where the three-dimensional culture is in contact with the culture vessel, specifically the bottom surface of the culture vessel. . In order to peel the surface of the three-dimensional culture that has been in contact with the culture surface from the culture surface, that is, the bottom surface of the culture vessel, the peeling method is not limited as long as the three-dimensional culture can be peeled from the culture vessel. For example, it may be physically peeled off using tweezers or the like, or peeled off by chemical treatment using a proteolytic enzyme or the like. In addition, after culturing the 3D culture placed on the semipermeable membrane laid on the bottom of the culture vessel so that no medium is supplied from below the semipermeable membrane, the 3D culture is lifted by lifting the semipermeable membrane. The lower surface may be exposed to the washing solution, or after culturing the three-dimensional culture using a special culture vessel laid with a thermosensitive polymer etc., the lower surface of the three-dimensional culture is peeled off by temperature control or the like. It may be exposed to the cleaning liquid. Alternatively, by providing a cut surface or a perforation in the three-dimensional culture and exposing the cleaning liquid to the cut surface or the perforation, at least a part of the removal target component from the three-dimensional culture is promoted to elute into the cleaning liquid. You may let them. Moreover, you may implement combining the acceleration | stimulation process by shaking mentioned above in the above-mentioned acceleration | stimulation process.
本発明の三次元培養物の精製方法において、前記除去対象成分は、培養対象となる細胞の起源となる動物と異なる種類の動物である異種動物から得られる成分、培養対象となる細胞の起源となる動物と同じ種類の動物である同種動物であって異なる個体から得られる成分及び抗生物質からなる群より選ばれる少なくとも１種であるとしてもよい。ここで、培養対象となる細胞の起源となる動物と異なる種類の動物である異種動物から得られる成分としては、例えば、ヒト細胞の培養において培地に添加したウシ胎児血清に含まれる種々のタンパク質などがこれに相当する。また、培養対象となる細胞の起源となる動物と同じ種類の動物である同種動物であって異なる個体から得られる成分としては、例えば、ヒト細胞の培養において培地に添加したヒトプール血清や同種血清に含まれる種々のタンパク質などがこれに相当する。抗生物質としては、例えば、微生物の産生物に由来する抗菌剤、抗真菌剤、抗ウイルス剤、抗腫瘍剤などが挙げられ、具体的には、ヒト細胞の培養においてコンタミネーションを防止するために培地に添加されるペニシリンやストレプトマイシンなどが挙げられる。これらの抗生物質が移植時において多量が投与されることによって、免疫系の疾患につながる可能性や耐性菌の出現などが懸念される。 In the three-dimensional culture purification method of the present invention, the component to be removed is a component obtained from a heterogeneous animal that is a different kind of animal from the animal that is the source of the cell to be cultured, the origin of the cell that is to be cultured, It is good also as at least 1 sort (s) chosen from the group which consists of the same animal which is the same kind of animal as a certain animal, and obtained from a different individual | organism | solid, and antibiotics. Here, examples of components obtained from a heterogeneous animal that is a different kind of animal from the animal that is the source of cells to be cultured include various proteins contained in fetal bovine serum added to the medium in the culture of human cells. Corresponds to this. In addition, as components obtained from different individuals of the same species that are the same type of animal as the source of the cells to be cultured, for example, human pool serum or allogeneic serum added to the medium in the culture of human cells. Various proteins contained therein correspond to this. Antibiotics include, for example, antibacterial agents, antifungal agents, antiviral agents, and antitumor agents derived from microbial products. Specifically, in order to prevent contamination in the culture of human cells. Examples include penicillin and streptomycin added to the medium. When these antibiotics are administered in large quantities at the time of transplantation, there is concern that they may lead to immune system diseases or emergence of resistant bacteria.
本発明の三次元培養物の精製方法において、洗浄液は等張液であることが好ましい。こうすれば、細胞の生存に好適な浸透圧を維持することができる。ここで、洗浄液としては、三次元培養物から除去対象成分が洗浄液に溶出可能な液体であって且つ除去対象成分や安全性が担保されていない物質が所定量以下又は全く含まれていない液体を適宜選定すればよい。例えば、生理食塩水、リン酸緩衝液（ＰＢＳ）、リンゲル（Ｒｉｎｇｅｒ）液などの塩類溶液や、ＤＭＥＭ（ダルベッコ変法イーグル培地）といった周知の基礎培地などを選定してもよい。さらに、除去対象成分を含まず、かつ培養対象となる細胞の起源となる個体の血清（自己血清）を含む溶液であってもよい。また、精製に用いる洗浄液の量は、洗浄容器の容積、培養対象となる細胞の種類、三次元培養物の大きさや生細胞数、細胞外マトリクスの量などによって適宜選定すればよい。例えば、三次元培養物が浸漬する量であれば足りるので最大量の限度は存在しないが、好ましくは、三次元培養物の体積に対して１０〜１０００倍量の洗浄液を用いることが好ましい。また、洗浄液は、精製中に交換しないとしてもよいが精製途中で新しい洗浄液に所定回数だけ交換するとしてもよい。この場合、洗浄液を交換しない場合に比べて洗浄液の量を少量としてもよい。 In the three-dimensional culture purification method of the present invention, the washing solution is preferably an isotonic solution. In this way, an osmotic pressure suitable for cell survival can be maintained. Here, as the cleaning liquid, a liquid that can be eluted from the three-dimensional culture into the cleaning liquid and that does not contain a predetermined amount or less of the target component or a substance whose safety is not guaranteed. What is necessary is just to select suitably. For example, a salt solution such as physiological saline, phosphate buffer (PBS), Ringer solution, or a well-known basic medium such as DMEM (Dulbecco's modified Eagle medium) may be selected. Further, it may be a solution that does not contain the component to be removed and contains the serum (autologous serum) of the individual that is the origin of the cells to be cultured. The amount of the washing solution used for purification may be appropriately selected depending on the volume of the washing container, the type of cells to be cultured, the size and number of living cells of the three-dimensional culture, the amount of extracellular matrix, and the like. For example, there is no limit on the maximum amount as long as the three-dimensional culture is immersed, but it is preferable to use 10 to 1000 times the amount of the washing solution with respect to the volume of the three-dimensional culture. The cleaning liquid may not be replaced during the purification, but may be replaced with a new cleaning liquid a predetermined number of times during the purification. In this case, the amount of the cleaning liquid may be small compared with the case where the cleaning liquid is not replaced.
本発明の三次元培養物の精製方法において、前記三次元培養物は、三次元スキャフォールドで培養対象となる細胞を保持させて培養することにより作製されるとしてもよい。このような三次元培養物としては、たとえば、三次元スキャフォールド上で培養対象となる細胞を培養した三次元培養物などが挙げられる。具体的には、シート状に敷設したフィブリンゲル上で角膜細胞を培養することによって得られる培養角膜や、シート形状のコラーゲンスポンジ上で線維芽細胞を培養することによって得られる培養真皮などが挙げられる。また、前記三次元培養物としては、ゲル状の三次元スキャフォールドを形成する溶液と培養対象となる細胞とを混合した混合液を前記培養面に配置し、該配置した混合液をゲル化させた後に培養することによって作製されることが好ましい。この場合、ゲル状の三次元スキャフォールドでは、微細格子が形成されているために洗浄液が浸透しにくいことから、三次元培養物から除去対象成分が抜けにくく、本発明を適用する意義が高い。具体的には、コラーゲンゲルに軟骨細胞を包埋して培養した培養軟骨などが挙げられる。ここで、三次元スキャフォールドとしては、接着や包埋などによって細胞を保持することにより細胞の足場としての役割を果たすものであればどのような材料によって構成されていてもよいが、例えば、コラーゲン、フィブリン、ヒアルロン酸といった天然高分子や、ポリグリコール酸（ＰＧＡ）、ポリ乳酸（ＰＬＡ）、ポリ乳酸グリコール重合体（ＰＬＧＡ）、ポリロタキサンといった合成高分子、ハイドロキシアパタイト、リン酸三カルシウムといった無機材料などの生体親和性材料で構成されていることが好ましい。この三次元スキャフォールドの形態としては、三次元構造であればいずれの形態であってもよく、ゲル状、スポンジ状、フィルム状、メッシュ状、不織布状、編織布状など、種々の形態とすることができる。殊に、密な網目構造（格子構造）で構成される三次元スキャフォールドに対しては、本発明は特に有用であり、このようなものとしてはゲル状やフィルム状の三次元スキャフォールドが挙げられる。 In the method for purifying a three-dimensional culture of the present invention, the three-dimensional culture may be prepared by holding and culturing cells to be cultured in a three-dimensional scaffold. Examples of such a three-dimensional culture include a three-dimensional culture obtained by culturing cells to be cultured on a three-dimensional scaffold. Specific examples include a cultured cornea obtained by culturing corneal cells on a fibrin gel laid in a sheet shape, and a cultured dermis obtained by culturing fibroblasts on a sheet-shaped collagen sponge. . In addition, as the three-dimensional culture, a mixed solution in which a solution that forms a gel-like three-dimensional scaffold and cells to be cultured is mixed is placed on the culture surface, and the placed mixed solution is gelled. It is preferable that it is produced by culturing after a while. In this case, in the gel-like three-dimensional scaffold, since the washing liquid is difficult to permeate because a fine lattice is formed, it is difficult to remove the component to be removed from the three-dimensional culture, and it is highly significant to apply the present invention. Specific examples include cultured cartilage in which chondrocytes are embedded in a collagen gel and cultured. Here, the three-dimensional scaffold may be composed of any material as long as it serves as a cell scaffold by holding cells by adhesion or embedding. , Natural polymers such as fibrin and hyaluronic acid, synthetic polymers such as polyglycolic acid (PGA), polylactic acid (PLA), polylactic acid glycol polymer (PLGA), polyrotaxane, inorganic materials such as hydroxyapatite and tricalcium phosphate It is preferable that the material is composed of a biocompatible material. The form of the three-dimensional scaffold may be any form as long as it has a three-dimensional structure, and various forms such as a gel form, a sponge form, a film form, a mesh form, a non-woven form, and a woven cloth form are used. be able to. In particular, the present invention is particularly useful for a three-dimensional scaffold composed of a dense network structure (lattice structure), such as a gel-like or film-like three-dimensional scaffold. It is done.
本発明の三次元培養物の精製方法において、前記三次元培養物は、培養対象となる細胞を二次元的に培養して得た二次元培養物を複数積層したのち更に前記培地を利用して培養することによって作製されるとしてもよい。ここで、二次元培養物を複数積層する際には、二次元培養物同士をゲル状の材料で接着して複数積層するとしてもよい。ゲル状の材料としては、上述の三次元スキャフォールドと同様の材質のものが利用でき、例えば、フィブリンゲルなどの生物学的接着剤が挙げられる。この場合、フィブリンゲル深部に除去対象成分がトラップされ、除去が困難なことが想定されるため、本発明の精製方法を適用する意義は高い。 In the three-dimensional culture purification method of the present invention, the three-dimensional culture is obtained by stacking a plurality of two-dimensional cultures obtained by two-dimensionally culturing cells to be cultured, and further using the medium. It may be produced by culturing. Here, when a plurality of two-dimensional cultures are stacked, the two-dimensional cultures may be bonded together with a gel-like material and stacked. As the gel material, the same material as the above-mentioned three-dimensional scaffold can be used, and examples thereof include biological adhesives such as fibrin gel. In this case, since the component to be removed is trapped in the fibrin gel deep part and it is assumed that the removal is difficult, it is highly meaningful to apply the purification method of the present invention.
本発明の三次元培養物の精製方法において、培養対象となる細胞は、その起源としては特に限定されず、例えば、ヒト、マウス、ラット、モルモット、ハムスター、ニワトリ、ウサギ、ブタ、ヒツジ、ウシ、ウマ、イヌ、ネコ、サルなどの温血動物が挙げられるが、ヒトであることが好ましい。この培養対象となる細胞としては、例えば、軟骨細胞、表皮細胞、上皮細胞、線維芽細胞、骨細胞、膵島細胞、肝細胞、心筋細胞、筋細胞又はこれらの前駆細胞や、間葉系幹細胞、胚性幹細胞（ＥＳ細胞）などが挙げられる。このうち、軟骨細胞や線維芽細胞などの間質細胞は、培養時に多くの細胞外マトリクスを産生するため、三次元スキャフォールドの表面を被覆し、除去対象成分が除去されにくい構造の三次元培養物を形成しやすい。したがって、本発明を適用する意義が高い。 In the method for purifying a three-dimensional culture of the present invention, the cell to be cultured is not particularly limited as the source, for example, human, mouse, rat, guinea pig, hamster, chicken, rabbit, pig, sheep, cow, Examples include warm-blooded animals such as horses, dogs, cats, monkeys, and the like, preferably humans. Examples of the cells to be cultured include chondrocytes, epidermis cells, epithelial cells, fibroblasts, bone cells, pancreatic islet cells, hepatocytes, cardiomyocytes, myocytes or their precursor cells, mesenchymal stem cells, Examples include embryonic stem cells (ES cells). Among these, stromal cells such as chondrocytes and fibroblasts produce a large amount of extracellular matrix during culture, so the surface of the 3D scaffold is covered and the 3D culture with a structure that makes it difficult to remove the components to be removed. It is easy to form things. Therefore, the significance of applying the present invention is high.
本発明の三次元培養物の精製方法において、培地は、培養対象となる細胞の種類によってその種類を適宜選定すればよい。例えば、周知のＤＭＥＭやα−ＭＥＮ、Ｍ１９９培地などの基礎培地を選定してもよい。 In the method for purifying a three-dimensional culture of the present invention, the type of medium may be appropriately selected depending on the type of cells to be cultured. For example, a basal medium such as a well-known DMEM, α-MEN, or M199 medium may be selected.
本発明の三次元培養物の精製方法において、精製の際の温度や時間は、培養対象となる細胞の種類や三次元培養物の構造などによって適宜選定すればよい。具体的には、精製工程後の三次元培養物において、所望の生細胞数が得られ、かつ除去対象成分を所望の値まで除去可能な温度と時間であれば、どのように設定してもよい。例えば、精製温度は４〜２０℃が好ましく、軟骨細胞の場合には殊に８〜１８℃が好ましい。また、精製時間は、２〜４８時間程度が好ましく、８〜２４時間であることが更にが好ましい。本発明の三次元培養物の精製方法では、対象となる三次元培養物に応じて、精製の条件、温度や時間等を設定することができるので、作業者間によるバラツキをなくすことができ、誰でも容易に、かつ安定して所望の三次元培養物を提供することができる。 In the method for purifying a three-dimensional culture of the present invention, the temperature and time for purification may be appropriately selected depending on the type of cells to be cultured, the structure of the three-dimensional culture, and the like. Specifically, in the three-dimensional culture after the purification step, any temperature and time can be set as long as a desired number of living cells can be obtained and the target component can be removed to a desired value. Good. For example, the purification temperature is preferably 4 to 20 ° C., and in the case of chondrocytes, 8 to 18 ° C. is particularly preferable. Further, the purification time is preferably about 2 to 48 hours, and more preferably 8 to 24 hours. In the three-dimensional culture purification method of the present invention, the purification conditions, temperature, time, etc. can be set according to the target three-dimensional culture, so that variations among operators can be eliminated, Anyone can provide a desired three-dimensional culture easily and stably.
以下に本発明の好適な実施形態を実施例を用いて説明する。 Preferred embodiments of the present invention will be described below using examples.
［実験例１］
（１）ウサギの培養軟骨の作製
日本白色家兎の膝、股、肩関節から関節軟骨を採取した。採取した関節軟骨をトリプシンＥＤＴＡ（エチレンジアミン四酢酸）溶液及びコラゲナーゼ溶液で酵素処理を行い、軟骨細胞を分離・回収した。得られた軟骨細胞を１０％ウシ胎児血清（ＦＢＳ）含有ＤＭＥＭで洗浄後、細胞密度が１×１０７個／ｍｌとなるように１０％ＦＢＳ含有ＤＭＥＭにより細胞懸濁液を調製した。次に、細胞懸濁液と３％アテロコラーゲンインプラント（高研社製）とが１：４（体積比）の割合になるように混合（包埋）した。この工程によって細胞密度は希釈される。すなわち、細胞懸濁液を１×１０７個／ｍｌの濃度で調製した場合、コラーゲンに包埋したときの細胞濃度は２×１０６個／ｃｍ３となる。続いて、この混合液１００μｌをポリスチレン製の培養容器の底面に略ドーム状となるようにマウントし、これを５％ＣＯ２、３７℃の条件下で１時間静置してゲル化させた。その後、培養容器に培地を加え、培養を開始した。培地は、５０μｇ／ｍｌアスコルビン酸（Ｌ−アスコルビン酸リン酸エステルマグネシウム塩ｎ水和物：Ｃ６Ｈ６Ｏ９Ｐ・３／２Ｍｇ・ｎＨ２Ｏ、日光ケミカルズ社製）を含むように調整した１０％ＦＢＳ含有ＤＭＥＭを使用した。また、培養は、３７℃、５％ＣＯ２の環境下で約４週間行った。この培養により、直径が約１０ｍｍ、厚みが約２ｍｍの培養軟骨が得られた。
（２）培養軟骨の洗浄
上記（１）で培養軟骨を作製した後、培養容器から培地を取り除き、培地と略同量のリンゲル液で上記（１）で得た培養軟骨を２回洗浄した。これを実験例１の培養軟骨とした。なお、このとき、培養軟骨は、培養容器の底面に接着した状態であった。
（３）測定試料の調製
上記（２）で洗浄した培養軟骨を５０ｍｌの遠心チューブに移し、この遠心チューブに適切な量のＤＰＢＳ（ダルベッコリン酸緩衝液）を加えた。続いて、ホモジナイザー（キネマティカ社製）を用いて培養軟骨を破砕し、懸濁液を得た。その後、この懸濁液を遠心分離機（ベックマン社製）を用いて１５，０００ｒｐｍで５分間遠心し、上清を回収して測定試料とした。
（４）ＢＳＡ（ウシ血清アルブミン）含有量の測定
培養軟骨に含まれるＦＢＳ由来タンパク質の除去効果の評価は、ＦＢＳに最も多く含まれるタンパク質であるＢＳＡの含有量を比較することにより行うこととした。このＢＳＡ含有量の測定は、周知のサンドイッチＥＬＩＳＡ法（Ｅｎｚｙｍｅ−Ｌｉｎｋｅｄ ＩｍｍｕｎｏＳｏｒｂｅｎｔ Ａｓｓａｙ）を利用したＢＳＡ測定キットを用いて行った。すなわち、まず、プレートに設けられたウェルに一次抗体を加え、室温で１時間反応させ、プレートを洗浄液で３回洗浄した後にブロッキング剤を３０分間反応させ、ブロッキングした。続いて、洗浄液で３回洗浄したあと、上記（３）で調製した測定試料を１〜１０００倍の範囲内で希釈し、１００μｌをウェルに加え、室温で１時間反応させた。反応後、洗浄液で５回洗浄したあと二次抗体を加え、室温で１時間反応させ、洗浄液で５回洗浄し、発色液を加えた。５〜３０分経過後、反応停止液として２Ｍ Ｈ２ＳＯ４を加え、プレートリーダー（モレキュラーデバイス社製）で波長（４５０ｎｍ）の吸光度を測定した。測定により得られた吸光度に基づいて検量線から測定試料のＢＳＡ濃度を読み取り、培養軟骨の単位体積当たりの数値に換算した。その結果を表１に示す。なお、上述の一次抗体、ブロッキング剤、洗浄液、二次抗体および発色液は、ＢＳＡ測定キットに付属しているものを使用した。
[Experimental Example 1]
(1) Production of rabbit cultured cartilage Articular cartilage was collected from knee, hip and shoulder joints of Japanese white rabbits. The collected articular cartilage was subjected to enzyme treatment with trypsin EDTA (ethylenediaminetetraacetic acid) solution and collagenase solution to separate and collect chondrocytes. The obtained chondrocytes were washed with DMEM containing 10% fetal bovine serum (FBS), and a cell suspension was prepared with DMEM containing 10% FBS so that the cell density was 1 × 10 7 cells / ml. Next, the cell suspension and 3% atelocollagen implant (manufactured by Koken Co., Ltd.) were mixed (embedded) so as to have a ratio of 1: 4 (volume ratio). This step dilutes the cell density. That is, when a cell suspension is prepared at a concentration of 1 × 10 7 cells / ml, the cell concentration when embedded in collagen is 2 × 10 6 cells / cm 3 . Subsequently, 100 μl of this mixed solution was mounted on the bottom of a polystyrene culture vessel so as to be substantially dome-shaped, and this was allowed to stand for 1 hour under the conditions of 5% CO 2 and 37 ° C. for gelation. Thereafter, a culture medium was added to the culture vessel, and culture was started. The medium was adjusted to contain 50 μg / ml ascorbic acid (L-ascorbic acid phosphate magnesium salt n hydrate: C 6 H 6 O 9 P · 3 / 2Mg · nH 2 O, manufactured by Nikko Chemicals). DMEM containing 10% FBS was used. The culture was performed for about 4 weeks in an environment of 37 ° C. and 5% CO 2 . By this culture, cultured cartilage having a diameter of about 10 mm and a thickness of about 2 mm was obtained.
(2) Washing of cultured cartilage After producing cultured cartilage in (1) above, the culture medium was removed from the culture container, and the cultured cartilage obtained in (1) above was washed twice with approximately the same amount of Ringer's solution as the culture medium. This was used as the cultured cartilage of Experimental Example 1. At this time, the cultured cartilage was in a state of being adhered to the bottom surface of the culture container.
(3) Preparation of measurement sample The cultured cartilage washed in (2) above was transferred to a 50 ml centrifuge tube, and an appropriate amount of DPBS (Dulbeccoline buffer) was added to this centrifuge tube. Subsequently, the cultured cartilage was crushed using a homogenizer (manufactured by Kinematica) to obtain a suspension. Thereafter, this suspension was centrifuged at 15,000 rpm for 5 minutes using a centrifuge (manufactured by Beckman), and the supernatant was collected to obtain a measurement sample.
(4) Measurement of BSA (bovine serum albumin) content Evaluation of the removal effect of FBS-derived protein contained in cultured cartilage was performed by comparing the content of BSA, which is the protein most contained in FBS. . The BSA content was measured using a BSA measurement kit using a well-known sandwich ELISA method (Enzyme-Linked Immunosorbent Assay). That is, first, a primary antibody was added to a well provided on a plate and allowed to react at room temperature for 1 hour. After the plate was washed 3 times with a washing solution, the blocking agent was reacted for 30 minutes for blocking. Subsequently, after washing with a washing solution three times, the measurement sample prepared in the above (3) was diluted within a range of 1 to 1000 times, 100 μl was added to the well, and reacted at room temperature for 1 hour. After the reaction, the reaction mixture was washed 5 times with a washing solution, a secondary antibody was added, allowed to react at room temperature for 1 hour, washed 5 times with the washing solution, and a coloring solution was added. After 5 to 30 minutes, 2MH 2 SO 4 was added as a reaction stop solution, and the absorbance at a wavelength (450 nm) was measured with a plate reader (Molecular Device). Based on the absorbance obtained by the measurement, the BSA concentration of the measurement sample was read from the calibration curve and converted into a value per unit volume of the cultured cartilage. The results are shown in Table 1. The primary antibody, blocking agent, washing solution, secondary antibody and color developing solution described above were those attached to the BSA measurement kit.
［実験例２］
実験例２では、上述の実験例１の（１）と同様にしてウサギの培養軟骨を作製し、実験例１の（２）と同様にしてリンゲル液で２回洗浄した。この培養軟骨を培養容器の底面に接着させたまま、培養容器に５０ｍｌのリンゲル液を加え、培養軟骨をリンゲル液に浸漬した。その後、蓋を閉めて密閉し、１３℃の条件下で２４時間静置した。これを実験例２の培養軟骨とした。実験例２の培養軟骨に含まれるＢＳＡ量についても、上述の実験例１の（３）と同様にして測定試料を調製して実験例１の（４）と同様にして測定した。その結果を表１に示す。
[Experiment 2]
In Experimental Example 2, cultured rabbit cartilage was prepared in the same manner as in Experimental Example 1 (1) above, and washed twice with Ringer's solution in the same manner as in Experimental Example 1 (2). While the cultured cartilage was adhered to the bottom surface of the culture container, 50 ml of Ringer's solution was added to the culture container, and the cultured cartilage was immersed in the Ringer's solution. Thereafter, the lid was closed and sealed, and the mixture was allowed to stand at 13 ° C. for 24 hours. This was used as the cultured cartilage of Experimental Example 2. The amount of BSA contained in the cultured cartilage of Experimental Example 2 was also measured in the same manner as in Experimental Example 1 (4) by preparing a measurement sample in the same manner as in Experimental Example 1 (3). The results are shown in Table 1.
［実験例３］
実験例３では、上述の実験例１の（１）と同様にしてウサギの培養軟骨を作製し、実験例１の（２）と同様にしてリンゲル液で２回洗浄した。この培養軟骨を上述の実験例２と同様にして５０ｍｌのリンゲル液に浸漬し、蓋を閉めて密閉した。その後、培養容器をバイオシェーカー（タイテック社製）に設置して１５０ｒｐｍ、１３℃の条件下で２４時間振盪した。これを実験例３の培養軟骨とした。実験例３の培養軟骨に含まれるＢＳＡ量についても、上述の実験例１の（３）と同様にして測定試料を調製して実験例１の（４）と同様にして測定した。その結果を表１に示す。
[Experiment 3]
In Experimental Example 3, rabbit cultured cartilage was prepared in the same manner as in Experimental Example 1 (1), and washed twice with Ringer's solution in the same manner as in Experimental Example 1 (2). This cultured cartilage was immersed in 50 ml of Ringer's solution in the same manner as in Experimental Example 2 above, and the lid was closed and sealed. Thereafter, the culture container was placed in a bioshaker (manufactured by Taitec Co., Ltd.) and shaken under conditions of 150 rpm and 13 ° C. for 24 hours. This was used as the cultured cartilage of Experimental Example 3. The amount of BSA contained in the cultured cartilage of Experimental Example 3 was also measured in the same manner as in (4) of Experimental Example 1 by preparing a measurement sample in the same manner as in (3) of Experimental Example 1 described above. The results are shown in Table 1.
［実験例４］
実験例４では、上述の実験例１の（１）と同様にしてウサギの培養軟骨を作製し、実験例１の（２）と同様にしてリンゲル液で２回洗浄した。続いて、ピンセットを用いて培養軟骨を損傷させないように注意しながら培養容器の底面から完全に引き剥がした。培養軟骨を剥離した後、５０ｍｌのリンゲル液を注入し、培養軟骨をリンゲル液に浸漬した。その後、蓋を閉めて密閉し、１３℃の条件下で２４時間静置した。これを実験例４の培養軟骨とした。実験例４の培養軟骨に含まれるＢＳＡ量についても、上述の実験例１の（３）と同様にして測定試料を調製して実験例１の（４）と同様にして測定した。その結果を表１に示す。
[Experimental Example 4]
In Experimental Example 4, rabbit cultured cartilage was prepared in the same manner as in Experimental Example 1 (1) above, and washed twice with Ringer's solution in the same manner as in Experimental Example 1 (2). Subsequently, the tweezers were used to completely peel the cultured cartilage from the bottom, taking care not to damage the cultured cartilage. After exfoliating the cultured cartilage, 50 ml of Ringer's solution was injected, and the cultured cartilage was immersed in the Ringer's solution. Thereafter, the lid was closed and sealed, and the mixture was allowed to stand at 13 ° C. for 24 hours. This was designated as cultured cartilage of Experimental Example 4. The amount of BSA contained in the cultured cartilage of Experimental Example 4 was also measured in the same manner as in Experimental Example 1 (4) by preparing a measurement sample in the same manner as in Experimental Example 1 (3). The results are shown in Table 1.
［実験例５］
実験例５では、上述の実験例１の（１）と同様にしてウサギの培養軟骨を作製し、実験例１の（２）と同様にしてリンゲル液で２回洗浄した。続いて、上述の実験例４と同様にして培養軟骨を培養容器の底面から完全に剥離し、５０ｍｌのリンゲル液に浸漬した後、蓋を閉めて密閉した。その後、培養容器をバイオシェーカー（タイテック社製）に設置して１５０ｒｐｍ、１３℃の条件下で２４時間振盪した。これを実験例５の培養軟骨とした。実験例５の培養軟骨に含まれるＢＳＡ量についても、上述の実験例１の（３）と同様にして測定試料を調製して実験例１の（４）と同様にして測定した。その結果を表１に示す。
[Experimental Example 5]
In Experimental Example 5, rabbit cultured cartilage was prepared in the same manner as in Experimental Example 1 (1) above, and washed twice with Ringer's solution in the same manner as in Experimental Example 1 (2). Subsequently, the cultured cartilage was completely peeled off from the bottom surface of the culture vessel in the same manner as in Experimental Example 4 described above, immersed in 50 ml of Ringer's solution, and then sealed with a lid closed. Thereafter, the culture container was placed in a bioshaker (manufactured by Taitec Co., Ltd.) and shaken under conditions of 150 rpm and 13 ° C. for 24 hours. This was used as the cultured cartilage of Experimental Example 5. The amount of BSA contained in the cultured cartilage of Experimental Example 5 was also measured in the same manner as in (4) of Experimental Example 1 by preparing a measurement sample in the same manner as in (3) of Experimental Example 1 described above. The results are shown in Table 1.
［実験例１〜５に関するＢＳＡ除去効果の評価］
（１）振盪によるＢＳＡ除去効果の評価
まず、振盪の有無によるＢＳＡの除去効果を検討する。培養軟骨を培養容器の底面に接着させたまま２４時間静置した実験例２と、培養軟骨を培養容器の底面に接着させたまま２４時間振盪した実験例３とを比較すると、表１に示すように、実験例２（４５２μｇ／ｃｍ３）は、洗浄しただけの実験例１（４８３μｇ／ｃｍ３）と比べてＢＳＡ含有量がほとんど変わらず、培養軟骨からＢＳＡがほとんど除去されていないことがわかる。これに対し、実験例３（３１７μｇ／ｃｍ３）は、実験例１のＢＳＡ含有量の約６５％であり、培養軟骨から約半分のＢＳＡが効率的に除去されたと言える。また、培養軟骨を培養容器の底面から剥離して２４時間静置した実験例４と、培養軟骨を培養容器の底面から剥離して２４時間振盪した実験例５とを比較すると、実験例４（９１μｇ／ｃｍ３）に対して、実験例５（４５μｇ／ｃｍ３）は約５０％であり、同様のことが言える。このように、静置した状態で培養軟骨を洗浄液に浸漬しておくよりも振盪した状態で浸漬しておく方がＢＳＡを培養軟骨から効率よく除去することができる。また、このことから、ＦＢＳ由来タンパク質のうちＢＳＡ以外のタンパク質についても、静置した状態で培養軟骨を洗浄液に浸漬しておくよりも振盪した状態で浸漬しておく方が該タンパク質を効率よく除去することができると推測される。
（２）剥離によるＢＳＡ除去効果の評価
次に、培養軟骨を培養容器の底面から剥離したか否かによるＢＳＡの除去効果を検討する。表１において、培養軟骨を培養容器の底面に接着させたまま２４時間静置した実験例２と、培養軟骨を培養容器の底面から剥離して２４時間静置した実験例４とを比較すると、実験例２（４５２μｇ／ｃｍ３）は、実験例１（４８３μｇ／ｃｍ３）とＢＳＡ含有量がほとんど変わらず、培養軟骨からＢＳＡがほとんど除去されていないことがわかる。これに対し、実験例４（９１μｇ／ｃｍ３）は、実験例１のＢＳＡ含有量の約１９％であり、培養軟骨からＢＳＡが効率的に除去されたと言える。また、培養軟骨を培養容器の底面に接着させたまま２４時間振盪した実験例３と、培養軟骨を培養容器の底面から剥離して２４時間振盪した実験例５とを比較すると、実験例３（３１７μｇ／ｃｍ３）に対して、実験例５（４５μｇ／ｃｍ３）は約１４％であり、同様のことが言える。このように、培養軟骨を容器に接着したまま洗浄液に浸漬しておくよりも容器から剥離して浸漬しておく方がＢＳＡを培養軟骨から効率よく除去することができる。すなわち、三次元培養物が洗浄液に接触する面積を大きくすることによって、より効果的に異種動物成分を除去することができる。殊に、実験例１〜５に使用した形態の培養軟骨においては、培地と境界をなしていた面（外周面）は、培地による栄養供給がリッチであるため細胞が活性化しやすく、これらの細胞により密に産生された細胞外マトリクスと増殖により増加した細胞とによって被覆されていることが考えられるため、この外周面からの除去対象成分の除去効率が悪いところ、このように培地と境界をなしていた面以外の面を洗浄液に露出させることによって、極めて効率的に培地由来の異種成分が除去できたことが分かる。また、このことから、ＦＢＳ由来タンパク質のうちＢＳＡ以外のタンパク質についても、培養軟骨を容器に接着したまま洗浄液に浸漬しておくよりも容器から剥離して浸漬しておく方が該タンパク質を効率よく除去することができると推測される。
（３）振盪・剥離によるＢＳＡ除去効果の評価
最後に、培養軟骨を培養容器の底面から剥離し、かつ振盪処理を加えたことによるＢＳＡの除去効果を検討する。表１において、培養軟骨を培養容器の底面から剥離して２４時間振盪させた実験例５（４５μｇ／ｃｍ３）は、他の実験例１〜４と比較するとＢＳＡ含有量が最も少ない。したがって、培養軟骨を培養容器の底面から剥離したあと洗浄液に浸漬して振盪したときに、培養軟骨からＢＳＡが非常に効率的に除去されたと言える。また、ＦＢＳ由来タンパク質のうちＢＳＡ以外のタンパク質についても同様のことが言えると推測される。なお、実験例１から実験例５のすべてにおいて、精製工程後も十分な量の生細胞が維持されていることを確認している。また、実験例１〜５では、ゲル状の三次元スキャフォールドを用いて作製した培養軟骨についてＢＳＡ除去効果を確認したが、ゲル状の材料を用いて作製した他の三次元培養物、例えば、細胞を二次元的に培養して得た二次元培養物をフィブリンゲルなどの生物学的接着剤で複数積層したのちに更に培養して作製することにより得た三次元培養物についても同様のＢＳＡ除去効果が得られると推測される。
[Evaluation of BSA removal effect for Experimental Examples 1 to 5]
(1) Evaluation of BSA removal effect by shaking First, the BSA removal effect by the presence or absence of shaking is examined. Table 1 shows a comparison between Experimental Example 2 in which the cultured cartilage was allowed to stand for 24 hours while adhered to the bottom surface of the culture container and Experimental Example 3 that was shaken for 24 hours while the cultured cartilage was adhered to the bottom surface of the culture container. Thus, it can be seen that Experimental Example 2 (452 μg / cm 3) has almost no change in BSA content as compared to Experimental Example 1 (483 μg / cm 3) which has only been washed, and BSA is hardly removed from cultured cartilage. In contrast, Experimental Example 3 (317 μg / cm 3) was about 65% of the BSA content of Experimental Example 1, and it can be said that about half of the BSA was efficiently removed from the cultured cartilage. Further, when Experimental Example 4 in which the cultured cartilage was detached from the bottom of the culture container and allowed to stand for 24 hours was compared with Experimental Example 5 in which the cultured cartilage was detached from the bottom of the culture container and shaken for 24 hours, Experimental Example 4 ( In contrast to 91 μg / cm 3), Experimental Example 5 (45 μg / cm 3) is about 50%, and the same can be said. Thus, BSA can be efficiently removed from the cultured cartilage by immersing the cultured cartilage in a washing state rather than immersing the cultured cartilage in a washing solution. In addition, for this reason, proteins other than BSA among FBS-derived proteins can be efficiently removed by immersing cultured cartilage in a washing state rather than immersing in cultured washing cartilage. It is speculated that you can.
(2) Evaluation of BSA removal effect by peeling Next, the removal effect of BSA depending on whether or not the cultured cartilage is peeled from the bottom surface of the culture container is examined. In Table 1, when comparing Experimental Example 2 in which the cultured cartilage was allowed to adhere to the bottom surface of the culture container for 24 hours and Experimental Example 4 in which the cultured cartilage was detached from the bottom surface of the culture container and allowed to stand for 24 hours, In Experimental Example 2 (452 μg / cm 3), the BSA content hardly changes from Experimental Example 1 (483 μg / cm 3), and it can be seen that BSA is hardly removed from the cultured cartilage. On the other hand, Experimental Example 4 (91 μg / cm 3) is about 19% of the BSA content of Experimental Example 1, and it can be said that BSA was efficiently removed from the cultured cartilage. Further, when Experimental Example 3 in which the cultured cartilage was adhered to the bottom surface of the culture vessel and shaken for 24 hours was compared with Experimental Example 5 in which the cultured cartilage was peeled from the bottom surface of the culture vessel and shaken for 24 hours, Experimental Example 3 ( Experimental example 5 (45 μg / cm 3) is about 14% of 317 μg / cm 3), and the same can be said. As described above, BSA can be efficiently removed from the cultured cartilage by detaching from the container and immersing the cultured cartilage, rather than immersing the cultured cartilage in the washing liquid while adhering to the container. That is, by increasing the area where the three-dimensional culture comes into contact with the washing liquid, it is possible to more effectively remove foreign animal components. In particular, in the cultured cartilage in the form used in Experimental Examples 1 to 5, the surface (peripheral surface) that bounded the medium was rich in nutrient supply by the medium, so that the cells were easily activated. It is thought that it is covered by the extracellular matrix produced more densely and the cells increased by proliferation, so the removal efficiency of the components to be removed from this outer peripheral surface is poor, so it forms a boundary with the medium in this way It can be seen that by exposing the surface other than the previously exposed surface to the cleaning liquid, the different components derived from the medium could be removed very efficiently. In addition, for this reason, proteins other than BSA among the FBS-derived proteins are more efficiently removed and immersed from the container than by immersing the cultured cartilage in the washing solution while adhering to the container. It is assumed that it can be removed.
(3) Evaluation of BSA removal effect by shaking / peeling Finally, the removal effect of BSA by peeling the cultured cartilage from the bottom surface of the culture vessel and adding a shaking treatment is examined. In Table 1, Experimental Example 5 (45 μg / cm 3) in which the cultured cartilage was peeled from the bottom surface of the culture container and shaken for 24 hours had the smallest BSA content compared to the other Experimental Examples 1 to 4. Therefore, it can be said that BSA was removed from the cultured cartilage very efficiently when the cultured cartilage was peeled off from the bottom surface of the culture container and then immersed in a washing solution and shaken. It is also speculated that the same can be said for proteins other than BSA among FBS-derived proteins. In all of Experimental Examples 1 to 5, it was confirmed that a sufficient amount of living cells was maintained after the purification step. Moreover, in Experimental Examples 1-5, although the BSA removal effect was confirmed about the cultured cartilage produced using the gel-like three-dimensional scaffold, other three-dimensional cultures produced using the gel-like material, for example, The same BSA can be applied to a three-dimensional culture obtained by two-dimensional culture obtained by culturing cells two-dimensionally with a biological adhesive such as fibrin gel and then further culturing. It is estimated that a removal effect is obtained.
［実験例６］
実験例６では、ヒトの関節軟骨から上述の実験例１の（１）と同様にして培養軟骨を作製した。なお、作製したヒトの培養軟骨は、ウサギの場合と同様、培養容器の底面に接着した状態であった。培養軟骨を作製後、上述の実験例１の（２）と同様にして培養軟骨をリンゲル液で２回洗浄した。これを実験例６の培養軟骨とした。実験例６の培養軟骨に含まれるＢＳＡ量についても、上述の実験例１の（３）と同様にして測定試料を調製して実験例１の（４）と同様にして測定した。
[Experimental Example 6]
In Experimental Example 6, cultured cartilage was produced from human articular cartilage in the same manner as in Experimental Example 1 (1) above. The prepared cultured human cartilage was in a state of being adhered to the bottom surface of the culture container as in the case of rabbits. After producing cultured cartilage, the cultured cartilage was washed twice with Ringer's solution in the same manner as in Experimental Example 1 (2) above. This was used as the cultured cartilage of Experimental Example 6. The amount of BSA contained in the cultured cartilage of Experimental Example 6 was also measured in the same manner as in Experimental Example 1 (4) by preparing a measurement sample in the same manner as in Experimental Example 1 (3).
［実験例７］
実験例７では、上述の実験例６と同様にしてヒトの培養軟骨を作製した。培養軟骨を作製後、上述の実験例５と同様にして培養軟骨を培養容器の底面から剥離してリンゲル液に浸漬し、バイオシェーカーで２４時間振盪した。これを実験例７の培養軟骨とした。実験例７の培養軟骨に含まれるＢＳＡ量についても、上述の実験例１の（３）と同様にして測定試料を調製して実験例１の（４）と同様にして測定した。その結果、実験例７の培養軟骨では、実験例６の培養軟骨のＢＳＡ含有量に対するＢＳＡ含有量は１０％未満であった。このことから、ヒトの培養軟骨においてもウサギの場合と同様、培養軟骨を培養容器の底面から剥離したあと洗浄液に浸漬して振盪することにより、培養軟骨からＦＢＳ由来タンパク質を効率的に除去することができる。また、実験例７では、細胞懸濁液とアテロコラーゲンインプラントとの混合液を１００μｌマウントして培養した培養軟骨を用いたが、その他に３００μｌ，１０００μｌ，１５００μｌの混合液をマウントして培養した培養軟骨のそれぞれについて実験例７と同様にして培養軟骨の剥離及び洗浄液内での振盪を行ったところ、いずれの培養軟骨でも実験例６の培養軟骨のＢＳＡ含有量に対するＢＳＡ含有量は１０％未満であり、同様の効果が得られた。
[Experimental Example 7]
In Experimental Example 7, human cultured cartilage was prepared in the same manner as Experimental Example 6 described above. After producing the cultured cartilage, the cultured cartilage was peeled off from the bottom surface of the culture container and immersed in Ringer's solution in the same manner as in Experimental Example 5 described above, and shaken with a bioshaker for 24 hours. This was used as the cultured cartilage of Experimental Example 7. The amount of BSA contained in the cultured cartilage of Experimental Example 7 was also measured in the same manner as in Experimental Example 1 (4) by preparing a measurement sample in the same manner as in Experimental Example 1 (3). As a result, in the cultured cartilage of Experimental Example 7, the BSA content with respect to the BSA content of the cultured cartilage of Experimental Example 6 was less than 10%. Therefore, as in the case of rabbits in human cultured cartilage, the FBS-derived protein is efficiently removed from the cultured cartilage by detaching the cultured cartilage from the bottom surface of the culture container and then immersing it in a washing solution and shaking. Can do. In Experimental Example 7, cultured cartilage cultured with 100 μl of a mixed solution of cell suspension and atelocollagen implant was used. In addition, cultured cartilage cultured with 300 μl, 1000 μl, and 1500 μl of mixed liquid. For each of the samples, the cultured cartilage was peeled off and shaken in the washing solution in the same manner as in Experimental Example 7. As a result, the BSA content of the cultured cartilage in any of the cultured cartilages was less than 10%. The same effect was obtained.
本発明は、例えば、三次元培養物が軟骨に損傷を受けた患者から採取した軟骨細胞を培養したものであるときには、この三次元培養物を患者の欠損部に戻すための再生医療製品として再生医療分野に利用することができる。 For example, when the three-dimensional culture is a culture of chondrocytes collected from a patient with cartilage damage, the three-dimensional culture is regenerated as a regenerative medical product for returning the defect to the patient. It can be used in the medical field.
Claims (10)
（ａ）洗浄容器内で前記三次元培養物を前記除去対象成分を含まない洗浄液に浸漬する工程と、
（ｂ）前記工程（ａ）の前、前記工程（ａ）のあと又は前記工程（ａ）と並行して前記三次元培養物から前記除去対象成分の少なくとも一部が前記洗浄液に溶出するのを促進させる工程と、
を含む三次元培養物の精製方法。 A three-dimensional culture purification method for removing the removal target component derived from the medium from a three-dimensional culture obtained by culturing cells to be cultured using a medium containing the removal target component,
(A) a step of immersing the three-dimensional culture in a cleaning solution that does not contain the component to be removed in a cleaning container;
(B) Before the step (a), after the step (a) or in parallel with the step (a), at least a part of the removal target component is eluted from the three-dimensional culture into the washing solution. Promoting the process;
A method for purifying a three-dimensional culture comprising
請求項１に記載の三次元培養物の精製方法。 The step (b) is a step of promoting the elution of at least a part of the removal target component from the three-dimensional culture into the cleaning liquid by shaking the cleaning container.
The method for purifying a three-dimensional culture according to claim 1.
請求項１又は２に記載の三次元培養物の精製方法。 In the step (b), at least a part of the component to be removed from the three-dimensional culture is exposed to the washing solution by exposing a portion of the three-dimensional culture other than the surface that has bounded the medium at the time of culture. Is a step of promoting elution into the cleaning solution,
The method for purifying a three-dimensional culture according to claim 1 or 2.
請求項３に記載の三次元培養物の精製方法。 In the step (b), the three-dimensional culture is peeled off from the culture surface on which the three-dimensional culture is produced, and the removed surface is exposed to the washing solution to expose the component to be removed from the three-dimensional culture. A step of accelerating at least a part of the elution into the cleaning solution,
The method for purifying a three-dimensional culture according to claim 3.
請求項３に記載の三次元培養物の精製方法。 In the step (b), a cut surface or a perforation is provided in the three-dimensional culture, and the cut surface or the perforation is exposed to the cleaning liquid, whereby at least a part of the component to be removed from the three-dimensional culture is transferred to the cleaning liquid. It is a process to promote elution,
The method for purifying a three-dimensional culture according to claim 3.
請求項１〜５のいずれかに記載の三次元培養物の精製方法。 The component to be removed is a component obtained from a heterogeneous animal that is a different type of animal from the animal that is the source of the cell to be cultured, or the same animal that is the same type of animal as the source of the cell to be cultured. It is at least one selected from the group consisting of components and antibiotics obtained from different individuals.
The method for purifying a three-dimensional culture according to any one of claims 1 to 5.
請求項１〜６のいずれかに記載の三次元培養物の精製方法。 The three-dimensional culture is produced by holding and culturing cells to be cultured in a three-dimensional scaffold.
The method for purifying a three-dimensional culture according to any one of claims 1 to 6.
請求項７に記載の三次元培養物の精製方法。 In the three-dimensional culture, a mixed solution obtained by mixing a solution that forms a gel-like three-dimensional scaffold and cells to be cultured is placed on the culture surface, and the placed mixed solution is gelled. Produced by
The method for purifying a three-dimensional culture according to claim 7.
請求項１〜６のいずれかに記載の三次元培養物の精製方法。 The three-dimensional culture is produced by stacking a plurality of two-dimensional cultures obtained by two-dimensionally culturing cells to be cultured, and further culturing using the medium.
The method for purifying a three-dimensional culture according to any one of claims 1 to 6.
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