WO2014119334A1 - 生体組織からの細胞の分離方法 - Google Patents
生体組織からの細胞の分離方法 Download PDFInfo
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Definitions
- the present invention relates to a method for separating cells from living tissue. More specifically, the present invention relates to a method capable of efficiently and stably separating cells having high biological activity by adjusting the amount of proteolytic enzyme used.
- Various organs and tissues such as liver, pancreas, kidney, periodontal tissue, skin, cartilage, bone and nerve tissue of multicellular animals such as mammals, birds, reptiles and fish, ES cell tissues, inducible pluripotent stem cells
- Enzymatic isolation of cells or cell aggregates (cell clusters) present in (iPS cell) material fibroblast tissue is useful for a wide range of applications such as cell transplantation, cell line establishment, treatment and diagnosis, and testing.
- the medical treatment which cures diabetes by transplanting the islet isolated from the pancreas to a patient is performed.
- islet transplantation it is essential to separate cell aggregates called islets that are present in the pancreatic tissue. At this time, it is necessary to decompose the pancreatic tissue and isolate the islets without damaging the islets.
- hepatic failure is treated by transplanting hepatocytes isolated from the liver into a patient.
- Living tissue is composed of cells and an intercellular matrix (intercellular substance). Intercellular substances are substances that connect cells, and there are structures and non-structures.
- the structure includes fibers such as collagen fibers, elastic fibers, and reticulofibers.
- An unstructured substance is a sol or gel-like substance called a substrate that fills the space between fibers, and includes glycoproteins and proteoglycans.
- a typical intercellular substance is a protein called collagen, which accounts for about 1/3 of the total protein weight in vivo.
- Collagen has a fibrous structure and is formally called collagen fiber or collagen fiber.
- the tissue is roughly classified into four types: epithelial tissue, support tissue, muscle tissue and nerve tissue.
- Epithelial tissue is a tissue that covers the surface of the body or organ, has a high cell density, and does not contain intercellular substances.
- the supporting tissue plays a role of supporting the body, organs, cells and the like, and includes connective tissue, cartilage tissue, bone tissue, blood and lymph.
- Muscle tissue is a fusion of differentiated cells for the purpose of contraction movement, and the proportion of intercellular substances is extremely small. Muscle tissue is composed of muscle cells, connective tissue, blood vessels and nerves, but the main structure is muscle fibers. Nerve tissue is mainly composed of inner lining and perineurium, both of which contain a lot of intercellular substances (collagen).
- the connective tissue is one type of supporting tissue, and refers to adipose tissue and fibrous connective tissue. As components of fibrous connective tissue, there are collagen fibers and elastic fibers. Fibrous connective tissue is roughly classified into hydrophobic connective tissue and tough connective tissue. Hydrophobic connective tissue refers to a fibrous connective tissue in which collagen is traveling irregularly, and is distributed in the subcutaneous tissue, mucosal tissue, nerves, outer vascular membrane, interlobular tissue, and the like.
- the amino acids constituting the collagen peptide chain have a primary structure in which “-glycine-amino acid X-amino acid Y” and glycine repeat every 3 residues.
- Human collagen is known to have about 30 molecular species. The most abundant in the body is type I collagen, which is fibrous. A large amount of non-fibrous collagen IV is also contained and is linked to each other through intermolecular disulfite bonds and is involved in the formation of a network (Non-patent Document 1). It has been reported that collagen VI exists between the islet and the endocrine tissue (Non-Patent Documents 2 and 3).
- various crude collagenases derived from Clostridium histolyticum contain various protease enzymes (collagen degradation activity and non-specific proteolytic activity) and non-protease components (phospholipase, etc.) in addition to collagenase. Contains. This crude collagenase provides enzymatic separation of cells and cell populations from most living tissues.
- the islet tissue is also a proteolytic enzyme selected from the group consisting of two types of collagenases (ColG and ColH) derived from Clostridium histolyticum and proteases (thermolysin, dispase or neutral protease (NP) derived from Histolyticus) It is known that it is decomposed
- the state of the intercellular matrix in the living tissue (particularly the collagen content) varies depending on the species, age, sex, tissue, living environment, and the like. It has not yet been clarified how much collagen is contained in which matrix of which tissue. Collagen exists widely from mammals to fish, and the difference in the constituent collagen by each species is not clear.
- the main object of the present invention is to provide a cell separation method capable of efficiently and stably separating cells having high biological activity from living tissue.
- the present invention provides the following method.
- a method for separating cells from living tissue wherein a certain amount of neutral protease and / or protease derived from Clostridium sp is added to the main protein according to the composition of the main protein of the living tissue.
- a method comprising using a decomposing enzyme composition obtained by adding an amount of decomposing enzyme.
- the method according to [1] comprising a procedure for preparing a decomposing enzyme composition by adding an enzyme amount and a procedure for treating the living tissue with the prepared decomposing enzyme composition.
- the type and amount of proteolytic enzyme to be used for cell isolation can be set from the composition of the main protein in the living tissue. For this reason, it is possible to efficiently isolate cells having high biological activity by adjusting the amount of proteolytic enzyme used.
- a method comprising using a decomposing enzyme composition to which collagen I and / or collagen III is decomposed in an amount corresponding to Clostridium sp. Collagenase H and / or collagenase G.
- the method according to [9] wherein a weight ratio (H / G) of the collagenase H and the collagenase G in the decomposing enzyme composition is 0.35 or more.
- the decomposing enzyme composition comprises a Clostridium.
- Sp neutral protease as a neutral protease and an ⁇ -N-benzoyl-L-arginine as a protease derived from the Clostridium sp.
- a protease having ethyl ester hydrochloride (BAEE) degrading activity [9] or [10].
- a method for separating hepatocytes from liver tissue, wherein a certain amount of neutral protease and / or protease derived from Clostridium sp is added to collagen I and / or collagen III of the pancreatic tissue A method comprising using a decomposing enzyme composition comprising collagenase I and / or collagen III degrading amount of Clostridium sp.
- Collagenase G and / or collagenase H are / or collagenase H.
- a weight ratio (H / G) of the collagenase H and the collagenase G in the decomposing enzyme composition is 0.25 or less.
- a method for isolating pancreatic islets from pancreatic tissue, comprising a Clostridium. Sp neutral protease and a protease having ⁇ -N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) degrading activity A method comprising using an enzyme composition.
- BAEE ⁇ -N-benzoyl-L-arginine ethyl ester hydrochloride
- biological tissue includes various organs and tissues of multicellular animals such as mammals, birds, reptiles, and fish without particular limitation.
- biological tissue includes ES cell tissue and fibroblast tissue for inducible pluripotent stem cells (iPS cells).
- cell includes cells and cell aggregates, and can include all types of cells and the like existing in the living tissue.
- the “cell” may be a hepatocyte, a pancreatic islet, a glomerulus, or the like.
- the present invention provides a cell separation method capable of efficiently and stably separating cells having high biological activity from a living tissue.
- ColH collagenase H
- ColG collagenase G
- the present inventors measured the abundance of collagen I and collagen III, which are the main proteins decomposed by ColH, in living tissue, thereby using the amount of protein differentiation enzyme suitable for cell separation from the living tissue. It has been clarified that cell separation can be performed without undue damage to cells (see Example 1). Furthermore, the inventors of the present invention have demonstrated that the isolation efficiency of Clostridium spp. Neutral protease (NP) as a neutral protease and a protease having BAEE degradation activity synergistically in isolation of islets from islet tissues. (See Example 2).
- NP Neutral protease
- a method for separating cells from living tissue which uses a decomposing enzyme composition in which the amount of degrading enzyme of the main protein is added according to the composition of the main protein in living tissue.
- the type and amount of the proteolytic enzyme to be used for cell isolation can be set from the composition of the main protein in the living tissue. For this reason, it is possible to suppress the usage-amount of a proteolytic enzyme and to isolate
- the main protein is a protein constituting an intercellular matrix, and may vary depending on the target biological tissue.
- collagen I, collagen II, collagen III, collagen IV, collagen V, laminin, fibronectin and vitronectin can be mentioned.
- the main proteins are preferably collagen I and collagen III.
- composition of major proteins is the ratio or amount of the main protein in the protein constituting the intercellular matrix.
- the composition of major proteins is collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, laminin, fibronectin, vitronectin, particularly collagen I and collagen III. Is the ratio or amount.
- the composition of the main protein can be determined by an enzyme immunoassay using a specific antibody or specific inhibitory peptide against the main protein. Specifically, for example, an antibody or an inhibitory peptide labeled with a tissue section of a living tissue by an immunohistological technique is reacted. Next, the signal intensity from the label of the antibody or inhibitory peptide bound to the main protein is visually confirmed under a microscope or detected by an apparatus, whereby the main protein is quantified and the composition is determined. In addition, the composition of the main protein can be rapidly quantified using an antibody, such as in-gel precipitation, immunoelectrophoresis, immunoturbidimetry, or enzyme immunoassay.
- an antibody such as in-gel precipitation, immunoelectrophoresis, immunoturbidimetry, or enzyme immunoassay.
- the main protein degrading enzyme may vary depending on the target biological tissue.
- the main protein degrading enzymes are ColH and ColG.
- ColH is important as a major proteolytic enzyme
- ColG is important as a major proteolytic enzyme.
- Decomposition enzyme composition It is preferable to use a degrading enzyme composition prepared by adding a certain amount of neutral protease and / or protease derived from Clostridium sp to the main protein degrading enzyme.
- neutral protease examples include thermolysin or Clostridium spp.
- Neutral protease (NP) examples include thermolysin or Clostridium spp.
- examples of the protease derived from Clostridium spp. include a protease having ⁇ -N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) degrading activity.
- the protease having BAEE degradation activity is preferably clostripain (CP). Since Clostridium spp. Produces many proteases, it is desirable to use enzymes produced as recombinant proteins for proteases from Clostridium spp.
- a degrading enzyme composition used for isolating islets from pancreatic tissue is composed of a Clostridium. Sp neutral protease (NP) as a neutral protease and a BAEE degrading activity as a protease derived from Clostridium. Sp. It is preferred to include both proteases having Proteases having NP and BAEE degrading activity have the effect of synergistically increasing the number of isolated islets in isolating islets from pancreatic tissue.
- the degrading enzyme composition is not particularly limited, but is, for example, 0.3 to 0.5 mg per 10 ml of the composition.
- the compounding quantity of the enzyme in a degradation enzyme composition can be suitably adjusted also according to the weight of the biological tissue made into a process target.
- the amount of the main protein degrading enzyme added to the degrading enzyme composition is set based on the composition of the main protein described above. Specifically, the amount of the main protein degrading enzyme is set based on the ratio or amount of the main protein in the protein constituting the intercellular matrix. More specifically, for example, when the proteins constituting the intercellular matrix are collagen III and collagen I, the usage amount of ColH and ColH is set based on the ratio or amount of collagen III and collagen I. Particularly preferably, based on the ratio or amount of collagen III and collagen I, the usage ratio of ColH and ColG in the degradation enzyme composition is set.
- the amount of ColH used is 0.35 times or more that of collagenase G Is preferred.
- separation efficiency falls, it is also possible to perform islet isolation
- the amount of ColH used is preferably 0.25 times or less that of collagenase G.
- the degradation enzyme composition in the case where islet isolation from pancreatic tissue is performed is performed by recombining high purity ColG 2 mg and ColH 1.1 mg (collagenase G) to 0.3 mg of thermolysin per 10 ml of the composition. And 0.55 times the amount).
- the degrading enzyme composition is 0.5 mg of thermolysin per 10 ml of composition, 14.4 mg of high purity ColG and 3.6 mg of ColH (0 against collagenase G). .25 times the amount).
- the degrading enzyme composition contains the main proteolytic enzyme in an amount necessary for the degradation (required minimum amount) based on the composition of the main protein in living tissue. For this reason, a desired cell can be separated from a living tissue without causing problems such as a decrease in physiological activity or a yield of separated cells due to the presence of an excessive or insufficient enzyme in the degradation enzyme composition.
- the composition of the main protein in the living tissue is determined, and a decomposing enzyme composition in which only a necessary amount of the main protein degrading enzyme is added is prepared.
- cells having high biological activity can be efficiently and stably separated from living tissue. Therefore, according to this method, it is possible to separate high-quality cells with a high yield by performing an optimal enzyme treatment on any target biological tissue.
- ⁇ Reference Example 1 Identification of major proteolytic enzymes used for separating cells from living tissue> [Identification of enzymes used to isolate islets from rat pancreatic tissue] Male Lewis rats (10-13 weeks old) were used. Prior to excising the pancreas, the duodenum was clamped. A degradation enzyme composition (10 ml cold Hanks solution (HBSS)) containing at least one of recombinant collagenase G 8.4 mg, collagenase H 2.9 mg as proteolytic enzyme, and thermolysin 0.3 mg as neutral protease is injected from the bile duct, 37 After 14 minutes of treatment at 0 ° C., density gradient centrifugation was performed to collect islets.
- HBSS cold Hanks solution
- the islet yield (IRQs) of the H group was 70% of the GH group.
- the islets could not be separated at all.
- ADP / ATP value FIG. 2
- ATP / DNA value FIG. 3
- Insulin / DNA value FIG. 4
- stimulation index FIG. 5
- a proteolytic enzyme composition (10 ml HBSS) containing at least one of recombinant collagenase G (14.4 mg), collagenase H (3.6 mg) as a proteolytic enzyme, and thermolysin (0.5 mg) as a neutral protease was prepared.
- Liver tissue (10-12 g) was treated with the prepared degrading enzyme composition at 37 ° C. for 7 minutes, followed by density gradient centrifugation to separate hepatocytes.
- the cells were incubated for 30 minutes in 50% acetonitrile supplemented with 10 ⁇ l of 100 mM TMPP (Sigma-Aldrich). Then, after acetone precipitation with cold acetone, the precipitate obtained by centrifugation was dried. The dried precipitate was digested with trypsin (10 ⁇ l / mg) overnight in 100 mM ammonium carbonate solution.
- FIG. 6 shows the amount of collagen I and collagen III converted to TMPP by collagenase H.
- the upper dotted circle indicates a collagen III data plot
- the lower dotted circle indicates a collagen I data plot.
- Example 1 Cell separation according to the composition of main protein in living tissue> [Quantification of collagen III and collagen I in rat pancreatic and liver tissues] Tissue sections of Lewis rats (10-13 weeks old) were prepared. The expression of collagen III and collagen I in pancreatic tissue and liver tissue was compared by enzyme immunoassay using labeled antibodies against collagen III and collagen I (Chemicon Merck Millipore). As a result, there were significantly fewer immunohistochemically stained images of collagen III and collagen I in liver tissue than in pancreatic tissue.
- liver tissue proteins other than collagen III and collagen I, and in the degrading enzyme composition for hepatocyte separation, the amount of collagenase H is expected to be smaller than that in the case of islet isolation. It was done.
- liver tissue (10 to 12 g) was obtained by decomposing enzyme composition (10 ml HBSS) containing 0.5 mg of thermolysin, 14.4 mg of recombinant collagenase G, 3.6 mg of collagenase H (0.25 times the amount of collagenase G).
- enzyme composition (10 ml HBSS) containing 0.5 mg of thermolysin, 14.4 mg of recombinant collagenase G, 3.6 mg of collagenase H (0.25 times the amount of collagenase G).
- Hepatocytes (5.02 ⁇ 2.11 ⁇ 10 8 cells) could be suitably isolated.
- a protease composition (10 ml HBSS) containing 0.3 mg of thermolysin as a proteolytic enzyme, 8.4 mg of recombinant collagenase G, and 2.9 mg of collagenase H (0.35 times the amount of collagenase G) was injected from the bile duct at 37 ° C.
- pancreatic islets (approx. 4000) were successfully isolated.
- an enzyme composition it is preferable to use an enzyme composition.
- the amount of collagenase H is relatively small (0.25 times the amount of collagenase G). It was found that it is preferable to use the decomposing enzyme composition.
- the amount of proteolytic enzyme suitable for cell separation from the tissue more specifically The amount ratio of collagenase H and collagenase G could be determined.
- Example 2 Synergistic effect of NP and CP in islet separation from islet tissue>
- a decomposing enzyme composition was prepared. Using the prepared degrading enzyme composition, islets were separated from islet tissues in the same manner as in Reference Example 1, and the number of isolated islets was compared.
- NP and CP an enzyme obtained by introducing and expressing an NP gene or CP gene derived from Clostridium histolyticum into Bacillus subtilis was used.
- the number of isolated islets decreased when using a degrading enzyme composition containing NP and using a decomposing enzyme composition containing thermolysin and CP, compared to using a decomposing enzyme composition containing thermolysin. .
- the decomposing enzyme composition containing NP and CP was used, the number of isolated islets increased significantly compared to the case using the decomposing enzyme composition containing thermolysin.
- an increase in the number of isolated islets was observed depending on the amount of NP and CP used.
- the cell separation method according to the present invention can efficiently and stably separate cells having high biological activity from biological tissue. Therefore, this cell separation method is useful for a wide range of applications such as cell transplantation, cell line establishment, treatment and diagnosis of diseases caused by specific cells, and examination.
- tailor-made cell separation that is optimized for donor tissue is possible by identifying a specific substrate for each enzyme component present on the tissue and preparing a kit.
- Conventional islet isolation has been forced to deal with a wide variety of pancreatic tissues having completely different matrix compositions for each donor using a single tool with a fixed composition.
- tailor-made that optimizes the donor pancreas by constructing highly pure, safe and stable enzyme components through the introduction of biotechnology, identifying specific substrates for each enzyme component on the pancreatic tissue, and creating a kit Type islet isolation becomes possible.
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Abstract
Description
[1]生体組織からの細胞の分離方法であって、一定量の中性プロテアーゼ及び/又はクロストリジウム属(Clostridium. sp)由来のプロテアーゼに、前記生体組織の主要タンパク質の組成に応じて該主要タンパク質の分解酵素量を加えてなる分解酵素組成物を用いることを特徴とする方法。
[2]前記生体組織の主要タンパク質の組成を決定する手順と、一定量の中性プロテアーゼ及び/又はクロストリジウム属(Clostridium. sp)由来のプロテアーゼに、決定された組成に応じて前記主要タンパク質の分解酵素量を加えて分解酵素組成物を調製する手順と、調製された分解酵素組成物により前記生体組織を処理する手順とを含む[1]記載の方法。
この方法によれば、生体組織の主要タンパク質の組成から、細胞の単離に使用すべきタンパク質分解酵素の種類や使用量を設定することができる。このため、タンパク質分解酵素の使用量を調整して、高い生物活性を有する細胞を効率的に分離することが可能である。
[4]前記主要タンパク質が、コラーゲンI及び/又はコラーゲンIIIである[1]~[3]のいずれかに記載の方法。
[5]コラーゲンI及び/又はコラーゲンIIIの組成に応じて、前記分解酵素組成物に加える前記コラゲナーゼH及び/又は前記コラゲナーゼGの量の比率を決定する[4]に記載の方法。
[6]前記中性プロテアーゼが、サーモリシン又はクロストリジウム属(Clostridium. sp)中性プロテアーゼである[1]~[5]のいずれかに記載の方法。
[7]前記クロストリジウム属(Clostridium. sp)由来のプロテアーゼが、α‐N‐ベンゾイル‐L‐アルギニンエチルエステル塩酸塩(BAEE)分解活性を有するプロテアーゼである[1]~[6]のいずれかに記載の方法。
[8]前記生体組織のタンパク質の組成を、酵素免疫定量法により決定することを特徴とする[1]~[7]のいずれかに記載の方法。
[10]前記分解酵素組成物中の前記コラゲナーゼH及び前記コラゲナーゼGの重量比(H/G)が0.35以上である[9]に記載の方法。
[11]前記分解酵素組成物が、前記中性プロテアーゼとしてのクロストリジウム属(Clostridium. sp)中性プロテアーゼと、前記クロストリジウム属(Clostridium. sp)由来のプロテアーゼとしてのα‐N‐ベンゾイル‐L‐アルギニンエチルエステル塩酸塩(BAEE)分解活性を有するプロテアーゼと、を含む[9]又は[10]に記載の方法。
[12]肝臓組織からの肝細胞の分離方法であって、一定量の中性プロテアーゼ及び/又はクロストリジウム属(Clostridium. sp)由来のプロテアーゼに、前記膵臓組織のコラーゲンI及び/又はコラーゲンIIIの組成に応じてコラーゲンI及び/又はコラーゲンIII分解量のクロストリジウム属(Clostridium. sp)コラゲナーゼG及び/又はコラゲナーゼHを加えてなる分解酵素組成物を用いることを特徴とする方法。
[13]前記分解酵素組成物中の前記コラゲナーゼH及び前記コラゲナーゼGの重量比(H/G)が0.25以下である[11]に記載の方法。
[14]膵臓組織からの膵島の分離方法であって、クロストリジウム属(Clostridium. sp)中性プロテアーゼ及びα‐N‐ベンゾイル‐L‐アルギニンエチルエステル塩酸塩(BAEE)分解活性を有するプロテアーゼを含む分解酵素組成物を用いることを特徴とする方法。
[15]前記BAEE分解活性を有するプロテアーゼがクロストリパインである[14]記載の方法。
本発明に係る細胞分離方法において、主要タンパク質は、細胞間マトリクスを構成するタンパク質であり、対象とする生体組織によって異なり得るが、例えばコラーゲンとして、コラーゲンI、コラーゲンII、コラーゲンIII、コラーゲンIV、コラーゲンV、ラミニン、フィブロネクチン、ビトロネクチンが挙げられる。例えば膵臓組織及び肝臓組織からの細胞分離が行われる場合、主要タンパク質はコラーゲンI及びコラーゲンIIIとされることが好ましい。
本発明に係る細胞分離方法において、主要タンパク質の組成は、細胞間マトリクスを構成するタンパク質に占める上記の主要タンパク質の比率あるいは量である。例えば膵臓組織及び肝臓組織からの細胞分離が行われる場合、主要タンパク質の組成はコラーゲンI、コラーゲンII、コラーゲンIII、コラーゲンIV、コラーゲンV、コラーゲンVI、ラミニン、フィブロネクチン、ビトロネクチン、特にコラーゲンI及びコラーゲンIIIの比率あるいは量である。
本発明に係る細胞分離方法において、主要タンパク質の分解酵素は、対象とする生体組織によって異なり得る。例えば膵臓組織及び肝臓組織からの細胞分離が行われる場合、主要タンパク質の分解酵素はColH及びColGである。膵島組織からの膵島分離では、主要タンパク質分解酵素としてColHが重要であり、肝臓組織からの肝細胞分離では、主要タンパク質分解酵素としてColGが重要である。
分解酵素組成物は、一定量の中性プロテアーゼ及び/又はクロストリジウム属(Clostridium. sp)由来のプロテアーゼに、上記の主要タンパク質の分解酵素を加えて調製されたものを用いることが好ましい。
分解酵素組成物による生体組織の処理は、従来手法と同様に行えばよい。分解酵素組成物には、生体組織の主要タンパク質の組成に基づいてその分解に必要な量(必要最小量)の主要タンパク質分解酵素が含まれている。このため、分解酵素組成物に過度なあるいは不足な酵素が含まれることによる、分離細胞の生理活性の低下や収量の低下という問題を生じることなく、生体組織から所望の細胞を分離できる。
[ラットの膵臓組織からの膵島分離に用いる酵素の同定]
雄Lewisラット(10~13週齢)を用いた。膵臓を摘出する前に、十二指腸をクランプした。タンパク質分解酵素としてリコンビナントコラゲナーゼG8.4mg、コラゲナーゼH2.9mg、中性プロテアーゼとしてサーモリシン0.3mgのいずれか一以上を含む分解酵素組成物(10ml冷Hanks液(HBSS))を胆管から注入し、37℃で14分間処理した後、密度勾配遠心分離を行って膵島を分取した。
タンパク質分解酵素としてリコンビナントコラゲナーゼG14.4mg、コラゲナーゼH3.6mg、中性プロテアーゼとしてサーモリシン0.5mgのいずれか一以上を含む分解酵素組成物(10ml HBSS)を調製した。肝臓組織(10~12g)を、調製した分解酵素組成物により37℃で7分間処理した後、密度勾配遠心分離を行って肝細胞を分離した。
[コラゲナーゼHの基質の同定]
100mgのLewisラットの膵組織片を、プロテアーゼ阻害剤カクテル(Roche)と1mM CaCl2を含む20mM HEPES(pH8.0)で37℃、1夜、処理した。その後、緩衝液で洗浄し、コラゲナーゼH0.1mg/mlを添加した緩衝液で、10時間、37℃で酵素分解を行った。
[ラットの膵臓組織及び肝臓組織のコラーゲンIIIとコラーゲンIの定量]
Lewisラット(10~13週齢)の組織切片を作製した。コラーゲンIIIとコラーゲンIに対する標識抗体(Chemicon Merck Millipore)を用いた酵素免疫定量法によって、膵臓組織及び肝臓組織のコラーゲンIIIとコラーゲンIの発現比較を行った。結果、肝臓組織におけるコラーゲンIIIとコラーゲンIの免疫組織染色像は、膵臓組織に比較して著しく少なかった。
肝臓組織の主要タンパク質は、コラーゲンIIIとコラーゲンI以外のタンパク質であり、肝細胞分離のための分解酵素組成物において、コラゲナーゼHの量は、膵島分離の場合に比して少なくてよいものと予想された。
コラゲナーゼG8.4mg及びコラゲナーゼH2.9mgに加えて、サーモリシン、クロストリジウム属(Clostridium. sp)由来の中性プロテアーゼ(NP)及びクロストリパイン(CP)のいずれか1つ又は2つを種々の添加量で含む分解酵素組成物を調製した。調製した分解酵素組成物を用い、参考例1と同様にして膵島組織から膵島を分離し、分離膵島数を比較した。
Claims (11)
- 生体組織からの細胞の分離方法であって、
一定量の中性プロテアーゼ及び/又はクロストリジウム属(Clostridium. sp)由来のプロテアーゼに、前記生体組織の主要タンパク質の組成に応じて該主要タンパク質の分解酵素量を加えてなる分解酵素組成物を用いることを特徴とする方法。 - 前記生体組織の主要タンパク質の組成を決定する手順と、
一定量の中性プロテアーゼ及び/又はクロストリジウム属(Clostridium. sp)由来のプロテアーゼに、決定された組成に応じて前記主要タンパク質の分解酵素量を加えて分解酵素組成物を調製する手順と、
調製された分解酵素組成物により前記生体組織を処理する手順と、を含む請求項1記載の方法。 - 前記主要タンパク質の分解酵素が、クロストリジウム属(Clostridium. sp)のコラゲナーゼH及び/又はコラゲナーゼGである請求項1又は2に記載の方法。
- 前記主要タンパク質が、コラーゲンI及び/又はコラーゲンIIIである請求項1~3のいずれか一項に記載の方法。
- コラーゲンI及び/又はコラーゲンIIIの組成に応じて、前記分解酵素組成物に加える前記コラゲナーゼH及び/又は前記コラゲナーゼGの量の比率を決定する請求項4に記載の方法。
- 前記中性プロテアーゼが、サーモリシン又はクロストリジウム属(Clostridium. sp)中性プロテアーゼである請求項1~5のいずれか一項に記載の方法。
- 前記クロストリジウム属(Clostridium. sp)由来のプロテアーゼが、α‐N‐ベンゾイル‐L‐アルギニンエチルエステル塩酸塩(BAEE)分解活性を有するプロテアーゼである請求項1~6のいずれか一項に記載の方法。
- 前記生体組織のタンパク質の組成を、酵素免疫定量法により決定することを特徴とする請求項1~7のいずれか一項に記載の方法。
- 膵臓組織からの膵島の分離方法であって、
一定量の中性プロテアーゼ及び/又はクロストリジウム属(Clostridium. sp)由来のプロテアーゼに、前記膵臓組織のコラーゲンI及び/又はコラーゲンIIIの組成に応じてコラーゲンI及び/又はコラーゲンIII分解量のクロストリジウム属(Clostridium. sp)コラゲナーゼH及び/又はコラゲナーゼGを加えてなる分解酵素組成物を用いることを特徴とする方法。 - 前記分解酵素組成物が、前記中性プロテアーゼとしてのクロストリジウム属(Clostridium. sp)中性プロテアーゼと、前記クロストリジウム属(Clostridium. sp)由来のプロテアーゼとしてのα‐N‐ベンゾイル‐L‐アルギニンエチルエステル塩酸塩(BAEE)分解活性を有するプロテアーゼと、を含む請求項9に記載の方法。
- 肝臓組織からの肝細胞の分離方法であって、
一定量の中性プロテアーゼ及び/又はクロストリジウム属(Clostridium. sp)由来のプロテアーゼに、前記膵臓組織のコラーゲンI及び/又はコラーゲンIIIの組成に応じてコラーゲンI及び/又はコラーゲンIII分解量のクロストリジウム属(Clostridium. sp)コラゲナーゼG及び/又はコラゲナーゼHを加えてなる分解酵素組成物を用いることを特徴とする方法。
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WO2024014517A1 (ja) * | 2022-07-15 | 2024-01-18 | デクセリアルズ株式会社 | 細胞の培養方法、治療用組成物の製造方法及び治療用組成物 |
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WO2024014517A1 (ja) * | 2022-07-15 | 2024-01-18 | デクセリアルズ株式会社 | 細胞の培養方法、治療用組成物の製造方法及び治療用組成物 |
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CA2898601C (en) | 2021-05-18 |
KR102144604B1 (ko) | 2020-08-14 |
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