JP6684252B2 - Method and device for separating stromal cells from living tissue without using enzyme - Google Patents
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Description
本発明は、生体組織から間質細胞を分離する方法及び装置に係り、詳細には、酵素を使用せず、生体組織から間質細胞を分離する方法及び装置に関する。 The present invention relates to a method and a device for separating stromal cells from living tissue, and more particularly to a method and a device for separating stromal cells from living tissue without using an enzyme.
動物の生体組織中に存在する間質細胞を生体組織から分離する方法としては、酵素を使用する方法及び酵素を使用しない方法がある。 As a method for separating stromal cells existing in a living tissue of an animal from the living tissue, there are a method using an enzyme and a method not using an enzyme.
酵素を使用して動物の生体組織から間質細胞を分離する方法は、初期分離段階で酵素を使用する方法、または収獲や継代(subculture)段階で酵素を使用する方法がある。 The method of separating stromal cells from the living tissue of an animal using an enzyme includes a method of using the enzyme in the initial separation stage, or a method of using the enzyme in the harvesting or subculture stage.
生体組織から間質細胞を分離する初期段階では、生体組織で間質細胞を取り囲んで強く付着しているコラーゲン(collagen)組織を、コラゲナーゼ(collagenase)のような酵素を使用することによって、生体組織から間質細胞を溶かし、このように溶かした間質細胞から酵素を洗浄した後、間質細胞を得る。しかし、その場合、酵素の毒性と費用、処理時間、異種ウイルスに係わる恐れなどの問題点が発生する。 In the initial stage of separating stromal cells from living tissue, collagen (collagen) tissue that surrounds stromal cells and is strongly attached to living tissue is used by using an enzyme such as collagenase (collagenase). The stromal cells are obtained by lysing the stromal cells from and lysing the enzyme from the thus lysed stromal cells. However, in that case, there are problems such as toxicity and cost of the enzyme, processing time, and fear of being associated with a heterologous virus.
生体組織から間質細胞を分離する前記初期段階で分離された間質細胞を培養器で増殖する場合、細胞密度(confluency)が高くなって継代を行わねばならないときには、トリプシン(trypsin)のような酵素を使用することで継代を行う。しかし、その際に使用される酵素は、異種動物の胃液から抽出された成分なので、安定性が劣る問題点が発生する。 When stromal cells separated in the initial stage of separating stromal cells from living tissue are grown in an incubator, when the cell density (confluency) becomes high and passaging is required, trypsin (trypsin) is used. Subculture is performed by using different enzymes. However, since the enzyme used in that case is a component extracted from the gastric juice of a different animal, it has a problem of poor stability.
酵素を使用せず、動物の生体組織から間質細胞を分離する方法は、初期分離段階では、超音波やレーザ、または強い負圧などを用いて生体組織を微細に切断することにより、コラーゲンを破壊し、遠心分離を通じて、間質細胞を分離する。しかし、この場合、間質細胞が、コラーゲンから完全に分離される確率が極めて低く、間質細胞の損傷が大きくなって、酵素を使用する方法に比べて、収率が5%未満であり、工程が複雑になる問題点がある。 The method of separating stromal cells from the living tissue of an animal without using an enzyme is to cut collagen in the initial separation stage by finely cutting the living tissue using ultrasonic waves, laser, or strong negative pressure. Disrupt and separate stromal cells through centrifugation. However, in this case, the stromal cells are extremely unlikely to be completely separated from collagen, the damage of the stromal cells is large, and the yield is less than 5% as compared with the method using the enzyme, There is a problem that the process becomes complicated.
酵素を使用せず、動物の生体組織から間質細胞を分離する方法は、収獲または継代段階では、分離された間質細胞を培養増殖するために、比重の異なるマイクロビードの表面に細胞を増殖し、このように細胞が増殖されたマイクロビードを液体と共に混合することを繰り返して、マイクロビードを互いに衝突させることにより、離脱する細胞を収去するか、または平面に間質細胞を培養増殖した後、スクレーパ(scraper)で間質細胞を掻き出す。しかし、この場合、マイクロビードが球状なので、互いに衝突過程においてマイクロビード表面に増殖された細胞離脱効果を極大化することができず、スクレーパで掻き出して離脱する細胞の数が多くなく、かつ掻き出す過程で細胞が損傷されるという問題点がある。 A method of separating stromal cells from animal living tissue without using an enzyme is to collect cells on the surface of microbeads having different specific gravities in order to culture and proliferate the separated stromal cells at the harvesting or passage stage. By repeating the mixing of the microbeads that have proliferated and thus proliferated with the liquid, and colliding the microbeads with each other, the detached cells are removed or the stromal cells are cultured and proliferated on a flat surface. After that, stromal cells are scraped out with a scraper. However, in this case, since the microbeads are spherical, it is not possible to maximize the cell detachment effect propagated on the surface of the microbeads in the collision process with each other, and the number of cells to be detached by scraping with a scraper is not large and the process of scratching There is a problem that cells are damaged by.
一方、脂肪組織のような生体組織を培養させたものは、培養液に浮かぶ特性があるという点を用いて、培養液でいっぱい満たされている容器に、脂肪組織を入れることにより、培養液に浮かんで培養容器の上部内側面に培養された脂肪組織の付着を誘導する。しかし、この場合、培養された脂肪組織の付着面は、平面状であって、培養された脂肪組織の付着面を極大化することができず、培養効率を極大化することができないという問題点がある。 On the other hand, what is obtained by culturing a biological tissue such as adipose tissue has the characteristic that the culture solution floats. Therefore, by putting the adipose tissue in a container filled with the culture solution, Floating induces the attachment of cultured adipose tissue on the upper inner surface of the culture vessel. However, in this case, the adhering surface of the cultured adipose tissue is flat, and the adhering surface of the cultured adipose tissue cannot be maximized, and the culture efficiency cannot be maximized. There is.
本発明が解決しようとする主要課題は、酵素を使用せず、微細に切断した生体組織を培養液に入れ、生体組織においてコラーゲンに取り囲まれている間質細胞が自発的マイグレーション(spontaneous migration)によって生体組織の外部への移動を誘導することにより、酵素の毒性と費用、処理時間、異種ウイルスに係わる恐れなどの問題点と、異種動物の胃液から抽出された成分を有する酵素の使用による不安定性の問題点を解消すると共に、生体組織から間質細胞を、酵素を使用せず、損傷無く、相対的に完全な自然状態の間質細胞として分離して、分離効率を向上させうる方法及び装置を提供することである。 The main problem to be solved by the present invention is to use, without using an enzyme, a finely cut living tissue put in a culture solution, and stromal cells surrounded by collagen in the living tissue are spontaneously migrated (spontaneous migration). By inducing migration of living tissue to the outside, problems such as toxicity and cost of the enzyme, processing time, fear of being associated with a heterologous virus, and instability due to the use of an enzyme having a component extracted from gastric juice of a heterologous animal And a device capable of improving the separation efficiency by solving the above problems and separating stromal cells from living tissue as relatively complete natural stromal cells without using enzymes and without damage. Is to provide.
本発明が解決しようとする他の主要課題は、培養液内で自発的マイグレーションによって生体組織の外部に移動した間質細胞を、生体組織で効率よくスクレイピング(scraping)することによって、酵素の毒性と費用、処理時間、異種ウイルスに係わる恐れなどの問題点と、異種動物の胃液から抽出された成分を有する酵素の使用による不安定性の問題点を解消すると共に、生体組織から間質細胞の分離効率を向上させうる方法及び装置を提供することである。 The other main problem to be solved by the present invention is to efficiently scrape stromal cells that have moved to the outside of the living tissue by spontaneous migration in the culture medium (scraping) in the living tissue, resulting in the toxicity of the enzyme. It solves problems such as cost, processing time, fear of heterologous virus, and instability due to the use of enzymes having components extracted from the gastric juice of heterologous animals, and the efficiency of separating stromal cells from living tissues. It is to provide a method and an apparatus capable of improving
前記課題を解決するための本発明の一実施例は、酵素を使用せず、生体組織から間質細胞を分離する方法であって、生体組織の間質細胞の自発的マイグレーション(spontaneous migration)を誘導して、間質細胞を生体組織の外部に移動させることを利用し、間質細胞の自発的マイグレーションの誘導は、生体組織を付着させうる素材の付着部材上に生体組織が付着した状態でなされ、間質細胞の自発的マイグレーションの誘導は、間質細胞が生存可能な培養液内でなされることを特徴とする方法を提供する。 One embodiment of the present invention for solving the above problems is a method for separating stromal cells from a living tissue without using an enzyme, which is a spontaneous migration of stromal cells of a living tissue (spontaneous migration). By inducing and migrating stromal cells to the outside of living tissue, induction of spontaneous migration of stromal cells is performed in the state where living tissue is attached to an attachment member of a material capable of attaching living tissue. Done, the induction of spontaneous migration of stromal cells provides a method characterized in that the stromal cells are made in a viable medium.
本実施例において、生体組織において間質細胞を取り囲むコラーゲンの間に間質細胞の少なくとも一部が外部に露出されるように生体組織を微細に切断することをさらに含んでも良い。 The present embodiment may further include finely cutting the living tissue so that at least a part of the stromal cells is exposed to the outside between the collagen surrounding the stromal cells in the living tissue.
本実施例において、付着部材の外部に移動した間質細胞を生体組織から分離することをさらに含んでもよく、間質細胞の分離は、付着部材に付着した間質細胞に物理的力を加えてなっても良い。 In this example, it may further include separating the stromal cells that have moved to the outside of the attachment member from the biological tissue, the separation of the stromal cells, by applying a physical force to the stromal cells attached to the attachment member. It can be.
本実施例において、生体組織の外部に移動した間質細胞に加える物理的力は、培養液内で生体組織の外部に移動した間質細胞を培養液と共に乱流運動させることにより発生する力であっても良い。 In this example, the physical force applied to the stromal cells that have moved to the outside of the biological tissue is the force generated by turbulent movement of the stromal cells that have moved to the outside of the biological tissue in the culture medium together with the culture medium. It may be.
本実施例において、生体組織から分離された間質細胞を収集することをさらに含んでも良い。 In this example, the method may further include collecting the stromal cells separated from the biological tissue.
前記課題を解決するための本発明の他の実施例は、酵素を使用せず、生体組織から間質細胞を分離する方法であって、(1)生体組織を微細に切断すること；(2)微細に切断した生体組織を培養液内でその生体組織が付着可能な素材の付着部材上に付着させること；(3)付着部材上で間質細胞の自発的マイグレーションを誘導して、間質細胞を生体組織の外部に移動させること;及び(4)生体組織の外部に移動した間質細胞を付着部材から分離すること；を含むことを特徴とする方法を提供する。 Another embodiment of the present invention for solving the above-mentioned problems is a method for separating stromal cells from a living tissue without using an enzyme, which comprises (1) cutting the living tissue into fine pieces; ) Attaching finely cut living tissue to an attachment member made of a material to which the living tissue can be attached in a culture solution; (3) Inducing spontaneous migration of stromal cells on the attachment member to form stroma And (4) separating the interstitial cells that have moved to the outside of the living tissue from the attachment member.
本実施例において、(1)の生体組織は、その生体組織において間質細胞を取り囲むコラーゲンの間に間質細胞の少なくとも一部が外部に露出されるように微細に切断されても良い。 In the present example, the living tissue of (1) may be finely cut so that at least a part of the stromal cells is exposed to the outside between the collagen surrounding the stromal cells in the living tissue.
本実施例において、(4)の間質細胞の分離は、生体組織の外部に移動した間質細胞が配列された複数の付着部材を培養液の乱流運動によって互いに衝突させて、間質細胞に物理的力を加えてなっても良い。 In the present example, (4) the separation of stromal cells, the stromal cells are caused to collide with each other by a turbulent motion of the culture solution of a plurality of attachment members in which the stromal cells that have moved to the outside of the biological tissue are arranged. You may add physical power to.
本実施例において、(5)生体組織から分離された間質細胞を収集することをさらに含んでも良い。 This example may further include (5) collecting the stromal cells separated from the biological tissue.
本実施例において、(2)ないし(4)を順次繰り返しても良い。 In this embodiment, (2) to (4) may be sequentially repeated.
本実施例において、生体組織、培養液、及び付着部材からなる群のうち、選択された少なくとも１つを入れ替えた後、(2)ないし(4)を順次繰り返しても良い。 In the present example, after replacing at least one selected from the group consisting of the biological tissue, the culture solution, and the attachment member, (2) to (4) may be sequentially repeated.
前記実施例において、生体組織は、皮膚、脂肪、軟骨、粘膜、血管、靭帯、心臓、脳、胎盤、臍帶、羊膜、筋肉、及び末梢神経からなる群のうち、選択された少なくともいずれか１つを含んでも良い。 In the above-mentioned embodiment, the biological tissue is at least one selected from the group consisting of skin, fat, cartilage, mucous membrane, blood vessel, ligament, heart, brain, placenta, umbilicus, amnion, muscle, and peripheral nerve. May be included.
前記実施例において、培養液は、DMEM(Dulbecco’s Modified Eagle’s Medium)及びウシ胎児血清(fetal bovine serum)からなる群のうち、選択された少なくともいずれか１つを含んでも良い。 In the above embodiment, the culture medium may include at least one selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium) and fetal bovine serum.
前記課題を解決するための本発明のさらに他の実施例は、酵素を使用せず、生体組織から間質細胞を分離する装置であって、培養液内で生体組織を付着させ、生体組織の間質細胞の自発的マイグレーションを誘導して、間質細胞を生体組織の外部に移動させる付着部材を備え、付着部材は、培養液よりもさらに小さな平均比重を有するか、または培養液よりもさらに大きな平均比重を有することを特徴とする装置を提供する。 Yet another embodiment of the present invention for solving the above problems is an apparatus for separating stromal cells from a living tissue without using an enzyme, wherein the living tissue is adhered in a culture solution, An attachment member that induces spontaneous migration of stromal cells to move the stromal cells to the outside of the biological tissue is provided, and the attachment member has an average specific gravity smaller than that of the culture medium, or more than that of the culture medium. Provided is a device having a large average specific gravity.
本実施例において、付着部材が培養液よりもさらに小さな平均比重を有する場合、その付着部材は、ポリプロピレン(polypropylene)、ポリエチレン(polyethylene)、ポリウレタン(polyurethane)、ECM(Extracellular Matrix)、コラーゲン(collagen)、ポリジオキサノン(polydioxanone)、ポリカプロラクトン (polycaprolactone)、PLLA(poly(L-lactide))、PLGA(poly(lactic-co-glycolic acid))、PLA(poly(lactic acid))、PGA(pterolyglutamic acid)、ヒアルロン酸(hyaluronic acid)及びシリコンからなる群のうち、選択された少なくともいずれか１つを含んでも良い。 In the present example, when the attachment member has an average specific gravity smaller than that of the culture solution, the attachment member is polypropylene (polypropylene), polyethylene (polyethylene), polyurethane (polyurethane), ECM (Extracellular Matrix), collagen (collagen). , Polydioxanone (polydioxanone), polycaprolactone (polycaprolactone), PLLA (poly (L-lactide)), PLGA (poly (lactic-co-glycolic acid)), PLA (poly (lactic acid)), PGA (pterolyglutamic acid), At least one selected from the group consisting of hyaluronic acid and silicon may be included.
本実施例において、付着部材が培養液よりもさらに大きな平均比重を有する場合、その付着部材は、テフロン（登録商標）(teflon)、ポリカーボネート(polycarbonate)、ポリエチレン(polyethylene)、フタレート(phthalate)、ポリスチレン(polystyrene)、ポリウレタン(polyurethane)、ECM(Extracellular Matrix)、コラーゲン(collagen)、ポリジオキサノン(polydioxanone)、ポリカプロラクトン (polycaprolactone)、PLLA(poly(L-lactide))、PLGA(poly(lactic-co-glycolic acid))、PLA(poly(lactic acid))、PGA(pterolyglutamic acid)、ヒアルロン酸(hyaluronic acid)及びシリコンからなる群のうち、選択された少なくともいずれか１つを含んでも良い。 In this example, when the attachment member has a larger average specific gravity than the culture solution, the attachment member is Teflon (registered trademark) (teflon), polycarbonate (polycarbonate), polyethylene (polyethylene), phthalate (phthalate), polystyrene. (polystyrene), polyurethane (polyurethane), ECM (Extracellular Matrix), collagen (collagen), polydioxanone (polydioxanone), polycaprolactone (polycaprolactone), PLLA (poly (L-lactide)), PLGA (poly (lactic-co-glycolic) acid)), PLA (poly (lactic acid)), PGA (pterolyglutamic acid), hyaluronic acid, and at least one selected from the group consisting of silicon.
本実施例において、付着部材は、生体組織において間質細胞を取り囲むコラーゲンの間に間質細胞の少なくとも一部が外部に露出されるように、微細に切断した生体組織を付着させるものであっても良い。 In the present Example, the attachment member attaches the finely cut biological tissue so that at least a part of the stromal cells is exposed to the outside between the collagen surrounding the stromal cells in the biological tissue. Is also good.
本実施例において、付着部材は、培養液内で生体組織を付着させ、生体組織の間質細胞の自発的マイグレーションを誘導して、間質細胞を生体組織の外部に移動させ、かつ該移動された間質細胞を生体組織から分離させるものであっても良い。 In the present example, the attachment member attaches the biological tissue in the culture medium, induces spontaneous migration of the stromal cells of the biological tissue, moves the stromal cells to the outside of the biological tissue, and The stromal cells may be separated from the living tissue.
本実施例において、付着部材は、自発的マイグレーションによって生体組織の外部に移動した間質細胞が配列される領域をなす本体と、本体から外側に延びて本体の厚さよりもさらに肉薄であって、他の付着部材に配列された間質細胞をスクレイピング(scraping)可能な形状を有するスクレイピング部と、を備えても良い。 In this example, the attachment member is a body that forms an area in which stromal cells that have moved to the outside of the biological tissue due to spontaneous migration are arranged, and extends outward from the body, and is thinner than the thickness of the body, And a scraping portion having a shape capable of scraping stromal cells arranged on another attachment member.
本実施例において、スクレイピング部は、該横断面のコーナー部の角度が鋭角をなしても良い。 In the present embodiment, the scraping portion may have an acute angle at the corner portion of the cross section.
本実施例において、培養液、生体組織、及び付着部材を内部に収容する容器をさらに備えても良い。 In the present embodiment, a container for accommodating the culture solution, the biological tissue, and the attachment member may be further provided.
本実施例において、容器は、培養液、生体組織、及び付着部材が収容される空間を形成し、回転による遠心分離が可能なように形成された傾斜部を有しても良い。 In this embodiment, the container may have a slanted portion that forms a space in which the culture solution, the biological tissue, and the attachment member are accommodated, and that can be centrifuged by rotation.
本実施例において、容器は、正転及び逆転による培養液の乱流を誘導可能なものであっても良い。 In this embodiment, the container may be capable of inducing a turbulent flow of the culture solution due to normal rotation and reverse rotation.
本実施例において、容器は、生体組織は透過されず、培養液は透過され、容器が静止された状態で培養液に浸漬される位置に配列され、生体組織が培養液に浮かぶことを遮断する遮断膜をさらに備えても良い。 In the present embodiment, the container is arranged at a position where the biological tissue is not permeated, the culture solution is permeated, and the container is immersed in the culture solution in a stationary state, and the biological tissue is prevented from floating in the culture solution. A blocking film may be further provided.
本実施例において、容器は、遠心力が最大である位置に形成され、生体組織から分離された間質細胞を遠心力によって収斂させる収斂部を有しても良い。 In the present embodiment, the container may have a converging portion which is formed at a position where the centrifugal force is maximum and which causes the stromal cells separated from the biological tissue to be converged by the centrifugal force.
本実施例において、容器は、収容空間から収斂部までの経路上に位置して、遠心力による間質細胞の移動を許容し、生体組織及び付着部材の移動を遮断するフィルタをさらに備えても良い。 In the present embodiment, the container is further provided with a filter that is located on the path from the accommodation space to the converging portion, allows the movement of stromal cells by centrifugal force, and blocks the movement of the biological tissue and the attachment member. good.
本実施例において、容器は収斂部に形成され、収斂部に収斂された間質細胞を外部に排出させる間質細胞排出部をさらに備えても良い。 In the present embodiment, the container may further include a stromal cell discharging part formed in the converging part and discharging the stromal cells converged in the converging part to the outside.
本実施例において、容器は、外部から収容空間に延びて培養液を注入するか、排出させる培養液貫通管をさらに備えても良い。 In this embodiment, the container may further include a culture medium penetrating tube that extends from the outside into the accommodation space to inject or discharge the culture medium.
本実施例において、容器は、内部消毒用のガスを注入させるガス注入部をさらに備えても良い。 In this embodiment, the container may further include a gas injection unit for injecting a gas for internal disinfection.
本発明の主要効果は、酵素を使用せず、微細に切断した生体組織を培養液に入れ、生体組織においてコラーゲンに取り囲まれている間質細胞が自発的マイグレーションによって生体組織の外側面に移動するように誘導することで、酵素の毒性と費用、処理時間、異種ウイルスに係わる恐れなどの問題点と、異種動物の胃液から抽出された成分を有する酵素の使用による不安定性の問題点とを解消すると共に、生体組織から間質細胞を、酵素を使用せず、損傷無く、相対的に完全な自然状態の間質細胞に分離して、分離効率を向上させる方法及び装置を提供することができる。 The main effect of the present invention is to put finely cut living tissue into a culture solution without using an enzyme, and stromal cells surrounded by collagen in the living tissue move to the outer surface of the living tissue by spontaneous migration. By inducing in this way, problems such as toxicity and cost of enzyme, treatment time, fear of being related to heterologous virus, etc. and instability due to use of enzyme having components extracted from gastric juice of heterologous animal are solved. In addition, it is possible to provide a method and a device for separating stromal cells from living tissue into relatively complete natural stromal cells without using an enzyme and without damage, thereby improving the separation efficiency. .
本発明の他の主な効果は、培養液内で自発的マイグレーションによって生体組織の外側面に移動した間質細胞を生体組織で効率よくスクレイピング(scraping)することで、酵素の毒性と費用、処理時間、異種ウイルスに係わる恐れなどの問題点と、異種動物の胃液から抽出された成分を有する酵素の使用による不安定性の問題点を解消すると共に、生体組織から間質細胞の分離効率を向上させる方法、及び装置を提供することができる。 Another main effect of the present invention is to efficiently scrape stromal cells that have moved to the outer surface of living tissue in the culture medium by spontaneous migration in the culture solution (enzyme toxicity and cost, treatment). Improves the efficiency of separating stromal cells from living tissues while solving problems such as time and fear of heterologous virus and instability due to the use of an enzyme having a component extracted from gastric juice of a heterologous animal. Methods and apparatus can be provided.
発明を実施するための具体的な内容を説明する。このような説明は、発明が属する技術分野で、通常の知識を有する者に発明を実施するための具体的な内容を理解させるために例示的に提供されるものであって、多様な他の形で変形可能なので、本発明の範囲が以下の説明によって限定されるものではない。 Specific contents for carrying out the invention will be described. Such description is provided as an example in order for a person having ordinary knowledge to understand specific contents for carrying out the invention in the technical field to which the invention belongs, and various other explanations are given. The scope of the present invention is not limited by the following description because it can be modified in shape.
1.酵素を使用せず、生体組織から間質細胞を分離する方法
本方法は、酵素を使用せず、生体組織から間質細胞を分離する方法であって、生体組織の間質細胞の自発的マイグレーション(spontaneous migration)を誘導して、間質細胞を生体組織の外部に移動させることを利用することを特徴とする。
1. Method for separating stromal cells from living tissue without using an enzyme This method is a method for separating stromal cells from a living tissue without using an enzyme, in which spontaneous stromal cells of living tissue are used. It is characterized by utilizing the fact that the stromal cells are moved to the outside of the living tissue by inducing spontaneous migration.
生体組織は、皮膚、脂肪、軟骨、粘膜、血管、靭帯、心臓、脳、胎盤、臍帶、羊膜、筋肉、及び末梢神経からなる群のうち、選択された少なくともいずれか１つを含むことができる。 The biological tissue can include at least one selected from the group consisting of skin, fat, cartilage, mucous membrane, blood vessel, ligament, heart, brain, placenta, umbilicus, amniotic membrane, muscle, and peripheral nerve. .
生体組織の間質細胞の自発的マイグレーションは、生体組織内で間質細胞を取り囲むコラーゲン(collagen)を突き抜いて、間質細胞が自発的に外部に移動することを意味する。このような間質細胞の自発的マイグレーションによって、生体組織から続けて間質細胞が伸びているイメージは、図1に例示したようである。 Spontaneous migration of stromal cells in living tissue means that the stromal cells spontaneously move to the outside by penetrating the collagen surrounding the stromal cells in the living tissue. The image of stromal cells continuously extending from the biological tissue due to such spontaneous migration of stromal cells is as illustrated in FIG.
このような間質細胞の自発的なマイグレーションによる間質細胞の生体組織外部への移動は、酵素を使用せず、生体組織から間質細胞を分離するための非常に重要な特性となりうる。本発明は、生体組織の間質細胞の自発的マイグレーションを誘導して、間質細胞を生体組織の外部に移動させることを利用することで、酵素を使用せずとも、生体組織から間質細胞を分離することができる方法を提供する。 The migration of stromal cells to the outside of living tissue due to such spontaneous migration of stromal cells can be a very important property for separating stromal cells from living tissue without using an enzyme. INDUSTRIAL APPLICABILITY The present invention utilizes spontaneous migration of stromal cells of living tissue to move the stromal cells to the outside of the living tissue. To provide a method by which can be separated.
間質細胞の自発的マイグレーションは、生体組織を付着させうる素材の部材に生体組織を付着させた状態で、さらに効率よくなされる。例えば、図2に例示されているように、クロミック縫合糸(chromic catgut)に脂肪組織が付着されたとき、脂肪細胞の自発的マイグレーションによって、脂肪組織の外部に脂肪細胞が移動して付着することができる。このように間質細胞の自発的マイグレーションは、生体組織を付着させうる素材の部材に、生体組織を付着させた状態で誘導されることにより、間質細胞の自発的マイグレーションをさらに効率よく誘導することができ、これにより、生体組織から間質細胞をさらに効率よく分離可能となる。ここで、生体組織を付着させうる素材の部材としては、多様な部材があり得る。例えば、クロミック縫合糸と同素材の部材となりうる。 The spontaneous migration of stromal cells is more efficiently performed in a state where the living tissue is attached to a member made of a material to which the living tissue can be attached. For example, as illustrated in Figure 2, when adipose tissue is attached to a chromic catgut, the spontaneous migration of adipocytes causes the adipocytes to move and attach to the outside of the adipose tissue. You can Thus, the spontaneous migration of stromal cells is more efficiently induced by the induction of the spontaneous migration of stromal cells by being guided to the member of the material to which the biological tissue can be adhered with the biological tissue attached. Therefore, stromal cells can be more efficiently separated from the living tissue. Here, various members can be used as the member made of the material to which the living tissue is attached. For example, it can be a member made of the same material as the chromic suture.
間質細胞の自発的マイグレーションの誘導は、間質細胞が生存可能な培養液内でなされることが望ましい。これにより、生体組織の外部に移動した間質細胞を培養液と共に分離することにより、間質細胞を損傷なしに収集して培養可能となる。 Induction of spontaneous migration of stromal cells is preferably performed in a culture medium in which stromal cells can survive. As a result, by separating the stromal cells that have moved to the outside of the living tissue together with the culture medium, the stromal cells can be collected and cultured without damage.
培養液は、DMEM(Dulbecco’s Modified Eagle’s Medium)及びウシ胎児血清(fetal bovine serum)からなる群のうち、選択された少なくともいずれか１つを含むことができる。 The culture medium may contain at least one selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium) and fetal bovine serum.
培養液は、生体組織と平均比重が同じものを使用することで、培養液に全体として生体組織を分布させて、自発的マイグレーションによって生体組織の外部に移動した間質細胞を、培養液と共に効率よく分離可能となる。 By using a culture medium that has the same average specific gravity as that of the living tissue, the living tissue is distributed as a whole in the culture medium, and the stromal cells that have moved to the outside of the living tissue due to spontaneous migration are efficiently used together with the culture medium. Well separable.
もし間質細胞の自発的マイグレーションが、培養液内で生体組織を付着させうる素材の部材に生体組織を付着させた状態でなされれば、培養液は、付着部材及び生体組織と平均比重が同じものを使用したり、または付着部材及び生体組織よりも平均比重がさらに大きなものを使用したりすることが望ましい。これは、この場合、培養液内で付着部材及び生体組織は、全体として分布されるか、または培養液の水面近くに付着部材及び生体組織が分布されることにより、付着部材に生体組織が接触する可能性がさらに大きくなり、付着部材に生体組織がさらに効率よく付着し、間質細胞の自発的マイグレーションがさらに効率よく誘導されるからである。 If spontaneous migration of stromal cells is performed in a state in which biological tissue is attached to a member of a material capable of attaching biological tissue in the culture medium, the culture medium has the same average specific gravity as the attachment member and the biological tissue. It is desirable to use a material having a larger average specific gravity than that of the attaching member or the living tissue. This means that in this case, the attachment member and the living tissue are distributed as a whole in the culture medium, or the attachment member and the living tissue are distributed near the water surface of the culture liquid so that the attachment member and the living tissue come into contact with each other. This is because the possibility of further increase is further increased, the biological tissue is more efficiently attached to the attachment member, and the spontaneous migration of stromal cells is induced even more efficiently.
生体組織は、間質細胞を取り囲むコラーゲンの間に間質細胞の少なくとも一部が外部に露出されるように、微細に切断した状態で付着部材に付着することがさらに望ましい。これにより、生体組織において、間質細胞の自発的マイグレーションをさらに誘導して、生体組織から間質細胞をさらに効率よく分離することができるからである。この際、かような生体組織の切断は、レーザなどを用いて行われる。 It is further desirable that the living tissue be attached to the attachment member in a finely cut state so that at least a part of the stromal cells is exposed to the outside between the collagen surrounding the stromal cells. Thereby, in the living tissue, the spontaneous migration of the stromal cells can be further induced, and the stromal cells can be more efficiently separated from the living tissue. At this time, such cutting of the living tissue is performed using a laser or the like.
間質細胞の自発的マイグレーションによって、間質細胞が生体組織の外部に移動した後には、間質細胞に物理的力を加えることで、間質細胞を生体組織から分離することができる。例えば、培養液内で間質細胞が自発的マイグレーションによって生体組織の外部に移動した後には、間質細胞を培養液と共に乱流運動させることにより、間質細胞に物理的力を加えて間質細胞を生体組織から分離することができる。もし培養液内の付着部材上で間質細胞が自発的マイグレーションによって生体組織の外部に移動した場合には、付着部材を培養液と共に乱流運動させることにより、付着部材上の間質細胞に物理的力を加えるか、または複数の付着部材を培養液と共に乱流運動させて、互いに衝突させることにより、付着部材上の間質細胞にさらに強い物理的力を加えて、間質細胞を生体組織から効率よく分離可能となる。 After the stromal cells migrate to the outside of the living tissue due to the spontaneous migration of the stromal cells, the stromal cells can be separated from the living tissue by applying a physical force to the stromal cells. For example, after the stromal cells move to the outside of the living tissue by spontaneous migration in the culture medium, the stromal cells are subjected to turbulent motion together with the culture medium to apply physical force to the stromal cells to cause the stromal cells to move. Cells can be separated from living tissue. If the stromal cells on the adherent member in the culture medium move to the outside of the living tissue due to spontaneous migration, the adherent member is turbulently moved together with the culture medium to physically transform the stromal cells on the adherent member. Mechanical force is applied to the stromal cells on the adherent members, and the stromal cells on the adherent members are subjected to turbulent motion together with the culture solution to cause stromal cells to turbulently move with each other. Can be efficiently separated from.
間質細胞は、生体組織から分離した後には、外部に収集される。かように収集された間質細胞は、培養液内で培養または継代培養を通じて増殖されうる。もし間質細胞が培養液内で生体組織から分離されるならば、間質細胞は、培養液と共に収集されて、さらに効率よく分離及び培養、または継代培養可能となる。 Stromal cells are collected outside after being separated from living tissue. The stromal cells thus collected can be grown in culture through culture or subculture. If the stromal cells are separated from the living tissue in the culture medium, the stromal cells can be collected together with the culture medium and can be further efficiently separated and cultured, or subcultured.
2.酵素を使用せず、生体組織から間質細胞を分離する装置
本装置は、酵素を使用せず、生体組織から間質細胞を分離する装置であって、培養液内で生体組織を付着させ、生体組織の間質細胞の自発的マイグレーションを誘導して、間質細胞を生体組織の外部に移動させる付着部材を備えることを特徴とする。
2.A device that separates stromal cells from living tissue without using an enzyme This device is a device that separates stromal cells from living tissue without using an enzyme and attaches living tissue in a culture solution. The present invention is characterized by including an attachment member that induces spontaneous migration of stromal cells of living tissue and moves the stromal cells to the outside of the living tissue.
付着部材の素材は、培養液内で生体組織を付着させ、生体組織の間質細胞の自発的マイグレーションを誘導して、間質細胞を生体組織の外部に移動させるように多様になされうる。例えば、付着部材は、生体組織を安定的に、または効率的に付着させるために、生体組織と同一であるか、類似した素材からなっても良い。もし付着部材の表面の少なくとも一部に生体組織を付着させた状態で付着部材を培養液内に配列させれば、培養液内に分布された生体組織は、同じ組織が付着した付着部材の表面にさらに容易に付着する。 The material of the attachment member may be variously made to attach the living tissue in the culture medium, induce spontaneous migration of the stromal cells of the living tissue, and move the stromal cells to the outside of the living tissue. For example, the attachment member may be made of the same material as or a similar material to the living tissue in order to attach the living tissue stably or efficiently. If the adhering members are arranged in the culture medium with the biological tissue adhered to at least a part of the surface of the adhering member, the biological tissues distributed in the culture liquid will be the surface of the adhering member to which the same tissue adheres. More easily attached to.
付着部材の形状は、培養液内で生体組織を付着させ、生体組織の間質細胞の自発的マイグレーションを誘導して、間質細胞を生体組織の外部に移動させるように多様になされうる。例示として図3に示されたように、付着部材110は、微細に切断した多数の生体組織120が付着し、付着した多数の生体組織120の間質細胞130が自発的マイグレーションによって生体組織120の外部に移動して配列される領域をなす表面を有する本体111からなる。このため、付着部材110は、全体として扁平な立方体の形状からなる。例示として図3に示されている付着部材110は、四角立方体の形状からなっているが、これに限定されず、付着部材は、円立方体、三角立方体、五角立方体など多様な形状からなりうる。 The shape of the attachment member may be variously configured so that the living tissue is attached in the culture medium, the spontaneous migration of the stromal cells of the living tissue is induced, and the stromal cells are moved to the outside of the living tissue. As shown in FIG. 3 as an example, the attachment member 110 has a large number of finely cut living tissues 120 attached thereto, and the stromal cells 130 of the plurality of attached living tissues 120 of the living tissue 120 are spontaneously migrated. It is composed of a main body 111 having a surface forming a region arranged to move to the outside. Therefore, the attachment member 110 has a flat cube shape as a whole. The attachment member 110 shown in FIG. 3 as an example has a rectangular cube shape, but the present invention is not limited thereto, and the attachment member may have various shapes such as a circular cube, a triangular cube, and a pentagonal cube.
付着部材110の厚さtは、微細に切断した多数の生体組織120が付着し、培養液の乱流運動に耐えられる剛性を有するように、厚くかつ培養液の乱流運動によって円滑に動くように薄くなされることが望ましい。 The thickness t of the attachment member 110 is thick and smooth so that a large number of finely cut biological tissues 120 are attached and have rigidity that can withstand the turbulent motion of the culture solution. It is desirable to make it thin.
もし付着部材の表面の少なくとも一部に凹凸形状が形成されれば、生体組織が付着部材の表面と接触する面積が大きくなり、さらにと容易に付着可能となる。 If the uneven shape is formed on at least a part of the surface of the attachment member, the area where the living tissue comes into contact with the surface of the attachment member increases, and the attachment becomes easier.
付着部材の比重は、培養液内で生体組織を付着させ、生体組織の間質細胞の自発的マイグレーションを誘導して、間質細胞を生体組織の外部に移動させるように多様になされうる。 The specific gravity of the attachment member may be varied so as to attach the living tissue in the culture medium, induce spontaneous migration of the stromal cells of the living tissue, and move the stromal cells to the outside of the living tissue.
もし、生体組織が培養液よりも平均比重がさらに小さく、培養液の水面近くにほとんど分布するようになれば、付着部材は、培養液よりも平均比重がさらに小さくすることが望ましい。これは、この場合、付着部材も生体組織と同様に、培養液の水面近くにほとんど分布するので、付着部材と生体組織との接触可能性がさらに大きくなり、付着部材上に生体組織の付着可能性がさらに大きくなるからである。したがって、この場合、生体組織の間質細胞が付着部材上で自発的マイグレーションによって生体組織の外部への移動可能性がさらに大きくなる。 If the biological tissue has an average specific gravity smaller than that of the culture medium and is almost distributed near the water surface of the culture medium, the attachment member preferably has an average specific gravity smaller than that of the culture medium. This is because, in this case, the adhesive member is almost distributed near the water surface of the culture solution as well as the biological tissue, so that the possibility of contact between the adhesive member and the biological tissue is further increased, and the biological tissue can be adhered to the adhesive member. This is because the sex becomes even larger. Therefore, in this case, the stromal cells of the living tissue are more likely to move to the outside of the living tissue by spontaneous migration on the attachment member.
付着部材が培養液よりも平均比重がさらに小さくなる場合、付着部材は、ポリプロピレン(polypropylene)、ポリエチレン(polyethylene)、ポリウレタン(polyurethane)、ECM(Extracellular Matrix)、コラーゲン(collagen)、ポリジオキサノン(polydioxanone)、ポリカプロラクトン (polycaprolactone)、PLLA(poly(L-lactide))、PLGA(poly(lactic-co-glycolic acid))、PLA(poly(lactic acid))、PGA(pterolyglutamic acid)、ヒアルロン酸(hyaluronic acid)及びシリコンからなる群のうち、選択された少なくともいずれか１つを含むものであり得る。 When the average specific gravity of the attachment member is smaller than that of the culture solution, the attachment member includes polypropylene (polypropylene), polyethylene (polyethylene), polyurethane (polyurethane), ECM (Extracellular Matrix), collagen (collagen), polydioxanone (polydioxanone), Polycaprolactone, PLLA (poly (L-lactide)), PLGA (poly (lactic-co-glycolic acid)), PLA (poly (lactic acid)), PGA (pterolyglutamic acid), hyaluronic acid And at least one selected from the group consisting of silicon and silicon.
もし、生体組織が培養液よりも平均比重がさらに大きく、培養液の底面近くにほとんど分布するならば、付着部材は、培養液よりも平均比重がさらに大きくすることが望ましい。これは、この場合、付着部材も生体組織と同様に、培養液の底面近くにほとんど分布するので、付着部材の生体組織との接触可能性がさらに大きくなり、付着部材上への生体組織の付着可能性がさらに大きくなるからである。したがって、この場合、生体組織の間質細胞が付着部材上で自発的マイグレーションによって生体組織の外部への移動可能性がさらに大きくなる。 If the biological tissue has an average specific gravity higher than that of the culture medium and is almost distributed near the bottom surface of the culture medium, the attachment member preferably has an average specific gravity higher than that of the culture medium. This is because, in this case, the adhesive member is distributed almost in the vicinity of the bottom surface of the culture solution as well as the biological tissue, so that the possibility of contact of the adhesive member with the biological tissue is further increased, and the adhesion of the biological tissue on the adhesive member This is because the possibility becomes even greater. Therefore, in this case, the stromal cells of the living tissue are more likely to move to the outside of the living tissue by spontaneous migration on the attachment member.
付着部材が培養液よりも平均比重がさらに大きくなる場合、付着部材は、テフロン(teflon)、ポリカーボネート(polycarbonate)、ポリエチレン(polyethylene)、フタレート(phthalate)、ポリスチレン(polystyrene)、ポリウレタン(polyurethane)、ECM(Extracellular Matrix)、コラーゲン(collagen)、ポリジオキサノン(polydioxanone)、ポリカプロラクトン (polycaprolactone)、PLLA(poly(L-lactide))、PLGA(poly(lactic-co-glycolic acid))、PLA(poly(lactic acid))、PGA(pterolyglutamic acid)、ヒアルロン酸(hyaluronic acid)及びシリコンからなる群のうち、選択された少なくともいずれか１つを含みうる。 When the average specific gravity of the attachment member is larger than that of the culture medium, the attachment member includes Teflon, polycarbonate (polycarbonate), polyethylene (polyethylene), phthalate (phthalate), polystyrene (polystyrene), polyurethane (polyurethane), ECM. (Extracellular Matrix), collagen (collagen), polydioxanone (polydioxanone), polycaprolactone (polycaprolactone), PLLA (poly (L-lactide)), PLGA (poly (lactic-co-glycolic acid)), PLA (poly (lactic acid) )), PGA (pterolyglutamic acid), hyaluronic acid, and at least one selected from the group consisting of silicon.
もし、生体組織が培養液と平均比重が同一であって、培養液の全体に分散して分布するならば、付着部材は、培養液と平均比重を同一にすることが望ましい。これは、この場合、付着部材も生体組織と同様に、培養液の全体に分散して分布されるので、付着部材の生体組織との接触可能性がさらに大きくなり、付着部材上への生体組織の付着可能性がさらに大きくなるからである。したがって、この場合、生体組織の間質細胞が付着部材上で、自発的マイグレーションによって生体組織の外部への移動可能性がさらに大きくなる。 If the biological tissue has the same average specific gravity as the culture medium and is dispersed and distributed throughout the culture medium, the attachment member preferably has the same average specific gravity as the culture medium. This is because, in this case, since the attachment member is also dispersed and distributed throughout the culture solution like the living tissue, the possibility of contact between the attachment member and the living tissue is further increased, and the living tissue on the attachment member is increased. This is because the possibility of adherence of is further increased. Therefore, in this case, the stromal cells of the living tissue are more likely to move to the outside of the living tissue on the attachment member due to spontaneous migration.
付着部材に付着する生体組織は、生体組織において間質細胞を取り囲むコラーゲンの間に、間質細胞の少なくとも一部が外部に露出されるように微細に切断した生体組織であることが望ましい。これにより、間質細胞の自発的マイグレーションがさらに望ましく誘導されて、間質細胞が生体組織でさらに望ましく分離されるからである。 The living tissue attached to the attachment member is preferably a living tissue finely cut so that at least a part of the stromal cells is exposed to the outside between the collagen surrounding the stromal cells in the living tissue. This is because the spontaneous migration of stromal cells is more desirably induced and the stromal cells are more desirably separated in the living tissue.
付着部材は、培養液内で生体組織を付着させ、生体組織の間質細胞の自発的マイグレーションを誘導して、間質細胞を生体組織の外部に移動させるだけではなく、自発的マイグレーションによって生体組織の外部に移動した間質細胞を生体組織から分離させうる。これにより、付着部材は、間質細胞が自発的マイグレーションによって生体組織の外部に移動するだけではなく、生体組織の外部に移動した間質細胞の生体組織からの分離も促進しうる。 The attachment member not only causes living tissue to adhere in the culture solution and induces spontaneous migration of stromal cells of the living tissue to move the stromal cells to the outside of the living tissue, but also the living tissue by spontaneous migration. Stromal cells that have migrated to the outside of the tissue can be separated from living tissue. Accordingly, the attachment member can promote not only the stromal cells to move to the outside of the living tissue by spontaneous migration but also the separation of the stromal cells that move to the outside of the living tissue from the living tissue.
付着部材が自発的マイグレーションによって生体組織の外部に移動した間質細胞を、生体組織から分離させることは多様になされうる。 The stromal cells that the attachment member has moved to the outside of the living tissue by spontaneous migration can be variously separated from the living tissue.
例えば、付着部材は、付着部材上で生体組織の外部に移動した間質細胞をスクレイピング(scraping)する役割をする。これにより、間質細胞は、物理的力を受けて生体組織から分離されて培養液内に移動する。このため、例示として図4に示されたように、付着部材210は、生体組織220が付着して自発的マイグレーションによって生体組織220の外部に移動した間質細胞230が配列される領域をなす本体211以外に、本体211から外側に延びて本体211の厚さtよりもさらに肉薄であって、他の付着部材に配列された間質細胞をスクレイピングすることができる形状を有するスクレイピング部212をさらに備えることができる。このようなスクレイピング部212は、該横断面のコーナー部の角度が90°よりも小さな鋭角からなりうる。このようなスクレイピング部212による自発的マイグレーションによって生体組織220から分離され、生体組織220の外側面に、または付着部材210上に配列されている間質細胞230を生体組織220、または付着部材210から容易に分離して培養液内に移動させうる。例示として図5に示されたように、第1付着部材210上で生体組織220から分離されて、生体組織220の外側面に、または付着部材210上に配列されている間質細胞230は、培養液の乱流運動と共に、乱流運動を行う隣接する第2付着部材210’との衝突過程で第2本体211’の厚さt’よりもさらに肉薄であって、外側に延びた第2スクレイピング部212’によって容易にスクレイピングされることにより、生体組織220または付着部材210から容易に分離されて培養液内への移動が可能となる。第1スクレイピング部212も他の付着部材上に配列されている間質細胞をスクレイピングする役割をする。第1スクレイピング部212と第2スクレイピング部212’は、それぞれ第1本体211の厚さtと第2本体211’の厚さt’よりもさらに小さな厚さを有するように、該横断面のコーナー部の角度A、A’が90°よりも小さな鋭角をなしている。但し、スクレイピング部の形状は、例示として図4及び図5に示されている形状に限定されず、付着部材上に配列された間質細胞を分離することができる他の多様な形状を有しても良い。 For example, the attachment member serves to scrape the stromal cells that have moved to the outside of the living tissue on the attachment member. As a result, the stromal cells receive physical force, are separated from the living tissue, and move into the culture medium. Therefore, as shown in FIG. 4 as an example, the attachment member 210 is a main body that forms a region in which the stromal cells 230 to which the biological tissue 220 is attached and moved to the outside of the biological tissue 220 by spontaneous migration are arranged. In addition to 211, a scraping portion 212 that extends outward from the main body 211, is thinner than the thickness t of the main body 211, and has a shape capable of scraping stromal cells arranged on another attachment member is further provided. Can be prepared. The scraping portion 212 may have an acute angle with a corner portion of the cross section smaller than 90 °. The stromal cells 230, which are separated from the living tissue 220 by the spontaneous migration by the scraping portion 212 and are arranged on the outer surface of the living tissue 220 or on the attachment member 210, are separated from the living tissue 220 or the attachment member 210. It can be easily separated and moved into the culture medium. As shown in FIG. 5 as an example, the stromal cells 230 that are separated from the biological tissue 220 on the first attachment member 210, are arranged on the outer surface of the biological tissue 220 or on the attachment member 210, Along with the turbulent motion of the culture solution, in the process of collision with the adjacent second adhering member 210 ′ that performs the turbulent motion, the second body 211 ′ is thinner than the thickness t ′ of the second main body 211 ′ and extends outward. By being easily scraped by the scraping section 212 ′, it can be easily separated from the living tissue 220 or the attachment member 210 and moved into the culture solution. The first scraping part 212 also plays a role of scraping the stromal cells arranged on another attachment member. The first scraping part 212 and the second scraping part 212 ′ have corners of the cross section so as to have a thickness smaller than the thickness t of the first body 211 and the thickness t ′ of the second body 211 ′, respectively. Angles A and A'of the part are acute angles smaller than 90 °. However, the shape of the scraping portion is not limited to the shape shown in FIGS. 4 and 5 as an example, and has various other shapes capable of separating stromal cells arranged on the attachment member. May be.
本装置は、培養液、生体組織、及び付着部材を内部に収容する容器をさらに備えることができる。 The apparatus can further include a container that accommodates the culture solution, the biological tissue, and the attachment member inside.
容器は、培養液、生体組織、及び付着部材が収容される空間を備える。例示として図6に示されたように、容器350は、培養液340、生体組織320、及び付着部材310、311が収容される収容空間351を備えることができる。但し、収容空間351は、これに限定されず、培養液340、生体組織320、及び付着部材310、311が収容される多様な形状を有することができる。 The container includes a space in which the culture solution, the biological tissue, and the attachment member are stored. As shown in FIG. 6 as an example, the container 350 can include a storage space 351 in which the culture fluid 340, the biological tissue 320, and the attachment members 310 and 311 are stored. However, the accommodation space 351 is not limited to this, and may have various shapes in which the culture solution 340, the living tissue 320, and the attachment members 310 and 311 are accommodated.
収容空間351は、回転による遠心分離が可能なように形成された傾斜部352を有しており、上方へ行くほど半径が大きくなる円形横断面を有する。収容空間351の上部は、蓋353で覆われている。蓋353は、収容空間351に収容された培養液340、生体組織320、及び付着部材310、311が外部に漏出されないようにする機能する。さらに、蓋353は、容器350の正転及び逆転による培養液340の乱流運動によって、培養液340の外部への漏出を遮断する機能する。蓋353は、収容空間351の傾斜部352とは反対の傾斜を有する傾斜部を有し、上方へ行くほど半径が小くなる円形横断面を有することが、回転による遠心分離に有利であり得る。 The accommodating space 351 has an inclined portion 352 formed so as to be centrifugally separated by rotation, and has a circular cross section in which the radius increases as it goes upward. The upper part of the accommodation space 351 is covered with a lid 353. The lid 353 functions to prevent the culture solution 340, the living tissue 320, and the attachment members 310 and 311 contained in the accommodation space 351 from leaking to the outside. Further, the lid 353 functions to block the leakage of the culture solution 340 to the outside due to the turbulent motion of the culture solution 340 due to the normal rotation and the reverse rotation of the container 350. The lid 353 may have an inclined portion having an inclination opposite to the inclined portion 352 of the accommodation space 351, and may have a circular cross section having a radius that becomes smaller as it goes upward, which may be advantageous for centrifugal separation by rotation. .
容器は、いずれか一方向への正転と反対方向への逆転を繰り返して、収容空間内の培養液の乱流を誘導することができる。このような培養液の乱流によって、生体組織及び付着部材も乱流運動をする。これにより、付着部材上に付着した生体組織で自発的マイグレーションによって生体組織の外部に移動することにより、付着部材上に配列されている間質細胞が生体組織または付着部材から分離されて、培養液内に分離される。また、容器は、いずれか一方向に回転して、培養液内に分布された間質細胞に遠心力を加えることにより、間質細胞を生体組織及び付着部材と分離させ、培養液と共に収集可能にする。 The container can repeat the forward rotation in any one direction and the reverse rotation in the opposite direction to induce a turbulent flow of the culture solution in the accommodation space. Due to such a turbulent flow of the culture solution, the living tissue and the attachment member also make a turbulent motion. As a result, the living tissue attached on the attachment member moves to the outside of the living tissue by spontaneous migration, so that the stromal cells arranged on the attachment member are separated from the living tissue or the attachment member, and the culture solution. Separated inside. In addition, the container can be rotated in any one direction and centrifugal force is applied to the stromal cells distributed in the culture medium to separate the stromal cells from the living tissue and the attachment member and collect them together with the culture medium. To
容器は、それが静止された状態で収容空間内の培養液に浸漬される位置に配列されている遮断膜をさらに備えても良い。遮断膜は、培養液よりも平均比重がさらに小さく、培養液に浮かぶ生体組織が培養液の水面上に浮かぶことを遮断することにより、生体組織が付着部材と接触して付着部材に付着可能にする。このため、遮断膜は、生体組織は透過されず、培養液は透過される貫通孔を備える。このような遮断膜の貫通孔を通じて、付着部材が透過されなくなる。 The container may further include a blocking membrane arranged at a position where the container is immersed in the culture solution in the storage space in a stationary state. The blocking membrane has an average specific gravity smaller than that of the culture medium, and blocks the living tissue floating in the culture medium from floating on the water surface of the culture medium, so that the living tissue can contact the attachment member and adhere to the attachment member. To do. For this reason, the barrier membrane has a through hole that does not allow permeation of biological tissue but permeation of the culture solution. The adhesive member is not permeated through the through hole of the barrier film.
例示として図6に示されたように、遮断膜360は、容器350が静止状態で収容空間351内部の培養液340の水面よりも下の水面近傍のように培養液340に浸漬される位置に配列される。遮断膜360には、複数の貫通孔が形成されているが、このような貫通孔は、培養液340は透過されるが、生体組織320及び付着部材310、311は透過されないように調節された大きさを有する。遮断膜360下部の培養液340内部に注入された生体組織320は、培養液340よりも平均比重が小さい場合、遮断膜360によって遮断されて培養液340の水面上に浮かばず、遮断膜360の下側近傍に集まる。遮断膜360の下部の培養液340内部に注入された付着部材310、311のうち、培養液340よりも平均比重の小さい付着部材311は、遮断膜360によって遮断され、培養液340の水面上に浮かばず、遮断膜360の下側近傍に集まる。もし、生体組織320が付着部材311よりも平均比重が小さい場合、付着部材311は、生体組織320が集まっている層の下側に集まる。したがって、生体組織320は、付着部材311と隣接するか、重畳して層を形成して、付着部材311との接触可能性が大幅増加し、付着部材311への付着可能性が大幅に増加する。結果として、付着部材311に付着した生体組織320において、間質細胞が自発的マイグレーションによって生体組織320の外部への移動可能性が大幅に増加する。 As shown in FIG. 6 as an example, the blocking film 360 is located at a position where the container 350 is immersed in the culture liquid 340 in a stationary state, as in the vicinity of the water surface below the water surface of the culture liquid 340 inside the accommodation space 351. Arranged. A plurality of through-holes are formed in the blocking film 360. The through-holes are adjusted so that the culture solution 340 can be permeated but the living tissue 320 and the attachment members 310 and 311 are not permeated. Has a size. When the average specific gravity of the biological tissue 320 injected into the culture solution 340 below the blocking film 360 is smaller than that of the culture solution 340, the biological tissue 320 is blocked by the blocking film 360 and does not float on the water surface of the culture solution 340. Collect near the lower side. Of the adhesive members 310 and 311 injected into the culture solution 340 below the blocking film 360, the adhesive member 311 having an average specific gravity smaller than that of the culture solution 340 is blocked by the blocking film 360 and is on the water surface of the culture solution 340. It does not float and gathers near the lower side of the blocking film 360. If the biological tissue 320 has an average specific gravity smaller than that of the attachment member 311, the attachment member 311 gathers under the layer where the living tissue 320 gathers. Therefore, the biological tissue 320 is adjacent to the adhesive member 311 or forms a layer in an overlapping manner to significantly increase the possibility of contact with the adhesive member 311 and significantly increase the likelihood of adhesion to the adhesive member 311. . As a result, in the living tissue 320 attached to the attaching member 311, the possibility of stromal cells migrating to the outside of the living tissue 320 due to spontaneous migration is significantly increased.
また、遮断膜は、培養液の乱流によって付着部材が乱流運動を行うとき、付着部材の培養液上部への離脱を遮断することにより、付着部材上で生体組織の外部に移動した間質細胞の培養液外部への離脱を遮断する役割もする。 In addition, the blocking film blocks the stroma that has moved to the outside of the biological tissue on the attachment member by blocking the detachment of the attachment member to the upper part of the culture solution when the attachment member performs turbulent motion due to the turbulent flow of the culture solution. It also serves to block the detachment of cells from the culture medium.
容器は、遠心力が最大である位置に形成され、生体組織から分離された間質細胞を遠心力によって収斂させる収斂部を有することができる。これにより、生体組織から分離された間質細胞を収斂して、外部に容易に排出可能となる。 The container may have a converging portion that is formed at a position where the centrifugal force is maximum and that causes the stromal cells separated from the biological tissue to converge by the centrifugal force. This allows the stromal cells separated from the living tissue to converge and be easily discharged to the outside.
例示として図6に示されたように、収斂部354a、354bは、収容空間351の上縁部のうち、遠心力が最大である位置に配列された第1収斂部354aと、収容空間351の上縁部のうち、第1収斂部354aと対称になりつつ、遠心力が最大である位置に配列された第2収斂部354bを備える。第1収斂部354a及び第2収斂部354bは、それぞれ培養液340と共に収斂された間質細胞を収容する空間を有する。このような第1及び第2収斂部354a、354bは、遠心力が最大である位置に配列されているので、容器350の回転による遠心分離がなされるとき、生体組織320から分離されて培養液340に分布されている間質細胞が培養液340と共に収斂されるように誘導する。収斂部は、図6の例示に限定されず、１つまたは三つ以上の収斂部からなりうる。 As shown in FIG. 6 as an example, the converging portions 354a and 354b include the first converging portion 354a arranged at a position where the centrifugal force is the maximum in the upper edge portion of the accommodation space 351, and the accommodation space 351. A second convergence part 354b is arranged at a position where the centrifugal force is maximum while being symmetrical with respect to the first convergence part 354a in the upper edge part. The first converging unit 354a and the second converging unit 354b each have a space for accommodating the stromal cells that have been converged together with the culture medium 340. Since the first and second convergent portions 354a and 354b are arranged at the position where the centrifugal force is the maximum, when the centrifugation is performed by rotating the container 350, the culture liquid is separated from the biological tissue 320. Stromal cells distributed in 340 are induced to converge with culture 340. The converging unit is not limited to the example of FIG. 6, and may include one or three or more converging units.
容器は、収容空間から収斂部までの経路上に位置して、遠心力による間質細胞の移動を許容し、生体組織及び付着部材の移動を遮断するフィルタをさらに備えることができる。これにより、収斂部には、間質細胞が分布された培養液は収斂され、生体組織及び付着部材は収斂されないように、調節可能となる。 The container may be further provided with a filter that is located on the path from the accommodation space to the converging portion, allows the stromal cells to move by centrifugal force, and blocks the movement of the biological tissue and the attachment member. As a result, the culture solution in which the stromal cells are distributed is converged in the convergent portion, and the living tissue and the attachment member can be adjusted so as not to be converged.
例示として図6に示されたように、フィルタ370a、370bは、収容空間351から第1収斂部354aまでの経路上に位置する第1フィルタ370aと、収容空間351から第2収斂部354bまでの経路上に位置する第1フィルタ370bを備える。第1フィルタ370aは、第1収斂部354aの入口に位置し、第2フィルタ370bは、第2収斂部354bの入口に位置する。第1及び第2フィルタ370a、370bには、複数の貫通孔が形成されているが、このような貫通孔は、間質細胞が分布された培養液340は貫通されるようにし、生体組織320及び付着部材310、311は貫通されないように調節されている大きさを有する。これにより、第1及び第2フィルタ370a、370bは、間質細胞が分布された培養液340の移動を許容し、生体組織320及び付着部材310、320の移動を遮断することにより、第1及び第2収斂部354a、354bに生体組織320及び付着部材310、311を除き、間質細胞が分布された培養液340を収斂させる。フィルタは、図6の例示に限定されず、収容空間から収斂部までの経路上の多様な位置に配列されうる。 As shown in FIG. 6 as an example, the filters 370a and 370b include a first filter 370a located on the path from the accommodation space 351 to the first converging portion 354a and a housing space 351 to the second converging portion 354b. A first filter 370b located on the path is provided. The first filter 370a is located at the entrance of the first convergence section 354a, and the second filter 370b is located at the entrance of the second convergence section 354b. A plurality of through holes are formed in the first and second filters 370a, 370b. The through holes allow the culture medium 340 in which the stromal cells are distributed to penetrate, and the biological tissue 320. Also, the attachment members 310 and 311 have a size adjusted so as not to be penetrated. Thereby, the first and second filters 370a, 370b allow the movement of the culture medium 340 in which the stromal cells are distributed, and by blocking the movement of the living tissue 320 and the attachment members 310, 320, the first and second The living tissue 320 and the attachment members 310 and 311 are removed from the second converging portions 354a and 354b to condense the culture solution 340 in which the stromal cells are distributed. The filter is not limited to the example of FIG. 6, and may be arranged at various positions on the path from the accommodation space to the converging unit.
容器は、収斂部に形成され、収斂部に収斂された間質細胞を外部に排出させる間質細胞排出部をさらに備えることができる。これにより、収斂部に収斂された間質細胞を外部に容易に排出させうる。 The container may further include a stromal cell discharge part formed in the convergent part and discharging the stromal cells converged in the convergent part to the outside. As a result, the stromal cells converged in the converging part can be easily discharged to the outside.
例示として図6に示されたように、間質細胞排出部355a、355bは、第1収斂部354aに形成され、第1収斂部354aに収斂された間質細胞を外部に排出させる第1間質細胞排出部355aと、第2収斂部354bに形成され、第2収斂部354bに収斂された間質細胞を外部に排出させる第2間質細胞排出部355bを備える。第1間質細胞排出部355aは、第1収斂部354aに収斂された間質細胞が分布された培養液340を外部に排出させ、第2間質細胞排出部355bは、第2収斂部354bに収斂された間質細胞が分布された培養液340を外部に排出させる。第1及び第2間質細胞排出部355a、355bは、第1及び第2収斂部354a、354bに形成された排出口に連結されたチューブからなりうる。間質細胞排出部は、図6の例示に限定されず、収斂部に形成され、収斂部に収斂された間質細胞を外部に排出させうる多様な構成からなりうる。 As shown in FIG. 6 as an example, the stromal cell discharge parts 355a and 355b are formed in the first convergent part 354a, and the stromal cells converged in the first convergent part 354a are discharged to the outside. A stromal cell discharge part 355a and a second stromal cell discharge part 355b formed in the second convergent part 354b and discharging the stromal cells converged by the second convergent part 354b to the outside are provided. The first stromal cell discharging part 355a discharges the culture solution 340 in which the stromal cells converged in the first converging part 354a are distributed to the outside, and the second stromal cell discharging part 355b is the second converging part 354b. The culture solution 340 in which the stromal cells converged on the cells are distributed is discharged to the outside. The first and second stromal cell discharge parts 355a and 355b may be tubes connected to discharge ports formed in the first and second convergence parts 354a and 354b. The stromal cell discharging part is not limited to the example shown in FIG. 6, and may have various structures formed in the converging part and capable of discharging the stromal cells converged in the converging part to the outside.
容器は、外部から収容空間に延びて培養液を注入するか、排出させる培養液貫通管をさらに備えることができる。これにより、収容空間に培養液を注入するか、排出させることを容易に行える。 The container may further include a culture medium penetrating tube that extends from the outside into the accommodation space to inject or discharge the culture medium. This makes it easy to inject or discharge the culture solution into the storage space.
例示として図6に示されたように、培養液貫通管380は、外部から蓋353を貫通して収容空間351内部に延びたチューブを備える。培養液貫通管380は、蓋353及び遮断膜360の中央部を順次貫通して、収容空間351の中央底面近くまで延びる。培養液貫通管380が貫通する蓋353の中央部には、ゴム材のシーリング部材390が配列されることにより、培養液貫通管380が貫通する収容空間351の中央部を堅固に密閉させうる。培養液貫通管380によって蓋353を開封せずとも、外部から収容空間351に培養液を容易に注入するか、排出することにより、必要に応じて、容易に培養液の注入または入れ替えが可能となる。培養液貫通管は、図6の例示に限定されず、外部から収容空間に延びて培養液を注入するか、排出させる多様な構成からなりうる。 As shown in FIG. 6 as an example, the culture medium penetrating tube 380 includes a tube that penetrates the lid 353 from the outside and extends into the accommodation space 351. The culture medium penetrating tube 380 sequentially penetrates through the central portions of the lid 353 and the blocking film 360, and extends to near the central bottom surface of the accommodation space 351. By arranging a sealing member 390 made of a rubber material in the central portion of the lid 353 through which the culture solution penetration tube 380 penetrates, it is possible to firmly seal the central portion of the accommodation space 351 through which the culture solution penetration tube 380 penetrates. Even if the lid 353 is not opened by the culture solution penetrating tube 380, it is possible to easily inject or replace the culture solution from outside by easily injecting or discharging the culture solution into the accommodation space 351. Become. The culture medium penetrating tube is not limited to the example shown in FIG. 6, and may have various configurations that extend from the outside into the accommodation space to inject or discharge the culture medium.
容器は、内部消毒用のガスを注入させるガス注入部をさらに備えることができる。これにより、容器内部の消毒を容易に行える。 The container may further include a gas injection unit for injecting a gas for internal disinfection. This makes it possible to easily disinfect the inside of the container.
3.実施例
本発明の一実施例は、酵素を使用せず、生体組織から間質細胞を分離する方法であって、(1)生体組織を微細に切断する段階と、(2)微細に切断した生体組織を培養液内でその生体組織が付着可能な素材の付着部材上に付着させる段階と、(3)付着部材上で間質細胞の自発的マイグレーションを誘導して、間質細胞を生体組織の外部に移動させる段階と、(4)生体組織の外部に移動した間質細胞を、生体組織から分離する段階と、(5)生体組織から分離された間質細胞を収集する段階と、を含む。
3.Examples One example of the present invention is a method for separating stromal cells from a living tissue without using an enzyme, comprising the steps of (1) finely cutting the living tissue, and (2) finely cutting it. The step of attaching the cut biological tissue in a culture solution onto an attachment member made of a material to which the biological tissue can attach, and (3) inducing spontaneous migration of stromal cells on the attachment member, A step of migrating to the outside of the living tissue, (4) a step of separating the stromal cells that have moved to the outside of the living tissue from the living tissue, and a step of collecting the (5) stromal cells separated from the living tissue. ,including.
(1)生体組織を微細に切断する段階は、生体組織において間質細胞を取り囲むコラーゲンの間に、間質細胞の少なくとも一部が外部に露出されるように微細に切断することを意味する。このような生体組織の切断は、レーザなどを用いて行われる。このように間質細胞を取り囲むコラーゲンの間に、間質細胞の少なくとも一部が外部に露出されるように生体組織を微細に切断することにより、生体組織から間質細胞の自発的マイグレーションを効率よく誘導することができる。 (1) The step of finely cutting the living tissue means finely cutting so that at least a part of the stromal cells is exposed to the outside between the collagen surrounding the stromal cells in the living tissue. Such cutting of living tissue is performed using a laser or the like. By thus cutting the living tissue finely so that at least a part of the stromal cells is exposed to the outside between the collagens surrounding the stromal cells, efficient migration of the stromal cells from the living tissue is efficiently performed. Can be well guided.
(2)微細に切断した生体組織を培養液内で、その生体組織が付着可能な素材の付着部材上に付着させる段階は、例示として図6に示されたように、生体組織、培養液、及び付着部材を入れた容器内でなされる。 (2) in the culture medium, the living tissue finely cut, the step of attaching the biological tissue on the attachment member of the material to which the biological tissue can be attached, as shown in FIG. 6 as an example, the biological tissue, the culture solution, And in a container containing the adhesive member.
まず、生体組織320、培養液340、及び付着部材310、311を収容する収容空間351を有する容器350を準備する。容器350の蓋353及び遮断膜360を容器350から分離した後、あらかじめ準備した微細に切断した生体組織320及び生体組織320が付着可能な素材の付着部材310、311を収容空間351に位置させる。次いで、蓋353及び遮断膜360を容器350と結合した後、蓋353及び遮断膜360の中央部を順次貫通して、収容空間351まで延びた培養液貫通管380を通じて間質細胞が生存可能な培養液340を注入する。この際、培養液340の水面が遮断膜360よりも高く位置するように培養液340を注入する。 First, a container 350 having a housing space 351 for housing the living tissue 320, the culture solution 340, and the attachment members 310 and 311 is prepared. After separating the lid 353 and the blocking film 360 of the container 350 from the container 350, the finely cut biological tissue 320 and the attachment members 310 and 311 of the material to which the biological tissue 320 can be attached are positioned in the accommodation space 351. Next, after the lid 353 and the blocking film 360 are coupled to the container 350, stromal cells can survive through the culture medium penetrating tube 380 that extends through the central portions of the lid 353 and the blocking film 360 to the accommodation space 351. Inject the culture medium 340. At this time, the culture solution 340 is injected so that the water surface of the culture solution 340 is located higher than the blocking film 360.
上記のように生体組織320と付着部材310、311とを容器350の収容空間351に位置させた状態で、培養液340を注入すれば、培養液340よりも平均比重の小さい生体組織320と付着部材311は、培養液340の水面側に上昇して遮断膜360の下側面、及びその近傍に層を形成して配列される。この際、付着部材311よりも平均比重の小さい生体組織320が付着部材311の上層に位置し、付着部材311は、生体組織320の下層に位置する。この場合、生体組織320が配列された層は、付着部材311が配列された層と一部重畳されるか、隣接するようになって、生体組織320は、付着部材311との接触領域が大幅に拡がる。培養液340よりも平均比重の大きい付着部材310は、培養液340の下側にある収容空間351の底面及びその近傍に配列される。培養液340よりも平均比重の大きい付着部材310の一部は、遮断膜360の上側面に配列されうる。このように培養液340、生体組織320、及び付着部材311が配列された状態で、経時的に生体組織320は、付着部材311に付着する。 When the culture solution 340 is injected in a state where the living tissue 320 and the attachment members 310 and 311 are positioned in the accommodation space 351 of the container 350 as described above, the culture medium 340 is attached to the living tissue 320 having an average specific gravity smaller than that of the culture solution 340. The members 311 are arranged so as to rise to the water surface side of the culture medium 340 and form a layer on the lower surface of the blocking film 360 and in the vicinity thereof. At this time, the living tissue 320 having an average specific gravity smaller than that of the attachment member 311 is located in the upper layer of the attachment member 311, and the attachment member 311 is located in the lower layer of the living tissue 320. In this case, the layer in which the biological tissue 320 is arranged partially overlaps with or is adjacent to the layer in which the adhesive member 311 is arranged, and the biological tissue 320 has a large contact area with the adhesive member 311. Spread to. The attachment members 310 having a larger average specific gravity than the culture solution 340 are arranged on the bottom surface of the accommodation space 351 below the culture solution 340 and in the vicinity thereof. A part of the attachment member 310 having an average specific gravity larger than that of the culture medium 340 may be arranged on the upper surface of the blocking film 360. With the culture solution 340, the biological tissue 320, and the attachment member 311 thus arranged, the biological tissue 320 adheres to the attachment member 311 over time.
(3)付着部材上で間質細胞の自発的マイグレーションを誘導して、間質細胞を生体組織の外部に移動させる段階は、例示として図6に示されたように、生体組織320が付着した付着部材311上で生体組織320内の間質細胞を、自発的マイグレーションによって生体組織320の外部に移動させることを意味する。 (3) Inducing the spontaneous migration of stromal cells on the attachment member, the step of moving the stromal cells to the outside of the biological tissue, as shown in FIG. 6 as an example, biological tissue 320 is attached. This means that stromal cells in the living tissue 320 on the attachment member 311 are moved to the outside of the living tissue 320 by spontaneous migration.
生体組織320内の間質細胞は、自発的マイグレーションによって生体組織320の外側面や付着部材311の表面、または培養液340内部に移動する。このような間質細胞の移動は、付着部材311上に付着した生体組織320のコラーゲンの間を通じて外部に露出された間質細胞によって活発に起こる。 Stromal cells in the living tissue 320 move to the outer surface of the living tissue 320, the surface of the attachment member 311, or the inside of the culture fluid 340 by spontaneous migration. Such migration of stromal cells is actively caused by the stromal cells exposed to the outside through the collagen of the living tissue 320 attached on the attachment member 311.
(4)生体組織の外部に移動した間質細胞を、生体組織から分離する段階は、例示として図6に示されたように、生体組織320が付着した付着部材311上で、生体組織320内の間質細胞の自発的マイグレーションによって生体組織320の外部に移動した間質細胞を生体組織320から分離して培養液340内に移動させることを意味する。 (4) the step of separating the stromal cells that have moved to the outside of the living tissue from the living tissue, as shown in FIG. 6 as an example, on the attachment member 311 to which the living tissue 320 is attached, inside the living tissue 320. This means that the stromal cells that have moved to the outside of the living tissue 320 due to the spontaneous migration of the stromal cells are separated from the living tissue 320 and moved into the culture medium 340.
生体組織320の外部に移動した間質細胞を、生体組織320から分離して培養液340内に移動させることは、例示として図6に示されたように、容器350の正転と逆転とを交互に繰り返して生体組織320の外部に移動した間質細胞に、生体組織320から分離させる物理的力を加えてなる。容器350の正転と逆転とを交互に繰り返せば、培養液340に乱流が発生して培養液340に浸漬された付着部材311が乱流運動をするので、付着部材311上で生体組織320の外部に移動した間質細胞を、生体組織320から分離させる物理的力を受ける。 Stromal cells that have moved to the outside of the biological tissue 320 are separated from the biological tissue 320 and moved into the culture solution 340, as shown in FIG. 6 as an example, the forward and reverse rotation of the container 350 is performed. A physical force for separating the stromal cells from the living tissue 320 is applied to the stromal cells that have moved alternately to the outside of the living tissue 320. By alternately repeating the normal rotation and the reverse rotation of the container 350, a turbulent flow is generated in the culture solution 340 and the attachment member 311 immersed in the culture solution 340 performs turbulent motion. The stromal cells that have moved to the outside of the body are subjected to a physical force that separates them from the living tissue 320.
また、多数の付着部材311の乱流運動によって付着部材311が互いに衝突しつつ、付着部材311に形成されたスクレイピング部(図5参照)によって、他の付着部材311上で生体組織320の外部に移動した間質細胞をスクレイピング(scraping)することにより、間質細胞の生体組織320から培養液340内部への分離をさらに促進させる。 Further, while the adhering members 311 collide with each other due to the turbulent motion of a large number of adhering members 311, the scraping portion (see FIG. 5) formed on the adhering members 311 causes the adhering members 311 to be outside the biological tissue 320 on the other adhering members 311. By scraping the migrated stromal cells, the separation of the stromal cells from the living tissue 320 into the culture medium 340 is further promoted.
また、多数の付着部材311の乱流運動によって、付着部材311が容器350の傾斜部352の内側面、平均比重が培養液340よりもさらに大きく、乱流運動をする付着部材310、培養液貫通管380の外側面または遮断膜360の下側面に接触ないし衝突しつつ、付着部材311上で生体組織320の外部に移動した間質細胞の生体組織320からの培養液340内部への分離を促進させる。 Further, due to the turbulent motion of a large number of adhering members 311, the adhering member 311 has an inner surface of the inclined portion 352 of the container 350, the adhering member 310 having an average specific gravity larger than that of the culture solution 340 and turbulent motion, and the penetrating culture solution. While contacting or colliding with the outer surface of the tube 380 or the lower surface of the blocking film 360, promoting separation of stromal cells that have moved to the outside of the living tissue 320 on the attachment member 311 from the living tissue 320 into the culture solution 340. Let
(5)生体組織から分離された間質細胞を収集する段階は、生体組織から培養液の内部に分離された間質細胞を生体組織及び付着部材と分離して収集するものであって、例示的に図7及び図8に示すように、(a)生体組織320から培養液340の内部に分離された間質細胞330を遠心分離によって収斂部354a、354bに収斂する段階と、(b)収斂部354a、354bに収斂された間質細胞330を外部に排出する段階からなる。 (5) The step of collecting the stromal cells separated from the living tissue is to collect the stromal cells separated from the living tissue inside the culture solution by separating the stromal cells from the living tissue and the attachment member. As shown in FIGS. 7 and 8, (a) a step of converging the stromal cells 330 separated from the living tissue 320 into the culture solution 340 into the converging sections 354a and 354b by centrifugation, and (b). The step of discharging the stromal cells 330 converged in the convergence parts 354a and 354b to the outside.
(a)生体組織320から培養液340の内部に分離された間質細胞330を遠心分離によって収斂部354a、354bに収斂する段階は、例示として図7に示すように、生体組織320から培養液340の内部に分離された間質細胞330を含む容器350を、正転または逆転の一方向に回転させて間質細胞330に遠心力を加えることにより、間質細胞330を、互いに対称になる位置に配列されて遠心力が最大に作用する第1収斂部354a及び第2収斂部354bにそれぞれ収斂させるものである。 (a) The step of converging the stromal cells 330 separated from the living tissue 320 into the culture solution 340 into the converging portions 354a and 354b by centrifugation, as shown in FIG. 7 as an example, the culture solution from the living tissue 320. By rotating the container 350 containing the stromal cells 330 separated inside the 340 in one direction of forward rotation or reverse rotation and applying a centrifugal force to the stromal cells 330, the stromal cells 330 are made symmetrical with each other. The first converging portion 354a and the second converging portion 354b are arranged at the positions and exert the maximum centrifugal force, respectively.
容器350が一方向に回転することにより、培養液340はもとより、生体組織320及び付着部材310、311も遠心力によって第1収斂部354a及び第2収斂部354bに移動する。この際、培養液340は、間質細胞320と共に第1収斂部354aの入口に配列された第1フィルタ370a、及び第2収斂部354bの入口に配列された第2フィルタ370bをそれぞれ透過して第1収斂部354a及び第2収斂部354bにそれぞれ収斂される。しかし、生体組織320と付着部材310、311は、いずれも第1収斂部354aの入口に配列された第1フィルタ370a、及び第2収斂部354bの入口に配列された第2フィルタ370bを透過できず、第1収斂部354a及び第2収斂部354bの何処にも収斂されない。これは、第1及び第2フィルタ370a、370bは、いずれも培養液340及び間質細胞320は透過するが、生体組織320及び付着部材310、311は透過できないように、貫通孔の大きさが調節されているからである。 When the container 350 rotates in one direction, not only the culture solution 340 but also the living tissue 320 and the attachment members 310 and 311 move to the first converging part 354a and the second converging part 354b by centrifugal force. At this time, the culture medium 340 permeates the stromal cells 320 through the first filter 370a arranged at the entrance of the first converging part 354a and the second filter 370b arranged at the entrance of the second converging part 354b. The first converging unit 354a and the second converging unit 354b are respectively converged. However, the living tissue 320 and the attachment members 310 and 311 can both pass through the first filter 370a arranged at the entrance of the first convergence part 354a and the second filter 370b arranged at the entrance of the second convergence part 354b. Therefore, it is not converged anywhere in the first converging part 354a and the second converging part 354b. This is because the first and second filters 370a, 370b are both permeable to the culture medium 340 and stromal cells 320, but impermeable to the biological tissue 320 and the attachment members 310, 311. Because it is adjusted.
上記のように第1及び第2収斂部354a、354bには、それぞれ培養液340と共に間質細胞320のみ収斂される。 As described above, only the stromal cells 320 are converged together with the culture medium 340 in the first and second convergence parts 354a and 354b.
(b)収斂部354a、354bに収斂された間質細胞330を外部に排出する段階は、例示として図8に示すように、第1及び第2収斂部354a、354bにそれぞれ収斂された培養液340及び間質細胞320を外部に排出することを意味する。 (b) the step of discharging the stromal cells 330 that have been converged in the convergent portions 354a and 354b to the outside, as shown in FIG. 8 as an example, the culture solutions that are respectively converged in the first and second convergent portions 354a and 354b. It means that 340 and stromal cells 320 are discharged to the outside.
容器350の一方向回転によって第1及び第2収斂部354a、354bにそれぞれ培養液340と間質細胞320のみ収斂されれば、容器350の回転を停止させる。次いで、第1収斂部354aに形成された第1間質細胞排出部355aを通じて第1収斂部354aに収斂された培養液340と間質細胞330とを外部に排出させ、第2収斂部354bに形成された第2間質細胞排出部355bを通じて、第2収斂部354bに収斂された培養液340と間質細胞330とを外部に排出させる。 The rotation of the container 350 is stopped when only the culture solution 340 and the stromal cells 320 are converged in the first and second convergence parts 354a and 354b by the one-way rotation of the container 350, respectively. Then, the culture solution 340 and the stromal cells 330 converged in the first convergent part 354a are discharged to the outside through the first stromal cell discharge part 355a formed in the first convergent part 354a, and the second convergent part 354b is discharged. Through the formed second stromal cell discharge portion 355b, the culture solution 340 and the stromal cells 330 converged by the second convergence portion 354b are discharged to the outside.
上記のような過程を通じて、生体組織320から分離された間質細胞330を最終的に収集する。 The stromal cells 330 separated from the living tissue 320 are finally collected through the above process.
上記(1)ないし(5)の段階を連続して行うことにより、生体組織から間質細胞を連続して分離することができて、分離効率を向上させうる。 By continuously performing the above steps (1) to (5), stromal cells can be continuously separated from the biological tissue, and the separation efficiency can be improved.
上記(2)ないし(5)の段階は、順に繰り返して実行されうる。これにより、容器350の収容空間351内に存在する同じ付着部材310、311や生体組織320または培養液340に残存する間質細胞330を繰り返して収集することができて、間質細胞の収率を向上させうる。 The steps (2) to (5) may be repeatedly performed in order. Accordingly, the stromal cells 330 remaining in the same attachment members 310, 311 and the living tissue 320 or the culture solution 340 existing in the accommodation space 351 of the container 350 can be repeatedly collected, and the yield of stromal cells can be increased. Can be improved.
上記(2)ないし(4)の段階において、生体組織320、培養液340、及び付着部材310、311のうち、少なくともいずれか１つを入れ替えた後、上記(2)ないし(5)の段階を順次に繰り返して行っても良い。これにより、容器350の収容空間351内に存在する生体組織320、培養液340、及び付着部材310、311のうち、少なくともいずれか１つを入れ替えつつ、間質細胞330を繰り返して収集することができて、収率を大幅に向上させうる。 In the above steps (2) to (4), after replacing at least any one of the living tissue 320, the culture solution 340, and the attachment members 310 and 311, the steps (2) to (5) above are performed. You may repeat in order. As a result, the stromal cells 330 can be repeatedly collected while replacing at least one of the living tissue 320, the culture fluid 340, and the attachment members 310 and 311 existing in the accommodation space 351 of the container 350. It is possible to improve the yield significantly.
本発明は、酵素を使用せず、生体組織から間質細胞を分離する方法、及び装置に利用可能である。 INDUSTRIAL APPLICATION This invention can be utilized for the method and apparatus which isolate | separate a stromal cell from a biological tissue, without using an enzyme.
110、210、210'、310、311 付着部材
120、220、320 生体組織
130、230、330 間質細胞
340 培養液
350 容器
353 蓋
354a 第1収斂部
354b 第2収斂部
360 遮断膜
370a 第1フィルタ
370b 第2フィルタ
380 培養液貫通管
390 シーリング部材
110, 210, 210 ', 310, 311 Adhesive member
120, 220, 320 Living tissue
130, 230, 330 Stromal cells
340 medium
350 containers
353 lid
354a 1st convergence section
354b 2nd convergence section
360 barrier
370a 1st filter
370b 2nd filter
380 Culture medium penetration tube
390 Sealing material
Claims (26)
生体組織の間質細胞の自発的マイグレーション(spontaneous migration)を誘導して、間質細胞を生体組織の外部に移動させることを利用し、
間質細胞の自発的マイグレーションの誘導は、生体組織を付着させうる素材の付着部材上に生体組織が付着した状態でなされ、
間質細胞の自発的マイグレーションの誘導は、間質細胞が生存可能な培養液内でなされ、
付着部材は、培養液よりも平均比重がさらに小さな第1の付着部材と、培養液よりも平均比重がさらに大きい第2の付着部材と、培養液と平均比重が同一の第3の付着部材を含めて、
培養液内で、第1の付着部材は培養液よりも平均比重がさらに小さな生体組織と分布する領域が重なり、第2の付着部材は培養液よりも平均比重がさらに大きい生体組織と分布する領域が重なり、第3の付着部材は培養液と平均比重が同一の生体組織と分布する領域が重畳されることを特徴とする方法。 A method for separating stromal cells from living tissue without using an enzyme,
Inducing spontaneous migration of stromal cells of living tissue (spontaneous migration) and utilizing the movement of stromal cells to the outside of living tissue,
Induction of spontaneous migration of stromal cells is performed in a state in which biological tissue is attached to an attachment member made of a material capable of attaching biological tissue,
Induction of spontaneous migration of stromal cells is performed in a culture medium in which stromal cells can survive ,
The attachment member is a first attachment member having a smaller average specific gravity than the culture solution, a second attachment member having a larger average specific gravity than the culture solution, and a third attachment member having the same average specific gravity as the culture solution. Including,
In the culture solution, the first attachment member overlaps with a region of the living tissue having an average specific gravity smaller than that of the culture medium, and the second attachment member has a region of distribution of the living tissue having an average specific gravity larger than that of the culture medium. And a region in which the third attachment member is distributed with living tissue having the same average specific gravity as the culture solution is overlapped .
間質細胞の分離は、付着部材に付着した間質細胞に物理的力を加えてなることを特徴とする、請求項１に記載の方法。 Further comprising separating the stromal cells that have migrated outside of the biological tissue from the biological tissue,
The method according to claim 1, wherein the stromal cells are separated by applying a physical force to the stromal cells attached to the attachment member.
(1)生体組織を微細に切断することと、
(2)微細に切断した生体組織を培養液内でその生体組織が付着可能な素材の付着部材上に付着させることと、
(3)付着部材上で間質細胞の自発的マイグレーションを誘導して、間質細胞を生体組織の外部に移動させることと、
(4)生体組織の外部に移動した間質細胞を付着部材から分離することと、を含めて、
付着部材は、培養液よりも平均比重がさらに小さな第1の付着部材と、培養液よりも平均比重がさらに大きい第2の付着部材と、培養液と平均比重が同一の第3の付着部材を含めて、
培養液内で、第1の付着部材は培養液よりも平均比重がさらに小さな生体組織と分布する領域が重なり、第2の付着部材は培養液よりも平均比重がさらに大きい生体組織と分布する領域が重なり、第3の付着部材は培養液と平均比重が同一の生体組織と分布する領域が重畳されることを特徴とする方法。 A method for separating stromal cells from living tissue without using an enzyme,
(1) finely cutting living tissue,
(2) attaching the finely cut living tissue onto an attachment member of a material to which the living tissue can be attached in the culture solution,
(3) Inducing spontaneous migration of stromal cells on the attachment member, and moving the stromal cells to the outside of the biological tissue,
(4) including separating the stromal cells that have moved to the outside of the biological tissue from the attachment member ,
The attachment member is a first attachment member having a smaller average specific gravity than the culture solution, a second attachment member having a larger average specific gravity than the culture solution, and a third attachment member having the same average specific gravity as the culture solution. Including,
In the culture solution, the first attachment member overlaps with a region of the living tissue having an average specific gravity smaller than that of the culture medium, and the second attachment member has a region of distribution of the living tissue having an average specific gravity larger than that of the culture medium. And a region in which the third attachment member is distributed with living tissue having the same average specific gravity as the culture solution is overlapped .
培養液内で生体組織を付着させ、生体組織の間質細胞の自発的マイグレーションを誘導して、間質細胞を生体組織の外部に移動させる付着部材を備え、
付着部材は、培養液よりもさらに小さな平均比重を有するか、または培養液よりもさらに大きな平均比重を有すること、
培養液、生体組織、及び付着部材を内部に収容する容器をさらに備えること、
容器は、遠心力が最大である位置に形成され、生体組織から分離された間質細胞を遠心力によって収斂させる収斂部を有すること、
容器は、収斂部に形成され、収斂部に収斂された間質細胞を外部に排出させる間質細胞排出部をさらに備え、
付着部材は、培養液よりも平均比重がさらに小さな第1の付着部材と、培養液よりも平均比重がさらに大きい第2の付着部材と、培養液と平均比重が同一の第3の付着部材を含めて、
培養液内で、第1の付着部材は培養液よりも平均比重がさらに小さな生体組織と分布する領域が重なり、第2の付着部材は培養液よりも平均比重がさらに大きい生体組織と分布する領域が重なり、第3の付着部材は培養液と平均比重が同一の生体組織と分布する領域が重畳されることを特徴とする、装置。 A device for separating stromal cells from living tissue without using an enzyme,
Attaching a living tissue in a culture solution, inducing spontaneous migration of stromal cells of the living tissue, and including an attachment member that moves the stromal cells to the outside of the living tissue,
The attachment member has an average specific gravity smaller than that of the culture medium, or has an average specific gravity larger than that of the culture medium,
Further comprising a container for containing the culture solution, the biological tissue, and the attachment member therein,
The container has a converging part formed at a position where the centrifugal force is maximum and concentrating the stromal cells separated from the biological tissue by the centrifugal force.
Container is formed in a converging portion, further example Bei stromal cells discharge portion for discharging the stromal cells converge to the converging portion to the outside,
The attachment member is a first attachment member having a smaller average specific gravity than the culture solution, a second attachment member having a larger average specific gravity than the culture solution, and a third attachment member having the same average specific gravity as the culture solution. Including,
In the culture solution, the first attachment member overlaps with a region of the living tissue having an average specific gravity smaller than that of the culture medium, and the second attachment member has a region of distribution of the living tissue having an average specific gravity larger than that of the culture medium. And a region where the third attachment member is distributed with living tissue having the same average specific gravity as the culture solution is overlapped with each other .
Priority Applications (1)
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