JPH06327462A - Formation of cell aggregate - Google Patents
Formation of cell aggregateInfo
- Publication number
- JPH06327462A JPH06327462A JP5119579A JP11957993A JPH06327462A JP H06327462 A JPH06327462 A JP H06327462A JP 5119579 A JP5119579 A JP 5119579A JP 11957993 A JP11957993 A JP 11957993A JP H06327462 A JPH06327462 A JP H06327462A
- Authority
- JP
- Japan
- Prior art keywords
- cell
- cells
- culture
- cell aggregates
- cell aggregate
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/12—Well or multiwell plates
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、肝細胞、乳腺上皮細
胞、腎細胞等の機能細胞を３次元の立体構造である細胞
凝集体の形態に培養し、その機能発現を維持する為の培
養法に関するものである。更に詳しくは、マルチプレー
トのように多数個のウエルを有するプレート内で、各ウ
エルに単一のほぼ同一サイズの細胞凝集体を形成させ、
あるいはチューブに１個の細胞凝集体を形成させる方法
に関するものである。The present invention relates to a culture for culturing functional cells such as hepatocytes, mammary gland epithelial cells and kidney cells in the form of a cell aggregate having a three-dimensional structure and maintaining the function expression. It is about law. More specifically, in a plate having a large number of wells such as a multiplate, a single cell aggregate of approximately the same size is formed in each well,
Alternatively, it relates to a method for forming a single cell aggregate in a tube.
【０００２】[0002]
【従来の技術】肝細胞等の３次元凝集体は、一般にスフ
ェロイド等の名称で呼ばれているが、スフェロイドの定
義が学術的にもやや不明確であるため、本発明では一般
にいわれているスフェロイドを含めて細胞凝集体と呼ぶ
ことにする。この細胞凝集体の形成方法としては、ポリ
ビニルベンジルラクトンアミドを塗布したポリスチレン
シャーレ（戸辺ら，人工臓器，２１巻，３号，ｐ．１０
４５−１０４９，１９９２）、アミノ基を導入したシャ
ーレであるプライマリア３００１（高畠ら，人工臓器，
１９巻，３号，ｐ．１１６５−１１６８，１９９０）、
セルロースにプロピルイソシアネートをグラフトした基
材（松田，生体材料，１０巻，１号，ｐ．１８−３５，
１９９２）等が盛んに研究されている。2. Description of the Related Art A three-dimensional aggregate such as a hepatocyte is generally called by a name such as spheroid. However, since the definition of spheroid is somewhat unclear academically, the spheroid generally referred to in the present invention. Will be referred to as a cell aggregate. Polystyrene petri dishes coated with polyvinylbenzyl lactone amide (Tobe et al., Artificial Organs, Vol. 21, No. 3, p. 10)
45-1049, 1992), an amino group-introduced petri dish, Primary A 3001 (Takahata et al., Artificial organ,
Volume 19, Issue 3, p. 1165-1168, 1990),
Base material obtained by grafting propyl isocyanate on cellulose (Matsuda, Biomaterials, Vol. 10, No. 1, p. 18-35.
1992) and the like are being actively studied.
【０００３】一方、ポリイソプロピルアクリルアミド
（ＩＰＡＡ）のように低温で水溶性、高温で非水溶性に
なる熱可逆型高分子を塗布した基材上で、３７℃で細胞
を培養し、室温に戻して低温で細胞を剥離し細胞凝集体
を形成させるような研究（森ら，Ｂio Ｔechnology ，
８巻，９号，１９９０）等も実施されている。On the other hand, cells are cultured at 37 ° C. on a substrate coated with a thermoreversible polymer that becomes water-soluble at low temperature and water-insoluble at high temperature such as polyisopropylacrylamide (IPAA), and is returned to room temperature. Study of detaching cells at low temperature to form cell aggregates (Mori et al., Bio Technology,
Vol.8, No.9, 1990) etc. are also implemented.
【０００４】このように細胞凝集体の形成に関する研究
は種々実施されてきたが、従来の技術で形成される細胞
凝集体は、いずれもその大きさが不均一であり、種々の
サイズの細胞凝集体が混在した培養系であった。従っ
て、培養のロット毎に細胞の数や細胞凝集体の大きさや
その分布が不均一であり、常に一定の条件で評価をする
ということは極めて困難であった。As described above, various studies on the formation of cell aggregates have been carried out, but the cell aggregates formed by the conventional techniques are not uniform in size, and cell aggregates of various sizes are formed. It was a culture system in which aggregates were mixed. Therefore, the number of cells, the size of cell aggregates, and the distribution thereof are non-uniform for each culture lot, and it was extremely difficult to always evaluate under constant conditions.
【０００５】例えば、ラット肝細胞におけるアルブミン
の合成能等の細胞機能の発現は、細胞凝集体内部と表面
部では異なる可能性が高く、細胞凝集体の大きさにより
細胞あたりのアルブミン合成能が異なってくる。又、細
胞の耐γ線性は細胞凝集体の内部と表面部では異なり、
内部ほど耐γ線性が高くなるため、細胞凝集体の大きさ
により耐γ線性の評価結果が異なってくる。[0005] For example, the expression of cell functions such as albumin synthesizing ability in rat hepatocytes is likely to be different inside and on the surface of the cell aggregate, and albumin synthesizing ability per cell is different depending on the size of the cell aggregate. Come on. Also, the γ-ray resistance of cells differs between the inside and the surface of cell aggregates,
Since the γ-ray resistance is higher toward the inside, the evaluation result of the γ-ray resistance varies depending on the size of the cell aggregate.
【０００６】一方、前述のＩＰＡＡを培養基材に用いる
方法では、まず細胞を培養容器にコンフルエント（敷石
状に、培養面に細胞が密に接着増殖した状態）に培養
し、それを低温下に移してＩＰＡＡを溶解することによ
って基材から剥離させ、細胞が自然に細胞凝集体を形成
するのを待つ方法であり、これは、培養容器の底面積に
よって細胞凝集体の大きさを制御できる可能性を唯一有
している。しかしながら、低温で溶解した不必要なＩＰ
ＡＡが培養液中に溶解しており、種々の悪影響の原因と
なる可能性が高い。また、このようにして形成した細胞
凝集体は、一旦溶解してＩＰＡＡのなくなった基材に接
着し、次第に細胞凝集体が崩れて細胞が再び基材に接着
し増殖を始めるようになってしまうという欠点を有して
いる。On the other hand, in the above-mentioned method using IPAA as a culture substrate, cells are first cultured in a culture vessel at a confluent state (paving stones, in which cells are closely adhered and proliferated on the culture surface), and the cells are kept at a low temperature. It is a method of transferring and dissolving IPAA to separate it from the substrate, and waiting for the cells to spontaneously form cell aggregates, which can control the size of cell aggregates by the bottom area of the culture vessel. Only has sex. However, unnecessary IP melted at low temperature
AA is dissolved in the culture medium and is likely to cause various adverse effects. In addition, the cell aggregate thus formed once dissolves and adheres to the base material free of IPAA, and the cell aggregate gradually collapses so that the cells adhere to the base material again and start to proliferate. It has the drawback of
【０００７】[0007]
【発明が解決しようとする課題】本発明は従来のこのよ
うな問題点を解決しようとするもので、均一なサイズの
細胞凝集体を多数、同時にかつ容易に形成させる方法を
提供しようとするものである。DISCLOSURE OF THE INVENTION The present invention is intended to solve the above-mentioned conventional problems, and to provide a method for easily forming a large number of cell aggregates having a uniform size at the same time. Is.
【０００８】本発明者らは先に、リン脂質で表面処理し
た基材の上では、足場依存性の接着性細胞やリンパ球等
の血液細胞が殆ど接着しないことを見出し、特願平３−
３３０９４０号に開示したが、この技術を底面がロート
状になった培養容器であるＵ型マルチプレート等に適用
すると、この基材上では細胞が培養中に自然に凝集し細
胞凝集体を形成することを見出し、鋭意研究を進めて本
発明を完成させるに至ったものである。The present inventors have previously found that anchorage-dependent adhesive cells and blood cells such as lymphocytes hardly adhere to a substrate surface-treated with phospholipids, and Japanese Patent Application No.
As disclosed in U.S. Pat. No. 330940, when this technique is applied to a U-shaped multiplate, which is a culture container having a funnel-shaped bottom surface, cells spontaneously aggregate on the substrate during culture to form cell aggregates. Based on this finding, the inventors have conducted earnest research and completed the present invention.
【０００９】[0009]
【課題を解決するための手段】即ち本発明は、底面が角
度１２０度以下のロート状、曲率半径が１０ｍｍ以下の
半球状、またはそれらの底面の中心部を平面にした形状
をなし、容器内面の少なくとも底面が水接触角３０度以
下の親水性を有する培養容器を使用し、該容器内に細胞
を播種し１２時間以上培養することによって、容器１個
あたりに１個の細胞凝集体を形成させることを特徴とす
る細胞凝集体の形成方法である。That is, according to the present invention, the bottom surface has a funnel shape with an angle of 120 degrees or less, a hemispherical shape with a radius of curvature of 10 mm or less, or a shape in which the central portion of the bottom surface is a flat surface, and the inner surface of the container is Using a culture vessel having a hydrophilicity of at least the bottom surface of which has a water contact angle of 30 degrees or less, cells are seeded in the vessel and cultured for 12 hours or more to form one cell aggregate per vessel. And a method for forming a cell aggregate.
【００１０】ここで重要な事は、培養容器の底面の形状
とその表面の親水性である。これら二つの条件のうち、
いずれが欠如しても目的を達成する事は不可能である。What is important here is the shape of the bottom surface of the culture vessel and the hydrophilicity of its surface. Of these two conditions,
It is impossible to achieve the purpose without any of them.
【００１１】本発明において使用する培養容器の基本的
な形状は、図１に示すような底面が角度１２０度以下の
ロート状（断面がＶ字型）（ｂ）、曲率半径が１０ｍｍ
以下の半球状（断面がＵ字型）（ａ）、またはそれらの
底面の中心部を平面にした形状（ｃ）であればいかなる
ものも使用可能であるが、改めて新型の培養容器を成型
しなくても、図２に示すような９６Ｕマルチプレート
（底面が半球状のウエル９６個を有する）や、テラサキ
プレート（底面が図１（ｃ）タイプのマルチプレー
ト）、さらにチューブ（底面が半球状の試験管）等市販
の製品も使用可能である。The basic shape of the culture container used in the present invention is a funnel shape (V-shaped cross section) (b) having a bottom surface of an angle of 120 degrees or less as shown in FIG. 1 and a radius of curvature of 10 mm.
Any of the following hemispherical (U-shaped in cross section) (a) or a shape (c) in which the central portion of the bottom surface thereof is a flat surface can be used, but a new type culture container is molded again. 96 U multiplate (having 96 wells with a hemispherical bottom) as shown in FIG. 2, Terasaki plate (multiplate of the type shown in FIG. 1C), and a tube (hemispherical bottom). Commercially available products such as (test tube of) can also be used.
【００１２】底面がロート状である場合には、その培養
面側の角度を１２０度以下、好ましくは９０度以下とす
るのが良く、１２０度を越えると単一の細胞凝集体形成
が困難になり、１つの容器内に複数個の細胞凝集体が形
成される確率が高くなってしまう。底面が半球状の場合
は、曲率半径が１０ｍｍ以下、好ましくは５ｍｍ以下と
するのがよい。１０ｍｍを越えるとやはり単一の細胞凝
集体の形成が困難になり、複数個の細胞凝集体が形成さ
れる確率が高くなってしまう。また、底面の中心部を平
面状にする場合、平面部が大きすぎると複数個の細胞凝
集体が形成される可能性が大きくなるので、平面部のサ
イズは５０〜１００μｍ以下とするのが好ましい。When the bottom surface is funnel-shaped, the angle on the culture surface side is preferably 120 degrees or less, preferably 90 degrees or less. Above 120 degrees, it becomes difficult to form a single cell aggregate. Therefore, the probability of forming a plurality of cell aggregates in one container increases. When the bottom surface is hemispherical, the radius of curvature is 10 mm or less, preferably 5 mm or less. If it exceeds 10 mm, it is still difficult to form a single cell aggregate, and the probability of forming a plurality of cell aggregates increases. Further, when the central portion of the bottom surface is made flat, the size of the flat portion is preferably 50 to 100 μm or less because if the flat portion is too large, a plurality of cell aggregates may be formed. .
【００１３】本発明における培養容器の材質は特に限定
しないが、培養に使用する少なくとも底面の親水性が水
接触角で３０度以下、好ましくは１０度以下であること
が必要である。３０度を越えると、一部の細胞が基材に
接着したり、複数個の細胞凝集体が形成される現象が発
生し好ましくない。このように培養容器の底面の接触角
を３０度以下にするためには、元々親水性の高いポリヒ
ドロキシエチルメタクリレートや、エチレンビニルアル
コール共重合体等で培養容器自体を成型するのが最も手
短かではあるが、容器全体の親水性が高すぎると、イン
キュベータ内で容器が白濁したり変形して好ましくない
場合もある。The material of the culture vessel in the present invention is not particularly limited, but it is necessary that at least the bottom surface used for culture has a water contact angle of 30 degrees or less, preferably 10 degrees or less. When it exceeds 30 degrees, some cells adhere to the substrate and a plurality of cell aggregates are formed, which is not preferable. Thus, in order to make the contact angle of the bottom surface of the culture container 30 degrees or less, it is the shortest way to mold the culture container itself with polyhydroxyethyl methacrylate, which has originally high hydrophilicity, or ethylene vinyl alcohol copolymer. However, if the hydrophilicity of the entire container is too high, the container may become cloudy or deform in the incubator, which is not preferable.
【００１４】また、親水性が必要なのは培養容器の特に
底面であるから、市販のＵ型マルチプレート等の各ウエ
ルの底面及び側面、少なくとも底面を表面処理して親水
化することによっても目的を達成することが可能であ
る。このための方法も特に限定はしないが、ポリヒドロ
キシエチルメタクリレートや、エチレンビニルアルコー
ル共重合体等を塗布することによって目的を達成するこ
とができる。また、リン脂質やその誘導体を塗布するこ
とも可能である。しかしながら、これらのポリマー等を
塗布する際には、基材が溶解したり変形したりするよう
な溶媒を使用しなことも重要である。市販のＵ型マルチ
プレートを使用する場合、溶媒はアルコール系が好まし
い。Since it is the bottom surface of the culture vessel that requires hydrophilicity, the objective is also achieved by surface-treating the bottom surface and side surfaces of each well such as a commercially available U-type multiplate, at least the bottom surface to make it hydrophilic. It is possible to The method therefor is not particularly limited, but the purpose can be achieved by applying polyhydroxyethyl methacrylate, ethylene vinyl alcohol copolymer, or the like. It is also possible to apply phospholipids or derivatives thereof. However, when applying these polymers and the like, it is also important not to use a solvent that dissolves or deforms the substrate. When a commercially available U-type multiplate is used, the solvent is preferably alcoholic.
【００１５】本発明における培養時間は、細胞の種類や
播種細胞濃度によって異なるが、少なくとも１２時間は
必要である。１２時間未満では、ウエル内での細胞の移
動や細胞同士の接着や増殖が不十分であり、単一の細胞
凝集体を形成することは困難である。The culturing time in the present invention varies depending on the cell type and the seeded cell concentration, but at least 12 hours is required. If it is less than 12 hours, the migration of cells within the well and the adhesion and proliferation of cells are insufficient, and it is difficult to form a single cell aggregate.
【００１６】このようにして培養容器に細胞を播種し細
胞凝集体を形成させる場合、細胞凝集体のサイズの制御
は、使用する細胞によって大きく二通りに分けることが
できる。まず、細胞として株化細胞を使用する場合は、
細胞が基材に全く接着しなくてもある程度自然に細胞が
増殖する。従って、細胞凝集体のサイズは細胞播種数と
培養期間の両方に依存するので、この二つを考慮する必
要がある。また、初代培養細胞を使用する場合には、特
に肝細胞のように、ほとんど増殖しない細胞が多い。こ
の場合には、細胞凝集体のサイズはほとんど細胞播種数
にのみ依存するので、播種数に留意する必要がある。When cells are seeded in the culture vessel in this way to form cell aggregates, the size control of the cell aggregates can be roughly divided into two types depending on the cells used. First, when using cell lines as cells,
Even if the cells do not adhere to the substrate at all, the cells grow to some extent spontaneously. Therefore, the size of the cell aggregate depends on both the number of cells seeded and the culture period, and thus these two must be taken into consideration. In addition, when primary culture cells are used, there are many cells that hardly proliferate, especially hepatocytes. In this case, it is necessary to pay attention to the seeding number, since the size of the cell aggregate almost depends only on the cell seeding number.
【００１７】一般に、細胞凝集体はそのサイズが大きく
なりすぎると、凝集体の中央部にまで培養液や酸素が十
分に供給されずに、凝集体の中央部の細胞が死滅すると
いわれている。このサイズを制御する事は極めて重要で
あるが、そのサイズは細胞の種類によっても、また細胞
凝集体の細胞密度によっても異なり、一概にこのサイズ
を決定することはできない。It is generally said that when the size of a cell aggregate becomes too large, the culture solution and oxygen are not sufficiently supplied to the central portion of the aggregate, and the cells in the central portion of the aggregate die. Although it is extremely important to control this size, it cannot be unconditionally determined because it depends on the cell type and the cell density of cell aggregates.
【００１８】しかし、本発明の方法によれば、使用する
細胞に応じてそのサイズを容易に制御できるため、最良
の細胞凝集体サイズを決定し、細胞の機能を最大限に維
持する事が可能である。更に、多数の一定サイズの細胞
凝集体をそのままの状態で、培地交換のみで永く維持す
ることも可能である。However, according to the method of the present invention, the size can be easily controlled according to the cells to be used, so that it is possible to determine the best cell aggregate size and maintain the cell function to the maximum. Is. Furthermore, it is also possible to maintain a large number of cell aggregates of a constant size as they are for a long time only by changing the medium.
【００１９】更に、ここで重要なことは、株化細胞、初
代培養細胞にかかわらず、播種直後には細胞は一箇所に
は集合しておらず、ランダムに底面に散在している点で
ある。ところが、経時的に、早い場合は１２時間程度、
遅くとも数日後には、細胞自らが自然に容器底面のほぼ
中央に集合し、ほぼ球形の３次元構造を構築し始める。
一般には、細胞浮遊液を遠沈管に入れて遠心し、遠沈管
の底で細胞の凝集塊を作成するセルパック法と呼ばれる
方法があるが、このように人為的に形成した細胞凝集体
はダメージが大きく、機能が低下している場合が多いの
に対し、本発明の方法で形成した細胞凝集体は、全くダ
メージを受けておらず、高い機能を維持している。Further, what is important here is that, regardless of the cell line or primary culture cell, the cells are not gathered at one place immediately after seeding, but are scattered randomly on the bottom surface. . However, over time, if it is early, about 12 hours,
After a few days at the latest, the cells themselves spontaneously aggregate at the center of the bottom surface of the container and start to construct a nearly spherical three-dimensional structure.
Generally, there is a method called cell pack method in which a cell suspension is put into a centrifuge tube and centrifuged, and cell aggregates are created at the bottom of the centrifuge tube.However, cell aggregates artificially formed in this way are damaged. In many cases, the cell aggregate formed by the method of the present invention is not damaged at all, and the high function is maintained.
【００２０】またさらに、例えば９６ウエルのＵ型マル
チプレートを使用して本発明の方法を実施すると、細胞
を播種するだけで、一定期間後には、ほぼ同一サイズ、
同一機能を有する細胞凝集体が、同時に９６個形成され
る。このようにして得られたプレート内の多数の細胞凝
集体を利用すると、プレートリーダー等によって、薬物
や化学物質の毒性の評価や濃度依存性、細胞増殖因子の
評価、研究等様々なアッセイに利用することが可能であ
る。種々の動物実験代替キット等への応用も可能であ
る。また、このようにして得られた多数の均一な細胞凝
集体を、合成高分子のネットワーク内等に組み込むこと
によって、人工臓器の開発や研究にも大きく寄与するも
のと期待される。Furthermore, when the method of the present invention is carried out using, for example, a 96-well U-type multiplate, cells are merely seeded, and after a certain period of time, almost the same size,
96 cell aggregates having the same function are simultaneously formed. By using a large number of cell aggregates in the plate obtained in this way, it can be used in various assays such as evaluation of drug and chemical toxicity, concentration dependence, cell growth factor evaluation, and research using plate readers. It is possible to It can also be applied to various animal experiment alternative kits. Further, by incorporating a large number of uniform cell aggregates thus obtained into a network of synthetic polymers, etc., it is expected to greatly contribute to the development and research of artificial organs.
【００２１】[0021]
【実施例】以下、実施例によって本発明をより具体的に
説明する。 （実施例１及び比較例１〜３）マルチプレート９６Ｕ
（住友ベークライト（株）製スミロンＭＳ−３０９Ｕ
Ｒ）の各ウエルに、レシチン（和光純薬（株）製）の５
％エタノール溶液（濾過滅菌済み）を０．３ｍｌずつ注
入し、溶液を排出後、２４時間風乾して、ウエル内表面
にレシチンを塗布したプレートを作成した（実施例
１）。以上の操作は全てクリーンベンチ内で無菌的に行
った。The present invention will be described in more detail with reference to the following examples. (Example 1 and Comparative Examples 1 to 3) Multi-plate 96U
(Sumitomo Bakelite Co., Ltd. Sumilon MS-309U
R), 5 wells of lecithin (manufactured by Wako Pure Chemical Industries, Ltd.)
0.3 ml of a% ethanol solution (filter sterilized) was injected, and the solution was discharged and air-dried for 24 hours to prepare a plate in which the inner surface of the well was coated with lecithin (Example 1). All the above operations were performed aseptically in a clean bench.
【００２２】一方、比較例として、レシチン末塗布のス
ミロンＭＳ−３０９Ｕ（比較例１）、及びウエル底面が
フラットで表面が細胞接着用処理されたプレート（同社
製スミロンＭＳ−３０９６Ｆ）（比較例２）、ウエル底
面がフラットなプレート（同社製スミロンＭＳ−８０９
６Ｒ）でその表面にレシチンを塗布したプレート（比較
例３）を用意した。On the other hand, as comparative examples, lecithin powder-coated Sumilon MS-309U (Comparative Example 1) and a plate having a flat bottom surface and a surface for cell adhesion treatment (Sumilon MS-3096F manufactured by the same company) (Comparative Example 2) ), A plate with a flat bottom surface (Sumilon MS-809 manufactured by the same company)
6R), a plate (Comparative Example 3) having its surface coated with lecithin was prepared.
【００２３】上記実施例及び比較例のプレートを用い
て、ＨｅＬａ細胞及びラット肝細胞で評価を行った。 １．ＨｅＬａ細胞での評価 各プレートの各ウエルに、１×１０3 ／ｍｌの濃度で、
ＨｅＬａ（株化ヒト子宮頸部癌細胞）を各０．２ｍｌず
つ播種した。３日後に、倒立顕微鏡で全ウエル内の細胞
の形態を観察するとともに、細胞数と細胞生存率を計測
した。Evaluation was carried out on HeLa cells and rat hepatocytes using the plates of the above Examples and Comparative Examples. 1. Evaluation in HeLa cells In each well of each plate, at a concentration of 1 × 10 3 / ml,
HeLa (human cervical cancer cell line) was seeded at 0.2 ml each. After 3 days, the morphology of cells in all wells was observed with an inverted microscope, and the number of cells and cell viability were measured.
【００２４】その結果は表１に示した通りで、単一の細
胞凝集体が全ウエルに形成できたのは本発明によるプレ
ートのみであった。また、ウエル間の細胞数のばらつき
も本発明のプレートが最も小さく、さらに、直径約２０
０μｍの細胞凝集体が形成されていたにもかかわらず、
その生存率は、他とほとんど変わらず極めて良好であっ
た。The results are shown in Table 1, and it was only the plate according to the present invention that a single cell aggregate could be formed in all wells. Further, the plate of the present invention has the smallest variation in the number of cells between wells, and the diameter is about 20.
Despite the formation of 0 μm cell aggregates,
The survival rate was very good, almost the same as the others.
【００２５】[0025]
【表１】 [Table 1]
【００２６】２．ラット肝細胞での評価 コラゲナーゼ潅流法によりラット肝細胞を採取し、プレ
ートの各ウエルに１．５×１０5 ／ｍｌの細胞濃度肝細
胞を０．２ｍｌ（３×１０4 ／ウエル）ずつ播種した。
２日毎に培地交換を行いながら培養を行い、７日後に倒
立顕微鏡で全ウエル内の細胞の形態を観察するととも
に、アルブミンの合成量を測定した。2. Evaluation in rat hepatocytes Rat hepatocytes were collected by the collagenase perfusion method, and 0.2 ml (3 × 10 4 / well) of 1.5 × 10 5 / ml cell concentration hepatocytes were seeded in each well of the plate. .
Culturing was performed while changing the medium every 2 days, and after 7 days, the morphology of cells in all wells was observed with an inverted microscope and the amount of albumin synthesized was measured.
【００２７】その結果は表２に示した通りで、各ウエル
のアルブミン濃度は、実施例では濃度が高く、かつばら
つきが小さく、各ウエルでの細胞機能が維持されてお
り、その発現状態がかなり均一であることを示してい
る。The results are shown in Table 2, and the albumin concentration in each well was high in the examples and had little variation, and the cell function in each well was maintained, and the expression state thereof was considerably high. It shows that it is uniform.
【００２８】[0028]
【表２】 [Table 2]
【００２９】[0029]
【発明の効果】本発明の方法によれば、培養の進行と共
に自然に細胞が容器底面の中央部に集合し、ほぼ球形の
細胞凝集体を形成するので、従来のように培養後に基材
から細胞を剥離する操作が不要で、細胞凝集体がダメー
ジを受けたり機能を低下することがなく、また、細胞凝
集体のサイズを容易に制御することが出来、複数個のウ
エルを有する培養容器（マルチプレート等）を用いれ
ば、ほぼ同一サイズ、同一機能の複数個の細胞凝集体を
得ることが出来て、これらは培地交換するだけでそのま
まの状態で永く維持することも可能であり、各種の分
析、評価、研究等広範囲の用途に利用出来るので、極め
て有用である。EFFECTS OF THE INVENTION According to the method of the present invention, cells are naturally aggregated in the central portion of the bottom surface of the container with the progress of culture to form a substantially spherical cell aggregate, so that the cells can be removed from the substrate after culturing in the conventional manner. There is no need to remove the cells, the cell aggregates are not damaged or their function is not reduced, and the size of the cell aggregates can be easily controlled. Multiple plates etc.) can be used to obtain multiple cell aggregates with almost the same size and function, and these can be maintained for a long time as they are by simply changing the medium. It is extremely useful because it can be used for a wide range of purposes such as analysis, evaluation, and research.
【図１】培養容器の底面の形状を示す断面図で、（ａ）
は底面が半球状、（ｂ）はロート状、（ｃ）はロート状
底面の中心部を平面にした形状を示す。FIG. 1 is a cross-sectional view showing the shape of the bottom surface of a culture container, (a)
Shows a bottom surface having a hemispherical shape, (b) shows a funnel shape, and (c) shows a funnel-shaped bottom surface having a flat central portion.
【図２】マルチプレートの一例を示す図で、（ａ）は斜
視図、（ｂ）は（ａ）図のＡ−Ａ′断面の一部拡大図で
ある。2A and 2B are views showing an example of a multi-plate, in which FIG. 2A is a perspective view, and FIG. 2B is a partially enlarged view of a cross section AA ′ in FIG.
Claims (5)
率半径が１０ｍｍ以下の半球状、またはそれらの底面の
中心部を平面にした形状をなし、容器内面の少なくとも
底面が水接触角３０度以下の親水性を有する培養容器を
使用し、該容器内に細胞を播種し１２時間以上培養する
ことによって、容器１個あたりに１個の細胞凝集体を形
成させることを特徴とする細胞凝集体の形成方法。1. A bottom surface has a funnel shape with an angle of 120 degrees or less, a hemispherical shape with a radius of curvature of 10 mm or less, or a shape in which the center of the bottom surface is a flat surface, and at least the bottom surface of the inner surface of the container has a water contact angle of 30 degrees. A cell aggregate characterized in that one cell aggregate is formed in each container by using the following culture vessel having hydrophilicity and inoculating cells in the vessel and culturing for 12 hours or more. Forming method.
チプレートであることを特徴とする、請求項１記載の細
胞凝集体の形成方法。2. The method for forming cell aggregates according to claim 1, wherein the culture container is a multiplate having a plurality of wells.
する、請求項１記載の細胞凝集体の形成方法。3. The method for forming cell aggregates according to claim 1, wherein the culture container is a tube.
ヒドロキシエチルメタクリレート、もしくはエチレンビ
ニルアルコール共重合体よりなることを特徴とする、請
求項１ないし請求項３のいずれかに記載の細胞凝集体の
形成方法。4. The cell aggregate according to claim 1, wherein at least the bottom surface of the inner surface of the culture vessel is made of polyhydroxyethyl methacrylate or ethylene vinyl alcohol copolymer. Forming method.
脂質またはリン脂質・高分子複合体を塗布したことを特
徴とする、請求項１ないし請求項３のいずれかに記載の
細胞凝集体の形成方法。5. The formation of cell aggregates according to claim 1, wherein at least the bottom surface of the inner surface of the culture container is coated with phospholipid or a phospholipid-polymer complex. Method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5119579A JP2716646B2 (en) | 1993-05-21 | 1993-05-21 | Method for forming cell aggregates |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5119579A JP2716646B2 (en) | 1993-05-21 | 1993-05-21 | Method for forming cell aggregates |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH06327462A true JPH06327462A (en) | 1994-11-29 |
| JP2716646B2 JP2716646B2 (en) | 1998-02-18 |
Family
ID=14764852
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5119579A Expired - Lifetime JP2716646B2 (en) | 1993-05-21 | 1993-05-21 | Method for forming cell aggregates |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2716646B2 (en) |
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