JP3877978B2

JP3877978B2 - Cell preservation method

Info

Publication number: JP3877978B2
Application number: JP2001148674A
Authority: JP
Inventors: 研一寺本; 譲原; 滋樹有井
Original assignee: Japan Science and Technology Agency; National Institute of Japan Science and Technology Agency
Current assignee: Japan Science and Technology Agency; National Institute of Japan Science and Technology Agency
Priority date: 2001-05-18
Filing date: 2001-05-18
Publication date: 2007-02-07
Anticipated expiration: 2021-05-18
Also published as: JP2002335954A

Description

【０００１】
【発明の属する技術分野】
本発明は、細胞、例えば肝細胞等の新規で且つ有効な冷蔵保存方法に関する。
【０００２】
【従来の技術】
従来、細胞の保存は培養による継代保存あるいは凍結保存が行われ、冷蔵保存の方法はなかった。しかし、継代保存は正常細胞では長期保存が出来ず、また凍結保存の場合は、手間がかかることと、凍結時の水の結晶化による細胞骨格のダメージが大きい等の欠点があり、改善が望まれていた。
冷蔵保存は、一般に利便性の点で有利であるが、細胞の保存、特に肝細胞の保存は難しく、プログラミングフリーザーを用いて凍結し、液体窒素中で保存しているのが現状である。ところが、この方法ではＤＭＳＯ、グリセロール等の凍結保存剤やシュークロースによる脱水処理を加えても長期保存後の細胞再生時生存率は１０％程度で実用性に耐えない。その原因は、細胞凍結保存法では、上でも述べたように、凍結時及び解凍時に細胞内骨格障害が水の結晶形成に伴って起こり生存率が低下するためである。
一方、自然界に存在する冬眠誘導物質（Hibernation induction trigger(HIT)）と非常に化学構造が似た冬眠誘導物質Ｄ−Ａｌａ^２，Ｄ−Ｌｅｕ^５−エンケファリン（ＤＡＤＬＥ）は、うさぎ冷阻血心臓に対し臓器保護作用のあることが報告されている（Transplantation,vol.63,326-329,1997)。また魚介類の鮮度保持、活魚輸送の方法としてエンケファリン誘導体が使用されている特許出願もある（特開平１１−２２０９７４号公報、特開平１１−３１８２７３号公報等）。
しかしながら、細胞そのものを冷蔵保存するためにこれらのエンケファリン誘導体を使用している例は未だない。
【０００３】
【発明が解決しようとする課題】
本発明は、上記した如き現状に鑑みなされたもので、従来知られている凍結細胞保存法における、細胞凍結時の水の結晶化による細胞骨格の破損を防止するため、肝細胞等の細胞を水が結晶化しない温度、例えば４℃前後の温度で冷蔵保存する方法を提供することを目的とする。
【０００４】
【課題を解決するための手段】
本発明は、細胞培養液中にエンケファリン誘導体を添加することを特徴とする細胞の保存方法に関する。
【０００５】
また、本発明は、細胞培養液中にエンケファリン誘導体を添加し、冷蔵保存してなる細胞に関する。
【０００６】
更に、本発明は、細胞移植に用いる上記細胞に関する。
【０００７】
更にまた、本発明は、組織又は細胞を、コラゲナーゼを用いた門脈灌流法より細胞分離を行った後、細胞培養して該細胞をスフェロイド化し、次いで、培養液中にエンケファリン誘導体を添加して、更に培養を行い、然る後、水が結晶化しない温度で所定時間冷蔵保存する細胞の保存方法に関する。
【０００８】
また、本発明は、エンケファリン誘導体を含有してなる細胞保存剤に関する。
【０００９】
更に、本発明は、エンケファリン誘導体を含有してなることを特徴とする細胞（培養）保存液に関する。
【００１０】
【発明の実施の形態】
本発明で用いられるエンケファリン誘導体としては、例えば、メチオニンエンケファリン、ロイシンエンケファリン、Ｄ−Ａｌａ^２，Ｄ−Ｌｅｕ^５−エンケファリン（ＤＡＤＬＥ）等が挙げられるが、ＤＡＤＬＥが特に好ましい。
【００１１】
本発明の保存方法を実施するには、水が結晶化しない温度で冷蔵保存することが望ましく、例えば４℃前後の温度で冷蔵保存するのが好ましい。
また、本発明の冷蔵保存した細胞は、水が結晶化しない温度で冷蔵保存したものであることが望ましく、例えば４℃前後の温度で冷蔵保存したものが好ましい。
【００１２】
本発明の保存方法を実施するには、組織又は細胞をコラゲナーゼ等により処理して細胞分離を行い、次いでこれをスフェロイド化した後、培養液中にエンケファリン誘導体を添加するのが好ましい。
また、本発明の冷蔵保存した細胞は、組織又は細胞をコラゲナーゼ等により処理して細胞分離を行い、次いでこれをスフェロイド化した後、培養液中にエンケファリン誘導体を添加し、冷蔵保存したものであることが望ましい。
【００１３】
本発明の方法により保存可能な細胞としては、特に限定されるものではないが、例えば、肝臓、肺、心臓、腎臓等の臓器の細胞や、神経細胞等が挙げられる。
中でも、これまで特に冷蔵保存が難しいとされていた肝細胞を冷蔵保存する場合に本発明の方法は極めて有効であり、肝細胞移植等の細胞移植における細胞保存方法として大いに期待されるものである。
更に、最近ＥＳ細胞や各臓器の前駆細胞が注目されているが、本発明の保存方法は、これらＥＳ細胞や各臓器の前駆細胞の保存にも大いにその効果が期待できるので、再生医療の臨床応用を考えたとき、本発明の方法は、極めて重要な技術となる。
【００１４】
本発明の保存方法を、肝細胞を保存する場合を例にして説明すると、概略以下の如くなる。
先ず、肝細胞又は肝組織をコラゲナーゼ等を用いて門脈灌流を行い、細胞分離を行う。
分離後、例えばポリ（Ｎ−ｐ−ビニルベンジル−ラクトアミド）（ＰＶＬＡ）等でコートしたディッシュ上で、例えば上皮増殖因子（ＥＧＦ）等の存在下、細胞培養を２日間行い浮遊肝細胞スフェロイド形成を誘導する。
この後、ＤＡＤＬＥ等のエンケファリン誘導体を培地中に添加し、更に２４時間通常の培養を行った後、４℃で所定時間冷蔵保存を行う。
【００１５】
本発明の細胞（培養）保存液は、それぞれの細胞の培養に適した、既存の或いは新規な培養液にＤＡＤＬＥ等の本発明に係るエンケファリン誘導体を添加したものであって、この培養保存液に細胞を加え、水が結晶化しない温度、例えば４℃に冷蔵するだけで簡単に大量の細胞の長期間保存が可能になる。
【００１６】
【実施例】
以下、実施例により本発明をより具体的に説明するが、本発明はこれら実施例により何ら限定されるものではない。
【００１７】
実施例１
下記方法により肝細胞の冷蔵保存を行った。
実験動物：６−７週齢、雄、ＳＤラット
肝細胞採取法：コラゲナーゼ・ＥＤＴＡによる門脈灌流法
スフェロイド形成：ポリ（Ｎ−ｐ−ビニルベンジル−ラクトアミド）（ＰＶＬＡ）コートディッシュ上で上皮増殖因子（ＥＧＦ）存在下に肝細胞培養を二日間行い浮遊肝細胞スフェロイド形成を誘導した。
保存実験：スフェロイド形成を行った後、ＤＡＤＬＥを培地中に添加し、更に２４時間通常の培養を行い、然る後、４℃で各種保存時間（１，３，５，７週間）冷蔵保存を行った。
冷蔵保存後、リン酸緩衝液にてスフェロイドを洗浄し、その後、再び通常の３７℃培養にて３日間培養を行い、各培養液中の尿素合成能、総胆汁酸分泌能を各ＤＡＤＬＥ濃度（０，８，３０，７５，２００μｇ/ｍｌ）について調べた。
結果を図１及び図２にそれぞれ示す。
図１及び図２中、■はＤＡＤＬＥ濃度０のときの、▲はＤＡＤＬＥ濃度８μｇ/ｍｌのときの、×はＤＡＤＬＥ濃度３０μｇ/ｍｌのときの、●はＤＡＤＬＥ濃度７５μｇ/ｍｌのときの、◆はＤＡＤＬＥ濃度２００μｇ/ｍｌのときの測定結果をそれぞれ示す。
【００１８】
図１から明らかなように、尿素合成能は、ＤＡＤＬＥ低濃度群において十分に維持され、７週間冷蔵保存後においても尿素合成能が認められた。
また、図２から明らかなように、総胆汁酸分泌能は７週間冷蔵保存においてもＤＡＤＬＥ添加群では認められた。
【００１９】
実施例２培養肝細胞に対するＤＡＤＬＥの影響
形態上の検討としてスフェロイド形成後の肝細胞冷蔵保存におけるＤＡＤＬＥの効果について調べた。
コラゲナーゼ肝灌流法による肝細胞採取後、インスリン、デキサメサゾン、アプロチニン、ＥＧＦ添加のＷＥ培地+２０％ＦＣＳ（牛胎児血清）にて３日間ＰＶＬＡコートディッシュ上培養を行い、スフェロイドを形成させた。３ｃｍディッシュ当たり５×１０^５個にて培養した。
ＤＡＤＬＥを各濃度（０，８，３０，７５，２００μｇ/ｍｌ）培地中に添加して、１日間培養後、４℃にて２週間保存した。この後、通常の３７℃培養系に戻し、インスリン、デキサメサゾン、アプロチニン、ＥＧＦ添加のＷＥ培地+２０％ＦＣＳにて再び３日間ＰＶＬＡコートディッシュ上培養を行い、観察した（×１００）。結果を図３に示す。
図３中、ＡはＤＡＤＬＥ濃度０のときの、ＢはＤＡＤＬＥ濃度８μｇ/ｍｌのときの、ＣはＤＡＤＬＥ濃度３０μｇ/ｍｌのときの、ＤはＤＡＤＬＥ濃度７５μｇ/ｍｌのときの、ＥはＤＡＤＬＥ濃度２００μｇ/ｍｌのときの結果をそれぞれ示す。
【００２０】
図３から明らかなように、通常分離した細胞と異なりスフェロイド形成された肝細胞塊では、明瞭にトリパンブルー色素法による細胞生存率の評価は困難であるが、７週間保存した細胞群においても充分細胞形態は保たれていた。ＤＡＤＬＥ濃度依存性にスフェロイドの保存状態が改善されていて、２００μｇ/ｍｌでは崩れて死滅したスフェロイドはまばらに観察されるのみであった。
【００２１】
【発明の効果】
本発明は、細胞培養液、就中、細胞浮遊液中にＤＡＤＬＥ等のエンケファリン誘導体を添加することにより細胞を長期間冷蔵保存可能としたもので、細胞を冷蔵保存できる利便性からも極めて有用な技術であり、細胞移植等における細胞の確保に有効な手段となる。更に、最近ＥＳ細胞や各臓器の前駆細胞が注目されているが、本発明の保存方法は、これらＥＳ細胞や各臓器の前駆細胞の保存にも大いにその効果が期待できるので、再生医療の臨床応用を考えたとき、本発明の方法は、極めて重要な技術となる。また、本発明は、ＤＡＤＬＥ等のエンケファリン誘導体を含んでなる新規な細胞培養保存液を提供するものであるが、この培養保存液に細胞を加え、水が結晶化しない温度、例えば４℃に冷蔵するだけで簡単に大量の細胞の長期間保存が可能になる。この点が本発明のこれまでにない革新的な点であり、臓器の保存液についてはこれまで多くの研究が行われてきたが、細胞保存液については未だ十分な研究がなされておらず、有効な細胞保存液がなかったと言うことからも、本発明は、当該分野における画期的な発明であると言うことが出来る。
【図面の簡単な説明】
【図１】図１は、実施例１で得られた冷蔵保存肝細胞培養液中の尿素合成能を各ＤＡＤＬＥ濃度（０，８，３０，７５，２００μｇ/ｍｌ）について調べた結果を示す。
【図２】図２は、実施例１で得られた冷蔵保存肝細胞培養液中の総胆汁酸分泌能を各ＤＡＤＬＥ濃度（０，８，３０，７５，２００μｇ/ｍｌ）について調べた結果を示す。
【図３】図３は、実施例２で得られた培養肝細胞に対するＤＡＤＬＥの影響を調べた結果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel and effective refrigerated storage method for cells such as hepatocytes.
[0002]
[Prior art]
Conventionally, cell storage has been performed by subculture or cryopreservation by culture, and there has been no method of refrigeration storage. However, passaging is not possible for long-term storage in normal cells, and in the case of cryopreservation, there are drawbacks such as time-consuming work and damage to the cytoskeleton due to water crystallization during freezing. It was desired.
Refrigerated storage is generally advantageous in terms of convenience, but it is difficult to store cells, particularly hepatocytes, and it is currently frozen using a programming freezer and stored in liquid nitrogen. However, in this method, even when a cryopreservation agent such as DMSO or glycerol or dehydration treatment with sucrose is added, the survival rate during cell regeneration after long-term storage is about 10%, which is not practical. This is because, in the cell cryopreservation method, as described above, an intracellular skeletal disorder occurs along with water crystal formation at the time of freezing and thawing, and the survival rate decreases.
On the other hand, hibernation inducer D-Ala ² , D-Leu ⁵ -enkephalin (DADLE), which has a very similar chemical structure to hibernation induction trigger (HIT) in nature, It has been reported to have an organ protecting action (Transplantation, vol. 63, 326-329, 1997). There are also patent applications in which an enkephalin derivative is used as a method for maintaining the freshness of seafood and transporting live fish (JP-A-11-220974, JP-A-11-318273, etc.).
However, there are still no examples of using these enkephalin derivatives for refrigerated storage of the cells themselves.
[0003]
[Problems to be solved by the invention]
The present invention has been made in view of the present situation as described above, and in order to prevent damage to the cytoskeleton due to crystallization of water during cell freezing in a conventionally known frozen cell storage method, cells such as hepatocytes are used. It is an object of the present invention to provide a method for refrigerated storage at a temperature at which water does not crystallize, for example, a temperature around 4 ° C.
[0004]
[Means for Solving the Problems]
The present invention relates to a method for preserving cells, which comprises adding an enkephalin derivative to a cell culture medium.
[0005]
The present invention also relates to cells obtained by adding an enkephalin derivative to a cell culture medium and refrigerated.
[0006]
Furthermore, this invention relates to the said cell used for a cell transplant.
[0007]
Furthermore, in the present invention, after tissue or cells are separated by the portal vein perfusion method using collagenase, the cells are cultured to spheroidize the cells, and then an enkephalin derivative is added to the culture solution. Furthermore, the present invention relates to a method for preserving cells by further culturing and then refrigerated for a predetermined time at a temperature at which water does not crystallize.
[0008]
The present invention also relates to a cell preservative comprising an enkephalin derivative.
[0009]
Furthermore, the present invention relates to a cell (culture) preservation solution characterized by containing an enkephalin derivative.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
Examples of the enkephalin derivative used in the present invention include methionine enkephalin, leucine enkephalin, D-Ala ² , D-Leu ⁵ -enkephalin (DADLE), and DADLE is particularly preferable.
[0011]
In order to carry out the storage method of the present invention, it is desirable to store in a refrigerator at a temperature at which water does not crystallize, for example, to store in a refrigerator at a temperature of around 4 ° C.
In addition, the refrigerated cells of the present invention are desirably those that have been refrigerated at a temperature at which water does not crystallize. For example, cells that have been refrigerated at a temperature of about 4 ° C. are preferred.
[0012]
In order to carry out the preservation method of the present invention, it is preferable to treat the tissue or cells with collagenase or the like to separate the cells, then spheroidize them, and then add the enkephalin derivative to the culture solution.
Further, the refrigerated cells of the present invention are those obtained by treating tissues or cells with collagenase or the like to separate cells, then spheroidizing them, adding an enkephalin derivative to the culture medium, and refrigerated. It is desirable.
[0013]
The cells that can be preserved by the method of the present invention are not particularly limited, and examples thereof include cells of organs such as liver, lung, heart, and kidney, and nerve cells.
Among them, the method of the present invention is extremely effective when refrigerated and stored for hepatocytes, which has been considered difficult to be refrigerated, and is highly expected as a cell storage method in cell transplantation such as hepatocyte transplantation. .
Furthermore, ES cells and progenitor cells of various organs have recently attracted attention, but the preservation method of the present invention can be expected to be highly effective in preserving these ES cells and progenitor cells of each organ. When considering application, the method of the present invention is a very important technology.
[0014]
The storage method of the present invention will be described as follows, taking the case of storing hepatocytes as an example.
First, hepatocytes or liver tissue is perfused with portal vein using collagenase or the like to separate cells.
After separation, cell culture is performed for 2 days in the presence of, for example, epidermal growth factor (EGF) on a dish coated with, for example, poly (Np-vinylbenzyl-lactoamide) (PVLA), etc. to form floating hepatocyte spheroids. Induce.
Thereafter, an enkephalin derivative such as DADLE is added to the medium, and further normal culture is performed for 24 hours, followed by refrigeration at 4 ° C. for a predetermined time.
[0015]
The cell (culture) preservation solution of the present invention is obtained by adding an enkephalin derivative according to the present invention such as DADLE to an existing or novel culture solution suitable for culturing each cell. A large amount of cells can be stored for a long period of time simply by adding cells and refrigeration to a temperature at which water does not crystallize, for example, 4 ° C.
[0016]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.
[0017]
Example 1
Refrigerated storage of hepatocytes was performed by the following method.
Experimental animals: 6-7 weeks old, male, SD rat hepatocyte collection method: portal vein perfusion method with collagenase EDTA spheroid formation: epidermal growth factor on poly (Np-vinylbenzyl-lactoamide) (PVLA) coated dish Hepatocyte culture was performed in the presence of (EGF) for 2 days to induce floating hepatocyte spheroid formation.
Preservation experiment: After spheroid formation, DADLE is added to the medium, and further normal culture is performed for 24 hours. After that, various storage times (1, 3, 5, and 7 weeks) are stored at 4 ° C. went.
After refrigerated storage, the spheroids were washed with a phosphate buffer solution, and then cultured again at normal 37 ° C. for 3 days. The urea synthesis capacity and total bile acid secretion capacity in each culture liquid were measured for each DADLE concentration ( (0, 8, 30, 75, 200 μg / ml).
The results are shown in FIGS. 1 and 2, respectively.
In FIG. 1 and FIG. 2, ■ is when DADLE concentration is 0, ▲ is when DADLE concentration is 8 μg / ml, × is when DADLE concentration is 30 μg / ml, ● is when DADLE concentration is 75 μg / ml, ◆ Indicates the measurement results when the DADLE concentration is 200 μg / ml.
[0018]
As is clear from FIG. 1, the urea synthesis ability was sufficiently maintained in the DADLE low concentration group, and the urea synthesis ability was recognized even after refrigerated storage for 7 weeks.
Further, as is clear from FIG. 2, the total bile acid secretion ability was recognized in the DADLE addition group even after refrigerated storage for 7 weeks.
[0019]
Example 2 Effect of DADLE on cultured hepatocytes As an examination of the morphology, the effect of DADLE in refrigerated storage of hepatocytes after spheroid formation was examined.
After collecting hepatocytes by the collagenase liver perfusion method, spheroids were formed by culturing on a PVLA-coated dish for 3 days in WE medium + 20% FCS (fetal calf serum) supplemented with insulin, dexamethasone, aprotinin and EGF. The cells were cultured at 5 × 10 ⁵ cells per 3 cm dish.
DADLE was added to each concentration (0, 8, 30, 75, 200 μg / ml) medium, cultured for 1 day, and stored at 4 ° C. for 2 weeks. Thereafter, the cells were returned to a normal 37 ° C. culture system, and cultured on a PVLA-coated dish again for 3 days in a WE medium + 20% FCS supplemented with insulin, dexamethasone, aprotinin and EGF, and observed (× 100). The results are shown in FIG.
In FIG. 3, A is when the DADLE concentration is 0, B is when the DADLE concentration is 8 μg / ml, C is when the DADLE concentration is 30 μg / ml, D is when the DADLE concentration is 75 μg / ml, and E is the DADLE concentration. The results at 200 μg / ml are shown respectively.
[0020]
As is clear from FIG. 3, it is difficult to clearly evaluate the cell viability by the trypan blue dye method in the spheroidized hepatocyte mass unlike the normal isolated cells, but it is sufficient even in the cell group preserved for 7 weeks. Cell morphology was preserved. The storage state of spheroids was improved depending on the DADLE concentration, and spheroids that collapsed and died at 200 μg / ml were only sparsely observed.
[0021]
【The invention's effect】
The present invention allows cells to be refrigerated for a long period of time by adding an enkephalin derivative such as DADLE to a cell culture solution, especially a cell suspension, and is extremely useful from the standpoint of refrigerated storage of cells. It is a technology and is an effective means for securing cells in cell transplantation and the like. Furthermore, ES cells and progenitor cells of various organs have recently attracted attention, but the preservation method of the present invention can be expected to have a great effect on the preservation of these ES cells and progenitor cells of each organ. When considering application, the method of the present invention is a very important technology. The present invention also provides a novel cell culture preservation solution comprising an enkephalin derivative such as DADLE. Cells are added to the culture preservation solution and refrigerated at a temperature at which water does not crystallize, for example, 4 ° C. This makes it easy to store a large number of cells for a long period of time. This point is an unprecedented innovative point of the present invention, and many studies have been conducted on organ preservation solutions, but sufficient research has not yet been conducted on cell preservation solutions. It can be said that the present invention is an epoch-making invention in the field because there is no effective cell preservation solution.
[Brief description of the drawings]
FIG. 1 shows the results of examining urea synthesis ability in chilled and stored hepatocyte culture solutions obtained in Example 1 for each DADLE concentration (0, 8, 30, 75, 200 μg / ml).
FIG. 2 shows the results of examining the total bile acid secretion ability in the refrigerated hepatocyte culture obtained in Example 1 for each DADLE concentration (0, 8, 30, 75, 200 μg / ml). Show.
FIG. 3 shows the results of examining the influence of DADLE on cultured hepatocytes obtained in Example 2.

Claims

A method for preserving hepatocytes, comprising adding D-Ala ² , D-Leu ⁵ -enkephalin (DADLE) to a cell culture medium.

The storage method according to claim 1, wherein refrigeration storage is performed at a temperature at which water does not crystallize.

The storage method according to claim 2, wherein the storage is refrigerated at a temperature of about 4 ° C.

After spheronizing liver tissue or liver ^{cells, D-Ala 2, D-} Leu 5 in the culture - storage method according to any one of claims 1 to 3 added enkephalin (DADLE).

Liver tissue or liver cells, after cell separation portal vein perfusion method using collagenase, and the cells cultured spheroids of the cells, then, D-Ala 2 in the culture medium, ^D-Leu 5 ^- enkephalin (DADLE) is added, culture is further performed, and after that, the cell is stored at a temperature at which water does not crystallize for a predetermined time.

A cell preservative comprising D-Ala ² , D-Leu ⁵ -enkephalin (DADLE).

A cell (culture) preservation solution comprising D-Ala ² , D-Leu ⁵ -enkephalin (DADLE).