JPS6363700A - Production of acid-soluble collagen having high gel strength - Google Patents
Production of acid-soluble collagen having high gel strengthInfo
- Publication number
- JPS6363700A JPS6363700A JP61208436A JP20843686A JPS6363700A JP S6363700 A JPS6363700 A JP S6363700A JP 61208436 A JP61208436 A JP 61208436A JP 20843686 A JP20843686 A JP 20843686A JP S6363700 A JPS6363700 A JP S6363700A
- Authority
- JP
- Japan
- Prior art keywords
- collagen
- solution
- acid
- soluble
- soluble collagen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108010035532 Collagen Proteins 0.000 title claims abstract description 99
- 102000008186 Collagen Human genes 0.000 title claims abstract description 99
- 229920001436 collagen Polymers 0.000 title claims abstract description 99
- 238000004519 manufacturing process Methods 0.000 title claims description 6
- 241001465754 Metazoa Species 0.000 claims abstract description 22
- 239000002253 acid Substances 0.000 claims abstract description 13
- 230000001133 acceleration Effects 0.000 claims abstract description 10
- 239000007787 solid Substances 0.000 claims abstract description 10
- 238000000605 extraction Methods 0.000 claims description 20
- 238000005119 centrifugation Methods 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 18
- 238000004113 cell culture Methods 0.000 abstract description 5
- 239000011159 matrix material Substances 0.000 abstract description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 abstract description 4
- 239000007788 liquid Substances 0.000 abstract description 3
- 239000006228 supernatant Substances 0.000 abstract description 3
- 241000282898 Sus scrofa Species 0.000 abstract 4
- 239000000126 substance Substances 0.000 abstract 1
- 210000002435 tendon Anatomy 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 41
- 210000001519 tissue Anatomy 0.000 description 19
- 239000002994 raw material Substances 0.000 description 15
- 239000000512 collagen gel Substances 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 10
- 235000002639 sodium chloride Nutrition 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 241000282887 Suidae Species 0.000 description 8
- 239000000835 fiber Substances 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 239000006185 dispersion Substances 0.000 description 6
- 239000010410 layer Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- 230000007935 neutral effect Effects 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000012136 culture method Methods 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108010050808 Procollagen Proteins 0.000 description 3
- 108010045569 atelocollagen Proteins 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 235000011121 sodium hydroxide Nutrition 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000001879 gelation Methods 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 235000015277 pork Nutrition 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- LCGISIDBXHGCDW-VKHMYHEASA-N L-glutamine amide Chemical compound NC(=O)[C@@H](N)CCC(N)=O LCGISIDBXHGCDW-VKHMYHEASA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108091006587 SLC13A5 Proteins 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical class [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 108010077465 Tropocollagen Proteins 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- -1 yields Substances 0.000 description 1
Abstract
Description
【発明の詳細な説明】
〔技術分野〕
この発明は、高ゲル強度の酸可溶性コラーゲンを得る方
法に関する。DETAILED DESCRIPTION OF THE INVENTION [Technical Field] This invention relates to a method for obtaining acid-soluble collagen with high gel strength.
生体内において、コラーゲンは細胞内で合成され、種々
の修飾反応をうけて３末鎖のプロコラーゲンができて細
胞外に分泌される。このプロコラーゲン分子は、コラー
ゲン分子より大きく、両末端に非コラーゲン性のかなり
大きなプロペプチドをもっている。生体内では、これら
のプロペプチドは、特異的なプロテアーゼにより僅かに
ペプチドを残して通常のコラーゲン分子となる。このよ
うにして生じたコラーゲン分子（トロポコラーゲン）は
、分子量約１０万のポリペプチド鎖が３本葉まってコラ
ーゲン特有のらせん構造を形成しており、長さ約３００
０人、直径１５人、分子量約３０万の棒状の分子である
。分子の両末端にはらせん構造をとらないペプチド鎖（
テロペプチド）が付いている。In vivo, collagen is synthesized within cells and undergoes various modification reactions to form 3-terminal procollagen, which is secreted outside the cells. This procollagen molecule is larger than a collagen molecule and has a fairly large non-collagen propeptide at both ends. In vivo, these propeptides are converted into normal collagen molecules by specific proteases, leaving only a small amount of peptide. The collagen molecule (tropocollagen) produced in this way has three lobes of polypeptide chains with a molecular weight of approximately 100,000, forming a helical structure unique to collagen, and has a length of approximately 300,000.
It is a rod-shaped molecule with a diameter of 0 and a diameter of 15 and a molecular weight of approximately 300,000. At both ends of the molecule there is a peptide chain that does not have a helical structure (
telopeptide).
プロコラーゲンがコラーゲン分子となると、初めて規則
性のある集合体を形成してコラーゲン線維となる。コラ
ーゲン線維内のコラーゲン分子は、加令により、分子内
架橋および分子間架橋が形成される。架橋の進行により
、コラーゲン組織は硬く丈夫なものに変化していくとと
もに、可溶性区分が減少し、不溶性区分が増大していく
。When procollagen becomes collagen molecules, it first forms regular aggregates and becomes collagen fibers. Collagen molecules within collagen fibers form intramolecular and intermolecular crosslinks due to aging. As crosslinking progresses, the collagen tissue changes to become harder and stronger, and the soluble segment decreases and the insoluble segment increases.
これらのコラーゲン線維を含む組織から、コラ−ゲン分
子の形状を全く変化させることなく、コラーゲンの可溶
性区分を得るには、大別して、■食塩等の中性塩溶液で
抽出する方法、■希酸溶液で抽出する方法
がある。■の方法で得られたコラーゲンは中性塩可溶性
コラーテーン、■の方法で得られたコラーゲンは酸可溶
性コラーゲンとそれぞれ呼ばれ葛。中性塩で抽出される
コラーゲンは、分子間架橋が形成されていないコラーゲ
ン（モノメリックコラーゲン）がほとんどである。しか
しながら、通常利用されうる動物組織を抽出原料として
用いた場合、中性塩で抽出されるコラーゲンの量が極め
て微量であるため、これを工業的に利用することは実質
的にほとんど不可能である。これに対し、酸で抽出を行
うと、モノメリックコラーゲンとともに分子間架橋によ
り生じるコラーゲン分子のオリゴマー（ポリメリックコ
ラーゲン）も抽出される。In order to obtain soluble collagen from tissues containing these collagen fibers without changing the shape of the collagen molecules at all, there are two methods: 1) Extraction with a neutral salt solution such as table salt, 2) Extraction with a neutral salt solution such as table salt, and 2) Dilute acid extraction. There is a method of extraction using a solution. The collagen obtained by method ① is called neutral salt-soluble collagen, and the collagen obtained by method ② is called acid-soluble collagen. Most of the collagen extracted with neutral salts is collagen in which intermolecular crosslinks are not formed (monomeric collagen). However, when normally available animal tissues are used as extraction raw materials, the amount of collagen extracted with neutral salts is extremely small, making it virtually impossible to use this industrially. . On the other hand, when extraction is performed with acid, oligomers of collagen molecules (polymeric collagen) produced by intermolecular crosslinking are also extracted together with monomeric collagen.
しかし、酸可溶性コラーゲンにおいても、幼若な動物組
織を用いた場合には、その収率（モノメリックコラーゲ
ンおよびポリメリックコラーゲンの収率）が高々数％以
下であることが知られているこのほかに、不溶性コラー
ゲンを可溶化するには、ペプシン等のプロテアーゼ（蛋
白質骨）・警￥素）で処理する方法、あるいは、飽和硫
酸ソーダの存在下で苛性ソーダで処理する方法が公知で
ある。これらの方法によって、コラーゲンのらせん構造
を保ったまま、はとんど１００％の収率で可溶化コラー
ゲンが得られる。しかじ、前者の方法では、テロペプチ
ド部分が消化されており、後者の方法では、さらにコラ
ーゲン分子中のアスパラギン、グルタミンのアマイドが
加水分解され得られたコラーゲンの等電点が低下してお
り、＋Ｂ１’Ｍな意味では元のコラーゲン分子の構造を
すべて保持しているものではない。However, even with acid-soluble collagen, it is known that when young animal tissue is used, the yield (yield of monomeric collagen and polymeric collagen) is at most a few percent or less. In order to solubilize insoluble collagen, it is known to treat it with a protease (protein bone) such as pepsin, or with caustic soda in the presence of saturated sodium sulfate. By these methods, solubilized collagen can be obtained with a yield of almost 100% while maintaining the helical structure of collagen. However, in the former method, the telopeptide portion is digested, and in the latter method, asparagine and glutamine amide in the collagen molecule are further hydrolyzed, lowering the isoelectric point of the resulting collagen. In the +B1'M sense, it does not retain all the structure of the original collagen molecule.
近年、コラーゲンゲルマトリックスを利用した紐胞培養
法が注目されている。コラーゲンゲルを支持基質として
細胞培養を行う７ケ法には、ゲル上に細胞を単層培養す
る方法と、ゲル内に細胞を包埋培養する方法とがある。In recent years, a cell culture method using a collagen gel matrix has been attracting attention. Seven methods for culturing cells using collagen gel as a support substrate include a method of culturing cells in a monolayer on gel and a method of culturing cells embedded in gel.
さらに、単層培養法は、ゲルが培養器に付着したままの
状態の付着コラーゲンゲル培養法と、ゲルが培養液中に
浮かんでいる状態の浮遊コラーゲンゲル培養法とに分け
られる。Furthermore, the monolayer culture method can be divided into an attached collagen gel culture method in which the gel remains attached to the culture vessel, and a floating collagen gel culture method in which the gel is floating in the culture medium.
浮遊コラーゲンゲル培養法は、これまで困難とされてい
た培養細胞における分化機能の維持を可能にした。すな
わち、この方法を応用すれば細胞培養により生体物質、
生理活性物質を大量に採取することも可能になってきた
。The floating collagen gel culture method has made it possible to maintain differentiation function in cultured cells, which was previously considered difficult. In other words, if this method is applied, biological materials,
It has become possible to collect large quantities of physiologically active substances.
また、コラーゲンゲル包埋培養法は、試験管内において
も形態形成、組織構築を観察することが可能になった。In addition, the collagen gel embedding culture method has made it possible to observe morphogenesis and tissue architecture even in vitro.
これらのように、細胞培養に極めて有効なコラーゲンゲ
ルは、上記のようにして得られた可溶性コラーゲンの溶
液のｐＨを中性とし、さらに塩類を加えて生体内の塩濃
度（ＮａＣｆで０．１４Ｍ）にした後、２５℃〜３７℃
に加温することによって得られる。これは、溶液中に分
散しているコラーゲン分子が生理的条件下で再び会合を
始め、コラーゲン線維が再構成され、網目状の線維の間
に液が閉じ込められるからである。Collagen gel, which is extremely effective for cell culture, is produced by adjusting the pH of the soluble collagen solution obtained as described above to neutrality, and then adding salts to the in-vivo salt concentration (0.14M NaCf). ), then 25℃~37℃
Obtained by heating to. This is because the collagen molecules dispersed in the solution begin to associate again under physiological conditions, the collagen fibers are reorganized, and the liquid is trapped between the networked fibers.
このようにして得られるコラーゲンゲルを細胞培養に用
いる場合、ゲルを調製する際に要求される特性としては
、線維再構成のスピード、すなわち、ゲル化速度が早い
こと、得られるゲルの強度が高いこと、および、ゲルの
透明度が高いことである。これらいずれの特性も得られ
る可溶性コラーゲンの中で酸可溶性コラーゲンが最も優
れており、他の方法によって得られるコラーゲンでは、
上記特性のすべてまたはいずれかを満足するものが得ら
れない。すなわち、酵素可溶化コラーゲンおよびアルカ
リ可溶化コラーゲンに比べ、酸可溶性コラーゲンは、元
のコラーゲン構造をほとんど損なうことなく保持してい
るために、線維再構成能が高く、したがって、その溶液
は、生理的条件下においた時、素早く、かつ、硬いゲル
を構成し、細胞の支持体として極めて優れたマトリック
スとなる。When using the collagen gel obtained in this way for cell culture, the properties required when preparing the gel include a fast fiber reorganization speed, that is, a fast gelation rate, and a high strength of the gel obtained. and that the gel has high transparency. Among the soluble collagens that can obtain both of these properties, acid-soluble collagen is the best, and among collagens obtained by other methods,
It is not possible to obtain a product that satisfies all or any of the above characteristics. In other words, compared to enzyme-solubilized collagen and alkali-solubilized collagen, acid-soluble collagen retains the original collagen structure without damaging it, and therefore has a higher fiber reconstitution ability. When placed under certain conditions, it quickly forms a hard gel, making it an excellent matrix for supporting cells.
しかし、これまでのところ、高いゲル強度をもつコラー
ゲンを高い収率、すなわち、工業的に利用できる程度の
収率でつくる方法は見出されていない。However, so far, no method has been found to produce collagen with high gel strength at a high yield, that is, at a yield that can be used industrially.
この発明は、以上のことに浴みて、ゲル強度の高いコラ
ーゲンゲルを与えるコラーゲンを高い収率で得ることが
できる高ゲル強度酸可溶性コラーゲンの製法を提供する
ことを目的とする。In view of the above, an object of the present invention is to provide a method for producing acid-soluble collagen with high gel strength, which can produce collagen with high gel strength in a high yield.
この発明は、上記の目的を達成するために、動物組織か
ら酸溶液により抽出して可溶性コラーゲンを得る方法に
おいて、前記動物組織として豚由来のものを用いるよう
にするとともに、抽出により得られた溶液を、前記動物
組織の乾燥固形分量１重量％以下、ｐＨ３以下、遠心加
速度３００００９以下の条件で遠心分離にかけて不溶性
コラーゲンを除くことを特１牧とする高ゲル強度酸可溶
性コラーゲンの製法を要旨とする。In order to achieve the above object, the present invention provides a method for obtaining soluble collagen by extracting animal tissue with an acid solution, in which the animal tissue is derived from a pig, and a solution obtained by the extraction is The gist is a method for producing acid-soluble collagen with high gel strength, which involves removing insoluble collagen by centrifuging the animal tissue under the conditions of dry solid content of 1% by weight or less, pH of 3 or less, and centrifugal acceleration of 300,009 or less. .
以下に、この発明の詳細な説明する。The present invention will be explained in detail below.
この発明では、抽出原料として、豚由来の動物組織を用
いることにしている。これは、豚由来の動物Ｍｉ織を抽
出原料として用いると、高ゲル強度のコラーゲンが高い
収率で得られるからである。In this invention, animal tissues derived from pigs are used as extraction raw materials. This is because when pig-derived animal Mi tissue is used as an extraction raw material, collagen with high gel strength can be obtained at a high yield.
豚由来の動物組織としては、たとえば、豚皮、４鍵など
が用いられるが、これらに限定されない。Examples of animal tissues derived from pigs include, but are not limited to, pig skin and four keys.
豚由来の動物組織は、通常の方法に従って積装されたの
ち酸溶液に分散し、抽出を行う。この抽出を行った後、
その抽出液は、遠心分離にかけ、可溶性コラーゲン両分
と不溶性コラーゲン画分に分離する。前記酸溶液として
は、酢酸、クエン酸などの有機酸、および、塩酸などの
無機酸のン容）夜が用いられるが、これらに限定されな
い。抽出時間は、特に限定されないが、通常どおり一夜
ないし三日間行うのがよい。Animal tissues derived from pigs are loaded according to conventional methods and then dispersed in an acid solution for extraction. After doing this extraction,
The extract is centrifuged and separated into soluble collagen and insoluble collagen fractions. The acid solution used includes, but is not limited to, organic acids such as acetic acid and citric acid, and inorganic acids such as hydrochloric acid. The extraction time is not particularly limited, but it is preferable to carry out the extraction for one night to three days as usual.
全操作は、コラーゲンの変性を避けるために、１〜１０
°Ｃの低温で行うことが好ましい。The whole operation was carried out at 1-10 min to avoid collagen denaturation.
Preferably, it is carried out at a low temperature of °C.
遠心分離は、以下のような条件で行われる必要がある。Centrifugation must be performed under the following conditions.
まず、動物組織の乾燥固形分の量を、酸溶液に対し１重
量％以下とすることである。前記乾燥固形分の量が酸溶
液に対し１重量％を上回ると、遠心分離の際、コラーゲ
ン溶液の粘性によって可溶性コラーゲン画分の分離が困
難となる。前記乾燥固形分の量としては、０．７重量％
以下が好ましく、０．５重量％以下がより好ましい。分
離に必要な乾燥固形分量は、遠心加速度によって異なる
が、たとえば、約２００００９の場合０．３重量％以下
が好ましい。つぎに、ｐＨを３以下とすることである。First, the amount of dry solid content of the animal tissue should be 1% by weight or less based on the acid solution. When the amount of the dry solid content exceeds 1% by weight based on the acid solution, it becomes difficult to separate the soluble collagen fraction during centrifugation due to the viscosity of the collagen solution. The amount of dry solid content is 0.7% by weight
It is preferably at most 0.5% by weight, more preferably at most 0.5% by weight. The amount of dry solids required for separation varies depending on the centrifugal acceleration, but for example, in the case of about 200009, it is preferably 0.3% by weight or less. The next step is to adjust the pH to 3 or less.
抽出ｐＨは、通常用いられるｐＨ（２〜４）でよいが、
遠心分離時のｐＨが高すぎると、可溶性コラーゲン両分
の分離が困難となる。The extraction pH may be a commonly used pH (2 to 4), but
If the pH during centrifugation is too high, it will be difficult to separate both soluble collagen components.
遠心分離時のｐＨは、２．７以下が好ましく、２．５以
下がより好ましい。遠心分離の遠心加速度は、３０００
０９以下とする。３００００９を上回ると、そのような
遠心装置は、処理量が小さく、工業的に利用することは
実質的にほとんど不可能である。The pH during centrifugation is preferably 2.7 or less, more preferably 2.5 or less. The centrifugal acceleration of centrifugation is 3000
09 or less. Above 300,009, such a centrifugal device has a small throughput and is virtually impossible to utilize industrially.
具体的操作としては、あらかじめ動物組織の乾燥固形分
の量が酸溶液に対して１重量％以下となるようにしてｐ
Ｈ３以下の酸溶液で抽出を行い、引き続き遠心分離を行
うか、もしくは、抽出は適当な条件で行い、遠心分離の
直前に遠心分離にかける溶液（抽出により得られた？８
液）を、動物組織の乾燥固形分量が酸溶液に対し１重量
％以下、ｐＨが３以下となるように調整して遠心分離を
行うことがあげられる。As a specific operation, the amount of dry solid content of the animal tissue is 1% by weight or less based on the acid solution in advance, and p
Extraction is performed with an acid solution of H3 or less, followed by centrifugation, or extraction is performed under appropriate conditions and the solution (obtained by extraction) is centrifuged immediately before centrifugation.
The centrifugal separation may be carried out after adjusting the dry solid content of the animal tissue to be 1% by weight or less relative to the acid solution and the pH to be 3 or less.
遠心分離にかけられる溶液が上記の乾燥Ｉ形分量および
ｐＨであれば、３００００９で少なくとも３０分間以上
遠心分離を行うことにより、上澄みの可溶性コラーゲン
画分と下層の不溶性コラーゲン画分に分離する。なお、
このとき、遠心分離の遠心加速度を高くする程、可溶性
コラーゲン画分中のモノメリックコラーゲンの割合が増
すが、特に細胞培養に適した高ゲル強度のコラーゲンゲ
ルを調製するのに用いるコラーゲンには、ポリメリック
コラーゲンが含まれていても差支えない。If the solution to be subjected to centrifugation has the above-mentioned dry Type I volume and pH, centrifugation is performed at 300009 for at least 30 minutes to separate it into a supernatant soluble collagen fraction and a lower layer insoluble collagen fraction. In addition,
At this time, the higher the centrifugal acceleration of centrifugation, the higher the proportion of monomeric collagen in the soluble collagen fraction. There is no problem even if polymeric collagen is included.
また、両画分を分離するには、高い遠心加速度を用いる
程、分離が容易に行えることはいうまでもないが、実際
に敵方Ｓ以上の遠心加速度が得られる、いわゆる超遠心
装置は、その処Ｆ１が小さく、これを工業的に利用する
ことが実質的にほとんど不可能である。このようなこと
から、この発明は、実際に工業的に利用可能な低遠心加
速度の遠心分離機（通常３００００９以下）を用いて、
酸可溶性コラーゲンを効率良く得る方法を研究した結果
、豚由来の動物ｍＷを用い、上記の条件で処理すること
により達成できることを見出し、完成するに至ったもの
である。In addition, it goes without saying that the higher the centrifugal acceleration used, the easier the separation between the two fractions. The area F1 is so small that it is virtually impossible to utilize it industrially. For this reason, the present invention uses a centrifugal separator with a low centrifugal acceleration (usually 300009 or less) that is actually available industrially.
As a result of research into a method for efficiently obtaining acid-soluble collagen, they discovered that it could be achieved by using animal mW derived from pigs and treating it under the above conditions, and the method was completed.
豚由来の動物組織を用い、上記のようにして遠心分離を
行うことにより、他の動物組織を原料にした場合に比べ
、大量の酸可溶性コラーゲンが得られる。By performing centrifugation as described above using animal tissues derived from pigs, a larger amount of acid-soluble collagen can be obtained than when other animal tissues are used as raw materials.
上記のようにして、遠心分離を行ったあと、上澄みの可
溶性コラーゲン画分を回収し、通常の方法にしたがって
精製を行えば、精製酸可溶性コラーゲンが得られる。After performing centrifugation as described above, the supernatant soluble collagen fraction is collected and purified according to a conventional method to obtain purified acid-soluble collagen.
このコラーゲン溶液（たとえば、３■／−濃度）８部を
水中で冷却しながら、この溶液にＮａＣｌを１．４Ｍ含
む０．１Ｍリン酸緩衝液１部およびＮａＯＨ溶液１部を
加えて、ｐＨ７，４に調整し、よく混合して３７℃に加
温すると、数分で高ゲル強度のコラーゲンゲルが形成さ
れる。While cooling 8 parts of this collagen solution (for example, 3/- concentration) in water, 1 part of 0.1M phosphate buffer containing 1.4M NaCl and 1 part of NaOH solution were added to the solution, pH 7. 4, mix well and heat to 37°C, a collagen gel with high gel strength will be formed in a few minutes.
この発明の高ゲル強度酸可溶性コラーゲンの製法は、酸
可溶性コラーゲンを高い収率で容易に得ることができる
。得られたコラーゲンから調製したコラーゲンゲルは高
いゲル強度を有しており、ゲル化速度および透明度など
の特性にも優れている。このため、このコラーゲンは、
上記のコラーゲンゲルマトリックスを利用した細胞培養
法において、マトリックスとして用いるのに適したもの
である。The method for producing acid-soluble collagen with high gel strength according to the present invention allows acid-soluble collagen to be easily obtained at a high yield. The collagen gel prepared from the obtained collagen has high gel strength and excellent properties such as gelation speed and transparency. Therefore, this collagen is
It is suitable for use as a matrix in the cell culture method using the collagen gel matrix described above.
（実施例１）
新鮮な豚皮の毛および付着物を取り除いて得た真皮層を
細断し、５％ＮａＣ１溶液および水でよく洗浄し、さら
にメタノール−クロロホルム混合溶液で脱脂し、ついで
よく洗浄して精製豚皮原料を得た。この原料１５ｇ（乾
燥重量３．８ｇ）をｐ）Ｉ２．４のＨＣＩ溶液溶液１７
００定Ｚ夜膨潤させ、ついでホモジナイザー（ポリトロ
ンＰＴＩＯ−３５型： Ｋ Ｉ ＮＥＭＡＴＩ ＣＡ社
製）を用い、よく分散し、約０．２２重量％の原料濃度
（乾燥重量基準、以下同様）の分散液を得た。得られた
分散液を時々攪拌しながら２４時間放置した後、遠心分
離機（日立高速冷却遠心機Ｈ１ＭＡＣ３ＣＲ２０Ｂ型：
日立１機社製：ＲＰＲ１２−２型ローター使用）を用い
、１２０００ｒｐｍ（最大遠心加速度２２２２０Ｘ９）
で１時間遠心分離した。なお、１回の処理量は約１１で
あった。遠心分離した溶液は、透明な上層と不透明な下
層に分離されており、上層の透明な部分を下層部が混入
しないように注意深く吸引回収し、酸可溶性コラーゲン
画分６６０−を得た。(Example 1) The dermal layer obtained by removing hair and deposits from fresh pig skin was cut into pieces, thoroughly washed with a 5% NaCl solution and water, further degreased with a methanol-chloroform mixed solution, and then washed thoroughly. A purified pork skin raw material was obtained. 15 g (dry weight 3.8 g) of this raw material was added to p) I2.4 HCI solution solution 17
00 constant Z overnight, and then well dispersed using a homogenizer (Polytron PTIO-35 type: manufactured by KINEMATI CA) to obtain a dispersion with a raw material concentration of approximately 0.22% by weight (on a dry weight basis, the same applies hereinafter). I got it. The obtained dispersion was left for 24 hours with occasional stirring, and then centrifuged (Hitachi high speed refrigerated centrifuge H1MAC3CR20B model:
12000 rpm (maximum centrifugal acceleration 22220x9)
The mixture was centrifuged for 1 hour. In addition, the amount of processing at one time was about 11. The centrifuged solution was separated into a transparent upper layer and an opaque lower layer, and the transparent upper layer was carefully collected by suction so as not to be mixed with the lower layer to obtain acid-soluble collagen fraction 660-.
次に、この溶液に１０％；；度になるように、Ｎａｃｌ
を加えて一夜放置し、生じた沈澱を遠心分離して回収し
、水洗後、ｐＨ３，０のＨＣ１溶液に再溶解した。さら
に、この溶液にＮａｏＨ溶液を加えてｐＨ７，０に調整
し、−夜装置して、生じた沈澱を回収し、よ（水洗した
後、ｐＨ３，ｏのＨＣ１溶液にコラーゲン濃度が３．０
■／ｍ／になるように溶解した。なお、上記の全操作は
すべて１０”Ｃ以下で行った。得られた精製酸可溶性コ
ラーゲン溶液は２０３ｍ１であり、収率は乾燥重量基準
で１６．０％であった。Next, add 10% NaCl to this solution.
was added and left overnight, and the resulting precipitate was collected by centrifugation, washed with water, and then redissolved in HC1 solution at pH 3.0. Furthermore, NaoH solution was added to this solution to adjust the pH to 7.0, and the resulting precipitate was collected at night. After washing with water, the collagen concentration was 3.0
It was dissolved in such a manner that the ratio was 1/m/. All of the above operations were performed at 10"C or lower. The volume of the purified acid-soluble collagen solution obtained was 203 ml, and the yield was 16.0% on a dry weight basis.
次に、前記精製酸可溶性コラーゲン溶液４０ｍＪを１０
０ｍｊビーカーにとり、氷冷しなからＮ ａ Ｃ２を１
．４Ｍ含む０．１Ｍリン酸緩Ｓｈ液５−を加えてよく混
合し、さらにＮ ａ ＯＨ溶液５−を加えてｐＨ７，４
に調整し、直ちに３７℃の恒温槽に入れ、１時間保持し
た。得られたコラーゲンゲルをレオメータ−（不動工業
社製、ＮＲＭ−２００２Ｄ型：径１″／２粘弾性用アダ
プター、浸入ａ度り０ｎ、浸入速度６ｃｍ／分で測定）
を用いてゲル強度を測定した結果、２３５ｇの高いゲル
強度であった。Next, 40 mJ of the purified acid-soluble collagen solution was added to
Place in a 0mj beaker and cool on ice, then add 1 ml of NaC2.
．． Add 4M of 0.1M phosphoric acid mild Sh solution 5- and mix well, then add NaOH solution 5- to adjust pH to 7.4.
The mixture was immediately placed in a constant temperature bath at 37°C and maintained for 1 hour. The obtained collagen gel was measured using a rheometer (manufactured by Fudo Kogyo Co., Ltd., NRM-2002D type: diameter 1''/2 viscoelastic adapter, penetration a degree 0n, penetration speed 6cm/min)
As a result of measuring the gel strength using , the gel strength was as high as 235 g.
（実施例２）
新鮮な豚足部から採取した鍵を実施例１と同様に細断し
、精製して得た精製豚皮原料１５ｇ（乾燥重量３．３ｇ
）をｐ Ｈ２，２のＨ（ｌ溶液１６５０ｍＺ（乾燥重量
で約０．２％濃度）に膨潤し、引き続き実施例１と同様
に処理して精製酸可溶性コラーゲン溶液（濃度３． Ｏ
ｑ／ ｍｊ） １９８ ａｎｔを得た。収率は１８．０
％であった。また、実施例１と同様にしてゲル強度を測
定したところ、２４６ｇであった。(Example 2) 15 g of purified pork skin raw material (dry weight 3.3 g
) was swollen in a solution of H (1650 mZ (about 0.2% dry weight) at pH 2.2) and subsequently treated as in Example 1 to obtain a purified acid-soluble collagen solution (concentration 3.0
q/mj) 198 ant were obtained. Yield is 18.0
%Met. Further, when the gel strength was measured in the same manner as in Example 1, it was 246 g.
（実施例３）
実施例２において、分散液の原料濃度０．２重量％およ
びｐＨ２，２を、原料濃度１．５重量％およびｐ Ｈ３
，５に変えて分散を行い、２４時間後ＨＣ１゜溶液を加
えて、原料濃度０．２重量％、ｐ Ｈ２，２に調整し直
ちに実施例１と同様にして遠心分離および精製を行い、
精製酸可溶性コラーゲン溶液１８１ ｍｌを得た。収率
は１６．５％であった。また、実施例１と同様にしてゲ
ル強度を測定したところ、２２０ｇであった。(Example 3) In Example 2, the raw material concentration of the dispersion was changed from 0.2% by weight and pH 2.2 to 1.5% by weight and pH 3.
After 24 hours, a 1° solution of HC was added to adjust the raw material concentration to 0.2% by weight and pH to 2.2, and immediately centrifugation and purification were carried out in the same manner as in Example 1.
181 ml of purified acid-soluble collagen solution was obtained. The yield was 16.5%. Further, when the gel strength was measured in the same manner as in Example 1, it was 220 g.
（比較例１）
実施例２において、分散液の原料濃度０．２重量％を１
．２重量％に変えた以外は、すべて実施例２と同じ条件
で処理したところ、遠心分離によって透明な酸可溶性画
分が得られなかった。(Comparative Example 1) In Example 2, the raw material concentration of the dispersion liquid was 0.2% by weight.
．． When the treatment was carried out under the same conditions as in Example 2 except that the concentration was changed to 2% by weight, no clear acid-soluble fraction was obtained by centrifugation.
（比較例２）
実施例２において、分散液のｐ Ｈ２，２を３．５に変
えた以外は、すべて実施例２と同じ条件で処理したとこ
ろ、遠心分離によって透明な酸可溶性画分が得られなか
った。(Comparative Example 2) In Example 2, all treatments were carried out under the same conditions as in Example 2, except that the pH of the dispersion was changed to 3.5. A transparent acid-soluble fraction was obtained by centrifugation. I couldn't.
（比較例３）
新鮮な牛皮を実施例１と同様に精製した後、実施例１と
同様の条件で処理したところ、精製酸可溶性コラーゲン
溶液が得られたが、収率は３．４％であった。(Comparative Example 3) Fresh cowhide was purified in the same manner as in Example 1, and then treated under the same conditions as in Example 1. A purified acid-soluble collagen solution was obtained, but the yield was 3.4%. there were.
（比較例４）
新鮮な手足部から採取した股を、実施例１と同様に精製
した後、実施例２と同様の条件で処理したところ、精製
酸可溶性コラーゲン溶液が得られたが、収率は４．９％
であった。(Comparative Example 4) Crotches collected from fresh limbs were purified in the same manner as in Example 1 and then treated under the same conditions as in Example 2. A purified acid-soluble collagen solution was obtained, but the yield was low. is 4.9%
Met.
Ｃ比較例５）
実施例１と同様にして得られたコラーゲン分散液にペプ
シン（乾燥重量に対し、４％）を加え、攪拌しながら２
０℃で４８時間処理し、原料コラーゲンを溶解してアテ
ロコラーゲン溶液を得た。C Comparative Example 5) Pepsin (4% based on dry weight) was added to the collagen dispersion obtained in the same manner as in Example 1, and 2% was added while stirring.
The mixture was treated at 0° C. for 48 hours to dissolve the raw collagen to obtain an atelocollagen solution.
この溶液を精製水を加えて２倍に希釈した後、グラスフ
ィルターで濾過し、残渣を除き、引き続き実施例１と同
様に精製処理を行って、精製アテロコラーゲン溶液を得
た。収率は８７．５％であった。この精製アテロコラー
ゲン溶液を実施例１と同様にしてゲル強度を測定したと
ころ、６５ｇと低いゲル強度であった。This solution was diluted twice by adding purified water, filtered through a glass filter to remove the residue, and then purified in the same manner as in Example 1 to obtain a purified atelocollagen solution. The yield was 87.5%. When the gel strength of this purified atelocollagen solution was measured in the same manner as in Example 1, the gel strength was as low as 65 g.
第１表に、実施例および比較例でそれぞれ得た可溶性コ
ラーゲンの原料、収率およびゲル強度を示した。Table 1 shows the raw materials, yields, and gel strengths of the soluble collagen obtained in Examples and Comparative Examples.
第 １ 表
第１表にみるように、同じ酸処理であっても、豚由来の
原料からは高い収率で可溶性コラーゲンが得られている
のに対して、牛山来の原料からは低い収率でしか可溶性
コラーゲンが得られていない。同じ豚由来の原料からで
も、酵素処理コラーゲンは、酸可溶性コラーゲンに比べ
て非常に高い収率で得られている。しかし、酵素処理コ
ラーゲンは、ゲル化した場合、酸可溶性コラーゲンに比
べてゲル強度が非常に小さい。Table 1 As shown in Table 1, even with the same acid treatment, soluble collagen can be obtained at a high yield from raw materials derived from pigs, but at a low yield from raw materials derived from Ushiyama. Soluble collagen can only be obtained in Enzyme-treated collagen can be obtained at a much higher yield than acid-soluble collagen even from the same raw material derived from pigs. However, when enzyme-treated collagen is gelled, its gel strength is much lower than that of acid-soluble collagen.
この発明にかかる高ゲル強度酸可溶性コラーゲンの製法
は、以上にみるように、豚由来の動物組織を用いるよう
にするとともに、抽出により得られた溶液を、前記動物
組織の乾燥固形分量１重量％以下、ｐＨ３以下、遠心加
速度３００００９以下の条件で遠心分離にかけて不溶性
コラーゲンを除くことを特徴とするので、ゲル強度の高
いコラーゲンゲルを与えるコラーゲンを高い収率で得る
ことができる。As described above, the method for producing acid-soluble collagen with high gel strength according to the present invention uses animal tissue derived from pigs, and adds a solution obtained by extraction to a dry solid content of 1% by weight of the animal tissue. Hereinafter, since insoluble collagen is removed by centrifugation under conditions of pH 3 or less and centrifugal acceleration 300009 or less, collagen that provides a collagen gel with high gel strength can be obtained at a high yield.
Claims (1)
ゲンを得る方法において、前記動物組織として豚由来の
ものを用いるようにするとともに、抽出により得られた
溶液を、前記動物組織の乾燥固形分量１重量％以下、ｐ
Ｈ３以下、遠心加速度３００００ｇ以下の条件で遠心分
離にかけて不溶性コラーゲンを除くことを特徴とする高
ゲル強度酸可溶性コラーゲンの製法。(1) In the method of obtaining soluble collagen by extraction from animal tissue with an acid solution, the animal tissue is derived from a pig, and the solution obtained by the extraction is mixed with a dry solid content of 1 Weight% or less, p
A method for producing acid-soluble collagen with high gel strength, characterized by removing insoluble collagen by centrifugation under conditions of H3 or less and centrifugal acceleration of 30,000 g or less.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61208436A JPH0774239B2 (en) | 1986-09-04 | 1986-09-04 | Manufacturing method of high gel strength acid soluble collagen for cell culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61208436A JPH0774239B2 (en) | 1986-09-04 | 1986-09-04 | Manufacturing method of high gel strength acid soluble collagen for cell culture |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6363700A true JPS6363700A (en) | 1988-03-22 |
| JPH0774239B2 JPH0774239B2 (en) | 1995-08-09 |
Family
ID=16556176
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61208436A Expired - Lifetime JPH0774239B2 (en) | 1986-09-04 | 1986-09-04 | Manufacturing method of high gel strength acid soluble collagen for cell culture |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0774239B2 (en) |
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| AU2006247317B2 (en) | 2005-05-16 | 2012-04-05 | Purdue Research Foundation | Engineered extracellular matrices |
| US20070269476A1 (en) | 2006-05-16 | 2007-11-22 | Voytik-Harbin Sherry L | Engineered extracellular matrices control stem cell behavior |
| AU2007297611B2 (en) | 2006-09-21 | 2013-02-07 | Purdue Research Foundation | Collagen preparation and method of isolation |
| WO2009076441A1 (en) | 2007-12-10 | 2009-06-18 | Purdue Research Foundation | Collagen-based matrices with stem cells |
| US9878071B2 (en) | 2013-10-16 | 2018-01-30 | Purdue Research Foundation | Collagen compositions and methods of use |
| US11919941B2 (en) | 2015-04-21 | 2024-03-05 | Purdue Research Foundation | Cell-collagen-silica composites and methods of making and using the same |
| CA3061428A1 (en) | 2017-04-25 | 2018-11-01 | Purdue Research Foundation | 3-dimensional (3d) tissue-engineered muscle for tissue restoration |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS50130798A (en) * | 1974-04-04 | 1975-10-16 | ||
| JPS60127364A (en) * | 1983-12-13 | 1985-07-08 | Watanabe Yakuhin Kogyo Kk | Production of liquid or gel of acid collagen fibril for forming coating film or formed coating film |
-
1986
- 1986-09-04 JP JP61208436A patent/JPH0774239B2/en not_active Expired - Lifetime
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS50130798A (en) * | 1974-04-04 | 1975-10-16 | ||
| JPS60127364A (en) * | 1983-12-13 | 1985-07-08 | Watanabe Yakuhin Kogyo Kk | Production of liquid or gel of acid collagen fibril for forming coating film or formed coating film |
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